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CANCER VACCINATION AND CHALLENGES – VOLUME 2
DELIVERY STRATEGIES FOR CANCER VACCINE AND IMMUNOTHERAPY
IN THE MANAGEMENT OF VARIOUS CARCINOMAS

Editors: Rishabha Malviya, PhD; Bhupendra Prajapati, PhD; Sonali Sundram, MPharm

Publisher: Apple Academic Press, 2025

DOCUMENT STRUCTURE:

• 11 Comprehensive Chapters
• Complete Text Extraction for RAG Processing
• All Sections, Subsections, Tables, Figures, and References Included

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CHAPTER 1
TITLE: Delivery System for Cancer Vaccine
AUTHORS: Sapna Rathod, Nisarg Patel, and Bhupendra Prajapati
LENGTH: 152,481 characters

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CHAPTER 1
Delivery System for Cancer Vaccine
SAPNA RATHOD,1 NISARG PATEL,1 and BHUPENDRA PRAJAPATI2
1APMC College of Pharmaceutical Education and Research,
Gujarat Technological University, Gujarat, India
2Shree S.K. Patel College of Pharmaceutical Education and Research,
Ganpat University, Kherva, Gujarat, India
ABSTRACT
The most valuable and cost-effective health measure to combat the rise
and spread of diseases is vaccination. Cancer vaccines (CV) serve as
highly promising and beneficial tools for clinical oncologists. Numerous
tumor-associated antigens offer excellent targets for immunotherapy and
vaccination. To ensure the sustained release of antigens to the immune
system, carriers are being utilized for vaccine administration. Researchers
have recently focused on vaccine delivery systems that can be specifically
targeted to tumor tissue, avoiding the challenges associated with systemic
effects. This chapter discusses studies that aim to develop more precise
vaccine delivery systems for neoplasm treatment. Antigen-loaded carriers
have shown promise in targeted therapy by activating the immune system
in a way that triggers a unique immune response to the disease’s antigen.
These carriers can be actively taken up by antigen-presenting cells (APCs).
Various carrier systems are available, including physical approaches and
non-physical approaches such as virus-like particles (VLP)/virosomes, poly­
meric micro- and nanoparticles (NPs), liposomes, archaeal lipid liposomes
(archaeosomes), immune-stimulating complexes, and cell-penetrating
peptides (CPP). The chapter will discuss their potential for future CV devel­
opment as well as their therapeutic potential.

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Cancer Vaccination and Challenges, Volume 2
1.1 INTRODUCTION

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1.1.1 INTRODUCTION TO CANCER
For several decades, cancer has been a leading cause of mortality. However,
developing a successful therapeutic approach for cancer treatment is quite
challenging [1]. Understanding the etiopathology of malignancies and the
processes involved in the spread of cancer cells is crucial. A wide range
of malignant diseases characterized by the uncontrolled proliferation of
abnormal cells are collectively referred to as “cancer.” It is recognized as the
second most common cause of mortality worldwide, following cardiovas­
cular diseases. In 2015, the World Health Organization (WHO) reported that
cancer alone accounted for 7.6 million deaths out of 58 million globally [1].
Studies suggest that by 2030, there will be 15.4 million cancer-related deaths
worldwide, representing an increase of 10 million cases per year.
Chemotherapy, radiation, and surgery are common and effective treat­
ments for cancer. The effectiveness of these treatments varies depending on
the type of cancer. One drawback of chemotherapy is that it targets rapidly
dividing cells without discrimination, resulting in the destruction of both
malignant and healthy cells. The main limitation of radiotherapy and surgery
is that they do not eliminate metastases. Therefore, there is still a need for
more effective and less harmful cancer therapies. Research indicates that
cancer vaccines (CVs) may be more effective than traditional chemotherapy
because they work with the patient’s immune system to inhibit the growth of
cancer cells [2].

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1.1.2 INTRODUCTION TO VACCINES
Since the first testing of Edward Jenner’s smallpox vaccine in 1798, vaccines
have significantly reduced disease morbidity and mortality, potentially
making them the most significant medical innovation ever [3].
While vaccinations have undeniably proven beneficial, advancements in
vector production, transportation, and usability would be highly advanta­
geous. Louis Pasteur’s “three is” paradigm—isolate, inactivate, and inject—
has historically served as the foundation for vaccine development [4, 5].
However, with our improved understanding of immunology, pathology, and
microbiology, vaccine development is shifting towards a more “rational
design” perspective [6, 7]. These rationally designed vaccines typically
consist of minimalistic components (such as subunits or peptides), which

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Delivery System for Cancer Vaccine
makes them less immunogenic but offers advantages in terms of safety and
production costs [8]. It is envisioned that integrating and optimizing these
approaches, along with the implementation of novel dosage and adjuvant
strategies, can help bridge this efficacy gap.

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1.1.3 VACCINES FOR CANCER
CVs function by activating the adaptive immune system, which ultimately
eliminates cancer cells while also providing prophylactic defense by inducing
an immune response against the tumor. This approach reduces the likelihood
of tumor recurrence in cancer cells originating from different organs or organ
systems within the body [9–11]. This is attributed to the innate ability of the
immune system to recognize tumor cells in the body. The antigens act as
immune system stimulators, priming the body to combat cancer cells.
Numerous investigations have identified a wide range of antigens associ­
ated with cancer, some of which are currently utilized as CVs in both clinical
trials and research [9]. Modern technology has made significant advance­
ments in identifying antigens that are specifically recognized by T cells in
tumors. Tumor antigens (TAs) can be categorized into two main groups:
tumor-specific common antigens and tumor-specific unique antigens. Shared
antigens expressed by multiple types of tumor cells are sometimes referred
to as tumor-associated antigens (TAAs). The most effective immunotherapy
agents and/or delivery systems work in conjunction with the most suitable
TAs, which are expressed to varying degrees in normal tissues, to achieve
optimal treatment outcomes [9]. Therefore, selecting an appropriate vaccine
delivery mechanism is crucial for the development of immunological strate­
gies for cancer therapy.

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1.1.4 THERAPEUTIC IMMUNOTHERAPY
The recognition of the necessity for therapeutic anticancer vaccines over
preventive ones has been acknowledged by researchers. Therapeutic vaccines
are administered after the onset of the illness and aim to either completely
halt the growth of malignant cells or control metastases and disease relapse.
The main agents involved in immunological activation against cancer are
antigen-presenting cells (APCs), particularly dendritic cells (DCs). By
targeting DCs in the tumor microenvironment (TME), it becomes possible
to redirect inflammation that promotes tumor growth towards a tumor-
killing mode. DC-specific antibodies and DC activators, in conjunction with

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Cancer Vaccination and Challenges, Volume 2
cancer-specific antigens, induce a robust antigen-specific CD4+ and CD8+
T-cell mediated immune response [12].
After triggering a cell-mediated immune response, cancer cells are
destroyed by interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF­
α), chemokines, and contact-mediated cytotoxicity. Many nanoparticles
(NP)/liposome formulations contain targeted molecules, such as antibodies
and immune modulators, along with tumor-specific antigens, to facilitate
the activation of APCs, primarily DCs. Due to their size and composition,
nanoformulations are readily taken up by DCs, resulting in a T-cell and
antibody response [13]. The majority of therapeutic vaccination techniques
lead to tumor regression through DC-mediated antigen-specific cytotoxic
T lymphocyte (CTL) responses [14]. The cargo (antigen/antibody/Toll-like
receptor (TLR) ligand/cytokines, see Figure 1.1) that these formulations are
designed to deliver is used to further categorize this section.
Over the past 20 years, research into tumor vaccines and immunology,
which are alluring replacements or complements to current cancer treat­
ments, has significantly increased [15]. These approaches aim to stimulate
the patient’s immune system to recognize and eliminate tumor cells. They
offer several important advantages, including the ability to:

• Induce targeted tumor cell death with minimal harm to healthy, non-
  tumor cells;
• Target both primary and secondary metastases by systemically
  inducing anti-tumor immune responses; and
• Generate immunological memory that provides long-lasting protec­
  tion against potential tumor recurrences in the future [16, 17].
  FIGURE 1.1 Mechanistic pathways involved in inducing the immunization by the cancer
  vaccines.

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Delivery System for Cancer Vaccine

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1.2 VACCINE DELIVERY SYSTEMS
1.2.1 PHYSICAL APPROACHES
The type of immunological response that a DNAvaccination induces can vary
depending on its administration method. DNA can be administered intrave­
nously (I.V.), subcutaneously (S.C.), intramuscularly (I.M.), or intranasally
(I.N.) [18]. Several studies have shown that cutaneous administration has
immunological advantages, such as stronger immune responses compared to
conventional delivery methods. However, the outcomes of skin immuniza­
tion have sometimes been unclear due to a lack of delivery systems that
can consistently and accurately deliver vaccines to the skin [19]. The nasal
route of vaccine administration is also receiving attention for its local and
systemic effects. However, there is a lack of efficient vaccine formulations

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and safe delivery methods for intranasal administration. Current research is
investigating particle systems based on polyacrylate polymers to enhance
mucosal immune responses [20]. Standard vaccines typically use intradermal
(I.D.) or subcutaneous (S.C.) inoculations. Intratumoral and/or intra-nodal
vaccination has shown to be more effective than other methods in various
animal models and clinical investigations. In a study reported in “Advances
in Cancer Research,” the sequential administration of primary immunization
S.C. followed by booster vaccination I.V. resulted in more potent anti-tumor
effects compared to either administration method alone [21].
The injection route may be influenced by several factors. Recent discov­
eries have shown that utilizing biolistic approaches, such as the Gene cannon
or Biojector 2000, can increase efficiency. Studies conducted on mice have
revealed that 100 times less DNA is required to elicit an antibody reaction
compared to needle injections [18]. Biolistic and needle injections can result
in different immunological reactions. Gene guns often induce T helper type 2
(Th2) responses with DNA vaccines, while needle injections typically elicit a
Th1 response. This discrepancy may be attributed to the use of higher inject­
able doses. It is important to note that this result is not commonly observed
[18]. Previous research has demonstrated that gene gun delivery using naked
DNA vaccines without any coating caused a Th1-biased immune response,
indicating that Au particles used in gene gun bombardment can alter the
immune response generated [22].
Furthermore, regardless of the route, certain antigens can influence the
immune responses [18]. Some techniques to increase the quantity of antigen
expressing DCs include I.D. injection before laser therapy, I.M. injection
before EP, and I.M. injection with microencapsulated vaccination. The

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following discussion addresses strategies for enhancing gene-based immu­
nization through physical delivery. These approaches are also depicted in
Figure 1.2.
FIGURE 1.2 Different approaches for cancer vaccine delivery by physical methods.
1.2.1.1 TATTOOING
Recently, tattooing has been suggested as a method of physically injecting
deoxyribonucleic acid (DNA) into epidermal cells. This approach shares
similarities with the effective strategy used for smallpox vaccination, as it
appears to expedite the development of robust host defense and effective
immunity. The rapid and sporadic synthesis of antigens following immuniza­
tion may account for this effect. Studies have shown that gene expression
after DNA tattooing is higher compared to intradermal injection and gene
gun delivery [23]. DNA transfer through tattooing seems to result in distinct
patterns of gene expression when compared to intramuscular injection. In
one experiment, injecting 100 g of DNA intramuscularly (I.M.) resulted in
gene expression peaks at least 10 times higher than tattooing 20 g of DNA.
Gene expression increased immediately after getting a tattoo and then gradu­
ally decreased over the next four days. In contrast, elevated levels of gene
expression were observed after I.M. injection of DNA, with a peak after
seven days that may have persisted for up to 30 days. Despite the lower
DNA concentration and reduced gene expression, tattooed DNA induced
more potent cellular and humoral immune responses to antigens compared
to I.M. DNA injection [24].

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Delivery System for Cancer Vaccine
Additionally, the effectiveness of DNA vaccination administered either
by tattoo or intramuscular needle injection has been investigated in rela­
tion to the effects of two additives: cardiotoxin and plasmid DNA (pDNA)
containing the mouse GM-CSF. The human papillomavirus type 16 (HPV16)
gene, encoding the L1 main capsid protein, was employed as a model antigen

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in this investigation. The results showed that intramuscular administration of
molecular adjuvants significantly increases the efficacy of the HPV16 L1
DNA vaccination. Furthermore, tattoo device administration of the HPV16
L1 DNA generated substantially stronger and quicker humoral and cellular
immune responses compared to intramuscular needle delivery paired with
molecular adjuvants. However, when more immediate and powerful immune
responses are required, tattoo delivery of DNA is a practical and affordable
technique that can be applied in the laboratory [23].
Getting a tattoo result in several minor mechanical injuries, followed
by bleeding, necrosis, inflammation, and skin regeneration, which uninten­
tionally boosts the immune system. As a result, tattooing “only” partially
replaces the role of adjuvants [24].

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1.2.1.2 GENE GUN
A gene gun is a biolistic device that allows DNA to directly enter cells by
hitting the target DNA using a nearby chamber. Through ex vivo or in vitro
gene gun technology, various somatic cell types, including primary cultures
and well-known cell lines, have been transplanted as suspension or adherent
cultures [25]. These events lead to the expansion and migration of DCs to
nearby lymphoid tissue, where they prime T cells to respond to the antigen
in a targeted manner [26]. Recently, skin vaccination for melanoma has been
conducted using TAA, human gpl00, and reporter gene assays as experi­
mental platforms [25].
The EGFR protein has been found to be highly expressed in different
types of cancer, including lung, breast, bladder, and colon carcinomas. In a
mouse experiment, immune and anticarcinogenic responses were evaluated
by delivering pDNA encoding the human EGFR extracellular domain using
three different techniques: intramuscular needle injection (I.M.), gene gun
with DNA coated in Au, and gene gun with uncoated DNA. Non-coating
pDNA gene gun injection showed the greatest anti-tumor effects and CTL
activity in an animal lung cancer study. These findings may guide the devel­
opment of a DNA vaccine for use in future human clinical trials. The creation

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Cancer Vaccination and Challenges, Volume 2
of an EGFR DNA vaccine to protect against malignancies that exhibit the
gene may require DNA immunization [22].
Additionally, it has been discovered that the CpG motif, consisting of
cytosine-phosphate-guanine, can modify the Th2-type cytokine environment
that is established in the draining lymph nodes (LN) as a result of gene-gun
bombardment. The findings indicate that the insertion of the CpG motif can
elevate the levels of Interleukin (IL)-12 mRNA in the local LN, regardless
of whether it is administered through intradermal injection, intramuscular

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injection, or gene-gun bombardment. These results suggest that CpG motif
injection induces a T helper type 1 (Th1)-biased environment in the localized
LN. The combination of DNA vaccination and CpG motif serves to activate
the Th1 immune response and functions as a “warning signal” [26].
In contrast to regular intramuscular (I.M.) injection, intradermal (I.D.)
administration using a gene gun has been found to be the most effective
method for administering the human papillomavirus (HPV) DNA vaccine.
Recently, it has been reported that uncarrier bare DNA can be delivered
using a gene pistol under low pressure. Compared to Au particle-coated
DNA immunization, the non-carrier unprotected prophylactic HPV DNA
vaccination effectively reduces local skin injury. By comparing this method
to the Au particle layered HPV DNA vaccine, researchers have determined
that it is also capable of enhancing T cell immunity to the HPV antigen and
improving anti-tumor effects [27].
Recent clinical investigations have utilized a Sig/E7detox/HSP70 DNA
vaccine, known as an HPV16 DNA vaccine, which encodes a signal peptide
linked to an inhibited variant of HPV16 E7 (E7 detox) and joined to HSP70.
In a previous study, the pNGVL4a-Sig/E7 (detox)/HSP70 vaccine was

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administered using three distinct delivery methods: needle intramuscular
injection, biojector, and gene gun. The purpose was to evaluate the antigenic
and anti-tumor responses. The gene gun method of DNA immunization
resulted in the highest number of E7-specific CD8+ T cells compared to
needle intramuscular injection and biojector administrations [28].
1.2.1.3 ULTRASOUND
To facilitate the fusion of DNA in cells, cell membranes can be temporarily
disrupted by ultrasound (US) [29, 30]. Furthermore, the combination of
therapeutic US and microbubble echo contrast agents may enhance the effi­
ciency of gene transfection [31]. This process enables direct and successful

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Delivery System for Cancer Vaccine
transfer of DNA into the cytoplasm. While proteins have been delivered into
cells using this technique [32], antigens have not yet been introduced into
DCs for cancer immunotherapy. In vitro and in vivo studies have demon­

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strated that the use of US technology can facilitate the introduction of naked
pDNA into colon cancer cells. Additionally, intra-tumoral injection of naked
pDNA prior to US treatment enhanced DNA transport and gene expression
in a mouse model of squamous cell cancer.
Currently, US is being investigated in a clinical setting. A CV developed
by Memgen was repeatedly administered intravenously (I.V.) to patients with
chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) in
a phase II study (University of California, San Diego; ID: NCT00849524).
1.2.1.4 ELECTROPORATION
In this method, electrical pulses are applied to the skin, causing temporary
pores to form, and facilitating DNA cellular entrance. The skin regains its
structure once electrical energy is removed and holds the immunogenic
substance due to pore closure.
Historically, milli- and microsecond pulses have been utilized in EP.
Recent investigations have explored direct DNA transfer to the nucleus using
extremely strong nanosecond electrical impulses (10–300 ns) at extremely
high intensities (10–300 kV/cm) [33].
EP can enhance immune responses by increasing protein expression,
releasing inflammatory chemokines and cytokines, and attracting APCs
like macrophages and DCs to the EP site [34]. Compared to levels achieved
by I.M. DNA injection alone, EP-mediated transport of pDNA increases
antigen-specific humoral and cellular immune responses. It has been demon­
strated that in vivo, EP reliably enhances immune responses in both small
and large animals, suggesting its potential application in humans [34, 35].
Subsequent comparisons between EP and US have shown the effectiveness
of transfecting skeletal muscle with exposed pDNA using EP [36].
In recent studies, it has been shown that administering pDNA by EP in
mice alone has proven to be successful as a boosting immunization. This
success may be attributed to the high level of antigen generation achieved by
the EP-booster vaccine. It is noteworthy that this approach has shown greater
success compared to using two DNA with EP doses [37]. This strategy is
particularly appealing as it eliminates the need for two distinct vaccine
types. For example, research has revealed that the dose and volume of the

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Cancer Vaccination and Challenges, Volume 2
vaccination significantly impact the efficiency of priming and tumor protec­
tion in mice when utilizing a DNA vaccine expressing the CTL epitope AH1
from colon cancer CT26 [38].
Animal models have been utilized to investigate an EP-driven DNA
vaccine strategy for the treatment of prostate cancer. To effectively stimulate
anti-tumor immune responses against tumors expressing prostate antigen,
a plasmid encoding the phPSA was administered in vivo via I.M. mediated

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EP [39]. The results demonstrated that the phPSA vaccination therapy
significantly delayed the progression of malignancies and prolonged animal
lifespan.
The researchers have employed a variety of strategies to develop the
optimal HPV DNA vaccine, including fusion of the E6 and E7 tumor anti­
gens (E6/E7), tissue plasminogen activator (tpa) signal sequence, inclusion
of CD40 ligand (CD40L), and Fms-like tyrosine kinase-3 ligand (Flt3L).
E6/E7 antigen-specific CD8(+) T cell responses were decreased when E6
and E7 were fused (E6/E7 (Co)), but the preventative antitumor impact was
increased. In contrast to CD40L-linked HPV DNA vaccination, the Flt3L­
fused HPV DNA vaccine demonstrated improved therapeutic anti-tumor
outcomes and increased E6- and E7-specific CD8+ T cell responses [40].
In phase I/II clinical investigations at Karolinska University Hospital,
Chron Vac-C, a curative DNA vaccine administered to patients previously
infected with the virus to hasten their recovery by boosting immune response,
showed good efficacy when delivered by EP [41]. This clinical trial was
conducted at the Gastroenterology and Infectious Disease Clinic at Sweden’s
Hospital. It was one of the earliest DNA vaccines against infectious diseases
to be administered to people using an EP technique.
To enhance antigen processing and delivery through the MHC class I
pathway and enhance the production of cytotoxic CD8+ T lymphocytes,
it is necessary to localize TAs subcellularly through their interaction with
immunostimulatory molecules such as calreticulin (CRT). Even with current
advancements, finding a reliable mechanism for DNA delivery remains

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crucial to ensure the effectiveness of immunotherapeutic methods. In a study
comparing three vaccination techniques – conventional intramuscular (I.M.)
injection, I.M. delivery mediated by electroporation (EP), and epidermal
gene gun-mediated particle delivery – the pNGVL4a-CRT/E7 (detox) DNA
vaccine was used. The results demonstrated that EP was the most efficient
method for immunizing against E7, as it generated the highest quantity of
E7-specific cytotoxic CD8+ T cells [42].

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Delivery System for Cancer Vaccine
Recently, several HPV DNA vaccines have been successfully adminis­
tered to rhesus macaques and mice model organisms using EP. One such
vaccine is VGX-3100, which contains plasmids targeting the E6 and E7
proteins of HPV subtypes 16 and 18. Currently, it is undergoing Phase I
clinical trials. It is recommended that individuals with a history of CIN 2 or
3 and those who have undergone surgery receive this vaccine [43].
Cyto Pulse is more efficient than previous I.M. EP techniques in specifi­
cally targeting skin cells. Cyto Pulse has developed two clinical vaccine
delivery devices, DermaVax and Easy Vax. Easy VaxTM focuses on the
epidermal layer of the skin and is widely used for prophylactic viral vaccina­
tion. On the other hand, Derma VaxTM primarily targets the dermis layer
of the skin. This method is suitable for situations where strong immune
responses and high doses are desired, such as in gene therapy and CVs.
Derma Vax will be utilized in ongoing or scheduled clinical investigations.
One such study, with the ID NCT01064375, aimed to create a DNA vaccine
for treating colorectal cancer using I.D. EP (El-porCEA). The study evalu­
ated the immunogenicity and safety of a CEA DNA immunization technique
in individuals with colorectal cancer.

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1.2.1.5 LASER
Studies conducted in vitro have demonstrated that a laser beam can impart
energy (for the first time as high as 20 mega eV) to a target cell, changing
the membrane permeability through a local thermal impact. For medicinal
uses, additional energy (up to 250 mega eV) is needed [44]. Recently, this
cutting-edge approach has been characterized as an efficient way to increase
the transfection effectiveness of plasmids injected I.D. and elicit an immune
response that is antigen-specific in both T cells and humoral immunity. This
novel approach was only used in a few trials to show that there is great
promise for developing a therapeutic HPV DNA vaccination [26].
1.2.1.6 JET INJECTORS
Jet injectors are used to drive nebulized medicinal solution or suspension
into the skin, using a spring mechanism or pressurized gases (usually carbon
dioxide, nitrogen, or helium) contained in a small cartridge or large canister.
This method allows the medication to reach the muscles directly or penetrate
deeper into the subcutaneous or intradermal layers.

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1.2.1.6.1 Liquid Jet Injectors
Liquid jet injectors are being developed as both single-use and multi-use
components. For chronic conditions like diabetes, reusable devices are
utilized to administer a daily dose for sustained effect. Replaceable devices,
pre-filled with medication, are used for office-based immunizations, mass
vaccinations, and emergency cases such as treating allergies and migraine
attacks.
These devices pressurize the liquid without the need for a needle, which
creates pores in the skin. Compressed gas or a spring can be used as the
power source. This results in a lower pressure profile, pushing the remaining
liquid into the skin. There is no need to reformulate the medication as it is
already in liquid form.
Walther et al. [45] have demonstrated that syringe, limited jet injection
of small amounts of exposed pDNA for galactosidase (LacZ), and green
fluorescent protein target gene paradigms can successfully and safely carry
out non-viral gene transfection in various preclinical cancer models.
An analysis of jet-injected tumors, both qualitatively and quantitatively,
revealed efficient gene transcription, increased intratumoral dispersion, and
deep DNA penetration. A phase I gene exchange experiment was conducted
using jet injection to deliver trace levels of pDNA intratumorally, based on
these intriguing preclinical data. The efficiency of gene transfer was assessed
through quantitative and qualitative examination of LacZ expression at the
mRNA and protein levels. The outcomes of this experiment demonstrate
the effectiveness of this method in treating locally accessible metastases
from solid tumors such as melanoma, breast neoplasm, and other types of
malignancies. Preclinical research on the use of jet injectors to deliver small
interfering RNA has also shown potential for additional cancer gene therapy
applications, including DNA vaccination, immunogene therapy, and gene
suppression techniques. Furthermore, the therapeutic in vivo jet-injection
delivery of human TNF-α as a suicide gene for CD significantly decreased
tumor progression.
However, liquid jet injectors have some drawbacks. They require precise
power source control to administer vaccines accurately and reliably to
distinct skin types or different areas of the same person’s skin. The high
velocity of the jet impacts on blood vessels and nerves, causing bleeding and
discomfort that may reduce patient compliance.

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1.2.1.6.2 Solid Dose Injectors
The stability of vaccines delivered in solid form, whether medicinal or
allergenic, eliminates the need for cold chain storage. This allows for the
administration of both the prime dose and booster doses simultaneously,
enhancing patient compliance.
One method for delivering solid formulations is the Powderject method
[46], which utilizes helium-powered devices to propel medication into the
outer layers of the skin at supersonic speeds. The drug particles are acceler­
ated by the pressurized gas in the helium microcylinder, enabling them to
penetrate the skin upon activation. The device is applied directly to the skin
during use. Corgentech and Pfizer are both exploring this technology for the
development of local anesthetics and the delivery of DNA vaccines on Au
carrier particles, respectively.
Another approach is the glide solid dosage injector method, where a hand
actuator pushes the medication. The actuator consists of a small solid rod
containing both the medicine and excipients [47]. When the predetermined
spring force is reached, the actuator automatically initiates and administers
the medication. The pushing motion is crucial, as it ensures consistent and
regulated delivery of the drug to the deep layers of the skin, regardless of
the skin type or location. This distinguishes it from the Powderject method,
where establishing a consistent and effective treatment rate may be chal­
lenging. In cases where multiple doses need to be administered, the actuator
can be designed as a reusable device, with preloaded medication cassettes
for convenience.

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1.2.1.7 MICRONEEDLES (MN)
A microneedle (MN) is composed of multiple microstructured projections
coated with medication or vaccines. It is applied to the skin to deliver
active substances intradermally, bypassing the stratum corneum. Unlike
transdermal administration systems that rely on diffusion, MNs manually
disrupt the epidermis temporarily, facilitating the entry of the medication or
vaccine to its target site. Due to their small size (1 µm in diameter and 1 to
100 µm in length), MNs differ from regular needles. They can be made from
various materials such as metals, silicon, silicon dioxide, polymers, glass,
and others. MNs can be designed to be short enough to avoid nerve endings
while still reaching the stratum corneum, reducing the risk of discomfort,
infection, or injury.

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Hollow MNs, solid MNs, MNs coated with pharmaceuticals, and
dissolving MNs are among the different designs available [48–50]. These
designs enable the administration of medications through various mecha­
nisms, allowing for the investigation of therapeutic timing control [51].
Solid MNs are often utilized for tissue pre-treatment in order to enhance
the transport of therapeutic medicines to the selected tissue [52, 53]. Here,

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the introduction of tiny needles and their subsequent removal create pores in
the tissue that are only a few nanometers wide, allowing the application of
hydrogels or medication solutions later on to reach deeper tissue layers [52,
54]. On the other hand, the MNs may have a layered structure, with the lower
levels transporting the therapeutic chemicals and the upper levels providing
mechanical support for penetration into the target tissue [55, 56]. In this way,
after the MNs are applied, the distinct MN layers will disintegrate, allowing
the therapeutic molecules to flow into the tissue [57, 58].
Cancer immune-based immunizations aim to mobilize the host’s innate
immunity to destroy the tumor tissue [59]. Such methods often focus on the
delivery of vaccines to the skin, the largest immune organ of the body, which
is abundant in APCs, such as macrophages, Langerhans cells, and DCs [60].
These APCs can activate T and B cells, thereby triggering a broader immune
response against tumors [61, 62].
In order to achieve this objective, researchers are investigating the use of
MN patches containing immunostimulatory adjuvants and/or antigens. By
combining methyl vinyl ether and maleic anhydride MNs with OVA-loaded
PLGA NPs, Zaric et al. [63] demonstrated a method to stimulate the immune
system against B16 melanoma tumors expressing OVA. The authors utilized
a water-in-oil double emulsion technique to generate OVA-loaded PLGA
NPs for the MNs in this procedure, followed by the addition of NPs to a
solution of methyl vinyl ether and maleic anhydride (19 by 19 array). These
researchers observed that the MNs were able to penetrate the mouse skin and
reach the dermal layer and nearby DCs in the ex vivo tests. The researchers
also observed that the transfected DCs were able to migrate to the proximal
LN, where they could activate CD8+ T cells and induce the production of
cytokines such as IFN-γ. Furthermore, this immune system activation, trig­
gered by the release of NPs from the MNs, resulted in a 13-day delay in
the growth of B16 melanoma tumors expressing OVA. Similarly, Kim and
colleagues developed an MN patch using Pluronic F127 and polyethylene
glycol (PEG) to co-deliver resiquimod (R848) and TAs simultaneously [64].
Human TLR7 and TLR8 serve as ligands for R848 and are expressed
on immune cells such as macrophages, dendritic cells, and B cells. The

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15
Delivery System for Cancer Vaccine
interaction between R848 and TLR7/8 leads to the production of IL-12,
IFN-γ, and TNF, which subsequently triggers humoral and Th1 immune
responses specific to the antigen [65, 66]. In this study, the use of lymphoma
cells expressing OVA allowed for the concomitant administration of OVA to
focus the immune response on tumor xenografts composed of E.G7-OVA.
A polydimethylsiloxane (PDMS) mold was utilized to deposit the combi­
nation of Pluronic F127/R848 and PEG/OVA, which was then cured at room
temperature under vacuum before being cut into an MN array consisting of
49 pyramid-shaped needles. It was discovered that the MNs could penetrate
the skin of mice and release their payload into nearby cells. The authors
also observed that the dissolved MNs formed nano-micelles, potentially
facilitating the distribution of R848 and OVA to RAW264.7 cells, thereby
inducing macrophage maturation and cytokine release. In vivo experiments
demonstrated that the R848 and OVA-loaded MNs could migrate to lymph
nodes and activate cutaneous APCs.
On the other hand, MNs can be designed to deliver immune therapies
based on antibodies that aim to overcome or bypass the immune suppres­
sive signals sent by tumor cells. Ye and colleagues developed an MN array
composed of hyaluronic acid (HA) to deliver 1-MT and anti-PD1 antibodies
to B16F10 melanoma tumors [67]. By specifically targeting the PD-1 recep­
tors expressed by T cells, aPD1 antibodies can evade the inhibitory signaling
from cancer cells, allowing T cells to activate [68].

— SECTION 33 —
1.2.2 VIRAL AND NON-VIRAL DELIVERY SYSTEMS (NONPHYSICAL)
1.2.2.1 VIRUS VECTORS
1.2.2.1.1 Viral Vector
Viruses can have their genetic material altered to deliver transgenes into
infected cells, as they are inherently immunogenic. However, the effective­
ness of using viral vectors in clinical trials has not yielded similar results as
infectious disease research. Recombinant viruses can express transgenes in
immune cells, particularly antigen-presenting cells (APCs) such as dendritic
cells (DCs) [69]. This leads to increased presentation of tumor antigens
(TAs) to cytotoxic T lymphocytes (CTLs), resulting in more frequent and
intense attacks on tumor cells. The antigens are expressed by the vaccination
vector [70].

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16
Cancer Vaccination and Challenges, Volume 2
Viruses can naturally infect human cells and elicit T cell and humoral

— SECTION 35 —
responses, making them suitable as vaccine delivery methods [71–73].
Certain viruses can be modified to specifically target tumor cells or possess
inherent oncolytic properties. Oncolytic viral therapy (OVT) is widely used
for cancer treatment, as it can selectively lyse tumor cells and induce an
immune response against viral and tumor antigens, leading to the genera­
tion of long-lived memory T cells [74]. Instead of directly lysing cells,
oncolytic viruses can be genetically modified to serve as delivery vehicles
for vaccination therapy. Adenovirus, Herpes simplex virus, Paramyxovirus,
Rhabdovirus, Vaccinia, and other viral strains are viable options for delivery
mechanisms [75].
The adenovirus has been extensively researched as an oncolytic viral plat­
form for treating breast cancer. This is due to the higher expression of E2F-1
in breast cancer cells compared to healthy tissue. The adenovirus genome is
strategically located downstream of the E2F-1 promoter, allowing for selec­
tive virotherapy through the integration of the immunologically regulatory
element IL-15 [76, 77]. Yan et al. demonstrated this replication selective
virotherapy by creating a recombinant adenovirus vector that integrates the
E2F-1 promoter and IL-15 [78]. Another recombinant adenovirus, developed
by Zhu et al. controls the adenoviral E1A gene through the hTERT promoter
and the adenoviral E1B gene through the hypoxia response element (HRE)
promoter. This recombinant adenovirus also includes the IL-24 gene, which
induces tumor-specific apoptosis and inhibits tumor cell growth [79].
Retroviruses, specifically RNA viruses, can be utilized as DNA delivery
vectors for managing malignancies when the viral proteins gag, pol, and
env are removed. Recombinant retroviral vectors have the ability to express
transgenes that are unique to tumor cells [80]. GDEPT (Gene-Directed
Enzyme Prodrug Therapy) employs recombinant retroviruses to convert
inactive medications into active, toxic metabolites specifically within tumor

— SECTION 36 —
cells [81]. The MetXia-P450 retroviral vector has shown promising results
in reducing the growth of malignant tissue in the MDA-MB-231 breast
tumor xenograft model and sensitizing T47D breast cancer cells to cyclo­
phosphamide (CTX) [82, 83]. Rexin-G, a separate vaccination regimen, is
used to treat various solid cancers, including breast cancer [84]. It is based
on recombinant replication-incompetent retroviral vectors and expresses a
synthetic human cyclin G1 transgene that promotes apoptosis and inhibits
angiogenesis [84, 85].
TLRs 3 and 9 can be utilized by parvovirus-H1 to stimulate the human
immune system, which triggers an NF-κB-dependent adaptive immunological

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17
Delivery System for Cancer Vaccine
response [86, 87]. Primary breast tumor cells isolated from breast cancer
patients’ tumors demonstrate a higher affinity for H1PV compared to normal
cells. Recombinant HIPV has also been employed in cancer immunotherapy.
Genetically modified HIPV with increased inflammatory cytokine (IFN-γ
and TNF-α) transgenes have shown therapeutic benefits [88].
The viral vector-DC vaccine and virus vector-CAR T are two crucial

— SECTION 38 —
methods of virus-mediated vaccine delivery. Targeting DCs with transgenic
technology has proven to be an effective strategy for directing the immune
response towards tolerance or immunity [89]. The key advantage of using
viral vectors to genetically modify DCs is the enhancement of DC matura­
tion achieved through this approach [90]. In an ex vivo study by Chen et al.
transmission of DCs with an Ad5 expressing the ErbB-2/neu gene resulted
in curative and protective immunity against a breast cancer cell line that
overexpressed this gene [91].
Recent studies on cancer therapy have focused a lot of attention on using
CAR-modified T cells together with oncolytic viruses to target and direct
solid tumors [92]. The potential to deliver gene-based vaccinations or thera­
peutic transgenes to the tumor microenvironment (TME) of solid malignant
cells has been explored. The interaction of viral vectors with tumor-specific
T lymphocytes has been found to enhance their effector capabilities. Patients
with hematological malignancies have shown significant benefits from CAR-
modified T cells [93]. According to findings by Bajgain et al. co-expression
of the inverted cytokine receptor (4/7ICR), which connects the IL4 receptor
exodomain and the IL7 receptor endodomain, enhanced the anti-cancer
activity of CAR T cells in a breast cancer model in vivo. This was achieved
by transforming the suppressive IL4 signal [94].

— SECTION 39 —
1.2.2.1.2 Bacterial Vectors
Busch and Fehleisen were the first to establish a connection between cancer
and bacteria (Streptococcus pyogenes). They found that erysipelas infection
by this organism in a cancer patient inhibited tumor growth [95]. In the 19th
century, William Coley utilized an attenuated combination of Serratia marc­
escens and Streptococcus to treat bone and soft tissue sarcomas. This concoc­
tion was named Coley’s poison [96]. This discovery motivated researchers
to focus on locating and utilizing bacterial strains or their products for the
treatment of different cancers [97]. In fact, certain bacterial constituents,
including exotoxins, have been found to initiate direct anticancer actions on

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18
Cancer Vaccination and Challenges, Volume 2
tumor cells, rather than having an indirect effect. Using bacteria as a delivery
system for transgenes with therapeutic value offers numerous benefits. In
the anaerobic conditions of malignant cells, specific bacterial species like
Clostridium are highly active and motile [98]. Due to their ability to reach
tumors in hard-to-access areas and the high metabolic activity of tumor
cells, which makes them resistant to chemotherapy, bacteria are considered
potential options for delivering anticancer drugs or gene-based vaccinations
to treat malignancies [99].
The most common species of bacteria used for breast cancer vaccination
are Salmonella typhimurium [100], Listeria monocytogenes [101], Clos­
tridium novyi [102], Clostridium acetobutylicum, Bifidobacterium longum,
Bifidobacterium adolescentis, and Escherichia coli.
Salmonella and Clostridium have reportedly been utilized as oncolytic

— SECTION 41 —
vectors [85, 103]. After the introduction of suppressed S. Typhimurium and
Bifidobacterium longum along with vectors for the synthesis of molecules
like TRAIL, which markedly slowed the growth of the neoplasm, it was
shown that the transcription of apoptotic genes (Suicide genes) in the host
genome increased [104, 105]. By disrupting the cell cycle and triggering
cell death [106] through the production of p53 and Bax, the Azurin protein
generated by Pseudomonas aeruginosa destroys malignant cells and stops
the growth of breast cancer and melanomas.
For the purpose of testing the effectiveness of the cytokine LIGHT (also
known as TNFSF14 or HVEM-L) in the D2F2 breast cancer model in Balb/c
mice, Loeffler et al. created a chimeric S. Typhimurium VNP20009 strain
[107]. Breast cancer growth and metastasis were prevented by S. typhimurium
RE88 in combination with DNA vaccines expressing Fra-1 and IL-18, which
activated immune cells (T cells, Natural killer (NK) cells, and DCs) and
blocked angiogenesis [108, 109]. The progression of 4 T1 breast cancer is
slowed down in vivo by oral administration of transgenic S. typhimurium
SL3261 and human granulocyte/macrophage colony-stimulating factor
(hGM-CSF) [109].
Salmonella strains that have undergone attenuation are commonly used
as live vectors in both human and veterinary medicine. It has been demon­
strated that humans respond favorably to attenuated forms of Salmonella (S.
typhimurium and S. typhi) used for vaccine administration [110].
Gram-positive bacteria Listeria monocytogenes can escape from the
phagosome and enter the cell cytosol by utilizing pore-forming enzymes
such as hemolysin, listeriolysin O, and phospholipase. This provides a
versatile vector for vaccine delivery and enables more efficient gene transfer.

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19
Delivery System for Cancer Vaccine
Numerous studies have shown the effectiveness of L. monocytogenes as a
delivery system for cancer treatment [111]. In a study by Wood et al., a
mouse model of breast cancer was treated using listeria-based vaccinations
targeting CD105 (Lm-LLOCD105A and Lm-LLOCD105B) [112].
The PROSTVAC vaccine, also known as PSA-TRICOM, includes booster
shots of recombinant fowl pox-PSA-TRICOM and a priming shot of recom­
binant vaccinia-PSA-TRICOM (prostate-specific antigen plus a TRIad of
co-stimulatory molecules). In phase II clinical trials, individuals with meta­
static, hormone-refractory prostate cancer received PSA-TRICOM [113].

— SECTION 43 —
1.2.2.1.3 Virus-Like Particles (VLPs)
The highly structured structures known as VLPs, which are extremely
repetitive, are packed with viral capsid proteins. This high density of capsid
proteins provides numerous conformational viral epitopes that can induce
potent immunological reactions [114]. Importantly, VLPs are self-assembled
by viral capsid proteins without any viral pathogenic nucleic acids. Due to
their inability to replicate, they offer a potentially safer alternative to the
attenuated viruses commonly used for immunization.
The first VLP-based vaccination targeted the Hepatitis B virus (HBV)
[115]. In the late 1960s, Dr. Baruch Blumberg serendipitously discovered the
distinctive Australia antigen in the serum of patients with acute and chronic
hepatitis B. He also provided the initial concrete evidence of VLP production
[116]. In the subsequent years, electron microscopy was employed to study
the virus morphology [117–119], and in the early 1980s, HBV VLPs were
assembled for the first time using a yeast expression system [115, 120].
Gamma herpes virus EBV, the first oncogenic virus discovered in
humans, attacks epithelial and B cells. EBV has been associated with various
malignancies, including lymphomas, gastric carcinoma, and nasopharyngeal
carcinoma, which can occur in both immune-competent and immune-compro­
mised individuals. EBV is also the primary cause of mononucleosis [121].
To develop another model of an EBV vaccine, the EBV gp350/220 was fused
with the fusion F protein of the novel disease virus, allowing the gp350/220
ectodomain to be presented in VLP particle form [122]. Chinese hamster
ovary cells were utilized to produce the EBV-VLPs, and BALB/c mice were
immunized with them. This resulted in a persistent and targeted antibody
response that could inhibit EBV infection in vitro, even without the use of
adjuvants [123]. In order to create a polyvalent vaccine, the glycoproteins

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Cancer Vaccination and Challenges, Volume 2
gH/gL or GB, which are essential for EBV entry, the EBV nuclear antigen
1, and latent membrane protein 2 (LMP2), expressed on the surfaces of all
EBV-infected cells, were also incorporated into VLPs. The gH/gL-EBNA1
and GB-LMP2 VLPs were successfully produced in CHO cells without the
use of adjuvants, and it was demonstrated that they induced high neutralizing
antibody titers or EBV-specific T cell responses in BALB/c mice. Therefore,
these EBV-VLPs can serve as an effective therapeutic vaccine for treating
EBV-associated malignancies and as a preventive vaccine against EBV
infection [124]. Some examples of VLPs as CV delivery agents are shown
in Table 1.1.
TABLE 1.1 List of VLP’s as a CV
Cancer Type

— SECTION 45 —
VLP
Antigen Used Adjuvant
References
Melanoma
Polyomavirus
OVA (model
With or without quila­
[125]
antigen), TRP2 saponin adjuvant
Melanoma
E. coli
gp100
–
[126]
Melanoma
CuMVT

— SECTION 46 —
LCMV-
Microcrystalline
[127]
TT830–843
tyrosine (MCT)
Melanoma
Bacteriophage
Melan-A
IFA (Montanide),
[128]
Qβ
topical imiquimod

— SECTION 47 —
+/– IFA
Pancreatic cancer
Baculovirus
mMSLN
–
[129]
(SHIV)
Cervical
Chimaeric
E7 and 16L1
–
[130]
intraepithelial
HPV16-VLPs
neoplasia
Breast cancer
Human

— SECTION 48 —
IGF-1R
–
[131]
parvovirus B19
Pancreatic cancer
SIV
Trop2
Alone or with
[129]
gemcitabine
Hepatocellular
E. coli

— SECTION 49 —
CLDN18.2
–
[132]
carcinoma
1.2.2.1.4 Virosomes
These extremely small, circular, unilamellar lipid membrane vesicles (150
nm) lack the nucleocapsids that typically contain the genetic material of the
original virus. However, they are packed with viral membrane proteins such
as hemagglutinin and neuraminidase, which are found in influenza. These
proteins enable virosome membranes to directly deliver their contents, which

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21
Delivery System for Cancer Vaccine
are specific antigens, to target cells by fusing with immune system cells. This
process triggers a specific immune response, even in the presence of weakly
immunogenic pathogens. Once the antigens have been delivered, the viro­
somes within the cells are completely eliminated. Unlike other liposomal and
proteo-liposomal carrier systems, the immunological characteristics of viro­
somes are strongly influenced by a viral protein that is sandwiched between
phospholipid bilayers. This intercalation not only provides structural stability
and uniformity to virosomal formulations, but also determines whether the
antigen’s epitopes are located on the surface of the virosome (PeviPROTM)
or inside it, thereby influencing the type of immune response induced by
virosome formulations (PeviTERTM) [133]. The use of PeviPROTM trig­
gers an immunological humoral response, resulting in the presentation of an
MHC II antigen due to the breakdown of the antigen in the cell’s endosomes.
In addition to eliciting a CD4+ and CD8+ positive response in vivo, antigens
produced by PeviTERTM can also generate a strong CTL response. The
virosomal encapsulation ensures correct antigen presentation via the MHC
I pathway, as the antigen is naturally delivered into the cytoplasm of the
antigen-presenting cell.

— SECTION 51 —
1.2.2.2 NON-VIRAL VECTORS
1.2.2.2.1 Cationic and Biodegradable Polymers
Pharmaceutical research and industry have recently utilized the capabilities
of cationic and other carbohydrate polymers to regulate the distribution
of drugs, proteins, antibiotics, peptides, DNA, or vaccines [134]. Various
systems, such as poly-lysine and its conjugates, chitosan, polyethylenei­
mine (PEI), lipopolyamines, diethylaminoethyl-dextran (DEAE-dextran),
polyamidoamine (PAMAM) dendrimers, dextranspermine polycations,
and dextranspermine polycations, have been developed in recent years
[134]. Numerous cationic polymers, including dendrimers (highly branched
PAMAM) and chitosans (a biodegradable linear amino polysaccharide),
have been examined for their gene transfer abilities [135].
Furthermore, chitosan can form complexes through electrostatic interac­
tions with negatively charged DNA (polyplexes). Chitosan and chitosan/
DNA nanospheres have recently been produced using an osmosis-based
method [134]. PEI and poly(L-lysine) (PLL) have been extensively studied
as polymers for gene therapy. It is possible to attach targeting ligands and/
or PEG, which provide steric stabilization to gene carriers, to the surfaces of

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22
Cancer Vaccination and Challenges, Volume 2
PEI polymers due to their properties [135]. When pegylated PEI polyplexes
were conjugated with a tumor-specific ligand and administered intravenously,
the transfection effectiveness increased fivefold compared to pegylated
(transferrin-free) PEI polyplexes [136]. Researchers investigated the effects
of cationic polyelectrolytes on malignant cells in vitro and in animals
transplanted with Ehrlich carcinoma or murine leukemia that causes ascites
(L5178Y). Administration of high-molecular-weight PEI, polyvinylamine,
and PPI at neutral pH, as late as 5 days after tumor transplantation, resulted
in a 10 to 40% reduction in tumor growth in solid Ehrlich carcinoma-bearing
mice. The only substance that extended the lifespan of mice with ascites
generated from leukemia cells was PPI. Therefore, cationic polymers may be
useful by directly electrostatically interacting with tumor cells and inducing
a generalized immunological response in the host [137].
In 2013, an oncolytic Ad (Ad-DB7-U6shIL8) (oAd/PNLG) was coated
with a polymer N-(2-aminoethyl)-2-aminoethyl methoxy poly(ethylene
glycol)-b-poly-N-L-glutamate (PNLG) polymer [138].
Another cationic polymer was created in 2014, this time with arginine
moieties specifically designed to promote lower levels and higher levels of
CAR expression [139]. mPEG-PEI-g-Arg-S-S-Arg-gPEI-mPEG (PPSA)
has a bio-reducible disulfide link to reduce cytotoxicity and several arginine
functional moieties to increase transgene expression. The oncolytic Ad
(DWP418) was delivered intratumorally to CAR-negative MCF7 xenograft
mice after being complexed with PPSA (DWP418/PPSA). The outcome
showed that, compared to bare DWP418, the DWP418/PPSA offered more
potent anti-tumor effects.

— SECTION 53 —
1.2.2.2.2 Microparticles (MPs) as Vaccine Delivery Carrier
Micro-scale particulates or capsules, as a vaccine delivery vehicle, offer
advantages over soluble antigens. By utilizing MPs, which can also provide
sustained antigen release, prolonged antigen presentation, and simulta­
neous transfer of the antigen and adjuvant to the same APC, large quanti­
ties of antigens can be delivered to APCs. Adjuvants and antigens can be
co-encapsulated in a single particle, spaced apart, or collectively attached to
its surface. The optimal size range for vaccine distribution that can enhance
and prolong an immune response remains ambiguous, as indicated by the
findings of the referenced studies [140].
The literature contains numerous preclinical studies that employ various
biodegradable particulate formulations as CVs. These particles for CVs have

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23
Delivery System for Cancer Vaccine
been created using well-known poly (a-hydroxy acid)-based polymers, such
as polylactic acid (PLA) and PLGA, as well as more experimental polymers
like polyanhydrides. MPs derived from polyanhydrides have been reported
to possess inherent immune adjuvant properties. Examples of such polyan­
hydrides include 2,6-dioxaoctane and 1,8-bis(p-carboxyphenoxy)-3,6-diox­
aoctane (p-carboxyphenoxy) hexane [141, 142]. Synthetic adjuvants CpG
and OVA were co-loaded onto polyanhydride particles (1–3 μm in diameter)
using a double emulsion solvent evaporation technique [143]. These MPs
provide additional prophylactic protection against an OVA-expressing tumor
threat, as evidenced by slower tumor growth and improved mice mortality
compared to mice vaccinated with soluble OVA and CpG. Mice administered
with these MPs also exhibit higher levels of OVA-specific CD8+ T cells than
mice vaccinated with soluble OVA and CpG [144].
In the context of tumor lysates, CpG demonstrates beneficial effects in
an alternative particle-based vaccination formulation. The crude TA prepara­
tion, resulting from repeated freezing and thawing of tumor cells, whether
they are autologous or allogeneic, can be utilized as a vaccine in soluble
form or encapsulated in particles. Potential cancer vaccines utilizing tumor
lysates are currently under investigation. According to De Temmerman
et al. mice were administered intraperitoneal injections of PLGA (75:25)
MPs containing tumor lysate (from B16.F1 melanoma) and CpG [145].
Consequently, the number of T cells in the spleen significantly increased.
The studies demonstrated that dendritic cells (DCs) in the peritoneal cavity
engulfed the MPs and transported them to the spleen, where they triggered
TH1 immunological reactions, as evidenced by elevated IFN-c production in
isolated splenocytes.
Calcium carbonate or silica-based inorganic MPs are being explored as
vaccine carriers [146, 147]. Human EGFR 2 antigen has been loaded onto
porous silicon MPs, measuring 1 µm in diameter and 400 nm in height [148].
Immunization with antigen-loaded silicon MPs stimulated the development
of a tumor-specific CTL response, extensive infiltration of CD11c+ DCs, and
increased intratumor type I IFN and MHC II expression.
Additionally, the porous silica MP vector (MSV) enables the simulta­
neous administration of the B16-derived antigen Tyrosinase-related protein
(TRP) 2 and the TLR agonists like CpG or MPLA into the same DC [149].
The immunization based on silica MPs may significantly increase the median
survival of tumor-bearing mice by coordinating effective host immune
responses. Inorganic CaCO3 MPs have also been developed to encapsulate
tumor lysates with the intention of using them for individualized anticancer

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24
Cancer Vaccination and Challenges, Volume 2
vaccination. Due to a higher rate of TLR agonist absorption by APCs, the
TLR ligand-adsorbed and antigen-encapsulated CaCO3 MPs outperform the
soluble mixture of TLR agonists and antigens in terms of activating APCs
and boosting the effectiveness of the CV.

— SECTION 56 —
1.2.2.2.3 NPs as Vaccine Delivery System
The utilization of nanoparticulate (NP) systems as a vaccine carrier has the
potential to enhance immunological responses in immune cells by increasing
the efficacy of antigen delivery to specific immune cells [150]. Currently, the
use of orally delivered nanoparticulate systems for vaccines is considered
the most promising approach. In fact, controlling the temporal release profile
may not be as crucial for immunological stimulation as it is for generating a
pharmacologic response at a higher dose [151]. Through chemical modifica­
tion, the vaccination antigen can be either compressed within or conjugated
on the surface of NPs [152, 153]. Antigens delivered using NPs have shown
to be more effective compared to other methods, which could lead to quick
antigen disintegration or a diminished immune response. Table 1.2 lists some
NPs used for cancer delivery. Furthermore, in addition to the site-directed
release of antigens, certain composite NPs also enable sustained release of
antigens to enhance exposure to the immune system. Moreover, several NPs
RNA interference treatments are currently undergoing human clinical studies
to evaluate their effectiveness and safety. Figure 1.3 illustrates different NPs
used for cardiovascular (CV) delivery.

1. Polymeric NPs: Chitosan, gamma polyglutamic acid (γ-PGA), HT,
   and other synthetic and natural polymers such as PEI, PLA, polypro­
   pylene sulfide, acrylic acid-based polymers, and PLGA are included.
   The utilization of polymers including PLGA, PEI, and chitosan has
   shown great potential in pre-clinical and clinical trials. Chitosan
   exhibited much lower toxicity compared to poly-l-lysine and PEI
   [175]. The primary benefits of PLGA/PLA particles for neoplasm
   vaccines and immunotherapies are their ability to act as a non-toxic,
   therapeutic vehicle for the concomitant delivery of antigens and adju­
   vants that can be dispersed over a long period of time. Chitosan NPs
   demonstrated tumor cell selectivity [176]. More studies are focusing
   on modifying chitosan to enhance the selectivity and bioavailability
   of chitosan NPs. Modified chitosan NPs exhibit characteristics such
   as targeting precision, pH sensitivity, and thermo-sensitivity [177]. In

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PAGE 44

Delivery System for Cancer Vaccine
25
TABLE 1.2 Summary of Nanoparticles as Carriers for Vaccine Delivery
Formulation
Antigen
Tumor Model
References
Polymeric NPs
Antigen-DC
Melanoma
[154]
OVA, MPLA, CpG
Breast cancer
[155]

— SECTION 58 —
OVA
E.G7-OVA
[156]
TRP2
Melanoma
[157]
OVA/Gp100/Trp1/Trp2Obsl1Kif18b/Def8/
Melanoma/colon [158]
Reps1/Adpgk/Dpagt1
cancer
Inorganic NPs (Au) OVA

— SECTION 59 —
EG7-OVA
[159]
SIINFEKL peptide
None
[160]
Inorganic NPs
OVA
CpG reduced
[161]
(silica)
B16-OVA
–
–
[162]
Inorganic NPs

— SECTION 60 —
OVA
Melanoma
[163]
(aluminum)
Microneedles
OVA
Melanoma
[164]
Lipid NPs
Synthetic long peptides
None
[165]

— SECTION 61 —
OVA
E.G7-OVA
[166]
MART-1
Melanoma
[167]
gp100 and TRP2
Melanoma
[168]
NY-ESO-1, MAGE-A3, tyrosinase, and TPTE Melanoma
[169]

— SECTION 62 —
L-BLP25, MUC1
Breast cancer
[170]
OVA
None
[171]
Virus-like particles PSA
Prostate
[172]
Virus vaccine
HPV
Cervical
[173]
Peptide or protein
E7 peptide/Trp2
TC-1 tumors/
[174]
conjugated
melanomas
FIGURE 1.3 Different NPs used for cancer vaccine delivery.

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Cancer Vaccination and Challenges, Volume 2
vitro anti-tumor testing of chitosan NPs revealed that the 500 mg/L
concentration inhibited cervical cancer Hela cells by 27%, liver
SMMC-7721 cells by 23%, gastric cancer BGC-823 cells by 29%,
and breast cancer MCF-7 cells by 55% [178].
When dendritic cells (DCs) were exposed to soluble antigens, PLGA-
modified particles, or PLGA NPs with encapsulated antigens, Shen et
al. investigated antigen absorption and CD8+ T cell activation [179].
Reddy et al. developed pluronic-stabilized polypropylene sulfide
NPs that activated DCs by activating the complement cascade and
generating an in-situ danger signal [180]. Liu et al. described a multi-
adjuvant whole-cell tumor vaccination based on NPs for cancer treat­
ment [181]. They also utilized PLGA NPs as a delivery system for
whole-cell tumor vaccination and demonstrated effective suppression
of tumor growth. To address these challenges, polymeric NP-encapsu­
lated curcumin, or nano-curcumin, is currently being explored [182].
In mouse models, intravenous injection of PLGA particles carrying a
TAA peptide (TRP-2) [180–188] and a TLR-4 ligand (7-acyl lipid A)
has shown inhibition of tumor growth with two doses.
Bourquin and colleagues expanded on their earlier research by
utilizing cationized gelatin NPs loaded with CpG [183, 184]. They
demonstrated that mice immunized with OVA and these NPs were
able to generate specialized and protective antitumor immune
responses [184]. Nanoemulsion vaccination was employed to
deliver tumor-specific antigens, resulting in the elicitation of strong
tumor-targeted antibody and CTL responses in mice. This ultimately
provided protection against tumor growth [185–188]. Wei and
colleagues developed melanoma-targeted nanoemulsion vaccines
encapsulating TAAs, MAGE-1, and/or MAGE-3. Additionally,
their formulations included A SEA and HSP70, which are believed
to enhance the development of tumor-specific immunity. Shi et
al. created an immunostimulatory vaccination to prevent stomach
cancer, delivering the CpG and MG7 antigens [185].
In a recent study, mice were vaccinated with γ-PGA NPs loaded with
the TAA EphA2 to assess the level of protection induced against
EphA2-expressing tumor cells [189]. Mice immunized with EphA2­
PGA NPs exhibited increased EphA2-specific CD8+ T lymphocyte
stimulation, target cell breakdown, and reduced overall liver growth,
serving as indicators of tumor protection [190].

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Delivery System for Cancer Vaccine

2. Inorganic NPs: Vaccines based on inorganic materials have received
   extensive research despite their poor biodegradability. Inorganic
   particles have been utilized as adjuvants and antigen delivery carriers
   to enhance immune response [191]. The four most commonly used
   inorganic particle types in vaccinations are aluminum, Ca3(PO4)2,
   silica, and Au.
   Studies have shown that aluminum particles can act as both carriers
   and adjuvants to stimulate the immune system, although they can
   securely bind to antigens and alter their structure, thereby reducing
   the effectiveness of vaccination [192, 193]. Li et al. used OVA and
   the Bacillus anthracis protective antigen protein as model antigens to
   develop aluminum hydroxide NPs that exhibited enhanced antigen-
   antibody responses.
   Calcium phosphate particles are an attractive choice for vaccina­
   tion applications due to their bioresorbability, nontoxicity, adjuvant
   properties, and ease of antigen loading [194].
   Colloidal gold, a solution composed of gold nanoparticles (AuNPs),
   has been utilized as an immunodiagnostic marker and a therapeutic
   agent for cancer treatment. As an effective delivery approach for
   cancer vaccines (CVs), Ojeda and colleagues developed an AuNP
   system with self-assembled monolayers of carbohydrate antigens,
   known as glyconanoparticles [195]. According to Lee et al., AuNP­
   based CVs can enhance the immune response by activating Toll-
   like receptor 9 (TLR-9) on dendritic cells (DCs) [196]. Kang et al.
   investigated the impact of OVA-carrying AuNPs on the transport
   of OVA to lymph nodes (LNs) and the activation of CD8+ T-cell
   responses [167]. Anionic poly I:C and cationic antigen peptides
   were self-assembled on gold NP templates (iPEM-AuNPs), forming
   an immune-potentiating nanosystem without the need for solvents or
   additional structural components, which significantly increased the
   number of antigen-specific CD8+ T cells [168].
   Silica-based particles are a common type of inorganic particle used
   in vaccine development due to their biocompatibility and versatility
   in terms of size and shape [197]. It has been demonstrated that
   mesoporous silica particles function effectively as persistent antigen
   carriers in vivo [198]. Kim et al. developed mesoporous silica rods
   using the OVA model antigen and CSF adjuvant as a controlled

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Cancer Vaccination and Challenges, Volume 2
vaccine delivery system [199]. Mesoporous silica NPs have
been studied for their interactions with various cell lines in vitro,
including HeLa cells [200, 201], 3T3 endothelial cells [202], human
mesenchymal stem cells [203], and human colon cancer cells [204].
The use of silica NPs as a vaccine delivery technology that enhances
the generation of antigen-specific B and T cell responses, thereby
inducing protective anticancer immunity, has been documented by
Myungi and colleagues [205] in a mouse tumor model.

3. Magnetic NPs: Hai-Yan Xie, Wei, and their team reported a
   magnetic cancer nanovaccine that exhibits significantly improved
   lymph node retention under the influence of a magnetic field,
   leading to enhanced vaccine effectiveness against carcinoma [206].
   When a magnetic field was applied, CD8+ DCs in the lymph nodes
   showed a significant increase in the uptake of anti-CD205-decorated
   magnetosomes [207].
4. Dendrimers: These are synthetic polymers that typically form
   spherical macromolecules and have a structure composed of chains
   that repeatedly branch. These dendrimers consist of three distinct
   domains of structure: terminal groups, recurrent branching units,
   and a central core that offers variable surface functionalities [208].
   Among the various dendrimer classes, PPI-based dendrimers and
   PAMAM are widely utilized and have garnered significant atten­
   tion [209]. Chahal et al. developed novel alkylated dendrimers by
   treating PAMAM G1 dendrimer with 2-tridecyl-oxirane, resulting in
   the production of nanoparticles encapsulating lipid-anchored PEG
   and mRNA. This technology facilitated the production of antibodies
   and CTLs that specifically recognize the antigen [210].
   By utilizing dendrimers, the G250 renal cell carcinoma specific
   antigen is fused to a GM-CSF chimeric molecule, creating an excep­
   tionally effective vaccine that elicits an immune response specifi­
   cally targeted against kidney cancers [211].

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1.2.2.2.4 Liposomes and Lipid Nanoparticles (LNP)
The phospholipid bilayers that constitute the spherical nanovesicles are referred
to as liposomes [174]. The head of the phospholipids is hydrophilic, while
the tail is hydrophobic, enabling them to self-assemble and form liposomes

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Delivery System for Cancer Vaccine
in aqueous environments [212]. Lipid-based technologies, such as liposomes,
are commonly employed in human clinical trials, particularly in cancer gene
therapy [37, 213]. By adjusting the lipid composition, charge, size, and surface
characteristics of liposomes, a versatile NP vaccine delivery system can be
created, offering a wide range of desirable properties [214–216]. Liposomes
have been utilized to transport RNA, DNA, and antigens, enhancing the
immune response to target antigens for cancer vaccines. Liposomal nano­
medicines hold great promise for advanced therapies in areas such as cancer
treatment, vaccine development, and cholesterol management [217].
In 1998, Longenecker’s research team described a 24-mer human mucin
1 (MUC1) peptide liposomal vaccine [218]. This vaccine, which utilized
MPLA as an adjuvant, elicited stronger Th1 responses and provided protec­
tion in mice compared to a KLH-conjugated vaccine. Furthermore, liposomal
co-encapsulation of HER-2/neu63-71 with CpG resulted in significant CD8+
T cell responses and regression of existing tumors [219].
In comparison to PLGA NPs, ISA-51, and emulsions, liposomes have
been found to be more effective in inducing functional antigen-specific
T cells, as indicated by another research [220]. Stimuvax, also known as
BLP25 liposome vaccine or L-BLP25, is a CV developed by Oncothyreon
in collaboration with Merck KGaA. It targets the exogenous core peptide
of MUC1 to stimulate the immune system. MUC1 is a type I membrane
glycoprotein that is extensively expressed in various tumors, including pros­
tate, breast, lung, and colorectal carcinoma [221]. VacciMax (VM) is another
liposome-based technology used in cancer peptide vaccinations [222]. The
AS01 liposomal formulation, which includes MPLA and QS-21, has the
ability to generate CD8+ CTL and antigen-specific antibody responses [223].
A three-part vaccination that combines a short MUC1 epitope, a Th epitope,
and a synthetic triacylated lipopeptide (Pam3CSK4) that activates TLR2 can
be integrated into liposomes and result in high IgG antibody titers in mice,
even without the use of an external adjuvant [224, 225].
Early trials against lung, liver, colon, and rectal cancers have shown
that traditional liposomes with a muramyl dipeptide derivative (ImmTher®)
enhance the cell destroying action of macrophage activity against Ewing’s
sarcoma [226]. A combination of non-UV active Sendai virus liposomes
and replicative protein antigens was developed. These liposomes enable the
addition of Sendai fusion protein to various types of mammalian cells, facili­
tating direct uptake into the cytoplasm [227]. Clinical tests comparing these
vaccinations to traditional liposomes demonstrated a lower risk of cancer.
Zhao et al. conducted experiments using protein antigen carriers to enhance

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Cancer Vaccination and Challenges, Volume 2
immune recognition in mouse models of malignancies using the DNA­
liposome-polycation complex. Due to its high efficiency, it has recently been
investigated as a vaccine adjuvant in experimental testing for delivering the
antibiotic protein HPV16 E7, which is associated with cervical cancer [228].
To create pH-sensitive fusogenic dioleoylphosphatidylethanolamine/egg
yolk phosphatidylcholine liposomes, Eiji Yuba and his colleagues utilized
two different types of poly(glycidol) derivatives that are pH-sensitive and
have either a linear (MGlu-LPG) structure or a hyperbranched (MGlu-
HPG) structure [229]. LNPs, similar to liposomes, are a crucial platform
for biocompatible and biodegradable drug administration [174]. Xu et al.
developed calcium-phosphate-lipid NPs with an uneven lipid bilayer and a
calcium phosphate core for use as a TRP2 peptide vaccine delivery approach
to treat skin cancer. Lipid-calcium-phosphate vaccination stimulated a
potent antigen-specific CTL response compared to free TRP2 peptide/CpG,
resulting in a greater reduction in tumor growth in lung carcinoma models as
well as B16F10 S.C. [159].

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1.2.2.2.5 Immuno-Stimulating Complex (ISCOM)
The particulate antigen delivery vehicles, known as immuno-stimulating
complex (ISCOM®) vaccines, are composed of phospholipids, cholesterol,
immunogens, and saponins. In this study, we demonstrate the advantages
of utilizing ISCOM® technology for developing vaccines with anti-cancer
properties. Through the use of animal models and a model cancer antigen, it
has been observed that ISCOM® vaccines can stimulate CD8 T cell responses
independently of CD4+ T cells, provide protection in three different tumor
models, and enhance Th1-biased immunity. Furthermore, it has been discov­
ered that coupling the vaccine antigen to the ISCOM® structure significantly
improves the first three stages of immune response. Notably, the activation of
CD8 T cells by the ISCOM® vaccine remains unaffected by the presence of
in vivo antibodies against the vaccination antigen. While immunization has
proven effective against tumors expressing the vaccine antigen, no activity
has been observed against neighboring tumor cells expressing the vaccine
antigen negatively. This suggests that significant anti-cancer action is not
achieved through determinant transmission or bystander activation.
As part of the third generation of anti-neoplastic vaccines, which have
yet to undergo substantial human testing, various synthetic non-living adju­
vants and delivery strategies are being developed. These include ISCOM®
vaccines, VLPs, liposomes, MF59, and heat shock proteins (HREs).

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Delivery System for Cancer Vaccine
A different category of immunostimulatory adjuvants consists of plant
extracts rather than chemicals linked to bacterial or viral pathogens. The
bark of Quillaja saponaria in South America yields saponins, which are
soluble in water and are structurally diverse compounds with potent pro-
inflammatory activities. The most commonly used RP-HPLC fraction of
Quillaja saponaria extracts for vaccine development is QS21 [230]. The
triterpene aldehyde group, assumed to be the active site of the adjuvant,
has been found in preclinical studies to induce a strong mixed T-helper 1
(Th1), humoral, and CD8 T cell response. The ASC/NALP3 inflammasome
pathway transforms pro-IL1 and pro-IL-18 into their bioactive forms, which
QS21 has been shown to largely activate. This justifies its combination with
a TLR4 ligand to trigger the upstream expression of the pro-forms. However,
large amounts of QS21 can cause cell membrane lysis and death of antigen-
presenting cells (APCs), suggesting that the intensity and significance of the
resulting immunological response are not directly associated with the extent
of inflammasome activation [231]. QS21 has undergone extensive testing
in cancer immunotherapy vaccine formulations using ganglioside antigens
(GD2, GD3, or GM2). Despite the frequently robust humoral response, there
is no definitive evidence of cell-mediated immunity in individuals. QS21 and
MPLA are combined in the adjuvant formulations AS01 and AS15, similar to
the MAGE-A3 cancer vaccines (GSK).

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1.2.2.2.6 Peptide and Protein Conjugated Delivery System
In comparison to other methods, peptide vaccines have several advantages.
Firstly, since Merrifield’s Nobel Prize-winning invention of solid-phase
peptide production in 1984, the processes of peptide fabrication and puri­
fication have continuously evolved and become automated. Consequently,
numerous contract manufacturing firms now offer high-quality synthetic
peptides for research and therapeutic purposes, which are thoroughly
described and reasonably priced.
Peptides are easier to synthesize and scale up compared to entire proteins
or most other types of antigens. Their synthesis is straightforward, allowing
for the creation of peptide vaccines tailored to specific antigen targets. This
eliminates concerns about proper protein folding or the location of the target
antigen, whether it is surface-bound or intracellular. On the other hand, CAR
T cell therapy requires a focus on surface antigens.
Secondly, peptides represent the simplest and most fundamental biolog­
ical component of a cancer epitope. By utilizing peptide vaccines, vaccine

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Cancer Vaccination and Challenges, Volume 2
strategies with clear mechanisms of action can be developed. Immune
responses can precisely target the desired epitopes. In contrast, identifying
the contents of vaccines such as tumor lysates, which contain a range of anti­
gens, other proteins, and additional tumor components, can be challenging
or even impossible.
However, the modest immunogenicity of peptide vaccines is likely their
greatest drawback. Peptides are often combined with delivery mechanisms
and/or adjuvants, as they are typically insufficient on their own to elicit
adequate immune responses. Despite ongoing research on these systems,
they still require improvement.
Tetanus toxoid is an additional carrier protein that has been used in the
development of peptide conjugate vaccines (CVs). Kunz’s research team
described a synthetic vaccine consisting of tetanus toxoid and a peptide
derived from MUC1. This vaccination significantly increased the level of
antigen-specific immune responses in mice [232]. Subsequently, researchers
combined MUC1 glycoprotein with the Thomsen-Friedenreich antigen and
a difluoro derivative of tetanus toxoid to generate MUC1-specific antibodies
that could bind to the MCF-7 breast cancer cell line [233]. According to
Tsuruma et al., a phase I clinical trial of a survivin-derived peptide vaccine
involved patients with advanced or recurrent colon and rectal cancer [234].
However, this vaccination alone, which contained a peptide derived from
survivin, did not show any discernible clinical effects. According to research
by Jaeger et al. the tumor vaccine utilized tyrosinase, melanoma-associated
peptides from Melan A/MART-1, gp100/Pme117, and influenza matrix
peptide [235].
Peptide sequences or modifications in self-assembling peptide delivery
systems can induce supramolecular assembly. For example, amphiphilic
peptides can be engineered to form micelles, nanoparticles, worm-like
micelles, fibrillary structures, or hydrogel networks [236]. Liu et al. devel­
oped amphiphilic peptide vaccines using TRP2 from melanoma or E7
peptides derived from HPV-16. These vaccines, when combined with the
adjuvant CpG, effectively inhibited the progression of melanoma tumors,
and induced sustained regression of large TC-1 tumors, respectively [237].
Compared to their parent compounds, these vaccines are less harmful to the
body and exhibit enhanced efficacy in combating tumors.
Zhu et al. generated albumin/vaccine nanocomplexes in situ by utilizing
endogenous albumin and self-assembling extrinsic vaccine components
(CpG and SIINFEKL). These nanocomplexes significantly enhanced
the accumulation of CpG and antigens in the lymph nodes [238]. In their

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Delivery System for Cancer Vaccine
research, Wang et al. concluded that albumin improved the stability of anti­
gens in acidic intracellular compartments and facilitated their internalization
by antigen-presenting cells (APCs). Additionally, albumin facilitated the
accumulation of antigens in draining lymph nodes [161].

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1.3 CHALLENGES AND DEVELOPMENTS
Despite numerous advancements and remarkable clinical outcomes, there
are still several challenges that need to be addressed. These include low anti­
tumor efficacy, costly procedures, and adverse effects [239, 240]. Controlled
vehicles (CVs) must possess qualities of being secure, efficient, and cost-
effective. Nanoparticles (NPs) significantly contribute to achieving these
objectives due to their enhanced immunogenicity, ability to target dendritic
cells (DCs), safety, improved antigen absorption, and increased bioavail­
ability. Various physical and chemical characteristics, such as size, targeting
ligand, particle rigidity, zeta potential, and intrinsic immunogenicity, have
been shown to have a significant impact on antigen presentation, cellular
absorption mechanisms, antigen distribution, and the types and strength of
immune responses. Considering these factors is crucial to maximize anti­
tumor responses [241].
However, tumor growth involves a delicate balance between the host
immune system’s induction of an anti-tumor reaction and the immune
system’s repressive microenvironment [242]. CVs and treatments that
reduce the cancer microenvironment (CME) can be combined to enhance the
effectiveness of immunotherapeutic outcomes [243]. Both preclinical and
clinical studies have been conducted in this regard. Treatment options include
modest kinase inhibitors, antibodies, or RNA interference that regulate the
suppressive immunological milieu, as well as the addition of chemotherapy
drugs such as doxorubicin, cisplatin, and docetaxel.
We have described various polymer-based systems in this chapter
that aim to enhance the overall effectiveness of cancer immunotherapies
mentioned earlier. These systems can address the aforementioned limita­
tions of immunotherapies by utilizing suitable polymers, which can further
improve therapeutic efficacy, biocompatibility, and specificity. Polymeric
systems offer a unique delivery strategy for conjugating immune ligands
and loading immunotherapeutic drugs, providing advantages such as low
toxicity, high biodegradability, and customizable surface and size. It is
worth noting that polymeric systems can protect bioactive molecules from

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Cancer Vaccination and Challenges, Volume 2
undesirable immune responses or promote favorable ones that benefit the
body’s condition. However, there are still challenges to overcome before
these polymeric system-mediated immunotherapy delivery approaches
can be implemented in clinical settings, including low therapeutic effects,
immunotherapy resistance, patient safety concerns, and high treatment
costs. Therefore, further research is needed to enhance current distribution
strategies. Additionally, more studies are required to explore the therapeutic
potential of nanomedicine, considering their biocompatibility and the effects
of different polymeric systems on various body parts [244].
Additionally, the future class of polymeric nanoparticle-based immuno­
therapy should be exceptionally multifunctional, possessing the capability
to target, exhibit intelligent responsiveness, and facilitate easy application.
Future research trends will primarily focus on and hold a particularly
optimistic outlook on personalized immune therapy utilizing polymer
nanoparticles.
Peptide-based cancer vaccines can be produced safely, inexpensively,
and effectively. They have demonstrated potential efficacy in targeting both
tumor-specific and tumor-associated antigens. Peptides are particularly
attractive due to their ability to be utilized in multiplexing techniques that
target multiple epitopes and convey the minimum number of biochemical
signals necessary to generate antigen-specific T cells. Addressing the issue
of low potency is one of the primary concerns that peptide drug carriers can
tackle to enhance immune response stimulation. The majority of preclinical
investigations on advanced peptide delivery systems have employed a signif­
icant subset of well-characterized peptide epitopes and cancer cell models.
In clinical and preclinical studies, efforts to standardize peptide dosage may
prove beneficial in facilitating direct comparisons [245].
Fresh peptide variants that exhibit enhanced TCR binding, and MHC
binding can also be synthesized. Utilizing peptide blends that elicit multiple
CD8+ T cell responses in addition to CD4+ responses can enhance T cell
immunity. In terms of MHC restriction, it is conceivable to identify larger
peptide sequences or pools of peptides that can accommodate a broader
range of MHC reactivity, potentially benefiting a larger patient population.
In the near future, we anticipate the persistence of certain patterns in cancer
peptide vaccinations. The development of novel peptide vaccine delivery
systems can be facilitated by the advancements in checkpoint blockade
techniques and neoantigen targeting. Currently, there are several phase I/II
clinical trials underway, investigating the use of a peptide jab in combination
with checkpoint blockade inhibition [246].

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Delivery System for Cancer Vaccine
Advancements in vaccine delivery systems have led to the development

— SECTION 76 —
of NPs, self-assembling peptides, and needle-free delivery methods. In our
phase 1 clinical trial, SCIB1 was administered using EP, as has been done
with previous cancer and infectious disease vaccines. These more recent
delivery technologies offer superior alternatives, although their implementa­
tion has been limited by the discomfort associated with EP administration
and the need for a specific vaccine administration system.
Due to their flexibility, high safety profile, and ability to store minuscule
drug molecules or RNA or peptide antigens, liposomes are being utilized as
delivery vehicles. Optimizing liposomes is necessary to achieve the correct
charge and particle size for effective integration as carriers and transport
across the cell membrane. Cancer antigens can be targeted through various

— SECTION 77 —
methods such as adding a peptide to an adjuvant, encapsulating the antigen
in NPs, or modifying genetic vectors to enhance the local immune response
and increase traffic to LN.
Another crucial technical challenge is the large-scale production of
nanocarriers for antigenic vaccine delivery [247]. Currently, most nanocar­
riers are produced in laboratories in small batches before being scaled up to
larger industrial capacities. Given the high cost of the scale-up process, it is
essential to closely evaluate scale-dependent variables that directly impact
treatment effectiveness. Additionally, the economic viability of the scale-up
process is carefully assessed to prevent any quality crisis [248, 249].
In addition to formulation challenges, significant regulatory barriers
hinder the use of genetic vaccinations for cancer treatment. To obtain regula­
tory approval, it is crucial to address the main formulation concerns related
to the excipients used in generating nanosystems for administering genetic
vaccines and their toxicity profile [249].

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1.4 CONCLUSION
The primary objective of gene therapy is the development of efficient and
safe gene carriers capable of delivering exogenous genetic components into
specific cell types, particularly tumor tissue. The development and evalu­
ation of vectors for delivering therapeutic genes into cancer cells include
both viral and non-viral approaches. Various viruses, such as retroviruses,
poxviruses, herpes simplex viruses, adenoviruses, and adeno-associated
viruses, have been modified to eliminate toxicity while maintaining their
strong gene transfer capabilities.

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Cancer Vaccination and Challenges, Volume 2
Non-viral vectors have gained attention as an alternative to viral vectors
due to the limitations associated with the latter. Biodegradable and cationic
polymers like PEI, PLL, MPs, NPs, peptides, and liposomes are examples of
non-viral vectors.
To enhance transfection efficiency, several innovative transport technolo­
gies and improvements to existing delivery systems have been developed.
The administration method of vaccination can also influence the interaction
between the vaccine and different antigen-presenting cells at the injection
site, thereby impacting the development of the immune system response.

— SECTION 80 —
KEYWORDS

• 
  cancer vaccine delivery systems
• 
  cell-penetrating peptides
• 
  immune-stimulating complexes
• 
  liposomes
• 
  polymeric microparticles
• 
  polymeric nanoparticles
• 
  tumor targeting
• 
  virus-like particles

— SECTION 81 —
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     cancer immunotherapy: A review. Front. Immunol., 13, 826876.
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     A. J., (2014). Thermal and pH sensitive multifunctional polymer nanoparticles for
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     cancer vaccines. J. Clin. Invest., 125(9), 3401–3412.
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     adjuvants, and delivery systems. Front. Immunol., 12, 627932.
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     vaccines. Adv. Therap., 1, 1800060–1800083.
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248. Ragelle, H., Danhier, F., Préat, V., Langer, R., & Anderson, D. G., (2017). Nanoparticle­
     based drug delivery systems: A commercial and regulatory outlook as the field matures.
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     delivery applications of micro- and nanoparticles. Int. J. Pharm., 532(1), 450–465.

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Cancer Vaccination and Challenges, Volume 2: Delivery Strategies for Cancer Vaccine and Immunotherapy in
the Management of Various Carcinomas. Rishabha Malviya, Bhupendra Prajapati, and Sonali Sundram (Eds.)
© 2025 Apple Academic Press, Inc. Co-published with CRC Press (Taylor & Francis)

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#

CHAPTER 2
TITLE: Current Knowledge and Prospects of Various Vaccine Delivery Systems for Lung Cancer Treatment
AUTHORS: Ritika Singh and Chanchal Deep Kaur
LENGTH: 43,530 characters

#

— SECTION 1 —
CHAPTER 2
Current Knowledge and Prospects of
Various Vaccine Delivery Systems for
Lung Cancer Treatment
RITIKA SINGH and CHANCHAL DEEP KAUR
Rungta College of Pharmaceutical Sciences and Research, Raipur,
Chhattisgarh, India
ABSTRACT
A typical occurrence of minor intragenic mutations is responsible for the
diverse characteristics and prognosis of the molecular mechanism under­
lying lung cancer. In this review, we highlight the pulmonary route as a
promising method of medication delivery for the treatment of pulmonary
cancer. Tumors exhibit all the hallmarks of neoplastic growth and are typi­
cally distinguished from other types of growth by their autonomy in devel­
oped indicators, insensitivity to antigrowth signaling, evasion of apoptosis,
genomic instability, ability to invade surrounding tissue and metastasize,
sustained angiogenesis, and involvement of surrounding stroma. Systemic
delivery is required to reach all lung tissues and the entire circulation for drug
access to both localized and distant metastatic lung cancer. The majority of
individuals who develop lung cancer today are either smokers or have had
prolonged exposure to occupational or environmental factors that disrupt the
organization of the epithelial cells in the lungs. Clinical oncologists possess
extensive experience in working with cancer vaccines (CVs). The advance­
ment of innovative techniques for vaccine administration holds the potential
to enhance the success of clinical trials. Cell-penetrating peptides, also
referred to as CPP, present promising candidates for utilization as carriers
in medication administration, gene transfer, and DNA vaccination. DNA

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Cancer Vaccination and Challenges, Volume 2
delivery serves as an effective method for studying gene structure, param­
eters, and functions. We assessed CVs based on cells, peptides, viruses, and
nucleic acids. Local drug delivery strategies aimed at tumor targeting have
the capacity to improve patient endurance and quality of life, enhance drug
accumulation at the targeted site, and reduce the likelihood of systemic side
effects. Currently, it is common practice to consider pulmonary administra­
tion of systemic medications for patients with respiratory and non-respiratory
conditions. Nanoparticles (NPs) that have been PEGylated, peptide- or
receptor ligands-conjugated can selectively deliver drugs to specific tissues
at lower concentrations and with reduced cytotoxicity compared to topical
drug administration. Investigation into local drug administration for lung
cancer.

— SECTION 3 —
2.1 INTRODUCTION
Since the first use of immunizations, medical professionals have discovered
a variety of innovative methods to prevent and treat infectious diseases. In
1796, Edward Jenner made the groundbreaking discovery that the cowpox
vaccination provided protection against smallpox infection [1]. This
discovery is often considered the starting point of the modern era of vaccina­
tions. The success of vaccines in preventing diseases that were previously
difficult or impossible to treat led to their application in combating cancer.
When people hear the term “vaccine,” they often think of anti-infective
vaccines used against viruses and bacteria. These vaccines have historically
prevented epidemics and saved numerous lives [1]. When administered to
a healthy individual, vaccines stimulate the immune system to develop an
artificial response against the pathogen. This is achieved by immunizing the
body with weakened or inactivated infectious organisms. Lung tumors are
currently one of the most frequently treated cancers and a significant contrib­
utor to cancer-related deaths globally [2]. It is one of the most commonly
detected cancers, and unfortunately, the mortality rate remains high despite
recent advances in therapy. The vaccine window for lung cancer is strategi­
cally positioned during the lengthy clinical latency phase to halt carcinoma
development and establish long-lasting anti-carcinoma immune surveillance
in high-risk patients [3]. Our specialized vaccination management can be
utilized to improve pulmonary cancer outcomes and prevent the disease. In
the past decade, significant progress has been made in understanding cancer
histology, utilizing prognostic biomarkers, and developing more effective

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Current Knowledge and Prospects of Various Vaccine Delivery Systems
treatments for lung cancer patients [4]. Despite the increasing number of
non-smokers, lung cancer still accounts for approximately 1.8 million deaths
and 3 million new cases each year [4].

— SECTION 5 —
2.1.1 TYPES OF LUNG CANCER
Based on histopathologial features, lung cancer can be classified into two:
i.
Non-small cell lung cancer (NSCLC); and
ii. Small-cell lung cancer (SCLC).
Lung cancer patients comprise 85%–90% of all cases, with non-small
cell lung cancer (NSCLC) accounting for over 90% of cases. Carcinoma
scientists prioritize NSCLC due to its higher prevalence. NSCLC is typi­
cally treated with radiotherapy, monoclonal antibodies (mAbs), and
surgery. However, NSCLC often exhibits resistance to chemotherapy and
radiotherapy, resulting in poor anticancer integration in distinct tissues and

— SECTION 6 —
undesirable side effects. To address this, simple drug carrier methods for
targeted tumor exposure have been explored to improve lifespan, quality of
life (QoL), local medicine concentration, and minimize systemic side effects.
Nanoparticles (NPs) have emerged as a promising nanostructure for
anticancer drug delivery. Their high surface-to-volume ratio and ability to
encapsulate a variable amount of drugs make them ideal for targeting specific
tissues. In the case of lung cancer, delivering NPs via inhalation has shown
significant advancements. However, conventional inhaled NPs have certain
limitations, which can potentially be overcome by using systemic polymeric
NPs with customized properties. These NPs can be delivered to lung cancer
cells through clathrin-treated endocytosis. Biomaterials, both natural and
synthetic, can be utilized to develop NP-based medication delivery systems.
In addition to NP-based delivery systems, cancer vaccines (CVs) have
shown promise as immune system modulators [5, 6]. This report focuses on
advancements in lung CV delivery technology. Specifically, we will discuss
new vaccine delivery technologies, molecular adjuvants, and immunomodu­
latory agents [7].

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2.2 MECHANISM OF LUNG CANCER ANTIGEN BEHAVING
Mechanism of lung cancer antigen behaviors is represented in Figure 2.1.

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FIGURE 2.1 Mechanism of lung cancer antigen behavior.

— SECTION 8 —
2.3 CONVENTIONAL CHEMOTHERAPY IN LUNG CANCER
Conventional chemotherapy is divided into two types [8, 9]:
i.
Tumor associated antigen (TAA) built carcinoma vaccine; and
ii. Non-mutant peptide-built carcinoma vaccine.
2.3.1 TUMOR ASSOCIATED ANTIGEN (TAA) BUILT CARCINOMA
VACCINE
Curative immunization of lung tumor patients with tumor-associated anti­
gens (TAA) such as cancer testis antigens, transformed antigens, and viral
antigens is a crucial aspect of modern carcinoma vaccines. The goal is to
activate tumor-specific T cells. Transformed antigens, also known as tumor-
specific antigens (TSA) or neo-antigens, are expressed exclusively in cancer
cells. They result from gene alterations that lead to the production of unique,
abnormal proteins. Tumor antigens can also be found in certain normal

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Current Knowledge and Prospects of Various Vaccine Delivery Systems
organs. The most effective curative vaccines are synthetic long peptide
(SLP) derivatives of antigens, delivered with a carrier, as well as DNA
and RNA vaccines targeting MHC-I and MHC-II molecules on specialized
antigen-presenting cells (APC), such as dendritic cells (DC). This approach
stimulates both CD8 and CD4 T-cell responses in affected individuals. The
advent of next-generation sequencing (NGS) technology and in silico epitope
prediction has ushered in a new era of therapeutic vaccinations. This allows
for the identification of patient-specific tumor antigens (TAs) resulting from
somatic alterations in specific malignancies.

— SECTION 10 —
2.3.2 NON-MUTANT PEPTIDE BUILT CARCINOMA VACCINE
The major antigenic peptide derivatives originate from TAAs and are
recognized by CD8 T lymphocytes, which are generated from cleavage in
the proteasome of intracellular proteins. Suppression of major histocompat­
ibility complex-I (MHC-I) molecules has been observed in 17 to 60% of
initial lesions from various forms of cancer in individuals. Furthermore,
between 40% and 89% of human cancerous nodules were found to be
deficient in histocompatibility complex I, including 74% of pulmonary
carcinoma, with a complete absence of group one molecules in 39% and a
locus failure. Previous studies have focused on the suppression of tumor-
associated antigen protein 1 and/or tumor-associated antigen protein 2 in
pulmonary cancer cells, resulting in resistance to T cell receptor-dependent
lysis. Tumor-associated antigen protein deficiencies have been analyzed in
a wide range of carcinomas, with up to 80% of non-small cell lung cancer
expressing lower levels of tumor-associated antigen protein 1 and/or tumor-
associated antigen protein 2 and are associated with uncontrolled growth
and resistance to immune system regulation. Therefore, the development of
alternative approaches for TAA processing may enhance anti-tumor T-cell
responses and T-cell-based immunotherapy strategies [10].

— SECTION 11 —
2.4 TARGETED THERAPY [11]
Dependence on inadequate treatment leads to unfavorable outcomes in
advanced stages of lung cancer. An analysis of treatment options that combine
clinical responses to chemotherapy and radiation is necessary. Additionally,
complementary treatments that target residual viruses after surgical removal
show promising results. New treatments should aim for improved outcomes

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while having a more favorable side effect profile compared to traditional
chemotherapy.
Selective treatment can be categorized into three types:

• Pharmacological agents, including enzyme suppressors and domi­
  nant-negative oligonucleotides;
• Gene transfer; and
• Immunotherapy (passive and active).

— SECTION 12 —
2.4.1 PHARMACOLOGICAL AGENT AND ENZYME INHIBITORS [11, 12]
A significant number of pharmacological agents used to date are gene-active
drugs, which have limited efficacy and significant toxicity. Pharmacological
agents that target various pathways responsible for mitogenesis, survival,
migration, invasion, and angiogenesis (the cancerous phenotype) have been
developed; some may also inhibit malignant transformation. Pharmaco­
logical tyrosine kinase inhibitors (TKIs) have shown remarkable potential
based on current medical research, such as the molecular agents gefitinib and
erlotinib. The cancer cell cycle suppressor pemetrexed and the proteasome
inhibitor bortezomib are both approved for use in other malignancies and
show some promise in the treatment of lung cancer [13]. Clinical trials are
evaluating numerous other molecular agents that target one or more path­
ways essential for cancer development and progression.

— SECTION 13 —
2.4.2 GENE THERAPY
Gene therapy is more of a novel carrier system than an actual therapy itself.
The ability of DNA delivery vectors to deliver a therapeutic gene to an organ,
region, or specific cell type (e.g., benign tumor) can be applied to various
different structures and targeted therapeutic approaches. A key aspect of
DNA-based therapy is the high production of a beneficial protein intracel­
lularly or its release into the local environment, thereby sparing non-targeted
tissues from the effects of the expressed protein. As the term “DNA therapy”
can be confusing, it is important to differentiate between DNA delivery
vectors and DNA-based therapeutic proteins [14].
2.4.3 IMMUNE CELL THERAPY
Immune cell therapy is a concept based on the understanding that the
immune system can differentiate between tumor cells and normal cells. It

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Current Knowledge and Prospects of Various Vaccine Delivery Systems
can be categorized as either passive or active. Passive immune cell therapy
involves the use of biologically active agents administered externally,
without relying on the body’s own machinery. In contrast, active immune
cell therapy utilizes the host’s immune cells and requires a fully functioning
immune system. Anti-inhibitor treatment is a specific type of immune cell
therapy used to counteract factors in both the host and the tumor that provide
a survival advantage in lung cancer.

— SECTION 15 —
2.5 CV PLATFORMS [15]
Cell-based vaccines, peptide-based vaccines, viral-based vaccines, and
nucleic acid-based vaccines, including DNA vaccines and mRNA vaccines,
are different types of cancer vaccines.
2.5.1 INITIAL FORM OF CANCER-BASED VACCINE
Cell-based CVs are created using whole cells or cell waste. These vaccines
carry malignant antigens and elicit a strong immune response [16].
2.5.2 PEPTIDE-BASED CV
Peptide-based vaccines involve the chemical and biosynthetic formulation
of specific tumor antigens (TAs). They induce a robust immune response
against the targeted TAs. When combined with adjuvants, peptide-based
vaccines can effectively enhance immune reactions, making them suitable
for preventing and treating infectious diseases.

— SECTION 16 —
2.5.3 VIRUS-BASED CV
Virus-based CVs utilize viruses that are known to be major contributors to
tumor development. These vaccines can include virus antiserum that lyses
cancer cells or virus vector antiserum. Approximately 13% of cancers are
associated with viral infections, with human gamma herpes virus, HBV,
Hepatitis C virus, and Human papillomavirus being the most common
cancer-related viruses [17]. Inactivated whole virus vaccines have demon­
strated efficacy in treating diseases like COVID-19 or Ebola. It is reasonable

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Cancer Vaccination and Challenges, Volume 2
to expect similar efficacy in the treatment of virus-associated cancers.
However, these vaccines are less frequently used in oncological diseases,
possibly due to challenges in manufacturing and safety concerns.
2.5.4 NUCLEIC ACID VACCINES CARRIER
Nucleic acid vaccines utilize genetic information to encode cancer-specific
antigens in the host, leading to the production of foreign particle proteins
through common physiological processes. These engineered antigens can
then elicit immune responses against cancer cells.

— SECTION 18 —
2.5.5 DNA VACCINES
DNA-based cancer vaccines are constructed using bacterial plasmids that
carry instructions for one or multiple cancer-specific antigens. This approach
aims to induce innate immune responses and modulate the immune reaction.
2.5.6 MRNA VACCINES
mRNA-based cancer vaccines represent a promising immunization strategy.
They involve the delivery of synthetic mRNA into cells to facilitate antigen
production. The expressed antigens are then presented on the surface of
antigen-presenting cells via MHC molecules, stimulating anti-tumor immune
responses [18] (Figure 2.2).
2.6 PHARMACEUTICAL TECHNOLOGIES FOR DIFFERENT

— SECTION 19 —
PULMONARY DRUG DELIVERY [19]
Recent years have witnessed an increase in research on utilizing the pulmo­
nary route for systemic medication delivery to treat both respiratory and
extra-pulmonary disorders. Chemotherapy for lung cancer benefits from the
potential of needleless release of smaller amounts of medication to specific
lung tissues, as well as its self-activating capacity.
2.6.1 LIPOSOME
Liposomes are vesicles with a bilayer structure, typically composed of phos­
pholipids as their building blocks. Depending on their size and composition,
liposomes can be either large (45–95 nm) or small (25–45 nm) unilamellar

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Current Knowledge and Prospects of Various Vaccine Delivery Systems
FIGURE 2.2 Schematic representation of CV platforms.
vesicles with a distinct bilayer. Multilamellar vesicles consist of multiple
concentric bilayers of phospholipids (1.0–5.0 m), while unilamellar vesicles
have a single bilayer. Liposomes can enhance the efficacy of chemothera­
peutics due to their phospholipid bilayer, which enables the transport of both
hydrophilic and hydrophobic drugs. Since phospholipids and cholesterol
are naturally found in lung surfactants and plasma membranes, liposomal
formulations composed of these two components are efficient and biocom­
patible for pulmonary medication administration [20].

— SECTION 21 —
2.6.2 MICELLES
Micelles are nano-scale structures that spontaneously form in aqueous envi­
ronments due to hydrophobic interactions. They consist of an amphiphilic

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Cancer Vaccination and Challenges, Volume 2
core surrounded by a hydrophilic shell. Micelles are created by dissolving
separate polymer chains in water at a concentration and temperature above
the critical point. The choice of polymeric materials is crucial in determining
micelle size, shape, stability, and drug retention. Micelles provide a unique
platform for the co-encapsulation of two different anticancer drugs, thereby
increasing the effectiveness of drug loading and resulting in a synergistic
anticancer effect.

— SECTION 22 —
2.6.3 DENDRIMERS
Dendrimers, also known as dendritic polymers, are tree-like nanoparticles.
The multifactorial exterior covering and hyper-branching tree-like core,
along with vacancies, aid in the encapsulation of medicinal ingredients.
Generation 4 (G4) dendrimers with NSCL cancer targeted peptides have the
potential to enhance drug delivery [21].
2.6.4 POLYMERIC NPS
Therapeutic elements in polymeric nanoparticles can either be encapsulated
within the polymeric matrix or located at the interface of the nanoparticles’
surface, making them a type of colloidal system. In nanospheres, the drug
is uniformly dispersed throughout the nanoparticle, while in nanocapsules,
the drug is enclosed within a hollow sphere surrounded by a polymer shell.
However, an effective drug delivery method requires a polymer that is – (i)
biocompatible; (ii) nonimmunogenic; (iii) easily synthesized and affordable;
(iv) solvent-free; and (v) environmentally friendly in vivo to minimize the
risk of toxicity from accumulating unmetabolized polymeric particles.

— SECTION 23 —
2.6.5 PEPTIDE-BASED NPS
Peptide-based nanoparticles involve attaching small peptides, such as growth
factors and medicinal drugs, to a soluble polymeric backbone through
ecological or separated joints or spacers. Numerous bodily processes rely
on neurotransmitters. The limited clinical application of these naturally
occurring peptide fragments, which contain 20 amino acids, is due to their
poor stability, short half-life in circulation caused by renal ultrafiltration
and serum protein breakdown. However, peptides are promising therapeutic
options as they are small molecules that can easily enter cells and release
medications in response to changes in tissue pH.

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Current Knowledge and Prospects of Various Vaccine Delivery Systems
2.7 AEROSOLES FOR LUNG CANCER TREATMENT
Direct delivery of anticancer medication via inhalation offers numerous
advantages. However, complete drug release through a nano-delivery
system proves to be more effective than predictable intravenous drug release
for pulmonary cancer. The ability of aerosols to deliver anticancer agents
directly to targeted cancer cells allows for increased therapeutic efficacy at
lower drug concentrations [22].

— SECTION 25 —
2.8 VACCINES FOR LUNG CANCER [22]
Table 2.1 summarized the status of the various lung cancer vaccines, while
Figure 2.3 present the mechanism of the lung cancer vaccine.
FIGURE 2.3 Different types and their mechanisms of vaccines for lung cancer treatment
include MAGE A3 (melanoma-associated antigen-A3), L-BLP25 (liposome-based lipopeptide
25), MUC1 (mucin 1), GVAX (granulocyte-macrophage colony-stimulating factor), and CD8
(cluster of differentiation 8).

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Types of Lung

Past Status of Cancer Vaccine

Current Status of Other Cancer

Future Prospects of Cancer

— SECTION 26 —
References
Cancers
Immunotherapy
Vaccine
Lung nodule
When managing the assessment of a
The increasing use of novel
After vaccination, lung nodule
[23]
carcinoma
patient with lung nodule carcinoma,
carrier and immunotherapeutic
carcinoma in the pulmonary
particular attention should be given to
agents has led to the emergence of area can occur in patients with
the source of the cancer.
unique imaging patterns, such as
gastrointestinal cancer, and
pseudo-progression.
the presence of duodenum
cancer can be an indication of
metastatic or stage IV disease.

— SECTION 27 —
NSCLC
Cluster of differentiation 4 (CD4) and
They did not express drug-limiting Vaccine-focused lung cancer
[24]
cluster of differentiation 25 (CD25)
toxicity, with a limited response at requires the transfer of immune
are T cells that have been exposed
the vaccination area.
stimulation and immune inhibi
to transforming growth factor-beta
tion stability to support immune
(TGF-β), which can inhibit immune
stimulation.
responses. This is particularly important
in immune disorders.
Small cell lung
Focusing on the melanoma-associated
The vaccine utilizes the immune
This CV investigation specifi-
[25]
cancer
antigen 3 and Mucin short variant
system to specifically target and
cally focuses on known and
S1 tumor antigen with a cell vaccine
eliminate tumor cells.
unknown tumor-related antigens.
enhances tumor endurance.
Mesothelioma
In the early period, medicine did not
VEGFR1 and VEGFR2 (vascular Continued research for novel
[26]
have satisfactory screening modalities
endothelial growth factor recep
agents and ongoing clinical trials
for the high-risk population.
tors) have been detected in the
are critical.
majority of mesothelioma cases,
and anti-VEGF rabbit polyclonal
antibodies inhibit the growth.
64
TABLE 2.1 Status of Lung CV
­
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Cancer Vaccination and Challenges, Volume 2

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PAGE 84

Types of Lung

Past Status of Cancer Vaccine

Current Status of Other Cancer

Future Prospects of Cancer
References
Cancers
Immunotherapy
Vaccine
Chest wall tumors The patient specified a mutated peptide There are numerous reports
A future challenge concerns
[25]
antigen assumed by a lineage series,
suggesting that a significant
the best way to increase the
which has only turned out to be liable
number of antitumor T-cell
potency of the chest wall tumor
to execute regularly in the particular
responses are directed towards
vaccine using a specific immune
patient.
somatically mutated peptides
adjuvant.
presented by human leukocyte
antigen class I-II.
Squamous cell
Fast and spectacular deterioration of
Focusing on numerous adapted
Immune evasion within the
[26]
carcinoma
numerous pulmonary tumors following peptide epitopes may have led to a tumor microenvironment also
neo-peptide vaccination.
broader immune response through presents a significant limitation
vaccination.
to any immune-based therapy.
Adenocarcinoma The enzyme-linked immunospot assay
The Kristen rat sarcoma vaccine
There was no similarity
[26]
indicated that the vaccinated patient
significantly increased the
observed in the cluster of differ
exhibited robust antigen-specific
number of intra-tumoral cluster of entiation 8+ tumor-infiltrating
interferon-γ-related immune responses. differentiation 4+ T cells compared lymphocytes.
to the adjuvant control.

65
TABLE 2.1 (Continued)
­
Current Knowledge and Prospects of Various Vaccine Delivery Systems

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Cancer Vaccination and Challenges, Volume 2

— SECTION 30 —
2.9 DELIVERY SYSTEMS FOR IMMUNOTHERAPY (LUNG CANCER)
2.9.1 ECOLOGICAL GENE CARRIER TECHNOLOGY
For the improvement of vaccines, the design of proficient direction and tumor
gene treatment is an area of extensive research. Live vectors such as vaccinia
virus, other of viruses, retroviruses, and bacillus Calmette-Guérin have been
specifically developed to deliver genes into cells and are the most commonly
used gene carrier tools in gene therapy [27, 28]. The major advantage of live
vectors is that they produce the foreign protein in its native conformation,
which is crucial for generating neutralizing antibodies and facilitating the
entry of foreign proteins into the MHC class I antigen presentation pathway
for CD8+ T cell priming.
The most effective vaccination strategy may involve combining one
type of immunogen with another, making it stronger. This approach can be
beneficial for several reasons:

• This method may be more efficient in priming naïve cells, while
  another technique may be more successful in stimulating memory
  responses.
• Both different arms of the immune system, such as CD4+ and CD8+
  T cells, can be enhanced using two different techniques.
• Some of the most effective vaccination approaches, such as the use
  of recombinant vaccinia virus or adenoviruses, may have a limited
  duration of effectiveness due to host immune responses.

— SECTION 31 —
2.9.2 NON-ECOLOGICAL GENE CARRIER TECHNOLOGY
Non-infectious vectors must possess the ability to efficiently condense and
protect DNA, target specific cell receptors, disrupt the endosomal membrane,
and deliver the DNA cargo to the nucleus [29]. Non-infectious vectors gener­
ally include naked DNA, DNA-liposome complexes, and DNA-polymer
complexes [30]. In other techniques, non-infectious molecular carriers used
for gene delivery are categorized into microparticles (MPs), nanospheres,
and liposomes [31]. Encapsulating recombinant DNA into micro- or nano­
spheres can provide protection from the environment prior to release and aid
in targeting a specific cell type for efficient delivery.

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2.9.3 PROTONATED LIPOIDS
Lipid-based formulations (e.g., liposomes) are commonly used in human
clinical trials, particularly in anticancer gene therapy [32]. Protonated lipids
are amphiphilic molecules composed of multiple fatty acid side chains, with
the cationic portion being lipid-containing. In aqueous media, protonated
lipids assemble into a lipid bilayer structure. The combination of micro­
spheres/chromosomes is often referred to as a lipoplex. Negatively charged
DNA can interact with protonated liposomes, resulting in aggregation and
stable complex formation over time. Due to their reduced stability, lipoplexes
are typically used immediately after their formation. The development of
new liposomal formulations has led to the production of smaller, more stable,
and uniform lipoplex molecules.

— SECTION 33 —
2.9.4 POLYSACCHARIDES AND PROTONATED GLYCANS
Protonated polyurethane and other related materials have recently been
employed in pharmaceutical assessment and manufacturing facilities due to
their ability to control the release of antibacterial medicines, amino acids,
proteins, oligonucleotides, and drugs [33]. They are also extensively studied
as non-viral DNA delivery systems for gene therapy.
2.9.5 MICRO/NANO-MOLECULE A FURTHER COME UP TO FOR DNA
VACCINE
Carriers utilizing micro-particle-based automation can effectively target
antigen-presenting cells [34]. Chromosome assembly or DNA encapsula­
tion has led to the enhancement of cytotoxic T lymphocyte responses to
engineered proteins [35]. Additionally, environmentally friendly and non-
antigenic poly-lactide micro-particles serve as advantageous immunization
carrier systems. These systems can elicit immune responses against foreign
particles encoded in or conjugated onto the carrier.

— SECTION 34 —
2.9.6 PROTONATED PEPTIDES/CELL INCISIVE PEPTIDES
Protonated peptides and cell-penetrating peptides (CPPs) have also emerged
as valuable tools in immunization strategies. Various natural or synthetic

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Cancer Vaccination and Challenges, Volume 2
CPPs have been identified as efficient agents for delivering therapeutic
cargo into biological compartments. CPPs facilitate the entry of hydrophilic
substances into cells by interacting with the cell membrane. This approach
has been successfully applied to transport drugs, micelles, polysaccharides,
lipoproteins, oligonucleotides, and fluorescent dyes into cells, showing
potential for future immunization techniques [35].

— SECTION 35 —
2.9.7 VIRUSES RESEMBLING MOLECULE AS A PROFICIENT
TRANSMISSION
Virus-like particles (VLPs) have gained increasing attention for their use
as immunization agents due to their repetitive foreign particle arrangement,
which is capable of efficiently stimulating the innate immune response. In
addition to their use as immediate immunogens, VLPs have the potential to
serve as carrier particles for the delivery of epitopes, DNA, and other small
particles targeting different diseases [36].
2.9.8 CARRIER TECHNOLOGY IN DENDRITIC CELL-BASED SYSTEM
Dendritic cells (DCs) are highly efficient macrophages capable of initiating
a primary immune response and have the ability to activate T cells and
promote the proliferation and differentiation of β cells. DCs provide a direct
link between the innate and adaptive immune responses and originate from
bone marrow (BM) precursors that reside in lymphoid tissues, where they
are capable of capturing antigens. DCs migrate from the lymphoid tissues to
the adjacent lymph nodes (LN) and initiate a cellular immune response [37].

— SECTION 36 —
2.9.9 CARRIER TECHNOLOGY IN PROTEIN PEPTIDE IMMUNIZATION
Nucleic acid glycoprotein polysaccharides and other similar units are inad­
equately immunogenic and require auxiliary transport systems or carriers
to enhance the natural immune response following vaccination [38]. For
optimal presentation, antigen delivery vehicles must accurately mimic the
function and immunological distribution of specific microorganisms. They
must actively or passively target antigen-presenting cells such as dendritic
cells, protect the antigenic protein from degradation, modulate the environ­
ment for subsequent immune reactions, and induce antigen-presenting cell

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maturation by interacting with components of the nonspecific immunity
such as Toll-like receptors (TLRs). Several strategies have been reported,
including direct conjugation of TLR agonists as immunostimulatory agents
[28, 40].

— SECTION 38 —
2.9.10 NON-ENVELOPING TECHNIQUE FOR MONITORING IN VIVO
ANTIGEN CAPTURE AND DELIVERY [41]
The main consideration for preventing adverse reactions to immunization
is the quantity of stimulated antigen-presenting cells that capture foreign
particles and transport them to the draining lymph nodes. The utilization
of magnetic resonance imaging (MRI) is an effective approach for these
purposes.
2.10 CHALLENGES AND LIMITATIONS OF LUNG CANCER
THERAPY
The main objective of recent therapeutic lung cancer vaccines is to create a
long-lasting immunological memory in the body against tumor cells, leading
to effective tumor regression while minimizing non-specific or adverse
events. Currently, the clinical focus is on developing effective therapeutic
vaccines that can stimulate a successful and sustained T cell response to
specific tumor antigens. The main challenge in vaccine development is the
identification of precise target antigens that are shared among multiple tumor
types and are uniquely expressed or overexpressed by tumor cells [39].

— SECTION 39 —
2.11 CONCLUSION
In this review, we have studied various types of drug delivery systems using
vaccines for lung (pulmonary) cancer, which pave the way for society as
well as rural areas.
CONFLICT OF INTEREST
We do not have any conflict of interest.

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— SECTION 40 —
KEYWORDS

• 
  cell penetrating peptide
• 
  cytotoxicity
• 
  dendrimers
• 
  nanoparticles
• 
  nanoshells
• 
  neoplastic growth
• 
  pulmonary route
• 
  tumors

— SECTION 41 —
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29. Martin, M. E., & Rice, K. G., (2007). Peptide-guided gene delivery. AAPS J. 9(1),
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32. Luo, D., & Saltzman, W. M., (2000). Synthetic DNA delivery systems. National
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35. Doria-Rose, N. A., & Haigwood, N. L., (2003). DNA vaccine strategies: Candidates for

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immune modulation and immunization regimens. Methods, 31, 207–216.

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38. Pokorna, D., Rubio, I., & Müller, M., (2008). DNA-vaccination via tattooing induces
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    current status and future prospects. Transl Lung Cancer Res. 3(1), 46–52. doi: 10.3978/j.
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41. Chadwick, S., Kriegel, C., & Amiji, M. (2009). Delivery strategies to enhance mucosal
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Cancer Vaccination and Challenges, Volume 2: Delivery Strategies for Cancer Vaccine and Immunotherapy in
the Management of Various Carcinomas. Rishabha Malviya, Bhupendra Prajapati, and Sonali Sundram (Eds.)
© 2025 Apple Academic Press, Inc. Co-published with CRC Press (Taylor & Francis)

— END OF CHAPTER 2 —

#

CHAPTER 3
TITLE: Advance Drug Delivery System for Targeting Carcinomas
AUTHORS: Hitesh Malhotra, Sweta Kamboj, Ashna Gupta, Kartik Babbar, and Rupesh Gautam
LENGTH: 51,998 characters

#

— SECTION 1 —
CHAPTER 3
Advance Drug Delivery System for
Targeting Carcinomas
HITESH MALHOTRA,1 SWETA KAMBOJ,1 ASHNA GUPTA,1
KARTIK BABBAR,1 and RUPESH GAUTAM2
1Guru Gobind Singh College of Pharmacy, Yamunanagar, Haryana, India
2Department of Pharmacology, Indore Institute of Pharmacy,
IIST Campus, Rau, Indore, Madhya Pradesh, India
ABSTRACT
The development of innovative vaccine delivery techniques, such as colloidal
immunostimulatory delivery systems, is a multidisciplinary scientific field
that is currently experiencing rapid progress through immunotherapy and
gene therapy-based treatment. Due to the weak immunogenicity of tumor
antigens (TAs), formulation strategies for cancer vaccines (CVs) play a
crucial role. A colloidal vaccine delivery system can alter the kinetics, body
distribution, uptake, and release of the vaccine. This chapter examines recent
studies focusing on the development of colloidal vaccine delivery systems
for more precise cancer therapies. Various carrier systems, including virus-
like particles (VLPs), polymeric micro- and nanoparticles (NPs), liposomes,
and archaeal lipid liposomes, are utilized. Additionally, several technologies
for drug delivery have been developed to enhance lymphatic channels and
lymph node uptake, resulting in a targeted immune response for cancer
control. Numerous antigens associated with malignancies serve as excellent
targets for immunotherapy and vaccine development. To achieve superior
therapeutic outcomes, optimally developed CVs should combine the most
effective TAs with the best immunotherapy drugs and delivery techniques.
Alongside NPs, platforms for vaccine delivery are gaining increasing

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interest. Furthermore, NP-based vaccine distribution technology holds
great potential for enhancing vaccine immune-gene city. Consequently, this
chapter encompasses a variety of dosage forms currently used to administer
chemotherapeutic drugs to patients with different types of carcinomas.

— SECTION 3 —
3.1 INTRODUCTION
A variety of illnesses are collectively referred to as cancer, which occurs
when malignant cells proliferate uncontrollably and have the ability to invade
or spread to other parts of the body. Cancer is one of the most dangerous
diseases globally, causing millions of deaths each year [1]. Surgical interven­
tion, radiation, chemotherapy, and targeted therapy are the most commonly
employed treatment modalities for cancer [2, 3]. Surgery is often the primary
approach for removing tumor masses, while radiation and chemotherapy,
considered standard treatment options, effectively inhibit tumor growth.
Following surgical removal of tumors, chemotherapy, and radiation are still
commonly used to prolong life or provide temporary relief from symptoms
[4]. However, both procedures can cause significant suffering for patients,
and in some cases, the intensity of pain leads to treatment discontinuation.
Additionally, contemporary chemotherapeutic drugs circulate throughout
the body and affect both cancer cells and normal cells [5]. This lack of selec­
tivity can result in undesired side effects in healthy tissues during treatment.
To selectively administer therapeutically effective concentrations of medi­
cine to the tumor area, targeted drug delivery holds immense potential for
enhancing cancer treatment. These strategies are based on the fundamental
principle of selectively eliminating cancer cells while minimizing toxicity to
normal cells. Despite recent advancements in cancer prevention, diagnosis,

— SECTION 4 —
treatment methods, and public awareness, cancer remains the leading cause of
death worldwide. Cancer is a complex disease that cannot be easily regulated
or tracked. Its progression is not limited to a single node or subset of nodes in
the network of the human body, but rather depends on the individual patient,
necessitating personalized therapies. Several factors hinder effective cancer
treatment and drug development, including inadequate delivery of therapeutic
drugs to the tumor site, non-specific distribution of anticancer agents leading
to severe side effects, and the development of acquired resistance by cancer
cells during chemotherapy, resulting in cross-resistance to multiple drugs [6].
These multifactorial conditions require the development of meticulously
planned strategies within the field of drug research. The conventional
concept of a “magic bullet” drug, which targets a single drug target, should

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Advance Drug Delivery System for Targeting Carcinomas
be replaced by the formulation of a guided vehicle that can deliver the medi­
cine at the precise time and location. This shift would alter the scientific
foundation of medication development. Therefore, the term “targeted drug
delivery” should be replaced with “navigated drug delivery,” as it involves
a vehicle that guides the route of the loaded medication to the intended site
of action, in addition to the cytotoxic agent that targets a specific cellular
area. To enhance the selective absorption of the cytotoxic agent by tumor
cells and spare healthy cells, these drug-loaded and guided vehicles should
have a multidimensional design. The current theme issue aims to address
the challenges posed by the complexity of cancer by presenting a range of
cutting-edge molecularly targeted cancer treatments and innovative drug

— SECTION 6 —
delivery methods [6].
Innovative nanoparticles (NPs) are continuously being developed to
address gaps in the delivery of therapeutic and imaging compounds for the
diagnosis and treatment of cancer. NPs have been considered as potential
carriers to provide an optimal platform for personalized approaches to cancer
diagnosis and therapy in cancer management. When searching for new targets
for cancer therapy, it is important to focus on epigenetics, as carcinogenesis
involves various genetic and epigenetic changes that contribute to the trans­
formation of normal cells into a malignant phenotype. Cancer is considered
both a genetic and an epigenetic disorder. The immune system is designed
to protect the body from microbial invasion and prevent diseases. Therefore,
cancer vaccinations can enhance the immune system’s ability to effectively
combat cancer. One of the main goals is to create vaccines that elicit strong
and targeted T-cell responses. This is achieved by exposing antigens to the
cell surface molecules of dendritic cells (DC), which effectively activate
T-cell responses.

— SECTION 7 —
3.2 DRUG DELIVERY PROCESS
To effectively deliver medications into solid tumors, nanocarriers must
overcome various biological obstacles [7]. For instance, a cancer nano-drug
system is introduced into the bloodstream through intravenous injection
and subsequently distributed throughout the body [8]. The nano-drug then
penetrates deeply into the malignant tissue by leveraging the Enhanced
Permeability and Retention (EPR) effect, leading to the aggregation of
nanoparticles around the tumor. The cancer cells ingest the medication,
causing the nanocarriers to release the cancer nano-drugs. Two factors
contribute to the difficulty of nano-drug penetration into tumor tissues:

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1. Nano-Drug Dimensions: Nano-drugs can range in size from a few
   nanometers to over 100 nanometers. Larger nanoparticles exhibit
   significantly lower diffusion capacity compared to small molecules,
   as diffusion rate is inversely proportional to particle size [9].
2. Pathological Properties of Tumor Tissue: Tumor tissues possess
   exceptionally thick stromal tissue, high cell density, elevated inter­
   stitial pressure, and lack of a capillary network due to uncontrolled
   cell growth. These factors make it extremely challenging for nano­
   materials to penetrate and disseminate within the tumor [10].
   Additionally, many tumors develop hypoxia due to their distance
   from the blood artery network. Hypoxic areas harbor tumor cells
   that exhibit high levels of treatment resistance and have the poten­
   tial to metastasize. Even small amounts of small-molecule medica­
   tions can be found in these areas. Understanding the mechanisms
   of penetration is crucial for improving the delivery of nano-drugs

— SECTION 9 —
into tumor tissues. Different nano-drug delivery methods can
penetrate tumor tissue through various means, such as paracellular
or transcellular transport. The mode of penetration is determined
by the characteristics of the nanocarrier, including its size, surface
charge, cellular uptake of the drug, and modified targeting poly­
peptide. All these factors influence how the drug penetrates tumor
tissues [11].
3.2.1 REMODELING THE MICROENVIRONMENT OF TUMOR TISSUE
The intercellular components of a cell’s cytoplasm and the surrounding
environment of neighboring cells constitute the cellular microenvironment
[12]. Both the environment and the tumor exhibit adversarial characteris­
tics and resist each other, but they are also interconnected and mutually
reinforcing. This understanding is crucial for comprehending the origin,
progression, and metastasis of malignancies, as well as their diagnosis,
prevention, and patient prognosis. Unlike healthy tissues, tumor tissue
demonstrates immunosuppressive conditions, hypoxia, a rigid extracel­
lular matrix (ECM), abnormal vasculature, and a slightly acidic pH [13,
14]. Nano-drugs penetrate tumor cells through the microenvironment of
the tumor tissue, which is of utmost importance. Within tumor microen­
vironments (TMEs), there is a significant presence of tumor-associated

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Advance Drug Delivery System for Targeting Carcinomas
fibroblasts (TAF). These fibroblasts secrete an ECM consisting of collagen,
laminin, and fibrin [15].
Nano-drugs are required to pass through this “barrier” in order to
enter tumor cells. Additionally, fibroblasts [16], immunological cells [17],
vascular endothelial cells [18], stellate cells, and other cells impact the
effectiveness of nano-drugs. Consequently, to enhance the anti-tumor
effect, researchers design carriers that utilize the characteristics of the
cancer tissue microenvironment. When nanomedicines exit the blood
vessels within tumors, the first obstacle they encounter is the dense ECM.
Nano-drugs do not deeply penetrate the tumor tissues due to the signifi­
cantly higher density of the tumoral stroma compared to normal tissue.
The excessive production of the tumor vascular endothelial growth factor
(VEGF) stimulates a high rate of new blood vessels with larger vessel
walls than normal blood vessels in the tumor tissue. Hypoxia, as indicated
by the study, also hinders the growth and spread of blood vessels, resulting
in increased drug resistance and the failure of radiation and photothermal

— SECTION 11 —
therapy (PTT). Various methods, such as increasing blood flow, providing
oxygen, generating oxygen on-site, and minimizing oxygen consumption,
can be employed to alleviate hypoxia. When combined with oxygen carriers,
PTT can enhance blood flow, reduce hypoxia, and address hypoxia-related
tumor radiation resistance. Tumors are characterized by a mildly acidic
pH, which facilitates their spread and migration. Moreover, tumor cells can
secrete immunosuppressive compounds, regulate the activity of immune
effector cells, and restrict the synthesis of regulatory cytokines. This shields
the tumors from cytotoxic T lymphocytes (CTLs) [14], thereby promoting
tumor growth.
3.2.2 NEED FOR TARGETED DRUG DELIVERY
The pursuit of therapeutic agent specificity is inherent in all treatment meth­
odologies (Figure 3.1). Chemotherapeutic and radiotherapeutic alternatives
have been developed to target and eliminate cancer cells, making the speci­
ficity of pharmaceutical action crucial in cancer treatment. These strategies
are based on the fundamental principle of selectively eradicating cancer cells
while minimizing toxicity to normal cells. Complete remission in individuals
with disseminated disease requires the eradication of all cancer cells. This
can be achieved either through direct pharmaceutical action or indirectly
through the bystander impact of therapy [19].

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Cancer Vaccination and Challenges, Volume 2
FIGURE 3.1 Different strategies for cancer treatment.
Chemotherapy regimens alone are ineffective in advanced carcinomas
and may only result in temporary responses. A combination therapy
involving high doses of radiation (60–70 Gy) and continuous infusions of
chemotherapeutic drugs (such as paclitaxel) has been studied for the treat­
ment of locally advanced cancers that cannot be surgically removed. Another
significant challenge is achieving therapeutically effective drug concentra­
tions in the tumor mass for a sufficient duration to allow the agent to work
effectively, particularly in solid tumors. Even after prolonged treatment with
these cytotoxic therapies, residual tumor cells remain due to the inability of
these drugs to penetrate the physiologically diverse tumor mass [20].
The high-dosage treatment required to maintain complete remission
produces unacceptable systemic side effects, leading many patients to
discontinue the medication. These adverse effects have a significant nega­
tive impact on patients’ quality of life (QoL). Due to the limited therapeutic
indices of various treatment alternatives, there has been a quest for effective

— SECTION 13 —
delivery methods for existing medications that can maximize therapeutic
effectiveness while minimizing undesired effects. The utilization of specially
designed drug delivery systems to target pharmaceuticals is a feasible
approach to enhance therapeutic efficacy and reduce systemic toxicity in
anti-cancer therapy. Therefore, the need to develop highly targeted drug

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Advance Drug Delivery System for Targeting Carcinomas
delivery systems (Figure 3.2) arises not only from a therapeutic standpoint
but also to assist patients in eliminating cancer before it becomes fatal [21].
FIGURE 3.2 Types of drug delivery system.

— SECTION 14 —
3.2.3 NPS IN CANCER THERAPY
Since the efficacy of nano-drug delivery and subsequent therapeutic success is
greatly influenced by the sizes, shapes, and surface features of NPs employed
in medical therapy (Table 3.1), these three parameters are frequently consid­
ered in NPs [22]. NPs with a diameter of 10 to 100 nm are commonly used
in cancer therapy because they can effectively transport drugs while also
achieving enhanced permeability and retention (EPR). However, particles
larger than 100 nm are likely to be removed from circulation by phagocytes.
Smaller particles (less than 1–2 nm) can easily leak from normal vasculature
and be filtered by kidneys (less than 10 nm in diameter) [23, 24]. The surface
characteristics of NPs can also impact their bioavailability and half-life. For

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example, NPs coated with hydrophilic substances like polyethylene glycol
(PEG) reduce opsonization and thus evade immune system clearance [25].
Consequently, NPs are often modified to become hydrophilic, prolonging
drug circulation and enhancing drug penetration and accumulation in tumors
[26, 27]. The diverse characteristics of NPs influence their therapeutic effec­
tiveness in cancer treatment.
TABLE 3.1 Different Nanoparticles and Their Applications
Types of Nanoparticles Structure
Applications
Polymeric nanoparticles Polymers such as chitosan,
Delivery system

— SECTION 16 —
PLGA
Tissue regeneration
Liposomes
Lipid bi-layer globules
Drug delivery for hydrophobic
and hydrophilic drugs
Dendrimers
Highly branched ends and
Delivery system, tissue
central core.
engineering, antimicrobials.

— SECTION 17 —
3.2.4 LIPOSOMES
To carry both hydrophobic and hydrophilic drugs, liposomes are vesicles
with one or more concentric phospholipid bilayers separated by water
compartments. They have long been the subject of extensive research [1, 28].
Liposomes have been used to deliver anticancer drugs that do not cause toxic
or allergic reactions, as they are physiologically inactive and biocompatible
[29–31]. Additionally, specific liposomes can be created using appropriate
ligands (peptides, antibodies, etc.) and utilize overexpressed receptors as
docking sites for delivering anticancer medications [28]. Cancer cells that
are EGFR-positive (epidermal growth factor receptor) absorb liposomes
more effectively when treated with cetuximab-ILs. Combining an anti-EGFR
antibody with a chemotherapeutic drug has revealed that an IL containing
5-FU modified by cetuximab can enhance the efficacy of Squamous cell
cancer (SCC). In addition to increasing the penetration of 5-FU into SCCs,
ionophoresis with ILs is a more efficient technique for reducing cell prolif­
eration and invasion compared to subcutaneous administration. This makes
it a more appealing therapeutic strategy for SCC.

— SECTION 18 —
3.2.5 EXOSOMES
Mammalian cells release extracellular vesicles known as exosomes. When
observed under a transmission electron microscope, these exosomes appear

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as cup-shaped nanosized structures (50–100 nm) composed of phospholipids
[32–34]. The surface of exosomes contains various labeled proteins and
ligand proteins, including ALIX, tetraspanins (CD9, CD63, and CD81),
integrins, and cell adhesion molecules (CAM). These proteins indicate that
the exosomes are internal endosomes and enable them to interact with target
cells for cargo delivery.
Exosomes, depending on their properties and origin, can be utilized
to target specific disease tissues and/or organs. They possess durability,
biocompatibility, low immunogenicity, and safety during circulation [32].
Recently, exosome-biomimetic NPs have gained attention as a reliable drug
delivery platform. These NPs are produced using exosomes as natural bioma­
terials and functionalized NPs. However, the repeated physical extrusion or
freeze/thaw cycles involved in the production of exosome-biomimetic NPs
may disrupt the protein integrity on exosome membranes and interfere with
cancer therapy.
Luminescent PSiNPs (silicon NPs) have been employed to create
biocompatible tumor cell-exocytosed exosome-biomimetic PSiNPs (doxo­
rubicin silicon NPs) as a drug carrier for targeted cancer treatment. These
PSiNPs exhibit excellent drug-loading capacity, high biocompatibility, and
biodegradability. The use of doxorubicin silicon NPs as a delivery vehicle for
DOX is possible. In vitro, doxorubicin silicon NPs can induce low expres­
sion of the multidrug-resistant protein P-glycoprotein, resulting in reduced
cell membrane fluidity and improved intracellular retention. Additionally,
the ability of doxorubicin silicon NPs to target tumor cells is regulated by
CD54, leading to significant cellular absorption. When comparing doxoru­
bicin silicon NPs to free doxorubicin, the latter exhibits greater cytotoxicity
against cancer stem cells (CSS). Intravenous administration of doxorubicin

— SECTION 19 —
silicon NPs results in enhanced tumor accumulation, tumor penetration,
and cross-reactive cellular absorption by bulk cancer cells and CSCs. This
leads to higher DOX enrichment in all tumor cells and side population cells,
further enhancing anticancer activity [35].
3.2.6 DENDRIMER
Donald A. Tomalia’s groundbreaking research was initially published in 1985
after he successfully created the first polyamidoamine (PAMAM) dendrimers
in 1979 [36, 37]. Dendrimers are a relatively new type of dendritic polymers
with a three-dimensional, branching, highly monodispersed, sequentially
synthesized macromolecular nanoscopic (1–100 nm) architecture [38, 39].

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Dendrimer architecture presents a novel strategy for solubilizing drugs
that are insoluble in water, with three main sites for drug entrapment using
various mechanisms, such as void spaces (through molecular entrapment),
branching points (via hydrogen bonding), and outside surface groups
(through charge-charge interactions). This offers a unique nanocontainer
feature for drug delivery [27, 30]. It also provides a polyvalent platform
for binding a range of biological targets, improving therapeutic effects,
which has been utilized to develop a novel nanodrug for antiviral and
anti-inflammatory therapy. Reports suggest that dendrimers can enhance
transdermal permeability and target specific medications. As a transdermal
permeation enhancer, dendrimers have successfully delivered indomethacin
via transdermal routes, offering an exciting delivery method for potential
anti-inflammatory treatment possibilities [26, 40]. The most widely used
critical tool for treating cancer, however, has been dendrimer-based targeted
delivery since it is highly biocompatible and has few side effects on healthy
cells, immunological function, and blood medicines. TZ-grafted dendrimers
were developed to efficiently distribute DTX to breast cancer cells that
express the human epidermal growth factor receptor 2 (HER2), which is
more selective and has higher anti-proliferation action than HER2-negative
cells. TZ-conjugated dendrimers showed better targeting and cellular
internalization than unconjugated dendrimers, as well as less hemolytic
toxicity and a longer circulatory half-life. The levels of ROS, mitochondrial
membrane potential, cell cycle distribution, and activation of caspases 3/7,
8, and 9 in PAMAM-DTX-TZ conjugates were also assessed and compared
to free DTX. These findings could help improve DTX’s therapy profile for
HER2-positive breast cancer [41–43].

— SECTION 21 —
3.3 NPS USED IN BIOMEDICAL APPLICATIONS
3.3.1 EXTENDED-RELEASE NP-DELIVERY SYSTEMS
Extended-release medication delivery systems utilize steady-rate drug release
or regulated release to provide a stable and increased therapeutic potential
while minimizing adverse side effects [44]. Extended-release NPs can retain
drugs either on their surface or adsorbed in a matrix to achieve continuous
release when used in a therapeutic environment [41]. Currently, hydrophobic
biodegradable polymeric NPs are commonly employed to continuously
deliver therapeutic medicines to the tumor site. To enhance longer circula­
tion, NPs can undergo specific cell-surface modifications. Nanomedicine

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extensively utilizes PEGylation, which involves conjugating PEG to a
nanopolymer. It has been demonstrated to increase the drug-hydrodynamic
radius, prolong plasma retention time, reduce proteolysis, decrease renal
excretion, and prevent immunodetection of antigenic determinants [45, 46].

— SECTION 23 —
3.3.2 HYDROGELS
In particular, polysaccharides can be physically or chemically crosslinked
to form hydrogels, which are hydrophilic 3D porous networks. The remark­
able properties of hydrogels, such as controlled degradation, sustained
release of cargo, low toxicity, functionality, and high drug loading capacity
with a reversible sol-gel transition, provide a platform for encapsulating
various materials [47]. Hydrogels can generally be classified in various
ways, including bioresponsive hydrogels, bioinspired hydrogels, static
hydrogels, dynamic hydrogels, and hybrid hydrogels. Additionally, there
are several therapeutic fields, such as ophthalmology, oral medicine,
gastroenterology, cardiovascular disease, and cancer, where hydrogels are

— SECTION 24 —
utilized, along with numerous delivery methods including peroral, rectal,
vaginal, ocular, transdermal, and implantable. Thermosensitive poloxamer
407 hydrogels, which undergo a rapid phase transition from liquid at room
temperature to solid at 37°C, enable prolonged release in body organs and
reduce off-target toxicity [48]. Some of the various polysaccharides and their
composites used to create hybrid hydrogels include alginate-cyclodextrin,
alginate-chitosan, alginate-keratin composite, alginate-Polyamidoamine
(PAMAM) hybrid nanogel, and alginate/liposome hydrogels. In this context,
hydrogels composed of alginate Ca++ and sodium carboxy-methyl cellulose
are crosslinked to construct complex hybrid dual-drug delivery systems
(DDDs). By utilizing the different pH conditions of the small intestine and
colorectum, this dual drug delivery system selectively transports aspirin and
methotrexate-loaded CaCO3 microspheres to their respective target organs.

— SECTION 25 —
Free radical polymerization methods were used to produce dual biorespon­
sive pH/thermo-sensitive hydrogels loaded with DOX and curcumin due to
the tumor microenvironment (TME) features of colorectal cancer (CRC),
which include high temperatures and acidic pH values [49]. These hydrogels
demonstrated improved loading capacity, effective drug release, and the
ability to induce apoptosis in colon cancer cells. Hyaluronic acid (HA) and
methylcellulose (MC) hydrogels were created as efficient drug delivery
systems for rectal distribution [50], taking advantage of MC’s thermosensi­
tive feature and HA’s inherent glycosaminoglycan composition.

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3.3.3 BIONICS
Innovative cell-based carriers have gained increasing popularity as conven­
tional targeted therapy’s drawbacks, such as toxicity and short half-lives,
have been exposed. These carriers possess fascinating properties, including
sustained drug release, slow clearance rates, effective targeting, and biocom­
patibility. In addition to exosomes, biological carriers known as bionics,
such as red blood cells (RBCs), macrophages, platelets (PLTs), neutrophils,
microbes, and stem cells, are utilized for drug delivery. Among them,
immune cells have the greatest applicability due to the surface membrane
proteins that enable interactions with cancer cells [51]. To achieve this,
the cell membrane needs to be separated, a core needs to be built, and a
shell-core structure needs to be formed. This process involves several steps,
such as cell membrane separation and fusion with core nanoparticles (NPs).
The unique properties of cell membranes make bionics useful for targeted
delivery and deep penetration into cells while evading immune surveillance.
Furthermore, bionics can serve as vectors for controlled drug release. Cell
membrane-disguised NPs have shown great promise in the treatment of
resistant cancer types when combined with chemotherapy, photothermal
therapy (PTT), photodynamic therapy (PDT), and immunotherapy [52].

— SECTION 27 —
3.4 DRUG DELIVERY SYSTEMS FOR RESISTANT COLORECTAL
CANCER
3.4.1 DRUG RESISTANCE MECHANISMS IN CRC
Exosomes are vesicles with a plasma membrane that are released by cells
and can be found in physiological fluids [53, 54]. These vesicles transport
genetic resources and proteins to distant regions in cancer cells, resulting
in tumor development, metastasis, and drug resistance. VEGF, fibroblast
growth factor (FGF), platelet-derived growth factor (PDGF), basic (FGF,
transforming growth factor (TGF), tumor necrosis factor (TNF), and inter­
leukin-8 (IL-8) have all been reported to induce angiogenesis, a process
that leads to the formation of new blood vessels. These molecules can all
be carried by exosomes from tumors. The persistence of CSCs (cancer
stem cells) after conventional therapy, which allows them to regain their
capacity for renewal and dedifferentiation, is one of the most challenging
problems in cancer recovery. Additionally, tumor dormancy allows tumor
cells to remain dormant but alive until they resume proliferating in response

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to the appropriate signals, leading to colorectal cancer recurrence [54]. The
classification of dormancy includes four subtypes with multiple processes:
basic cancer dormancy, metastatic dormancy, therapy-induced dormancy,
and immunologic dormancy. Autophagy, by preserving the viability of
cancer cells, promotes cancer dormancy and is necessary for tumor cells
to progress into the proliferative stage. Several drugs used to treat cancer
induce autophagy, resulting in resistance. There is a significant link between
the presence of circulating tumor cells and tumor resistance with the capacity
of EMT (epithelial-mesenchymal transition). Furthermore, these circulating
tumor cells can acquire stem cell characteristics, leading to colorectal cancer
recurrence and metastasis.
Studies utilizing whole genome sequencing have identified significant
genetic variability in tumors, which can lead to treatment resistance, recur­
rence, and a poor prognosis [55]. Chromosomal instability (CIN) is the most
common type of instability observed in malignancies, including colorectal
cancer. CIN and subsequent cancer development can be triggered by various
pathways, such as mutations in the TP53 gene, defects in DNA repair genes,
overexpression of AURKA, and GINS1. Copy number alteration (CNA)
investigations on organoid cultures have revealed the de novo formation
of whole-chromosome and sub-chromosomal modifications during tumor
growth, with errors in chromatin serving as the underlying causes [56]. For
example, lagging chromatin leads to whole-chromosomal CNAs, while
chromatin bridges result in sub-chromosomal CNAs. Aneuploidy is a char­
acteristic feature of several malignancies.

— SECTION 29 —
3.4.2 ADVANCED STRATEGIES FOR CRC DRUG DELIVERY
Researchers have shown increasing interest in colon targeting due to the
development of drug delivery systems as a new tool, the presence of many
adverse effects and poor patient survival rates associated with conventional
therapies, and the emergence of alternative therapies that utilize more focused
targeted drug delivery systems [57]. When aiming for optimum delivery of
colorectal cancer (CRC), factors such as the CRC microenvironment qualities,
tumor heterogeneity profile, chemo-physical properties of medications, and
colon transitory duration should be taken into consideration. Various vectors
and techniques, such as neural tube defects (NTDs), can be employed [58].
The use of single-cell technology enables the creation of novel thera­
peutic strategies against resistant cancers, making it a promising technique
in the fields of cancer therapy and precision medicine. It provides a profile of

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heterogeneities in tumors and their environments. Due to the heterogeneity
and variable response of the same malignancies to a given therapy, it is
crucial to utilize single-cell analysis [59]. Several steps are involved in using
single-cell RNA sequencing, starting with the preparation of tissue samples
from cancerous tumors. The next step is to choose a platform, such as 10X
Genomics Chromium, Nadia (Dolomite Bio), Illumina’s Bio-Rad ddSEQ
Single-Cell Isolator, BD Rhapsody Single-Cell Analysis System (BD), or
Fluidigm C1, to sequence the RNA in those samples. In the end, scientists

— SECTION 31 —
employ a variety of methods to assess their data, including cell cycle phase
assignment, normalization, batch effect correction, and quality control. They
also cluster cells and reconstruct the trajectory of each cell using pseudo time
to determine whether any regulatory genes control gene expression patterns
throughout the cell cycle phase [60, 61]. Finally, they search for enrichment
patterns among genes and gene regulatory networks to infer which genes are
involved in regulating specific aspects of cell function [62]. Additionally,
single-cell analysis can unveil the phylogeny of a tumor and the evolution
of clones. For example, single-cell sequencing forms the basis of Simulated
Annealing Single-Cell Inference (SASC) [63].
3.5 CONCLUSION
Many different types of NPs, both organic and inorganic, have already
been extensively utilized in the clinical treatment of various types of
cancer. NP-based drug delivery systems offer superior pharmacokinetics,
biocompatibility, tumor targeting, and stability compared to conventional
medications. They also play a crucial role in reducing systemic toxicity and
combating drug resistance. Due to these advantages, NP-based medications
are commonly employed in gene therapy, radiation therapy, targeted therapy,
hyperthermia, and chemotherapy. Furthermore, nanocarrier delivery tech­
nologies provide enhanced platforms for combination therapy, which helps
overcome drug resistance mechanisms. The use of nanotechnology in cancer
treatments has ushered in a new era characterized by the overexpression of
efflux transporters, compromised apoptotic pathways, and hypoxic tumor
microenvironments (TMEs). According to various multidrug resistance
(MDR) pathways, NPs loaded with targeted agents in addition to cytotoxic
agents can reverse drug resistance. With further investigation, many hybrid
NPs have garnered interest and demonstrated improved delivery capabilities.
More comprehensive research on the molecular characteristics of specific

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tumors will lead to more targeted approaches for these medications. Further
investigation is warranted regarding the precise targeting of cancer cells. It
is important to note that complex interactions exist between NPs and the
immune system. The size, shape, composition, and surface of the NPs all
influence their interaction with the immune system. Despite nano-vaccines
and synthetic APCs demonstrating greater success compared to conventional
immunotherapy, the clinical effectiveness of this therapy remains unsatisfac­
tory, necessitating further research to determine the safety and tolerability
of these innovative techniques. The incorporation of immunomodulatory
factors into NPs may also enhance the potency of immunotherapy vaccines.
Therefore, a deeper understanding of the TME and additional research on the
interaction between NP-based drug delivery systems and tumor immunity
are necessary for the development of hybrid NPs that are more suitable for
cancer therapy and the engineering of NPs that target.

— SECTION 33 —
KEYWORDS

• 
  cancer
• 
  cancer therapy
• 
  drug delivery system
• 
  hydrogels
• 
  hypoxic tumor microenvironments
• 
  immune
• 
  immune system
• 
  immunomodulatory factors
• 
  nanoparticle
• 
  vaccine

— SECTION 34 —
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    T., et al., (2019). CD44/CD133-positive colorectal cancer stem cells are sensitive to
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60. Frank, M. H., Wilson, B. J., Gold, J. S., & Frank, N. Y., (2021). Clinical implications
    of colorectal cancer stem cells in the age of single-cell omics and targeted therapies.
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    1095–1112. https://doi.org/10.1111/JCMM.17164.
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    Yousefi, M., et al., (2017). Nanoparticles and targeted drug delivery in cancer therapy.
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the Management of Various Carcinomas. Rishabha Malviya, Bhupendra Prajapati, and Sonali Sundram (Eds.)
© 2025 Apple Academic Press, Inc. Co-published with CRC Press (Taylor & Francis)

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#

CHAPTER 4
TITLE: Correlation Between Cancer Vaccine and Immunity
AUTHORS: Neelakanta Sarvashiva Kiran, Chandrashekar Yashaswini, Gaonkar Lowkesh, and Bhupendra G. Prajapati
LENGTH: 113,009 characters

#

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CHAPTER 4
Correlation Between Cancer Vaccine and
Immunity
NEELAKANTA SARVASHIVA KIRAN,1 CHANDRASHEKAR YASHASWINI,1
GAONKAR LOWKESH,1 and BHUPENDRA G. PRAJAPATI2
1Department of Biotechnology, School of Applied Sciences, REVA
University, Kattigenahalli, Yelahanka, Bangalore, Karnataka, India
2Shree S.K. Patel College of Pharmaceutical Education and Research,
Ganpat University, Kherva, Gujarat, India
ABSTRACT
The human immune system is responsible for both recognizing and eliminating
various antigenic molecules and compounds that possess both endogenous
and exogenous characteristics. The onset of various forms of cancer has been
known to be triggered by multifactorial antigens and antigenic mutations over
several years. In recent years, there has been a significant increase in interest
within the scientific community regarding cancer immunology, leading to
major breakthroughs, primarily through the development of cancer vaccines
(CVs). Cancer vaccines have been extensively studied worldwide and have
been found to initiate various immunological responses within organisms,
which have been mechanistically proven to suppress cancer growth. Despite
the extensive research and development of such vaccines, the success and
comprehensive understanding of their immunological effects and their fate
within the human system remain limited. This chapter aims to provide insight
into the factors linking immunology and cancer, focusing on the outcomes
of CVs. Additionally, the chapter will discuss the mechanistic aspects of
CV efficacy and their immunological effects in the treated host, providing a
structural basis for future advancements in the field of cancer immunology.

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4.1 INTRODUCTION
Cancer is initiated by the proliferation of a clonal cell population within
tissues. Carcinogenesis and the onset of malignancy can be analyzed and
characterized in various ways. Cancer can result from chromosomal defects,
which are induced by abnormalities in genes that regulate cellular func­
tions, particularly cell proliferation and reproduction. One way to describe
this process is by highlighting the defining factors of cancer tissues: the
“hallmarks” of tumors [1]. Cancer progression requires the acquisition of
six key features: self-sufficient growth, insensitivity to anti-proliferative
signals, evasion of apoptosis, unlimited replicative potential, maintenance
of vascularization, and, in the case of malignancies, invasion, and metastasis
[2]. However, a single gene alteration is insufficient for the full development
of cancer. Additional genetic mutations are necessary for the progression
towards malignancy and invasion. Therefore, the likelihood of developing
the disease depends not only on the initial mutations that initiate tumorigen­
esis but also on subsequent mutations that drive tumor growth [3].
Cancer can be conceptualized as a sequential step functionally divided
into three stages: initiation, promotion, and development. The first stage in
the two-stage model of cancer progression is initiation. Initiators are often
rendered electrophilic by drug-metabolizing enzymes in the body and can
then affect DNA if they are not already reactive with mutated DNA. Many
initiators are specific to certain tissue types or species, as they require oxida­
tive metabolism to become active. Once a cell is impacted by an initiator, it
becomes susceptible to promotion until it dies. Initiators have irreversible
effects. Since initiation is the result of a permanent genetic mutation, all
daughter cells produced when the defective cell divides will always carry
the mutation [4]. In contrast to initiators, promoters do not form a covalent
bond with the cell’s DNA or other biomolecules. To modify intracellular
pathways that result in enhanced cellular proliferation, many molecules bind
to the cell’s surface receptors. Specific promoters, which interact with recep­
tors on or in target cells of specified tissues, can be divided into two main
categories: nonspecific promoters that influence gene expression without
the involvement of an identified receptor. Promoters are frequently selected
for a particular tissue or species due to their interactions with receptors that
are present in many tissue types at varying levels [5]. Benign papillomas
develop when the promoter is applied frequently to initiator-exposed skin.
After therapy is terminated, a significant number of these papillomas regress,
but some transform into cancer. Papillomas that progress into cancer have

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a second, spontaneous mutation, according to the frequency of progres­
sion. Leslie Foulds’ term “progression” describes the slow transition from
a benign tumor to a neoplasm and then to cancer. Progression is linked to
a karyotypic mutation (having the wrong number of chromosomes) since
nearly all progressing cancers are aneuploid. Along with this karyotypic
shift, the tumor also exhibits altered physiology, morphology, metastasis,
and a faster pace of development [6].
The investigation of interactions between the immune response and
malignancies or other abnormalities is known as cancer immunology. At the
core of this field is the precise detection of malignant cells and the antigens
they express. This concept is the basis of spontaneous immunosurveillance,
which was proposed by Burnet and Thomas in 1957. According to their
proposal, lymphocytes act as sentinels that detect and eliminate continu­
ously emerging, immature altered cells. The stepwise immunosurveillance
process begins with the recognition and removal of dysfunctional cells, and
both innate and adaptive immune cells work together to detect and eradicate
tumors in their early stages of development. It is conceivable to infer that
the proliferation of cells and tissues is maintained in a balanced equilibrium
state. However, over time, cellular escape from naturally defensive responses
can occur, potentially leading to the development of cancer [7]. As a result of
this process, tumor cells function as a constant source of novel and modified
proteins that are recognized as antigens by the adaptive immune system. The
recognition of tumor antigens (TAs) by the human body’s immune system can
trigger an inflammatory response, characterized by the production of various
cytokines, among other factors. Tumor cells will be completely removed if
the immune response is adequate. If this does not occur, a prolonged equilib­
rium will be established where tumor progression and immune surveillance
will dynamically balance each other. Due to the significant genetic instability
of cancer cells that resist immune-mediated destruction, malignant cell
clones eventually develop into clinically evident malignancies. Antitumor
effector cells play a crucial role in cancer immunology. Tumor-infiltrating
lymphocytes (TILs) primarily consist of T-cells, characterized by the pres­
ence of the cluster of differentiation 3 (CD3) surface protein. They infiltrate
various tumor types and are considered one of the most important specific
antitumor effector cells. Within the TIL population, two subgroups of T-cells
can be identified: CD8 þ and CD4 þ T-cells [8].
The significance of tumor cell immunotherapy has largely been the
notion that vaccination can initiate an immune response to cancer. Cancer
vaccines (CVs) may differ in several crucial ways from more “conventional”

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vaccinations used to prevent infectious diseases, although they share a similar
concept. CVs are considered therapeutic rather than preventive, as they are
intended to be administered after the onset or detection of the disease [9].
CVs can be designed to induce cell-mediated immunity, such as cytotoxic
T cells, rather than humoral responses, since many tumor antigens may
be intracellular proteins rather than cell surface molecules. Lastly, unlike
foreign antigens, self-molecules could be the targets of CVs [10].
The past several decades has witnessed remarkable advancements in
immunotherapeutics, which complement the available treatment options. In
contrast to other therapeutic approaches, immunotherapy primarily aims to
enhance the quality of life (QoL) for those affected by the disease. The tech­
nique of immunotherapy involves boosting or supplementing the immune
system using a wide range of substances, including antibodies, vaccines, in
vitro-stimulated effector cells of the immune system, and lymphokines [11].
CVs are classified into two main categories: preventive and therapeutic
CVs. Therapeutic CVs are designed to activate the patient’s adaptive immune
system against specific tumor antigens (TAs), with the aim of halting the
progression of existing tumors, slowing tumor growth, and eliminating any
remaining disease. The key elements necessary for effective therapeutic
vaccination against malignancies include the delivery of substantial amounts
of high-quality antigens to dendritic cells (DCs), optimal stimulation of DCs,
generation of strong and long-lasting CD4+ T helper cell and cytotoxic T
lymphocyte (CTL) responses, infiltration of the tumor microenvironment
(TME), and durability of the immune responses.
In the fight against cancer, a preventive CV, especially one that targets
multiple types of cancer, would be a significant milestone. The potential to
use immunotherapies for cancer treatment has been demonstrated in recent
times, although progress in developing cancer vaccines is still in its early
stages. Scientists in both academia and industry are exploring various
approaches to using vaccines for targeting cancers, but only a small fraction
of these approaches have entered clinical trials [12].

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4.2 UNDERSTANDING OF THE IMMUNE SYSTEM
Dynamic interactions between organs, tissues, cells, and molecules that
constitute the immune system are crucial for protecting against infectious
diseases. The immune system is composed of three main host defenses
and can be considered a stratified system. External barriers include ciliated

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epithelia, mucous, epidermis, and biochemical barriers such as stomach
acids and destructive enzymes in secretions. Innate and adaptive immune
responses are the second and third lines of defense, respectively [13]. The
term “immune system” refers to a group of cells and proteins that work
together to defend the epidermis, nasopharynx, gastrointestinal system, and
other regions against external antigens such as pathogens (toxins, viruses,
bacteria, fungi, and parasites). Innate immunity and adaptive immunity
are the two lines of defense of the immune system. Innate immunity is the
primary defense against bacterial invasion [14].

— SECTION 6 —
4.2.1 INNATE IMMUNE SYSTEM
The protection provided by physiological and anatomical barriers is enhanced
by innate immunity. The innate immune system focuses on a constrained
set of receptors to recognize invasive infections, but it compensates for this
lack of invariant receptors by focusing on conserved microbial elements that
are shared by several pathogen subgroups. One distinguishing feature of the
innate immune system is its speed; minutes after exposure to a pathogen, the
innate immune system begins producing a protective inflammatory response.
Furthermore, the ensuing adaptive immune response is greatly influenced by
innate immunity [15]. Because of the extensive breadth of innate immunity,
it can occasionally be intimidating to distinguish between the innate immune
system and the rest of the host. This is partly because innate immune systems
change during evolution. The selection forces imposed by microorganisms
on the host population shape it and help it survive. On occasion, infections
are killed by proteins that were co-opted to execute an activity unrelated
to host protection [16]. The fundamental components of the innate immune
system are, for the most part, established in the tree of life, which has existed
for hundreds of millions (and in some cases, billions) of years. As a result,
they are widely distributed. The innate immune system has been honed for a
longer amount of time than the body’s immune response and is more flaw­
less in practically every regard, even though it is frequently thought of as
“primitive” or “crude” in comparison to adaptive immunity. The fundamental
components and roles of the immune system:

• 
  The ability to identify a wide variety of diseases;
• 
  Destroying these pathogens when they have been identified;
• 
  Preserving host tissues (i.e., self-tolerance is required).

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Physiological and anatomical barriers, intracellular internalization
processes, and inflammatory reactions that are quickly triggered by the pres­
ence of an antigen are all included in innate immunity. Innate immune systems
block pathogen entry, halt the spread of disease, and eliminate microbial
and host waste. While some innate defenses are completely antigen-neutral,
others utilize broadly specialized pattern recognition molecules (PRMs)
that assist in the elimination of a specific subset of pathogens. Some PRMs
are soluble molecules that mark pathogens for removal, while others are
pattern recognition receptors (PRRs) secreted on the surfaces of effector
cells. Innate immunity either successfully eradicates the pathogen or keeps
the infection under control until delayed, lymphocyte-mediated adaptive
immune responses can take effect [17]. Recent research has provided new
insights into the cellular and molecular mechanisms that directly interact
with inflammation and cancer.
Inflammation and cancer are connected through two mechanisms. The
activation of several classes of oncogenes in the intrinsic pathway drives the
release of inflammation-related proteins, which in turn create an inflamma­
tory environment. Inflammatory conditions also promote cancer development
through the extrinsic route, such as intestinal cancer associated with colitis.
Transcriptional factors like NF-kB, Stat3, and HIF, as well as cytokines like
TNF and chemokines, play crucial roles at the intersection of the extrinsic
and intrinsic pathways. Consequently, inflammation is a critical aspect of the
tumor’s microenvironment and can be targeted with medications [18].

— SECTION 8 —
4.2.1.1 IMPORTANT CELLULAR ELEMENTS IN INNATE IMMUNITY
Innate immunity is composed of a variety of cell types that serve as the
body’s “first line of defense” against microbes. While the functions of each
cell type are distinct, there is overlap in the cellular machinery, including
the production of PRRs and the inflammatory response to the detection of
PAMPs/DAMPs.
4.2.1.2 DENDRITIC CELLS (DCS)
Antigen-presenting cells (APCs) belonging to the DC family play a crucial
role in the development of antigen-specific immunity and resistance. The
Major Histocompatibility Complex (MHC), a co-stimulatory molecule
necessary for T cell activation, and CCR7 expression are both upregulated

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Correlation Between Cancer Vaccine and Immunity
by the diverse array of PRRs that DCs express in response to the detection of
PAMPs and DAMPs. The latter is a crucial chemokine receptor that instructs
migration into tumor-draining lymph nodes (TDLN). As a component of the
cancer immunity cycle, DCs in TDLNs present tumor-associated antigens
and produce CD8+ T cells that are specific for those antigens [19]. Common
myeloid progenitor cells are the source of DCs, which may develop into
four distinct kinds of DCs: conventional type I DCs (cDC1s), conventional
type 2 DCs (cDC2s), plasmacytoid DCs (pDCs), and monocyte-derived DCs
(MoDCs). Due to their skilled antigen manufacturing and cross-presentation,
cDC1s—which exhibit BATF3 and IRF8 expression—are the most potent
inducers of cell-mediated immunity. Intra-tumoral CD4+ T cell frequency
has been observed to be enhanced by DC2s, supporting CD8+ T cell activity
[20]. When subjected to viral stimuli, pDCs, a rare subtype of DCs, may
create interferons (IFN) and activate innate immunity, earning them the
name interferon-producing cells (IPCs). In addition to directly controlling
T cell activity, pDCs also have a limited ability to prime naive T cells in
comparison to other DC subtypes. According to a more recent study, pDCs
may have a pro-tumor function because their infiltration into tumors is a bad
predictor of prognosis in many malignancies. MoDCs have been found to
help maintain immunity after chemo- or radiotherapy-induced cell death,
albeit their function in anti-tumor immunity is less apparent [21].

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4.2.1.3 MACROPHAGES
Macrophages, known as myeloid cells, are present in healthy tissues
throughout the body and play a crucial role in coordinating innate immune
responses and maintaining tissue homeostasis. TAMs (tumor-associated
macrophages) exist on a spectrum of polarization states, ranging from
protumorigenic M2 macrophages to antitumorigenic M1 macrophages,
which correspond to distinct gene expression programs. In many cancer
types, macrophages are directed towards an M2 functional program that
promotes tumor development. M2 TAMs induce local immunosuppression
through various mechanisms, including the synthesis of IL-10 and TGF-β,
extracellular arginine depletion, enrichment of regulatory T cells, and
regulation of T cell proliferation [22]. Hypoxia and inhibitory cytokines
like IL-4, IL-10, and IL-13 drive the differentiation of monocyte precursors
into M2 macrophages. On the other hand, antitumorigenic M1 macrophages
contribute to tumor control through processes such as phagocytosis and the

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release of pro-inflammatory cytokines (PICs) like IFN-α, IFN-β, and IFN-γ.
Recent studies using single-cell RNA sequencing have identified multiple
subclusters of TAMs in solid tumors, suggesting that macrophage function
exists along a continuum of states and cannot be simply categorized [23].
For example, research on pancreatic cancer has revealed five subgroups of
TAMs, each with unique gene expression patterns that correspond to different
immunosuppressive or immune-stimulatory behaviors. Histological analysis
of clinical specimens has shown that tumor-associated macrophages (TAMs)
are associated with worse overall survival (OS). Depletion of TAMs has been
shown to enhance the effectiveness of radiation, chemotherapy, and immune
checkpoint inhibitors (ICI) in preclinical animal models [24].

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4.2.1.4 NEUTROPHILS
The innate immune system’s reaction to bacterial infection is carried out
by neutrophils, which are circulating myeloid cells. Numerous solid tumors
have been found to include large amounts of neutrophils in the immune
infiltrate and increases in both tumor-associated neutrophils (TANs), as well
as peripheral blood neutrophils, are linked and associated with decreased
outcomes [25]. The pro-neoangiogenesis, pro-tumor migration and invasion,
and pro-local immunosuppression pathways of TANs are among their tumor­
igenic properties. Contrarily, TANs have been found to have antitumorigenic
effects through direct cytotoxicity of tumor cells, the formation of reactive
oxygen species, and the release of PIC [26]. Due to the functional flexibility
of TANs, protumorigenic N2 TANs and antitumorigenic N1 TANs have been
classified in a bipolar manner, similar to TAMs. According to research, early
tumor formation is governed by N1 TANs. TGF-, IL-10, and IL-6 signaling,
however, encourage the differentiation of tumorigenic N2 TANs over time.
Despite recent developments, the tumor immunobiology of TANs is still
substantially a subject of current investigation [22].

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4.2.1.5 MYELOID-DERIVED SUPPRESSOR CELLS (MDSCS)
A subset of myeloid cells known as MDSCs is distinguished from other types
of myeloid cells by their predominantly immunosuppressive characteristics.
The differentiation of myeloid cells into the MDSC phenotype is linked to
chronic inflammation and brief exposure to tissue factors and inflamma­
tory mediators necessary for proper development. MDSCs consist of two
morphological subgroups, namely Mo-MDSC and Polymorphonuclear,

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which are mononuclear or polymorphonuclear [27]. Compared to conven­
tional neutrophils and monocytes, MDSCs express higher levels of immu­
nosuppressive chemicals such as nitric oxide (NO), IL-10, and arginase-1,
while also exhibiting poorer phagocytic capacities. MDSCs have been found
to inhibit both the innate and adaptive immune systems in various types of
solid tumors, contributing to a “cold” tumor microenvironment (TME). The
infiltration of MDSCs into the tumor site is associated with a poorer prog­
nosis, higher cancer stage, and increased tumor load. These characteristics
underscore the significance of MDSCs in therapeutic strategies aimed at
combating an immunosuppressive TME [22].

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4.2.1.6 MAST CELLS
Mast cells, which are granulated innate immune cells residing in peripheral
tissues, play a crucial role in muscle repair and local immunological responses
by releasing various signaling chemicals. The presence of mast cells in the
tumor stroma of different solid malignancies has been associated with a poor
prognosis. However, in breast cancer, the presence of mast cells has been
identified as a favorable predictive marker [28]. The precise location of mast
cells within the tumor microenvironment (TME) may potentially impact
tumor growth. Mast cell-secreted molecules, such as VEGF and matrix
metalloproteases, have been found to enhance tumor development and meta­
static potential by promoting angiogenesis, lymph angiogenesis, and altering
the extracellular matrix (ECM). Mast cell tryptase, acting as an agonist for
proteinase-activated receptor-2 (PAR-2), promotes endothelial cell prolifera­
tion and has been linked to tumor cell migration. Tumor-associated mast cells
exhibit anticancer properties, including mediating tumor cell death through
inflammatory cytokines and the peroxidase system. Ongoing studies aim to
fully comprehend the mechanisms by which mast cells operate in the tumor
microenvironment [22].

— SECTION 15 —
4.2.1.7 NATURAL KILLER (NK) CELLS
Innate lymphocytes known as natural killer (NK) cells have a shorter half-life
compared to B and T lymphocytes and require continuous replenishment from
bone marrow (BM) progenitors. NK cells undergo a linear differentiation
process, transitioning from highly proliferating, functionally inferior, imma­
ture cells to a community of potent big granular effectors, and eventually
reaching a non-neuronal, hypo-functional state. This process, which spans

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several weeks, ensures a constant supply of circulating cytotoxic and inflam­
matory antitumor lymphocytes that are poised for spontaneous activation
upon stimulation. Many characteristics of NK cells resemble those of CD8+
T cells, which are the target of immunological checkpoint blockade (ICB)
therapies that are currently revolutionizing clinical cancer treatment [29].

— SECTION 16 —
4.2.2 ADAPTIVE IMMUNE SYSTEM
The adaptive immune system, often referred to as the acquired immune
system, is a component of the immune system composed of systemic,
specialized cells, and processes that destroy or suppress infections. Adaptive
defenses have evolved to provide a broader and more precise repertoire of
self- and non-self-antigen recognition. Adaptive immunity involves a well-
controlled interaction between T and B lymphocytes and APC, which promote
the development of immunologic effectors specific to pathogen pathways,
immunologic memory formation, and the regulation of a healthy host immune
system. The acquired immune system is one of the two fundamental types of
immunity found in vertebrates [30]. After an initial response to a particular
pathogen, adaptive immunity develops immunological memory, resulting in
an enhanced response upon subsequent encounters with the same pathogen.
Antibodies are a critical component of the adaptive immune system [31].
Adaptive immunity can provide long-lasting protection, potentially lasting a
person’s entire lifespan. However, in certain cases, such as with chickenpox,
it does not confer permanent protection. For example, a person who survives
measles is now protected from measles for the remainder of their life. This
mechanism of adaptive immunity forms the basis of vaccination [32]. The
lymphatic system consists of various lymphoid organs where lymphocytes
mature and become activated. During development, genomic sequences
are rearranged and assembled to generate the genes that encode the distinct
antigen receptors of B and T cells. This rearrangement process generates an
incredibly diverse array of receptor specificities capable of recognizing all
possible components of pathogens. In addition to specificity, the develop­
ment of immunologic memory is a key characteristic of adaptive immunity.

— SECTION 17 —
4.2.2.1 CELLULAR IMMUNITY AND T-CELLS
4.2.2.1.1 T-Cell Development
BM precursors give rise to T lymphocytes, which are transported to the
thymus for maturation, selection, and export to the periphery. The peripheral

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T cell subsets include regulatory T (Treg) cells, which regulate immune
responses, memory T cells, which develop from previously activated anti­
gens and provide long-term immunity, and naïve T cells, which can respond
to new antigens [33]. When naïve T cells encounter a pathogen and receive
costimulatory signals from DCs, interleukin 2 (IL-2) is produced. Effector
cells proliferate, differentiate, and migrate to various locations to assist in
pathogen clearance by generating effector cytokines and cytotoxic mediators.
Activated effector cells have a limited lifespan, but some differentiate into
memory T cells that can migrate, localize to different tissues, and undergo
self-renewal [34]. Although their lineage and ancestry are still unclear,
memory subsets contribute to long-term immunization and protective
responses. Common lymphoid progenitors from the fetal liver or BM give
rise to T cells in the thymus. Platelet selectin glycoprotein 1 in progenitors
and the integrin P-selectin on thymic epithelium interact to promote thymus
seeding. Various cytokine receptors, including IL-2, IL-4, IL-9, IL-15, and
IL-21, share the common γ chain, which is encoded on the X chromosome.
Under the influence of IL-7, newly arriving cells rapidly expand [35]. Muta­
tions in this polypeptide cause X-linked SCID disorder, characterized by a
lack of T lymphocytes. This early thymocyte proliferation is accompanied by
the stimulation of Notch-1, as well as other transcriptional factors that bind
cofactors to all T-cell lineages and stimulate the expression of genes crucial
in T-cell receptor (TCR) development. A coordinated set of genomic rear­

— SECTION 18 —
rangements results in the production of functioning genes that encode the a
and b or g and d domains of the TCR during the subsequent maturation of the
increased reservoir of T-cell precursors or pro-T lymphocytes in the thymus.
This process is antigen-independent [36]. If gene-segment rearrangements
result in a gene generating a full-length TCR polypeptide without intro­
ducing stop codons, they are referred to as productive sequentially effective
rearrangements resulting in the expression levels of ab or gd from two TCR
genes. Pre-T cells change into double-positive T cells, which exhibit both
CD4 and CD8, at the TCR. At the cell surface, the TCR chain is put together
dynamically with the proteins that comprise CD3, including g, d, e, and z
chains. Both negative and positive selection incidents surrounding antigens
and MHC molecules control the further maturation of these double-positive
lymphocytes, which are situated in the thymic cortex, into single-positive T
lymphocytes, which are located in the medulla. When self-MHC (complexed
to self-peptides) on the thymic epithelium is bound by the TCR of the double
T cells with low avidity, positive selection takes place. The TCR-positive
double-positive cells that do not bind to self-MHC are destroyed [37]. The

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T-cell receptor (TCR) of double-positive T cells, on the other hand, binds to
the self-MHC peptide with a very high affinity, preventing the maturation
of autoreactive T-cell progenitors (central tolerance). The activity of the
autoimmune regulator (AIRE) gene, which promotes the expression of genes
with broad tissue specificity in the thymic epithelium, facilitates the elimina­
tion of T-cell clones interacting with peptides ordinarily produced in distant
organs [38]. Dysfunction of this gene is directly responsible for certain
self-reactive T cells escaping and can result in autoimmune polyendocrine
disorders. By further interacting with lymphoid cells’ epithelial MHC class I
proteins, double-positive thymocytes that successfully navigate both positive
and negative selection grow into CD81 single-positive T cells, while those
selected on MHC class II develop a CD41 single-positive morphology [39].

— SECTION 20 —
4.2.2.1.2 T-CELL ACTIVATION
The concurrent interaction of the T-cell binding site and a co-stimulatory
protein, such as CD28 or ICOS, mostly on T cells by the complex of
major histocompatibility (MHCII) polypeptide and co-stimulatory proteins
on the APC, results in the activation of CD4+ T cells. In the absence of
co-stimulation, the T cell receptor activity alone causes anergy; both are
necessary for the generation of an efficient immune response [40]. The PI3K
pathway, which generates PIP3 at the plasma membrane and recruits PH
domain-containing signaling molecules like PDK1 that are necessary for
the activation of PKC and eventual synthesis of IL-2, is a typical signaling
pathway engaged downstream from co-stimulatory molecules. CD4+
signaling is necessary for the optimal CD8+ T cell response. CD4+ cells
help maintain the memory of CD8+ T lymphocytes after an acute infection
and in the first antigenic stimulation of naïve CD8 T cells [41]. As a result,
CD4+ T cell activation may enhance CD8+ T cell activity. The T cell recep­
tor’s interaction with its relevant peptide displayed on MHCII on an APC
generates the initial signal. Nerve cells, B lymphocytes, and macrophages,
to name a few, are examples of professional APCs that express MHCII.
The peptides presented to CD4+ cells by MHC class II molecules are typi­
cally longer, ranging from 12 to 25 polypeptides in length. This is because
the binding sites of MHC class II molecules are open [42]. On the other
hand, the peptides presented to CD8+ T-cells by MHC class I molecules
are shorter, around 8 to 13 peptides in length. The second signal for T-cell
activation is provided through co-stimulation, where a small number of

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Correlation Between Cancer Vaccine and Immunity
stimuli, mostly pathogen products but sometimes cell breakdown products
like necrotic aggregates or heat shock proteins (HREs), activate surface
proteins on the antigen-presenting cells (APCs). Naive T-cells constitutively
express the co-stimulatory receptor CD28, and co-stimulation is provided by
the APC’s CD80 and CD86 proteins, collectively known as the B7 protein
(B7.1 and B7.2, respectively). OX40 and ICOS are additional receptors that
are expressed upon T-cell activation, but their expression heavily relies on
CD28. The second signal, provided by co-stimulation, authorizes the T-cell
to respond to an antigen. Without this signal, the T-cell becomes anergic and
finds it more difficult to activate in the future. Self-peptides are typically not
accompanied by sufficient co-stimulation, which helps prevent inappropriate
responses to self-antigens [43]. Upon proper activation (i.e., after receiving
signals one and two), a T-cell undergoes changes in the expression of many
proteins on its cell surface. HLA-DR, CD69, CD71, CD25 (also a marker
for Treg cells), and CD25 are all markers of T-cell activation. Additionally,
activated T-cells produce more CTLA-4, which competes with CD28 for
binding to the B7 molecules. This serves as a checkpoint mechanism to
prevent excessive T-cell activation. Multiple proteins constitute the complex
that forms the T cell receptor [44]. The T cell receptor consists of distinct
alpha and beta genes, which create two distinct peptide chains. The complex
also includes CD3 proteins, such as CD3 homodimer, CD3 homodimer
with six ITAM motifs, and CD3 heterodimer. Lck phosphorylates the ITAM
patterns on CD3, attracting ZAP-70. Other molecules like CD28, LAT, and
SLP-76 can also have their tyrosine residues phosphorylated by Lck and/
or ZAP-70. This enables the formation of signaling complexes involving
these proteins. When LAT is phosphorylated, SLP-76 is recruited to the
membrane, where it can subsequently recruit PLC-, VAV1, Itk, and perhaps
PI3K. On the innermost layer of the membrane, PLC-breaks down PI [4,
5] P2 to produce the active mediator’s diacylglycerol (DAG) and inositol­
1,4,5-trisphosphate (IP3). Additionally, PI3K phosphorylates PIP2 to
generate phosphatidylinositol-3,4,5-triphosphate (PIP3). Some PKCs are
bound and activated by DAG. PKC-, which is crucial for triggering the
transcription factors NF-B and AP-1, is particularly significant in T cells.
When PLC-releases IP3 from the membrane, it diffuses quickly and acti­
vates calcium channel sensors on the ER, causing calcium to be released into
the cytosol. Low calcium levels in the rough ER enable STIM1 to cluster on
the ER membrane and activate CRAC channels on the cellular membrane,
allowing more calcium from the external environment to enter the cytosol.
When calmodulin binds to the aggregated cytosolic calcium, calcineurin can

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Cancer Vaccination and Challenges, Volume 2
be activated. NFAT is then activated by calcineurin and binds to the nucleus.
IL-2, a cytokine that promotes the long-term proliferation of activated T
cells, is one of several genes whose transcription is induced by the transcrip­
tion factor NFAT. Additionally, PLC-B can initiate the NF-B pathway [45].
PKC is triggered by DAG, which phosphorylates CARMA1, causing it to
unfold and serve as a scaffold. Through the CARD domains, the cytosolic
domain contacts the adaptor BCL10, which subsequently binds TRAF6,
leading to ubiquitination at K63. Target proteins are not degraded by this
type of ubiquitination. Instead, it recruits NEMO, IKK, and TAB1-2, and
TAK1 to enable K48 ubiquitination, resulting in proteasomal degradation.
TAK1 phosphorylates IKK-, IKK-, and IB. Rel A and p50 can then bind to
the NF-B response element inside the nucleus. This fully activates the IL-2
gene when combined with NFAT signaling. While TCR recognition of the
antigen is typically required for activation, other activation pathways have
been reported. For instance, it has been demonstrated that Cd8 can stimulate
cytotoxic T cells, leading to their tolerance. Reactive oxygen species regulate
the activation of T cells [46].

— SECTION 23 —
4.2.2.1.3 Subsets of T-Cell Effectors
Despite the fundamental ideas guiding thymic evolution, most T cells follow
the same activation pathways. However, there is a surprising variety of
effector actions induced in response to activated T cells. These actions can
directly contribute to the eradication of infected target cells and pathogens.
T cells can also serve as helper cells, providing cognate or cytokine signals
to enhance both T-cell and B-cell responses by triggering mononuclear
phagocyte activation. T cells play a crucial role in controlling immuno­
logical responses, preventing tissue damage caused by highly inflammatory
or autoreactive immune reactions. The CD41 ab TCR population constitutes
the majority of activating T cells in the body [38]. These cells are classi­
fied as T-H cells, as the majority of them have a helper function. During
activation, TH cells produce a variety of cytokines. Immunologists Robert
Coffman and Tim Mossman discovered about 20 years ago that not every
CD41 TH cell has the potential to produce the entire spectrum of cytokines
known to be part of the T-cell repertoire. Instead, they showed that there are
two primary types of TH cells, TH1 and TH2 cells, each of which mainly
produces distinct panels of cytokines. This was demonstrated through the
analysis of T-cell clones [35]. The CD41 ab TCR fraction of lymphocytes

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Correlation Between Cancer Vaccine and Immunity
also plays a crucial role in controlling T-cell responses, and it is likely that
numerous regulatory cell types are involved in this process. It has been
demonstrated that IL-10-producing regulatory T (TR1) lymphocytes, along
with naturally occurring and induced CD251 CD41 T cells expressing the
transcriptional forkhead box protein 3, suppress T-cell reactions. A severe

— SECTION 25 —
multisystem proinflammatory illness results from the lack of forkhead box
protein 3, which is encoded on the X chromosome. Recent literature has
conducted a comprehensive evaluation of the intricacy of the T-Lymphocyte
system [34]. NKT cells, a distinct subtype of T cells, recognize nonpeptide
pathogens presented by non-classical MHC class of the CD1 family, similar
to gd T cells. NKT cells are characterized by the simultaneous expression of
NK cell antigens and T-cell (CD3, TCRab) antigens (CD56). The majority
of NKT cells are classified as “invariant NKT cells” because they exhibit
a single distinct TCRa arrangement, Va24-Ja18 with Vb11. Activated NKT
cells have been associated with the etiology of allergies and are capable of
rapidly producing cytokines, such as IL-4, in large quantities. The explo­
ration of natural and pathogen-derived molecules that may promote NKT
growth and activation is currently a highly researched topic [47].

— SECTION 26 —
4.2.2.2 HUMORAL IMMUNITY AND B-CELLS
4.2.2.2.1 B-Cell Growth and Development
Humoral immunity, also known as antibody-mediated immunity, is medi­
ated by B lymphocytes. When a B cell encounters an antigen, it undergoes
transformation into a specific cell that secretes antibodies. These antibodies,
produced by plasma cells, closely resemble the receptors on progenitor B cells.
Once released into the lymphatic system and blood, the antibodies attach to the
target epitope and initiate neutralization or destruction. Antibody production
can last for days or even months until the antigen is neutralized [48].
Memory B cells, distinct from other B cells, do not develop into plasma
cells but play a crucial role in providing long-lasting immune memory.
B-cell receptors (BCRs) are expressed on the cell membrane of B cells,
distinguishing them from T lymphocytes and NK cells, the other two groups
of lymphocytes. The BCRs enable B cells to recognize unfamiliar antigens
and mount an antibody response. B cells originate from the bone marrow at
the immature stage and undergo development to reach the mature or naïve
stage [49]. The presence of IgD, in addition to IgM, on the surface of the cell
indicates this. There is no interaction with any exogenous antigen during the

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Cancer Vaccination and Challenges, Volume 2
entire development process. Therefore, it is referred to as B-cell develop­
ment independent of antigen. Any genetic changes that disrupt the structural
aspects of the pre-B cell receptor or the signaling pathways connected to it
result in immunodeficiency with agammaglobulinemia, as well as a lack of B
cells [50]. Immunoglobulin genes are assembled in a similar manner to TCR
genes, with light chains being assembled from three segments and heavy
chains being assembled from four segments (VH, D, JH, and CH). The k and
l genes are located on chromosomes 2 and 22, respectively, while the heavy
chain sequences are located on chromosome 14. The chapter “Structure and
Function of Immunoglobulins” provides a detailed examination of immuno­
globulin structure [51].

— SECTION 28 —
4.2.2.2.2 Activation of B-Cells
After activation and exposure to an antigen, the second stage of B-cell devel­
opment, known as the antigen-dependent phase, occurs. The activated cell
will either develop into a memory cell, which can be triggered again in the
future, or it will develop into a plasma cell that generates enormous amounts
of antibodies, depending on the numerous interactions and cytokine stimuli
it receives.

1. T-Cell Dependent Activation: Foreign proteins are examples of T
   cell-dependent (TD) antigens, to which B cells respond and become
   activated. They are called TD antigens because they cannot elicit
   a host response in organisms lacking T cells. B cell reactions to
   these antigens take several days, but the antibodies produced have a
   higher affinity and greater functional versatility compared to those
   produced through T cell-independent (TI) activation. Once a B cell
   receptor (BCR) binds to a TD antigen, the antigen is internalized
   through receptor-mediated endocytosis, destroyed, and presented
   to T cells as peptide fragments bound to MHC class II molecules
   on the cell membrane. T helper (TH) cells, particularly follicle T
   helper (TFH) cells, recognize and bind to these MHC-II-peptide
   complexes through their T cell receptor (TCR). T cells also express
   the membrane protein CD40L, as well as cytokines such as IL-4
   and IL-21, following TCR-MHC-II-peptide interaction. CD40L,
   by binding to the B cell surface receptor CD40, acts as a crucial
   co-stimulatory component for B cell activation. It promotes B cell
   proliferation, class switching to IgG, biological hypermutation, and
   also plays a role in T cell development and differentiation [53]. T

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Correlation Between Cancer Vaccine and Immunity
cell-derived cytokines not only promote B cell differentiation, but
also induce immunoglobulin class switching, stimulate somatic
hypermutation, and enhance B cell proliferation. These signals are
believed to activate B cells. After activation, B cells undergo a two-

— SECTION 30 —
step process that results in the production of short-lived plasmablasts
for immediate protection, as well as the generation of long-lived
cells and memory B cells for sustained protection. The extrafol­
licular response, which is the initial stage, takes place outside of
lymphoid follicles but within the secondary lymphoid organs (SLO).
Activated B cells undergo proliferation, potential class switching of
immunoglobulins, and differentiate into plasmablasts. These plas­
mablasts primarily produce early, low-affinity antibodies of the IgM
class. In the second stage, activated B cells enter a lymphoid follicle
and form the germinal center (GC), a specialized microenvironment
where B cells undergo extensive proliferation, class switching of
antibodies, and affinity maturation through somatic hypermutation.
TFH cells within the GC provide crucial support for these processes.

— SECTION 31 —
This results in the generation of long-lived plasma cells and high-
affinity memory B cells. Plasma cells secrete significant quantities
of antibodies, which can either remain in the SLO or selectively
migrate to the bone marrow [40].

2. T-Cell Independent Activation: Foreign polysaccharides and
   unmethylated CpG DNA are examples of TI antigens, which acti­
   vate B cells without the assistance of T cells. These antigens can
   induce an antibody response in individuals lacking T cells, hence
   the given name. Although the B cell response to these pathogens is
   rapid, the antibodies produced tend to have lower affinity and less
   functional diversity compared to those generated by TD activa­
   tion. Similar to TD antigens, TI antigens also require additional
   signals from B cells for full activation. However, these signals
   are not delivered by T cells, but rather through the binding and
   recognition of prevalent microbial components to toll-like receptor
   sites (TLRs), or by strong binding of BCRs to specific epitopes
   on bacterial cells [55]. Following activation by TI antigens, B
   cells may undergo antibody class switching, proliferate in external
   lymphatic tissue follicles while remaining within SLOs, and
   differentiate into short-lived plasmablasts that produce early, weak
   antibodies, primarily of the IgM class, but also a small population
   of long-lived plasma cells [56].

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Cancer Vaccination and Challenges, Volume 2

3. Memory B-Cell Activation: The target antigens, which are also
   recognized and bound by the parent B cell, trigger the activation of
   memory B cells. While certain memory B cells, such as those that
   are virus-specific, may be triggered without the aid of T cells, others
   require it. Upon primary antibody, the memory B cell internalizes
   the antigen via receptor-mediated endocytosis, breaks it down, and
   then presents the fragments to T cells on the cell membrane in asso­
   ciation with MHC-II molecules. These MHC-II-peptide complexes
   are recognized and bound by memory T helper (TH) cells, typically
   memory follicle T helper (TFH) cells, which have been generated
   from T cells stimulated with the same antigen [57]. The memory B
   cell is activated following TCR-MHC-II-peptide binding as well as
   the relay of additional signals from the memory TFH cell, and then
   undergoes differentiation into either plasmablasts or plasma cells
   through an extrafollicular reaction, or into plasma cells and more
   memory B cells via a GC reaction. It is unknown if memory B cells
   continue to develop their affinities within certain additional GCs.
   Memory B cells can be stimulated in vitro using a variety of activa­
   tors, such as monoclonal antibodies (mAbs); however, researchers
   have discovered that the most effective activator is a combination of
   R-848 with recombinant human IL-2 [58].

— SECTION 33 —
4.3 CANCER IMMUNOTHERAPY AND CANCER VACCINES (CVS)
The remarkable success of immuno-oncology therapies in stimulating the
innate autoimmune process towards tumor recurrence has rendered them
effective therapeutic approaches. In addition to novel immunotherapies
currently undergoing clinical and experimental investigations, the Food and
Drug Administration (FDA) of the USA has approved a significant number
of successful immunotherapies (Table 4.1), evaluated through various
parameters of vaccine development and drug toxicokinetics, which have been
utilized in clinical settings over the past decade (Figure 4.1). Promising cancer
immunotherapies that have the potential to elicit potent antitumor immunity
with minimal off-target effects in clinical scenarios include immune check­
point blockades (ICB), chimeric antigen receptor T cells (CAR-T), cancer
vaccines (CVs), and cytokine treatments. CVs, for instance, exemplify immu­
notherapies that can initiate tumor-specific immune responses by introducing
antigens to lymph nodes (LN) containing antigen-presenting cells (APCs). All
of these vaccines have demonstrated highly effective anti-tumor immunity, as

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Correlation Between Cancer Vaccine and Immunity
well as long-term immunological memory that inhibits tumor development,
recurrence, or metastasis to other organs. Furthermore, CVs often incorporate
immunologic adjuvants that enhance the activity of APCs and augment the
body’s natural anti-tumor immune responses. However, due to the inefficiency
of the anti-CV cascade, the clinical translation of these vaccines remains chal­
lenging. The specific recognition of antigens, co-encapsulation of the antigen
and adjuvant, LN-targeted antigen delivery, internalization, and transfer of
the antigen, and cross-presentation of the antigen to T cells constitute the core
five components of the cascade [59].
TABLE 4.1 List of Approved Cancer Vaccines and its Target
Vaccine Market
Name
Vaccine
Constitution
Country of
Manufacture
Target Type of
Cancer
Type of
Vaccine

— SECTION 35 —
References
HybriCell
Dendritic cell Brazil
Renal cell carcinoma;
melanoma.
Therapeutic
vaccine
[98]
CIMAVax EGF® Protein
Cuba
Lung cancer
Therapeutic
vaccine
[99]
M-Vax™
Tumor cell

— SECTION 36 —
USA
Melanoma
Therapeutic
vaccine
[100]
DCVax®-Brain
Dendritic cell
Glioblastoma
Preventive
vaccine
[54]
Oncophage®
Peptide
Renal cell carcinoma Preventive
vaccine
[94]
FIGURE 4.1 Complexity of onco-vaccine development.

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Cancer Vaccination and Challenges, Volume 2
Due to remarkable progress in cancer detection and knowledge, conven­
tional treatments have largely remained unchanged. Unfortunately, there are
instances where conventional medicines, such as surgery, chemotherapy,
and radiotherapy, prove to be ineffective, resulting in the spread or recur­
rence of cancer cells. Recent studies indicate the need for a strategy like
immunotherapy. Cancer immunotherapy has emerged as a key element in
cancer treatment. One type of therapy, immuno-oncology, enhances the
body’s natural immune response to cancer by generating highly aggressive
T lymphocytes that target and eliminate tumors. The most commonly used
medications in cancer immunotherapy, immune checkpoint inhibitors, often
have response rates of less than 30% in patients. Therefore, it is crucial to
improve existing cancer immunotherapies [60].

— SECTION 38 —
4.3.1 CANCER VACCINES (CVS) THAT INFLUENCE THE PRODUCTION
OF COMMON ANTIBODIES
The selection of the target antigen is arguably the most crucial factor in the
development of a vaccine. In contrast to non-specific vaccinations, which use
tumor lysate, the majority of studied vaccinations are specifically designed
to stimulate T cell reactions, rather than targeting common TAs or antigens
produced by cancerous cells and non-vital surrounding tissues. Typically,
malignant cells activate these shared antigens, while healthy tissue or early
embryogenesis express them in lower quantities. Examples include antigens
commonly expressed during embryogenesis, antigens associated with the
testes, and antigens that differentiate melanoma. As these proteins produce
unaltered self-antigens, high avidity T cells that recognize them are likely
eliminated during development due to the thymus’s detection of the antigen.
The limited pool consists of naive T cells that can respond to the vaccination.
Targeting such antigens puts the specific vaccination system at an immediate
disadvantage [61].
Therapeutic research, however, has demonstrated the feasibility of acti­
vating T cells in response to related antigens. Previous studies on patients
with melanoma have shown that immunization with gp100, a common prac­
tice, only slightly increased the strength of gp100-reactive T-cell responses.
On the other hand, modifying the peptide’s immunogenicity through anchor
residues significantly enhanced its binding affinity to MHC-I. This approach
was employed in clinical research, where a modified gp100 peptide vaccina­
tion was administered with IL-2. As a result, gp100-reactive T lymphocytes
were detected in the peripheral blood of melanoma patients [62].

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Correlation Between Cancer Vaccine and Immunity
Furthermore, in a phase III study investigating the same strategy, indi­
viduals who received the vaccination in combination with IL-2 showed
significantly greater therapeutic responses compared to those who received
IL-2 alone. These individuals also experienced a somewhat longer progres­
sion-free survival, with a median of 2.2 months compared to 1.6 months for
the IL-2 alone group. It is worth noting that the use of vaccinations in cancer
treatment extends beyond peptide vaccines [63]. DCs have been utilized as a
vaccine to activate T cell responses against tumors. Early research conducted
by Banchereau et al. demonstrated that DCs generated from CD34+ progeni­
tors can elicit measurable T-cell responses to various antigens when loaded
with peptides from Melanie/MART-1, MAGE-3, gp100, and tyrosinase
[64]. Building upon this research, Palucka et al. showed that allogeneic DCs
stimulated by monocyte-derived tumor lysis of cells can induce T cell reac­
tions against common malignant cells, leading to partial or full recovery in
some patients among a population of 20 immunized individuals [65]. Larger-
scale clinical studies are currently underway to assess the overall therapeutic
efficacy of this vaccination approach in treating various malignancies [66].

— SECTION 40 —
4.3.2 NEOANTIGEN-TARGETING ANTIBODIES
Neoantigens are a distinct category of tumor-associated antigens that have
been the focus of recent research. The “non-self” peptides in this type of
antigen are generated through alterations in the tumor genome that are not
synonymous. Typically, these alterations are specific to the patient’s tumor,
making them a key aspect of personalized medicine. However, under­
standing “shared” neoantigens, which are multiple mutant epitopes observed
in a patient, is crucial for the practicality of neoantigen vaccines. Several
studies have linked the clinical effectiveness of checkpoint blockade and
various types of adoptive cell treatment to neoantigens. Tumor cells are
the sole producers of neoantigens, which exhibit a strong resistance to T
lymphocytes that recognize the thymus as their most likely origin, evading
deletion. Consequently, vaccines targeting these antigens overcome a signifi­
cant barrier that hinders vaccines against shared antigens. Vaccines targeting
neoantigens not only provide protection against them but also reduce off-
tumor, on-target autoimmunity due to the tumor-specific expression of these
antigens [67].
Targeting immunotherapies to neoantigens is effective in significant
mouse model research. Castle et al. demonstrated the efficacy of a specific

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Cancer Vaccination and Challenges, Volume 2
peptide immunization against mutant regions such as Actn4 and Kif18b,
which inhibited the growth of B16 tumors in a highly immunosuppressive
mouse melanoma model. However, the mice could not be cured by immu­
nization alone. Yadav et al. also showed that immunization with a peptide
targeting mutants like Reps1, Adpgk, and Dpagt1 inhibited tumor develop­
ment in a mouse colon cancer MC38 model [68]. This study found that
inhibitory receptors PD-1 and Tim3 were highly expressed by neoantigen­
specific T lymphocyte cells within the tumor, indicating T cell dysfunction.
The expression of tumor receptors was decreased, although this may have
been caused by the study’s adjuvant. Duan et al. demonstrated that neoepi­
tope vaccination targeting Tnpo3 resulted in tumor shrinkage in a preventive
scenario using a novel method to identify immunogenic neoantigens and
combined treatment [69]. The response to neoantigen immunization was
enhanced by anti-CD25 and anti-CTLA4 antibodies. Preclinical studies
are increasingly focused on precisely determining whether certain tumors
include immunogenic neoantigens [70].
Neoantigen-specific vaccinations have been previously tested on several
human subjects. Carreno et al. immunized individuals with three malignant
tumors using neoantigen peptide-containing DCs. They demonstrated that
this approach increased the quantity and range of neoantigen-specific T
lymphocytes [71]. Two groups have more recently attempted this strategy
with RNA and peptide vaccinations. Ott et al. administered 13–20 lengthy
polypeptides with tumor-specific mutations that were predicted to bind
human leukocyte antigen (HLA-A or HLA-B) to six high-risk melanoma
patients. Contrary to expectations, CD4+ T cells predominated among the
vaccine-stimulated T cells instead of the predicted CD8-positive T cells [72].
However, none of the six patients showed any signs of tumor recurrence
25 months later. Pembrolizumab was administered to two patients who
still had the illness, resulting in full recoveries. Sahin et al. administered
a cocktail of synthetic RNAs with anticipated neoantigens to patients with
stage III and stage IV melanoma [73]. Unlike the study by Ott et al. [72],
this investigation focused on selecting mutated sequences that could interact
with both HLA class II molecules and HLA class I molecules. Both studies
demonstrated that CD4+ T cells were preferentially activated in response
to vaccination. Additionally, two patients showed clinically significant
responses to immunization. One patient demonstrated disease progression
after receiving the immunization. However, following pembrolizumab
therapy, they experienced complete clinical recovery. This study provides
compelling evidence that tumor-specific vaccinations do indeed elicit patient

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Correlation Between Cancer Vaccine and Immunity
immunity. The integration of immunization with the potential for immuno­
therapy significantly enhances the efficacy of the medication. Furthermore,
several clinical trials are currently assessing the safety and feasibility of
administering neoantigen vaccines, such as NCT02287428, NCT02510950,
NCT02427581, and NCT02348320, to patients with glioblastoma or breast
cancer who are concurrently undergoing chemotherapy or radiation therapy.
These immunization efforts target homologous antigens for the respective
antigen category treatment, potentially altering the benefits of immunoge­
nicity prediction algorithms as they become more precise [74].
The perfect vaccine formulation is still unknown, even though finding the
best therapeutic approaches is a top priority for researchers. Several orga­
nizations are currently investigating different techniques such as microbial
vectors, recombinant peptides, or nucleotide vaccinations, each with varying
degrees of success. However, due to the unique nature of antigens in each
specific tumor type, it is challenging to directly compare different vaccine
platforms. The multinational Human Immunome Project aims to bridge this
knowledge gap by evaluating various delivery strategies targeting the same
antigen, in order to determine the most effective immunogenic vaccination

— SECTION 43 —
systems for cancer patients. The results of such trials have the potential to
significantly enhance the therapeutic potential of cancer vaccines. Addition­
ally, the Human Immunome Project also aims to define the innate immune
capabilities in cancer patients. Early research has shown that, compared
to cells from healthy individuals, developing cancer patients have fewer T
cell stimulators in their DCs. A more recent study revealed that melanoma
patients have higher concentrations of MDSCs and an immune-suppressive
DC fraction that is BDCA1+CD14+, which inhibits T cell activation through
an antigen-based system. These investigations shed light on the widespread
immunological dysfunction experienced by cancer patients. Further charac­
terization of this immunological dysfunction may lead to improvements in
vaccination responses within the patient population [75].

— SECTION 44 —
4.3.3 DCS AS A COMPONENT OF A CANCER VACCINATION
DCs are indeed the main cell types that the aforementioned vaccinations
target, as they play a crucial role in initiating the immune response against
tumors. Therefore, the “immunization” of DCs is much safer than adminis­
tering vaccinations, even if only a small number of vaccinations come into
contact with DCs. The DC vaccines undergo this treatment and the distinct

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Cancer Vaccination and Challenges, Volume 2
DCs from a patient are isolated to create them. TAAs are added after the
cells have been co-cultured with antigens such as proteins, peptides, mRNA,
and DNA, which have reached maturity [75]. These adjuvants include TLR
agonists or cytokines. After treatment, the patient receives the DCs again,
which are then transferred to the LN, where the CD8 T cells are activated
to initiate the anti-tumor immune response. The fundamental advantage of
DC vaccinations is that, unlike traditional vaccines that require immediate
delivery of vaccine components inside patients, off-target effects are much
less of a concern because DCs are treated in vitro. The difficulty and high
cost of producing cell procedures, as well as the batch-to-batch variation
across vaccinations for different patients, pose significant obstacles to the
standardized production of DC vaccines [76]. Although it has been approved
by the FDA, its commercialization has only been achieved since Sipulencel
T, the first DC-based CV, was created in 2010 to treat metastatic prostate
cancer in a limited number of developed nations. This is mainly due to
the expensive and stringent production requirements of vaccine factories.
Comparing clinical studies and extrapolating their findings is challenging
due to the wide variations in manufacturing processes in DC vaccines and
the resulting composition. Silence T has been successful, and phase I and

— SECTION 45 —
II clinical studies are producing encouraging preliminary results, although
there have also been some significant failures. The dismal brief patients’
condition is assessed, whereas encouraging findings from past studies have
forced Argos Therapeutics to halt a DC vaccine for renal cell carcinoma that
is now in the third phase of clinical trials. Similar findings from a phase
III, DC melanoma vaccination clinical study revealed the drug’s absence
of an appreciable effect on patient mortality or recovery-related indicators.
However, it is possible that the challenging procedure for acquiring, growing,
and treating DCs [77, 78].
4.3.3.1 DCS’ ROLE IN PROMOTING ANTI-TUMOR IMMUNITY
Multiple diverse subsets, including four main populations, make up DCs.
These subgroups are most frequently categorized by ontogeny. These
include MoDCs, plasmacytoid DCs, and conventional or traditional DCs,
which were divided into types 1 and 2 (MoDC). The propensity for antigen
presentation, migration, and cytokine release varies across each group.
Therefore, choosing the appropriate DC vaccine development depends on
the cell type [79].

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— SECTION 47 —
4.3.3.2 DCS ARE PRODUCED FROM MONOCYTES
APCs, also known as MoDCs, are a diverse population that primarily
develops in response to inflammation. Monocyte progenitors are generated
from CD14+ monocytes or CD34+ HSPCs, allowing for the production of
large numbers of mature MoDCs with antigens derived from peripheral
blood through treatment with GM-CSF and IL-4. Most clinical trials utilize
ex vivo-produced MoDCs (IL-4). Extensive testing of ex vivo-produced
MoDCs in immunization trials has demonstrated their capacity for cross-
priming T cells and the release of anti-tumor cytokines, including IL-12 [80].
A subset of patients who received this therapy exhibited anti-tumor
activity, highlighting the potential of MoDCs as an effective component
of vaccines. However, the clinical data for the majority of individuals’
responses have been mild, possibly due to the fact that ex vivo differentia­
tion techniques only partially replicate the physiological growth of MoDCs.
It has been observed that GM-CSF and IL-4 treatment of both murine and
human precursors leads to MoDCs that differ from their naturally occur­
ring (primary) counterparts in terms of transcription and phenotype. This
disparity may be attributed to limitations in ex vivo-produced MoDCs, such
as reduced binding ability to CD11c+ DCs isolated from circulating blood.
Additionally, MoDCs generated in vivo may have a diminished capacity to
migrate into lymph nodes, which could explain the suboptimal efficacy of
vaccines formulated with such MoDCs [81].

— SECTION 48 —
4.3.3.3 TRADITIONAL DCS OF TYPE 1
A growing body of research indicates that cDC1s are a crucial component of
tumor immunity and could serve as a potential alternative cell type for vacci­
nation. Although there are inconsistent definitions regarding cDC1-specific
gene signatures, which may introduce some variability in our study, bulk
whole cancer gene expression analyses reveal that cDC1 gene signatures
are associated with improved outcomes in various types of cancer. Recently,
strong evidence was published in a single-cell RNA sequencing (scRNA­
seq) study of non-small-cell lung cancer (NSCLC), demonstrating that CDC
clusters correlate with improved survival. In this analysis, immune cells were
categorized using unmonitored grouping, despite the bias of reference genes.
Numerous pre-clinical studies using cDC1 depletion models based on mice
deficient in Batf3, a transcription factor essential for cDC1 development,

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have shown the necessity of cDC1s for immunogenic tumor cell rejection
and responses to immunotherapies like ICB and adoptive T cell transfer [82].
It appears that cDC1s mediate cancer immunity through various special­
ized tasks involving different processes. One of the most crucial processes
is the ability of CD8+ T lymphocytes, a cell type closely associated with
prognosis in multiple types of cancer, to cross-present exogenous antigens
on major histocompatibility class I (MHCI) molecules to cDC1s. It has been
established that tumor rejection requires cross-presentation, particularly via
cDC1s. By utilizing a clustered regularly interspaced short palindromic repeats
(CRISPR) screen, it was discovered that the BEACH domain-containing
protein WDFY4 is essential for the cross-presentation of cDC1s in both
primary splenic cDC1s and ex vivo-differentiated cells. Wdfy4-/- mice also
exhibited an inability to mediate the rejection of a specific antibody fibrosar­
coma tumor. Theoretically, WDFY4 modifies vesicular trafficking pathways
that play a crucial role in antigen processing, although the exact mechanisms
remain unclear. It is worth noting that the loss-of-function characterization
of cell type specificity did not affect the presentation of TAAs by cDC2s or
MoDCs, and only allowed for cross-presentation, as it had no adverse effects
on cDC1 formation, chemokine production, or MHCII antigen presentation.
This suggests that cross-presentation, particularly via cDC1s, is required for
tumor rejection in the fibrosarcoma model. These research findings align
with previous studies that describe various interactions involving DCs and
the cDC1 subunit in cross-presentation processes [83].
According to multiple studies, the impact of cDC1s on the immune
response against tumors goes beyond cross-presentation alone. Interferon
regulatory factor 8 (Irf8VENUS), a crucial transcription factor necessary for
cDC1 lineage commitment, which is typically regulated by BATF3-mediated
autoactivation, can be expressed as a transgene to restore cDC1 development

— SECTION 50 —
in Batf3-/- mice. This results in functional cDC1s that can be utilized in
both cases, but fibrosarcomas are not effectively eliminated. Although some
BATF3-dependent activity that is necessary for cross-presentation does not
appear to be related to tumor rejection in cDC1s, the precise processes are not
well understood. One mechanism involves the influence that cDC1s have on
immune cell migration, which is facilitated by interactions between chemo­
kines and receptors that bring different immune cells together within tumors
and towards lymph nodes. For example, the infiltration of CXCR3+ T cells
into the tumor is promoted by chemokine (C-X-C motif) ligands 9 and 10
produced by cDC1s (CXCL9), while XCR1+ cDC1s are attracted to XCL1
produced by tumors. This highlights the importance of tumor-produced

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chemokines and their effects on the secretion and behavior of cDC1s. These
chemokines, found within the tumor environment, activate cDC1-dependent
cellular signaling and anti-cancer responses against various tumor types. DCs
need to express C-C chemokine receptor 7 (CCR7) in order to be transported
to lymph nodes that drain tumors. In melanoma models, where effector T cell
priming occurs and leads to anti-tumor activities, cDC1s must express CCR7
for tumor-associated antigens (TAAs) to be transported to lymph nodes.
Overall, these findings demonstrate the critical role of cDC1s in migrating
towards lymph nodes, where they can present TAAs to effector T cells and
infiltrate the tumor, thereby attracting more DCs and lymphocytes through
the release of chemokines [84].
According to two recent investigations, the effectiveness of immunity
checkpoint inhibition in colon cancer MC38 models adenocarcinoma is
determined by cytokines secreted by cDC1s. One study utilized scRNA-seq
in combination with intravital live cell imaging to demonstrate that cDC1­
like cells secreted IL-12 after the release of interferon (IFN) from a CD8+
T cell-coated surface with an anti-programmed cell death protein 1 (PD-1)
antibody. This reactivation of exhausted T lymphocytes, followed by anti­
tumor responses, required interaction. The requirement for cDC1-derived
CXCL9 for tumor regression on PD-1 blocking was also discovered. In
contrast to its traditional role as a T cell chemoattractant, CXCL9, in this
study, only served to promote CD8+ T cell proliferation and reactivation.
Previous findings using anti-TIM-3, a different checkpoint blockade treat­
ment, are consistent with this (also known as mucin-domain containing-3 and
T-cell immunoglobulin). cDC2s did not affect CXCL10, a distinct chemoat­
tractant that interacts with CXCR3 and predominantly increases the tumor’s
susceptibility to checkpoint inhibitors. These diverse findings demonstrate
the crucial role of cDC1s in triggering the immunological generation of
cytokines to activate [85].

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4.3.3.4 TRADITIONAL DCS OF TYPE 2
Additionally, although the data’s breadth is less substantial compared to
MoDCs and cDC1, the immunotherapeutic potential of DC subsets is also
diminished. cDC2s are experts at priming CD4+ T cells via antigen presen­
tation on MHCII (Box 1), effectively polarizing TILs toward anti-tumor T
helper 1 (Th1) or Th17 phenotypes. Furthermore, humans, as evidenced
by cDC2s, can release IL-12, and under specific circumstances, CD8+ T
lymphocytes can cross-present antigens. scRNA-seq analysis has shown that

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intra-tumoral levels of cDC2s are favorably associated with NSCLC patient
survival rates [86].
In a B16-F10 melanoma model, the presence of Tregs or regulatory T
cells, which inhibit the migration of cDC2s, has been shown to stimulate
CD4+ anti-tumor immunity and activity upon their removal. The anti-tumor
effects of Treg depletion were not reliant on cDC1s, suggesting that cDC1s are
not actively suppressed by Tregs in this context. Conversely, in the presence
of PD-1 inhibition in the same tumor model, IL-12 produced by cDC1 was
found to be necessary for an anti-tumor response, indicating the involvement
of distinct and potentially synergistic cDC subsets in therapeutic processes.
Another example is the potential of tumor-derived cDC2s to polarize CD4+
T cells towards a Th17 phenotype, which subsequently leads to a phenotypic
shift of tumor-associated TAMs from pro-tumor M2 to anti-tumor M1. This
elicited a potent anti-tumor response in a lung cancer model with significant
TAM infiltration. However, the impact on cDC1s was less pronounced in a
B16 model with low TAM invasion. If present, cDC1s exhibited a stronger
anti-tumor response in the absence of TAMs, likely due to enhanced CD8+
T cell priming. Primary cDC2s derived from primary cDC2s were clinically
evaluated in studies investigating vaccination against human blood mela­
noma, demonstrating their anti-tumoral effects. These findings have been
observed in certain malignant tumors, leading to improvements in immuno­
genicity and patient survival in numerous cases [87].

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4.3.3.5 THE PLASMACYTOID DCS
In response to viral infections, plasmacytoid dendritic cells (pDCs) are well
known for producing significant quantities of type-I IFNs. On one hand,
pDCs have been associated with immunosuppression in cancer, primarily
through promoting pro-tumor Treg production of inducible costimula­
tory ligand (ICOS-L). This relationship between intra-tumoral tumors and
pDC-level development in various malignancies is likely explained by this
function. However, pDCs also exhibit a valuable early response to cancer
vaccinations. It has been demonstrated that following the administration of
an RNA vaccine in nanoparticle (NP) form, pDC-derived type-I IFNs are
necessary for the maturation of activated effectors through cDC1s anti­
tumor responses. This finding once again suggests that utilizing different DC
subsets may be advantageous. Indeed, pDC-cDC1 crosstalk is beneficial in
an anti-viral setting. Primary pDCs have shown the ability to produce type-I
IFN, as well as release IL-12 and cross-prime T-cells. Therefore, in melanoma

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Correlation Between Cancer Vaccine and Immunity
patients, IFN-driven T-cell responses were induced by pDCs isolated from
human blood. According to preclinical and clinical research, pDCs may
serve as beneficial components for anti-cancer vaccination, despite potential
concerns regarding PDC-mediated immunosuppression [88].

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4.3.3.6 COMBINATION OF DC VACCINATIONS
The relative importance of each DC subset in anti-tumor responses will
inevitably vary depending on the type of cancer (as demonstrated by cDC2­
based model-dependent vaccinations) and the therapeutic approach. To fully
leverage their complementary activities and intercellular communication, it
is reasonable to hypothesize that an ideal vaccine would incorporate multiple
DC subsets. Further research is needed to elucidate the specific functions
through which each DC subgroup effectively prevents tumor growth; this
knowledge could guide the development of multiplexed DC vaccinations.
Additionally, improvements in ex vivo differentiation techniques are neces­
sary to enable the generation of DC subsets with intact immunogenic capa­
bility [85].

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4.4 ELEMENTALS OF CANCER VACCINES (CVS) AND
IMMUNOTHERAPY
Through the reactivation of innate defense mechanisms, which include
immune cells, tumor immunotherapy aims to treat cancers. Innate immunity
cells and macrophages have the ability to directly eliminate pathogens in
innate immunity. T lymphocytes and B lymphocytes require activation by
antigens in adaptive immunity to generate effector cells or antibodies that
initiate an immune response. Antigen-presenting cells (APCs) process tumor
cell-derived antigens before presenting them. Tumor cell death and antigen
release serve as the starting point, leading to an enhanced immune response and
further tumor cell death. The “cancer immunity cycle” refers to the progres­
sive processes resulting from this closed-loop system, which theoretically
creates positive antitumor feedback regulation. Based on this cycle, several
tumor immunotherapies, including immune checkpoint inhibitors (ICBs) and
CAR-T cell immunotherapy, have entered clinical studies. First-generation
ICBs, such as anti-PD or PD-L1, rely on the presence of cytotoxic T cells
(CTLs) that infiltrate tumors. However, the presence of myeloid-derived
suppressor cells (MDSCs) with immunosuppressive properties, regulatory
T lymphocytes, and tumor-associated macrophages near the tumor site

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significantly impede the therapeutic benefits. The aforementioned approaches
demonstrate effectiveness at the later stages of the cancer immunity cycle. By
delivering and ingesting antigens prior to the cancer-immunity cycle, tumor

— SECTION 59 —
vaccines, which are considered to be one of the most effective methods of
enhancing immunity, induce CTLs and humoral immunological responses.
The development of a tumor vaccine primarily involves four essential steps:
selecting the vaccine’s active ingredients, precise vaccine administration,
antigen release or presentation, and T-cell immunological infiltration. The
initial phase focuses on choosing the crucial vaccine ingredients, including
the appropriate adjuvants and antigens. The immunogenicity of the adjuvants
and antigens, particularly TA, plays a vital role in the anti-tumor immu­
nological activity. In the second phase, effective distribution of the tumor
vaccine relies on the ability of antigen-presenting cells (APCs) to promote
endocytosis in lymphoid organs and minimize systemic side effects. The third
phase involves intracellular delivery of the vaccinations, reducing the amount
of antigen destroyed in lysosomes and endosomes. To maximize CD8+ T
cell activation against solid tumors, the immunological response triggered by
the vaccine must cross-present more foreign antigens through MHC class I
molecules. The cancer vaccines precisely target the antigen-presenting cells
(APCs), which are quickly phagocytosed and processed to enhance cross-
presentation. Tumor vaccines can effectively improve the immunosuppres­
sive environment, promote lymphocyte infiltration, and enhance the efficacy
of tumor vaccines when combined with other therapies to eradicate the tumor
microenvironment. The tumor’s dependence on a gradual cascade response
triggers an immune response against the tumor. Several guiding principles
are laid out to guide the development, design, and ongoing improvement of
successful tumor vaccines, including the conditions required to prime the
immune response. To maximize the CD8+ immune response and eliminate
tumor tissues, adjuvants, and antigens must be delivered simultaneously via
dendritic cells (DCs) and used as intelligent reactive elements. This approach
enables cross-presentation and facilitates T lymphocyte infiltration [89].
The loop between cancer and immunity during tumor immunotherapy.
Step 1: In step 2, APCs endocytose the relevant antigens after the tumor
cells have died and released their antigens. DCs are among the APCs that
are most proficient at consuming and handling cancer antigens at this stage.
Step 3: Mature DCs are formed when the lymph nodes are the destination
of immature DCs that have absorbed the antigen. DCs then activate antigen-
specific cytotoxic T cell responses by presenting antigen fragments to CD8+
or CD4+ T lymphocytes through the MHC I or MHC II pathway of the MHC.

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Correlation Between Cancer Vaccine and Immunity
Step 4: Effector T cells that recognize an antigen migrate towards tumor
tissues. Step 6 follows step 5 to recognize, or rather, bind to tumor cells. Step
7 involves the release of granzyme and the destruction of tumor cells [89].

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4.5 TYPES OF CANCER VACCINES (CVS) AND THEIR TARGET
For cancer patients, immunotherapy has always been a desirable and poten­
tially effective treatment option. Vaccines are medications that support the
body’s immune system. They can educate the immune system to recognize
and eliminate harmful microorganisms and cells. Throughout your life, you
receive various vaccinations to protect against common illnesses. There are
also additional vaccinations available for cancer. Both cancer prevention and
cancer treatment vaccines are available [90].
4.5.1 THERAPEUTIC CANCER VACCINES (CVS)
Therapeutic cancer vaccinations are designed to enhance the patient’s adaptive
immunological response to a specific tumor, aiming to regain control over
tumor progression, induce shrinkage of existing tumors, and eliminate minimal
residual disease. The key principles for effective therapeutic vaccination
against a tumor include delivering substantial amounts of high-quality antigens
to dendritic cells (DCs), optimal stimulation of DCs, induction of robust and
persistent CD4+ T helper cell and CTL (cytotoxic T lymphocyte) responses,
infiltration of the tumor microenvironment (TME), and ensuring durability and
maintenance of the immune response. These goals can be achieved through
various approaches, such as utilizing immune checkpoint inhibitors (ICIs) to
reverse tumor-induced immune exhaustion, administering cancer antigens
with adjuvants to activate DCs and effector T cells, or employing autologous
DCs loaded with specific tumor antigens for vaccination [91].

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4.5.2 PREVENTIVE CANCER VACCINES (CVS)
Other potential antigens for preventative vaccinations must be examined
as objectives for malignancies, as the infectious underlying etiology is
either unclear or non-existent for the majority of cases. Since most tumor-
associated antigens (TAs) are composed of self-molecules, inducing a strong
immune response against them has been considered to violate self-tolerance
and increase the risk of developing autoimmunity. As a result, most efforts
to generate cancer vaccines have been focused on treating advanced cancer,

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Cancer Vaccination and Challenges, Volume 2
where the risks associated with the choice of target molecules have been
found to be more manageable [92].

— SECTION 64 —
4.6 MECHANISM OF ACTION AND IMMUNOLOGICAL EFFECTS OF
CANCER VACCINES (CVS)
The immunological mechanism of CVs and their approach to tumor cell
death has not been clearly understood, yet the process of immunogenic cell
death (ICD) has been deduced in various cancer research. Adequate commu­
nication between immunological and non-immune components of the cancer
microenvironment (CME) is necessary for effective antitumor immunity
(Figure 4.2). Antigen-presenting cells (APCs), such as dendritic cells (DCs),
collect and cross-present the peptides produced by cancerous cells, acti­
vating T lymphocytes. In the CME, natural killer (NK) cells, neutrophils,
and macrophages of the innate defense mechanism play a critical role in the
early identification and destruction of tumor cells. When cancer cells die
spontaneously or as a result of therapies like certain forms of chemotherapy,
they produce tumor-associated antigens (TAs) and molecular patterns that
are generally associated with various risk factors [93].
Predominantly, CVs involve the exogenous infusion of specific TAs
along with functionally active DCs or DCs activating proteins. The objec­
tive of immunotherapeutic vaccines is to stimulate the patient’s adaptive
immune response against specific TAs, thereby halting tumor development,
causing existing tumors to regress, and eliminating any minimal residual
disease. Key elements required for effective therapeutic vaccination against
cancers include significant doses of high-quality cancer peptide delivery
to DCs, optimal activation of DCs, induction of potent and long-lasting
CD4+ T helper and CTLs responses, infiltration of the CME, robustness,
and restoration of the immune response [75]. This can be achieved through
various approaches, such as using immune checkpoint antagonists to reverse
tumor-induced immune depletion, introducing cancer proteins with mole­
cules to activate DCs and effector T lymphocytes, or utilizing autologous
DCs loaded with specialized tumor antigens for vaccination. Alternatively,
the tumor’s endogenous immune system can be extensively stimulated to
induce malignant cell destruction and enhance the accessibility of cancer
antigens through in situ vaccines (ISVs). The in-situ approach generates the
vaccine within the CME by harnessing antigens from dead or injured cancer
cells. This differs from traditional immunization, where antigens are care­
fully selected, purified, or prepared before administration to patients [95].

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Correlation Between Cancer Vaccine and Immunity
FIGURE 4.2 General mechanism of action of cancer vaccines (CVs).

— SECTION 66 —
4.7 FUTURE PERSPECTIVE OF CANCER VACCINES (CVS) IN CANCER
IMMUNOLOGY
Although a vast number of cancer-related antigens have been discovered,
questions have been raised about their limited immune activation and suit­
ability for use in the creation of cancer vaccines. The security of these
vaccinations will continue to be an issue. While the current technical trend
of using pure recombinant proteins instead of whole-cell or subunit patho­
gens has increased vaccination safety, it has also resulted in decreased
immunogenicity due to the absence of recognizable entire pathogens [96].
However, the utilization of recombinant vectors could potentially offer a
platform. It is necessary to investigate new targets and routes for producing
efficient immune responses. Overall, there is enormous promise for devel­
oping innovative paths for more effective immune adjuvants, particularly
those that are effective against the devastating illness of cancer [97].

— SECTION 67 —
4.8 CONCLUSION
One of the biggest successes of immunotherapy so far has been its accelera­
tion of developments in cancer research, significantly influencing anticancer
therapy. Numerous new insights into how the immune response affects cancer
have been revealed through research, justifying the FDA’s authorization of

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Cancer Vaccination and Challenges, Volume 2
immunotherapeutic drugs. Oncologists now utilize novel immunotherapies
that modify immunological checkpoints, utilize infections to trigger an effi­
cient immune reaction, employ CVs to stimulate the immune system, and
develop T-cell treatments for specific antigens. Importantly, individuals who
previously had limited treatment options have shown dramatic improve­
ments in their prognoses due to these immunotherapy techniques. Despite
the fundamental change in anticancer therapy and its emergence as the fifth
element of anticancer drugs, immunotherapy still faces challenges. These
obstacles, including immunosuppressive CME, T-cell stimulation, and loss
of responsiveness, reduce the effectiveness of immunotherapy. However,
they also provide opportunities for unique medication interactions and
novel drug designs. Ongoing research may lead to the development of novel
immunotherapy delivery systems, the identification of fresh immunotherapy
potentiation pathways, a deeper understanding of the immune-cancer interac­
tion, and most importantly, continued improvement in clinical achievements.

— SECTION 68 —
ACKNOWLEDGMENTS
The authors sincerely acknowledge and express our deep gratitude to REVA
University and Ganpat University for providing us with an opportunity and
platform for research.
CONFLICT OF INTEREST
The authors declare that there is no conflict of interest.
KEYWORDS

• 
  antigenic stimulation
• 
  cancer vaccine
• 
  immunological checkpoints
• 
  multifactorial antigens
• 
  mutations
• 
  pattern recognition molecules
• 
  tumor-infiltrating-lymphocytes
• 
  vascular endothelial growth factor

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Cancer Vaccination and Challenges, Volume 2: Delivery Strategies for Cancer Vaccine and Immunotherapy in
the Management of Various Carcinomas. Rishabha Malviya, Bhupendra Prajapati, and Sonali Sundram (Eds.)
© 2025 Apple Academic Press, Inc. Co-published with CRC Press (Taylor & Francis)

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#

CHAPTER 5
TITLE: Cancer Vaccine for Lung Cancer
AUTHORS: Ankita Panigrahi, R. Mythreyi, T. S. Gopenath, Kanthesh M. Basalingappa, and K. Gobianand
LENGTH: 96,923 characters

#

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CHAPTER 5
Cancer Vaccine for Lung Cancer
ANKITA PANIGRAHI,1 R. MYTHREYI,1 T. S. GOPENATH,2
KANTHESH M. BASALINGAPPA,1 and K. GOBIANAND3
1Division of Molecular Biology, School of Life Sciences, JSS Academy of
Higher Education and Research, Mysore, Karnataka, India
2Department of Biotechnology and Bioinformatics, JSS Academy of
Higher Education and Research, Mysore, Karnataka, India
3Department of Microbiology, Noorul Islam College of Dental Sciences,
Aralummoodu, Thiruvananthapuram, Kerala, India
ABSTRACT
Cancer is the leading cause of death worldwide, claiming the lives of
millions of people each year. The most prevalent types of cancer include
lung, breast, colorectal, prostate, and pancreatic cancer. Treatment options
for cancer encompass chemotherapy, radiotherapy, surgery, and the use of
inhibitors that can impede cancer signaling pathways. However, cancer
remains a significant health concern. In the past decade, scientists have made
advancements in the development of cancer vaccines (CVs) that stimulate
the body’s immune system. These CVs fall under the category of immu­
notherapy, which triggers an immune response against tumor-associated
antigens (TAAs) and tumor-specific antigens. They are classified as either
preventative or therapeutic vaccines based on the timing of administration.
Preventative vaccines, for instance, have shown a 70% reduction in the
risk of cervical and hepatic cancer when used against HPV and HBV. On the
other hand, therapeutic vaccines enhance the recognition of tumor antigens
(TAs) while reducing immune tolerance. Although CVs hold promise, the
development of lung CVs has been challenging due to the constant exposure

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of the lungs to antigens, dust, and microbes, as well as the difficulty in identi­
fying the target antigen, among other factors. However, when combined with
other conventional therapies, vaccines such as MAGE-A3 and L-BLP25 have
shown benefits for patients. Combinations of vaccines and targeted drugs,
such as immune checkpoint inhibitors (ICIs) and antiangiogenic therapy,
have proven to be more effective. It is crucial to understand the tumor micro-
environment (TME), pathways, and utilize personalized approaches in the
development of vaccines to ensure the success of CVs.

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Therapeutic vaccines have shown more promising results. To enhance
the immune response of patients to viral vectors, multiple antigens, and
boost strategies, new clinical trials are being standardized.
5.1 INTRODUCTION
Cancer is the leading cause of death worldwide, accounting for over 10
million deaths in 2020 alone. According to the World Health Organization
(WHO), one out of every six deaths in the world is due to cancer. Among
all cancers, lung cancer is the second most common, affecting people glob­
ally. In females, it is the second most common after breast cancer, while in
males, it is the most common. The American Cancer Society states that the
causes of cancer can be genetic, dietary, related to exposure to radiation or
chemicals, or due to infection.
The lungs are vital organs in the human body, especially in the respiratory
system. However, smoking is the major cause of lung cancer, accounting for
more than 80% of cancer diagnoses. Non-smokers are also at risk of devel­
oping lung cancer due to factors such as secondhand smoke exposure and
other factors [1, 2]. Accurate identification of primary bronchial carcinoma
is crucial for treatment purposes. Despite significant progress in the develop­
ment of treatment options over the last century, widespread effectiveness has
not been achieved. Understanding and classifying cancer is essential for the
development of effective treatments.
There are two main types of lung cancer: small cell lung carcinoma
(SCLC) and non-small cell lung cancer (NSCLC). Furthermore, NSCLC and
SCLC are further classified into various subgroups, including squamous cell
carcinoma, adenocarcinoma, large cell carcinoma, and more [3]. When exam­
ined under a microscope, specific types of tumors such as Pancoast tumors
(upper sulcus tumors) and carcinoids, which are composed of specialized
cells called neuroendocrine cells, can be identified. We will discuss these in
more detail in the upcoming paragraph. Approximately 10–15% of tumors

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Cancer Vaccine for Lung Cancer
are SCLC, while 85–90% are NSCLC, identified through histopathology of
the tumor [4].
The chances of detecting cancer at an early stage are about 15%, and
more than 75% of lung cancer cases are detected at the metastasis stage. The
treatment for the primary stage includes surgery in addition to chemotherapy,
but for the advanced stage, combined therapy is required, which includes
targeted therapy, pathway inhibition, radiotherapy, and immunotherapy [5].
Advanced strategies, along with conventional approaches, are being used,
such as mutational activation targeting the epidermal growth factor receptor
(EGFR) and the ROS proto-oncogene treatment. However, the outcome is
disappointing mainly due to delays in diagnosis and low response to current

— SECTION 5 —
approaches [6, 7]. Screening methods for lung carcinoma include lung biopsy,
chest X-ray, low dose computed tomography, and sputum cytology. Low-
dose CT is highly sensitive but may expose patients to radiation. Sputum
exfoliative cytology is a rapid, specific, practical, and economical diagnosis
method, although its sensitivity is reduced [8, 9]. The need of the hour is to
find the right diagnostic tool with high specificity and sensitivity. In the year
2000, Hanahan & Weinberg discovered the six hallmarks of cancer, which
include inducing angiogenesis, activating invasion and metastasis, resisting
cell death, unlimited replicative potential, evading apoptosis, and sustaining
proliferative signaling. In 2011, two more hallmarks were added due to our
understanding of epigenetics and crosstalk between cancer cells, which are
reprogramming cellular metabolism and avoiding immune destruction.
In recent years, the most debated topic in the field of cancer research has
been cancer immunotherapy, which has led to the discovery of new approaches
for its treatment. With the increased use of immunotherapy in cancer, such as
Monoclonal antibodies (mAbs) like trastuzumab and alemtuzumab, there is
enormous potential in using the cellular arm as a cancer vaccine (CV) [10].
Initially, there were mainly three types of cancer treatment, but Professor
Lloyd J. Old noticed that cancer cells are different from normal cells in our
body. He predicted in the early 1900s that cancer immunotherapy, along with
surgery, chemotherapy, and radiotherapy, would treat cancer more effectively
[11]. After decades of extensive research and intense clinical trials, cancer
immunotherapy was recognized and became the fourth pillar of cancer treat­
ment. Immunotherapy utilizes the body’s own defense mechanism to fight
against the disease, revolutionizing the way cancer is treated. There are four
main types of immunotherapies based on the type of cell used for treatment:
checkpoint inhibitors, cytokines, CAR T-cell therapy, and CV. The use of
nanotechnology to develop nano-vaccines has also shown great potential,

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although there is still a long way to go. We will discuss their mechanisms of
action on our body and current research in later paragraphs. With the inven­
tion and development of vaccines, global pandemics have been significantly
reduced, saving millions of lives. Vaccines teach our immune system to
fight against all types of foreign microorganisms. Although vaccines have
prevented certain types of cancer, such as the Human Papilloma vaccine
(HPV) and Hepatitis B vaccine (HBV), which have the potential to cause
hepatocellular carcinoma (HCC), intensive research is ongoing to develop
vaccines targeting existing cancers, known as CV. Cancer cells have the
property of escaping from immune cells and preventing our immune system
from attacking the tumor by producing immunosuppressive checkpoints
[12]. Vaccines given after the onset of infection are called therapeutic
vaccines. In the case of cancer, they are mainly divided into autologous and
analogous CV. The first type is taken from the patient, processed, multiplied,
and injected, whereas the second one is grown from non-self-cells [13].
Autologous CV provides numerous benefits, such as being a source of tumor
antigen (TA), activating CTLs and Th cells, and even offering personalized
treatment options for the development of personalized vaccines.
In this chapter, we will discuss the role of CV therapy in detail for lung
cancer. As lung carcinoma progresses, the survival rate decreases, high­
lighting the importance of early diagnosis. In stage III and IV, systematic
cytotoxic chemotherapy is the primary treatment option. Although targeted
therapies based on mutations have been utilized, their success is limited.
Therefore, cancer immunotherapy remains the most powerful approach
[14]. CV therapy, which aims to induce T cell infiltration into the tumor, is
considered the most effective strategy. Various types of vaccines, including
peptide vaccines, dendritic cell (DC) vaccines, vector-based antigen-
specific vaccines, whole cell vaccines, and vaccines targeting tumor-specific
T cells, have been developed [15]. Tumor eradication can be achieved by
stimulating the immune system to produce CD4+, CD8+, and cytotoxic
T lymphocytes (CTLs) that target tumor-associated antigens (TAAs). An
example of a therapeutic vaccine is CIMAvax-EGF, developed in Cuba for
NSCLC, as EGFR is present in more than 50% of patients. This vaccine,
licensed in 2008, induces antibodies against autologous EGF [15]. Recent
approaches involve the use of adjuvants that enhance the immunogenic
response to vaccines. These adjuvants further activate antigen-presenting
cells (APCs), which in turn activate tumor-specific T cells [16]. Finally, we
will explore the causes of lung tumors and how mutations contribute to the
development of cancer.

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5.2 LUNG CANCER
In the case of lung cancer, the cells of the lung behave abnormally and divide
uncontrollably, forming a tumor that can metastasize to other organs of the
body. The accumulation of molecular abnormalities over a prolonged period
of time is involved in the pathogenesis of cancer. External factors, such as
tobacco smoking, account for approximately 80% of all cases [17]. Other
factors include air pollution and exposure to harmful gases. Understanding
the molecular characterization of lung cancer will aid in the development of
better clinical approaches.
There are two different types of small cell lung cancer (SCLC): combined
SCLC and small cell carcinoma. These types are classified based on their
appearance under a microscope. SCLC is commonly associated with
smoking and is typically treated with chemotherapy. On the other hand, non-
small cell lung cancer (NSCLC) accounts for a higher percentage of cases,
grows slowly, and can metastasize to different parts of the body. NSCLC is
classified into adenocarcinoma, which is usually found in the outer lining
of the lung; squamous cell carcinoma, which is found near the bronchus in
the center of the lung; and large cell carcinoma, which can develop in any
part of the lung but grows faster. Under a microscope, specific types of lung
cancer, such as Pancoast tumors, can be identified. These are rare and can
develop due to certain disease conditions, such as tuberculosis or lymphoma.
Carcinoid tumors are also uncommon and grow slowly. They are composed
of specialized cells called neuroendocrine cells. Treatment for lung cancer
often involves surgery. Let’s understand the types of lung cancer and the
molecular genetic behind cancer development (Figure 5.1).

— SECTION 8 —
5.2.1 TYPES OF LUNG CANCER
Mainly lung cancer is classified into two types of SCLC and NSCLC.
About 15% of lung cancers are attributed to SCLC, which exhibits a high
proliferation rate and poor prognosis. SCLC can be further classified into
two subtypes: small cell carcinoma and combined mixed cell carcinoma.
These subtypes are distinguished based on their microscopic appearance.
The primary cause of SCLC is strongly associated with smoking [18]. By
examining the genetic profile, it is primarily caused by the inactivation of
two tumor suppressor genes, namely RB1 and TP53 (p53), which is different
from NSCLS where oncogenic mutations are essential for tumor formation.

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FIGURE 5.1 Two main types of lung cancer (NSCLC and SCLC) and its further classification.
Changes in the tumor microenvironment also contribute to the development
of SCLC [19]. Overall, the mechanisms by which extrinsic and intrinsic
factors contribute to the formation and proliferation of SCLC are not yet fully
understood. SCLC is characterized by early metastasis and rapid growth.
Let’s look at the gene which contribute to the formation of tumor along
with their occurrence in SCLC (Table 5.1).
Another gene that is observed in SCLC patients, although very rare, is
FAT1, NF1, and APC, occurring in 4% [20]. The prognosis for a patient with
SCLC is generally poor. However, 30% of patients have LS-SCLC, which
refers to limited stage disease where the tumor is confined to one side of the
chest and can be treated with radiation. The remaining 70% of patients have
ED-SCLC, which indicates an extensive stage diagnosis.
Now, let us delve into the causes of NSCLC, a complex and the most
common type of cancer. It is classified into three major types: adenocarci­
noma, which originates from secretory cells and is predominantly found in
the outer lining of the lungs, especially in smokers; squamous cell carci­
noma, which arises from squamous cells located in the central part of the
lung; and large cell carcinoma, which is challenging to treat due to its rapid
spread throughout the body, large size, and being referred to as large cell
undifferentiated carcinoma [21].

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Cancer Vaccine for Lung Cancer
TABLE 5.1 Types of Gene Mutation Which is Responsible for Causing SCLC Cancer and
Their Function in Normal Condition
Gene Name Occurrence
(%)
Type of
Mutation
Function

— SECTION 11 —
References
TP53
89%
Deletion
Stress response, transcription
regulation, tumor suppressor.
[20]
RB1
64%
Deletion
Transcription repression, tumor
suppression, cell cycle regulation.
[20]

— SECTION 12 —
KMT2D
13%
Deletion
Tumor suppression, chromatin
modeling, histone modification.
[20]
PIK3A
7%
Activate
mutation
Oncogene, PTEN-mTOR.
[20]

— SECTION 13 —
PTEN
7%
Deletion
Tumor suppressor, m-TOR.
[20]
NOTCH1
6%
Inactivate
mutation
Tumor suppression.
[20]

— SECTION 14 —
CREBBP
5%
Deletion
Tumor suppressor, chromatin
remodeling.
[20]
The most common mutation in NSCLC is TP53, which occurs in approxi­
mately 50% of patients. Additionally, mutations in EGFR account for about
10%–35% of cases, leading to irregularities in the AKT and MAPK pathways,
further promoting cell proliferation. The increasing molecular heterogeneity
in NSCLC tumors necessitates complex treatment procedures. Let’s examine
the different genes responsible for NSCLC mutations in patients (Figure 5.2)
(Table 5.2).
TABLE 5.2 Genes Causing Mutations in NSCLC Patients and Their Occurrence Percentages
Gene
Causes
Occurrence

— SECTION 15 —
References
TP53
Mutation
50%
[22]
EGFR
Mutation
10–35%
[22]
KRAS
Mutation
15–27%
[22]

— SECTION 16 —
ALK
Fusion
5–10%
[22]
HER2
Overexpression
20%/2%
[22]
PIK3CA
Mutation
2%–8%
[22]

— SECTION 17 —
AKT1
Mutation
1%
[22]
BARF
Mutation
5%
[22]
MET
Mutation
5%
[22]

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FIGURE 5.2 The most common type of lung cancer is adenocarcinoma, which typically
grows slowly. Large cell carcinoma, on the other hand, is fast-growing and mainly found
in the outer part of the lung. Squamous cell carcinoma begins in the bronchi, usually in the
center of the lung. Small cell carcinoma has a lower occurrence compared to other non-small
cell lung cancers.
Let’s look at the differences between different types of cancer along with
their histology of all types of cancer (Table 5.3).
TABLE 5.3 Lung Cancer is Characterized by Uncontrolled Cell Division, and There are
Various Types of Lung Cancer with Different Histology and Additional Symptoms Beyond
the Common Ones
Type
Location
Causes
Histology
Presentation

— SECTION 19 —
References
Adenocarcinoma Periphery
KRAS, ALK,
Pneumocytes type II Clubbing
[23]
EGFR mutation and Clara cell.
Squamous cell
Periphery
Mainly smoking Pneumocytes (giant
Gynecomastia [23]
carcinoma
and undifferentiated)
Large cell
Central part Mainly smoking Intercellular bridge
Hypercalcemia [23]
carcinoma
and keratin pearls
Small cell
Central part Smoking
Kulchitsky cell
Crushing
[23]
carcinoma
(undifferentiated)
syndrome, A

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— SECTION 21 —
5.3 CANCER IMMUNOTHERAPY
The tumor microenvironment (TME) in the case of cancer contains different
cells such as immune cells, endothelial cells, stromal cells, etc. However,
through a series of different mechanisms, cancer cells evade immune
surveillance. Based on this, several approaches have been developed to
modify immune cells that can recognize and attack cancer cells [24]. Cancer
immunotherapy is a type of treatment that involves using our body’s immune
system to fight against cancer. In the last decade or so, it has shown remark­
able results in cancer patients. Successful clinical trials have been conducted
against both NSCLC and SCLC. However, the percentage of responders
is low. To overcome this issue, non-genetic and genetic determinants of
patients should be evaluated. The genetic profile can be accessed using next
generation sequencing along with some advanced technology [24].
Moreover, cancer immunotherapy can be classified into four main
different categories:

• 
  Checkpoint inhibitors;
• 
  Cancer vaccine;
• 
  Cytokines; and
• 
  CAR T-cell therapy.
  Let’s discuss about them in detail and their effect on cancer cells (Figure 5.3).

— SECTION 22 —
5.3.1 CHECKPOINT INHIBITORS
Cancer cells, noncancerous cells such as stromal cells, immune cells, and
various other cells, along with the extracellular matrix (ECM), constitute
the tumor microenvironment (TME). The growth of cancer is dependent on
the interaction between tumor cells, such as tumor-associated macrophages
(TAM), T cells, natural killer cells (NK cells), tumor-associated neutrophils
(TANs), innate lymphoid cells (ILCs), and mast cells. These cells influence
the process of angiogenesis by producing enzymes such as cytokines and
chemokines and establish immunosuppression [25].
The communication between immune cells occurs due to molecules such
as cytokines, which play a crucial role in modulating cancer progression.
Immune checkpoint inhibitors (ICIs) release T cells, which act as brakes
and further activate the immune system by generating antitumor immune
responses. Additionally, they activate the adaptive and innate immune
systems, leading to an effective response against tumors and peripheral

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FIGURE 5.3 There are different types of cancer immunotherapy used to treat tumors,
such as checkpoint inhibitors, CART T-cell therapy, vaccines, and cytokines. Additionally,
monoclonal antibodies and oncolytic viruses are also used as immunotherapies to target
specific types of cancer.
organs like blood and LN [25]. To illustrate their role, let’s consider an
example: in the case of myeloma bone disease, mesenchymal stem cells,
stromal cells of the bone, and other bone cells contribute to the development
of the disease [26]. The interaction between cytokines and cell adhesion
leads to the formation of new blood vessels, promoting further proliferation
[27]. In vivo models have demonstrated that defective immunosurveillance

— SECTION 24 —
results in cancer proliferation [28]. Cancer-associated fibroblasts (CAFs)
also facilitate tumor formation by secreting cytokines and chemokines that
interact with the tumor microenvironment (TME) and contribute to immune
evasion by increasing checkpoint activity and immunosuppressive cytokines,
thereby promoting cancer proliferation [28]. Recent progress in both clinical
and preclinical settings has shown that checkpoint inhibitors are an effective
method of cancer therapy.
The Food and Drug Administration (FDA) has approved three molecules
for use in humans. Ipilimumab is the first drug approved for metastatic mela­
noma, acting as an antibody against CTLA-4 (CTLA associated protein 4).

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The second drug blocks the inhibitory receptor for programmed cell death-1
(PD-1), which interacts with PD-L1 and PD-L2. Two drugs, pembrolizumab
and nivolumab, have been approved as anti-PD-1 antibodies. The third
category of drugs consists of anti-PD-L1 antibodies, including avelumab,
atezolizumab, and durvalumab. These PD-L1 blocking agents are used for
different types of cancer such as non-small cell lung cancer (NSCLC), Merkel
cell carcinoma, and urothelial carcinoma. These drugs are often combined

— SECTION 26 —
to achieve higher efficacy and better results [29, 30]. Despite these efforts,
many patients do not respond to these therapies and develop resistance,
although the mechanism behind this is not fully understood.
5.3.2 CAR-T CELL THERAPY
CAR-T, or Chimeric Antigen Receptor-T cell therapy, has demonstrated
effective clinical responses. These engineered receptors have the ability
to target and eliminate cancerous cells that express tumor-specific anti­
gens. The therapy involves two crucial components: autologous T cells,
which are isolated from the patients’ peripheral blood, and CARs created
through genetic engineering. The patients’ T cells are isolated and geneti­
cally modified to express CARs that specifically target the tumor-specific
antigens. Subsequently, the modified CAR-T cells are reintroduced into the
cancer patients. These cells can identify and bind to tumor cells expressing
the particular antigen, leading to their elimination [31]. CAR-T cells are
primarily composed of four parts: an intracellular signaling domain, a
spacer/hinge domain, an extracellular antigen binding domain, and a trans-
membrane domain. Each component is designed to optimize CAR activity
[32, 33]. The intracellular domain facilitates signal transduction and T cell
proliferation, while the extracellular signaling domain targets the cancer cell-
specific antigen. Additionally, CAR-T cells are engineered to overcome the
immunosuppressive tumor microenvironment (TME) by secreting various
cytokines [34].
There are several surface antigens currently undergoing preclinical trials.
Let’s discuss them one by one.

1. EGFR: These are receptor tyrosine kinases that belong to the HER/
   ErbB family. They function by transducing extracellular signals.
   Approximately 60% of NSCLC patients exhibit EGFR mutations,
   making it possible to target these EGFRs for CART cell therapy. In
   vitro experiments have shown a significant decrease in tumor size
   and a promising potential [35].

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2. Mesothelin (MSLN): It is a tumor-associated antigen (TA) that is
   expressed at very low levels in normal cells compared to its overex­

— SECTION 28 —
pression in solid cancers. Results have shown that it decreases cancer
cell invasion and proliferation. In a mouse model, it demonstrated
slow tumor growth when injected into the tail vein [36].

3. ROR1: Or receptor tyrosine kinase-like orphan receptor 1, sustains
   pro-apoptotic and survival signals in cases of lung carcinoma. Over-
   expression of ROR1 is observed in lung carcinoma. In vitro testing
   has shown its anti-cancer activity [37].
   There are various other targeted antigens. Let’s examine them in
   Table 5.4.
   TABLE 5.4 Targeting Antigens for SCLC and NSCLC Lung Cancer for CAR-T Cell
   Therapy in Their Clinical Phase
   Type of Cancer
   Target Antigen
   Phase
   Status

— SECTION 29 —
References
NSCLC
MUC 1
Phase 1/2
Unknown
[36]
NSCLC
TnMUC1
Phase 1
Recruiting
[36]

— SECTION 30 —
NSCLC
PD-L1
Phase 1
Unknown
[36]
NSCLC
Mesothelin
Phase 1
Recruiting
[36]
NSCLC

— SECTION 31 —
PD-L1
Early phase 1 Terminated
[36]
NSCLC
Alpha-PD1, MSLN Phase 1
Recruiting
[36]
NSCLC
ROR 1
Phase 1
Recruiting
[36]

— SECTION 32 —
NSCLC
MUC1, PD-L1
Phase 1/2
Recruiting
[36]
SCLC
DLL3
Phase 1
Suspended
[36]
SCLC

— SECTION 33 —
GPC3
Phase 1
Recruiting
[36]
Lung cancer
CEA
Phase 1/2
Recruiting
[36]
Lung cancer
CD276
Phase 1
Not yet recruiting [36]
Lung cancer

— SECTION 34 —
HER2
Phase 1
Recruiting
[36]
CAR-T cell therapy has wide potential. However, patients face some
issues including toxicities on targets, evasion of antitumor response, and
off-tumor toxicity. The treatment of solid tumors is even more challenging
due to factors such as immunosuppressive TME and difficulty in infiltration
of CAR-T cells into tumor sites. Successful implantation has enhanced the
effectiveness of treating solid tumors through continuous modification and
evolution in CAR structures, leading to reduced toxicities [37]. However,
more clinical trials are required to enhance the efficacy and efficiency of
CAR-T therapy in lung cancer. Now, let’s turn our attention to the third type
of cancer immunotherapy, which is cytokines.

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— SECTION 36 —
5.3.3 CYTOKINES
Cytokines are produced by immunocytes and non-immunocytes as messenger
molecules to communicate with other cells. These proteins have a molecular
weight below 30 kDa and play various roles in cancer immunotherapy,
including T cell activation, infiltration, and presentation. They also assist in
the differentiation of CD4+ cells into regulatory T cells and helper T cells,
which upregulate the activity of tumor progression [38]. This type of immu­
notherapy is based on extracting cytokines that enhance the activity of our
immune system to suppress cancer. IL2 was the very first cytokine used for
cancer treatment. IL-2, IL-12, IFN type I colony-stimulating factor (CSF),
ligand (CCL)21, and chemokine (C-C motif) are all types of cytokines with
properties that suppress cancer. However, some cytokines, such as IFN-
gamma, IL-1, and TNF-alpha, have dual roles [39].
Recent research shows the use of various other types of cytokines, but
Th1 or helper T cells have the most potent antitumor activity when tested in
animal models. Combination immunotherapy can be used, such as combining

— SECTION 37 —
it with CV to achieve effective results [40].
Let’s look at different cytokines that are being used (Table 5.5).
TABLE 5.5 Different Types of Cytokines and Their Different Types Which are Being
Produced by Immune Cells Mostly Having Anti-Cancerous Activity
Type
Receptor
Produced by
Activity
References
IL-18 TLR
Macrophages, DC,
Activate T-cell and
[40]
epithelial cell.
increase activity of Th1.

— SECTION 38 —
IL-2
CD-25
T cell, DC, and NK cell. Promote T-cell growth.
[40]
IL-12 IL-12R beta 1 Macrophages and DC.
Differentiation of Th 1.
[40]
The efficacy of cytokines as drugs on clinical outcomes is limited. One
possible reason is the short lifespan of cytokines in the blood, requiring high
doses to achieve the desired effect. For example, the effective dose of IL-2 is
6,00,000 IU/kg, which needs to be administered every 8 hours for a period
of 5 days. This high dosage often leads to adverse conditions in patients,
including musculoskeletal pain, fatigue, fever, and chest pain. More serious
conditions can include cardiac arrest, immune system disorders, and gastro­
intestinal disorders. To address these drawbacks, researchers are exploring
the use of nanomaterials as carriers to target oncogenic tissues. Nanomate­
rials in chemotherapy offer significant benefits, such as improved circulation
time, water solubility, and targeted deposition at the site of cancer [41]. For

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example, PjME/GM-CSF coated with chitosan showed greater effective­
ness in DC recruitment compared to naked Pjme/GM-CSF. Encapsulation
of IFN-alpha with poloxamer blend microspheres allowed for sustained
release in the blood over a month [42]. The growth of tumors was delayed
when IL-2 and TGF-beta inhibitors were loaded with liposomal polymeric
gels. Encapsulation of PEG liposomes with TNF-alpha and BN175 has also
shown potential in reducing toxicity [43]. Additionally, studies have shown

— SECTION 40 —
that adjuvants can enhance the activity of cytokines, although clinical results
have not been consistently encouraging. Careful design of cytokine-based
anti-tumor immunotherapy is critical, as cytokines can also have suppres­
sive and anti-inflammatory effects in certain cases. Lastly, we will discuss
CV, which is the main focus of this chapter. Before delving into CV, it is
important to note that there are other types of immunotherapies, including
the use of monoclonal antibodies, oncolytic viruses, metabolic inhibitors,
molecular agonists, and antibody-drug conjugates.
5.4 CANCER VACCINE (CV)
The CV on which this chapter is based involves the exogenous administra­
tion of specific tumor antigens (TAs) combined with certain proteins to acti­
vate our body’s immune system. This stimulation of the biological system
activates the adaptive immune system, which is specific to a particular
TA. Successful vaccination against cancer requires large and high-quality
dendritic cells (DCs), their activation, long durability of their response,
strong and sustained activation of CD4+ CTL T helper cells, infiltration of
the tumor microenvironment (TME), and a maintained response.

— SECTION 41 —
Other methods can be used to achieve these mechanisms, such as using
immune checkpoint inhibitors (ICIs) or utilizing the TME to induce cancer
cell death. Another approach called in situ vaccine (ISVs) can be used to
activate tumor cell death. In the recent approach of vaccination, specific
antigens are selected, purified, and administered into the host [44, 45]. To
increase effectiveness, TAs are combined with adjuvants, which further acti­
vate DCs to stimulate the patient’s adaptive immune system. This helps gain
control over the growing tumor by inducing regression, establishing long-
lasting anti-tumor memory, eradicating the disease, and avoiding adverse
or non-specific reactions. However, these approaches pose challenges such
as monoresistance and immunosuppression, which will be discussed in the
upcoming paragraph.

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The CV demonstrates significant potential in inducing long-lasting and
specific antigens against a specific cancer known as TA. The primary objec­
tive of CV is to educate the patient’s immune system to eliminate tumor cells
and specifically identify them. The targets for immune response can be TAAs,
which are antigens present in both normal and oncogenic cells, or tumor-
specific antigens (TSAs), which are exclusively found on tumor cells. TSAs
exhibit high specificity in their action and consist of mutant proteins resulting
from somatic mutations in the normal protein sequence. These antigens provide
a major advantage as they are crucial for oncogenesis and tumor progression
[46], but personalized treatment is required for each cancer type due to the
varying nature of these mutations. On the other hand, TAAs are expressed
in different types of tumors that share a common origin and some histology.
However, TAAs are immunologically weak due to tolerance developed by our
immune system during the developmental phase [47] (Table 5.6).
TABLE 5.6 TAs Types and Examples [49]*
TA Type
Subtype
Example

— SECTION 43 —
References
TAAs (tumor
associated antigens).
Product of silent genes
Universal tumor ag
Oncoviral TAAs
MAGE-1, NY-ESO1
Her2/neu, telomerase,
survivin
[48]
Differentiation
HPV E6, E7, EBV-latent
antigens
Gp100, tyrosinase, Melan-A
Mutational antigen
Neoantigen
Case-specific mutation
[48]
(TSA) Product of
mutated oncogene
P53, Ras, Bcr-Abl
*TAs are classified into two categories: TAA and mutational antigens. To better understand,
let’s compare these two types of TAs.

— SECTION 44 —
5.4.1 TYPES OF CVS
There are different types of CVs that have been developed to elicit an
immune response in order to prevent its occurrence and progression. CVs
can be either composed of cell-based or non-cell-based products. Cell-based
vaccines include DC, whole cell vaccines with adjuvants, peptides, vaccines
pulsed with nucleic acid, DC pulsed with immune stimulators, whole cell
genetically modified tumor vaccines (GMTVs), and tumor cells fused with B
cells or DCs or TAAs. Non-cell-based CVs include particle-based vaccines,
viral vector-based vaccines, DNA vaccines, or anti-idiotypic antibody
vaccines [50].

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5.4.1.1 BACTERIAL/VIRAL-BASED CVS
To combat bacterial infections, bacteria were the first to be utilized as
vaccines to stimulate cellular or humoral immune responses. Killed or
weakened bacteria have long been employed as prophylactic vaccines. With
recent advancements in the field of molecular biology, the lifestyle of patho­
genic bacteria, as well as genetically engineered bacteria, has been modified
to enable them to serve as antigen delivery vectors. Attenuated bacteria
have significant potential to induce a broad range of immune responses and
elicit strong danger signals through their pathogen-associated molecular
patterns (PAMPs) such as flagellin, CpG, lipopolysaccharides (LPS), and
lipoproteins. PAMPs further bind to pathogen recognition receptors (PRRs)
including C-type lectin-like receptors (CLRs), RIG-like receptors (RLRs),
NOD-like receptors (NLRs), and Toll-like receptors (TLRs). These interac­
tions directly or indirectly activate antigen-presenting cells (APCs), which
in turn direct adaptive immune responses. Additionally, toxins produced by
bacteria reinforce effector or memory responses [51] (Figure 5.4).
FIGURE 5.4 For the treatment of lung cancer, viral or bacterial vaccines with biological or
non-biological gene delivery vectors are used to protect foreign DNA. These vaccines have
very high immunogenicity and are easier to produce, but they also possess potential risks and
the possibility of unwanted infections.

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Several viruses have been used in the cancer vaccine (CV) process, such
as poxvirus, alphavirus, and adenovirus. The benefit of using a virus-based
vaccine is that it activates both the adaptive and innate immune systems,
resulting in a strong and long-lasting effect. However, the anti-viral immune
response can neutralize the vector, requiring repeated doses of the cancer
vaccine. To address this issue, a heterologous prime-boost strategy is
employed. In this strategy, the patient is first injected with one virus, followed
by a boost using a different viral vector, such as a DNA plasmid. An example
of this approach is PROSTVAC-VF/Tricom, where the priming is done with
a vaccinia virus encoding PSA, followed by six booster doses of PSA with
fowlpox virus [52]. During phase II clinical trials, 125 men with metastatic
colorectal cancer (mCRC) were given PROSTVAC in combination with

— SECTION 47 —
GM-CSF. Unfortunately, the results could not be replicated in phase III, and
the trial was stopped [53]. The reason for this could be that the vaccine alone
was not able to suppress the immunosuppressive tumor microenvironment
(TME) and was not potent enough. Currently, trials are ongoing to combine
PROSTVAC with various checkpoint inhibitors (CPI) to enhance its efficacy.
To further increase the potency of the cancer vaccine, various vaccine-based
immunotherapy regimens (VBIR) are being developed. These regimens
consist of heterologous prime-boost vaccines, where the priming is achieved
using non-human specific viruses and replication-defective chimpanzee
adenovirus. Boosting is done through intramuscular electroporation [54].
5.4.1.2 CELL-BASED CV
The concept of cancer cell vaccines previously assumed that tumor cells
would provide TAAs, which can elicit a specific type of immune response.
However, immune cells such as B cells, which secrete antibodies, T cells, and
DCs, play a crucial role in the development of cell-based cancer vaccines. B
cells not only produce antibodies but also serve as a source of chemokines
and cytokines, thus regulating the immune response. Only when B cells are
appropriately activated, they become professional antigen-presenting cells.
The first time CD40 and its ligand CD40L were explained, they were iden­
tified as the most important stimuli for B cell activation. Activated CD4+
cells express CD40L and further activate B cells, enhancing the humoral and
cell-mediated immune response [55]. Preclinical trials conducted on CD40
B cell-based cancer vaccines have shown strong rationale. For example,
LCMV antigen-pulsed CD40B cells have demonstrated a delay in tumor
growth in C57BL/6 mice and many more [56].

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Now, let’s discuss DCs. They induce a strong immune response against
both self and non-self-antigens. DCs have various roles, such as micropino­
cytosis, phagocytosis, detection of tumor cells or pathogenic cells through
environmental sensors, co-stimulation of T cells, and providing crosstalk
between macrophages, B cells, and NK cells. Based on the potential of DCs,
the first clinical trials were conducted between 1995 and 2004 using GM-CSF

• IL-4, which are derived from DCs. They were tested in various ways to
  promote antitumor activities, such as in the case of melanoma patients or the
  characterization of shared TAs like gp100 and MART-1 [57]. These were also
  tested on leukemia, myeloma, or hepatocellular cancer. Provenge, an FDA-
  approved cancer vaccine targeting prostate cancer, is based on GM-CSF [58]
  (Figure 5.5).
  FIGURE 5.5 Cell-based CVs, such as DCs, play a role in activating the innate and adaptive
  immune systems. They can control antigen presentation and are highly immunogenic, but
  they are expensive to produce.

— SECTION 49 —
5.4.1.3 GENE-BASED CV
Type vaccines are a great tool for eliciting an immune response, even in
the case of TAs. Gene-based vaccines are classified into two main types:
viral vaccines and DNA plasmid vaccines. With advancements in the field
of immune-oncology, DNA CVs are considered the best CVs for activating
our immune system [59]. They activate a wide and broad range of immune
responses, but in clinical trials, their effect was modest, possibly due to the
immunosuppressive nature of developing tumors. To increase response effi­
cacy, optimizing the antigens is required, or DNA therapy can be combined

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with other therapies to attenuate the immunosuppressive nature of the TME.
Various clinical trials focus on enhancing these two strategies to overcome
limitations in CV (Figure 5.6).
FIGURE 5.6 Gene-based vaccines, such as RNA and DNA, have the ability to deliver
multiple antigens and induce both cellular and humoral immunity. However, RNA-based
vaccines require specific transportation and storage conditions.

— SECTION 51 —
5.4.1.4 PROTEIN/PEPTIDE-BASED CVS
Most of the peptide vaccines are based on epitope peptides that stimulate
CD4+ or CD8+ T helper cells, targeting tumor-specific antigens or TAAs.
There are numerous nanomaterials and adjuvants that can enhance the effi­
cacy of the peptides, offering several benefits such as ease of production,
lower cost, high stability, reduced carcinogenicity, and resistance to harmful
pathogens. These peptides typically consist of 8–12 amino acids derived
from TAAs, which are overexpressed during tumorigenesis. They can be
internalized into DCs and further assembled into peptides that bind to HLA.
Antigens used in peptide-based therapeutic vaccines for lung cancer include
CDCA1, KIF20A, Lengsin, and URLC10, which are all tumor-specific
antigens. TAAs such as STEAP, TERT, SOX2, and VGFR are also included.
Peptide-based vaccines for different types of cancer are being tested in
clinical trials. The first peptide-based cancer vaccine approved by the FDA
is sipuleucel-T, used for prostate cancer. These vaccines often combine
multiple epitopes with multiple targets [60] (Figure 5.7).

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FIGURE 5.7 Protein or peptide-based cancer vaccine have low toxicity, but they have low
to moderate immunogenicity.

— SECTION 53 —
5.5 BIOLOGICAL RATIONALE FOR USING LUNG CANCER VACCINE
The spread of tumor growth is not only dependent on the characteristics of
cancer cells, but also on their interaction with our immune system [61]. Dr.
William B. Coley was the first to explain the use of immunotherapy in the
treatment of cancer. He used a streptococcal vaccine, later named ‘Coley’s
toxin,’ which was used to treat malignant diseases. A disease-free condition
was observed in more than 50% of patients suffering from soft tissue sarcoma
[62]. Despite these successes, immunotherapies in lung cancer have not been
explored until recently, which has shown survival benefits [63]. With the posi­
tive response, there has been an interest in lung cancer immunotherapy, which
works by helping cancer cells release the immune response or modulating
the interaction of antigen-presenting cells and T cells. Additionally, cellular
immune response can be induced by CTLs, but the success rate is very low.
The limitations can be attributed to a variety of reasons, such as the lack of
function or physical disabling of primed tumor-specific T cells, high infiltra­
tion of lymphocytes that are immunosuppressing the regulatory T cells such as
CD25+ and CD4+, as they further secrete CTLA-4 (cytotoxic T-lymphocyte
antigen-4) and TGF-beta, and priming of tumor-specific T cells, etc. [64].
Recent approaches in CV development mainly focus on immunogenic
adjuvants that can be coupled with TAs or tumor cells and then administered
into patients. These adjuvants induce an immune response by enhancing the
APC response in the body. Furthermore, these APCs present antigens to T
cells, resulting in the activation of tumor-specific T cell response.

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Other methods of strengthening the immune system include genetic
modification of allogenic cell lines or autologous tumor cells to secrete
co-stimulatory molecules. Additionally, naturally secreted immune modula­
tors can be added to vaccines. Another approach is to express TA as a viral
vector to elicit the expression of cytokines and co-stimulatory molecules.
Priming of the patient’s immune system can also be achieved by using
CVs that contain autologous DCs loaded with TAs to elicit a specific CTL
response.
In the case of NSCLC and SCLC, various therapies can be used in vaccine
development. These include target protein-specific vaccines against MUC-1
(mucin 1), melanoma-associated antigen A3 (MAGE-A), epidermal growth
factor (EGF), and whole cell vaccines such as belagenpumatucel-L [65].

— SECTION 55 —
5.5.1 RATIONALE FOR IMMUNOTHERAPY IN SMALL CELLS LUNG
CANCER (SCLC)
SCLC, or small cell lung cancer, is an immunogenic tumor with a very poor
prognosis despite extensive research. It primarily affects individuals who
directly or indirectly consume tobacco, leading to somatic mutations and
a suppressed immune response. This is attributed to a high level of non-
synonymous somatic mutations known as TMB, or tumor mutational burden
[60], which impacts the adaptive immune system. The presence of a specific
mutation accounts for the highest rate of somatic mutation, even affecting
DNA repair mechanisms and increasing the likelihood of developing tumor-
specific neoantigens that can trigger the adaptive immune system. Defects in
tumor mismatch repair genes can result in MSI, or microsatellite instability,
which benefits PD-1 ICI and immunotherapy in various cancers [66].
Despite the high somatic mutation rate in SCLC, it exhibits an immuno­
suppressive phenotype characterized by low expression of major histocom­
patibility antigens, beta-2 microglobulin, and HLA. The loss of expression
of different types of HLA-A, B, and C allows cancer cells to evade host
immunosurveillance [67]. Additionally, SCLC shows low levels of TILs
(tumor-infiltrating lymphocytes), myeloid cells in an immunosuppressive
state, and a very low CD8/CD3 ratio.
With advanced research in the field of immunotherapy, the suppressed
immune response can be elevated with the help of checkpoint inhibitors such
as nivolumab, durvalumab, pilimumab, atezolizumab, etc. These inhibitors
have shown anti-cancerous activity and improved survival rates in patients
suffering from SCLC. Atezolizumab, when combined with chemotherapy,

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has set a milestone in the field of cancer immunotherapy as it became the
first drug to increase chances of survival in patients suffering from extensive
stages of SCLC. Due to the success of checkpoint inhibitors, clinical trials
were conducted for CVs, TLR9 inhibition, and the use of cytokines, among
others. For CV, a different approach was taken, such as stimulating anti­
tumor immunity to generate an adaptive immune response. This is achieved
by exposing the tumor antigens to the immune cells. Various approaches are
being tested in clinical trials for CV, which will be discussed in the upcoming
paragraph. Different types of CVs have been tested in SCLC lung cancer
patients.
A common cell membrane ganglioside called GD3 is mostly expressed
in SCLC cancer patients and in a limited number of normal tissues. Around
15 patients who were given a dose of BEC2 (an anti-idiotypic monoclonal
antibody that has shown to induce anti-GD3 antibodies) with bacillus
Calmette Guerin (BCG) vaccine showed increased immunogenicity [60]
when administered intradermally on weeks 0, 2, 4, and 6. All of them
developed anti-BEC2 antibodies, while five of them developed anti-GD3
antibodies. In a randomized phase III clinical trial, a total of 515 patients
with LS-SCLC were administered a combination of BEC2 and GD3 after
chemotherapy. Unfortunately, there was no observed improvement in quality
of life (QoL), overall survival (OS), and progression-free survival (PFS).
Among the patients, 33% developed a humoral immune response, while
others experienced prolonged survival [69]. Another anti-idiotypic mono­
clonal antibody, CV 1E1O, was developed to elicit an immune response
against GM3 in SCLC, breast, and melanoma cancer [70]. Phase I clinical
trials were conducted on nine patients after their initial chemotherapy. The
treatment was administered every two weeks, followed by six doses every
28 days, resulting in significantly extended survival. The modified version
of 1E1O, now known as racotumomab, is primarily used in NSCLC and has
been discontinued for SCLC patients.
In the case of SCLC patients, N-polysialic acid (polySA), a side chain
attached to the neural cell adhesion molecule, is commonly found [71].
During phase I clinical trials, polySA was conjugated with the keyhole
limpet hemocyanin (KLH) vaccine to induce an increased antibody response
in SCLC patients. Additionally, MHC class I expression is induced by
mutated p53 protein in SCLC patients, which can serve as an efficient target
for immune recognition [69]. A tumor vaccine consisting of the wild-type
p53 gene and DCs, carried by adenovirus, was tested in phase I and II trials
involving 29 patients. After the initial round of chemotherapy in ES-SCLC

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patients, the gene specific to p53 was found to be detached in approximately
57% of cases [73]. This vaccine was combined with all-trans retinoic acid
(ATRA), which suppresses the growth of myeloid-derived cells, and tested
in clinical trials. Positive responses were observed in 20% of the patients and

— SECTION 58 —
43.3% of those who received both treatments [74] (Table 5.7).
TABLE 5.7 In Case of SCLC Various Agents Such as Bec2 and Others as Illustrated Above
Studied on Clinical Trials
Agents
Phase Study
No. of
Endpoint
Result
References
Patients
Adjuvant Bec2/

— SECTION 59 —
III
Randomized
515
OS
14.3 and 16.4 month [75]
BCG
in vaccination and
observation arm.
KLH-conjugated II
Single group
20
Antibody
8/9 with IgG and
[75]
N-propionylated
response
9/9 with IgM.
polysialic acid.
Autologous DC

— SECTION 60 —
II
Palatinum-based 29
P53-specific 57.1% with p53
[75]
adenovirus p53
chemotherapy
response.
specific response.
vaccine.
then vaccine.
5.5.2 RATIONALE FOR IMMUNOTHERAPY IN NSCLC
Other approaches to immunotherapy, such as checkpoint inhibitors, have
revolutionized the field of cancer immunotherapy, especially in the case of
NSCLC. Unfortunately, not all patients exhibit the same durability, and the
prognosis is very poor. To further enhance effectiveness, strategies need to
be improved. One way is to use therapeutic oncogenic vaccines to achieve a
long-lasting effect against specific tumor antigens with minimal side effects.
The main benefit of using these vaccines is that they can boost anti-cancer
tumor activity and work synergistically with immune checkpoint inhibitors
(ICIs). Therapeutic vaccines can be either preventive or therapeutic, used
for early treatment of cancer or in combination with various other types
of cancer drugs during treatment. They are designed to act against tumor
antigens, increase immunosurveillance of cancer cells, and primarily trigger
T cell responses. Recently, it has been found that neoantigens play a crucial
role in developing therapeutic vaccines, and targeting these antigens is of
utmost importance. Many clinical trials are being conducted based on this
concept. Now, let’s discuss the different types of therapeutic vaccines in
the case of NSCLC and their mechanisms of action. As we have already

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discussed, there are different types of therapeutic vaccines, such as cellular,
viral vector, protein, and peptide-based vaccines. To enhance the delivery of
these vaccines, more immunogenic antigens, such as neoantigens, are being
selected. These antigens can broaden tumor-specific T cell responses and
enhance anti-cancer activity [76].
Now let’s discuss the interaction between immune cells and lung cancer
TME. It primarily involves the suppression of immune cells that aid in the
growth, expansion, and metastasis of tumor cells. Various interactions take
place in the TME to stimulate the heterogeneity of tumor cells, including
mechanical pressure, induction of hypoxia, hypoglycemia, and various
biochemical signaling such as auto, para, and endo to interact with cancer
cells [77]. The activation of the patient’s body to fight against cancer has
been the most appealing method, but the restrictions imposed on the TME
can affect immunotherapy. In the pre-malignant stage of cancer, preventive
measures are given, such as in the case of diseases like HBV and HPV, which
can decrease the risk of cancer, such as HCC or cervical cancer caused by
these viruses. Despite the development of many therapeutic vaccines, the
outcomes are unsatisfactory. The reasons for the low efficacy can be attrib­
uted to the immunosuppressive nature of the TME, difficulty in identifying
TA, overexpression of self-antigens, low affinity with T cells, and many
more [78, 79].
Let’s explore various CVs for NSCLC along with their latest updates.
These CVs can serve as adjuvants in both unresectable and resectable
NSCLC by providing a response to various immunotherapies.
One such NSCLC therapeutic vaccine is CIMAvax-EGF, which was
developed in Cuba. The research for this vaccine began in 1995 and it was
licensed by the Cuban Regulatory Agency in 2008. CIMAvax-EGF consists
of a recombinant protein carrier from Neisseria Meningitidis B, human
EGF conjugated with P64K, and Montanide ISA51 as an adjuvant. Upon
induction, it produces antibodies against EGF, preventing it from binding
to its receptor and inhibiting tumor growth. So far, it is considered safe for
NSCLC patients. To achieve better control over cancer growth, a combina­
tion of CIMAvax and ICIs is being administered to patients in clinical trials.
Another therapeutic vaccine is UV1, which is composed of three long
polypeptides covering an epitope-rich sequence containing the active cata­
lytic site of human telomerase reverse transcriptase (hTERT). hTERT is a
TAA that is overexpressed in approximately 85% of solid tumors [80]. It
is crucial for the immortality of tumor cells, making it an important target
for anticancer therapy. UV1 peptides have been extensively analyzed in the

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Cancer Vaccine for Lung Cancer
bloodstream over a long period of time. When the first-generation hTERT
vaccine called GV1001 was clinically tested, it demonstrated a high amount
of CD4+ responses against hTERT peptides [81]. Upon induction of UV1
peptides, it elicits a wide spectrum of immune responses, such as Th1, inter­
leukin-2 (IL-2), interferon-gamma (IFN-γ), and tumor necrosis factor alpha
(TNF-α), which collectively stimulate and activate other cells like CD8+ T
cells and effector cells [82]. Phase I clinical trials were also conducted using
UV1 and GM-CSF (granulocyte-macrophage colony stimulating factor) for
melanoma and prostate cancer [83].
As we discussed earlier, there are four types of cancer vaccines based on
their composition: peptide/protein, viral, genomic, and cell-based vaccines.
Now, let’s categorize these vaccines based on their types specifically for
NSCLC (Table 5.8).
TABLE 5.8 In the Case of NSCLC, Clinical Trials Have Been Conducted for Vaccine
Development Using Different Compositions Such as Virus, Cell-based, and Peptides
Name
Composition TAA
Phase Patients
No.
Drug
Stage
Endpoint

— SECTION 63 —
References
TG4010
Virus-based
MUC1
II
65
IV
Tumor
[84]
response
LV305
Virus-based

— SECTION 64 —
NY-ESO-1 I
47
IV
Safety
[84]
GVAX
Cell-based
Autologous
tumor cells.
II
86

— SECTION 65 —
IV
Safety
–
1650-G
Cell-based
Allogenic
cell
II
12
I-IIB
Immunogenic
response
[84]
Belagenpumatucel-L Cell-based
Allogenic
cell

— SECTION 66 —
III
532
IIIA-B/
IV
OS
[84]
PRAME
Peptide based PRAME
I
60
IB, II,

— SECTION 67 —
IIIA
OS
[84]
Tecemotide
(L-BLP25)
Peptide-based MUC 1
III
1513
III
OS
[84]
Racotumomab alum Peptide-based NeuGcGM3 III
1082

— SECTION 68 —
IIIA-IV OS
[84]
CIMAvax-EGF
Peptide-based EGF
III
579
IIIB-IV OS
[84]
MAGE-A3
Peptide-based MAGE-A3 III
2312

— SECTION 69 —
IB-IIA PFS
[84]
5.6 MECHANISM OF ACTION OF CANCER VACCINE ON LUNG
CANCER
CV induces a cell-mediated and humoral immune response against specific
tumor antigens (TA). Upon injecting CV, it triggers cell-mediated immune

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responses that begin with antigen-presenting cells such as dendritic cells (DC)
and uptake of TAAs. First, the TAAs are processed and displayed on the surface
of cells associated with major histocompatibility complex (MHC) class I and
class II. The interaction of processed TA on MHC complexes with T cell recep­
tors (TCR) further activates T helper cells such as CD4+ and CD8+, which
secrete interleukins like IL-2, IL-12, and INF-γ. Collectively, they secrete
cytotoxic T lymphocytes (CTLs), which recognize antigens on cancer cells and
initiate cellular immune attack leading to apoptosis of tumor cells. Additionally,
CD4+ T cells enhance the activity of natural killer (NK) cells, which engage
in phagocytic activity and stimulate B cell response. These B cells produce
antigen-specific antibodies, resulting in a humoral immune attack (Figure 5.8).
FIGURE 5.8 An example of how a therapeutic CV works in the case of lung cancer: Tumor
antigens (TAs) from cancer cells are taken up by antigen-presenting cells (APCs), which
process these antigens and present them on the surface of MHC Classes I and II molecules.
In the case of lung cancer, the interaction between TCR (T cell receptor)
and MHC I complex in the tumor lymph node activates CD8+ T helper
cells, CTLs, and antigens that recognize CTLs. Furthermore, CTLs secrete
pro-inflammatory mediators (IFN-gamma and TNF-alpha) via different
pathways such as perforin and granzyme. The interaction between MHC

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class II and TA further stimulates the release of different subsets of T cells
and, in addition, activates CD4+ cells which produce antibodies against TA
by B cells and enhance tumor clearance.

— SECTION 71 —
5.7 OPTIMIZING VACCINE STRATEGIES FOR LUNG CANCER
The main goal of a cancer vaccine is to stimulate the immune response against
a specific set of antigens. This can be achieved by enhancing the function
of antigen-presenting cells, which in turn triggers a T cell response leading
to antibody production and activation of T cytotoxic cells. This approach,
known as immunotherapy, has already shown promise in CAR-T cell therapy
and immune checkpoint inhibitors (ICIs). It is now time for cancer vaccines
to play a role in eradicating cancer, particularly focusing on lung cancer.
There are two keyways to leverage the cellular immune response and
unlock the potential of vaccines in cancer treatment. The first is to induce a
T cell response with higher specificity and efficacy, ensuring that the vaccine
effectively reaches the site of the cancer and performs its intended function.
Various approaches can be used to optimize tumor antigen (TA) targeting,
including the use of whole tumor-derived mRNA, allogeneic tumor cell
lines, autologous tumor cells, and irradiated autologous tumor cells [85].
However, these approaches present challenges in terms of standardization
and require adherence to good manufacturing practices (GMP).
Adjuvants offer numerous benefits in vaccine development. These
substances are used in combination with target antigens to elicit a strong
immune response. However, finding the right adjuvant for CV poses several
challenges. They are poorly immunogenic and must pass the polarized cell-
mediated immunity. Adjuvants are further classified into two major types:

• Particulate adjuvants, which act on antigens as delivery systems or
  depots; and
• Immunomodulatory adjuvants, which can trigger the innate immune
  response.
  Immunomodulatory molecules possess the capability to mimic
  pathogen-associated patterns and interact with Toll-like receptors (TLRs)
  on antigen-presenting cells (APCs) such as B cells, dendritic cells (DCs),
  and macrophages. Several noteworthy adjuvants for optimizing vaccine
  candidates (CVs) are discussed below. TLR4 is an immunostimulatory
  adjuvant that aids in APC activation [86]. It is used in combination with
  ASo4 adjuvant, primarily in vaccines against HPV and HBV. Stimulation

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of TLR also activates homeostatic counterregulatory mechanisms, including
IL-10, PD-L1, and others. Another adjuvant is Saponin, derived from the
soapbark tree (Quillaja Saponaria), which contains a pro-inflammatory
molecule called QS21. It is widely used in vaccine development to elicit a
robust Th1 and humoral response. Clinical trials have shown no convincing
evidence of cell-mediated immunity, but humoral immunity was observed
[82]. Another example is the stimulator of interferon genes (STING), located
in the endoplasmic reticulum, which triggers IFN responses. Activation of
STING by bacterial cyclic dinucleotides in preclinical trials resulted in apop­
tosis of tumor cells at high doses, although other studies have unfortunately
shown that its activation also leads to apoptosis of T cells [87]. Therefore, its
implementation in CVs should be approached cautiously or used in combina­
tion with other therapies. Other examples include granulocyte-macrophage
colony-stimulating factor (GM-CSF), the most commonly used ingredient in
CV development. It is beneficial for DC recruitment, maturation, and antigen
presentation [84]. It is also utilized in monocyte-derived immunotherapy. Heat
shock proteins (HSPs) are endogenous proteins with immunogenic activity
that are released during stress and can be delivered to DCs, which can cross-
present and induce T cell-based immune responses [89]. There are several
ongoing trials combining HSPs with other therapies to combat cancer. An
example of a particulate adjuvant is alum, which has been used for decades
and is known to induce innate immune responses. In the case of cancer
vaccines (CVs) aiming for sustained long-term immune responses, they are
being used in combination with type 1 polarizing agents such as recombinant
IL-12, IISA 51, and MPL (GSK’s ASo4) [90]. Other particulate adjuvants
include Freund’s adjuvant, which becomes less toxic when combined with
squalene or oleate. One therapeutic CV that combines all these components
is Montanide ISA-51, also known as incomplete Freund’s adjuvant. Another
effective approach to achieve this goal is through virosomes, microspheres,
liposomes, and ISCOMs [91]. Virus-like particles (VLPs) can fuse with the
membrane of target cells. One successful VLP-based vaccine is Gardsil®,
which is composed of capsid proteins of HPV serotypes and induces a long-
term humoral immune response. Other strategies for optimizing targeted
CVs include improving effector T cell access into the tumor, combating
immunosuppressive cells in the tumor microenvironment (TME) that elicit
T cell immune responses, enhancing tumor visibility to the immune system
for rapid induction of immune responses, releasing T cells from negative
checkpoint signals, and addressing the immunosuppressive metabolic tumor
environment [92].

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— SECTION 74 —
5.7.1 INFUSING NANOTECHNOLOGY IN LUNG CANCER VACCINE
Nanotechnology has various effects on lung cancer treatment. Due to
its good biocompatibility, it can carry cancer drugs without causing any
toxicity. Nanoparticles (NPs) with high surface area to volume ratio enhance
the presentation of antigen-presenting cells (APCs) to tumor antigens (TA).
For example, CE6-doped mesoporous silica NPs, modified with PEG and
glycol chitosan (GC) via a linker, were loaded with CpG oligonucleotide.
When these NPs were applied to the tumor, the linker dissociated, releasing
CpG/GC into the tumor environment. Subsequently, photodynamic therapy
(PDT) generated reactive oxygen species (ROS), which released the TAAs.
These TAAs were internalized by dendritic cells (DCs), enhancing antigen
presentation [93].
Another method of developing cancer vaccines involves fabricating
nano-vaccines based on fluoropolymer, which activate the toll-like receptor 4
(TLR-4) signaling pathway, inducing rapid maturation of DCs. Personalized
cancer vaccines can be developed by grafting polyethyleneimine (PEI) with
fluoroalkane, combined with cancer cell membrane isolated from autologous
tumors. These cell membranes express tumor-specific neoantigens, which
can also be used to produce nano-vaccines [67].

— SECTION 75 —
5.8 CURRENT PERSPECTIVE AND FUTURE DIRECTION
It is important to develop anti-cancer immunotherapy composed of various
different types of treatment methods so that more effective anti-cancer
therapies can be developed, which are effective on a large scale of patients.
The cancer immune cycle can be corrected using a combination of multiple
therapies that revolve around various immune cells, disorder development,
and their causes [72]. To achieve success in anti-cancer immunotherapies,
it is essential to build and assess the immune status of each cancer and the
therapies that can be induced. In the case of SCLC cancer, adoptive cellular
therapy (ACT), i.e., the infusion of T cells into cancer patients, has shown
various potential in specific immune response. A variation of ACT known
as chimeric-antigen receptor T cell or CAR-T therapy has been widely used.
Various clinical trials are being conducted for SCLC patients to evade cancer
[68]. Combining immunotherapy with CV is used to develop next-generation
vaccines, and the coupling of checkpoint inhibitors with antitumor signals
has shown interest in immunotherapy. Examples under clinical trials include
the galinpepimut-S vaccine in combination with pembrolizumab and the Ad.
p53 DC vaccine with ipilimumab and nivolumab. New predictive biomarkers

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— SECTION 76 —

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Cancer Vaccination and Challenges, Volume 2
are under development as a low response in immunotherapy is a major
challenge as it can guide and enhance immune response. One well-known
biomarker is PD-L1, its expression and tumor mutational burden (TMB) are
best characterized and significant in NSCLC compared to SCLC [69]. Its
expression was measured using the tumor proportion score (TRS) and the
percentage of positive staining by immunohistochemistry. Response rates to
nivolumab monotherapy or with ipilimumab were compared between PD-L1
negative and PD-L1 positive expression.
As we are aware, the immunosuppressive ability of tumors weakens the
body’s immune control. However, recent vaccines primarily focus on antigen
presentation rather than immunosuppression of the lung cancer microenvi­
ronment (CME) [67]. There are various pathways responsible for DC anergy
in lung tumors, such as the expulsion of cDCs from the lung cancer lesion,
molecular expression, and downregulation of functional markers. In the
future, vectors could be utilized to deliver DC activation genes. For instance,
a lung cancer patient was administered a DC-targeting lentiviral vector that
can deliver a gene capable of effectively responding to T cells [72]. Despite
the potential applications of DC vaccines, their adaptation for clinical use
remains a challenge [68]. Clinical trials II were conducted after immuniza­
tion with TP53-transfected DC vaccine in SCLC patients, but no survival
difference was observed. As mentioned, antigen-loaded DC vaccines fail
to migrate to the lymph nodes and induce a T cell immune response due
to the immunosuppressive environment observed in patients. However, the
common blockade of these immune checkpoints includes PD-1 or PD-L1,
which have been extensively explored. In a study conducted by Chen et al.
PD-1 blockade-activated DC-CIK showed great potential in solid tumors,
even in the case of NSCLC patients. Understanding the physiology of
DCs and how to generate an immune response through novel approaches
is the future requirement, in combination with other immunotherapies.
Non-immunotherapies and immunotherapies, such as checkpoint inhibitors,
CAR-T therapy, and CV, have been utilized in numerous clinical trials to
enhance the efficacy and treatment of lung cancer.

— SECTION 77 —
5.9 CONCLUSION
The complexity of the tumor-associated immunosuppressive microenviron­
ment poses challenges for the delivery of CV. Data from clinical and preclin­
ical settings suggest that vaccines have the potential to eliminate cancer
cells. One of the challenges we face with CV is developing a roadmap for

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our vaccine to overcome the immunosuppressive environment. Additionally,
we need to address the challenge of developing technologies in immunomics
that provide a wide range of biomarkers for designing therapeutic vaccines.
Both NSCLC and SCLC are highly challenging diseases, particularly
SCLC, where immunotherapy can help patients by inducing antitumor
responses. However, the immunosuppressive phenotypic environment of
SCLC hinders vaccine development. The combination of nanotechnology
and nano-vaccines offers unique advantages, such as eliminating half-life,
achieving peak drug concentration, improving clearance rates, and ensuring
biocompatibility. This makes nanotechnology a powerful tool for vaccine
development.
Furthermore, we should explore the role of CD4+ T cells in anti-CV
development and investigate whether gut microbes hinder the efficacy of
immunotherapy. We also need to determine the optimal therapy to improve the
survival and quality of life of patients. These challenges are difficult to address,
but the rewards can revolutionize immune therapy by establishing long-term
immunological memory and transforming the way we treat lung cancer.

— SECTION 78 —
KEYWORDS

• 
  cancer vaccine
• 
  combination therapy
• 
  immune response
• 
  immunotherapy
• 
  lung cancer
• 
  non-cell small cell lung cancer
• 
  reactive oxygen species
• 
  tumor antigens

— SECTION 79 —
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the Management of Various Carcinomas. Rishabha Malviya, Bhupendra Prajapati, and Sonali Sundram (Eds.)
© 2025 Apple Academic Press, Inc. Co-published with CRC Press (Taylor & Francis)

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#

CHAPTER 6
TITLE: Synergistic Effect of Different Vaccines and Chemotherapeutic Agents in Combating Carcinomas
AUTHORS: Hitesh Malhotra, Rohit Kamboj, Amrit Sarwara, Sumit Arora, and Rupesh K. Gautam
LENGTH: 53,042 characters

#

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CHAPTER 6
Synergistic Effect of Vaccines and
Chemotherapeutic Agents in Combating
Different Carcinomas
HITESH MALHOTRA,1 ROHIT KAMBOJ,1 AMRIT SARWARA,1
SUMIT ARORA,1 and RUPESH K. GAUTAM2
1Guru Gobind Singh College of Pharmacy, Yamunanagar, Haryana, India
2Department of Pharmacology, Indore Institute of Pharmacy, IIST
Campus, Rau, Indore, Madhya Pradesh, India
ABSTRACT
Cancer vaccines (CVs) are agents that assist the body in combating cancer.
They stimulate the immune system to identify and eliminate harmful patho­
gens and cells. There are two types of vaccines: (i) Preventive vaccines (such
as the HPV vaccine and Hepatitis B vaccine); and (ii) Therapeutic vaccines
(such as the Sipuleucel-T vaccine and BCG live for bladder cancer). Chemo­
therapy is a treatment that involves the use of one or more anticancer drugs
to halt the growth of tumor cells, either by killing them or preventing them
from dividing. The potential of using vaccines in conjunction with traditional
chemotherapeutic medicines to treat neoplasms is being investigated through
various animal and human studies. T-cells face challenges in infiltrating
large tumor masses due to the lower expression of Major Histocompatibility
Complex (MHC) class 1 molecules in these tissues, and the tumor cells them­
selves outcompete the antigen-specific T-cells produced within the host cell
system. The combination of chemotherapy and immunotherapy has opened
new avenues in cancer management. Depending on the cytotoxic agent and
the specific vaccination used, this synergy can be achieved through various
mechanisms. Chemotherapeutic drugs can alter the phenotype of tumor cells

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by modifying the expression of TAAs, MHC-I, ICAM-1, and APM, making
them more susceptible to immune-mediated attack. The combination of these
two approaches may therefore be more effective than using each strategy
alone. Vaccination against a specific target structure can specifically target
cancer cells that are resistant to chemotherapy. In this chapter, we discuss
the synergistic effect demonstrated by chemotherapy in combination with
immunotherapy for the effective treatment of cancers.

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6.1 INTRODUCTION
One of the most significant public health issues in the world is cancer. With
the evolving nature of the disease and the aging population, the global burden
of cancer has become increasingly prominent. Cancer is responsible for one-
fourth of deaths in the United States, and many cases are not effectively
treated by current conventional therapies. The primary causes of cancer are
believed to be mutations in oncogenes, tumor suppressor genes, and genes
related to genome stability. Cancer is commonly seen as a cell-autonomous
genetic disease. There are two types of cancer: benign and malignant, with
malignant cancer having the ability to metastasize. The process of metas­
tasis, which involves the transformation of cells into a malignant form, is
driven by various genetic and epigenomic changes due to increased genomic
instability. These changes lead to abnormal cell cycles, which in turn affect
the characteristics of other cells and tissues [1].
Cancer vaccines (CVs) have the potential to complement standard cancer
treatments in a non-overlapping manner by inducing a therapeutic host anti­
tumor immune response. Currently, primary neoplasms are managed with a
variety of treatments, including surgery, local radiation, immunotherapy, and
chemotherapy in the majority of cases. Recent preclinical and clinical research
has validated the rationale for combining vaccinations with chemotherapy
in patients with advanced tumors. Patients who receive anticancer vaccina­
tion monotherapy only demonstrate modest clinical effectiveness in terms of
tumor burden. Chemotherapy, immunotherapy, and radiation are examples
of palliative cancer treatments that have limited efficacy [2]. To improve
survival and reduce mortality during cancer therapy, new strategies need to
be developed [3]. Depending on the type of cancer, chemotherapy has been
used to reduce tumor burden, accelerate tumor shrinkage, limit metastasis,
and prevent cancer relapse. Alkylating drugs, antimetabolites, mitotic spindle
inhibitors, and topoisomerase inhibitors are among the most commonly used

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Synergistic Effect of Vaccines and Chemotherapeutic Agents
therapeutic agents [4]. Chemotherapy has adverse effects on both healthy
and malignant cells, which means it can also impact normal cellular activi­
ties. Chemotherapeutic agents may have drawbacks such as rapid clearance
and poor pharmacokinetics, in addition to various side effects, due to their
nonspecific distribution and multidrug resistance (MDR) [5].

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6.2 IMMUNOTHERAPY
Immune-oncology, also known as tumor biotherapy, aims to utilize the
precise strength and precision of the immune system to treat tumors by
stimulating the person’s own immunity. Cancer is fought by the body’s
defense system with the assistance of immunotherapy. The body’s immune
system helps defend against infections and other disorders. It consists of
organs in the lymphatic system and white blood cells. Based on the condition
of the patient’s immunity and the mechanisms of immunotherapeutic drugs,
there are two types of immune cell therapy: active and passive [6].
6.2.1 HOW DOES IMMUNOTHERAPY WORK AGAINST CANCER?
The body’s defense system recognizes and removes aberrant cells, stopping
or slowing the progression of many malignancies. Immune cells, such as
TILs, are occasionally observed in and around tumors, indicating that the
body’s defense system is responding to the presence of tumors. TIL-positive
patients often have better outcomes in their fight against cancer compared
to TIL-negative patients. There is great potential for CVs to complement
conventional cancer treatments by inducing a therapeutic host anti-tumor
immune response without interfering with them.
However, cancer cells have their own defenses against destruction by
immune cells. For instance, tumor cells:

• They have genomic modifications that reduce their visibility to the
  body’s defense system;
• They have polypeptides on their outside that prevent leucocytes;
• Change the normal cells in the tumor’s vicinity so that they interfere
  with the immune system’s response to the tumor cells.
  Biotherapy improves the body’s defense system’s capability to fight
  tumor. Types of immunotherapies (Figure 6.1):

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• 
  Immune checkpoint inhibitors;
• 
  T-cell transfer therapy;
• 
  Monoclonal antibodies;
• 
  Immune system modulators.
  FIGURE 6.1 Different types of immunotherapies.

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6.3 CHEMOTHERAPY
Paul Ehrlich, a German scientist who studied the use of pharmaceuticals
to treat infectious disorders, coined the term “chemotherapy.” The purpose
of chemotherapy is to prevent tumor invasion and metastasis by inhibiting
cell proliferation and tumor development. However, chemotherapy also
has harmful effects on normal cells. Tumor development can be inhibited
at various levels within the cell and its surroundings. Chemotherapy drugs
generally disrupt the production or function of neoplastic cell proteins by
interfering with DNA, RNA, or macromolecular synthesis, or by altering the
proper functioning of preformed molecules [7].
6.3.1 IMMUNOSUPPRESSIVE ADVERSE EFFECTS OF

— SECTION 8 —
CHEMOTHERAPEUTICS
Many of the current tumor chemotherapy drugs are also used as immunosup­
pressants to treat severe systemic autoimmune disorders [8]. Protein tyrosine

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Synergistic Effect of Vaccines and Chemotherapeutic Agents
kinase inhibitors (TKIs) can also affect the T-cell component of acquired
immunity. Imatinib mesylate, which specifically inhibits the interaction
between the macromolecules KIT, c-ABL, and its tumorigenic combination
derivative BCR-ABL, inhibits the protein tyrosine kinase LCK7. This is
believed to be the mechanism by which imatinib mesylate inhibits T-cell
proliferation and stimulation at high doses. Animal model studies have
shown that imatinib mesylate preferentially inhibits the growth of memory
CTLs but has no effect on initial T and B-cell responses [9].
Glucocorticoids are important components of chemotherapy regimens
used to treat various lymphoproliferative diseases due to their ability to
induce death in white blood cells (WBCs). Corticosteroids significantly
disrupt dendritic cell (DC) development and antigen presentation, despite
stimulating the expression of template receptors Toll-like receptor 2 (TLR2)
and TLR4. Corticosteroids also suppress the transcription of genes encoding
the TCR genes TCRA, TCRB, TCRD, and TCRG, as well as message trans­
mitters and activators involved in the innate immune response. Glucocorti­
coids increase the expression of several TGF family members, which inhibits
the effector functions of T-cells and natural killer (NK) cells. In addition to
inhibiting the cytostatic property of NK cells promoted by the NK (p46),
NK(G2D), or 2C4 receptors, glucocorticoids also decrease the growth of NK
cells triggered by interleukin-2 (IL-2) and IL-15 and block the cell-surface
expression of mainly two natural cytotoxicity receptors [10] (Table 6.1).
TABLE 6.1 Prominent Side-Effects of Cytotoxic Agents
Sl. No. Cytotoxic Agent
Side-Effect

1. 
   Irradiation
   Lymphocytopenia
2. 
   Alkylating agent’s high dose Lymphocytopenia
3. 
   Steroids
   Suppression in production of cytokines and
   chemokines.
4. 
   Taxanes
   Thymocytes and NK cell expansion and stimulation
   retardation.

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6.4 CHEMOIMMUNOTHERAPY (CIT)
Chemoimmunotherapy (CIT) can integrate and apply both classic chemo­
therapy and modern immunotherapy methods to limit tumor development,
metastasis, and relapse, even when a cure or symptom relief is not achievable
in palliative care. Chemotherapy, in conjunction with surgery, is the primary
treatment for tumor patients. To defeat cancer, one must not only devise

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plans for effectively eliminating whole tumor cells by employing the ideal
ratio and timing of anticancer medicines but also make an effort to elicit an
immunological response so that the body’s defense system can control any
remaining cancer cells.
However, as the tumor grows, it develops treatment resistance, limiting
the effectiveness of chemotherapy. The most common causes of resistance
expansion are gene amplification by targeted drugs and point mutation in
certain tumor cells, which allow the therapeutic agent’s deadly impact to
escape (e.g., T790M mutation in EGFR). Until just a few years ago, coupling
chemotherapy with active immune treatment was unthinkable. The idea was
that concurrent active immune treatment with chemotherapy would be useless
because chemotherapy would impair any immunological responses, partly
because dividing immune cells are susceptible to chemotherapeutic agents
[11]. However, new therapeutic options for cancer have been made possible
by combining immune cell therapy and drug therapy for cancer treatment,
and early research shows that anti-cancer vaccinations and chemotherapy
have a synergistic impact, as depicted in Figure 6.2.
FIGURE 6.2 Mechanistic approach of CIT.
Chemotherapeutic drugs can induce various biological reactions that
impact the survival and proliferation of tumor cells. Programmed cell death, a

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Synergistic Effect of Vaccines and Chemotherapeutic Agents
crucial natural process for regulating healthy cell numbers during growth and
disease, plays a significant role. It is evident that different medications can
trigger typical apoptotic pathways, leading to the destruction of tumor cells.
Currently, all cytotoxic anticancer drugs used in clinical settings, including
nucleosides, DNAdamaging agents, and microtubule-targeting drugs, promote
apoptosis in cancer cells. However, the effectiveness of chemotherapy in
treating cancer is limited by antineoplastic resistance. In addition to resistance
to the specific treatment being used, tumors may develop cross-resistance to
other drugs with different mechanisms of action [12]. This acquired resistance
is a disappointing aspect of chemotherapy. Most chemotherapy agents elimi­
nate target cells by inducing programmed cell death, also known as apoptosis,
which was previously considered non-stimulatory or tolerable. Consequently,
treatments that induce cell death may render cytotoxic T lymphocytes (CTLs)
insensitive, even though CTLs are normally responsible for eliminating tumor
cells. Vaccination against a specific target structure could selectively target
chemotherapy-resistant tumor cells. Immunotherapy possesses the capability
to address the shortcomings associated with significant drug resistance and
limited specificity of chemotherapy drugs, while improving the responsive­
ness of tumor cells to chemotherapy medicines. Therefore, the synergistic
effects of this combination system might boost treatment efficacy. Addition­
ally, it has been shown that some chemotherapeutic medicines alone can
have immunomodulating effects by causing immunogenic cell death (ICD),
making tumor cells more susceptible to immune attack, and eliminating the
cells that reduce immunity [13].
In an investigation of FDA-sanctioned goods for drug therapy, immune
cell therapy, and CIT approaches, the importance of using and developing
CIT for the prospect of increasing therapeutic application in cancer treatment
was emphasized. Cancer immunotherapies work by immunomodulating the
immune system, which involves boosting T cell activity, removing immune
cells that suppress the immune system, and then boosting endogenous
immunity to stop tumor growth. The use of immunomodulators is one of the

— SECTION 11 —
potential cancer immunotherapy methods (Tables 6.2 and 6.3). The immuno­
modulation stops the spread of cancer by increasing cell activity by utilizing
antibodies to inhibit or activate regulatory receptors. Currently, antibody-
based immunotherapy focuses on leukocytes instead of tumor cells. The
immunostimulatory effects of chemotherapy medicines, such as increasing
alteration load and antigen expression burden, promoting thymocyte stimula­
tion along with selection to a tumor, and increasing Major histocompatibility
complex (MHC) expression to promote protein expression, may supplement

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the immunomodulatory effects of Immune checkpoint inhibitors (ICIs).
Clinical studies have demonstrated that adding ICIs to chemotherapy can
increase anti-tumor efficacy in some cancer types [14].
TABLE 6.2 Combination of Chemotherapeutic Agent with Immune Adjuants
Chemotherapeutic Immune
Tumor Type
Synergistic Action
Agent
Adjuvant

— SECTION 13 —
DOX
Arginine
Breast cancer
Increased effectiveness of
treatments and suppression of
carcinoma cells.
PTX
aGC+PD-L 1
Melanoma and lung Enhanced antimetastatic effect.
metastasis.
DOX
TLR 2 agonist
Breast cancer.
High therapeutic efficacy.

— SECTION 14 —
PTX
(TLR)7 agonist Melanoma, lung
Prevent cancer cell
cancer and cervical development.
cancer.
DTX
TLR-9 agonist Colon carcinoma
Highest effectiveness against
tumors and minimal adverse
effects that are not intended.
TABLE 6.3 Combination of Some Chemotherapeutic Agents and Monoclonal Antibodies
Chemotherapeutic Agent
mAbs
Synergistic Action

— SECTION 15 —
DOX
Anti-PD-1
Regression of colon carcinoma.
PTX
PD1\PD-L1 inhibitor
Significant anticancer activity.
DTX
Anti-PD-L1
Substantial cancer suppression.
6.5 CHEMOIMMUNOTHERAPY’S IMMUNE-BOOSTING
TECHNIQUES
Biotherapy is divided into two categories: active and passive, focusing on
the state of the individual’s immune system and the mechanisms of immu­
notherapeutic medications. Passive immunotherapy, instead of inducing
cancer cell death, enhances the body’s defense system to fight tumor cells.
This is achieved by using immunotherapeutic agents such as monocytes,
specific monoclonal antibodies (mAbs) targeting malignant cells, autolo­
gous cell transplant therapy (ACT), and immune adjuvants. Passive immune
cell therapy involves the frequent administration of molecules that bind to
malignant cells, including certain immunotoxins, which can be used alone or
in combination with drugs, toxins, or radionuclides, delivering them directly

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Synergistic Effect of Vaccines and Chemotherapeutic Agents
to tumor cells. In contrast, active immunotherapy aims to generate long-
term immunity. The most effective method of immunotherapy is ACT, also
known as cell-based immunotherapy. It involves extracting tumor-specific
lymphocytes from cancer patients, modifying, activating, and expanding
them outside the patient’s body. As a result, T lymphocytes can now target
cancer cells. Cytokines, a diverse group of small soluble macromolecules, are
released by various cells including mastocytes, thymocytes, B lymphocytes,
and MPs. They play a crucial role in cell communication, stimulating the
growth and differentiation of lymphocytes, and regulating the inflammatory
or anti-inflammatory responses of different cell types. In the early stages
of carcinogenesis, pro-inflammatory cytokines (PICs) exhibit anti-cancer
effects by enhancing antigen activation, activating immune-enhancing cells,
and increasing the quantity and antitumor effect of the immune system in the
tumor microenvironment (TME) [15].

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6.6 IMPACT OF ANTICANCER THERAPY ON HEALTHY CELLS
Until now, there has been significant focus on utilizing chemicals or cells that
can enhance the body’s immune response in immune cell therapy. However,
there is now a growing emphasis on suppressor mechanisms due to the
recognition that counterproductive cells play a role in defense mechanisms.
MDSC and Tregs are two major contributors in cancer. MDSCs are innate
immune suppressor cells that can hinder both the inherent and adaptive
natural defenses of the body. Similarly, Tregs from the adaptive immune
system can dampen immunological responses. Both types of cells can be
found in significant numbers at the tumor site in cancer patients. Moreover,
scientific evidence suggests that these two cell types are therapeutically
meaningful and reliable indicators of patient survival rate [16].

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6.7 CHEMOTHERAPY RESISTANCE AND IMMUNE TARGETING
Drugs used for cancer treatment can trigger a series of biological reactions
that influence the growth and survival of tumor cells. Among these reac­
tions, programmed cell death, a natural process that regulates cell counts
during growth and illness, has been extensively studied. Many clinically
available medications induce tumor cell death by activating similar apoptotic
pathways [17]. Consequently, most cytotoxic anticancer treatments, such
as drugs that bind to tubulin, compounds that impair DNA, and nucleoside

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monophosphates, lead to the demise of carcinoma cells. However, the effec­
tiveness of these treatments is often limited due to the development of drug
resistance in cancer cells. One challenging aspect of adaptive resistance in
cancer cells is that resistance to one type of anticancer therapy can lead to
resistance to other drugs with different mechanisms of action [18]. Drug
resistance significantly hampers the efficacy of chemotherapeutic treatments
in managing metastatic tumors. Abnormalities in apoptotic pathways associ­
ated with carcinoma play a crucial role in resisting the drugs used for cancer
treatment and radiation. Overexpression of RAP, a protein responsible for
the survival of T-lymphocyte antigens and macromolecules from the Burkitt
lymphoma-2 family, is a major contributor to compromised apoptosis.
Another mechanism of drug resistance involves tumor antigen (TA) cyto­
chrome P1B1, which can deactivate anticancer or cytostatic drugs, thereby
affecting the effectiveness of treatment. ATP transporters, which remove
drugs from the cell, also contribute to drug resistance [19, 20] (Figure 6.3).
FIGURE 6.3 General mechanism of chemotherapy resistance.

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6.8 VACCINE PLUS CHEMOTHERAPY
Recent research has demonstrated that, despite what might seem contradic­
tory, vaccination treatment may not only be compatible but also beneficial
when combined with certain chemotherapy regimens administered at the
appropriate schedule. Medications such as interferon (INF) can upregulate
several tumor-associated antigens (TAAs) and human leukocyte antigen
(HLA) on the surface of cancer cells.

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Furthermore, various medications commonly used in tumor treatment
have shown the ability to overexpress tumor immunotoxins and/or histocom­
patibility immunogens. For example, Tegafur has been shown to upregulate
Carcinoembryonic antigen (CEA) and HLA in tumor cells. In an experi­
mental melanoma model, systemic cyclophosphamide (CTX) combined
with locally and intratumorally injected DC completely regressed the tumor
[21]. Preclinical mouse experiments have demonstrated that chemothera­
peutic drugs such as chlorambucil, daunorubicin, paclitaxel, and vinorelbine
enhance the anticancer immune response to total tumor-cell vaccination [22].
Moreover, cancer patients with a high tumor burden have been found to
have higher levels of immunological regulatory T cells (CD4+/CD25 high).
It is conceivable that systemic chemotherapy used to eliminate these Tregs
may enhance the effectiveness of cancer vaccinations. Additionally, the
death of cancer cells caused by certain chemotherapy drugs may promote
DC uptake and subsequent activation of cytotoxic CD8+ T cells. It has been
demonstrated that daunorubicin-induced CPP32-mediated cell death in
colon carcinoma lines leads to a potent immune response. In contrast, cells
destroyed with mitomycin C did not elicit an immune reaction [23].
Combining chemotherapy with vaccination presents several significant
issues. Firstly, it is evident that patients with metastatic cancer who have under­
gone multiple administrations of various anticancer drugs have compromised
immunity as a result. Therefore, combining vaccination with chemotherapy
early in the course of the illness, when the body’s defense system is still
relatively functional, may be advantageous. Secondly, not all chemotherapy
drugs may be used in conjunction with vaccines. Lastly, the timing of dosage
may be crucial when combining vaccination with chemotherapy [24, 25].
To optimize the administration of anticancer medications and immunization
concurrently, further research is undoubtedly necessary. A recent publication
by Arlen et al. reported a phase II human trial involving individuals with
cancerous AIPC who were randomly assigned to receive either the vaccina­
tion alone or the vaccine in combination with low-dose tesetaxel. The vacci­
nation schedule included an initial priming shot with rV-B7-1 and rV-PSA
combined, followed by periodic booster shots with rF-PSA. The vaccinations
were administered locally using colony stimulating factor-2 (CSF-2). The
main objective of the study was to determine whether concurrent tesetaxel
therapy had any impact on the development of an immunological response
to the vaccination, as measured by the relative shift of PSA-specific CD8

— SECTION 21 —
T-cell progenitors from day 0 to day 80. Clinical results and the safety of the
combined therapy were considered secondary objectives. After 3 months of

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treatment, both groups showed a 3.33-fold median increase in thymocytes
precursor to gamma-seminoprotein or kallikrein-3 (KLK3). Additionally, an
immunogenic reaction to other tumor-associated antigens (TAs) linked to
prostate cancer was discovered. Eleven patients who progressed on vaccina­
tion alone were allowed to switch to receiving tesetaxel upon progression.
This investigation demonstrated, for the first time in clinical studies, the safety
of combining tesetaxel with vaccination without suppressing vaccine-specific
thymocyte responses. Furthermore, the findings of this study provided initial
evidence that prior immunization prolongs patients’ response to tesetaxel
compared to tesetaxel alone. As a result of these investigations, the NCI
has recently initiated a randomized phase II trial to evaluate the therapeutic
benefit of tesetaxel + biotherapy versus tesetaxel alone for individuals with
metastatic breast carcinoma [26].
The most intriguing component of vaccination treatment is its potential to
initiate a strong immunogenic response in the host, which can be utilized in
subsequent treatments. Clinical experiments have demonstrated this tendency
in multiple cases. Out of 10 patients who did not develop immunity from the
initial vaccination, nine went on to receive additional treatments. In contrast,
five individuals who did establish vaccination immunity surprisingly exhibited
significant responses to salvage treatment administered after disease progres­
sion. Salvage treatment typically lasted for at least a year. Other researchers
have also discovered that inducing or enhancing the immunogenic response
to immunotherapy can improve the clinical response to chemotherapy.

— SECTION 22 —
6.9 VACCINE WITH HORMONE THERAPY
Mixing androgen-deprivation therapy (ADT) with vaccination to treat pros­
tate carcinoma is gaining popularity. Research by Kwon et al. demonstrated
that T-cell infiltration was easily detected in benign prostate hyperplasia
and malignancies in the prostatic gland. Thymocyte in the prostate showed
limited thymocyte receptor use, indicating a local oligoclonal reaction that
became noticeable after 1 to 3 weeks of treatment. Other studies have shown
that ADT causes the thymus to increase, expands the T-cell repertoire,
and eliminates the body’s defense system’s tolerance to the prostate [27].
These findings have significant implications for administering vaccination
in combination with hormone therapy for treating hormone-dependent
cancers, including prostate carcinoma. Currently, there is no standard of
care for patients with prostate cancer [26]. Patients who have not undergone
castration surgery continue to receive ADT and are randomly assigned to

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Synergistic Effect of Vaccines and Chemotherapeutic Agents
either vaccination or ARA treatment with flutamide. Patients with increasing
PSA levels and no metastases after six months may receive both therapies
concurrently. The main objective of the trial was to compare the duration of
therapeutic failure between those who received the immunization and those
who received ARA. Secondary outcomes included vaccination immuno­
logical response, immunotherapy efficacy, and the impact of combining the
two modes in individuals with developing physiological insufficiency but
lacking metastases. At the time of PSA development, flutamide was given
to 12 individuals in the vaccination arm. The combination therapy had a
mean duration to therapeutic failure of 426.116 days, for a total of 791.359
days from the beginning of treatment. In comparison, vaccination was
administered to 8 participants in the flutamide arm at the time of gamma­
seminoprotein or kallikrein-3 (KLK3) development. The average duration
of the combined treatment was 158.272 days, and the overall trial period
was 486.99 days. Although both the vaccination plus flutamide seemed to
have therapeutic efficacy, individuals appeared to react more favorably to
flutamide after receiving the immunization. To our knowledge, this was the
first clinical research that focused on prostate cancer patients and provided
detailed support for the idea that vaccination and hormone treatment may
work better together than separately. According to a post-five-year review of
this trial, patients who received the vaccination first and then the flutamide
supplement had a 60-month survival rate of 74.9%, compared to patients
who underwent the vaccination earlier and then the flutamide supplement
and had a five-year survival rate of 43% [28].
As a result of the previous study, the NCI has recently initiated a clinical
trial in prostate cancer patients that combines PSA-TRICOM vaccinations +
GM-CSF with second-line androgen antagonist (flutamide). Patients will be
randomly assigned to receive either the vaccination with androgen antagonist
or the androgen antagonist alone. Flutamide will be discontinued if prostate
specific antigen (PSA) levels increase, and patients will either resume the
vaccine trial or be removed from the regimen due to excessive toxicity. The
objective of the vaccine test is to determine if a combination of vaccine and
androgen antagonist prolongs the duration until therapy failure compared to
the androgen antagonist alone. The secondary goals of the trial include eval­
uating toxicity and the response of PSA-specific thymocytes in individuals
with PSA precursor. The study will also investigate immunological patterns
that may be altered by the treatment, such as the immunogenic effects of
discontinuing flutamide. The findings of this investigation may provide the
basis for a definitive phase III trial using this therapeutic approach.

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6.10 BIOTHERAPY WITH ANTI-CTL 4 MAB
An insufficient impulse from the thymocyte macromolecule is not enough
to achieve optimal activation of T-cells for most weak antigens, such as
TAAs [7]. Activation of the T-cell specific to the target antigen requires
a secondary immune-modulatory signal transmitted from B7 on the APC
through CD28 on the T-cell. Two to three days after activation, CTLA-4
is also produced on the surface of the T-cell and binds to B7. This results
in a negative signal due to the increased affinity of CTLA-4 binding to
CD28, thereby suppressing the immune response. In addition to blocking
CTLA-4’s inhibitory effect, anti-CTLA-4 mAb also promotes T-cell clones
with higher affinity [2, 38]. In murine tumors that were highly immunogenic
or moderately immunogenic, anti-CTLA-4 mAb demonstrated anticancer
effects. However, the development of weakly immunogenic tumors like
MC38 is not significantly influenced by anti-CTLA-4 mAb alone [29].
A clinical investigation using anti-CTLA-4 as a treatment approach was
published by Hodi et al. We evaluated the biological efficacy and toxicity
of vaccination therapy in nine advanced cancer patients who had previously
received treatment for either ovarian carcinoma (n=2) or melanoma (n=7).
A dose of 43.3 mg/kg of MDX-CTLA-4 was administered. T-cell reac­
tions against normal melanocytes were observed, but no significant harm
occurred. Three patients with melanoma, who had previously undergone
treatment with an autologous GM-CSF secreting tumor cell vaccination,
exhibited significant tumor regression accompanied by immune cell infil­
tration. This study revealed that the use of an anti-CTLA-4 antibody may
enhance past immunological memory responses. Recent preclinical research
has explored the potential of anti-CTLA-4 mAb to modulate the number and/
or avidity of antigen-specific T cells when combined with vaccination. In
order to enhance T-cell responses, preliminary experiments were conducted
to determine the optimal dosage regimen of anti-CTLA-4 mAb in combina­
tion with both rV-CEA-TRICOM and rF-CEA-TRICOM. Vaccination of
mice with rV-CEA-TRICOM alone or in combination with anti-CTLA-4
mAb resulted in T cells with comparable frequencies of tetramer-positive
precursors. Although mice vaccinated with rV-CEA-TRICOM exhibited
a 2-fold increase in CEA-specific T cells compared to those receiving
rV-CEA-TRICOM plus anti-CTLA-4 mAb, there was a significant differ­
ence in tetramer dissociation and a 10-fold increase in functional avidity
in T cells receiving both rV-CEA-TRICOM and anti-CTLA-4 mAb. The
combination of rV-CEA-TRICOM, anti-CTLA-4 mAb, and rF-GMCSF
resulted in a synergistic reduction of CEA-expressing tumors in preclinical

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Synergistic Effect of Vaccines and Chemotherapeutic Agents
mouse tumor studies. In clinical trials involving patients with melanoma,
the combination of anti-CTLA-4 mAb (ipilimumab; Medarex, Princeton,
NJ) with a peptide vaccination demonstrated anticancer efficacy, along with
severe but transient immunological breakthrough events such as colitis and
panhypophysitis [30]. Additionally, preliminary findings were revealed from
a complete tumor-cell vaccination combined with ipilimumab. Patients with
asymptomatic metastatic AIPC who had not undergone chemotherapy were
recruited and treated with GVAX® (Cell Genesys, South San Francisco, CA)
every 2 weeks, along with ipilimumab dosage escalation in groups of three
patients every four weeks for up to 24 weeks. PSA levels decreased by more
than 50% in five out of six individuals who received the top two dosage levels
(3 mg/kg and 5 mg/kg). One patient, with a prestudy PSA level of 50 ng/ml,
was among the responders who experienced an immunological breakthrough
event. After two months of therapy, this patient’s retroperitoneal adenopathy
resolved, and their PSA level dropped to 0.5 ng/mL. Another patient showed
considerable improvement in bone scan lesions when their PSA dropped by
more than 50%. Compared to each treatment method alone, the combination
therapy resulted in a higher percentage of individuals with PSA reductions.
It is interesting to note that some of these individuals initially experienced
a deterioration in their condition, as indicated by PSA, before mounting
a significant response that coincided with the onset of an immunological
breakthrough event.
Additionally, new research supports the idea that clinical responses are
less frequent in patients with rapidly advancing diseases who have already
undergone chemotherapy, as their immune systems are comparably less
functional than those who have not. This is more likely to occur after several
months of therapy. Patients who have not previously undergone anti-cancer
therapy were much more likely to experience a clinical response and partici­
pate in the NCI’s ongoing ipilimumab and vaccination trial (05-C-0167) for
a longer period of time.

— SECTION 27 —
6.11 THERAPEUTIC CANCER VACCINES
Specified Antigens: The decision to target a specific neoplasm antigen has
received a lot of attention in the context of improving anticancer vaccines.
Targeted antigens continue to be the focus of discussions, despite the extensive
investigation of several cancer vaccination techniques. They can be divided
into two primary groups. Tumor-specific immunotoxins, such as CTA,
upregulated immunotoxin (Her2/neu, survivin, MUC-1), and differentiation

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antigens, serve as indicators for both healthy and tumor cells. Examples of
tumor-specific immunotoxins include Mart1, PSA, and PAP. While normal
cells do express certain TAAs, their immunogenicity leads to specific T-cell
reactions. However, TAAs are subject to a certain level of self-tolerance. This
central thymic tolerance is bypassed when it comes to tumor-associated anti­
gens (TAAs), as the immune system interprets them as foreign antigens. This
includes the carcinogenic viral genome in virally induced malignancies and
specific antigens created by non-silent genetic variations of healthy genes.

— SECTION 28 —
6.12 SYNERGISTIC EFFECTS OF VACCINES AND
CHEMOTHERAPEUTIC AGENTS
Vaccines used to treat cancer are known as treatment vaccinations or thera­
peutic vaccines. These vaccinations are part of an immunotherapy cancer
treatment. To activate the host defenses, treatment vaccinations primarily
include tumor-associated antigens (TAAs) and tumor-specific immunotoxins.
In theory, the vaccine might induce both specific cellular immunity and a
humoral immune response, preventing tumor development and eventually
eradicating tumor cells.
6.13 COMBINATION OF DC WITH ANTI-CANCER THERAPY TO
ACHIEVE A SUPRA-ADDITIVE EFFECT
Anti-cancer therapy, along with surgery, is the primary treatment for tumor
patients. However, as the tumor grows, it develops treatment resistance,
limiting its effectiveness. The most common causes of resistance expan­
sion are gene amplification by targeted drugs and point mutation in certain
tumor cells, which enable the therapeutic agent to evade its deadly impact.
A combination of chemotherapy and immunotherapy is employed to combat
tumor drug resistance and immune evasion [31].

— SECTION 29 —
6.13.1 WHAT CAUSES ANTICANCER THERAPY AND DC TO WORK
TOGETHER SYNERGISTICALLY?
The anti-neoplastic efficacy of chemotherapeutic drugs can be enhanced
through immunomodulation. Immunomodulation can be achieved through
various means, including (Figure 6.4 and Table 6.4):

• Promotion of immunogenic tumor cell death to facilitate DC
  phagocytosis (cyclophosphamide, cisplatin, doxorubicin, irinotecan,
  5-FU).

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Synergistic Effect of Vaccines and Chemotherapeutic Agents

• Inhibition of tumor-induced immune suppression by Tregs and
  MDSCs (Capecitabine, busulfan, tesetaxel).
• Activation of cytotoxic T-cells such as NK LGL, macrophages, and
  tumor-specific cytotoxic thymocytes (busulfan, tesetaxel, oxali­
  platin, capecitabine).
  In one trial, low-dose tesetaxel was combined with DC vaccination
  administered within a tumor to treat mouse lung cancer. In terms of tumor
  development suppression, T cell tumor cell collection, and production of
  anti-cancer immunological reaction in local lymph glands, the therapeutic
  impact can be improved compared to the vaccination or paclitaxel alone treat­
  ment groups. Non-toxic levels of chemo agents influenced the endogenous
  molecules. Changes in DC activity was associated with changes in GTPase
  levels. Another study discovered that paclitaxel, doxorubicin, mitomycin C,
  and methotrexate increased DCs’ capacity to deliver antigens to Ag-specific
  T lymphocytes. The enhanced secretion of IL-12p70 expression was related
  to activated DC activity [32, 33].
  FIGURE 6.4 Probable immuno-mechanism of chemotherapeutic agents.

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TABLE 6.4 Chemotherapeutic Agents and Their Impact on the Immune System
Agent
Class
Immunogenic Effect
Busulfan
Alkylating agent
By reducing the immunological inhibition brought on
by Tregs, increase CD8 T cell activation.
Daunorubicin Anthracyclines
Expanding tumor immunogenicity.
Tesetaxel
Taxanes
Increase DC activation and mannose-6-phosphate
receptor expression on tumor cells, both of which are
necessary for granzyme B-mediated death.
Capecitabine Anti-metabolites
Lowers Tregs, improves CD8 activation, and reduces
tumor-induced immunosuppression.
Cisplatin
Platinum compound Enhance tumor immunogenicity.

— SECTION 32 —
5-FU
Antimetabolites
Enhance tumor immunogenicity.
Vincristine
Taxol
Overexpression of DC maturation factors like CD40,
CD80, CD86, MHC-II, secretion of IL-6, IL-12.
6.14 CONCLUSION
A variety of recombinant vaccines have been created as a result of research
in molecular biology and immunology. These vaccines consist of viral-
based immune adjuvants, along with thymocytes co-activation molecules or
lymphokines, for active biotherapy. There is increasing evidence that vacci­
nations can complement conventional cancer treatments such as radiation,
chemotherapy, and surgery. Therefore, it is necessary to conduct relevant
non-human and early human research to investigate these strategies. In order
to better understand how they enhance cancer immunity and validate specific
tests that correlate with human study responses, future human studies will
also need to incorporate more comprehensive immune response monitoring.
Additionally, individuals with advanced-stage diseases have been included in
almost all clinical studies for cancer vaccinations. Further research is needed
to determine if these vaccinations can improve survival rates in individuals
with early-stage disease and minimal tumor burden.

— SECTION 33 —
KEYWORDS

• 
  cancer
• 
  major histocompatibility
• 
  cancer vaccinations
  complex
• 
  chemotherapy
• 
  T-cells
• 
  dendritic cells
• 
  tumor
• 
  immunotherapy
• 
  vaccines

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18. Shankar, S., Ganapathy, S., Chen, Q., & Srivastava, R. K., (2008). Curcumin sensitizes
    TRAIL-resistant xenografts: Molecular mechanisms of apoptosis, metastasis, and
    angiogenesis. Mol. Cancer, 7, 16. [CrossRef] [PubMed].
19. Sharma, A., Koldovsky, U., Xu, S., Mick, R., Roses, R., Fitzpatrick, E., Weinstein, S.,
    et al., (2012). HER-2 pulsed dendritic cell vaccine can eliminate HER-2 expression and
    impact ductal carcinoma in situ. Cancer, 118(17), 4354–4362.
20. Shimizu, J., Yamazaki, S., & Sakaguchi, S., (1999). Induction of tumor immunity
    by removing CD25+CD4+ T cells: A common basis between tumor immunity and
    autoimmunity. J. Immunol., 163, 5211–5218.
21. Tong, Y., Song, W., & Crystal, R. G., (2001). Combined intratumoral injection of bone
    marrow-derived dendritic cells and systemic chemotherapy to treat pre-existing murine
    tumors. Cancer Res., 61, 7530–7535. [PubMed: 11606390].
22. Leach, D. R., Krummel, M. F., & Allison, J. P., (1996). Enhancement of antitumor
    immunity by CTLA-4 blockade. Science, 271, 1734–1736. [PubMed: 8596936].
23. Casares, N., Pequignot, M. O., Tesniere, A., Ghiringhelli, F., Roux, S., Chaput, N., et al.,
    (2005). Caspase-dependent immunogenicity of doxorubicin-induced tumor cell death. J.
    Exp. Med., 202, 1691–1701. [PubMed: 16365148].
24. Emens, L. A., & Jaffee, E. M., (2005). Leveraging the activity of tumor vaccines with
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25. Nigam, A., Yacavone, R. F., Zahurak, M. L., Johns, C. M., Pardoll, D. M., Piantadosi,
    S., et al., (1998). Immunomodulatory properties of antineoplastic drugs administered in
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    [PubMed: 9454900].
26. Arlen, P. M., Pazdur, M., Skarupa, L., Rauckhorst, M., & Gulley, J. L., (2006). A
    randomized phase II study of docetaxel alone or in combination with PANVAC-V
    (vaccinia) and PANVAC-F (fowlpox) in patients with metastatic breast cancer (NCI
    05-C-0229). Clin. Breast Cancer, 7, 176–179. [PubMed: 16800982].
27. Drake, C. G., Doody, A. D., Mihalyo, M. A., Huang, C. T., Kelleher, E., Ravi, S., et al.,
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    antigen. Cancer Cell, 7, 239–249. [PubMed: 15766662].
    Arlen, P. M., Gulley, J. L., Todd, N., Lieberman, R., Steinberg, S. M., Morin, S., et al.,
    (2005). Antiandrogen, vaccine, and combination therapy in patients with nonmetastatic
    hormone refractory prostate cancer. J. Urol., 174, 539–546. [PubMed: 16006888].
28. Madan, R., Gulley, J., Dahut, W., Steinberg, S., Liewehr, D., & Schlom, J., (2007).
    5-year overall survival (OS) in non-metastatic androgen independent prostate cancer

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(AIPC) patients (pts) treated with nilutamide (N), vaccine (V), and combination therapy

— SECTION 39 —
[abstract]. Multidisciplinary Prostate Cancer Symposium Program/Proceedings (p.
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29. Hodge, J. W., & Schlom, J., (1999). Comparative studies of a retrovirus versus a
    poxvirus vector in whole tumor cell vaccines. Cancer Res., 59, 5106–5111. [PubMed:
    10537283].
30. Maker, A. V., Phan, G. Q., Attia, P., Yang, J. C., Sherry, R. M., Topalian, S. L., et
    al., (2005). Tumor regression and autoimmunity in patients treated with cytotoxic T
    lymphocyte-associated antigen 4 blockade and interleukin 2: A phase I/II study. Ann.
    Surg. Oncol., 12, 1005–1016. [PubMed: 16283570].
31. Ghiringhelli, F., et al., (2005). CD4+CD25+ regulatory T cells inhibit natural killer
    cell functions in a transforming growth factor-β-dependent manner. J. Exp. Med., 202,
    1075–1085. The first demonstration of the inhibitory effect of Treg cells on NK-cell
    responses in mice and humans in vitro and in vivo.
32. Lim, S., Park, J. H., Chang, H. (2023). Enhanced anti-tumor immunity of vaccine
    combined with anti-PD-1 antibody in a murine bladder cancer model. Investig Clin
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33. Weir, G. M., Liwski, R. S., & Mansour, M., (2011). Immune modulation by chemotherapy
    or immunotherapy to enhance cancer vaccines. Cancers (Basel), 3(3), 3114–3142.

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the Management of Various Carcinomas. Rishabha Malviya, Bhupendra Prajapati, and Sonali Sundram (Eds.)
© 2025 Apple Academic Press, Inc. Co-published with CRC Press (Taylor & Francis)

— END OF CHAPTER 6 —

#

CHAPTER 7
TITLE: Cancer Immunotherapy Using mRNA
AUTHORS: Umang Shah, Aashka Thakkar, Alkesh Patel, Sandip Patel, Mehul Patel, and Rajesh Maheshwari
LENGTH: 49,294 characters

#

— SECTION 1 —
CHAPTER 7
Cancer Immunotherapy Using mRNA
UMANG SHAH,1 AASHKA THAKKAR,1 ALKESH PATEL,1 SANDIP PATEL,2
MEHUL PATEL,1 and RAJESH MAHESHWARI3
1Ramanbhai Patel College of Pharmacy, Charotar University of Science
and Technology (CHARUSAT), CHARUSAT Campus, Changa, Gujarat, India
2Department of Pharmacology, L. M. College of Pharmacy, Ahmedabad,
Gujarat, India
3Department of Pharmacy, Sumandeep Vidyapeeth (Deemed to be
University), Piparia, Vadodara, Gujarat, India
ABSTRACT
Cancer immunotherapy has gained tremendous momentum in the past few
years due to its wide range of benefits and its ability to overcome the limitations
of conventional treatments. mRNA vaccines have emerged as a promising
new option in the battle against cancer. This is because they activate the innate
immune system and deliver antigens through co-stimulation. One advantage
is that mRNA vaccines cannot enter the nucleus, thus leaving genetic expres­
sion unaltered. In the initial stage of mRNA therapy, genetic mutations in a
patient’s tumor cells can lead to the identification of neoantigens. In-silico
approaches then determine which neoantigens are likely to elicit a response
from the immune system by binding to T cell receptors (TCRs). Synthetic
mRNA has proven effective in immunizing patients against cancer using this
method. One of the main goals of personalized cancer immunotherapy with
mRNA vaccines is to stimulate T cells that can recognize specific malignant
cells based on the unique molecular characteristics of the disease. However, a
significant obstacle to its widespread therapeutic application is the instability
of mRNA and its inefficient transport to the target region. Recent advances

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suggest that protecting mRNA from degradation by RNases can be achieved
through nanoparticulate formulations, which ultimately improve the uptake
of antigen-presenting cells in vivo. This chapter provides an overview of
the background and current advances in mRNA-based immunotherapy for
cancer treatment, as well as insights into future efforts to enhance mRNA
vaccines for cancer treatment.

— SECTION 3 —
7.1 BIOLOGY OF SYNTHETIC MRNA
Messenger ribonucleic acid (mRNA) is a subtype of RNA found in the
nucleus. It is responsible for carrying genetic information from DNA to
proteins through the processes of transcription and translation. Synthetic
mRNA, made possible by technological advancements and research invest­
ments, exhibits remarkable stability when introduced into mammalian cells.
It can be effectively translated into proteins and possesses exceptional prop­
erties such as fluorescence emission, ligand attachment via click chemistry,
and photocross-linking. There have been reports indicating the effectiveness
of synthetic mRNA against various biological conditions. These include
restoring deficient proteins to mitigate severe diseases, immunizing cancer
patients, and making specific alterations in DNA through genome engi­
neering [1].

— SECTION 4 —
7.1.1 STRUCTURE
The three fundamental components that make up the core structure of
mRNA are as follows: (i) the middle part is an open reading frame (ORF)
that codes for protein. It is flanked on both ends by untranslated sections
that are 5′ and 3′ in length (UTRs); (ii) a section called “a cap” that is
located in the front, which is structurally a 7-methyl-guanosine residue
attached to the 5′-end by a 5′-5′-triphosphate; and (iii) the 3′-end of the
poly(A) tail, the ORF region improves protein expression and codon opti­
mization with sequence changes. The UTRs are considered the regulatory
elements that improve the efficacy of the translation process with their
structure and length. The 5′ moiety increases the overall stability of the
mRNA molecule. The length of the poly(A) tail influences translational
efficacy significantly [2]. The mRNA structural elements have been
presented in Figure 7.1.

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Cancer Immunotherapy Using mRNA
FIGURE 7.1 mRNA structural elements.

— SECTION 6 —
7.1.2 PROCESSING SYNTHETIC MRNA
By utilizing complementary DNA (cDNA) as a template, the SP6 in vitro
transcription (IVT) of cDNA can generate synthetic mRNAs that are
highly valuable. Plasmid DNA (pDNA) is specifically employed for this
purpose, along with RNA polymerase as a bacteriophage [3]. The structure
of mRNA consists of an open reading frame (ORF), multiple untranslated
regions (UTRs), a 5′ cap motif, and a poly(A) tail, as previously discussed.
The pDNA template contains an ORF, a bacteriophage promoter, and a
poly(d(A/T)) sequence that is translated into the poly(A) tail to facilitate
IVT. Additionally, a unique restriction site is present for the linearization
of the plasmid, enabling precise specification of the transcription endpoint.
In the initial step of the procedure, the linearized pDNA is transcribed
into mRNA using recombinant RNA polymerase in a mixture containing
nucleoside triphosphates. To obtain capped mRNA during transcription, a
cap analog such as the dinucleotide m7G(5′)-ppp-(5′)G can be added to the
reaction mixture. This is crucial for achieving the desired outcome. When
the cap analog is abundant compared to the amount of GTP, transcription
commences with the cap analog instead of GTP, resulting in the production
of capped mRNA [4]. Following transcription, DNase digestion is performed
on both the pDNA template and bacterial DNA. The sample, which contains
numerous additional components along with mRNA, is purified through

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precipitation and extraction processes [5]. The strategies for the capping
reaction are illustrated in Figure 7.2.
FIGURE 7.2 Strategies for capping reaction.

— SECTION 8 —
7.1.3 HOW CAN SYNTHETIC MRNA CONTRIBUTE TO THE CANCER
TREATMENT?
Cancer immunotherapy studies have shown that mRNA is a promising
vaccine candidate. The main aim behind this approach is to activate the host’s
immunity against tumor suppression, ultimately increasing the survival rate
among cancer patients [6]. The mRNAvaccines that specifically target tumor-
specific antigens have the ability to combat malignant cells that overexpress
these antigens, leading to tumor regression through immunologic memory.
Functional mRNA vaccines primarily enhance immunological reactions
through adaptive immune responses. Upon exposure to synthesized mRNA,
antigen presentation cells (APCs) present the translated encoded proteins
to CD4+ T cells via the major histocompatibility complex-II (MHC-II).
Subsequently, CD4+ T cells present them to CD8+ T cells via MHC-I,
triggering a robust immunological response [7]. The major advantages of
immunotherapy using synthetic mRNA are that these vaccines can activate
both B cell-mediated immunity (humoral immunity) and CD4+/CD8+ T
cell-mediated immunity. This aids in the active elimination of malignant
cells from the tumor microenvironment (TME). Furthermore, mRNA poses
no genetic risks as it cannot integrate into the genome sequence, making it
highly tolerable [8].

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— SECTION 10 —
7.1.4 POSSIBLE LIMITATIONS
The degradation of mRNA and the hindrance of protein synthesis are the
primary obstacles to the treatment of cancer through mRNA-based immuno­
therapy. Additionally, the protein expression of mRNA may be diminished
due to inadequate in vivo delivery of mRNA-based vaccines. These limita­
tions have severely restricted the clinical use of mRNA vaccines for cancer
immunotherapy. Other challenges that impede the successful implementa­
tion of this approach include excessive immunogenicity (which unneces­
sarily triggers the immune response) and a lower safety profile compared to
inactivated vaccines. Furthermore, it is less effective than DNA vaccines [9].
7.2 MODIFICATION OF SYNTHETIC MRNA
In order to overcome the aforementioned limitations, scientists worldwide
have consistently addressed these issues and have developed synthetically
modified mRNA. Significant modifications have been made to the nucleo­
bases, RNA terminals, and ribose structure. One approach involves creating
cap mimics at the 5′ position to enhance stability, translational efficiency,
and reduce immunogenicity. Another modification includes substituting
oxygen with sulfur at the β position of the triphosphate bridge in m7GpppG,
resulting in reduced sensitivity to the decapping complex DCP1/DCP2 and
increased affinity of the cap for eIF4E [10]. Furthermore, the tetraphosphate
bridge has shown greater affinity for eIF4E compared to the triphosphate
moiety. Chemical alterations of 0.2–0.4% in the pyrimidine bases have been
found to confer resistance against RNase cleavage and activation of toll-like
receptors (TLRs). For instance, replacing natural uracil/cytosine (U/C) with
m5C improves resistance to RNase [11]. However, replacing some or all
of the A with m6A reduces the binding affinity of TLR3/7/8 with synthetic
mRNA, enabling it to evade recognition by the innate immune system. Addi­
tionally, modifying the ribose component of synthetic mRNA with 2′-OMe
or 2′-O-fluoro may reduce inflammatory reactions. In vitro studies have
demonstrated that 2′-O-fluoro-modified synthetic mRNA exhibits higher
affinity for the RIG-I receptor compared to regular mRNA, as observed
through a comparative analysis of the two RNA types [12].

— SECTION 11 —
7.2.1 FOR ANTIGENIC RECEPTORS
In the early 1980s, high dosages of IL-2 infused into melanoma patients
alongside tumor infiltrating lymphocytes were shown to be curative for the

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disease [13]. As conventional cancer treatments have proven ineffective,
scientists have been working to improve outcomes by engineering T cells to
express cancer-specific T cell receptors (TCRs) or chimeric antigen receptors
(CARs). Several years of research have shown that electroporation of CAS9
mRNA into human T cells is a powerful alternative to direct integration of
CD19-specific CAR to the T cell receptor constant (TRAC) locus [14]. This
increased the potency of T cells and led to the uniform expression of CARs.
In addition to this, when chemically modified synthetic mRNA, i.e., 1 mψ
mRNA, was electroporated with murine T cells, it was found that upregula­
tion of major checkpoints such as PD-1 and LAG-3 was drastically reduced.
The primary advantage of mRNA encoded CARs or TCRs is that they can
prevent the development of cytokine release syndrome [15].

— SECTION 12 —
7.2.2 IMMUNOMODULATORS
Synthetic mRNAs serve as a highly effective tool for delivering a wide range
of immunomodulators, thereby enhancing the immune response of T cells
in malignant conditions. For instance, the delivery of stimulatory cytokines
such as IL-12, IL-15, and GM-CSF, in conjunction with mRNA encoding
tumor antigens (TA), can effectively counteract immune suppression caused
by cancerous cells. Moreover, the co-expression of TA mRNA with immu­
nostimulatory molecules like constitutively active TLR4, CD40L, and CD70
in melanoma patients triggers T cell activation and facilitates dendritic cell
(DC) maturation [16]. Furthermore, intra-tumoral administration of mRNA
encoding two cytokines, namely IL-36γ (an alarm signal against foreign
pathogens) and IL-23 (a coordinator of immune responses to danger signals),
elicits a robust immune response against various tumor models. Combining
mRNA-based vaccines with immune checkpoint inhibitors (ICIs) such as
anti-CTLA4 and anti-PD-1 targeting antibodies has been demonstrated
to suppress tumor development by reducing PD-1 expression on antigen-
presenting cells (APCs), enhancing T cell activation and proliferation, and
restricting tumor growth [17].

— SECTION 13 —
7.2.3 ANTIBODIES
Monoclonal antibodies (mAbs) are extensively utilized as potent therapeutic
agents against cancer. This is due to their ability to primarily target stromal
or vascular growth factors that deprive tumor cells of essential oxygen and
nitrogen, ultimately leading to their destruction. However, the main challenges

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Cancer Immunotherapy Using mRNA
associated with their usage are the high production cost and the site of action
of these proteins. Research suggests that in vitro transcribed mRNA presents
a viable solution to overcome these challenges. It is evident that mRNA is
capable of encoding any given protein, including fragments of antibodies
and full-size mAbs [18]. The subsequent distribution of this mRNA to cells
and the synthesis of these proteins ensures post-transcriptional modification
and optimal assembly. Over the years, significant effort has been dedicated
to determining whether messenger RNA (mRNA) can effectively function
as a vector for delivering mAbs. This strategy holds utmost importance as
a suitable immune response against the tumor is crucial to trigger a cascade
of immune reactions that contribute covalently to the fight against cancer,
thereby improving the quality of life (QoL) for cancer patients [19].

— SECTION 14 —
7.3 DELIVERY STRATEGIES OF SYNTHETIC MRNA FOR THE
CANCER IMMUNOTHERAPY
Significant technical advances over the past few decades have allowed for
the development of medicines that utilize mRNA as a revolutionary step
towards cancer immunotherapy treatment. The development of more effi­
cient materials and delivery systems has assisted in removing significant
barriers to eliciting the necessary immune response to combat a particular
form of cancer [16]. Once the host cell has taken up the synthetic mRNA by
endocytosis or the cell membrane, it will begin translating the mRNA into
the mature form of the target peptide in the cytoplasm. Post-transcriptional
changes cause these protein constructions, which are indistinguishable

— SECTION 15 —
from the results of endogenous mRNA, to be degraded by intracellular
compartments. Antigen-presenting cells (APCs) use major histocompat­
ibility complex (MHC) presentation to transfer these peptides to effector
cells present in the immune system of hosts, where they activate helper T
lymphocytes and NK cells while also stimulating the production of cancer-
specific killer T cells [20]. Exogenous mRNA not only generates a cancer-
specific immune response but also aids in maintaining an immune-friendly
tumor microenvironment (TME) by activating retinoic acid-inducible gene
I (RIG-I) and TLR, which results in the secretion of type I interferon (IFN)
and other inflammatory cytokines (RIG-I) [21]. The production of CD8+
cytotoxic T cells that are specific to antigens can be stimulated. The ratio
between active CD8 cells and immune suppressive Tregs can be increased,
and memory T cells can be induced for a long-standing immune response by
engineering mRNA constructs to express proinflammatory cytokines such

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— SECTION 16 —

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Cancer Vaccination and Challenges, Volume 2
as IL-2, IL-7, IL-12, and IL-15 [22–24]. Furthermore, mRNAs are used to
encode monoclonal antibodies (mAbs), which have been demonstrated to

— SECTION 17 —
play a crucial role as passively targeted immunotherapy methods for various
cancers, including lymphomas and breast cancers. In-vivo research suggests
that mRNA encoded monoclonal antibodies (mAbs) can indeed elicit a more
prolonged antitumor effect than their recombinant equivalents in animal
models. In vivo research suggests that mRNA-encoded mAbs can indeed
elicit a more prolonged antitumor effect than their recombinant equivalents
in animal models [25, 26].
7.3.1 DELIVERY STRATEGIES FOR MRNA
The utilization of mRNA technology in cancer immunotherapy has demon­
strated potential, yet there remain numerous challenges to be addressed in
order to generate an effective immune response. The large RNA molecule
carries a negative charge and must first penetrate the cell membrane, which
poses a significant obstacle to intracellular release due to its negative charge.
Ribonucleases, which are abundant in the skin and bloodstream, degrade
mRNA upon reaching the cell. Naked mRNA can be delivered intradermally,

— SECTION 18 —
subcutaneously, or intramuscularly (I.M). However, these methods have limi­
tations as they cannot efficiently enter intracellular compartments, resulting in
a short half-life, rapid breakdown, and inadequate immune response. There­
fore, the establishment of a robust delivery system is crucial for achieving an
optimal therapeutic effect. In order to ensure adequate translational capacity,
advancements in mRNA technology have been linked to the development of
innovative nanotechnology-based delivery techniques [6, 27, 28].
7.3.2 SYNTHETIC SYSTEMS
7.3.2.1 DELIVERY SYSTEMS BASED ON LIPIDS
The most studied delivery strategies for RNA-based therapies are lipid-
based materials. These structures, known as lipid nanoparticles (LNP), are
composed of a cationic or, more recently, a pH-dependent lipid layer (ioniz­
able); a polyethylene glycol (PEG) component; cholesterol; and phospho­
lipids [29]. The ionizable amino lipid coating of the liposome is designed to
acquire a positive charge as the pH decreases, facilitating easier endosomal
absorption while maintaining the encapsulation of the negatively charged
mRNA molecule. In addition to its role as a stabilizer with cholesterol,
the PEG molecule also plays a crucial role in preventing breakdown by

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— SECTION 19 —

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Cancer Immunotherapy Using mRNA
macrophages [30–32]. It is well-known that the construction of the amino
lipid component greatly influences delivery efficiency, tolerability, and
tissue clearance [33]. Attempts to enhance mRNA vaccine delivery through
LNP have achieved effective RNA release in cell lines and robust, long-term
humoral immune responses for various viral infections in mouse models
[34–36]. Although the follow-up period for clinical trials of two mRNA
vaccines based on LNP for SARS-CoV-2 during the COVID pandemic
was relatively short, positive outcomes were confirmed, indicating good
effectiveness in diverse populations, and ultimately leading to regulatory
approval [37, 38]. However, the design of LNPs needs to be improved to
ensure that the mRNA cargo specifically targets antigen-presenting cells
(APCs) in cancer vaccines (CVs). This will help prevent degradation and
maintain optimal translation processes [39–42].

— SECTION 20 —
7.3.2.2 DELIVERY SYSTEMS BASED ON POLYMERS
mRNA vaccines against deadly viruses such as Ebola, HIV, Zika, and H1N1
influenza have gained popularity due to the use of polymeric polymers
and dendrimers modified with nanotechnological fatty side chains. These
modifications help reduce harmful effects and prevent enzymatic breakdown
in vivo [43–47]. Studies using murine models have shown that an antiangio­
genic RNA sequence delivered through polymeric structures, enclosed in a
PEG outer shell, can inhibit pancreatic cancer growth [48]. Similar mRNA
vaccines have demonstrated the ability to effectively produce tumor-related
antigens in vivo [49]. In a castration-resistant prostate cancer model, admin­
istration of an RNA vaccine based on a polymer encoding PTEN resulted in
decreased tumor growth, as reported by Islam et al. [50]. This was achieved
by restoring PTEN’s normal function.

— SECTION 21 —
7.3.2.3 DELIVERY SYSTEMS BASED ON PEPTIDES
Cell-penetrating peptides (CPPs) must be cationic in order to transport
proteins, small chemical molecules, nanoparticles (NPs), and oligonucle­
otides through the cell membrane. CPPs are a potential family of non-viral
delivery vectors due to their low toxicity and high transfection efficiency
[51–53]. However, the limited selectivity of cells and tissues, as well as
inefficient cargo absorption across multiple layers of cells, are limitations
to the widespread implementation of this technique in a medical context

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Cancer Vaccination and Challenges, Volume 2
[54]. Recent research has focused on discovering the most effective CPPs
to enhance immunological activation. Cationic peptides like protamine can
protect RNA from degradation in lysosomes during transport. Activation of
TLR 7 with a protamine-based delivery system has been shown to induce
a robust immunological response [55, 56]. Biotech innovations have also
made progress in mRNA release using peptides. For example, a powdered
form of the cationic KL4 peptide complex, formed by PEGylation, is
effectively utilized as an aerosol-based delivery system to the lungs [57].
Effective uptake and delivery of mRNA encoding the Ova peptide linked to
the GALA peptide by antigen-presenting cells (APCs) resulted in increased
antigen-specific T cell activation and DC maturation, compared to naked
RNA or other peptide complexes [58].

— SECTION 23 —
7.4 SUCCESS STORIES OF MRNA IN TERMS OF CLINICAL TRIAL
Many successful milestones have been achieved in the translation of mRNA­
based immunotherapeutics from bench to bedside. However, each milestone
has presented new challenges in order to make it more suitable and conve­
nient for patient care.
There has been significant interest in cancer immunotherapies following
the approval of six immune checkpoint blockers and CAR-T cell immuno­
therapies. Notably, the USFDA approved an immune cell-based vaccine
(PROVENGE) for the treatment of hormone-refractory prostate cancer in
2010 [59]. Personalized cancer vaccines are being investigated in combi­
nation with checkpoint blockers or cytokine therapies for various solid or

— SECTION 24 —
metastatic cancers, showing promising clinical results [60].
The majority of mRNA-based immunotherapeutics based in Washington,
D.C. were explored in clinical trials. Additionally, various biotech compa­
nies such as CureVac, BioNTech, and Moderna investigated promising anti­
tumor responses from mRNA-based immunotherapies delivered by non-viral
vectors in pre-clinical studies. To suppress the tumor microenvironment
(TME), mRNA-based immunotherapeutics encoding immunostimulants
such as IL-12, IL-32, OX40L, CD40L, and CD70 were injected at the target
site [6].
Numerous studies were conducted to analyze the platform, antigens,
delivery methods, clinical end goals, and preliminary clinical data.
Significant corporations carried out clinical trials of mRNA treatments.
Table 7.1 illustrates some of the clinical investigations that have been
conducted [62].

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TABLE 7.1 ClinicalTrials.gov Registered mRNA-based Cancer Immunotherapy Trials
NCT No.
Sponsors
Antigen
Delivery System
Indications
Combination
Status
Platform
Therapy, If Any

— SECTION 26 —
NCT03289962
BioNTech and
Neoantigen
RNA lipoplex, i.v. Multiple solid
Atezolizumab
Phase I, recruiting,
Genentech
mRNA
tumors.
2020 completion.
NCT03815058
BioNTech and
Neoantigen
RNA lipoplex, i.v. Melanoma
Pembrolizumab
Phase 2 recruiting,
Genentech
mRNA
2022 completion).

— SECTION 27 —
NCT03897881
Moderna and Merck
Neoantigen
Lipid nanoparticu-
Multiple solid
Pembrolizumab
Phase 1, recruiting,
mRNA
late system; i.m.
tumors.
2022 completion.

— SECTION 28 —
NCT03948763
Moderna and Merck
TAAs mRNA
Lipid nanoparticu-
Colorectal, NSCLC, Pembrolizumab
Phase 1 recruiting,
late system; i.m.
pancreatic.
2024 completion.
NCT03380871
Neon and Merck
Neoantigen
Polymeric, s.c.

— SECTION 29 —
NSCLC
Pembrolizumab, Phase 1 active, not
peptides
pemetrexed,
recruiting, 2021
carboplatin.
completion.
NCT02897765
Neon therapeutics
Neoantigen
Polymeric, s.c.
Solid tumors
Nivolumab
Phase 1 active, not
and BMS.
peptides
recruiting, 2021
completion.

— SECTION 30 —
NCT03639714
Gritstone oncology
Neoantigen
Viral vector, i.m.
NSCLC, colorectal
Nivolumab
Phase 1/2 recruiting,
and BMS.
and self-
cancer, endometrial
ipilimumab
2022 completion.
amplifying
cancer, breast
mRNA.
cancer.

— SECTION 31 —
NCT03953235
Gritstone oncology
Neoantigen
Viral vector, i.m.
NSCLC, colorectal
Nivolumab
Phase 1/2 recruiting,
and BMS.
cancer.
ipilimumab
2023 completion.

— SECTION 32 —
NCT03164772
CureVac, Boehringer Neoantigen
Lipid nanopar-
NSCLC, colorectal
Durvalumab,
Phase 1/2 recruiting,
Ingelheim, and
mRNA.
ticles, intradermal
cancer.
tremelimumab
2024 completion.
MedImmune
injection.
Cancer Immunotherapy Using mRNA

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7.5 POSSIBILITIES OF PERSONALIZED ONCOTHERAPEUTICS
WITH THE MRNA-BASED VACCINE
Cancer development is a unique process, and this characteristic could be
utilized to personalize treatment based on individual needs. The develop­
ment of tumor resistance can lead to therapeutic failure with immunotherapy,
radiotherapy, and chemotherapy. The identification of specific neoantigens,
tumor microenvironment (TME), and cancer stem cell (CSC) markers can
aid in stratifying the population and targeting treatment accordingly [63].
Considering the flexibility in designing mRNA-based vaccines, tailoring
them to the specific stage of cancer becomes feasible. To achieve effective
antitumor effects, the mRNA encoding specific antigens must be delivered
properly and translated within dendritic cells (DCs). Activation of TLR4
on DCs triggers the adaptive immune response, including the generation of
targeted T cell subsets and subsequent production of tumor-specific anti­

— SECTION 35 —
bodies [64]. mRNA-based oncotherapeutics have shown promising results
in lung adenocarcinoma [65], bladder cancer [66], and other malignan­
cies. The mRNA-based vaccine development process can deliver various
types of neoantigens suitable for highly heterogeneous tumors. It can even
compensate for the limitations of immunotherapy in treating solid tumors
[67]. The synthesis of mRNA-based vaccines can be achieved through an
in vitro transcription (IVT) process, which delivers relatively stable mRNA
after specific chemical modifications [68]. IVT can optimize the immu­
nogenicity of mRNA and prevent early degradation by the innate immune
system [61]. Ongoing development of personalized mRNA vaccines by
Moderna, Argos, and BioNTech holds promise for positive outcomes in
the future.

— SECTION 36 —
7.6 CONCLUSION AND FUTURE PERSPECTIVES
As we have observed, mRNA serves as a highly effective and adaptable
platform for the development of CVs. Prior to 2020, no mRNA vaccine
had received global approval. However, in response to the rapid spread of
the deadly COVID-19 disease, the USFDA authorized the use of multiple
mRNA vaccines against it, highlighting the advantages of swift and
efficient mRNA vaccine production in combating newly emerging infec­
tious diseases. In a rapid response to the pandemic, therapeutic CVs were

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Cancer Immunotherapy Using mRNA
created by leveraging the scientific, clinical, manufacturing, and regula­
tory knowledge accumulated over decades of mRNA research. Studies
have demonstrated that mRNA-based vaccinations elicit a favorable
immune response in cancer cells, leading to apoptosis and inhibition of
tumor growth. Notably, mRNA-based CVs have shown superior efficacy
and reduced toxicity compared to conventional cancer treatments such
as chemotherapy and radiation. Unlike vaccines for infectious diseases,
which target well-characterized antigens for preventive immunization,
most tumor-targeting antigens exhibit significant variation among indi­
viduals, are rare, and remain poorly understood. Consequently, concerns
have been raised regarding the safety and efficacy of mRNA CVs. More
and more clinical trials, particularly those focusing on tailored vaccina­
tions, are opening the door to the creation of mRNA vaccines against a
variety of malignancies. The therapeutic potential of mRNA vaccines
has been hindered by their instability and the need for in vivo delivery.
Despite significant progress made in addressing these limitations over the
past few decades, the design of mRNA vaccines still faces challenges. It
is crucial to intensify efforts to enhance the stability of mRNA vaccines in
order to facilitate their widespread use as cancer treatments. While there
is still much work to be done, mRNA therapy shows great promise. If this
research is successfully translated into clinical practice, it will signifi­
cantly enhance our ability to combat cancer.
Investigating and harnessing the seemingly contradictory innate immu­
nity of mRNA, developing novel and well-tolerated delivery systems to
improve antigen expression and presentation efficiency, and engineering
mRNA structures to achieve prolonged and controlled expression are all
areas that require further investigation. Future studies should focus on
technological research and clinical development. Cancer immunotherapy
will benefit from the knowledge gained by advancing mRNA through all
stages of drug development for the first time. This has sparked increased
interest in mRNA-based vaccines as cutting-edge immunotherapies.
mRNA vaccines have the potential to make a significant contribution to
the fight against cancer. Moving forward, the scientific community will
explore the potential of mRNA vaccines in conjunction with standard
cancer treatments.

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— SECTION 39 —
KEYWORDS

• 
  antigen presenting cells
• 
  cancer immunotherapy
• 
  chimeric antigen receptors
• 
  immune mediated adverse events
• 
  immune response
• 
  messenger ribonucleic acid
• 
  neoantigens
• 
  T-cell
• 
  tumor microenvironment
• 
  vaccine

— SECTION 40 —
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Cancer Vaccination and Challenges, Volume 2: Delivery Strategies for Cancer Vaccine and Immunotherapy in
the Management of Various Carcinomas. Rishabha Malviya, Bhupendra Prajapati, and Sonali Sundram (Eds.)
© 2025 Apple Academic Press, Inc. Co-published with CRC Press (Taylor & Francis)

— END OF CHAPTER 7 —

#

CHAPTER 8
TITLE: Autologous Vaccines for Prostate and Pancreatic Cancer
AUTHORS: Nayankumar C. Ratnakar, Beenkumar R. Prajapati, and Bhupendra G. Prajapati
LENGTH: 66,647 characters

#

— SECTION 1 —
CHAPTER 8
Autologous Vaccines for Prostate and
Pancreatic Cancer
NAYANKUMAR C. RATNAKAR,1 BEENKUMAR R. PRAJAPATI,1 and
BHUPENDRA G. PRAJAPATI2
1L.M. College of Pharmacy, MG Science Institute, Opp. Gujarat University,
University Area, Navrangpura, Ahmedabad, Gujarat, India
2Shree S.K. Patel College of Pharmaceutical Education and Research,
Ganpat University, Ganpat Vidyanagar, Gujarat, India
ABSTRACT
Vaccines are medicines that help the body fight against diseases and train
the immune system to identify and eliminate harmful cells. Throughout their
lives, people receive multiple vaccines. Cancer vaccines (CVs) provide a
similar level of protection against these viruses. There are vaccines avail­
able for both treating and preventing cancer. CVs offer potent and clinically
viable therapeutic approaches to reduce tumor burden, eradicate residual
cancer cells, and prevent relapse. The development of new CVs has made
remarkable progress in recent years, with techniques that induce strong,
long-lasting, and cancer-specific immune responses, while also improving
therapeutic efficacy and minimizing systemic side effects. For both solid
tumors and blood cancers, autologous CVs are potential personalized immu­
notherapeutic treatments that rely on the patient’s own cells to stimulate
an immune response. Using autologous tumor cells as a source of tumor
antigens (TAs), vaccination with these cells offers several advantages. This
type of vaccine can be tailored to the patient and contains antigens that can
activate both Th cells and Cytotoxic T lymphocytes (CTLs). As a result,
they are considered personalized vaccines. However, the effectiveness of the

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vaccine will be limited if the tumor does not provide enough antigens. The
current standard systemic treatment for prostate cancer consists of a prostate
cancer vaccine combined with anti-hormonal and cytostatic medications. An
autologous pancreatic cancer vaccine is created by genetically modifying
pancreatic cancer cells unique to the patient to produce a cytokine called
granulocyte-macrophage colony-stimulating factor (GM-CSF), which has
potential immunostimulatory and anti-tumor effects.

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8.1 INTRODUCTION OF AUTOLOGOUS CANCER VACCINE (CV)
The development of vaccinations has opened up new avenues for the
prevention and treatment of infectious illnesses. The first vaccination was
discovered in 1796 by Edward Jenner, who discovered that the cowpox
vaccine may prevent smallpox infection [1]. Recent advancements in
immune system-based tumor treatment methods have made immunotherapy
a viable option for cancer treatment [2]. With the expansion of antibody-
mediated immunotherapies like checkpoint blockade, cancer vaccines are
once again emerging as potential therapeutics. Both in pre-clinical and
clinical settings, T-cell responses to modified tumor epitopes are often
associated with the success of immunotherapy [3]. Personalized cancer
immunotherapy encompasses various techniques, but autologous tumor
cell-based vaccines (ATVs) have shown significant promise in inducing
potent antitumor immune responses [4]. Research into autogenous vaccines
dates back to the early 1900s. The first autogenous vaccine was introduced
by Sir Almroth Edward Wright in 1903, and various case reports about its
manufacturing and applications were published in the years that followed
[5]. An autologous vaccination is created by removing tumor cells from a
person and using those cells to create a vaccine preparation at lab scale. Later
on, it is given to the person whose tumor cells were removed. An autologous
cell vaccination may induce a cytotoxic T-lymphocytic immune reaction to
cell surface-expressed tumor-associated antigens (TAAs), leading to tumor
cell death. This reaction is typically accompanied by an adjuvant immu­
nostimulant [6]. An autogenous (autologous) vaccine, also known as an
autovaccine, is a therapeutic vaccine created specifically for a patient with a
history of recurring or chronic illnesses. In veterinary medicine, autogenous
vaccines are frequently used to treat infectious illnesses that affect domestic
and agricultural animals (stable or herd-specific vaccinations) [7]. In order
to treat a variety of chronic illnesses, including skin infections, infections of

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— SECTION 4 —

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the respiratory system, infections of the colon, and infections of the urinary
tract, autogenous vaccinations were administered to adults, children, and
newborns. Additionally, autogenous vaccinations were applied in cases of
bronchial asthma, sepsis, gonorrhea, candidiasis, and osteomyelitis, among
others [7].
Vaccines used for cancer treatment are different from those used for virus
prevention. These vaccines aim to stimulate a normal immune response to
eliminate cancer cells in humans. Their purpose is to enhance the body’s
defenses against diseases rather than simply avoiding illness [8]. Some
cancer therapy vaccines contain cancer cells, components of cancer cells, or
purified antigens. In order to produce the vaccine, occasionally a patient’s
own immune cells are taken and exposed to these substances in a laboratory.
Once the vaccine is prepared, it is administered to the body through an injec­
tion to enhance the immune system’s ability to fight against cancer cells [9].

— SECTION 5 —
8.2 APPROVED CV AND ITS PROPOSED MECHANISM
CVs function by enhancing antigen presentation pathways to induce the T
cell response. Antigen selection is a crucial stage in the development of CVs.
The effectiveness of cancer vaccinations heavily relies on the T lymphocytes’
ability to recognize tumor antigens (TAs) [10]. An ideal antigen for a CV
should be highly immunogenic, specifically expressed in cancer cells (but not
in normal cells), and essential for the survival of cancer cells [1]. There are
two categories of TAs: tumor-specific antigens (TSAs) and tumor-associated
antigens (TAAs). TSAs are a group of proteins exclusively found in tumor
cells. They are also known as neoantigens, which are non-self-proteins
resulting from specific mutations in tumor cells [11]. Neoantigens are solely
produced by tumor cells, leading to a targeted T-cell response against the
tumor with minimal “off-target” damage [12]. In recent years, the focus of
tumor vaccination has shifted towards CVs targeting neoantigens. Promising

— SECTION 6 —
results have been observed in recent clinical trials exploring neoantigen
vaccinations, with improved patient survival [13]. The widespread use
of next-generation sequencing (NGS) techniques enables the rapid and
cost-effective identification of personalized neoantigens. Additionally, the
development of epitope prediction algorithms for MHC I binding has greatly
facilitated the discovery of potential novel immunogenic epitopes [14]. In
recent years, researchers have expanded the pool of antigens for immuni­
zation by combining common antigens with neoantigens to enhance the

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— SECTION 7 —

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Cancer Vaccination and Challenges, Volume 2
effectiveness of vaccines. For example, the APVAC1/2 vaccines have been
successful in stimulating T-cell responses in the treatment of glioblastoma,
as they contain both common tumor antigens (TAs) and patient-specific
neoantigens [15]. Personalized neoantigen vaccines, when combined with
PD-1 or PD-L1 inhibitors, have also shown promising anti-tumor effects in
early clinical investigations. Tumor-associated antigens (TAAs) are versatile
and can be used in a variety of patients. Initially, TAA-based cancer vaccines
primarily targeted TAAs. However, T cells that recognize TAAs and poten­
tially other autoantigens may be eliminated during development due to
central immunological tolerance in the thymus, which can affect the efficacy
of vaccination [16]. Therefore, TAA-based cancer vaccines need to be potent
enough to “break the tolerance.” Despite years of research on TAAs, clinical
studies of TAA-based cancer vaccines have only achieved limited success, as
shown in Figure 8.1. Additionally, the expression of TAAs in non-cancerous
tissues poses the risk of vaccine-induced autoimmune damage [17].
FIGURE 8.1 Types of CVs.

— SECTION 8 —
8.3 DEVELOPMENT OF AUTOLOGOUS CV
Over the past five decades, autogenous CVs have been extensively studied
in clinical trials as exploratory immunotherapies with the goal of inducing or
enhancing CD8+ cytotoxic T cell lymphocyte (CTL) TA-specific responses
[18]. In the past, numerous pivotal studies and hundreds of autogenous CV

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Autologous Vaccines for Prostate and Pancreatic Cancer
clinical trials were mainly unsuccessful in proving a distinct therapeutic
advantage [19]. Several factors, including but not limited to: [1] insufficient
antigens; [2] a lack of effective adjuvants; [3] insufficient immunogenic plat­
forms; and [4] a lack of CTLs attempting to enter the tumor as a consequence of
immunosuppression caused by a high burden of disease, insufficient immune
wellness, or an immunosuppressive tumor cell microenvironment [20].
Neoantigen-based vaccines are customized treatments for specific
tumors, often focusing on several tumor-associated antigens (TAs) that
are unique to each patient [21]. A biopsy of the tumor is collected for
whole-exome plus RNA sequencing to identify and confirm the presence of
quasi-genetic abnormalities expressed in the tumor, which are then included
in an autologous vaccine. Major histocompatibility class I (MHCI) motif
prediction techniques are used to prioritize mutations after analysis. Ranked
lists of candidate antigens are further refined based on the outcomes of an in
vitro binding assay, where synthesized peptides are tested for their affinity
to a relevant class I leukocyte antigen gene of interest [22]. Neoantigen­
specific T lymphocytes are less likely to be eliminated during the acquisition
of immune self-tolerance compared to T cells that are specific to tumor-
associated antigens (TAAs), as some neoantigens are specific to the tumor.
This enhances their ability to induce potent T cell responses, increases their
immunogenicity, and expands and diversifies the immune responses [23].
Variant mutations can be targeted by neoantigen-based vaccines. These muta­
tions can include frameshift mutations caused by the deletion or insertion of
numerous nucleotides in the genome, as well as single nucleotide variants
resulting from a shift of a single nucleotide from one base to another. The
selected neoantigens can be clonal, present in all tumor cells, or subcloned,
found only in a portion of tumor cells. Both types of neoantigens have an
impact on immunoreactivity [24]. Furthermore, mutations can be catego­
rized based on their effect on tumor development. Driver mutations, which
provide growth advantages selected during tumor evolution, and passenger
mutations, which lack inherent growth benefits, can be utilized to create
customized cancer vaccines [25].

— SECTION 9 —
8.4 TREATMENT PROTOCOL BY CVS
8.4.1 RECENT TREATMENT PROTOCOL FOR PROSTATE CANCER
Due to tissue-specific antigens present only in the prostate gland, prostate
cancer appears as an ideal target for this immunotherapy [26]. Additionally,
the disease often progresses slowly because cells are constantly proliferating

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Cancer Vaccination and Challenges, Volume 2
[27]. This allows enough time for the immune system to respond to the
vaccine, especially considering that repeated vaccinations are required for
the best anti-tumor immunological response [28].
In terms of total cancer incidence, prostate cancer ranks fourth overall
and second among males. In 2020, there was an increase in prostate cancer
diagnoses to over 1.4 million [29]. Age and prognosis variables affect the
initial treatment of patients with prostate cancer. Surgery or radiation treat­
ment can be used to treat individuals with localized cancer when screening
tests are not an option. However, disease recurrence can occur in up to 30%
of patients, which is often indicated by a steadily rising blood level of pros­
tate-specific antigen (PSA) [30]. Several novel hormone treatment strategies
for metastatic or advanced prostate cancer have been authorized for clinical
usage in recent years [31]. Despite hormone treatment’s temporary ability
to stabilize the disease, recurrent prostate cancer inevitably progresses to
metastatic castrate-resistant prostate cancer (mCRPC). Patients with mCRPC
have limited treatment options. Docetaxel was approved in 2004 due to its
demonstrated survival advantage, despite its significant side effects. However,
newer therapies such as cabazitaxel, abiraterone, and enzalutamide, which
have been approved since 2004, are currently only indicated for patients who
have already received docetaxel treatment. These therapies do not address
the need for less toxic treatments in cancer patients without cancer-related
pain who choose to avoid chemotherapy [32]. Furthermore, studies have
shown that enzalutamide and the medication apalutamide can reduce the
incidence of metastases in males with CRPC that has not yet spread to other
parts of the body. Additionally, darolutamide has been shown to prolong the
period of survival without cancer progression in men [33].
Two forms of immunotherapy are being explored for prostate cancer: vaccines
and checkpoint inhibitors. Treatment vaccines are injections that stimulate the
immune system to recognize and fight against tumors [34]. Only Sipuleucel-T,
which received FDA clearance in 2010 for use in patients with asymptomatic or
slightly symptomatic mCRPC, is a cancer immunotherapy vaccine that has been
approved for use in clinical oncology [35]. Immune checkpoint inhibitors (ICIs)
are a type of medication that disable proteins on immune cells, enhancing the
immune system’s ability to eliminate cancer cells [36].

— SECTION 10 —
8.4.1.1 CELL-BASED VACCINE
Autologous (patients’ own tumor cells) or tumor cell lines are used to create
cell-based vaccinations (allogeneic). In this case, a human GM-CSF gene

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Autologous Vaccines for Prostate and Pancreatic Cancer
was transfected into two prostate cancer cells, LNCaP and PC-3, to produce
the granulocyte macrophage colony-stimulating factor-transduced allograft
prostate cancer cell vaccine (GVAX). However, in two clinical phase 3
studies, both when used alone and in combination with docetaxel, GVAX
proved to be ineffective (VITAL-1; VITAL-2) [37].

— SECTION 11 —
8.4.1.2 PEPTIDE-BASED VACCINE
The creation of peptide vaccines requires the use of specific TAA epitope
subunits. Similar to cell-based vaccines, techniques for peptide vaccinations
can be based on either generic TAAs or specific peptide structures. Several
peptide vaccines have undergone testing in early-stage 1/2 clinical studies,
and the preliminary findings are highly positive. For example, a directed
Ras homolog gene family member C (RhoC) elicited a strong and persistent
T cell response in the majority of patients who had undergone radical pros­
tatectomy (NCT03199872) [38]. In the majority of mHNPC patients who
were not selected based on their HLA type, the human telomerase reverse
transcriptase (hTERT) peptide vaccine UV1, in conjunction with GM-CSF,
induced targeted immune responses with manageable side effects. Additional
participants are needed for early-stage therapeutic trials using the B-cell
lymphoma extra-large protein (Bcl-xl) Bcl-xl 42, a peptide fragment, and
42-CAF09b, an adjuvant that enhances immunostimulation [39].

— SECTION 12 —
8.4.1.3 VIRAL/BACTERIAL-BASED VACCINES
PROSTVAC (PSA-TRICOM), a human PSA vaccine, utilizes two distinct
poxvirus vectors (PROSTVAC-Vand -F). These vectors incorporate three Tcell
co-stimulatory molecules (TRICOM) to enhance the immunological response.
However, the promising results observed in a phase 2 study (NCT01322490),
which examined patients receiving PROSTVAC (n = 432) or PROSTVACplus
macrophage colony-stimulating factor (n = 432) compared to placebo (n
= 433), did not carry over to the phase 3 setting. There was no difference
between the two treatment approaches in terms of overall survival (OS) or the
percentage of patients who maintained their health [40].
8.4.2 RECENT TREATMENT PROTOCOL FOR PANCREATIC CANCER
One of the cancers with the highest mortality rates is pancreatic cancer.
In the US, there were 45,750 fatalities and 56,770 diagnoses recorded in

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— SECTION 13 —

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Cancer Vaccination and Challenges, Volume 2
2019 [6]. According to one research, by the year 2030, pancreatic cancer
will surpass breast, prostate, and colon cancers as the leading cause of
cancer-related death in the US [42]. These figures demonstrate the urgent
need for new and innovative treatment options for individuals with this fatal
condition. In fact, several initiatives are underway to alter the course of the
disease and achieve better outcomes, similar to those seen in other cancers.
Chemotherapy remains the primary treatment approach for pancreatic cancer
at all stages of the disease. Based on recent evidence and approvals from
the US Food and Drug Administration (FDA), the three main therapeutic
groups are adjuvant, unresectable or metastatic, and neoadjuvant or induc­
tion treatments [42].Whole-cell vaccinations, peptide-based vaccines, DC
vaccinations, new vaccines (plasmid immunizations, virus-based vaccina­
tions, bacteria vectors, as well as fermentation recombinant vaccines), and
mRNA immunization are all types of cancer vaccines that are now available
on the market. However, these vaccines face significant challenges today
due to the severity of pancreatic cancer (PC), the side effects of chemo­
therapy or radiation, and the weakened immune systems of patients. Some

— SECTION 14 —
of the methods used in cancer vaccination to enhance anti-cancer immunity
include the introduction of tumor antigens (TAs), often in combination with
antigen-presenting cells (APCs) such as dendritic cells (DCs) and other
immunological modulators that can alter the tumor. The main methods used
to treat metastatic PC involve cytotoxic drugs or antibodies that target CD-8+
T cells, which can kill tumor cells or inhibit their reproduction. Nearly all
cancer vaccines work by stimulating tumor-specific CD-8+ cytotoxic T cells
through the injection of MHC class I-restricted peptide epitopes derived from
antigens expressed exclusively on the tumor cells. In a recent multicenter
Phase II study, patients with PC (n=30) who received the peptide cocktail
vaccine OCV-C01 in combination with gemcitabine, the recommended first-
line treatment, had a mean Disease-Free Survival (DFS) of 15.8 months,
which was an improvement over gemcitabine monotherapy [43]. Therefore,
therapeutic strategies that combine chemotherapy and vaccines may increase
the number of melanoma-specific T cells in immunogenic tumors, leading to
more favorable outcomes [44].
Clinical studies have demonstrated the safety and effectiveness of
DC-based vaccinations for prostate immunotherapy in inducing tumor-

— SECTION 15 —
specific immune responses. The clinical results of DC vaccination, a potential
immunological treatment for prostate cancer with metastatic spread, are often
promising. Studies have investigated the effects of a DC vaccine targeting
prostate cancer with metastatic disease. For example, in a stage one pilot

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Autologous Vaccines for Prostate and Pancreatic Cancer
study on the MUC1-peptide DC vaccine in patients with metastatic prostate
cancer, Rong et al. found that the vaccine increased the immune response
to the cancer antigen MUC1 in patients who had metastatic prostate cancer
without causing significant harm [45]. According to Mehrotra et al., a poten­
tial therapeutic approach to prevent prostate cancer metastases by activating
CD8+ T cells involves immunization with peptide-pulsed DCs and the TLR-3
agonist poly-ICLC [46]. Liang et al. investigated the CTL responses induced
by DC immunization for pancreatic cancer using longitudinal evaluation of
therapy responses, and they demonstrated that DC vaccinations can signifi­
cantly increase CTL response and inhibit the migration of cancer cells [47].
The following are some ICIs for pancreatic cancer.

1. Ipilimumab: A fully human anti-CTLA-4 IgG1 monoclonal anti­
   body, received clinical authorization for use in the United States
   and Europe in 2011 [48]. However, the outcomes for patients with
   localized and advanced or metastatic pancreatic adenocarcinoma
   (PAC) have been poor. Ipilimumab therapy in PAC patients has
   also been associated with increased toxicity [49]. Interestingly,

— SECTION 16 —
combination chemotherapy has shown more promising results. In
fact, gemcitabine, a common therapy for advanced PAC, has demon­
strated enhanced immunological response by boosting naive T cell
activation [50].

2. Tremelimumab: In a stage II study (open label), the anti-CTLA-4
   inhibitor tremelimumab, a human IgG2 mAb, was evaluated as a
   monotherapy (NCT02527434) [51]. Patients who met the eligibility
   criteria experienced tumor progression after receiving a 5-FU
   gemcitabine regimen in the cohort of patients with metastatic pancre­
   atic carcinoma. Tremelimumab yielded unsatisfactory results, with
   disease progression observed in 18 out of 20 individuals. However,
   in a phase I trial (NCT00556023), Aglietta et al. assessed the efficacy
   of Tremelimumab in combination with gemcitabine [52].
   Different vaccine therapy for pancreatic cancer:

— SECTION 17 —

1. GVAX:
   Granulocyte-macrophage
   colony
   stimulating
   factor
   (GM-CSF) is a protein expressed by pancreatic cancer cells in the
   irradiation allogeneic whole tumor cell vaccination known as GVAX
   [53]. This process stimulates T cell priming and APC antigen uptake
   [54]. In a phase I study, GVAX was evaluated in patients with local­
   ized PAC after resection but before adjuvant chemoradiotherapy.

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— SECTION 18 —

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Cancer Vaccination and Challenges, Volume 2
The safety and efficacy of GVAX in promoting the development
of anti-tumor immunity were assessed by examining enhanced
delayed-type hypersensitivity reactions to autologous tumors after
immunization [55].

2. Survivin-2B 80-88 (SVN-2B) and KIF20A-66 Peptides: These
   are derived from the tumor-associated antigens (TAAs) kinesin
   family member 20A (KIF20A), which are up-regulated in pancreatic
   adenocarcinoma (PAC). These peptides represent HLA-A24-re­
   stricted cytotoxic T cell epitopes. The epitope peptide KIF20A-66
   was developed as a cancer vaccine. In a phase I/II clinical study,
   patients with metastatic pancreatic cancer who received KIF20A-66
   peptides as second-line therapy showed a better prognosis compared
   to the control group receiving best supportive care after gemcitabine
   chemotherapy failure [56–58].
3. Algenpantucel-L: It is a whole-cell vaccine composed of two
   genetically modified allogeneic pancreatic cell lines (HAPa-1 and
   HAPa-2) that express the murine enzyme (1,3)-galactosyltrans­
   ferase (GT), which is the main barrier to xenotransplantation and the
   cause of hyperacute rejection. Ongoing clinical trials are comparing
   algenpantucel-L to chemotherapy and chemoradiotherapy in indi­
   viduals with borderline resectable and metastasized unresectable

— SECTION 19 —
PAC [59, 60].

4. Mucin 1 (MUC-1): The type I transmembrane protein with
   significant O-linked glycosylation, known as mucin 1 (MUC-1), is
   essential for normal cell signal transduction as well as oncogenic
   signaling that promotes invasion, angiogenesis, and metastasis. In
   solid tumors, including PAC, it is highly expressed in cancerous
   cells [61, 62]. A phase I trial using the MUC-1 peptide in PAC found
   that mucin vaccination is safe and may enhance responses to tumor
   antigens (TAs). A MUC1 peptide-loaded DC immunization used as
   adjunct therapy was shown to be well tolerated and had no harmful
   side effects. Four out of 12 individuals who received the vaccine
   have been observed for more than four years and show no signs of
   recurrence [63].
5. Dendritic Cell Vaccine (DC): Antigen-presenting cells (APCs)
   known as DCs have the capability to activate immature T cells and
   enhance the immune system’s ability to combat tumors [64]. Ex-vivo

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— SECTION 20 —

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Autologous Vaccines for Prostate and Pancreatic Cancer
DCs obtained from patients are cultured with a specific antigen to
generate DC vaccines, which are then activated and matured before
being reintroduced into the patient. In a recent study, researchers once
again administered autologous DCs separated from the peripheral
blood of HLA-A2 positive patients. Three different A2-restricted
peptides, namely survivin, carcinoembryonic antigen (CEA), and
human telomerase reverse transcriptase (hTERT, TERT572Y)
(SRV.A2), were loaded into the DCs. Additionally, Poly-ICLC was
administered intramuscularly (I.M) to the patients [64]. The most
common side effects of the treatment were fatigue and flu-like

— SECTION 21 —
symptoms. Early experiments have shown promising results with
other synthetic peptides derived from pancreatic melanoma antigens
that could potentially be loaded into DCs [65].

6. K-Ras Vaccine: According to the K-Ras vaccine, the K-Ras onco­
   gene was found to be mutated in 95% of people with pancreatic
   cancer. A previous study conducted in 1995 discovered a tempo­
   rary Ras-specific T-cell response in 2 out of 5 patients with PAC
   and a KRAS mutation who were vaccinated with a synthetic Ras
   peptide that matched the K-RAS mutation found in the tumor tissue.
   Surprisingly, individuals with advanced illness who responded to
   the peptide vaccine had a longer overall survival of 4.9 months
   compared to non-responders who had 2 months. Peptide-specific
   immunity was generated in 58% of patients. However, a more recent
   study included patients with localized PAC who had undergone
   adjuvant chemotherapy [66, 67].

— SECTION 22 —
8.5 PIPELINE OF CV
Patients, their families, medical professionals, researchers, and the general
public can easily access information on officially and privately funded clin­
ical studies for various illnesses and conditions through ClinicalTrials.gov,
a web-based resource. Websites provide information on medical research
involving human volunteers. Clinical trials are primarily documented
on ClinicalTrials.gov as interventional studies. In a clinical trial, human
volunteers are randomly assigned to interventions, such as medical products,
behaviors, or surgeries, according to a protocol or plan. The results of these
interventions on biomedical or healthcare outcomes are then evaluated.
Information on the pipeline of many prostate and pancreatic CVs can be
found on the webpage [68] (Tables 8.1 and Table 8.2).

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— SECTION 23 —

PAGE 241

NCT Number,
Out Come
Start Date
Completion References
Phase, and Status
Date
NCT05104515

— SECTION 24 —
NRA
01–11–2021
31–01–2023 [69]
Phase I – R
NCT05010200
NRA
05–01–2022
01–12–2027 [70]
Phase I – R
NCT04701021
NRA
07–02–2021
31–12–2023 [71]
Phase I – R

— SECTION 25 —
NCT04090528
NRA
21–10–2019
01–12–2025 [72]
Phase II – R
NCT03532217
NRA
14–09–2018
25–07–2022 [73]
Phase I – C
NCT03493945

— SECTION 26 —
NRA
01–05–2018
31–12–2023 [74]
Phase I–II – R
NCT03481816
• Castration-resistant metastatic prostate cancer (mCRPC) progresses due
24–07–2018
09–03–2020 [75]
Phase I – C
to low levels of testosterone.
• Vaccinations are being developed to enhance the immune response in
identifying and eliminating cancer cells.
• The effectiveness of ETBX-071, ETBX-061, and ETBX-051 is currently
being evaluated against mCRPC.

— SECTION 27 —
NCT03315871
NRA
20–03–2018
01–12–2023 [76]
Phase II – R
222
TABLE 8.1 Clinical Trial Studies of Different Vaccines for Prostate Cancer, as per USFDA, from 2015 to 2022
Cancer Vaccination and Challenges, Volume 2

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— SECTION 28 —

PAGE 242

NCT Number,
Phase, and Status
Out Come
Start Date
Completion
Date
References
NCT02485964

— SECTION 29 —
NA-C
NCT01706458
Phase II – C
NCT01696877
Phase I-II – C
NCT01487863
Phase II – C
•
•
•
•
•
•
PSA is considered the ideal antigen for the treatment of prostate cancer.
These vaccines have the ability to enhance Langerhans cells, thereby
promoting T-cell activity. They also exhibit antigenicity by containing a
large number of T-cell aptamers and demonstrate high bioavailability in
a single solution.
For individuals who have not responded to hormone therapy, this
treatment is intended to address issues within the body.
Vaccines may help the body mount a potent immune response to
eradicate tumor cells.
Men who will undergo a procedure to remove their cancerous prostate
glands and who have previously received standard hormonal therapy are
administered a single intravenous injection of the drug cyclophospha
mide and the investigational prostate cancer vaccine GVAX.
The study determines the effectiveness and safety of sipuleucel-T in
males with castrate-resistant metastases of prostate cancer.
01–08–2015
20–05–2013
18–01–2013
01–12–2011
01–12–2018
13–08–2020
18–12–2018
01–06–2016
[77]
[78]
[79]
[80]
*R: Recruiting; **C: Completed; *** NRA: Non-recurring; and **** NA: Not applicable.

223
TABLE 8.1 (Continued)
­
Autologous Vaccines for Prostate and Pancreatic Cancer

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— SECTION 30 —

PAGE 243

NCT Number
Outcome Received
Start
End
References
NCT05111353
The safety of the vaccination was assessed by counting the number of patients who
10–Oct–22
31–Dec–27
[81]
Phase I – R
experienced each type of adverse event. Vaccine immunogenicity was assessed by the amount
of neoantigen-specific T cells (both Arm 1 and Arm 2).

— SECTION 31 —
NCT03956056
Vaccine immunogenicity is determined by ELISPOT analysis.
13–Feb–20
21–Jun–23
[82]
Phase I – R
Vaccine immunogenicity is determined by multiparametric flow cytometry.
NCT05013216
The study evaluates the enhanced performance of transformation in interferon (IFN-Î3)
11–Apr–22
01–May–26 [83]
Phase I – R
generating mutant-KRAS-specific CD8 and CD4 T cells, the number of study participants
who experienced adverse effects related to the study drug, and the fold change in interferon-
producing mutant-KRAS-specific CD8 and CD4 T cells at week five.

— SECTION 32 —
NCT05116917
The following statistics are calculated: overall survival (OS), progression-free survival (PFS), 05–Nov–21
01–Dec–24
[84]
Phase II – R
disease control rate (DCR), disease management rates (DCR), EORTC QLQ-C30, and adverse
treatment events.
NCT04117087

The percent change in interferon (IFN)-Î3-producing mutant-KRAS-specific CD8 and CD4 T
29–May–20
01–Jun–24
[85]
Phase I – R
cells, as well as the disease-free survival (DFS) rate, is calculated.

— SECTION 33 —
NCT04810910
Participant numbers for adverse clinical and laboratory events (AEs), relapse-free survival
30–Mar–21
30–Mar–25
[86]
Phase I – R
(RFS), and overall survival (OS) are determined.
NCT04161755
Substance-related toxicity.
11–Nov–19
11–Nov–23
[87]
Phase I – R

— SECTION 34 —
NCT04799431
The progression-free survival (PFS) according to RECIST 1.1, the disease control rate (DCR), 01–Jan–23
01–Jan–28
[88]
Phase I – R
and the overall survival rate (OS) are calculated.
NCT04157127
MTD of DC vaccine, dose limiting toxicities (DLTs) reported, time to recurrence, and overall
03–Aug–20
Nov–25
[89]
Phase I – R
survival

— SECTION 35 —
NCT04627246
Analysis of the carbohydrate antigen 19-9, a tumor marker (CA19-9).
11–Sep–20
Sep–28
[90]
Phase I – R
Immunological response gene and cell analysis, and their link with both immune-related
adverse reactions and recurrence, free existence.
224
TABLE 8.2 Clinical Trial Studies of Different Vaccines for Pancreatic Cancer, as per USFDA, from 2015 to 2022
Cancer Vaccination and Challenges, Volume 2

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— SECTION 36 —

PAGE 244

NCT Number
Outcome Received
Start
End
References
NCT05438667
Time to progression; overall survival; progression-free survival, peak plasma concentration;
07–Jun–22
30–Jun–25
[91]
Early Phase I – R Perivascular TCR-T cell count, maximal plasma concentration (C
), peak timing, and peak
max
value of cytokines (T
).
max

— SECTION 37 —
NCT04111172
Adverse event (AE) frequency | guanylyl cyclase C-specific T-cell response (GCC).
10–Nov–20
Dec–24
[92]
Phase II – R
NCT03953235
Please determine how the immune system reacts to the neoantigens expressed by the
18–Jul–19
Dec–23
[93]
Phase I-II – R
GRT-C903 and GRT-R904 genes.
Phase 1 – Objective response rate (ORR) with RECIST v1.1

Clinical benefit rate (CBR) using RECIST v1.1 and duration of response (DOR) using RECIST
v1.

— SECTION 38 —
NCT04246671
Patients with toxic dose limitations.
10–Aug–20
Jan–26
[94]
NCT04807972
The study includes the patients’ global impressions of severity (PGIS) and the patient global
28–May–21
15–Jun–25
[95]
Phase II – R
impression of changes (PGIC) measures to assess participants’ overall perception of the
changes in their pancreatic cancer symptoms.

— SECTION 39 —
NCT03948763
Dose-limiting side effects, frequency of adverse events, frequency of objective responses,
26–Jun–19
–
[96]
Phase I – C
mutant KRAS-specific T cells, and the proportion of patients who discontinued study therapy
due to an adverse event.
NCT03767582
Participants who experienced study drug-related side effects, and the proportion of patients
12–Dec–19
Mar–23
[97]
Phase I–II – R
who responded to immunotherapy.

— SECTION 40 —
NCT02757391
The level of tetramer-positive T-cell population that develops after T-cell infusion determines
09–Aug–19
02–Oct–20
[98]
Phase
whether an immune response will last, the quantity of intracellular cytokines determines
I – Terminated
whether an immune response will last.
NCT04853017
Evaluate the safety of ELI-002.
04–Oct–21
Dec–24
[99]
Phase I – R
Please find the decrease and clearance rate of circulating tumor DNA (ctDNA).

225
TABLE 8.2 (Continued)
*R: Recruiting; **C: Completed; *** NRA; **** NA: Not applicable.
Autologous Vaccines for Prostate and Pancreatic Cancer

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8.6 FUTURE OF CV
There are currently more treatment options available for prostate and pancre­
atic cancer patients compared to the past. However, the prognosis for these
diseases remains poor and they are falling behind in a rapidly developing
field. In order to address this, changes must be made to the design of clinical
trials, and the participation of patients in these trials should always be consid­
ered. There are still unanswered questions regarding the role of radiotherapy
in cancer treatment and the specific population that will benefit from it.
Additionally, new concerns have been raised regarding resistance to immu­
notherapy and the effectiveness of focused therapy. Nevertheless, progress
is being made, from innovative pharmacological approaches to increased
studies involving elderly patients, those with poor performance status, and
individuals with refractory diseases. These initiatives provide hope that more
effective treatment strategies are within reach [42]. Numerous clinical trials
are currently underway to evaluate the efficacy of immunotherapy in treating
prostate and pancreatic cancer, including the use of immune checkpoint
inhibitors, adoptive cell transfer, and combinations with chemotherapy,
radiotherapy, or other targeted molecular treatments. It is well established
that prostate and pancreatic cancer have a low tumor mutational load and
low immunogenicity. Limited mutation rate hinders neoantigen formation
and release, resulting in a low number of tumor-infiltrating lymphocytes
(TILs). Moreover, the stroma of pancreatic cancer tissue exhibits a highly
desmoplastic nature and harbors a significant population of immunosup­
pressive fibroblasts. Further research should focus on overcoming immu­
notherapy resistance in pancreatic cancer patients by addressing multiple
immune vulnerabilities through combination immunotherapy and cytotoxic
approaches. Additionally, the identification of reliable biomarkers will guide
the selection of optimal treatment strategies to improve patient outcomes.
The integration of diverse therapeutic modalities with immunotherapy holds
promise for patients with pancreatic cancer [6].
Currently, this research is undergoing rigorous testing and evaluation
in clinical studies conducted in Madison, Wisconsin, under the supervision
of the U.S. FDA. McNeel and his colleagues will assess antigen-specific
DNA vaccines and gain insights into the interaction between prostate cancer
and the immune response through the clinical trial process. According to
McNeel, immunotherapies are expected to expand rapidly in the coming
years, potentially including vaccines for the majority of individuals with
prostate cancer. “If we can develop tumor-specific immunizations, prostate

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cancer may become similar to high blood pressure, a chronic condition,”
he said. This approach could potentially lead to earlier interventions and
improved outcomes across the board [41].

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KEYWORDS

• 
  autologous tumor cell-based vaccines
• 
  chronic condition
• 
  cytotoxic T-lymphocytes
• 
  dendritic cell
• 
  immunotherapy resistance
• 
  pancreatic cancer
• 
  prostate cancer
• 
  tumor-infiltrating lymphocytes

— SECTION 44 —
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    Pancreatic Cancer that Can Be Treated with Surgery—Full text view—Clinicaltrials.
    Gov. (n.d.). Retrieved from: https://clinicaltrials.gov/ct2/show/NCT04161755 (accessed
    on 14 March 2024).
88. Neoantigen-Targeted Vaccine Combined with Anti-pd-1 Antibody for Patients with Stage
    IV MMR-p Colon and Pancreatic Ductal Cancer—Full text view—Clinicaltrials. Gov.
    (n.d.). Retrieved from: https://clinicaltrials.gov/ct2/show/NCT04799431 (accessed on
    14 March 2024).
89. Th-1 Dendritic Cell Immunotherapy Plus Standard Chemotherapy for Pancreatic
    Adenocarcinoma—Full text view—Clinicaltrials. Gov. (n.d.). Retrieved from: https://
    clinicaltrials.gov/ct2/show/NCT04157127 (accessed on 14 March 2024).
90. Personalized Vaccine with SOC Chemo Followed by Nivo in Pancreatic Cancer—Full
    text view—Clinicaltrials. Gov. (n.d.). Retrieved from: https://clinicaltrials.gov/ct2/
    show/NCT04627246 (accessed on 14 March 2024).
91. TCR-T Cell Therapy on Advanced Pancreatic Cancer and Other Solid Tumors—Full
    text view—Clinicaltrials. Gov. (n.d.). Retrieved from: https://clinicaltrials.gov/ct2/
    show/NCT05438667 (accessed on 14 March 2024).

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92. A
    Vaccine
    (Ad5.
    F35-hgcc-padre)
    for
    the
    Treatment
    of
    Gastrointestinal
    Adenocarcinoma—Full text view—Clinicaltrials. Gov. (n.d.). Retrieved from: https://
    clinicaltrials.gov/ct2/show/NCT04111172 (accessed on 14 March 2024).
93. A Study of a Personalized Cancer Vaccine Targeting Shared Neoantigens—Full text
    view—Clinicaltrials. Gov. (n.d.). Retrieved from: https://clinicaltrials.gov/ct2/show/
    NCT03953235 (accessed on 14 March 2024).
94. TAEK-VAC-HerBy Vaccine for Brachyury and her2 Expressing Cancer—Full text
    view—Clinicaltrials. Gov. (n.d.). Retrieved from: https://clinicaltrials.gov/ct2/show/
    NCT04246671 (accessed on 14 March 2024).
95. https://classic.clinicaltrials.gov/ct2/show/NCT04807972 (accessed on 14 March 2023).
96. A Study of mRNA-5671/v941 as Monotherapy and in Combination with Pembrolizumab
    (V941-001)—Full text view—Clinical trials. Gov. (n.d.). Retrieved from: https://
    clinicaltrials.gov/ct2/show/NCT03948763 (accessed on 14 March 2024).
97. Trial of Neoadjuvant and Adjuvant Nivolumab and BMS-813160 with or without
    GVAX for Locally Advanced Pancreatic Ductal Adenocarcinomas. – Full text
    view—Clinicaltrials. Gov. (n.d.). Retrieved from: https://clinicaltrials.gov/ct2/show/
    NCT03767582 (accessed on 14 March 2024).
98. Cd8+ t Cell Therapy and Pembrolizumab in Treating Patients with Metastatic
    Gastrointestinal Tumors—Full text view—Clinicaltrials. Gov. (n.d.). Retrieved from:
    https://clinicaltrials.gov/ct2/show/NCT02757391 (accessed on 14 March 2024).
99. A Study of eli-002 in Subjects with KRAS Mutated Pancreatic Ductal Adenocarcinoma
    (PDAC) and Other Solid Tumors—Full text view—Clinicaltrials. Gov. (n.d.). Retrieved
    from: https://clinicaltrials.gov/ct2/show/NCT04853017 (accessed on 14 March 2024).

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Cancer Vaccination and Challenges, Volume 2: Delivery Strategies for Cancer Vaccine and Immunotherapy in
the Management of Various Carcinomas. Rishabha Malviya, Bhupendra Prajapati, and Sonali Sundram (Eds.)
© 2025 Apple Academic Press, Inc. Co-published with CRC Press (Taylor & Francis)

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#

CHAPTER 9
TITLE: Combined Therapy of Cancer with Vaccines and Chemotherapeutic Agents
AUTHORS: Dipa K. Israni and Ramesh K. Goyal
LENGTH: 89,205 characters

#

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CHAPTER 9
Combined Therapy of Cancer with
Vaccines and Chemotherapeutic Agents
DIPA K. ISRANI1 and RAMESH K. GOYAL2
1Department of Pharmacology, L.J. Institute of Pharmacy, LJ University,
Ahmedabad, Gujarat, India
2Department of Pharmacology, Delhi Pharmaceutical Sciences and
Research University (DPSRU), Delhi, India
ABSTRACT
Cancer is the most vulnerable group due to the potential of malignant cells
to evade and exploit the host immune system. According to the WHO,
cancer was the leading cause of death globally in 2020, accounting for
approximately 10 million deaths. Cancer prevention is a crucial public health
concern in the 21st century, as it plays a vital role in the fight against cancer.
Despite advancements in oncogenic drug development, there are still limita­
tions such as early blood clearance, resistance to cytotoxic drugs, toxicity
associated with chemotherapy, and site-specific drug delivery. In recent
years, therapeutic cancer vaccinations have gained significant popularity
with the development of novel treatments for specific oncologic indications.
While cancer immunizations are routinely administered to previously diag­
nosed cancer patients, resistance to single-agent immunotherapy often leads
to treatment failure and toxicity, including neurotoxicity, immunotherapy
resistance, and hyper-progressive disease. Only a small portion of patients
experience long-term benefits. Due to these limitations, a paradigm shift is
necessary in cancer therapy. The growing body of evidence from preclinical
and clinical studies supports the potential of certain chemotherapy drugs
to synergize with vaccines, offering a successful approach known as

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“chemoimmunotherapy” (CIT). The purpose of studying therapeutic cancer
vaccines (CVs) in conjunction with chemotherapy is to improve immune
response, treatment effectiveness, and reduce resistance to single-agent
immunotherapy. In this review, we will highlight the combination of CVs
and chemotherapeutic agents, focusing on the latest combination tactics,
synergistic effects, related mechanisms, and associated processes.

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9.1 INTRODUCTION
Despite significant advances in cancer detection and understanding, typical
treatment options have remained largely unchanged. Surgical resection,
chemotherapy, and radiation therapy are commonly used therapies. However,
in many cases, these treatments prove ineffective and lead to tumor recur­
rence or metastasis, necessitating an alternative approach to treatment
[1]. Conventional anticancer treatment is believed to work by selectively
destroying tumor cells or permanently halting their proliferation. Cytotoxic
medications either disrupt DNA synthesis or cause chemical damage to DNA,
resulting in the death of tumor cells [2]. According to findings in cellular
biology, most chemotherapy drugs currently in use eventually activate the
proapoptotic suicide machinery of cancer cells [3]. Despite the increased
effectiveness and improved survival rates provided by modern treatments,
the side effects and long-term consequences of chemotherapy remain a major
concern for both patients and clinicians. The most dreaded adverse effects for
cancer patients undergoing therapy are chemotherapy-induced nausea and
vomiting (CINV) [4]. Mucositis of the mouth and gastrointestinal tract can
lead to local ulceration and pain, resulting in malnutrition, impaired absorp­
tion, weight loss, anemia, fatigue, and an increased risk of sepsis. Diarrhea
and constipation are other common side effects caused by chemotherapy;
severe diarrhea can be fatal due to dehydration and electrolyte imbalance
[5]. Studies have shown that platinum-based chemotherapies can cause
hypersensitivity reactions. Evidence suggests that chemotherapy can cause
persistent, subclinical musculoskeletal muscle injury, as well as central and
peripheral neurotoxicity that can persist for many years after treatment is
completed, significantly impacting the functional ability and quality of life
of cancer survivors. Chemotherapy-induced side effects include peripheral
neuropathy with many anticancer drugs, such as proteasome and angiogen­
esis inhibitors, vinca alkaloids, taxanes, and platinum-based therapies. Long-
term chemotherapy-induced peripheral neuropathy (CIPN) is associated

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Combined Therapy of Cancer with Vaccines and Chemotherapeutic Agents
with a high rate of morbidity, including depression, ataxia, and sleepless­
ness. Chemotherapy-induced gastrointestinal neuropathy has been linked to
severe gastrointestinal impairment in cancer survivors [5, 6]. Due to adverse
drug reactions, hypersensitivity, toxicities, and relapse in cancer patients
undergoing chemotherapy, new treatment strategies are needed. Recently,
cancer immunotherapy has become a crucial component of cancer treatment.
Immunization options include DNA, peptide, protein, viral, recombinant
vector (including yeast- and bacterial-based), whole tumor cells, and DC.
In summary, cancer immunotherapy is a therapeutic approach that enhances
the immune system’s response to cancer by increasing the number of highly
active cancer T cells capable of lysing tumor cells and curing malignancies
[1]. However, therapeutic efficacy, uncertainty in prognosis, toxicity, and
cost are the primary concerns of immunotherapy. Recent evidence suggests
that combined immunotherapies will be necessary for effective cancer vacci­
nation programs to overcome tumor immune evasion [7].

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9.2 CANCER VACCINES (CVS)
Several cancer medications do not induce long-term immune cells and
instead treat the patient quickly, necessitating the need for this treatment to
be improved so that it elicits robust responses that help prevent a recurrence.
Cancer vaccinations could help to tackle this problem. Cancer therapeutic
vaccinations may target a variety of cancer cell-produced antigens. The
early CVs combined whole cell preparations with an adjuvant or virus to
stimulate a more robust immune response. Numerous potential CV targets
have been identified through recent research. Modern vaccines either target
tumor-specific antigens (TSAs), which are only found in cancerous cells, or
tumor-associated antigens (TAAs), which are present at low levels in both
cancer cells and healthy cells. By employing recently found antigens, such
as brachyury, a stimulator of the epithelial-mesenchymal transition, vaccines
can also target tumor stem cells and interfere with the invasion and meta­
static processes. Along with TAAs, patients can also be protected against
their neoepitopes with tailored therapeutic CVs as a stand-alone treatment or
in conjunction with other medicines (altered antigens generated by a specific
tumor). Effector T cells may eventually get activated as a result and elimi­
nate cancerous tissues. The benefit of CVs is that they can utilize a person’s
complete immune system, producing robust and long-lasting responses [8,
9]. In 1990, the Intravesical BCG vaccine (TheraCys), which was licensed

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Cancer Vaccination and Challenges, Volume 2
by the FDA for the efficacy and prophylaxis of initial or recurrent invasive
non-muscle urothelial carcinoma after transurethral surgical removal, was
the first CV ever compared to CIS DFS of four months and nine days and Ta/
T1 DFS of 10 months and five days. TheraCys extended DFS to 30 months
in bladder carcinoma in situ (CIS) patients and 22 months and five days in
patients with urothelial carcinoma [10].

— SECTION 7 —
9.2.1 DNA IMMUNIZATION
Vaccines utilizing DNA are typically bacterial plasmids with tumor antigen
(TA)-encoding genes attached to Cytomegalovirus promoters. These vaccines
aim to elicit a non-specific immunogenic response that complements the
adaptive immunogenic response to cancer containing these antigens. Conse­
quently, DNA vaccines aim to enhance the generation of T lymphocytes,
such as CD8+ and CD4+ cells, that specifically target tumors [11]. Another
objective of DNA vaccines is to overcome immunological tolerance and
induce immune memory, particularly when DNA immunization is employed
to stimulate T cells against an antigen to enhance the efficacy of other thera­
peutic approaches [12].

— SECTION 8 —
9.2.2 PEPTIDE IMMUNIZATION
Vaccines made from peptides are not the genetic components of the disease
being treated, such as a tumor or an infectious pathogen, and are designed
to induce an immune response [13]. Peptide immunization does not over­
burden the immune system since only the epitope of interest is administered
to the individual’s immune system. These vaccinations are cost-effective
and easy to manufacture. However, due to the presence of immunosup­
pressive chemicals in the tumor microenvironment (TME), they often fail
to trigger an immune response [14], and the effectiveness of peptide-based
vaccines when combined with adjuvants is unknown. Therefore, the safety
of adjuvant use should be evaluated and ensured to guarantee the efficiency
of peptide-based vaccines. IMLYGIC or T-VEC, an FDA-approved peptide­
based vaccine that is not strictly peptide-based but can be considered as one,
is a modified herpes simplex virus-1 that replicates in the tumors of recurrent
melanoma patients, causing cellular injury and death. This virus is classi­
fied as a peptide vaccine because it is engineered to express a granulocyte-
macrophage colony-stimulating factor, which enhances the anti-tumor

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Combined Therapy of Cancer with Vaccines and Chemotherapeutic Agents
response and T-cell priming [15]. T-VEC was an FDA-approved genetically
modified oncolytic viral melanoma therapy for metastatic disease in 2015
[16]. T-VEC, a type of therapeutic tumor vaccine, belongs to a novel class
of medicines that utilizes a genetically engineered, GM-CSF-expressing live
attenuated herpes virus. T-VEC has a dual mechanism of action, allowing
it to induce both local and systemic immune responses. It is administered
via injection into unresectable, nodal, subcutaneous, or cutaneous tumors in
melanoma patients with recurrent disease, where it acts as a local cytotoxic
agent by causing viral replication and cell death. GM-CSF produced during
viral replication promotes T-cell priming by antigen-presenting cells (APCs)
presenting tumor antigens (TAs) generated after tumor rupture caused by the
virus. Tumor-antigen-loaded dendritic cells (DCs) then move systemically
and elicit a distant immunogenic response, even though the immune reac­
tions in the injected tumor are higher than those in distant metastases [17].

— SECTION 10 —
9.2.3 DC IMMUNIZATION
In 1973, Robert Steinman first described dendritic cells (DCs) due to their
high MHC expression and diverse form. DC-based vaccines are produced by
collecting patient DCs and ex vivo transfecting them with the target antigen
(TA); these cells are then reintroduced into individuals with cancer, which
can be done through various routes such as intralymphatic, intravenous,
intranodal, and intradermal routes. DCs readily recognize and destroy tumor
cells. DCs have the ability to influence adaptive immunological responses,
making them effective stimulators of memory and immune cells. Addition­
ally, DCs activate naive T cells. However, due to the limited number of DCs
in peripheral blood and tissues, their population must be increased in vivo
or ex vivo prior to vaccine administration [18]. The advantages of DC-based
vaccines include their personalized nature, the ease of inducing an immune
response as DCs are primarily responsible for initiating adaptive responses,
the ability to tailor a response to a specific antigen or set of antigens, and
their relative safety as they do not cause adverse effects [19]. Sipuleucel T,
also known as PROVENGE, is an FDA-approved DC-based cancer vaccine
used to treat castration-resistant prostate cancer that has spread beyond the
prostate gland and is unresponsive to hormone therapy. While DC-based
vaccines for other types of cancer have not yet been licensed, current efforts
are focused on prostate cancer, metastatic melanoma, and breast cancer
[20]. Sipuleucel-T (PROVENGE) cellular immunotherapy is utilized for the

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treatment of asymptomatic or mildly symptomatic mCRPC. This therapy
involves the extraction of autologous peripheral blood mononuclear cells,
including APCs, through leukapheresis. These cells are then expanded using
a fusion product of TA PAP called PA2024 and GM-CSF, which activates
the APCs. During a 40-hour incubation period, the recombinant antigen is
transformed into peptides by the APCs, affecting Major Histocompatibility
Complex signaling and T-cell stimulation. Each dose of the vaccine contains
at least 50 million activated autologous CD541 cells (also known as ICAM-1
cells) with PAP-GMCSF [15].

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9.2.4 WHOLE CELL CANCER IMMUNIZATION
Whole-cell CVs are composed of whole tumor cells that have been modified
ex vivo. Despite the lack of significant results in CVs, whole-cell CVs offer
several advantages. They have the advantage of expressing both tumor-
associated and tumor-specific antigens. Additionally, whole-cell CVs elicit
both cellular and humoral immune responses [15].
9.3 FACTORS THAT INFLUENCE TUMOR IMMUNOTHERAPY
Many factors impact the effectiveness of tumor immunotherapy. First, it is
linked to humoral immunity, which is connected to inheritances and internal
microbiota [21, 22]. Second, it is related to malignant cells. Patients with a
high number of clonal sources of neoantigens and minimal tumor neoantigen
intra-tumor heterogeneity benefit more from treatment. Other factors that
significantly impact therapeutic outcomes include tumor mutation burden and
the target of tumor cell mutation. Third, environmental elements such as daily
routine, food, bacterial illness, and medication usage are associated with it [23].

— SECTION 13 —
9.3.1 IMMUNOTHERAPY DRUGS RESISTANCE MECHANISMS
Cancer immunity encompasses the observation, balance, and evasion of
immune cells [24]. Tumor cells have the ability to evade immune recognition
and attack by interacting with immune regulatory cells, downregulating the
expression of tumor antigens, producing immunosuppressive substances, and
employing other mechanisms to evade the host immune response, allowing
them to proliferate and eventually form visible tumor lesions. Furthermore,
tumor development can be induced by deliberately upregulating proteins that
prevent immune detection, such as Programmed death-ligand 1 (PD-L1),

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arachidonic acid lipoxygenase, and Indoleamine 2,3-dioxygenases (IDO-1
and IDO-2) [23].

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9.3.2 PRIMARY IMMUNOLOGICAL RESISTANCE MECHANISMS
Tumor-specific T cells produce interferon (IFN), which detects tumor cells
and associated antibodies in cells that display antigens, having an anticancer
impact on Antigen Presenting Cells (APCs). This result can directly obstruct
the proliferation of tumor cells and cause tumor cell death. It can also attract
additional immune cells, stimulate TA presentation, and increase the produc­
tion of antigen presentation proteins, such as the major histocompatibility
complex (MHC) molecule [25]. If the pathway is altered, programmed death­
ligand one (PD-L1) expression is exposed, and the expression of PD-L1 in
cancer cells increases, inhibiting PD-L1 or Programmed cell death protein
1 (PD-1) immunotherapy. Alternatively, cancerous cells directly express
PD-L1, which interacts with PD-1 on T cell surfaces to inhibit active CTL,
reduce the immunological response, and result in T cell reduction, leading to
primary drug resistance [23].

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9.3.3 THE ACQUIRED IMMUNE RESISTANCE MECHANISMS
Invading lymphocytes from tumors have the ability to unleash a significant
number of IFN-γ mediated cancer cells that express PD-L1 when the body
mounts an anti-cancer immune response. When combined with PD1 from
CTL, PD-L1 can decrease the immunological killing action of effector T
cells on malignancies [23].
9.3.4 INTERNAL AND EXTRINSIC IMMUNOTHERAPY RESISTANCE
MECHANISMS
Internal and extrinsic mechanisms of immunotherapy resistance can be
distinguished. Primary resistance can be caused by alterations in the
antigen survival process mechanism, cancer-related gene expression and
antigen mutagenesis, insufficient HLA expression, and more. Mechanism
modifications in the antigen manufacturing process include MAPK pathway
activation and PI3K pathway augmentation due to persistent WNT/-catenin
signal pathway expression, loss of PTEN expression, or highly expressed
mixed PD-L1 expression. Acquired resistance can result from the loss of IFN
signaling, target antigens, HLA, and T-cell activity. The extrinsic route may

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be influenced by alterations in the immunological checkpoints CTLA-4,
PD-1, or others, T-lymphocyte letdown and phenotypic changes, immuno­
suppressive cell populations such as Regulatory T-cells, myeloid-derived
suppressor cells (MDSCs), and macrophages-II, and tumor microenviron­
mental cytokines and metabolites like CSF-1 [26].

— SECTION 18 —
9.3.5 THE SELF-NEUTRALIZATION MECHANISM OF CANCER CELLS
Programmed cell death protein 1 is highly expressed in the cancerous cells
of certain individuals. However, Immune Checkpoint Inhibitors PD-1/
PD-L1 still have a limited effect. According to research, this condition arises
because these tumor cells express both Programmed death ligand 1 (PDL-1)
and Programmed cell death protein 1 (PD-1). Prior to the action of Immune
Checkpoint Inhibitors (ICIs) PD-1/PD-L1, the interaction between tumor
cells’ Programmed Death Ligand 1 and its Programmed Cell Death Protein
1 leads to the loss of the ICIs PD-1/PD-L1 target. This process is known as
self-neutralization [23].
9.3.6 ANTIGEN-PRESENTING CELLS SELF-NEUTRALIZATION

— SECTION 19 —
MECHANISM
Antigen Presenting Cells decrease the capacity of T cells to transmit signals
of Programmed cell death protein 1 by blocking the binding of Programmed
cell death protein 1 and Programmed death ligand 1 to each other, resulting
in reduced antitumor activity [23].
9.3.7 EXOSOME RELEASE FROM CANCER CELL SUPPRESS
IMMUNITY
Exosomes, which contain the protein programmed death ligand-1 (PD-L1)
and inhibit T cells from entering the tumor, are released by cancer cells
to suppress the immune response [22]. Colorectal cancer, breast cancer,
lung cancer, and metastatic melanoma release exosomes containing
PD-L1. The immune system employs various mechanisms to inhibit
Programmed death ligand 1 (PDL-1) and Programmed cell death protein
1 (PD-1), rendering them ineffective in adhering to the receptor or having
any impact on the receptor site. In summary, a combination of intrinsic
immune system features such as the degree of mutation, cytokine compo­
sition, and external environmental factors like gut flora and pathogenic

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bacteria contribute to the total escape of the immune system, including
programmed death ligand 1 (PDL-1) and programmed cell death protein 1
(PD-1) co-expression [23].

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9.3.8 TREATMENT MEASURES AGAINST TUMOR RESISTANCE
Avoiding tumor cell resistance is challenging throughout the therapy
process, but patients can achieve the most outstanding treatment results
through proper approaches. Firstly, it is crucial to prioritize immunotherapy
for the appropriate group prior to treatment. Factors such as the presence of
PBRM1 gene deficiencies, intestinal flora, as well as the type and quantity
of tumor markers, are all assessed to determine the patient’s eligibility for
therapy. Subsequently, personalized treatment strategies and combination
therapy are designed for each patient to enhance cancer cell sensitivity to
immunotherapy, accelerate tumor cell death, and minimize the development
of drug resistance in tumor cells [23].

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9.4 RATIONALE FOR COMBINING CHEMO- AND
IMMUNOTHERAPIES
Full-dose chemotherapy is known to affect immune system cells that replicate.
In fact, high-dose chemotherapy can induce immunosuppression, and medica­
tions developed for cancer treatment are now primarily used for autoimmune
diseases or to prevent organ transplant rejection [3]. Increasing evidence
suggests that certain chemotherapeutic drugs are more effective against
tumors in immune-competent hosts compared to immunodeficient ones.
Conventional chemotherapy has the potential to enhance the immune system.
Targeted chemotherapeutic drugs, which are designed to target biochemical
pathways essential for tumor cell survival and proliferation, also possess
immunomodulatory properties. Chemotherapeutic agents enhance both the
innate and adaptive arms of the immune system through various mechanisms
[27]. Chemotherapy alone often falls short of curing cancer patients, and
recurrence is common due to clinically undetected micrometastases. Therapies
that can prevent recurrence are actively being explored in combination with
chemotherapy to improve treatment outcomes. Cancer immunotherapy holds
significant potential for treating various malignancies as it aims to enhance the
body’s inherent ability to fight tumors by improving the effectiveness of its
own defense mechanisms. A successful anti-tumor immune response requires
multiple crucial stages, starting from the production of antigens by cancer cells

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and culminating in the destruction of cancer cells [28]. Combining immuno­
therapy and chemotherapy, known as chemoimmunotherapy (CIT), explores
the concept that CIT may have a synergistic effect against certain malignancies,
as depicted in Figure 9.1. However, CIT has not made significant progress
in recent years due to the discovery that several chemotherapy agents often
induce immunosuppression, especially at high doses. The notion that chemo­
therapy and immunotherapy are conflicting paradigms has been disproven by
extensive research on anticancer immune responses, leading to the emergence
of CIT as a new field of cancer research. The distinct immunological reac­
tions induced by immunotherapy have a significant prognostic and predictive
impact on cancer patients undergoing chemotherapy in these combination
systems. By eliminating regulatory T cells (Tregs) and MDSCs, chemotherapy
can reduce immunosuppression [29], while also increasing the susceptibility
of cancer cells to the lethal action of T lymphocytes (CTLs), thereby enabling
more effective immunotherapy. Recently, various immunotherapies such as
immunization, TLR agonists, monoclonal antibodies (mAbs), adoptive immu­
notherapy, and immune checkpoint inhibitors have been proposed in conjunc­
tion with chemotherapy to combat cancer. These immunotherapies may target
different stages of the cancer-immunity cycle. In this chapter, we will discuss
the combined treatment of vaccines and chemotherapeutic agents [30].
FIGURE 9.1 Combined effect of chemotherapy and vaccine on cancer-cell immunity cycle.
Note: Steps 1–7 shows the cancer immunity cycle. Chemotherapeutic agents act on Step 7
where it kills cancer cells, but the antigen that is released from it may cause relapse. Vaccine
activates APCs and DCs to kill cancer cell but may not be effective alone. Combining the
vaccine and chemotherapeutic agent has a synergistic effect as well as it prevents relapse.

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9.4.1 EFFECTS OF CIT ON SYNERGY
One reason tumors grow is their ability to evade immune surveillance.
Based on the results of preclinical and clinical studies, the combination
of immunotherapy and chemotherapy has shown remarkable efficacy.
This includes enhanced anti-cancer activity, inhibition of cancer infil­
tration and metastasis, reversal of drug resistance, and prevention of
recurrence [31].
9.4.2 INCREASED ANTI-TUMOR ACTIVITY
Numerous research studies have validated the significant impact of
synergistic chemotherapy and immunotherapy on tumor burden exten­
sion. As previously mentioned, Doxorubicin treatment increased the
expression of CD80 on mature monocytic myeloid cells, granulocytic
myeloid cells, and DCs. The combination of effective Doxorubicin
dosages and cancer immunotherapies led to the maturation of tumor-
infiltrating APCs, resulting in the formation of a co-stimulatory pheno­
type that induced anticancer T-cell activity in both CT26 and MCA205
tumor models [32].
CVs have been utilized in chemoimmunotherapy, demonstrating the
extensive infiltration of vaccine-induced, multifunctional CD8+ T cells that
synergize with chemotherapy drugs to enhance tumor cell killing. Instead
of a single therapy, the combination treatment of long synthetic peptides
(SLP) and chemotherapeutics effectively reduced tumor cell growth. SLP
vaccinations only caused a temporary decrease in tumor growth in the
majority of animals, with complete tumor elimination observed in 16% of
the mice. Conversely, survival rates were improved by combining chemo­
therapy drugs such as topotecan, carboplatin, Gemcitabine, or Cisplatin.
Particularly, Cisplatin exhibited strong synergy with SLP immunization
in tumor elimination, without the need for a maximum tolerable dose
(MTD). TUNEL analysis of living cells revealed that mice treated with
the vaccine were heavily infiltrated with TNF-producing T cells, which
may be responsible for the beneficial effect. This was further supported
by the fact that the TNF inhibitor, etanercept, completely eliminated this
synergy. Surprisingly, the combination of the two therapies did not cause

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any additional systemic adverse effects. In subsequent research, Paclitaxel
and a safe lipopolysaccharide (LPS) were co-encapsulated to create a
chemo-immune therapeutic nanoparticle delivery system (TLNP) as the
immunostimulant. TLNP-treated mice exhibited a significantly higher
number of activated CD8+ T cells, CD4+ T cells, and DCs compared to
mice treated with Paclitaxel and LPS alone, which correlated with improved
tumor shrinkage. The presence of significant levels of TNF and IL-12, both
critical cytokines released by active APCs, suggested that the combina­
tion treatment successfully promoted the infiltration of macrophages and
DCs into the TME, activating an immunological stimulatory state. Seventy
percentage of TLNP-treated mice with tumors survived 30 days after
therapy, compared to 50% of Paclitaxel-treated mice and 30% of LPS-
treated animals, indicating the restoration of a functionally active state in
the immunosuppressive TME. The robust cancer-specific immunological
response to LPS may be enhanced by paclitaxel-induced tumor-associated
antigens (TAs) via Immunogenic cell death (ICD), thereby enhancing the
destructive impact of paclitaxel [33].

— SECTION 27 —
9.4.3 PREVENTING INVASION AND SPREADING
More than 90% of cancer-related deaths are caused by tumor metastases,
which are the most common type of cancer. Tumor metastases are a highly
complex process characterized by significant tumor heterogeneity. Several
significant risks associated with tumor heterogeneity, such as vascular
endothelial growth factor A (VEGFA), Tumor Growth Factor, matrix metal­
loproteinase (MMP), cancer-associated fibroblasts (CAFs), and Tumor
Associated Macrophages, contribute to tumor invasion and metastasis [34].
Bevacizumab, the initial licensed anti-angiogenic mAb, has shown improved
patient survival when administered in combination with a chemotherapeutic
agent for kidney, colorectal, and metastatic lung malignancies. Preclinical
studies have proposed combined targeting of Tumor Associated Macrophages
(TAMs) and cancer cells as an aggressive approach to reducing metastatic
lung nodules. As previously mentioned, combination therapy has resulted
in a significant reduction in mRNA expression of matrix metalloproteinase
(MMP-9) and VEGFAcompared to monotherapy equivalents. The concurrent
delivery method has effectively limited tumor development and metastasis in
various mouse breast and colon cancer models. Additionally, a prodrug hyal­
uronic acid-PLGLAGG-DOX (HA-Psi-DOX), which is MMP-2 sensitive,

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has demonstrated increased tumor accumulation ability while causing fewer
systemic adverse effects. PD-1 inhibition during HA-Psi-DOX therapy has
led to the recruitment of tumor-infiltrating lymphocytes (TILs) to the cancer
site, inhibiting cancer spread. The robust immune response and cytotoxicity
induced by HA-Psi-DOX treatment are primarily responsible for its high
anti-metastasis impact [25].

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9.4.4 MEDICATION RESISTANCE REVERSAL
Chemotherapy alone may temporarily slow tumor development, but it can
eventually eliminate the chemo-sensitive population. Cancer cells that
develop resistance to chemotherapy drugs often lead to chemotherapy
failure. The overexpression of the protein wingless-type MMTV integration
site (Wnt) family, for instance, is believed to hinder cell lysis and the infiltra­
tion of cancer-promoting immune cells in CAFs [35]. Accidental exposure
to cisplatin nanoparticles (NPs) increases Wnt16 production, resulting in
stromal remodeling and resistance to tumor cell treatment. In this study,
the research team [35] developed a quercetin phosphate prodrug and trans­
formed it into lipid-coated calcium phosphate NPs. Intravenous delivery
of lipid-coated calcium phosphate nanoparticles (LCP NPs) significantly
downregulated Wnt16 expression, leading to a 50% reduction in collagen
compared to the untreated control group. In a stroma-rich bladder cancer
model, it exhibited a synergistic anti-tumor effect when combined with
cisplatin NPs. P-glycoprotein (P-gp) and multidrug resistance-associated
protein (MRP-1) are two examples of ATP-binding cassette (ABC) trans­
porters that play a crucial role in the development of multidrug resistance
(MDR) in cancer therapy. A preclinical study demonstrated that the sequen­
tial administration of TLR3 agonist polylysine-polycytidylic acid (PIC) and
a limited dose of cisplatin inhibited the expression of P-glycoprotein (P-gp)
and MRP-1, thereby enhancing the effectiveness of chemotherapy. CAFs,
tumor-associated macrophages (TAMs), and MDSCs are examples of immu­
nosuppression networks that were suppressed in vivo and were positively
linked to the synergism of the combination treatment as well as a reduction in
unwanted effects [36]. Cytotoxic chemicals react with the electrophilic prop­
erties of glutathione and become inactive as a result. Thus, the high levels
of glutathione and cysteine in fibroblasts promote resistance to antibiotics.
In a related development, it was found that fibroblasts reduce the buildup
of cisplatin in ovarian carcinoma cells by releasing thiols and increasing

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intracellular glutathione levels. Additionally, the scientists found that CD8+
T cells and IFN-γ particularly targeted stromal fibroblasts and altered
their thiol metabolism, significantly affecting the cancer cells’ response to
chemotherapy. Therefore, exploring the interaction between immunotherapy
and chemotherapy may undermine chemotherapeutic resistance and restore
cancer sensitivity to chemotherapy medicines. Furthermore, the combina­
tion with ICIs highlighted the critical relevance of overcoming medication
resistance in patients with mesothelioma in practical practice. The associ­
ated research has shown that treating gemcitabine with immune checkpoint
blockers significantly increased the overall survival (OS) of patients who
had previously been resistant to gemcitabine or anti-PD-1 monotherapy [31].

— SECTION 31 —
9.4.5 AVOIDING RECURRENCE
Due to the frequent occurrence of local recurrence or metastases else­
where, cancer treatment is hindered by tumor reoccurrence after an initial
decline following chemotherapy. Imbalances in co-stimulatory and inhibi­
tory signals in metabolism are directly linked to tumor recurrence and are
associated with a poor prognosis. Adoptive transfer (AT) of tumor-specific
CD4+ T cells after cyclophosphamide (CTX) therapy resulted in a signifi­
cant initial anti-tumor immune response, but it also led to an increase in
inflammatory monocytes (CD11b+Ly6ChiCCR2hi) in mice with advanced
lymphoma [37]. The programmed death protein 1 (PD-1) and its ligand
PD-L1 pathway mediate the induction of functional toleration of anti­
cancer CD4+ effector T cells. These immunosuppressive monocytes hinder
long-term tumor control and enable recurrence. Due to the high rate of
monocyte proliferation, it was predicted that reducing chemotherapy doses
would favor an unrestricted anti-cancer immunogenic response. As a result,
the combination therapy eliminated the suppressive role of inflammatory
monocytes and prevented tumor recurrence. It was also observed that
administering Dacarbazine the day before peptide immunization, along
with IFNα, increased cancer cell death activity. The treatment demonstrated
an increased anti-tumor activity due to the co-production of TNF, IFNα,
and Granzyme B, which reduce late tumor recurrence following CIT [38].
Furthermore, the hypoxia-inducible factor (HIF)-regulated gene network
influences several factors, including chemotherapeutic drug resistance,
tumor metastasis, and recurrence. Based on this, bio-similar core-shell
nanoplatforms were created to deliver catalase and Doxorubicin (mZCD)

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for relieving tumor hypoxia. It has been proven that oxygen production is
critical in downregulating the expression of HIF1 IF1α, which can enhance
the therapeutic benefits of chemotherapy and reduce the expression of
PD-L1. When combined with ICIs co-delivery, the dual suppression of the
Programmed death protein 1 (PD-1) and its ligand PD-L1 induces a robust
immunological reaction and has a superior effect on suppressing cancer
spreading and extending cancer recurrence time [31].

— SECTION 33 —
9.5 COMBINING VACCINES AND CHEMOTHERAPEUTIC AGENTS
The therapeutic effectiveness of standard chemotherapy schedules
primarily depends on the cytotoxicity of cancer cells. Until recently, it was
commonly believed that chemotherapy would inevitably decrease vaccine-
mediated immune responses and anticancer efficacy when used alongside
cancer vaccination. However, there is mounting evidence that several
chemotherapeutic medications possess immunomodulatory properties that
can enhance immunization-mediated anticancer benefits. This synergy
can be achieved through various mechanisms, depending on the specific
chemotherapeutic medication, vaccine, and dosage regimen. Chemothera­
peutic drugs can alter the transcription of tumor-associated antigens, major
histocompatibility complex, and intracellular cell adhesion molecule-1,
thereby modifying the phenotype of tumor cells and making them more
susceptible to immune-mediated attack [39–41]. These drugs can also
induce apoptosis in tumor cells, leading to IL-12-mediated stimulation
of dendritic cells, antigen presentation, and cross-presentation to T cells.

— SECTION 34 —
This results in the stimulation of cytotoxic T-lymphocytes with a more
potent and effective cancer lytic capability. Chemotherapeutic drugs can
also modify immunomodulatory cells such as Regulatory T (Treg) cells
and MDSCs, induce leukopenia, and work in conjunction with vaccina­
tion to enhance effector immune responses to various TAAs. Table 9.1
demonstrates the immunomodulatory effects of chemotherapeutic agents.
Recent research has shown that specific chemotherapy regimens, when
combined with certain cancer vaccinations, may reduce tumor growth
rates in cancer patients. Detailed analyses of the synergistic effects of
cancer chemotherapy and immunotherapy treatments have already been
conducted. Numerous preclinical studies have investigated the impacts of
combining established vaccination platforms with chemotherapy, some of
which have been implemented in clinical settings [42].

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TABLE 9.1 Chemotherapeutic Agents and Their Immunomodulatory Mechanism
Drugs
Mechanism of Action
Activation/Enhancement
Inhibition/Reduction
Cisplatin
CD4+ T cell; effector T-cells;
MDSC; Treg, IL-1; IL-6; IL-8;
NK; CTL; APCs and macro
IL-10; IL-12; TNF-α; TNF-β.
phage; IL-1α; IL-2; TNF-α.
Carboplatin
Macrophage; CTL, IL-1α; IL-2;
IL-10; VEGF; TGF-β; CCL2.
TNF-α; IFN-γ, CD8+ T cell.
Paclitaxel
Macrophage; DC; effector
Treg; MDSC, IL-1; IL-6; IL-8;
T-cells, IL-2; IL-12.
IL-10; IL-12; TNF-α; TNF-β;
TGF-β; VEGF; CCL2; CCL22.
Gemcitabine
NK; CTL; memory T-cells.
MDSC; Treg; macrophage,
TGF-β1.
Docetaxel
CTL; NK; LAK; IFN-α; IL-6;
MDSC; Treg, TNF; TGF-β; IL-1.
GM-CSF; IFN-γ; TNF-α.
Cyclophosphamide CD41 T-cell development, DC
activation.
Treg function

— SECTION 36 —
5-FU
IFN-γ production by intratumoral MDSC
CD81 T cells.
­
9.5.1 CHEMOTHERAPY PRIOR TO VACCINATION
Certain chemotherapeutic medications may exhibit enhanced efficacy when
combined with vaccinations in specific sequences. The underlying cause of
this phenomenon remains unknown. Antimetabolites such as 6-mercapto­
purine, azathioprine, methotrexate, and 5-FU only inhibit proliferation after
lymphocytes have been activated by the antigen. On the other hand, alkyl­
ating agents like CTX and busulfan can inhibit proliferation either before or
after the host’s exposure to an antigen. Clinical trials have demonstrated the
effectiveness of administering CTX and paclitaxel prior to vaccination. There
are several potential mechanisms that could explain this observation. Firstly,
pre-immunotherapy chemotherapy may eliminate enough tumors to alleviate
the inhibitory impact on immune effectors caused by the presence of tumors.
Secondly, by reducing the tumor burden, the effector T cell to tumor ratio
may be improved, enhancing the ability of effector T cells to eliminate the
remaining tumor cells. Thirdly, a decrease in regulatory T cells may poten­
tiate a more robust immune response upon activation. Lastly, chemotherapy
may enhance the production of tumor antigens or modify their expres­
sion, thereby facilitating more effective recognition by immune effectors.
Nowak et al. conducted a study involving gemcitabine in combination with

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immunotherapies to investigate some of these processes [43]. As previously
mentioned, gemcitabine significantly reduces the humoral immunological
response to tumor antigens. However, it does not impede TA-specific cellular
priming, as it promotes TA cross-presentation, T-cell proliferation, and tumor
infiltration. Nowak and his team [43] hypothesized that because gemcitabine
induces apoptosis in tumor cells, the immune system would be exposed to a
large quantity of tumor antigens. While this exposure alone may not initiate
a robust anti-tumor response, it could prime the host immune system for
subsequent adjuvant immunotherapy. The timing of adjuvant treatment is
crucial. Based on the understanding of gemcitabine’s action, Nowak and
his team investigated the synergy between gemcitabine and immunotherapy
(CD40 ligation using FGK45). In mice with established tumors, pretreat­
ment with gemcitabine followed by FGK45 resulted in tumor regression,
while mice treated with FGK45 followed by gemcitabine experienced
faster tumor regrowth compared to those treated with gemcitabine alone.
Several trials using CTX have administered a single intravenous dose prior
to vaccination, prioritizing the immunomodulatory impact, particularly the
suppression of suppressor or regulatory T cells, over the anticancer effect.
In these trials, patients who received CTX before immunization showed
higher levels of peripheral blood cytotoxic T lymphocyte (CTL) precursor
survival compared to those who received the vaccine alone. Intravenous
CTX (or intraperitoneal in animal trials) appears to be more effective than
oral medication, although not all researchers have observed a benefit from
CTX pretreatment [44]. Studies with doxorubicin, melphalan, paclitaxel,
and 5-FU have also demonstrated that administering the chemotherapeutic
before immunization increases the immunogenicity (particularly CD8+
T-cell responses) of various vaccines, while others have found that doxoru­
bicin is more effective when given after immunization. Chemotherapeutics
are typically administered between 1 and 3 days before vaccination. The
identification of the timing of medicine and immunotherapy poses a chal­
lenge when analyzing the function of CTX and potentially other chemo­
therapeutic drugs. In Berd’s research, the first and second vaccinations were
preceded by low-dose CTX therapy. This therapy utilized autologous tumor
cells modified with the hapten dinitrophenyl (DNP) as a vaccine, which
was administered with bacilli Calmette-Guérin as an adjuvant [45]. Prior to
the research immunizations, a delayed tumor hypersensitivity skin test was
conducted using autologous tumor cells modified with DNP. Notably, it was
observed that patients who underwent baseline skin testing 3 to 8 days before
CTX administration exhibited significantly larger DTH responses and had a

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higher five-year overall survival rate compared to patients who underwent
baseline skin testing on the day of or the day after CTX administration.
The hypothesis was that administering the DTH test would stimulate the
surge of regulatory T cells, which would then be eliminated by subsequent
CTX administration. This elimination would allow for the expansion of
effector T cells without inhibition by regulatory T cells three days later when
immunized with the intended vaccine. In summary, our findings demonstrate
that pretreatment with chemotherapy, particularly CTX and paclitaxel,
enhances the immunogenicity of future immunizations. The activity of
CTX is believed to be related to the regulation of suppressor or regulatory
cells, although the mechanism of action for other drugs remains unknown.
Additionally, it cannot be completely ruled out that tumor elimination by
the chemotherapeutic agent may lead to antigen release and subsequent
formation of an immune response against the released antigens. Neverthe­
less, the authors suggest that clinical studies combining chemotherapy and
vaccinations should include immunizations administered in close proximity
to chemotherapy treatment (1–3 days) [46].

— SECTION 39 —
9.5.2 VACCINE PLUS CYCLOPHOSPHAMIDE AND ITS COMBINATIONS
A variety of malignancies are treated using alkylating medications, such as
CTX. These compounds have applications in cardiovascular-based treat­
ment regimens due to their immunomodulatory properties in addition to
their direct cytotoxic effects on malignant cells through DNA alkylation.
Studies have demonstrated that CTX increases the expression of HLA and
cytokine release in tumor cells, leading to enhanced maturation of dendritic
cells (DCs) and improved cytotoxic T lymphocyte (CTL) killing. The direct
effects of CTX on DCs and other components of the host immune system are
well-documented. For instance, CTX treatment enhances the lytic capacity
of CD8+ T cells. The role of regulatory T cells (Tregs) is crucial in tumor
immune tolerance, as evidenced by emerging data, as they significantly
impede the generation of effective anti-tumor immune responses in cancer
patients. It has been discovered that CTX can reverse Treg suppression,
enabling the stimulation of vaccine-induced solid immunity. In an experi­
mental melanoma model, systemic CTX combined with DC vaccination
resulted in increased anticancer effects. In the treatment of metastatic breast
cancer, a study investigated the administration of 72 metronomic doses of
CTX in conjunction with a sialyl-Tn-keyhole limpet hemocyanin vaccine

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Combined Therapy of Cancer with Vaccines and Chemotherapeutic Agents
(THERATOPER, Biomira) [47]. CTX selectively abrogates Tregs, reduces
residual Treg function, enhances DC maturation, and boosts memory T-cell
responses, making it a promising option for cancer immunotherapy. Addi­
tionally, CTX is utilized as an angiogenesis inhibitor. These effects of CTX
have been observed even at dosages lower than those required for cytotox­
icity. In mouse models of melanoma, mesothelioma, and prostate cancer,
low-dose CTX combined with DC vaccinations (GMCSF-secreting vaccine)
significantly reduced tumor size compared to vaccines alone. Pretreatment
with a lower dose of CTX has been well received in recent clinical studies.
It has been observed that this approach enhances the immunogenicity of
peptide CVs while significantly reducing the concentration of Treg cells
[48]. Another study demonstrated that the immune response to whole tumor
cell vaccination in a preclinical model of breast cancer can be enhanced by
carefully timing the administration of CTX, doxorubicin, and paclitaxel.
In this particular case, the combined anti-cancer effects of vaccination and
chemotherapy were attributed to the improved effectiveness of the vaccine
rather than the direct apoptotic impact of chemotherapy on cancer cells. A
recent investigation focused on the interaction between CTX, doxorubicin,
and a GM-CSF producing HER2/neu-expressing whole tumor cell vaccine
in patients with metastatic breast cancer [42, 49].

— SECTION 41 —
9.5.3 VACCINES PLUS GEMCITABINE
Gemcitabine was observed to increase immune-supportive macrophages M1
and circulating CD4 and CD8 T cells while reducing MDSCs and Regulatory
T-cells (MDSC and Tregs) in preclinical studies. These results were obtained
from patients with ovarian cancer participating in a phase I and phase II
study that included gemcitabine, Pegintron, and a p53 synthetic long peptide
(SLP) vaccination. However, the effects of the vaccination in combination
with gemcitabine on outcomes have been inconclusive. Algenpantucel-L
combined with gemcitabine and 5-fluorouracil-based conventional adjuvant
chemoradiotherapy resulted in phase II research with a 12-month Progressive
Free Survival (PSF) of 62% and an OS rate of 86% for surgically removed
pancreatic cancer [50]. Phase III research in metastatic pancreatic cancer that
randomly assigned patients to either concurrent chemotherapy plus immu­
nization, concurrent chemotherapy plus telomerase vaccination (GV1001),
or chemotherapy alone in a 1:1:1 ratio did not achieve its primary OS goal,

— SECTION 42 —
even though these results were favorable compared to historical data [10].

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— SECTION 43 —
9.5.4 VACCINES PLUS PACLITAXEL AND DOCETAXEL
One of the most popular chemotherapy drugs for cancer is taxanes, which
have been used to treat numerous cancers, such as lung, prostate, and breast
carcinomas. It has been shown that docetaxel decreases MDSCs and Tregs
while increasing CTL responses. Recombinant viral vaccines were admin­
istered to tumor-bearing mouse models, followed by docetaxel therapy, to
enhance vaccine-induced T-cell responses to both vaccine-delivered antigens
and tumor-derived antigens [49]. Docetaxel reduces tumor size in mouse
models and is dependent on antigen-specific CD8 T-cells [51]. Additionally,
other preclinical results indicate that docetaxel may increase calreticulin
(CRT) production in tumor cells, thereby enhancing their susceptibility to
CD8 T-cell-mediated cytotoxicity. Findings from a clinical trial on docetaxel
and PANVAC vaccination as a combined treatment for breast cancer have
demonstrated positive clinical outcomes. When combined with a DNA
vaccination at a low dose, paclitaxel, another taxane medication, increased
the CD8+ T cell/Treg ratio more than when the vaccine was used alone.
Patients who received docetaxel alone also exhibited lymphocyte responsive­
ness to tumor-associated antigens, indicating the drug’s proposed impact on
immunity. Docetaxel and other vaccinations in prostate cancer are currently
undergoing evaluation in multiple active studies. These include the treatment
of castration-sensitive prostate cancer with PROSTVAC and docetaxel (phase
II; NCT02649855), as well as the treatment of metastatic castration-resistant
disease with docetaxel and DCVAC/PCa (phase III; NCT02111577) [10].
It has been demonstrated that paclitaxel increases the expression of antigen
processing and presenting machinery (APM) proteins, such as calmodulin,
low molecular mass polypeptides 2 and 7, and transporter 1. This raises the
possibility of greater recognition by cytotoxic T lymphocytes (CTL) [52].
Similarly, treatment with sublethal doses of paclitaxel has been shown
to elevate the expression of ICAM-1 on NK cells, thereby promoting NK
cell-mediated lysis [53]. Furthermore, taxanes can directly influence various
aspects of the host immune system, thereby impacting tumor immunogenicity.
For instance, they can enhance macrophage activation and stimulate the
intra-tumor production of inflammatory cytokines, leading to enhanced tumor
lysis. It has been demonstrated that sublethal doses of paclitaxel improve
IL-12-dependent antigen presentation by dendritic cells (DCs), accompanied
by higher expression of APM components and improved costimulation.
Additionally, prior treatment of paclitaxel in tumor cells exposed to DCs has
been observed to result in the production of CD8+ T cells with a stronger lytic
capacity [52]. Increasing data indicate that the short window of T-lymphocyte

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recovery after lymphopenia associated with chemotherapy presents a unique
opportunity to enhance the effectiveness of anti-tumor immunotherapy.
For example, Docetaxel has been shown to modify the subsets of T, B, and
NK cells, as well as to enhance CD8+ activity while eliminating Tregs. In
preclinical experiments conducted on CEA-Tg mice implanted with CEA+
tumor cells, a combination of Docetaxel with an rV/F-CEA/TRICOM vacci­
nation regimen demonstrated improved anti-tumor efficacy compared to the
responses elicited by the vaccine or Docetaxel alone. Following immuniza­
tion, Docetaxel significantly increased immune responses to recombinant
viral vaccinations, including antigen-specific T cell responses to both the
Tumor-associated antigen (TAA) supplied by the vaccination and to cascade
antigens generated by the tumor. Docetaxel has been or is currently undergoing
combined assessment with various platforms for cancer vaccination, such as
– (i) a vaccination producing PSA and B7.1 (rV-PSA/B7.1; PROSTVACR);
(ii) a prime/boost vaccination employing vaccinia and fowlpox viruses that
express MUC-1, CEA, and TRICOM (PANVACR); and (iii) the DC vaccine
Provenge R (Dendreon Corp.) [10, 42, 49].

— SECTION 45 —
9.5.5 VACCINE AND IRINOTECAN
Irinotecan inhibits DNA repair and induces a complex immune response
involving the activation of tumor-suppressor proteins, as well as the enhance­
ment of tumor immunosurveillance by Natural Killer (NK) cells and acti­
vated CD8 T-cells. The combination of G17DT (a vaccine that combines the
N-terminus of the growth factor gastrin with a diphtheroid toxin to stimulate
the production of anti-gastrin 17 antibodies) with irinotecan was investigated
in a phase II multicenter study involving metastatic colorectal cancer patients
who had progressed on irinotecan. Among the 161 patients, a partial response
was observed in 3%, stable disease in 32%, and progressive disease in 65% of
the treated individuals. Adverse reactions were comparable to those of irino­
tecan monotherapy, except for more severe injection site reactions reported by
52% of patients. Anti-gastrin 17 antibodies were detected in 62% of patients
and were associated with a survival advantage [10].

— SECTION 46 —
9.5.6 VACCINE PLUS PLATINUM-BASED CHEMOTHERAPY
In preclinical investigations, the combination of carboplatin and paclitaxel
demonstrates immunomodulatory anti-tumor effects. A recent study discov­
ered that the combination of carboplatin, paclitaxel, and HPV16 peptide

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vaccination improved survival in mouse tumor models, which was associ­
ated with a decrease in the frequency of myeloid cells in both the tumor
and peripheral circulation. Additionally, carboplatin and paclitaxel together
enhanced ex vivo T-cell activity for antigen recall. In a phase IIb/III study
for stage IV NSCLC, TG4010 (modified Ankara vaccination expressing
MUC-1 and IL-2) supplemented with platinum-based chemotherapy showed
modest benefits. Patients were randomly assigned to either TG4010 plus
chemotherapy or placebo plus chemotherapy. The combination group
exhibited a longer median progression-free survival and more confirmed
responses. Surprisingly, the TG4010 group experienced delayed reactions
as well as longer-lasting responses [10]. When administered after vaccina­
tion, certain cytotoxic drugs such as doxorubicin and cisplatin enhance the
cytotoxic impact of vaccine-generated CTLs, likely due to their modification
of the immunogenic phenotypes of cancer cells. These medications render
tumor-reactive T cells more susceptible to them and permeabilize cancer cell
membranes to granzyme B, an enzyme produced by CTLs [49].

— SECTION 47 —
9.5.7 VACCINE PLUS DOXORUBICIN
DNA-intercalating medications known as anthracyclines, such as doxoru­
bicin, have been associated with a decrease in the occurrence of various types
of carcinomas, including lung, bladder, breast, and ovarian. Unlike other
DNA-damaging medications, doxorubicin induces the rapid translocation of
ERp57 and CRT to the cellular membrane, leading to caspase-dependent
immunomodulatory cell death, effectively killing cancer cells when admin­
istered in lethal doses. Noncytotoxic doses of doxorubicin promote antigen
presentation by DCs in an IL-12-dependent manner, which is linked to the
regulation of APM components and increased activity of effector T-cells
[42]. One study reported that durable CD8+ T-cells were enhanced by a
combination of limited doxorubicin, INF-α, and autologous DC immuniza­
tion treatment. Furthermore, it was observed that one out of the five animals
that survived for 16 months without cancer after the recorded remission of
recurrent illness had a median overall survival of 109 days [54].

— SECTION 48 —
9.5.8 VACCINE PLUS 5-FLUOROURACIL, GEMCITABINE, AND
METHOTREXATE
A variety of malignancies, including pancreatic and colon cancers, as well
as HNSCC, can be treated using antimetabolites. These medications have

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demonstrated various immunomodulatory characteristics. Gemcitabine,
for example, can enhance MHC-I expression in cancer cells, leading to
increased susceptibility to cytotoxic T-cell-mediated lysis [42]. A unique
therapeutic approach for advanced pancreatic cancer involves combining
gemcitabine with an oncolytic measles vaccine virus (MeV), resulting in
a mutual enhancement of each component’s efficacy. In comparison to
single-agent therapy, the combination of inadequate doses of both medi­
cations significantly increased cytotoxicity in three tumor cell lines [55].
Similarly, 5-FU therapy of colon cancer cell lines can enhance ICAM-1
and Fas expression, rendering them more susceptible to the lethal actions
of CD8+ T lymphocytes [42]. Geary et al. [56] reported that mice treated
with low-dose 5-fluorouracil and an adenoviral tumor vaccine together had
a 95% survival rate, compared to 7% with Ad5-OVA alone and 30% with
5-fluorouracil alone. Furthermore, the presence of 5-fluorouracil resulted in
increased amounts of OVA-specific CD8+ T cells in the spleens, improving
the immunogenic response to cancer cells while also reducing tumor mass. It
is possible that the ability of 5-FU to reduce MDSC populations is what has
led to the observed therapeutic advantage [56]. Methotrexate, for instance,
has been demonstrated to have direct effects on T-cell cytotoxicity. Anti-
metabolites can enhance DC performance both directly and indirectly. One
study found that direct methotrexate treatment of DCs improved antigen
presentation to T lymphocytes. Overexpression of interleukin-12 (IL-12) and
antigen-processing machinery elements has been associated with stimulation
of DC activity, and improved DC performance has been observed following
contact with gemcitabine-treated cancer cells. Additionally, gemcitabine has
been shown to reduce MDSCs in tumor-bearing rodents without affecting
other immune cell subtypes [42].

— SECTION 49 —
9.6 CHEMO-IMMUNOTHERAPY – MECHANISMS OF ACTION IN
GENERAL
9.6.1 DEBULKING OF TUMORS
Tumor debulking is one of the most significant advantages of cytotoxic
treatment. The main source of immunosuppressive tumor microenvironment
(TME) is tumor cells. Therefore, reducing the bulk of cancer cells decreases
the production of immunosuppressive substances. Additionally, reducing the
bulk of cancer cells decreases the number of cancerous cells that need to be
eliminated by immune cells. This can have negative effects, especially in
tumors with limited infiltration of immunological cells near the TME [27].

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— SECTION 51 —
9.6.2 IMMUNOGENIC CELL DEATH (ICD)
Multiple studies have demonstrated that conventional chemotherapy
enhances immunotherapy and promotes immunogenic cell death (ICD).
Cytotoxic therapy leads to the production and translocation of damage-
associated molecular patterns (DAMPs), which increases the adjuvanticity
of cancer cells [57]. The release of intracellular chemicals such as ATP
enhances antigen-presenting cell (APC) recruitment, while cytoplasmic
annexin A1 from cancer cells interacts with formyl peptide receptor 1 to
facilitate contact between dendritic cells (DCs) and injured cancer cells.
Moreover, heat shock protein 70 (HSP70), heat shock protein 90 (HSP90),
and calreticulin (CRT) exposed from the endoplasmic reticulum assist DCs
in phagocytosing stressed cancer cells. Inflammatory cytokines such as type
I interferon (IFN-I) are released from intracellular DNA and RNA through
the Toll-like receptor 3 (TLR3), cyclic GMP-AMP synthase (cGAS)/stimu­
lator of interferon genes (STING) pathway, and TLR9 receptor. C-C motif
chemokine ligand 2 (CCL2), C-X-C motif chemokine ligand 1 (CXCL1),
and CXCL10 promote T-cell recruitment, while IFN-I and other molecules
like high mobility group box 1 (HMGB1), generated by stressed cancer cells,
facilitate DC maturation and T-cell antigen presentation [27].

— SECTION 52 —
9.6.3 ENHANCEMENT IN THE ANTIGENICITY OF CANCER CELLS
Anthracyclines, CTX, platinum, and taxanes are just a few of the commonly
used cytotoxic medications that target cell cycle progression and eliminate
developing cells. Once tumor cells have perished, antigen-presenting cells
(APCs) take up their contents and deliver tumor neoantigens to immune
cells. Further research has demonstrated that cytotoxic drugs stimulate the
antigen-presenting machinery. Gemcitabine can significantly enhance the
expression of HLAs-A, -B, and -C and alter the repertoire of peptide anti­
gens expressed in HLA class I by increasing β2-microglobulin production
[58]. Topotecan, by activating the NF-κB/IFN-β/MHC-I signaling axis, also
upregulates HLA class I expressions [27].

— SECTION 53 —
9.6.4 DEPLETION OF IMMUNOSUPPRESSIVE CELLS
Cytotoxic treatments, such as gemcitabine, 5-fluorouracil, CTX, and
platinum, can reduce MDSCs in both humans and animals. Trabectedin
induces caspase-8-dependent apoptosis to selectively decrease monocytes
and macrophages [59]. Human Treg cells are more susceptible to the effects

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of CTX compared to other immune cells due to their deficiency in the expres­
sion of the CTX generating transporter ABCB1. Additionally, chemotherapy
alters the tumor microenvironment (TME) and promotes the development of
immune cells, thereby enhancing anticancer immunity. For instance, CTX
and doxorubicin facilitate the differentiation of tumor-associated macro-
phages into macrophage 1 [27].

— SECTION 54 —
9.6.5 MODULATION OF GENE EXPRESSION
DNA methylation, histone modification, chromatin remodeling, and the
readout of these changes are examples of epigenetic modifications that
have a significant impact on oncogenesis and are crucial events in various
cancers, such as the elimination of tumor suppressor genes brought on by
DNA methylation. Consequently, epigenetic modulators are a growing class
of anti-tumor medications. In addition to the direct activation of ICD and
enhancement of anti-tumor immunity reported with HDAC inhibitors vori­
nostat and Panobinostat [60], gene expression alteration is another important
mechanism that contributes to the synergy between epigenetic modulators
and immunotherapy. It has been shown that both HDAC and DNMT inhibi­
tors increase the activity of the antigen processing and presentation system.
Epigenetic modulators increase the expression of both HLA class molecules
and tumor-associated antigens. Epigenetic modulators have also been
demonstrated to have an immediate impact on the immune system, poten­
tially increasing anticancer immunity. They can stimulate co-stimulatory
molecules, including CD80, CD86, and ICAM-1, as well as immunological
checkpoints like CTLA4, PD1, and PD-L1. Furthermore, cytokines can be
generated, and epigenetic modulators can enhance immunotherapy response.
Epigenetic modulators can also affect innate immunity. HDAC inhibitors can
activate the activating receptor NKG2D on the surface of NK cells as well as
the stress-inducing ligands MICA and MICB on cancer cells to promote NK
cell destruction of cancer cells [27].

— SECTION 55 —
9.6.6 ENHANCEMENT AND RE-ESTABLISHMENT OF
CHEMOTHERAPY RESPONSIVENESS
Several studies have found that immunotherapy and cytotoxic chemotherapy
are mutually beneficial. Following cancer progression on anti-PD1 treatment,
some patients with chemoresistant malignancies responded to the challenge

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of chemotherapy. After disease progression on immune checkpoint inhibi­
tion, both non-small cell type cancers and Hodgkin’s lymphoma showed
enhanced responsiveness to salvage chemotherapy [27].

— SECTION 56 —
9.6.7 CHEMOTHERAPY’S DISADVANTAGES OF IMMUNOTHERAPY
Lymphodepletion is one of the most detrimental adverse effects of chemo­
therapy on the immune system, with the potential to induce immunosuppres­
sion. Certain immunosuppressive medications used in clinical settings for
the treatment of autoimmune diseases are cytotoxic chemotherapeutic agents
employed to target cancer cells, albeit with different dosages and regimens.
The extent to which chemotherapy-induced lymphodepletion suppresses anti­
cancer immunity remains a subject of debate. Typically, lymphocyte counts
recover following lymphodepletion caused by cancer treatment, allowing the
immune system to “reset.” A study has shown varying degrees of recovery
in different immune cell subpopulations with regards to anticancer immunity
[61]. Additionally, chemotherapy may impact tertiary lymphoid structures
(TLS). TLS are ectopic lymphoid aggregates that form in non-lymphoid
tissues, including cancerous tissues, and resemble secondary lymphoid
organs such as lymph nodes in terms of their anatomy. There is substantial
evidence supporting the notion that TLS attract lymphocytes into tumors,
similar to how lymph nodes do, thereby eliciting localized and systemic
immune responses against malignancies. The presence and density of TLS in
tumors are generally associated with prognosis across various cancer types,
sometimes independent of pathogenic tumor-lymph node-metastasis (TNM)
staging. The lymphodepletion effect of chemotherapy has the potential to
impair TLS, either directly or through related treatments such as corticoste­
roids [27].

— SECTION 57 —
9.7 CONCLUSION
The research discussed here emphasizes advancements in CV development,
as well as the synergies provided by combining cancer vaccinations with
chemotherapy. The combination of chemotherapy and immunotherapy has
been shown to promote cytolysis and reduce cancer cell growth through
various mechanisms. Clinical investigations have demonstrated that
combining chemotherapy and immunotherapy can be more beneficial than
either therapy alone. Further analysis is still underway to evaluate the

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findings of more extensive research on combined CIT. The innate immune
condition of cancer patients will influence their response to chemotherapy;
therefore, the dosage and sequence of combination therapies should be
determined based on preclinical research and clinical viability. When
developing rationale-based CIT pairings, these and other variables must
be taken into account. Extensive investigations in preclinical studies and
on patients have indicated that T cells can be trained to recognize tumor
cells, supporting the idea that vaccination could be used for cancer preven­
tion or recurrence. Vaccination has proven highly effective in reducing the
incidence of malignancies with a microbial origin. However, vaccination
against existing cancers has been largely ineffective. Until recently, it
was widely believed that cytoreductive treatment, when combined with
a CV, would inevitably impair vaccine-mediated immune responses and
anticancer effectiveness. However, recent studies suggest that it may be
possible to enhance vaccine-mediated anticancer effects by utilizing
the immunomodulatory characteristics of cytoreductive regimens. This
synergy can be achieved through various pathways, depending on the
specific cytotoxic chemotherapy and vaccine used, the dosage regimen
for each modality, and other factors. As a result, an increasing body of
clinical and preclinical evidence supports the adoption of a combined
strategy that incorporates immunotherapy and primary chemotherapeutic
medications as the standard approach for treating various types of cancer.
Clinical studies have demonstrated that therapeutic cancer vaccines (CVs)
can be immunogenic, and some have shown efficacy in at least a small
subset of patients. DC therapeutic CVs have been approved for use in
clinical trials, and checkpoint inhibitors in combination with them are well
advanced. Tumors can be heterogeneous and develop clinical resistance to
monotherapies. Therefore, effective anticancer treatments need to target
multiple sites simultaneously. The diversity of the host and the disease are
often associated with limited options and poorer outcomes. Combination
approaches must consider the specific genetic, epigenetic, and complex
nature of cancer. By modifying inhibitory molecules, regulatory immune
cells, and the metabolic resources and demands of T cells, vaccine-activated
T cells can be promoted to be fully functional within the immunosuppres­
sive tumor microenvironment (TME). Identifying reliable biomarkers for
patient selection, standardized measures for toxicity monitoring, and a
comprehensive understanding of the schedule and dosage of combination
treatments will all be crucial for successful therapeutic development and
decision-making.

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— SECTION 60 —
KEYWORDS

• 
  cancer
• 
  chemo-immunotherapy
• 
  chemotherapeutic agents
• 
  decision-making
• 
  immunotherapy
• 
  monotherapies
• 
  therapeutic development
• 
  vaccine

— SECTION 61 —
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Cancer Vaccination and Challenges, Volume 2: Delivery Strategies for Cancer Vaccine and Immunotherapy in
the Management of Various Carcinomas. Rishabha Malviya, Bhupendra Prajapati, and Sonali Sundram (Eds.)
© 2025 Apple Academic Press, Inc. Co-published with CRC Press (Taylor & Francis)

— END OF CHAPTER 9 —

#

CHAPTER 10
TITLE: Role of Various Cancer Vaccine Adjuvants in Cancer Management
AUTHORS: Nazneen Siddique, Himanshu Solanki, Brijesh Parlekar, and Bhupendra Prajapati
LENGTH: 113,905 characters

#

— SECTION 1 —
CHAPTER 10
Role of Various Cancer Vaccine
Adjuvants in Cancer Management
NAZNEEN SIDDIQUE,1 HIMANSHU SOLANKI,1 BRIJESH PARLEKAR,2
and BHUPENDRA PRAJAPATI3
1Department of Pharmaceutics, SSR College of Pharmacy, Silvassa,
UT of Dadra & Nagar Haveli and Daman & Diu, India
2Director, Pro Regulatory Biosciences Ltd., Saskatoon, Saskatchewan,
Canada
3Professor, Department of Pharmaceutics, Shree S.K. Patel College of
Pharmaceutical Education and Research, Ganpat University, Kherva,
Gujarat, India

— SECTION 2 —
ABSTRACT
Basically, cancer is a group of diseases caused by abnormal cells that
grow out of control, surpassing their normal boundaries and invading
nearby body parts or spreading to other organs. Metastasis is one of the
most significant reasons for death in cancer patients. The Food and Drug
Administration (USFDA) has approved the first cancer vaccine (CV) along
with other therapeutic benefits. Patients have indeed been tested for other
CVs that enhance the immune system’s response against cancer. Emerging
vaccine technologies include adjuvants for DNA vaccines, cancer, and
autoimmune vaccines, as well as mucosally administered vaccinations.
Initially, alum was used as an adjuvant, but it had severe local and systemic
adverse effects. However, it was able to generate a good antibody (Th2)
response and was approved for human usage. Adjuvants are incorporated
into vaccine formulations to facilitate potent and long-lasting immune

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responses. Several CV adjuvants exist, including Monophosphoryl lipid A
(MPLA), CpG ODN (which increase the efficiency of antigen-presenting
cells (APCs) and enhance the production of cell-mediated immune
responses), toll-like receptor (TLR) cytokines (which enhance antitumor
effects), and IL-4 and IL-13 (Interleukin) cytokines’ receptors (which
inhibit the development of functional Th1 immunity).
This chapter includes various adjuvants for cardiovascular diseases (CVs),
such as Granulocyte-macrophage colony-stimulating factor (GM-CSF). The
combination of adjuvants, such as a mixture of TLR agonist and α galacto­
sylceramide, shows synergistic effects in CVs. Additionally, certain body
cells, like Dendritic cells (DCs), play a role in boosting the immunity of CVs
as antigen-presenting cells. Polymers like chitin, chitosan, and glycolated
chitosan are also used as adjuvants in CVs to balance the immune response.
Furthermore, the differentiation of adjuvants is related to their substances
and mechanism of action.

— SECTION 4 —
10.1 INTRODUCTION OF ADJUVANTS
In the past, vaccines were primarily developed based on the detection
of antigens (Ag) that induce specific immune responses. The objective
of immunotherapeutic agents is to enhance the antitumor response while
minimizing unintended adverse effects [1]. An immune response is trig­
gered and activated by two components of vaccination: the adjuvant
and the antigen, which are proteins and carbohydrates derived from
the pathogen against which an adaptive immune system is required [2].
Immunology, including the understanding of T and B lymphocyte recep­
tors and their ligands, conjugates, and even peptide vaccines, has been
developed as a result of this strategy, which improves safety, increases
effectiveness, and facilitates manufacturing. With the exception of the
recently approved vaccine that prevents tumors caused by HPV infection,
cancer vaccines (CVs) aim to treat severe disease rather than prevent it.
Developing therapeutic CVs has been challenging because several early
tumor vaccines showed promise in preclinical studies but were not effec­
tive in clinical trials. This chapter provides a list of adjuvants, delivery

— SECTION 5 —
methods, and vaccination innovations that are currently being researched
or authorized [1].
Adjuvants come from the Latin term “adjuvare,” which means to
improve or support. They are small chemicals used in vaccines that work

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Role of Various Cancer Vaccine Adjuvants in Cancer Management
non-specifically to enhance the immune system’s response to certain cell
types. These adjuvants increase the immune response by inducing the
immune system to react to them. There are various types of adjuvants,
including cytokines, non-biological substances, synthesized small mole­
cules, natural products derived from bacteria or other microorganisms,
and biological products. Adjuvants are classified into two categories based
on their predominant modes of action: immunopotentiatory adjuvants
that boost innate immunity by activating pattern-like receptors (PRPs),
specifically toll-like receptors (TLR), and delivery system adjuvants that
promote the uptake of antigens by antigen-presenting cells (APCs) [3].
Emerging vaccine technologies include adjuvants for DNA vaccines,
cancer vaccines, autoimmune vaccines, and mucosally administered vacci­
nations. When used alone, adjuvants have limited toxicity and elicit long-
lasting immunological responses. However, they significantly enhance
the size, breadth, quality, and durability of a specific immune response
to antigens [4]. Table 10.1 provides the classification of adjuvants. The
substances that have adjuvant activities include oil in water emulsion,
natural and synthetic surfactants, mineral gel, and bacterial derivatives. In
live vaccines, endogenous adjuvants are used to stimulate innate immunity
by expressing molecular patterns connected to pathogens (PAMPs). For
example, it has been proven that the innate immune system is activated
by live attenuated yellow fever vaccines (YF-17D) through the signaling
of numerous TLRs. Various mechanisms of adjuvant action are depicted
in Figure 10.2. The incorporation of various TLR agonists such as TLR
type-3, 4, 5, 7, and TLR type-9 has been the most important advancement
in CV adjuvants. Multi-adjuvanted strategies that simultaneously promote
immunity and prevent inhibition will be advantageous for future vaccines
[5]. Macrophages, dendritic cells (DCs), cells generated from myeloid
suppressor, as well as lymphocytes, are shown to be immune cell types
that can either directly or indirectly promote or suppress gastric cancer by
controlling immunological responses [6]. Finally, the researchers provide
a brief overview of adjuvant safety before highlighting some noteworthy
developments in various immunotherapy and a variety of additional treat­
ments involving nanoadjuvants [7]. A list of various adjuvants, along with
examples of adjuvants that have received FDA approval or not and are used
to treat various cancers, their uses, and the manufacturers of each adjuvant,
is shown in Table 10.1.

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270
TABLE 10.1 List of Different Adjuvants, Including Examples of Adjuvants That Have Received FDA Approval or Not and are Utilized in the
Treatment of Different Types of Cancers, Along with Their Respective Uses and Manufacturers
Type of Adjuvants
Adjuvant
Types of
Marketed
FDA Approval Name of
Adverse Events

— SECTION 7 —
References
Cancers
Manufactured Yes/No
Inventor
Dendritic-based
IL-12, cytosine
Prostate cancer Sipuleucel-T
Yes
Dendreon
Fatigue, fever, back pain,
[63]
phosphorothionate
headache, vomiting, anemia,
guanine and MPL.
constipation, chills.
Cytokine-based
Granulocyte-
Prostate cancer, Sipuleucel-T
Yes
Dendreon
Fatigue, fever, back pain,
[73]
macrophage stimulating gastric cancer
headache, vomiting, anemia,
factors.
constipation, chills.
Virus like particle
Major capsid (LI
Cervical cancer, Gardasil
Yes
Merck
Pain, swelling, redness,
[13], www.
protein)
vulvar cancer,
itching, bleeding, headache, gardasil.com
vaginal cancer.
fever, nausea, dizziness.
–

— SECTION 8 —
MPL
Cervical cancer, Cervarix
Yes
Glaxosmith Headache, myalgia, redness, [26, 95], European
breast cancer
Kline
swelling, fatigue.
medicine agency.
com
–
HPV
Cervical cancer –
–
–
–
–
–
HPV + CpG
Genital cancer –
–
–
–
[83]
oligodeoxynucleotides.
Oil in water emulsion. TRL
Not specific

— SECTION 9 —
QS-21,
No
–
Problems with stability
[18, 95]
ISCOMS,
and difficult degradation;
ISCOMATRIX
concern over surfactant
safety.
CPG

— SECTION 10 —
TAL
Non-small cell –
No
–
–
[13]
oligodeoxynucleotides.
lung cancer
(NSCLC).
Cancer Vaccination and Challenges, Volume 2

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TABLE 10.1 (Continued)
Type of Adjuvants
Adjuvant
Types of
Marketed
FDA Approval Name of
Adverse Events

— SECTION 12 —
References
Cancers
Manufactured Yes/No
Inventor
mRNA-based
OVA
Non-small cell
–
No
–
–
[104]
lung cancer

— SECTION 13 —
(NSCLC).
–
TLR4
Breast cancer
Trimix
No
Sheck
Pain or bruising, Temporary [104], https://
Exley
swelling of the penis skin.
healthcare.utah.
Inorganic adjuvant
Silver, calcium
Not specific
–
–
–
In vivo degradation is
[18]
phosphate, silica,
aluminum compounds,
difficult.
zinc compounds, iron
oxide, etc.
Liposomes
Phospholipids
Not specific
–
–
Glenny
Problems with liquid
[18]
and cholesterol;
stability and potential
nonphospholipid
breakdown of unsaturated
amphiphiles.
liposomal lipids.
Polymeric particle
Chitosan, albumin,
Breast cancer
–
–
–
Difficulties to creating
[18, 69]
adjuvants
alginate, fibrinogen,
polymeric particles with
collagen.
predictable and stable
characteristics.
Yeast-derived
GalCer
Lung cancer,
Heplisav-B,
–
–
–
[30, 82]
(glycosphingolipids)
liver cancer.
Engerix-B
Role of Various Cancer Vaccine Adjuvants in Cancer Management

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— SECTION 15 —
10.1.1 CHARACTERISTICS OF ADJUVANTS
There are different types of adjuvants that help boost the immunity of cancer
vaccines to treat various types of cancers. It is important that these adju­
vants are compatible with vaccination and do not affect the diagnostic or
therapeutic effect. Table 10.2 describes the merits and demerits of adjuvants
utilized in cancer vaccines to enhance the immune response.
TABLE 10.2 Merits and Demerits of Adjuvants Used in Various CVs
Merits
Demerits
Reduce the amount of Ag/number of
High cost for formulation.
vaccinations necessary to generate the
optimal immunological response.
Increase effectiveness.
Do not have FDA approval for use as
standalone items.
Greater ease of manufacture.
Amyloidosis, autoimmune arthritis, anterior
uveitis, and other conditions are caused by
hypersensitization of the host tissue.
Improve safety.
Birth abnormalities (teratogenesis), cancer,
and other genetic diseases.
–
Reduced effectiveness of immune response.
To minimize the risk of adjuvant and vaccine component degradation,
it is necessary to assess the adjuvant effects and compatibility. One evalua­
tion focuses on various aspects of mRNA cancer immunotherapy, including
antigen and target selection, vector and adjuvant usage, different adminis­
tration routes, and evaluation of clinical studies. This evaluation aims to
identify the current status and challenges associated with these vaccines [8]
(Figure 10.1).
Some of the main characteristics which are required for evaluation are:

• An effective group is essential for achieving optimal effect and
  stability. Factors such as oil in water, particle size, charge, etc., play
  a significant role in this regard.
• Should be safe when administered inside the body.
• Should be stable.
• Biodegradable and easy to remove once the desired effect has
  diminished.
• To promote Ag-specific immune response.
• Economic to produce.
• Must be economical.

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Role of Various Cancer Vaccine Adjuvants in Cancer Management
FIGURE 10.1 Adjuvant classifies as per used in modern vaccine formulation.
10.2 APCS (ANTIGEN PRESENTING CELLS) ROLE IN T-CELL

— SECTION 17 —
ACTIVATION
Adjuvants primarily provide three functions in cancer vaccination: antigen
provider, APC uptake/activation promoter, and cross-presentation activator
[9]. The effective elicitation of immune responses by adjuvants involves
three steps: antigen delivery, APC activation, and antigen cross-penetration.
By concentrating on lymph nodes (LN) along the lymphatic circulation, the
adjuvant demonstrates a considerable depot impact during the first stage of
antigen delivery, enabling ongoing recruitment and stimulation of APCs at
the site of injection [9]. In consideration, recent contributions to the develop­
ment of effective T-cell and B-cell-based therapeutic cancer vaccines are
emphasized. Researchers focus on adjuvants as the essential element for
manageable APC activation and T-cell priming in T-cell-based vaccina­
tions [10]. T-cell response can also be divided into CD4+ “helper” T-cell
(Th) reactions and CD8+ cytotoxic T lymphocyte (CTL) reactions. CD4+
“helper” T-cell (Th) reactions are vital for secreting accessible immune
regulators such as cytokines that support CD8+ T cell and B cell responses.
Additionally, there are two types of helper T cells, Th1 and Th2, which
promote B cell production, growth, and CTL-mediated destruction. Mean­
while, the secondary structure of B-cell receptors (BCRs) binds antigenic
macromolecules such as proteins and polysaccharides (or, in some cases,
synthetic materials) [11]. Natural killer cells (iNKT), due to their ability to
stimulate various cell types, play a crucial role as a bridge between the innate

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Cancer Vaccination and Challenges, Volume 2
and adaptive immune systems. A potent iNKT cell agonist, the glycolipid­
galactosyl ceramide (GC), induces the release of Th-1 and Th-2 type
cytokines [12]. Enhancing the effectiveness of activated immune responses
can be achieved through hydrogel/magnetic control. In the second step of
APC activation, antigens from vaccines with a particulate adjuvant are more
easily taken up by APCs. The third step, Ag cross penetration, involving
TLR agonists, can promote DC activation, leading to increased exposure of
foreign antigens by MHC I and MHC II molecules (major histocompatibility
complex), thus promoting the development of robust cellular immunity [9].
Bone marrow (BM)-derived progenitor cells undergo rigorous positive and
negative selection in the thymus before leaving the organ and acquiring
specific T-cell receptors (TCR) capable of recognizing foreign peptides
presented by MHC molecules on APCs, as shown in Figure 10.3. The three
signals crucial for T-cell activation by MHC molecules, as shown in Figure

— SECTION 19 —
10.4, are also involved [13]. In addition to the cognate antigens, or signal 1,
costimulatory cues from ligands on the surface of the APC, such as CD80,
CD86, and CD40, as well as pro-inflammatory cytokines (PICs), or signal
2 and 3, are also essential for the effective activation of certain T cells. For
the expression of these chemicals involved in signals 2–3, DCs must be fully
mature (Figures 10.2 and 10.3).
FIGURE 10.2 Various mechanisms of adjuvant action.

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Role of Various Cancer Vaccine Adjuvants in Cancer Management
FIGURE 10.3 Signal 2 activates ligands for specific immune receptors that are gathered
with Signal-1 to activate T-cells. Native CD8 T-cells require Signal-3 engagement to undergo
clonal proliferation and establish signal transduction. The addition of adjuvants to the complex
will increase the provision of both Signals 1 and 2.
Collectively, Signals 1–3 guarantee CD4+ T cell Type 1 immunological
polarization and effective cytotoxic activities by CD8+ T cells (also known
as cytolytic T lymphocytes, or CTLs). In contrast, signal 1 is activated
when signal 2 is not present, and the presence of either immunosuppressive
cytokine (Signal 3) or proinflammatory cytokines (Signal 1) induces type 2
immunity (through Th2 cells) or immunosuppression (via Tregs) [1].
FIGURE 10.4 Distinct signals which come from APCs (Ag presenting cells) for activation
of T-cell.

— SECTION 20 —
10.3 INTRODUCTION OF ALUM
Alum was initially used as an adjuvant due to its safety for human consump­
tion, ability to generate a positive Ab (Th2) response, and lack of local and

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systemic adverse effects [3]. Alum, a salt of aluminum, has a large surface
area and high Ag capacity due to its hydroxyl (OH) and phosphate (PO4)
derivatives. It enhances Ab responses to an immunogen and stimulates
T-helper type 2 responses. The structure, surface area, charge, and adsorp­
tive characteristics of aluminum salts are influenced by various parameters,
making their production challenging. For example, hemagglutinin from the
influenza virus does not adhere well to alum [14].
In modern vaccines, antigens are added to pre-prepared aluminum salts,
known as aluminum-adsorbed vaccines [15]. Alum is a special adjuvant that
has been approved by the USFDA. It significantly enhances antigen persis­
tence, absorption, and local immunostimulatory response [16]. Adjuvanted
aluminum-based substances do not promote greater and more effective Th1/
cytotoxic T-lymphocyte responses; instead, they induce lesser Th2 responses
with the formation of IgM, IgE, and specific cytokines. While some alums
directly act on MHC class-II for Ag presentation, others interact with Ag
to form multimolecular Ag/adjuvant aggregates that facilitate uptake by
APCs. When used in human vaccinations according to current immunization
schedules, all aluminum salt adjuvants are considered safe [17].
Alum, which contains inorganic adjuvants such as aluminum hydroxide
and aluminum phosphate/aluminum hydroxyl phosphate, was identified
by Glenny in 1926 as having an excellent safety record for treating cancer.
Nanoalum, including aloxide nanoparticles (NPs), aluminum oxyhydroxide
particles, and metal oxide particles like calcium phosphate and zinc phos­
phate, are naturally found in the body. Calcium phosphate is used to treat
influenza and herpes simplex virus [9]. Currently, TLR agonists such as
Monophosphoryl lipid A (MPLA) and cytosine-phosphate-guanine (CpG)­
1018 have been incorporated into licensed vaccines for their adjuvant
activity. Additional TLR agonists are being developed for this purpose [18].

— SECTION 21 —
10.3.1 ALUM SUPPRESSES TUMOR GROWTH IN-VIVO
Aluminum adjuvants trigger the Nalp3 system, an intrinsic innate immune
response pathway. In-vitro studies have demonstrated that aluminum particles
from a variety of aluminum adjuvants can agglomerate transforming them
into insoluble particles that are quickly phagocytosed by macrophages and
cause the release of IL-1 and IL-18 [16]. One study was conducted to show
that macrophages can still produce inflammasome-dependent IL-1 while
being inhibited from actin/tubulin polymerization by cytochalasin and IL-1
synthesis by LPS (LPS alum) [19]. A novel approach for enhancing anti-tumor

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Role of Various Cancer Vaccine Adjuvants in Cancer Management
immune responses uses aluminum salts (alum), CpG oligodeoxynucleotides
(ODN), and the innate defense regulator peptide HH2 [16]. Systems based on
NP present a promising approach to efficiently deliver various payloads, such
as adjuvants to enhance therapeutic efficacy, in addition to mRNA. Adjuvant
pulses could increase immune stimulation by restoring innate immune activa­
tion in the presence of these synthesized mRNAs in an anti-tumor vaccination
[20]. The use of alum in human vaccinations has been licensed, however, it
can have harmful local or systemic adverse effects, including myofascitis,
abscesses, and sterile eosinophilia. Luteolin triggered TH1 immunity as
opposed to the effects of the alum adjuvant, that also triggered TH2 immunity
and may have a stronger tumor-killing effect. In contrast to alum, luteolin
boosted the synthesis of IL-12, which reduces IgE expression [21].

— SECTION 23 —
10.3.2 GRAPHENE OXIDE NANOCOMPLEXES WITH ALUMINUM
FUNCTIONALIZATION FOR EFFECTIVE CANCER VACCINATION
Applications of graphene oxide (GO) in biomedicine are extensive due to
its high surface area, superior adsorption capacity, and biocompatible char­
acteristics. GO can be employed as delivery systems for cytosolic drugs,
thanks to its ability to disrupt lipid membranes. GO-Al (OH) nanocomplexes
were prepared using the chemical precipitation method, and their surface
morphology was determined using SEM and EDS. GO-Al (OH)/ovalbumin
vaccine (OVA) formulations were created using a simple combining proce­
dure. These formulations showed improved cellular uptake of Ag in DCs and
a better ability to activate Th1 and Th2 responses [14].
FITC was covalently linked with OVA and subsequently loaded onto GO
and GO-AlO to examine cellular absorption. However, without an adjuvant,
these secure vaccination antigens often have poor immunogenicity. There­
fore, there is a growing interest in creating effective and secure vaccine
adjuvants. The combination of alum and CpG oligodeoxynucleotides
successfully slowed the growth of tumors in mice when used as vaccine
adjuvants [16]. A novel formulation containing sodium chloride and alum
simultaneously improves cellular and humoral immunity [22].

— SECTION 24 —
10.3.3 MONOPHOSPHORYL LIPID A (MPLA) IS INTRODUCED AS AN
ADJUVANT TO CANCER VACCINES
Monophosphoryl lipid (MPL) is an adjuvant made of aluminum that is used
in approved prophylactic vaccinations for contagious diseases. MPL®, a

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metabolite of Salmonella Minnesota lipopolysaccharide (LPS), is a potent
activator of antibody and T-cell responses. MPL is the only TLR ligand
used in approved human vaccinations. In Europe, MPL® is authorized for
use as Pollinex Quattro® for the management of allergies due to its ability
to reduce Th2 reactivity to allergens. GlaxoSmithKline (GSK) Biologicals
is developing multiple MPL® formulations and mixtures (AS01, AS02, and
AS04) as part of clinical studies with vaccines for cancer, HIV, vesicular
stomatitis virus (VSV), leishmania, malaria, TB, and tuberculosis [23].
However, the hydrophobicity of MPLA poses significant challenges in its
advancement as a prophylactic CV. The absence of the antigen in the APCs
prevents MPLA alone from being sufficient to stimulate a unique adap­
tive immune system response to malignancy. To address this, a successful
CV called “vacosome” was created by using a TLR 4 agonist such as
phosphatidylcholine and reconstructing the cancer cell membrane. This
vacosome promoted an antitumor reaction against breast cancer 4T1 cells
in vitro and initiated and improved BM DC maturation [24]. Developing
cancer immunotherapy vaccines that target tumor-associated carbohydrate
antigens (TACAs) specifically can be beneficial. However, their low
immunogenicity presents a significant challenge. To overcome this, the
development of structurally specified completely synthetic glycoconjugate
vaccines using a monophosphorylated version of Neisseria meningitides
lipid A was investigated [25]. Novel therapeutic CVs were created by cova­
lently joining a GM2 derivative to keyhole limpet hemocyanin (KLH) and
MPLA. Furthermore, the self-adjuvant feature of the GM2-MPLA combi­
nation was demonstrated by its ability to trigger potent immune responses
without the use of an adjuvant. The cancerous cell line that expresses GM2,
MCF-7, was subjected to identical complement-dependent cytotoxicity
by the specific antibodies of both conjugates, which demonstrated robust
binding [26]. We determined whether co-administration of a DNA vaccine
with a TLR-4 ligand, MPLA, and natural killer T (NKT) cell ligand –
galactosylceramide (-GalCer) adjuvants might impact the effectiveness
of DNA vaccines against tumors in order to increase the effectiveness of
DNA vaccines targeted against human papillomavirus (HPV) tumors. In
the current research, we proposed that NKT cell antigen-GalCer and MPL
might enhance each other’s anticancer effects in the field of cancer immu­
notherapy. It has been established that concurrent stimulation of several
immunological targets by a mixture of adjuvants may result in producing
an immune response that is more potent and lasts longer against tumors
[27]. In order to examine the manufacture and immunological assessment

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Role of Various Cancer Vaccine Adjuvants in Cancer Management
of an anti-CD205-tailored PLGA-based nanoparticulate CV, ovalbumin
was employed as a model antigen and Monophosphoryl lipid was used.
The adjuvant was enclosed within the NP using a double emulsification
solvent evaporation process [28].

— SECTION 26 —
10.3.4 ALUM-CONTAINING APPROVED HUMAN VACCINES
There are several licensed human vaccines containing alum as shown in
Figure 10.5. Despite their high reactogenicity, pertussis vaccinations that
contained diphtheria and tetanus toxoid proteins along with inactivated
Bordetella pertussis germs first became widely utilized in the 1940s [29].
According to a study on the adjuvant effects of aluminum in pertussis and
HPV vaccines, an acellular form of the pertussis vaccine with lower reac­
togenicity was developed in Japan in 1981 after painstakingly identifying
endotoxin-minimized protein fractions that conferred protective immunity.
These were combined with the toxoids that cause diphtheria and tetanus
and adsorbed on alum. The resulting subunit vaccine, known as diphtheria,
tetanus, pertussis (DTP), was quickly accepted in Japan as a replacement
for DTP, which ultimately reduced reactogenicity and also led to a signifi­
cant decrease in whooping cough cases. MPL, developed by Edgar Rubi in
the 1970s, is derived from the LPS portion of the gram-negative bacterial
cell wall. MPL has been used to modify LPS through acid and base hydro­
lysis to create a detoxified version of “colay’s toxin” for cancer therapy.
Currently, MPL is used in combination with Alum and other adjuvants to
enhance therapy by targeting TLR ligands, and this combination provides
strong evidence in trials of the HPV vaccine [30]. The mechanism of alum
involves the creation of a depot that allows for continuous release of Ag, the
formation of particle structures that promote phagocytosis of Ag by APCs
(DCs, macrophages, and B cells), an increase in MHC class-II expression,
and Ag presentation. Alum enhances adaptive immunity by inducing tissue
damage that activates inflammatory DCs through uric acid. Alum also
exhibits a “depot effect,” resulting in a gradual release of Ag from the
vaccination site [31]. Two prophylactic CVs, Gardasil, and Cervarix, have
been approved for specific types of cancer. These vaccines are produced
through a fermentation process that utilizes salts, carbohydrates, vitamins,
and amino acids as a medium for yeast growth, followed by further physical
or chemical purification [3].

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FIGURE 10.5 Alum is an ingredient in several approved human vaccines that protect
against viral infections that might cause cancer.

— SECTION 28 —
10.3.4.1 DPT VACCINE
It is a monovalent vaccine. Vaccination for pertussis was approved in 1914.
The first instance of alum-precipitated diphtheria toxoid was noted in 1926.
In 1937, a tetanus toxoid with adsorption was first sold [32]. The long-
lasting immune response observed in humans after a natural sickness is not
produced by the current acellular pertussis immunizations [33]. Since 1981,
Japan has employed vaccinations against pertussis, diphtheria, and tetanus
toxoids, precipitated with alum (DTaP) including both initial and booster
vaccinations. The acellular DTaP vaccine, in contrast to the whole cell DPT
vaccination, contains an alum adjuvant to boost the immunogenicity of the
pertussis toxin and filamentous hemagglutinin, as well as the diphtheria
toxoid, tetanus toxoid, and other vaccine components [34]. Contrarily,
TLR-independent adjuvants have been used empirically to boost antibody
responses in alum-based vaccinations, such as the triple vaccine diphtheria,
pertussis, and tetanus (DPT), HPV, and hepatitis vaccines. Aluminum salts
(alum) and their gel forms are examples of such adjuvants [35].

— SECTION 29 —
10.3.4.2 HBV (HEPATITIS B VIRUS)
HBV is particularly important as an etiologic agent in regions with a high
incidence of liver cancer. Chronic hepatitis, liver cirrhosis, and hepatocel­
lular carcinoma (HCC) can all originate from HBV infection. Hepatitis B
and C are the main causes of HCC. Differentiating between an adjuvant and
an enhanced treatment can be challenging. The use of adjuvants can reduce
the frequency of therapy in many lung cancer patients [29]. The adjuvant
in the HBV vaccination is 3-o-desacyl-4-MPLA, aluminum phosphate. The
addition of adjuvants can enhance immunogenicity [36]. In recent studies, a

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— SECTION 30 —

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Role of Various Cancer Vaccine Adjuvants in Cancer Management
new immunostimulatory adjuvant is used in the two-dose, inactivated, yeast-
derived vaccination known as Heplisav-B. Another example of an adjuvant
used for immunostimulation is a recombinant vaccination called Engerix-B,
which contains aluminum [37]. Intravenously administered synthetic-GalCer
(glycosphingolipids) and HBV protein antigen are combined to protect
subjects from HBV [38].

— SECTION 31 —
10.3.4.3 HUMAN PAPILLOMAVIRUS (HPV)
Women who receive the AS04-HPV-16/18 vaccine prior to their first sexual
encounter are afforded over 90% protection against cervical intraepithelial
neoplasia grade 3 or higher, regardless of the HPV type, as indicated by data
from clinical trials and real-world experience [39]. The vaccine does not
have any impact on pathogen removal.
Adjuvant HPV vaccination has the potential to reduce the incidence of
recurrent cervical cancer. Following surgery, a decrease in the occurrence
of vaginal intraepithelial neoplasia (VaIN), vulvar intraepithelial neoplasia
(VIN), and genital warts has been observed with adjuvant HPV vaccination
[40]. Additionally, other adjuvant components include proteins derived from
yeast (HPV L1 produced in yeast cells for large-scale protein production),
polysorbate 80 (an emulsifier), and amorphous aluminum hydroxyphosphate
sulfate (AAHS) [41]. Recent studies have demonstrated that subcutaneous
injection of the human HPV peptide vaccine with CpG oligodeoxynucleo­
tides as an adjuvant can prevent orthotopic genital cancer in mice [42]. Three
distinct vaccines, Cervarix, Gardasil, and Gardasil 9, have shown efficacy in
reducing the prevalence rates of cervical cancer [41].

— SECTION 32 —
10.4 CYTOSINE-PHOSPHODIESTER-GUANINE-OLIGONUCLEOTIDE
(CPG-ODN)
Recombinant immunostimulatory oligonucleotides, known as CpG oligo­
nucleotides (ODN), are specifically designed to activate Toll-like receptors
(TLRs) 9. Subsequent research has identified TLR agonists with improved
safety profiles, with the TLR9 agonist CpG ODN standing out for its ability
to strongly stimulate Th1 immune responses. Furthermore, CpG activation
promotes the expression of co-stimulatory molecules such as class II MHC,
CD80, CD86, and the Fcy receptor in B cells [43]. The interaction of CpG
with TLR9 (TRAF6) triggers a signaling pathway involving the activation

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— SECTION 33 —

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Cancer Vaccination and Challenges, Volume 2
of MyD88, IL-1R-associated kinase (IRAK), and tumor necrosis factor
receptor-associated factor 6. This signaling cascade leads to the activation
of certain mitogen-activated kinases (MAPK) and transcription factors,
including NF-kB and AP-1, resulting in the production of pro-inflammatory
chemokines and cytokines. CpG ODN has been shown to enhance humoral
and cellular immune responses, including Th1 cells and CTL, induced by
vaccines against infections, allergies, and malignancies. Simultaneous
administration of ODN and Ag to the same antigen-presenting cell (APC)
accelerates production, increases the concentration, and prolongs the dura­
tion of the induced immune response [43]. CpG ODNs exhibit adjuvant char­
acteristics that persist in immunosuppressed individuals when administered
mucosally or systemically. Clinical studies have demonstrated the safety
of CpG ODNs and their ability to enhance the effectiveness of concurrent
immunizations [43].

— SECTION 34 —
10.4.1 DELIVERY OF CPG-ODN FROM A CRYOGEL CANCER VACCINE
AT THE RIGHT TIME IS EXHIBITED BY ULTRASOUND-TRIGGERED
RELEASE
For scaffold-based CVs, it remains unclear when to administer adjuvants,
particularly the commonly used CpG-ODN. In order to investigate the
hypothesis that timely administration of CpG-ODN can enhance immune
responses, we developed a cryogel vaccination technique that enables
on-demand release of CpG-ODN through ultrasound (US) stimulation.
Upon US stimulation, a controlled burst release and sustained release of
CpG-ODN were observed, with the latter continuing at an accelerated rate
even after the US was turned off. Four days after vaccination, the cytotoxic
T-lymphocyte (CTL) response to US stimulation in live mice was signifi­
cantly higher compared to the control group. Furthermore, eight days after
immunization, US stimulation resulted in a significantly higher IgG2a/c
antibody titer compared to all other groups, except for those that received US
stimulation. The peak accumulation of DCs in the cryogels coincided with
the US-triggered release, which was optimal. This technology provides a
promising platform for investigating the optimal timing of immunostimula­
tory drug delivery for tumor vaccination, as it allows for periodic control
of vaccine components through on-demand delivery [44]. The development
of a subcutaneous composite system, which utilizes on-demand medication
administration repeatedly activated by ultrasound (US), is proposed. Model

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Role of Various Cancer Vaccine Adjuvants in Cancer Management
drugs (dye) encapsulated in liposomes are in direct contact with gas-filled
microbubbles, allowing for enhanced release triggered by US-induced events
through the use of an in-situ cross-linking hydrogel. In vitro studies demon­
strate negligible cytotoxicity of the composite when administered subcutane­
ously to rats, with only mild tissue reaction observed. Furthermore, in vivo
experiments show that drug release can be triggered by the application of US
two weeks after injection [45].
To facilitate in situ picture-guided melanoma vaccination, a novel blend
of multipurpose nanoadjuvants (M-NA) was synthesized. The M-NAconsists
of an iron oxide/gold core and a cationic polymer shell, with CpG-ODN
electrostatically fused on its surface through multilayer synthesis. Following
intratumoral delivery, the M-NA can be absorbed by antigen-presenting cells
(APCs) and retained in the dense extracellular matrix (ECM) of gliomas for
an extended period. This method holds promise for imaging-guided locally
tailored therapy [46].

— SECTION 36 —
10.5 TOLL-LIKE RECEPTOR (TLR)
Toll-like receptor (TLRs) are epidermal receptors that are primarily expressed
by innate immune cells. They can be divided into TLRs that are expressed
on endosomal membranes and those that are on the cell surface (TLR-1, 2,
4, 5, and TLR-6) and intracellular (TLR-3, 7, 8, and TLR-9). TLR agonists
are gaining significance by employing them as adjuvants in vaccinations
for severe and difficult diseases, including cancer, AIDS, and malaria,
because they can connect the innate and adaptive immune system responses.
TLR4 agonists have undergone testing as immunotherapy against cancer,
anti-allergic medications, and adjuvants in vaccinations against pathogenic
disorders. TLR9 agonists have also been researched for use in antitumor
immunomodulation and as elements of vaccines against infectious illnesses.
It may be easier to understand why TLR4 and TLR9 agonists underwent a
more creative process as vaccine adjuvants if you consider that they were
first identified as such [47]. Additionally, each TLR comprises a transmem­
brane domain, a leucine-rich repeat (LRR) portion for recognizing PAMP/
DAMPs, and a TIR domain for subsequent signaling pathways and inducing
an inflammatory response. TLRs are an excellent target for adjuvants as they
can serve as a “danger” signal to initiate an immune response that promotes
long-lasting defense [47]. TLR agonists can be combined with other adju­
vants, either competitive or non-TLR, to create adjuvants with antitumor

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activity or synergistic effects [48]. TLR3 activation has also gained recent
attention [49]. Polyinosinic-polycytidylic acid stabilized by lysine and
carboxymethylcellulose, known as poly-ICLC (Hiltonol), is one of the TLR
agonists being investigated as a potential adjuvant for CVs. There are several
other TLR agonists, such as Imiquimod and MPLA, a TLR4 agonist [1]. A
three-cohort trial is noteworthy, in which ovarian cancer patients underwent
frequent subcutaneous administrations of overlaying long peptides that could
be delivered individually, mixed in Montanide, or combined with Montanide
and Poly-ICLC. T cells had to be induced by Montanide, and Poly-ICLC
made these responses stronger and more stable [49]. The development
of a vaccine against the tumor-associated antigen mucin 1 (MUC1) is a
significant challenge due to its poor immunogenicity. To enhance immune
responses against MUC1, one study aims to create a three-component drug
by linking a relatively low-molecular toll-like receptor 7 agonist (TLR7a) to
carrier protein BSA via the MUC1 glycopeptide (BSA-MUC1-TLR7a). To
investigate their synergistic effects, we additionally added an Alum adjuvant
to the three-component mixture. According to immunological tests, anti­
MUC1 antibody responses were synergistically boosted by the alum adjuvant
and the built-in TLR7a, and immune responses were biased towards Th1.
Meanwhile, the vaccine candidate’s antibodies successfully detected tumor
cells and led to complement-dependent cytotoxicity [50]. In addition to regu­
lating infection and tissue healing and boosting antitumor effects through the
stimulation and regulation of innate immune responses, mounting evidence
suggests that TLRs also play a role in maintaining tissue homeostasis. TLR
agonists are widely used to increase the effectiveness of numerous cancer
medicines [51]. To demonstrate that TLRs can be used in conjunction with
other adjuvants to enhance the effectiveness of DNA vaccines designed to
prevent HPV infection, the anti-tumor effectiveness of DNA vaccination was
tested in relation to the effects of monophosphoryl lipid A (MPLA), a TLR4
ligand, and the adjuvant galactosylceramide (-GalCer), an NKT cell ligand
[27].

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10.6 IL-4 AND IL-13 CYTOKINES’ RECEPTORS
The Th1/Th2 paradigm has identified IL-4 and IL-13 as the archetypal
Th2-expressed cytokines that counter-regulate Th1 responses necessary
for the effective treatment of numerous intracellular infections. Interleukin
(IL)-4 and IL-13 have been extensively studied for their roles in innate and

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adaptive Th2-mediated immunity; nevertheless, it is still unclear how these
cytokines alter the cellular connections required to prevent the develop­
ment of functional Th1 immunity. HIV-specific vaccine efficacy can be

— SECTION 39 —
significantly increased by IL-4/IL-13 suppressed vaccine methods. When
the wealth of latest discoveries on IL-4 and IL-13 activity at the mucosae
is taken together, IL-4/IL-13 is believed to be a cytokine. The capture
vaccine technique to inhibit cytokine activities has significant potential to
be used against numerous severe mucosal infectious diseases, particularly
those that require protection from high-quality T and B cell immunity. As
a result, cancer trials have suggested that targeting IL-13Ra2 may be an
effective anti-cancer therapy. An immune toxin and an IL-13Ra2-targeted
DNA vaccination have recently been evaluated in mouse tumor models as
a potential treatment for advanced malignancies [52]. Immunosuppressive
Th2 autophagy-restraining cytokines like IL-4 and IL-13 or protective Th1
autophagy-promoting cytokines like IFN are produced by today’s vaccines
and adjuvants. Another feature of TB infection is a low IFN-/IL-4 ratio and
relatively high amounts of Th2 cytokines, which suppress Th1 responses and
hinder efficient protective immunity. A secure and non-toxic immunization or
adjuvant is necessary to reduce the pre-existing subversive Th2 autophagy­
restraining cytokines and increase Th1 autophagy-promoting cytokine (IFN-)
secretion (IL-4 and IL-13) [53]. Myeloid-lymphatic endothelial cell progeni­
tors (M-LECP), a subset of BM-derived cells, display M2-type macrophage
markers. We hypothesize that the immunosuppressive Th2 cytokines IL-4,
IL-13, and IL-10 would promote pro-lymphatic specification of M-LECP
during their formation from BM myeloid progenitors due to their ability to
induce the M2 type in macrophages. By blocking Th2 pathways, immuno­
suppression may be reversed, and this may also prevent the development of
pro-lymphatic progenitors, which eventually aid in the spread of cancer [54].
It is believed that IL-4 and IL-13 effectively function in pancreatic cancer
cells through the IL-13R1/IL-4R signaling pathway [55].
It has become increasingly evident that the establishment of a perfect,
long-lasting anti-tumor immune response relies on the generation of CD4+
T helper (TH) cells and cluster of differentiation (CD)8+ CTL cells. The
production of TH1 (retinoic acid receptors) and gamma orphan receptors for
IFN, IL-2, IL-12, and TNF (ROR-) is thought to be stimulated by peptide­
based vaccines, while TH17 (IL-17) cytokine profiles are associated with
this stimulation, as opposed to TH2 (IL-4, 6, 13, and IL-13, excluding IL-5)
and forkhead box P3 (Foxp3) regulatory T cell (IL-10 and TGF-) production
in vivo and in vitro [56].

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10.7 GM-CSF (GRANULOCYTE-MACROPHAGE COLONY­
STIMULATING FACTOR)
Granulocyte-macrophage colony-stimulating factor (GM-CSF) is used
for the treatment of melanoma. Excision is the primary method of cancer
treatment, but it is not effective for metastatic disease. Immunotherapy has
shown promise in certain melanoma patients. Melanoma is a suitable target
for immunotherapy due to the production of tumor-associated antigens by
melanoma cells. Preclinical studies have been conducted to investigate the
adjuvant properties of GM-CSF in limiting tumor formation and inducing
growth inhibition. In October 2015, Ipilimumab was approved for adjuvant
therapy in the USA for cutaneous melanoma patients who had undergone
complete excision and total lymphadenectomy with pathologic involvement
of regional lymph nodes larger than 1 mm in diameter [57]. GM-CSF can
be delivered as an adjuvant by injecting it at the injection site, ingesting it
systemically, or by injecting a viral or plasmid vector expressing GM-CSF.

— SECTION 42 —
However, the results of this human trial have been inconsistent. When
GM-CSF and IFA (incomplete Freund’s adjuvant) were used as adjuvants for
a vaccine containing 12 melanoma peptides, patients with surgical stage III/
IIIB/IV melanoma showed a similar immune response. The development of
other strategies utilizing GM-CSF continues, such as GM-CSF-expressing
oncolytic vaccinia viruses, autologous DCs, allogeneic whole tumor cells,
adjuvant GM-CSF following immunization with melanoma peptides or
RNA, whole carcinoma cell vaccines, and GM-CSF DNA vaccines [58].
Early studies indicate that GM-CSF stimulates humoral and cellular anti­
tumor responses and acts as an immune adjuvant. Patients who received the
vaccine with QS-21 had a T-cell response rate of 44.4%, while those who
received the vaccine with GM-CSF had a T-cell response rate of 50.0%. In
independent investigations, only 26.7% (4/15) of patients who received
GM-CSF as an adjuvant to a peptide vaccination showed a cytotoxic T-cell
response [59]. A small increase in the number of natural killer (NK) cells and
significant increases in CD4+ (P 0.001) and CD8+ (P = 0.007) cells from base­
line were observed when GM-CSF was used as a chemotherapy adjuvant [59].
Patients with high-risk neuroblastoma (NB) have experienced success
with adjuvant therapy using anti-GD2 monoclonal antibodies (mAbs) and
GM-CSF [60]. The level and duration of gene expression in DCs increase
when biomaterials and GM-CSF are utilized to create potential delivery
vectors for genetically modified DCs or when host DCs are genetically modi­
fied in situ for immunization and the treatment of autoimmune disease [61].

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10.7.1 GM-CSF USE AS AN ADJUVANT WITH CANCER VACCINES:
USEFUL OR HAZARDOUS
The appeal of GM-multifactorial CSF’s causes lies in its potential use as
a vaccination adjuvant. Considering the numerous immune system conse­
quences, GM-CSF has been utilized as a vaccination adjuvant or anticancer
treatment in a significant trial conducted over the past decade. GM-CSF is
employed as a standalone agent, as a side effect of whole-cell vaccinations
using mutated tumor cells, as well as an adjuvant, safely and effectively in
vaccinations composed of peptides and DCs that are used to treat various
malignancies. A significant portion of current clinical research focuses on
anticancer treatment using peptide-based vaccinations. However, when used
as an immunological adjuvant, GM-CSF has the potential to inhibit rather

— SECTION 44 —
than stimulate the immune system. Recent results from trials using GM-CSF
as a therapy for melanoma have shown that immune adjuvants used in
vaccines can have adverse and immunosuppressive effects. GM-CSF has
also demonstrated potential as a vaccination adjuvant in studies involving
peptide-based, DC-based, and whole-cell vaccines for the treatment of
breast, prostate, pancreatic, renal cell, ovarian, and other malignancies [62].
In one of the phase II studies comparing median dose interferon Alpha 2B to
adjuvant immunotherapy using the CSF-470 vaccination, bacillus Calmette-
Guerin, and recombinant human (rh) GM-CSF in stages II B, II C, and III
cutaneous melanoma patients, the CSF-470 vaccine plus BCG and rhGM-
CSF given as adjuvant therapy to stages IIB, IIC, and III CM IFN-2b have
been the subject of a randomized research (interferon). Treatment was less
effective at delaying or stopping the development of distant metastases [58].

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10.7.2 A ROLE FOR G-CSF AND GM-CSF IN NON-MYELOID CANCERS
However, the differentiation of progenitor cells in the bone marrow into
differentiated granulocytes, macrophages, and T lymphocytes is regulated
by G-CSF and GM-CSF, which stand for granulocyte and granulocyte-
macrophage colony-stimulating factors. Our aim is to summarize current
research on the adjuvant functions of G-/GM-CSFs in the pathophysiology
of solid malignancies and provide insights into the complexity of their
therapeutic applications due to the increasing usage of these agents in cancer
treatment. The prospective mechanisms of progression are similar to those
seen with the adjuvant use of recombinant cytokines, such as induction of

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immunological tolerance and angiogenesis. However, caution should be
exercised when using recombinant cytokines as an adjuvant treatment in
patients with lung cancer, as G-/GM-CSF may accelerate cancer progression
and distant metastases. Tumors that secrete GM-CSF include lung tumors,
gliomas, bladder tumors, melanoma, and skin carcinomas, colorectal tumors,
and bone metastases in prostate and breast tumors [62]. Although adjuvant
IFN-2b does not improve overall survival (OS), it increases disease-free
survival (DFS); however, it is accompanied by significant toxicity and is not
always considered a gold standard therapy [58].

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10.8 DENDRITIC CELLS (DCS) AS AN ADJUVANT-BASED
THERAPEUTIC CANCER VACCINE
Dendritic cell (DC) activation is required for the initiation of T-cell responses
to infections and malignancies [67]. Due to their ability to gather, analyze, and
transport antigens to T cells, DCs play a vital role in vaccination. The cellular
changes that accompany DC maturation include decreased antigen-capture
activity, increased expression of costimulatory molecules and surface MHC
class II molecules, the development of chemokine receptors (such as CCR7)
that regulate their migration, and the capacity to secrete various cytokines
(such as interleukin-12 [IL-12]) that regulate T cell differentiation. DCs can
be utilized for cancer vaccination in a variety of methods, including in vivo
non-targeted peptide, protein, and nucleic acid-based vaccines, vaccines
consisting of antigens that selectively bind to DC antibodies, and vaccina­
tions employing ex-vivo generated DCs that have been loaded with antigens.
Adjuvants for vaccines work by stimulating DC maturation [63]. DCs are
well-known for their special capacity to activate both innate and adaptive
immune pathways [64]. Combinations of adjuvants that target several
pathways may work in concert to increase the effectiveness of immune
responses because they can synergistically activate DCs [23]. Adjuvant DC
vaccine therapy is more effective than first-line therapy for primary solid
tumors. The majority of DC-based vaccinations are now being developed
in an adjuvant approach to work together with current cancer treatments.
Clinical trials are now being conducted on some enhanced DC vaccines,
some of which are anticipated to receive FDA approval. We also hope that
DC vaccines will be created as a cancer-prevention vaccine. But the only
DC vaccine available now is Dendreon’s Provenge against Prostate Cancer,
which obtained FDA approval in 2010 [65]. To achieve the highest level of

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clinical success, new cancer vaccination techniques (Box 2) take into consid­
eration adjuvants that stimulate cross-presenting DCs [66]. Tumor lysates
or recombinant proteins are likely to function optimally in the context of
relatively young DCs. However, preprocessed peptides that are MHC class
I or MHC class II-restricted are less reliant on being engulfed by macro-
phages or DCs. Additionally, in the innovative realm of biotechnological,
multiformat antibodies, such as bispecific antibodies, are being exploited
to bring target cells into close proximity with DCs, thereby enhancing their
reactivity or facilitating the delivery of antigenic or stimulatory payloads to
CD40-expressing cells, such as DCs [67]. Recent studies have demonstrated
positive clinical outcomes of DC vaccinations targeting hTERT and Survivin
antigens in metastatic prostate carcinoma [68]. The efficacy of this antigen
delivery technique in DC-based cell therapy was investigated in a study, along
with the effects of antigen delivery using perfluoropropane gas-entrapping
liposomes (Bubble liposomes, BLS) and ultrasound exposure on MHC class
I presentation levels in DCs [69]. Other strategies, such as genetic vaccine
strategies and peptide vaccine strategies, are also employed as adjuvants to
enhance the effectiveness of DC-based cell vaccines [64]. All melanoma
patients involved in the study exhibited distinct T cell responses following
RNA-induced DC vaccinations. Glioblastoma can also benefit from the use
of mRNA-transfected DCs in clinical trials [64].

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10.9 COMBINATION OF ADJUVANT
Most vaccines currently available on the market only contain a single adju­
vant. However, due to their inherent limitations, no single adjuvant can elicit
all the necessary immune responses required for different vaccinations. As a
result, researchers are exploring the potential of using vaccine formulations
that incorporate multiple adjuvants. According to a new approach, care­
fully selecting combinations of adjuvants may enhance immune reactions
to vaccines in complementary and even synergistic ways. This strategy
is promising and provides numerous opportunities for vaccine scientists
to tailor immune responses to specific vaccines [64]. The selection of the
targeted antigen(s) and the need for effective adjuvants are two crucial factors
in enhancing their innate immunogenicity [49]. A new theory suggests that
the careful choice of adjuvant combinations can improve immune responses
to vaccines in complementary and synergistic ways [64]. As observed so
far, combining vaccination with effective adjuvant therapy should result in

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heightened immune responses [70]. Therefore, current research combines
peptides with adjuvants, aiming to achieve three main objectives: preventing
rapid degradation and ensuring sustained release of the antigen, promoting
efficient uptake of the antigen by native antigen-presenting cells (APCs),
and stimulating full activation of APCs to initiate potent anti-Th1/CTL
responses and long-term immunological memory. It is common practice to
form an oil-in-water emulsion with Montanide ISATM51 (an incomplete
Freund’s adjuvant analog typically administered subcutaneously) to preserve
the peptides and ensure their delayed release [71]. In individuals with
melanoma, it has recently been shown that CpG has a potent adjuvant effect
when paired with a long New York esophageal squamous cell carcinoma-1
(NY-ESO-1) peptide [72]. Imiquimod may be an extremely inefficient adju­
vant on its own, but it could be highly useful when combined with other TLR
ligands. Therefore, our objective is to identify a single preferred combina­
tion that can elicit potent anti-vaccine T cells and durable clinical responses
in cancer patients by administering peptides (the antigen), montanide (for
depot impact), and a TLR agonist (for APC activation). We believe that the
most immunogenic combinations should be selected for larger studies to test
for anti-tumor activity. Small-scale clinical studies intended to investigate
peptide, adjuvant, and immunomodulatory combinations should assess the
immunogenicity of these combinations. Synergistic combinations may lead
to more unfavorable occurrences, so it is crucial to carefully monitor any
resulting toxicities [49]. When the combined therapy was less effective,
the addition of a tumor-antigen-based peptide, primarily linked to -GalCer,
significantly enhanced the antitumor activity in tumor models [73].

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10.9.1 TOLL-LIKE RECEPTOR (TLRS) AND ALPHA
GALACTOSYLCERAMIDE (GALCER) COMBINATION THERAPY
Pathogen-associated molecular patterns are distinctive molecular markers
for pathogens that Toll-like receptors (TLRs) can recognize. TLR3 agonists
with varying degrees of efficiency have been previously utilized as adjuvants
in cancer treatment to induce an IFN-mediated antitumor immune response
[74]. TLRs play a crucial role in innate immune responses by detecting
pathogenic antigens. Va14 natural killer T (iNKT) cells have been shown
to recognize the glycolipid -GalCer as a ligand. TLR agonists can enhance
the production of IFN-c in immune cells that have been administered
GalCer injections to heighten their sensitivity. In vitro, GalCer-pre-treated

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splenocytes were stimulated along with other TLR agonists including CpG
ODN, Poly-inosinic acid: cytidylic acid (I:C), Lipopolysaccharides (TLR4
agonist), and Imiquimod (TLR7 agonist, TLR9 agonist), resulting in a
significant increase in the production of IFN-c mRNA [75]. TLRs assist in
the innate recognition of pathogen-associated molecular patterns (PAMPs)
and the initiation of immune responses in dendritic cells (DCs) [76]. Overall,
the treatment of NKT agonists, TLR ligands, and melanoma antigens via
a multivalent nano-vaccine demonstrated synergistic anti-tumor innate
immune efficacy in the B16F10 melanoma mouse model [77].

— SECTION 53 —
10.9.2 ALPHA GALACTOSYLCERAMIDE (GALCER) AND
MONOPHOSPHORYL LIPID A (MLA) COMBINATION THERAPY
A lipid known as Monophosphoryl lipid A (MLA), derived from the LPS
of Salmonella Minnesota R595 [78], is the first TLR4 agonist approved for
use as a human immunization adjuvant. In this study, the effectiveness of
the adjuvants GalCer and MPL in enhancing antitumor immune responses
was examined when combined with an HPV-16 E7 DNA vaccination. The
results showed that using a combination of adjuvants for the DNA vaccina­
tion was significantly more effective than using separate adjuvants in terms of
lymphocyte proliferation, CTL activity, IFN-, IL-4, and IL-12 responses, as
well as tumor protection against TC-1 cells [76]. The combination of -GalCer
and MPL’s powerful adjuvanticity may play a crucial role in the development
of an effective and novel cancer immunotherapy. Researchers also predicted
that the combination of NKT cell antigen GalCer and MPL could enhance the
anticancer benefits for immunotherapy of cancer [27]. Two vaccines, Cervarix
and Fendrix, already contain MPLA adjuvant and are used to prevent diseases
such as HCC and cervical cancer, respectively [79].

— SECTION 54 —
10.9.3 ALPHA GALACTOSYLCERAMIDE (GALCER) AND CPG
COMBINATION THERAPY
APC activation, cytokine release, and adaptive immunity can all be enhanced
by intravenous infusion of α-GalCer and TLR ligands, as we have previ­
ously demonstrated. Furthermore, studies have indicated that administering
the NKT cell agonist a few hours prior to the TLR agonist significantly
enhances cytokine release. To investigate various intratumoral procedures,
either a single dose of 2 g of α-GalCer was administered simultaneously

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with the initial dose of CpG or six hours earlier (by delaying the first CpG
dose by 6 h). Intratumoral administration of α-GalCer significantly improves
intratumoral CpG-induced local and distant antitumor immune responses
[73]. However, compared to each treatment alone, the combination treat­
ment exhibited a lower rate of tumor growth, and the majority of the animals
experienced complete rejections.

— SECTION 56 —
10.10 POLYMERS ADDED AS AN ADJUVANT TO CANCER VACCINES
Particulate adjuvants have shown promise in facilitating the delivery of anti­
gens to lymphatic organs and enhancing immunogenicity. Examples of such
adjuvants include inorganic particles, polymeric particles, liposomes, emul­
sions, and exosomes. A recent study by Sun and colleagues demonstrated the
creation of AlO(OH)-polymer nanoparticles (APNs), which are nano-sized
vaccine carriers with improved lymphatic motility. The research findings
revealed that APNs effectively facilitated cytosolic transport and cross-
presentation of antigens upon internalization by antigen-presenting cells
(APCs), resulting in the development of adaptive cell-mediated immunity.
Polymeric particle adjuvants can be derived from natural or synthetic
sources such as plants, animals, and microbes. Common examples include
albumin, fibrinogen, alginate, chitosan, and collagen. These polymers offer
advantages such as high loading capacity, efficient antigen protection in
vivo or in vitro, affordability, biocompatibility, biodegradability, and ease
of production. However, they also have limitations in terms of stability and
controlled properties [9]. Biologically derived polymers with adjuvant prop­
erties can be directly obtained from biosources or chemically synthesized.
These polymers can enhance antigen presentation, create depots, improve
endocytosis, and bind to pattern recognition receptors (PRRs) to modulate
the immune response when utilized in vaccines and immunotherapies [12].

— SECTION 57 —
Numerous methods, including conjugation, simple mixing, encapsulation, or
adsorption based on charge or hydrophobic interactions, can be employed to
produce antigens from polymeric particles [110]. Adsorption and encapsula­
tion have traditionally been considered the two main strategies for loading
antigens onto polymeric adjuvants [9]. While biologics and small compounds
still dominate the adjuvant industry, recent research is beginning to support
the use of immunomodulatory polymers in therapies. When studying the
immunology of next-generation polymeric adjuvants that target PRRs,
it is important to consider the polymers’ ability to establish biophysical

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connections with cells [11]. In order to generate potent and long-lasting
T-cell responses in combined tumor immunotherapy, a pH-responsive multi-
vesicular polymeric NPs are utilized to deliver the STING agonist cGAMP,
as well as peptide oncoviral antigens and neoantigens [80]. The phospho­
lipid bilayer shell structure is filled with a polymeric polyplex mRNA core.
Dendritic cells (DCs) efficiently uptake the resulting lipopolyplex mRNA
vaccine through macropinocytosis. DCs treated with lipopolyplex mRNA
vaccination exhibit enhanced antigen presentation capabilities, effectively
stimulating an anticancer immune response [81].

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10.10.1 POLYMERIC NANOPARTICLES
The majority of these characteristics, such as cellular activity, higher
bioavailability, protection of the antigen against deterioration, and controlled
antigen release, appear to be present in nanoparticles (NPs). The most well-
known type of polymeric NP used as an antigen delivery technique is poly(D,
L-lactic-co-glycolide) nanoparticles (PLGA-NPs), sometimes referred to as
PEG. Data from flow cytometry and confocal laser scanning microscopy
(CLSM) demonstrate that human CD14+ monocytes and the mouse Hepa
1–6 hepatoma cell line are more receptive to PLGA/PEI (polyethyleneimine)
NPs. In one study, it was shown that the therapeutic vaccination’s anticancer
effects are enhanced by the addition of an HPV E7 SLP to extremely tiny
polymeric nanoparticles (NPs) [68]. Both melanoma cancer and HPV cancer
are treated with the use of these polymeric NPs. In order to create a vaccine
technology for acute myeloid leukemia (AML) cell membrane, the leukemic
cell membrane material is coated on immune-stimulating adjuvant-loaded
nanomaterials [83]. Toll-like receptor agonist (TLRa)-loaded NPs have the
power to increase tumor suppression and destruction while reducing the
danger of immune-related damage.

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10.10.2 ALBUMIN AS AN ADJUVANT IN CANCER VACCINES
Pulmonary immunization using albumin-binding surfactant molecules
adducts of peptide antigens and CpG adjuvant (amph-vaccines) elevated
vaccination levels in the lungs and mediastinal lymph nodes (MLNs) [84]. We
incorporated aluminum ions into NPs using bovine serum albumin (BSA),
which has been shown to chelate inorganic ions like Au and Gd into NPs
under benign conditions. The NPs’ albumin may aid in supplying the quickly

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expanding tumor with energy [85]. Al-BSA-Ce6 NPs is an innovative and
distinctive alumina adjuvant delivery system that is useful in the treatment
of cancer. An efficient vaccination adjuvant that boosts antigen-specific
CD8+ T cell-mediated antitumor immunity is albumin and interferon-fusion
protein. This composition lengthens the half-life of IFN in vivo, delivers it
to LN, and boosts innate immunity to tumors [86]. Nanocomplexes made
of albumin or AlbiVax are easily adaptable to immunogenic vaccination
for individualized tumor immunotherapy. Exogenous neoantigen vaccines
delivered by albumin/AlbiVax nanocomplexes can improve tumor therapy
and neoantigen-specific immunity [87].

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10.10.3 CHITOSAN AS AN ADJUVANT IN CANCER VACCINES
Due to their immunostimulatory effects and structural similarities to glucans,
chitin, and chitosan have been the subject of several studies investigating
their adjuvant properties. The preservation of the vaccine in the nasal cavity
via mucoadhesion, as well as the opening of endothelial cell junctions for
paracellular administration of the vaccine, is thought to be the mechanism
of vaccine augmentation by chitosan through mucosal delivery. Small,
phagocytosable chitin particles caused alveolar macrophages to release
cytokines such as TNF, IL-12, and IL-18, which primarily induced NK cells
to produce INF [88]. The complexation of a nucleic acid-based adjuvant with
chitosan regulates immune adjuvant activity (CTS). Chitosan is negatively
charged, making it more difficult for it to pass through cell membranes. The
TLR3-type adjuvant action of the RNA adjuvant was significantly enhanced
through complexation with CTS [89].

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10.10.4 COLLAGEN AS AN ADJUVANT IN CANCER VACCINES
The benefit of collagen anchoring in extending the intratumoral retention
of IL-2 has been observed. The addition of MSA improved the molecular
weight of the design, resulting in reduced diffusive flux away from the tumor
and ensuring electrostatic access of IL-2 receptors when bound to collagen
fibrils. Clinical evidence has revealed the buildup of collagen type I along
the aggressive margin of breast, colorectal, and skin cancers. This margin,
in contrast to the tumor’s core, typically contains a higher concentration of
immune cells, particularly T cells [90]. Collagen is also utilized as a marker
for detecting presurgical margins in the eradication of basal cell carcinoma

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[91]. In human breast cancer, the presence of aligned collagen serves as a
predictive indicator of survival [92].
10.11 NANO-ADJUVANT IN CANCER VACCINES
Because cancer vaccination utilizes delivery vehicles or adjuvants to induce
potent cellular and humoral immunity, it is an effective technique for both
preventing and treating cancers. Innovative delivery methods and vaccine
adjuvants have been developed more frequently in immunotherapy as they
can stimulate immune responses with fewer doses and adverse effects.
Thanks to nanotechnology, investigators now have complete control over
the production of gadgets at the molecular level. In this regard, pathogen-
specific antigens are combined with artificial or natural nanostructures
to create nano-vaccines, which have shown to elicit manageable immune
responses. This approach recommends utilizing crucial pathogen sub-units
such as peptides, proteins, membranes, polysaccharides, and capsules to
produce vaccines that are more flexible and secure [93]. To enhance immu­
nity, various metals can be employed as effective adjuvants in the creation
of nano-vaccines. Additionally, antigens can be successfully delivered by
metal-based nanoparticles to antigen-presenting immune cells [94]. Nano­
based vaccines that are currently being developed to treat cancer include
those based on proteins, peptides, nucleic acids, DNA, RNA, bacteria or
viruses, liposomal vaccines, polymeric vaccines, and more.

— SECTION 64 —
10.11.1 IMMUNOSTIMULATORY NANO-ADJUVANT
CVs contain immune adjuvants that can promote the immune system,
improve the immune responses evoked by antigens, and target specific
immune responses. Small molecule antigens must possess these adjuvant
qualities because they are typically not very immunogenic [95]. Immuno­
therapy is currently an effective clinical approach for treating cancer and
infectious disorders. Potential immunostimulatory medicines for use in
immunotherapy include small synthetic compounds that bind to TLR7 [93].
Examples of immunostimulatory nanoadjuvants include porous silicon
microparticles (pSiMPs) and Selenium nanoparticles (SeNPs). It is now
understood how immunomodulatory and nanotherapeutic approaches can be
utilized to develop breast cancer vaccines.

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10.12 DIFFERENT DELIVERY SYSTEMS OF ADJUVANTS
Significant advancements have been made in refining vaccination adminis­
tration methods to overcome poor cellular absorption. DNA vaccines have
proven to be more effective in both pre-clinical and clinical investigations
due to advances in delivery and plasmid design. As shown in Figure 10.6,
there are various vaccine delivery systems, but DNA vaccination holds
promise for eliciting both cellular and humoral immune responses [96].
Some of the delivery systems include Electroporation and Gene gun vaccine
delivery used in prostate cancer, NP vaccine delivery system, Self-assembly
peptides used for gene therapy and bone regeneration in vivo, and genetic
vaccines (DNA, RNA, viral vector, and peptide vaccine) as shown in Figure

— SECTION 67 —
10.7. Delivery devices that resemble viruses and virosomes are frequently
utilized to treat infectious disorders. Examples of commercially available
vaccinations that have been adjuvanted with virus-like or virosome delivery
methods include the influenza vaccine, the HBV vaccine, and the HPV
vaccine. Additional clinical research on VLP and virosome adjuvants is also
underway at various stages, showing promising results in producing high
antibody titers and inducing adaptive immunity [38].
10.12.1 GENE GUN DELIVERY
In 1990, the first study on a gene delivery method was published by labo­
ratory researchers [5]. The epidermal Langerhans and keratinocytes can
be directly transfected with DNA using a gene gun, which is a transgenic
device. This delivery method utilizes transgenic vectors such as plasmid
DNA (pDNA) [5]. Direct DNA transfer is advantageous for the expression
of transgenic proteins using mammalian gene expression vectors. These
instances promote the development of DCs and their migration to the local
lymphoid tissue, where they serve as the starting point for T cells to initiate
antigen-specific immune responses used for melanoma vaccination in the
skin. In lung cancer, non-coated DNA exhibited the highest levels of cyto­
toxic T-lymphocyte activity and the most effective anti-tumor effects [96].
In one of the investigations, GM-CSF gene-transfected autologous tumor
cells were used for vaccination to achieve optimal therapeutic outcomes
[97]. The FDA has set the intensity level up to 0.3–3 w/cm2, which raises
the temperature by no more than 1°C [98]. In one of the studies, ultrasound
microbubbles (USMB) were used for cardiovascular therapy. This technique

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has been employed to treat various cancers such as breast cancer, brain
cancer, pancreatic cancer, melanoma cancer, and HCC. Focused ultrasound
breaks through the blood-brain barrier, improving the delivery of chemo­
therapy drugs for the treatment of glioblastoma [99]. A recent study has been
carried out which states that, in the TME, US simultaneously stimulated LA
and BMT, resulting in the production of singlet oxygen and NO gas. Addi­
tionally, after being exposed to US, cells showed strong CD80, CD86, and
MHCII expression [100]. Another study titled “Focal Therapy with High-
Intensity Focused US: Efficacy in 1379 Men Without Metastatic Prostate
Cancer” was also conducted [101].

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10.12.2 ULTRASOUND
By briefly rupturing cell membranes with ultrasound (US), DNA can be
effectively and immediately transported into the cytosol and incorporated
into the cells. This method is employed to introduce naked plasmid DNA
(pDNA) into colon cancer cells [54]. The frequency of US ranges from kHz
to MHz, depending on the model organism selected for the preclinical test
and the tissue types. The frequency level is lower for therapeutic uses, which
results in deeper penetration due to less attenuation, leading to the best thera­
peutic results. The FDA has set the intensity level up to 0.3–3 W/cm2, which
raises the temperature by not more than 1°C [8]. In one of the studies, USMB
is used for a CV. This technique has been used to treat various cancers such as
breast cancer, brain cancer, pancreatic cancer, melanoma cancer, and HCC.
The Focused United States enhances the delivery of chemotherapeutic drugs
for the treatment of Glioblastoma by disrupting the Blood-Brain Barrier
[57]. A recent investigation has been conducted which affirms that, within
the Tumor Microenvironment, US concurrently stimulated Local Anesthesia
and Bone Marrow Transplantation, resulting in the generation of singlet
oxygen and Nitric Oxide gas. Furthermore, cells exhibited robust expression
of CD80, CD86, and MHCII after exposure to US [20]. The effectiveness of
Focal Therapy with High-Intensity Focused US has been demonstrated in
1379 men with Non-Metastatic Prostate Cancer [53].

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10.12.3 ELECTROPORATION
Nucleic acid molecules are delivered to the target cells using this technique.
The technique involves electroporation of a plasmid containing an antigen

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into a dendritic cell (DC). Due to the penetration of water molecules through
the lipid bilayer, the cell membrane can be reorganized to orient the polar
heads of the phospholipids towards the water. As a result, nanopores are
formed in the cell membrane [102], allowing large compounds such as DNA
or RNA, typically encoding human IL-12, to pass through and enter the cell’s
cytoplasm. This method is employed to electroporate muscles in living organ­
isms, effectively stimulating anti-tumor immune responses against cancers
expressing the prostate antigen. The vaccination is both safe and effective
[96]. It represents a potential approach for gene delivery to enhance cardio­
vascular immunity [102]. In mouse cancer models, intratumoral electropora­
tion of self-amplifying RNA, which produces IL-12, along with an adjuvant
to boost vaccine immunity, has demonstrated anticancer effects [103].
FIGURE 10.6 Classification of various vaccine delivery systems of adjuvant containing
cancer vaccines.

— SECTION 71 —
10.12.4 LIPOSOMES DELIVERY SYSTEM
Gregoriadis and Allison were the first to describe the capacity of liposomes
to elicit immunological responses to integrated or linked antigens [104].
Liposomes were one of several innovative novel drug delivery methods that
demonstrated a breakthrough in the ability to transfer active compounds to
the target of action. Currently, numerous formulations are being used in clin­
ical settings [105]. Liposomes, which are used as vaccine delivery vehicles,

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Role of Various Cancer Vaccine Adjuvants in Cancer Management
can have antigenic stimuli incorporated in the bilayer, adsorbed onto their
surface, or engrafted onto them [104]. A liposomal antigen delivery method
that has been altered with a pH-sensitive dextran alternative has been shown

— SECTION 72 —
to improve antigen cytosolic delivery [106]. Vaccine delivery methods based
on liposomes are very flexible and adaptable [107]. In 2014, cyclical di-GMP/
YSK05 liposomes were identified as a new adjuvant delivery mechanism for
cancer immunotherapy. In this study, CD80, CD86, and MHC class I expres­
sions were found to be significantly higher [108]. It appears that liposomes
have a significant advantage over other antigen delivery methods due to
their capacity to be modified by a variety of ligands in order to effectively
and specifically target APCs. A VADS made of liposomes has already been
successfully used to create vaccines that have been licensed for clinical
vaccination against a variety of illnesses, including those brought on by the
most difficult pathogens, like shingles and malaria [109]. Liposomes are a
type of carrier that fall under the first category of adjuvants; they have the
advantage of carrying insoluble antigens and greatly enhancing the immuno­
genicity of synthetic peptides or weak protein antigens. Liposomes are now

— SECTION 73 —
being researched as antigen delivery methods for tuberculosis, hepatitis A,
malaria, influenza, and non-small cell lung cancer (NSCLC) [17].
FIGURE 10.7 (a) Electroporation delivery system; (b) gene gun delivery system; and (c)
liposomes delivery system, these all method are used to deliver Ag in vivo.

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— SECTION 74 —
10.13 CONCLUSION
We continue to heavily rely on aluminum-based chemicals for human adjuvants,
despite the abundance of information on immune function that has emerged in
recent decades. The efficacy of these adjuvants was initially recognized over
80 years ago. While there are promising indications that new adjuvants could
address some of the limitations associated with aluminum-based adjuvants,
there is concern that many of these potential alternatives may not receive
approval for human use due to logistical or financial considerations, rather than
concerns for the greater good. The regulatory standards for human adjuvants
have significantly increased since the introduction of alum as an adjuvant. In
fact, it is likely that current products would not exist if alum had not been
in use for such a long time and had not received regulatory approval. The
recent discoveries regarding IL-4/IL-13 and their receptors raise additional
questions, particularly regarding their impact on infection and immunity in the
innate and adaptive compartments. For example, the primary cellular sources
and catalysts for the production of IL-4 and IL-13 remain unknown. Several
vaccines, such as Fendrix for hepatitis B, Shingrix for shingles, and Cervarix
for cervical cancer, have been authorized with adjuvants containing TLR4
agonists. Similar to this, Heplisav-B, another Hepatitis B vaccination, contains
a TLR9 agonist. The earlier discovery of TLR9 agonists may account for their

— SECTION 75 —
early development as vaccine adjuvants. In conclusion, TLR agonists have
become highly effective immunostimulatory agents against both infectious
diseases and cancer, as well as vaccine adjuvants. To ensure effective anticancer
action, DC vaccinations require a variety of molecular or immunological
markers, depending on the context (e.g., cancer type, antigen heterogeneity/
immunogenicity, tumor immune microenvironment, overall immunological
fitness). Particularly for certain types of cancer, next-generation DC vaccines
can be used in combination with adjuvant therapies. To make DC vaccines
more accessible to patients, technological advancements that reduce the
barriers to regulation-compliant manufacturing are also required, especially
when combined with already expensive ICIs and the current financial pres­
sures on healthcare systems. GM-CSF was used as an adjuvant in the design of
Sipuleucel-T. In a phase III trial, the Montanide ISA-51 peptide vaccine with

— SECTION 76 —
modified gp100 and incomplete Freund’s adjuvant showed excellent results.
Additionally, as the group of PRRs (Pattern Recognition Receptors) expands,
more research is being conducted on their potential use as adjuvants in thera­
peutic cancer vaccines. There are now multiple clinical trials targeting TLR3
agonists and TLR9 agonists that focus on combining them with GM-CSF
or other TLR agonists. Nanoparticles (NPs), self-assembling peptides, and

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— SECTION 77 —
needle-free delivery methods have all been developed as a result of improve­
ments in vaccine delivery methods. Electroporation delivery system, as it has
been with previous cancer and infectious disease vaccines. Although these
more contemporary delivery methods offer improved substitutes, their use has
been constrained by the discomfort of electroporation administration and the
requirement for specialized vaccine delivery equipment. Due to their flexibility,
ability to store tiny drug candidates or antigens in the form of RNA or peptides,
and high safety profile, liposomes are being exploited as delivery systems.
Liposomes must be tuned to have the proper charge and particle size for incor­
porating their contents and transporting them across the cell membrane. To
enhance local immune response and trafficking to LN, cancer antigens can be
targeted through peptide-adjuvant linkage, antigen incorporation into NPs, or
genetic vector modification, such as the ImmunoBody® platform.

— SECTION 78 —
KEYWORDS

• 
  alum
• 
  antigens
• 
  bovine serum albumin
• 
  dendritic cells
• 
  granulocyte-macrophage colony-stimulating factor
• 
  graphene oxide
• 
  lipopolysaccharide
• 
  mediastinal lymph nodes
• 
  toll-like receptor
• 
  Α-galactosylceramide

— SECTION 79 —
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the Management of Various Carcinomas. Rishabha Malviya, Bhupendra Prajapati, and Sonali Sundram (Eds.)
© 2025 Apple Academic Press, Inc. Co-published with CRC Press (Taylor & Francis)

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#

CHAPTER 11
TITLE: Cancer Vaccine and Chemotherapeutic Agent: Advances in Cancer Management
AUTHORS: Alkeshkumar Patel, Samir Patel, Sanika Dongre, Priyanka Srivastava, Hemangini Vora, Dhruti Patel, and Krishna Jani
LENGTH: 175,849 characters

#

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CHAPTER 11
Cancer Vaccine and Chemotherapeutic
Agent: Advances in Cancer Management
ALKESHKUMAR PATEL,1 SAMIR PATEL,1 SANIKA DONGRE,1
PRIYANKA SRIVASTAVA,2 HEMANGINI VORA,3 DHRUTI PATEL,4 and
KRISHNA JANI5
1Ramanbhai Patel College of Pharmacy, Charotar University of Science
and Technology (CHARUSAT), CHARUSAT Campus, Changa, Gujarat, India
2M.S. Patel Cancer Center, Shree Krishna Hospitals, Pramukhswami
Medical College, Karamsad, Anand, Gujarat, India
3Cancer Biology Department and Immunohematology, The Gujarat
Cancer and Research Institute, Ahmedabad, Gujarat, India
4Sabra Dipping Company, Colonial Heights, Virginia, USA
5Cinical Research Coordinator, ViSafe Research, Vadodara, Gujarat, India

— SECTION 2 —
ABSTRACT
Carcinogenesis is a multifactorial chronic process that encompasses a
variety of factors. Monotherapy of chemotherapy would not be sufficient
to eradicate resistant tumor cells and cancer stem cells (CSCs). As it solely
relies on cytotoxicity and apoptosis induction, the potential of the immune
system as a weapon against cancer remains untapped. Vaccine develop­
ment has played a crucial role in saving mankind from deadly infections.
The fundamental mechanism of any vaccine is based on activating specific
immune cells in the short term while leaving memory cells to prevent disease
recurrence. Each type of cancer has its specific antigen on tumor cells, such
as carcinoembryonic antigen (CEA) and MUC1 present in colorectal cancer

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tumors. The FDA has endorsed the HPV vaccine to prevent vaginal, cervical,
and anal cancer caused by human papillomavirus (HPV). The success of
the Hepatitis B vaccine for liver cancer and sipuleucel-T (Provenge) for
metastatic prostate cancer has sparked interest among immunologists
and cancer biologists to develop vaccines for many other types of cancer.
Cancer vaccines (CVs) can be considered as part of immunotherapy-based
anticancer treatment. However, there are several challenges that need to be
addressed for the proper efficacy of anticancer vaccines. These challenges
include the systemic immunosuppressive environment, loss of antigen
from tumor cells or deficits in major histocompatibility complex (MHC),
the presence of glycocalyx on tumor cells, larger tumor size at advanced
stages, and the application of vaccines in immunocompromised patients.
So, it would be considered as synergism if we can combine chemotherapy
with vaccine treatment. The combined strategy may appear like abscopal
effect mediated by radiation therapy. Preclinical study of cyclophosphamide
(CTX) and doxorubicin delayed the growth of breast cancer in mice when
combined with GM-CSF/HER2/neu complete-tumor cell. The application
of therapeutic CVs in conjunction with chemotherapy and the utilization of
the immunomodulatory effects of chemotherapy to enhance the anticancer
effects of vaccinations are the main foci of this review. The immunomodu­
latory effects include the induction of cancer cell death, which stimulates
antigen-presenting cells (APCs) like macrophages and dendritic cells (DCs),
increases tumor antigen (TA) expression, adhesion molecules, and leuko­
penia, and favors the development of T cells that are specific for the tumor.

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11.1 INTRODUCTION
According to a report from the Centers for Disease Control and Preven­
tion, cancer ranks as the second leading cause of mortality. Cancer can
be considered an important source of premature death worldwide [1]. It
becomes crucial to diagnose cancer at an early stage, as the number of cases
rises drastically, and advanced stage patients have a high mortality rate. The
GLOBOCAN 2020 assessment reported 10 million cancer-related deaths
globally in 2020, with approximately 19.3 million new cases diagnosed.
By 2040, it is projected that there will be 28.4 million new cancer cases
worldwide, representing a 47% increase compared to 2020. This increase
can be attributed to lifestyle modifications, environmental pollution,
and demographic changes [2]. The escalating risk factors associated with

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globalization and expanding wealth may further exacerbate this situation [3].
The highest mortality rates due to cancer are found in lung cancer, followed
by colorectal, liver, stomach, and female breast cancer [4]. Cancer occurs as
a result of multiple factors that chronically disturb the body’s homeostasis.
It is characterized by abnormal high cell proliferation, disturbed cellular
differentiation, high nuclear to cytoplasmic ratio, mitochondrial defects,
angiogenesis, chronic inflammation, immune evasion, and apoptosis evasion
[5]. All of these abnormalities are caused by multiple defective genetic muta­
tions [6] and epigenetic changes [7]. Early diagnosis remains a significant
challenge due to lack of public awareness and socioeconomic factors. A
majority of cases are detected at advanced stages of the disease when they
reach the clinics. Nowadays, there are various therapeutic modalities avail­
able for cancer treatment. These include chemotherapy, hormonal therapy,
radiation, surgical ablation, and immunotherapy [8]. Additionally, comple­
mentary medicine such as naturopathy and acupuncture are used as palliative
support during cancer treatment [9].
The origins of chemotherapeutic agents can be traced back to World War
II. Nitrogen mustard, initially used as a chemical weapon, was found to cause
a significant decrease in leukocyte counts. In 1940, two renowned scientists,
Louis Goodman and Alfred Gilman, explored the potential of this agent in
lymphoma treatment. Since then, numerous alkylating agents, cytotoxic
drugs, and antimetabolites have been discovered to save the lives of cancer
patients [10]. The main mechanisms of chemotherapy involve halting the cell
cycle, DNA alkylation, disrupting nucleotide synthesis, deforming microtu­
bules, interfering with topoisomerase function, and inducing apoptosis in
rapidly dividing tumor cells. However, the application of chemotherapy is
limited by its toxicities and the emergence of resistance in many cases. Dose-
limiting toxicities include mucositis, neuropathy, damage to vital organs,
diarrhea, myelosuppression, and the development of secondary tumors as
late side effects [11].
There are reports available on the failure of chemotherapy as monotherapy in
cancer treatment. Following the use of platinum-based chemotherapy for meta­
static cervical cancer, the patient experienced recurrence [12]. Even the treat­
ment of advanced stages of cancer requires high doses of chemotherapy, which
increases the vulnerability to irreversible toxicities. The use of anthracyclines
and doxorubicin for childhood cancer causes cardiotoxicity at a late onset and
raises the risk of mortality due to the disruption of the cardioprotective network
of Vascular Endothelial Growth Factor A (VEGFA) and Signal Transducer and
Activator of Transcription 3 (STAT3) signaling [13]. Myelosuppression is one

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of the main concerns of chemotherapy, further promoting anemia, immuno­
suppression, and hemorrhage [14]. Immunosuppression is an essential element
for the initiation and spread of cancer, responsible for tumor aggression and
resistance. Numerous cancer microenvironments release immunosuppressive
cytokines such as tumor necrosis factor alpha (TNF-α), interleukin (IL)-8,
and IL-6, exacerbating clinical conditions and leading to resistance [15].
Transforming growth factor (TGF-), an essential immunoregulatory cytokine
secreted by tumor cells and stromal fibroblast cells, causes the formation of
cancer-associated fibroblasts (CAFs). These cells further promote cancer
development, invasion, cancer stemness, and resistance [16]. All these indica­
tions lead to the suppression of antitumor immunity, which is the main key
player in halting tumor growth. While chemotherapy acts on other hallmarks
of cancer to prevent its growth, the unnecessary suppression of immunity may
flourish the tumor and make it a more stubborn, heterogenous resistance cell
factory day by day [17]. Therefore, there is a need for combined treatment with
different therapeutic agents that curb tumor growth through immunomodula­
tion and maintain vigilance through natural body defense mechanisms [18].
However, the use of immune-based therapy alone would not be sufficient to
effectively control cancer progression.
Despite the use of monoclonal antibody (mAb) conjugates, in the case
of HER2-positive breast cancer, more than 33% of patients with metastasis
develop resistance [19]. The surface of most tumors shows the presence
of different neoantigens, which can be explored to target the tumor more
specifically and precisely through immune modalities [20]. Vaccine devel­
opment can be considered the greatest lifesaving regimen for mankind. It
has protected humans against numerous deadly infections, from smallpox to
SARS-CoV-2. Sipuleucel-T (Provenge) received FDA approval in 2010 as
the first therapeutic cancer vaccination to treat metastatic castrate-resistant
prostate tumors. Subsequently, many vaccines have been tested in clinical
trials as monotherapy or in combination to improve treatment outcomes and
prevent cancer recurrence [21]. However, vaccines have their limitations and
need to be used rationally and in combination with chemotherapy to achieve
synergistic effects.

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11.2 CARCINOGENESIS – ITS HALLMARKS AND IMMUNE
DYSFUNCTION
The emergence of genetically abnormal and overproliferation of cells in the
tissue, with dysregulation at multiple biological processes such as energy,

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metabolism, respiration, cellular death, and differentiation, is referred to
as carcinogenesis, which is the development of cancer. Carcinogenesis can
be initiated by the transfer of hereditary mutated genes such as BRCA1
and BRCA2 in breast cancer [22], APC for colorectal cancer [23], KRAS
in many cancers [24], and BCR-ABL for leukemia [25]. Environmental
factors and inappropriate lifestyle choices often trigger the conversion of
proto-oncogenes to oncogenes or defects in DNA mismatch repair genes and
the deactivation of tumor suppressor genes, leading to the development of
cancer over time. Carcinogenesis can be caused by physical, chemical, or
biological carcinogens (cancer-causing agents).

— SECTION 8 —
11.2.1 GENETIC AND EPIGENETIC MANIPULATION AT THE ROOT OF
THE DISEASE
The genetic insult caused by hereditary or environmental factors promotes
the overactivation of oncogenes, which disrupt cell cycle regulators and allow
for excessive cell proliferation. Genetic mutations act as drivers and confer
significant advantages to tumor cells compared to normal cells, including
enhanced proliferation, high metabolism, respiration, immune evasion, and
apoptosis evasion [26]. Integrative OncoGenomics (IntOGen), developed by
a group of researchers, provides a comprehensive identification of mutated
genes in different tumors [27]. FMS-like epidermal growth factor receptor
(EGFR), KRAS, tyrosine kinase 3 (FLT3), platelet-derived growth factor
(PDGF) receptor, MYC, and BRAF are among the several oncogenes that
undergo alterations. Adenomatous polyposis coli (APC), BRCA1 and 2,
PTEN, TP53, and transforming growth factor receptor-2 (TGFRB2) are
among the tumor-suppressor genes involved. Certain alterations such as
translocation, amplification, and deletion lead to genetic mutations that
initiate chronic carcinogenesis [28]. The knowledge of genomics can help in
the development of personalized medicine for cancer patients. Recent theory
also supports the involvement of epigenetics, in addition to genetics, in
initiating and promoting tumorigenesis. Epigenetic changes do not involve
alterations in the DNAsequence. The regulation of genetic expression through
epigenetic modifications includes processes such as methylation, acetyla­
tion, and histone modification. Abnormal epigenetic modifications sustain
tumor growth by promoting autophagy, which further enhances the survival
of cancer stem cells (CSCs). These modifications activate cellular signaling
systems such as Hedgehog, Notch, and Wnt-catenin, which maintain CSC

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dormancy and contribute to resistance and recurrence [29]. Additionally, the
role of micro-RNA (miRNA) has been reported in controlling transcription,
apoptosis, and cell proliferation [30].

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11.2.2 HALLMARKS OF THE CANCER – RECENT UPDATES
During carcinogenesis, the mutated cells acquire changes in functional char­
acteristics. These capabilities are required for the transformation of normal
to neoplastic state, known as hallmarks of cancer [31]. The vital hallmarks
involve immune evasion, immortality, chronic inflammation, metastasis,
invasion, angiogenesis, apoptosis resistance, hypoxia, metabolism dysregu­
lation, and sustained proliferative signaling pathways [32]. The prevalent
genetic mutations and epigenetic reprogramming during tumorigenesis
support the cancer hallmarks that eventually develop tumors and promote
metastasis through epithelial mesenchymal transition [33]. The presence of
microbiomes can affect tissue homeostasis. The inhabitants of colon, lungs,
skin, and urogenital mucosal microbiomes can halt or promote inflammatory
changes based on chemicals released by a variety of populations. This carries
a potential role in the development of the cancer phenotype [34].

— SECTION 11 —
11.2.3 STRONG CORRELATION OF CANCER OCCURRENCE AND
IMMUNE ABNORMALITIES
At the initial stages of cancer evolution, the innate and adaptive immune
systems work together to distinguish and eradicate tumor cells [35]. The
immune system receives warning signals for the manifestation of cancer
cells, including type I interferons (INF), damage-associated molecular
patterns (DAMP), and altered expression of stress-induced ligands such as
ULBP1, MICA/B, and NKG2D [36]. Innate immune cells such as γδ T cells,
natural killer (NK) cells, natural killer T (NKT) cells, and macrophages are
activated in inflammatory situations and identify and eliminate tumor cells
through various pathways. These pathways include antibody-dependent cell
cytotoxicity, generation of pro-inflammatory cytokines (PICs), phagocy­
tosis, TRAIL-induced and FasL-induced apoptosis, and cytotoxic molecules
[37]. Dendritic cells (DCs) carrying tumor fragments migrate to lymph
nodes (LN) where they activate tumor-specific CD8+ T and CD4+ Th1 cells.
Upon arrival at the tumor site, the activated CD4+ and CD8+ T lymphocytes
work together to effectively eliminate tumor cells [38]. Tumor Necrosis
Factor (TNF), IFN, and IL-12 are examples of Th1 effector cytokines that

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CD4+ T helper cells produce together with their innate complements ILC1s,
enhancing the actions of macrophages and cytotoxic cells [39]. Important
functions in the elimination of tumors are also played by tumor-infiltrating
B lymphocytes. They can develop the effector phenotype (Be1), which is
demonstrated by the production of Th1 cytokines and cytotoxic chemicals
and serving as tumor-specific antigens [40]. When B cells differentiate
into antibody-secreting plasma cells, tumor cells that express antigens are
susceptible to cytotoxicity and phagocytosis facilitated by antibodies [41].
The residual tumor and immune cells enter a phase of dynamic equilibrium
if the tumor is not completely destroyed during the elimination phase. Novel
tumor variants persist and develop during this phase, but they are eventu­
ally eradicated. This period continues until the tumor expands and develops
mutations that allow it to evade immune recognition and eradication, leading
to tumor advancement known as the escape phase [42]. Immune escape can
be attributed to various factors, including down-regulation of MHC class I
molecules, loss of tumor antigen (TA) expression, chronic activation-induced
immune cell exhaustion, and the emergence of ligands for co-inhibitory
receptors (PD-L1/PD-1, CD80/CTLA-4, HLA-E/NKG2A-CD94, MHC
class I/KIR, HVEM/BTL) [43–45]. Additionally, the expression of CD47 by
tumor cells can block the process of phagocytosis, as CD47 acts as a ‘do not
eat me’ signaling molecule [14]. The role of T and B lymphocytes has been
exemplified in Figures 11.1 and 11.2.
FIGURE 11.1 Role of tumor associated with T-lymphocyte.

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Tumor associated T-lymphocytes in tumor micro-environment of
colorectal cancer:

• The TH1 and TH2 CD4+ T cell subsets become dysfunctional as a
  result of upregulation of IL-4, IL-6, TNF-α, and INF-α, and down-
  regulation of IL-2.
• Tumor-derived IL-6, IL-1, and TGF-β contribute to the development
  of Th17 in the tumor microenvironment (TME).
• TH-17 activates the Wnt pathway by activating IL-17A and
  suppressing IFN-ɑ in tumors, which leads to enhanced proliferation
  and inhibited apoptosis in colorectal cancer.
• IL-22 produced by TH22 involves increasing proliferation and anti­
  apoptotic activity in colorectal cancer that is suppressed by STAT-3
  inhibitor.
• PGE2 tumor is responsible for suppressing T cells by downregulating
  T cell receptors (TCRs) such as CD3 and SLP63, as well as IL2R. It
  induces activation of the STAT-5 pathway in colorectal cancer.
• PD-L1 is present in the tumor microenvironment (TME) and binds
  to PD-1 on cytotoxic T-cells, resulting in evasion of the immune
  response.
• Through the stimulation of the STAT1 and STAT3 signaling
  pathways, IL-35 generated by tumor cells can evade the immune
  response by enhancing the number of Treg cells and suppressing the
  proliferation of CD4+CD25-T effector cells within the tumor.
• Extra cellular vesicles derived from colorectal cancer cells alter T
  cells into Treg cells via TGF-β/Smad, which suppresses the immune
  system.
• The generation of CCL17, CCL5, and CXCl12 by Treg cells
  induces epithelial mesenchymal transition. The secretion of IL-10
  and TGF-β by accumulated TAM, DCs, and MDSCs leads to cancer
  progression.
• Treg prevents the activation of CD8+ T cells, CD4+ T cells, NK
  cells, NKT cells, B cells, and APC cells.
  Tumor associated B-lymphocytes:
• The B-regulatory cells CD19LOW and CD27HIGH secrete high
  levels of IL-10, inhibit INF-gamma and TNF-alpha secreted by T
  cells, and reduce TH17 activity, all of which contribute to the devel­
  opment and spread of colorectal cancer.

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317
FIGURE 11.2 Role of tumor associated with B-lymphocyte.

• 
  Production of IgA+ B cells increases by decreasing the level of
  microRNA15A and microRNA16-1, which suppress the immune
  response by evading the proliferation and activation of cytotoxic T cells.
• The immune-suppressive environment was formed by the tumor
  cells’ production of IL-35, which had a positive correlation with
  Treg and MDSC cells and a negative correlation with CD3+ T cells.
• Chemokine CCL7 and its receptor CCR2 are produced in high
  amounts in colorectal cancer, which suppresses immunoglobulins
  secreted by B-cells.
• The Wnt/β-catenin pathway, which is responsible for progression,
  invasion, and EMT, is activated by the decreased expression of
  BTG3 and BTG1 genes. The diverse mechanisms through which
  tumorous cells bypass immune surveillance pave the way for the
  establishment of immunotherapy approaches. Immunotherapy aims
  to manipulate T-cell function and block tumor metabolism [46]. It
  also involves targeting immunosuppressive checkpoints (PD-1 and
  CTLA-4 axes), immunosuppressive modulators TGF-β and IDO,
  CD47 signaling, and CCL2-mediated recruitment of immunosup­
  pressive cells [47].

— SECTION 15 —
11.3 CHEMOTHERAPY – THE CONTROVERSIAL GOLD STANDARD
The tumor cells are much more stubborn compared to normal cells. They have
advantages in terms of proliferation, respiration, metabolism, telomerase

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enzymes, and apoptosis inhibition. Except for mitochondrial defects, they
possess all the characteristics that sustain tumor growth. It would be inap­
propriate to consider cancer treatment without the cytotoxic potential of
chemotherapy. However, the failure of the therapy to discriminate between
rapidly dividing normal cells and tumor cells leads to life-threatening toxici­
ties [35, 48–50].

— SECTION 16 —
11.3.1 MECHANISMS OF TUMOR CELLS DEATH BY
CHEMOTHERAPEUTIC AGENTS
Chemotherapy targets tumor cells mostly based on their rapid cell proliferation
feature. The different mechanisms of chemotherapy involve the formation of
DNA lesions by interacting with specific nucleotides [51], suppression of the
Wnt/-catenin, MEK/ERK, and PI3K/Akt signaling pathways that promote
cancer stemness [52], activation of apoptotic pathways [53], microtubule
disaggregation [54], and impairment or intervention in nucleotide synthesis
or integration [55]. The mechanisms, along with the therapeutic application
and toxicities of first-line chemotherapeutic agents for the most prevalent
cancer, are given in Table 11.1.

— SECTION 17 —
11.3.2 TUMOR MICROENVIRONMENT AND RESISTANCE TO
CHEMOTHERAPY
Tumors consist not only of malignant cells, but also various non-malignant
cells. These include adipocytes, dendritic cells (DCs), fibroblasts, compo­
nents of the vasculature, and tumor-infiltrating lymphocytes. The tumor
microenvironment (TME) plays a crucial role in the initiation and progres­
sion of tumors through continuous communication with the surrounding
tissue [106]. By manipulating the TME, tumor cells can evade immune
responses [85]. Elevated levels of VEGF-A contribute to abnormal angio­
genesis, characterized by disorganized and leaky blood vessels that result
in hypoxia [107]. Conversely, increased expression of VEGF-C/D promotes
lymphangiogenesis, which stimulates tumor progression and dissemination
[108]. Moreover, tumors induce stromal transformation by releasing VEGF,
TGF-β, and PDGF-BB, leading to the conversion of healthy fibroblasts into
cancer-associated fibroblasts (CAFs), lymphatic endothelial cells, pericyte
detachment from blood vessels, and alterations in blood vessel structure [109].
The aberrant stromal components release immunosuppressive molecules

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PAGE 338

Chemotherapeutic Mechanism of Action
Dose Limiting Toxicities
Therapeutic Application
Agent
5-Fluorouracil
By inhibiting the transformation of deoxyuridylic acid to thymidylic Hepatotoxicity (serum
In the treatment of basal cell
[56–61]
acid through the action of the enzyme thymidylate synthetase, it
aminotransferase elevations, coma carcinomas as well as palliative
interferes with DNA synthesis.
with hyperammonemia, lactic
cancer care, it is administered
acid elevations, and respiratory
as an injection.
alkalosis), gastrointestinal
(diarrhea, stomatitis),
myelosuppression.
Doxorubicin
To bind to nucleic acids, it selectively intercalates the planar
Cardiac toxicity includes
Used to produce degeneration
[62–64]
anthracycline nucleus with the DNA double helix. This prevents
reversible myopericarditis,
in proliferative cancers –
DNA replication and transcription.
left ventricular dysfunction,
acute lymphoblastic and
arrhythmias, and congestive
myeloblastic, Hodgkin’s
heart failure (CHF). BM can

disease, Wilms’ tumor,
cause myelosuppression,
neuroblastoma, sarcomas
while dermatology poses an
of bone as well as soft
extravasation hazard as a vesicant. tissues, carcinomas (ovarian,
transitional cell bladder, gastric,
breast, thyroid, bronchogenic),
malignant lymphoma.
Paclitaxel [65–67] Paclitaxel attaches itself to the β subunit of tubulin, thus preventing
Cardiotoxicity, nervous system
It is used in the treatment
the normal development and functioning of microtubules.
(peripheral neuropathy),
of breast, lung, and ovarian
Additionally, it causes cancer cells to undergo programmed cell
hematologic (neutropenia), bone
cancer, along with its additional
death (apoptosis) by attaching to the inhibitor of apoptosis Bcl-2
marrow (myelosuppression),
use in Kaposi’s sarcoma.
(B-cell leukemia 2) and halting its activity.
musculoskeletal, and connective
tissue (arthralgia/myalgia).
TABLE 11.1 The Use of Chemotherapy is Common in the Treatment of the Most Prevalent Types of Cancer, as well as Some That Have
Immunomodulatory Properties
Cancer Vaccine and Chemotherapeutic Agent: Advances in Cancer Management
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Chemotherapeutic Mechanism of Action
Dose Limiting Toxicities
Therapeutic Application
Agent
Cyclophosphamide Three mechanisms of action: (i) Alkyl groups bind to DNA bases,
Bone marrow (myelosuppression), It is used to treat mycosis
[68–70]
causing DNA fragmentation. This prevents the damaged DNA from cardiac (cardiac necrosis or
fungoides, lymphomas,
being used for DNA synthesis or RNA transcription; (ii) cross-
hemorrhagic myocarditis,
myelomas, leukemia,
linking triggers DNA injury by preventing DNA from being divided pericardial tamponade),
ovarian adenocarcinoma,
for synthesis or transcription; and (iii) the process of generating
gastrointestinal (nausea,
neuroblastoma, breast
nucleotide mismatches that result in mutations.
vomiting), Renal (hemorrhagic
cystitis).
carcinoma, and retinoblastoma.
Methotrexate
Cell division is prevented by inhibiting the nucleotide synthesis
Hepatotoxicity (fibrosis,
It is used in the treatment
[71–76]
enzymes. This leads to increased levels of adenosine and adenosine cirrhosis, enzyme elevations),
of several cancers, severe
triphosphate in the extracellular space, resulting in an anti-
gastrointestinal (diarrhea,
psoriasis, juvenile rheumatoid
inflammatory effect on rheumatoid arthritis.
ulcerative stomatitis),
arthritis, and severe rheumatoid
methotrexate-induced
arthritis.
lung disease, malignant
lymphomas, blood (neutropenia,
thrombocytopenia), bone marrow
(myelosuppression), renal
(azotemia), Pulmonary toxicity
Etoposide [77, 78]

Etoposide inhibits DNA topoisomerase II, hindering DNA
Bone marrow (myelosuppression), It is applied to the management

— SECTION 20 —
re-ligation that results in essential DNA synthesis errors during the
hematologic (leukopenia,
of testicular and small cell lung
prophase of cellular division and ultimately leading to the death of
neutropenia), and mucosal
cancers.
malignant cells through apoptosis.
toxicity.
Irinotecan [79–81] Prevents replication and induces death by inhibiting the activity of
Hematologic (neutropenia),
Used in the treatment of
topoisomerase I.
gastrointestinal (diarrhea,
metastatic carcinoma (rectum
nausea, vomiting), constitutional
symptoms (asthenia).
or colon).

320
TABLE 11.1 (Continued)
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Chemotherapeutic Mechanism of Action
Dose Limiting Toxicities
Therapeutic Application
Agent
Cisplatin [82–85]
Three mechanisms: (i) Alkyl groups bind to DNA bases, causing
Renal (nephrotoxicity), neurology It is used to treat various,
DNA breakage. This prevents the damaged DNA from being used to (neurotoxicity), auditory
carcinomas, sarcomas, germ

create new DNA or produce RNA; (ii) cross-linking induces DNA
(ototoxicity), Bone marrow
cell tumors like ovarian and
damage by inhibiting DNA division for synthesis or transcription;
(myelosuppression)
testicular cancers, lymphomas.
and (iii) the formation of nucleotide mispairing leads to mutations.
Levamisole [86–88] Levamisole’s ability to inhibit parasite growth may be attributed to
Bone marrow (myelosuppression), It is used to treat some skin and
its agonistic effect at nicotinic receptors in nematode muscle, which neurotoxicity, vertigo.
helminth infections and as an
causes spastic paralysis. Levamisole improves T-cell response by
adjuvant in treatment of colon
promoting the induction and progression of T-cells, boosting the
cancer.
activities of monocytes and macrophages, and recovering impaired
immunological function.
Prednisolone
Prednisone impedes polymorphonuclear leukocyte movement
Hematologic (neutropenia),
It is used to treat some cancers,
[89–91]
and diminishes the increased capillary permeability to lessen
skeletal wasting.
inflammatory conditions, and
inflammation. It suppresses NF-Kappa B and other inflammatory
adrenocortical insufficiency.
transcription factors, impedes neutrophil death and demargination,
restrains phospholipase A2, which lessens the production of
arachidonic acid derivatives, thereby promoting anti-inflammatory
genes such as interleukin-10.
Lenalidomide
Lenalidomide affects cytokine generation, controls co-stimulation
Hematologic (anemia,
In low to intermediate risk
[92–94]
of T-cells, and increases NK cell-mediated cytotoxicity to manifest
neutropenia, thrombocytopenia),
myelodysplastic syndrome, it is
its immunomodulatory effects. By preventing the growth and
bone marrow (myelosuppression), used to treat multiple myeloma
promoting death in tumor cells, lenalidomide directly combats
thyroid (hypothyroidism),
and anemia.
tumors. By preventing the release of angiogenic growth factors by
non-neutropenic sepsis.
tumor cells, it has anti-angiogenic effects.

TABLE 11.1 (Continued)
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322
TABLE 11.1 (Continued)
Chemotherapeutic Mechanism of Action
Dose Limiting Toxicities
Therapeutic Application
Agent
Gemcitabine
Following tumoral transformation into bioactive triphosphorylated
Bone marrow (myelosuppression), It is used as the only
[95–97]
nucleotides that interfere with DNA synthesis and target
hematologic (thrombocytopenia),
medication to treat pancreatic
ribonucleotide reductase, gemcitabine exerts its antiproliferative
asthenia.
cancer. Several types of cancer,
effect. Additionally, it has self-potentiating pharmacological effects
such as non-small cell lung
that improve the chances of gemcitabine triphosphate successfully
cancer, metastatic breast cancer,
binding to the DNA chain.
and ovarian cancer, benefit
from its usage as adjuvant
therapy.
Vincristine [98, 99] The antitumor activity of vincristine is primarily attributed to its
Neurology (neurotoxicity,
Used in treatment of acute
suppression of mitosis metaphase through its association with
peripheral neuropathy), bone
erythremia, acute panmyelosis,
tubulin. Additionally, vincristine may impact various processes,
marrow (myelotoxicity).
Hodgkin’s disease, malignant
including cellular respiration, ATPase activity, calmodulin-
dependent Ca2+-transport, glutathione metabolism, cyclic AMP, as
well as amino acid, nucleic acid, and lipid biosynthesis.
lymphoma, and acute leukemia.
Docetaxel
Docetaxel hinders the normal function of microtubule growth by
Bone marrow suppression,
It can treat several
[100–102]
attaching to the β-subunit of tubulin. Additionally, it attaches to the
hematologic (neutropenia), skin
malignancies, such as gastric
apoptosis inhibitor protein Bcl-2, causing neoplastic cells to undergo and neurosensory toxicity.
adenocarcinoma, head and neck
apoptosis.
cancer, locally advanced breast
cancer, metastatic (breast and
prostate).
Oxaliplatin
Oxaliplatin is converted non-enzymatically by displacement of
Nervous system (neurotoxicity,
It is used to treat stage III colon
[103–105]
the labile oxalate ligand to its active metabolites. After activation,
peripheral sensory neuropathy).
cancer as well as rectum or
oxaliplatin predominantly binds to the guanine and cytosine moieties
colon carcinoma.
of DNA, causing cross-linking and inhibiting the synthesis of new
DNA as well as the transcription of existing DNA.
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323
such as prostaglandin (PG) E2, IL-6, and TGF-β, which significantly hinder
cell cytotoxicity. Additionally, they recruit and activate immunosuppressive
cells through chemokines like CCL2, CCL5, and CCL22 secreted by tumor
cells [110]. The transition from a pro-inflammatory Th1 response to an
immunosuppressive Th2 response is brought on by infiltrating regulatory
cells (Treg, Breg, and Treg cells), M2-like tumor-associated macrophages,
immature DCs, CD4+ Th2 and ILC2s cells, and M2-like tumor-associated
macrophages [111], facilitated through the discharge of IL-4, IL-5, IL-10,
IL-35, IL-13, and TGF-β [112]. Also, tumor cells hamper cytotoxic T
lymphocyte (CTL) because of metabolic exhaustion [113]. Tumor cells
outrun T-cells to acquire nutrients like amino acids and glucose as well
as oxygen [114]. Immature DCs or MDSCs and Treg cells, expression of
ectonucleotidases (CD39 and CD73), Indoleamine 2,3-dioxygenase (IDO),
arginase-1 by tumor cells, further attribute to T-cell nutritional deficiency
and exhaustion by diminishing the tryptophan levels as well as rising its
immunosuppressive catabolite kynurenine concentration, through restricting
arginine’s accessibility and by hydrolyzing ATP to an extremely immuno­
suppressive adenosine [115]. Additionally, Treg cells defend IL-2, which is
essential for T-cell viability and function, by upregulating the expression of
the CD25, an IL-2 receptor alpha-chain [116].

— SECTION 24 —
11.3.3 RESISTANCE OF CANCER STEM CELLS (CSC) TO
CHEMOTHERAPY
According to two distinct hypotheses, tumors are constituted by hetero­
geneous cell subpopulations. One hypothesis is the stochastic or clonal
evolution type, while the other is the CSC model or hierarchical [117,
118]. In the hierarchical model, tumor growth is initiated by a small cell
subpopulation known as tumor initiating cells (TICs) [119]. According to
the hierarchical assumption, CSCs are a minor subpopulation of cells within
tumors that possess assets such as multipotency, self-renewal, and differen­
tiation capabilities. Furthermore, cues from the stromal milieu, pre-existing
somatic mutations, cell type source, and stage of malignant progression can
all influence CSC-like properties [120]. Resistance to chemotherapy [121],
immunotherapy [122], and radiotherapy [123] are displayed by these cells
and their TICs [124].
The chemotherapeutic resistance of CSCs/TICs, which have undergone
an epithelial to mesenchymal transformation, appears to be higher [125].

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Cancer Vaccination and Challenges, Volume 2
An increase in aldehyde dehydrogenase (ALDH) activity in these cells
seems to mediate resistance to certain chemotherapeutic agents [126].
The ability of CSCs/TICs to present tumor antigens (TAs) to T cells for
immune detection or immune response is determined by the expression
of antigen presentation molecules such as Programmed death receptor 1
(PD-1)/-1L], coinhibitory molecules [e.g., cytotoxic T-lymphocyte antigen
4 (CTLA4), B7-H2, and B7-H3], co-stimulatory molecules (e.g., CD80,
CD86), as well as MHC-I [127]. Resistance to conventional therapies is
significantly associated with the intrinsic mechanisms involved in CSC/
TIC resistance to chemotherapy or radiation therapy. Immunosuppressive
chemicals produced by CSCs and TICs can impair the effectiveness of the
immune system [128].

— SECTION 26 —
11.4 PARTIALLY USED CRITICAL WEAPON AGAINST CANCER
Although chemotherapy acts on multiple hallmarks of cancer, the immu­
nosuppression induced by cytotoxicity to the bone marrow (BM) makes
patients more vulnerable to cancer recurrence and chemo resistance. This
situation also increases the susceptibility of immunocompromised cancer
patients to other infectious diseases.
11.4.1 IMMUNOTHERAPY
The outcomes achieved in terms of robust positive response through immune-
based therapies surpassed the expectations. For their groundbreaking break­
throughs in the field of immunotherapy, two outstanding scientists, namely
James Allison and Tasuku Honjo, have just been awarded the 2018 Nobel
Prize in medicine. Their findings shared the same approach of utilizing the
immune system to combat cancer. Since 2011, the FDA has approved several
medications based on the suppression of immunological checkpoints [129].
Most of these treatments have utilized antibodies that target the Programmed
cell death 1 (PD-1) or CTL-associated protein 4 (CTLA-4) pathways, either
singly or in combination [130]. Based on the expression of the tumor-
associated antigen, which indicates the type of “stemness” of a cell, T cells
can recognize CSCs/TICs. Because somatic point mutations in tumor cells
might result in the creation of completely new epitopes, the immune system
is able to identify altered antigens [131].

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— SECTION 28 —
11.4.2 CAR T-CELL THERAPY
Research on the transfer of chimeric antigen receptor (CAR) T cells is
currently being conducted [132] and has fantastic potential for the manage­
ment of both liquid and solid tumors [133]. A single chain fragment on the
antigen receptor of CAR T cells links it to an intracellular region, and it is
typically complemented by a co-stimulatory protein. Due to their capacity
to differentiate any antigen present on the surface of CSCs, CAR T cells
provide a good substrate for the creation of selective CSC therapies [134].
11.4.3 IMMUNE-BASED MONOCLONAL ANTIBODIES AND CHECK
POINTS INHIBITORS
The presence of immune-based abnormalities in cancer presents an oppor­
tunity to develop therapy aimed at resolving it and utilizing it as a natural
defense against cancer. Monoclonal antibodies (mAbs) derived from plasma
cells bind to the epitope part of the antigens, activating tumoricidal effects
simultaneously with other cellular immune and complement mechanisms.
The use of mAbs offers the advantage of long-term memory, which is lacking
in chemotherapy [135]. Among the five types of natural antibodies used to
produce mAbs, IgG is the most commonly employed due to its extensive
interactions with monocytes, NK cells, neutrophils, and DCs [136]. Targeted
tumor-associated antigens (TAs) are typically overexpressed on the surfaces
of cancer cells. For example, cetuximab suppresses the growth factor receptor
EGFR, leading to the death of tumor cells [137]. Trastuzumab prevents the
overexpression of Human Epidermal Growth Factor Receptor 2 (HER2)
from internalizing and heterodimerizing [138]. Rituximab, used to treat non-
lymphoma Hodgkin’s and myeloma, demonstrates more efficacy and less
toxicity compared to conventional therapy by specifically targeting CD20 on
tumor cells [139]. Immune checkpoints are proteins that regulate costimula­
tory and coinhibitory immune responses, acting as brakes. They are present
on immune cells to prevent the development of autoimmune conditions.
When counter proteins are present on opposing cells, immune activation
is inhibited, making immune checkpoints function as inhibitory receptors
[140]. Different proteins with checkpoint functions include Programmed
death receptor-1 (PD-1), which is linked to apoptosis of T cells; Cytotoxic
T lymphocyte antigen-4 (CTLA-4), expressed by Treg cells, NK cells, and
Treg cells; and lymphocyte activation gene 3 (LAG3), responsible for T

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cell activation and secretion of immune response-related cytokines. Other
less explored checkpoints are T cell immunoglobulin and immunoreceptor
tyrosine-based inhibitory motif domain (TIGIT), V-domain Ig suppressor
of T cell activation (VISTA), as well as T cell immunoglobulin and mucin
domain-containing 3 (TIM3) [141]. FDA-approved checkpoint inhibitors
include Ipilimumab, Pembrolizumab, Nivolumab, Cemiplimab, Atezoli­
zumab, and Opdualag. Recent developments in the field of drug discovery
have led to the invention of antibody drug conjugates (ADC), which can be
considered as an amalgamation of two different therapies. ADCs utilize the
specificity of monoclonal antibodies and the toxicity of chemotherapeutic
agents [142] (Figure 11.3).
FIGURE 11.3 Different mechanisms of antitumor immunity.
Pathways to tumoricidal destruction by immunity:

• Generation of tumor associated antigens (TAAs) from tumor cells.
• Stimulation of DC by TAAs.
• DCs and the major histocompatibility complex (MHC) [MHC-I,
  MHC-II] complex function as antigen-presenting cells (APCs) to
  activate helper T (Th) cells and cytotoxic lymphocytes (CTLs).

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— SECTION 30 —

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327

• Activation of Th-17 cells leads to the release of interleukin 17
  (IL-17), which induces tumor cells to secrete chemotactic factors
  – CXCL9 and CXCL10, followed by the subsequent recruitment of
  cytotoxic effectors – NK cells and CD8+ cells.

— SECTION 31 —

• Stimulation of Th-9 cells results in the release of Interleukin 21
  (IL-21) along with Interleukin 9 (IL-9), which also leads to the
  activation and generation of cytotoxic cells.
• Interferon (INF-γ), IL-2, and tumor necrosis factor (TNF-α) are
  released upon activation of Th-1 cells, triggering the STAT-3
  pathway to produce antitumor antibodies.
• Cytotoxic cells, together with B cells, exert their antitumor effect
  and induce apoptosis.
• Immunological memory aids in the generation of cytotoxic cells
  during tumor remission.
• Memory T cells contribute to the development of an immune
  response against re-challenged tumor cells.
  To date, the FDA has approved a total of 12 antibody-drug conjugates
  (ADCs) for various types of cancer. Despite significant advancements in
  monoclonal antibody (mAb) therapy, the issue of resistance persists, similar
  to chemotherapy. The development of resistance can be considered as
  a form of therapeutic resistance. This can be attributed to factors such as
  the emergence of new mutations in the targets (e.g., CD20 in the case of
  rituximab, S492R in the case of cetuximab), the generation of new vari­
  ants, immunoediting of the tumor, epithelial to mesenchymal transition
  (EMT), immune selection processes, and the activation of secondary growth
  signaling pathways by tumors [143, 144].

— SECTION 32 —
11.5 THE BOON OF MEDICAL PRE-INTERVENTION TO THE
MANKIND-VACCINE
The term chemotherapy is used to describe the elimination of cancerous or
infectious cells, which have different surface proteins compared to normal
cells. A British physician demonstrated the effectiveness of vaccines by
discovering the smallpox vaccine. According to the CDC, more than 18
deadly and highly disabling diseases are now under control thanks to the
invention of vaccines. Recent reports have shown positive outcomes in
reducing fatality rates for COVID-19 vaccinations [145].

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— SECTION 34 —
11.5.1 VACCINE FOR CHRONIC DISEASES THAT INCREASE THE RISK
OF CANCER DEVELOPMENT
As per the National Cancer Institute (NCI), many infectious agents chroni­
cally alter the tissue milieu and provoke cancer development. The infec­
tious agents, such as bacteria and parasites, secrete certain inflammatory
molecules, while viruses can modify the genotype of the host DNA. Even
the secretion of immunosuppressive chemicals also favors the initiation of
cancer chronically. Many virus infections chronically lead to carcinogen­
esis, such as Epstein-Barr Virus (EBV) linked to lymphoma, Hepatitis B
and C linked to liver cancer, Human Immunodeficiency Virus (HIV) linked
to Kaposi sarcoma, liver and lung cancer, and Human Papillomaviruses
(HPVs) responsible for reproductive tract, head and neck cancer. Addition­
ally, infection with Helicobacter pylori increases the occurrence of gastric
cancer [146]. A list of specific vaccines that not only prevent the occurrence
of infectious diseases but also halt carcinogenesis in the long run are given
in Table 11.2.

— SECTION 35 —
11.5.2 AVAILABILITY OF SPECIFIC ANTIGENS ON THE TUMOR
CELLS
Opportunities to develop cancer vaccines (CVs) are increased by the presence
of specific tumor-associated antigens (TAs). The mutated genes transcribe
mRNA that encodes peptides or proteins, which can be detected in blood or
cancerous tissues. A single amino acid change in the peptide or frameshift
mutation can be considered abnormal and create a neoantigen. Immune cells
can recognize these altered patterns. Aberrant proteins (antigens) are present
due to abnormal genetic mutations. The causative genes include CDK4,
CTNNB1, CASP8, KRAS, BCR-ABL, and P53 [197]. The proteins encoded
by these mutated genes confer uncontrolled cell proliferation, resistance to
apoptosis, and sometimes immune evasion. All of the aforementioned genes
encode highly specific TAs. Other low-specific TAs include prostate-specific
antigen (PSA) and carcinoembryonic antigen (CEA) [198]. Overexpres­
sion of certain proteins on the tumor surface beyond a threshold level also
promotes recognition by cytotoxic T lymphocytes (CTL). Different genes
responsible for overexpression include RAGE-1, PRAME, and ERBB2
(HER2/NEU) [199]. There are antigens like Mucin-associated sialyl-Tn (sTn)
antigens that interact with immune subsets and create an immunosuppressive

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PAGE 348

FDA Approved
Types of Vaccine
Mechanism
Immunization Potential
Application
Brand of Vaccine
ENGERIX-B
Hepatitis B vaccine
Produces targeted humoral
Antibody levels against HBsAg of less than
Active immunization against all subtypes
[147–150]
(recombinant).
antibodies against the
10 mIU/mL are recognized as providing
of hepatitis B virus infection.
hepatitis B viral surface
antigen (HBsAg).
protection from hepatitis B virus infection.

— SECTION 37 —
GARDASIL 9
Human papillomavirus
Produces highly pure virus-
It was found to be >90% effective and
Indicated for the inhibition of
[148, 151, 152]
9-valent vaccine
like particles (VLPs) of the
generated antibody response against 9
oropharyngeal, genital, as well as head and
(recombinant).
primary capsid L1 protein
subtypes of HPV.
neck cancers, precancerous or dysplastic
that initiate the humoral
immune response.
lesions caused by HPV subtypes.

— SECTION 38 —
TICE BCG
BCG live
Generates antibodies
The effective success rate of TICE BCG was
For the management and prevention of
[154–157]
and antigen-specific
found to be 90% for bladder cancer.
urinary bladder carcinoma in situ (CIS),
T-cell response against
as well as for the prevention of initial or
tuberculosis and acts as an
recurring Ta and/or T1 papillary tumors
immunotherapy against
bladder cancer.
following transurethral resection (TUR).

— SECTION 39 —
PREHEVBRIO
Hepatitis B vaccine
Produces targeted humoral
It is known that antibodies against HBsAg at
To prevent infection induced by all known
[158, 159]
(recombinant).
antibodies against the
concentrations of less than 10 mIU/mL offer
hepatitis B virus strains.
hepatitis B viral surface
antigen (HBsAg).
protection from hepatitis B virus infection.

— SECTION 40 —
CERVARIX
Human papillomavirus
Stimulates the production

Provides 80% protection against HPV
It is recommended for the prevention of
[160–163]
bivalent (types 16 and 18)

of antibodies against HPV
subtypes known to cause female genital
conditions such as anal, vulval, cervical,
vaccine (recombinant)
types 16 and 18.
oncogenic cancer.
and vaginal cancers caused by carcinogenic
HPV types 16 and 18.
TABLE 11.2 The Potential of Vaccines in the Prevention of Dangerous Diseases That can Chronically Transform into Cancer Development
Cancer Vaccine and Chemotherapeutic Agent: Advances in Cancer Management
329

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PAGE 349

FDA Approved
Types of Vaccine
Mechanism
Immunization Potential
Application
Brand of Vaccine
VAXELIS
Diphtheria, tetanus, and
Induces the production of
Antibody levels of ≥1.0 µg/mL against
It offers active immunization against
[164–167]
polio toxoids, inactivated specific antibodies against
polyribosylribitol phosphate (PRP) are
haemophilus influenzae type b and
poliovirus, adsorbed
influenzae type b, diphtheria, considered as a safeguard against haemophilus diphtheria-related invasive infections
acellular pertussis
haemophilus, pertussis,
influenzae type b, ≥0.1 IU/mL against
(Hib), tetanus, pertussis, poliomyelitis, and
vaccine, haemophilus B
hepatitis B, tetanus.
diphtheria, ≥0.1 IU/mL against tetanus, and
hepatitis B.
conjugate, and hepatitis B
≥10 mIU/mL against HBsAg are considered as
vaccine.
a safeguard for hepatitis B virus infection.

— SECTION 42 —
RECOMBIVAX
Hepatitis B vaccine
Produces specialized
Antibody levels of ≥10 mIU/mL against
Vaccination is intended to prevent infection
HB [168, 169]
(recombinant)
humoral antibodies against
HBsAg are known to offer protection against
caused by established subtypes of the
the hepatitis B surface
antigen (HBsAg).
hepatitis B virus infection.
hepatitis B virus.
GARDASIL [170, Human papillomavirus
Produces humoral immune
It was found to be >90% effective and
This is used to provide active immunity
171]
quadrivalent (types 6, 16, response initiated from
generated antibody response against 4
against anal, vaginal, vulvar, cervical
11, as well as 18) vaccine highly purified virus-like
subtypes of HPV.
cancer, as well as precancerous or
(recombinant)
particles (VLPs) of the main
capsid L1 protein.
dysplastic lesions and genital warts.

— SECTION 43 —
HEPLISAV-B
Hepatitis B vaccine
Creates specific humoral
Antibody levels of ≥10 mIU/mL against
For preventing infection brought on by any
[172, 173]
(recombinant),
antibodies to the hepatitis
HBsAg are regarded as offering safeguard
known hepatitis B virus subtype.
adjuvanted.
B virus surface antigen
(HBsAg).
against hepatitis B virus infection.

— SECTION 44 —
TWINRIX [174,
Hepatitis A inactivated
Generates antibodies to
Antibody titer values for hepatitis A depend
Provides active immunization of people
175]
and hepatitis B
hepatitis A virus (anti-HAV) upon the assay used, whereas antibody
aged 18 and older against infection with
(recombinant) vaccine
and antibodies to fight
concentration against HBsAg of ≥10 mIU/
all known hepatitis B virus subtypes and
against hepatitis B virus
mL is known to provide protection against

diseases brought on by the hepatitis A
surface antigen (HBsAg).
hepatitis B virus infection.
virus.

330
TABLE 11.2 (Continued)
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PAGE 350

FDA Approved
Types of Vaccine
Mechanism
Immunization Potential
Application
Brand of Vaccine
Liquid PedvaxHIB Haemophilus B conjugate Stimulates production
The antibody concentration of >0.15 to >1.0
Infants and children between the ages
[176–179]
vaccine (meningococcal
of antibodies against
g/mL against PRP of the haemophilus B
of 2 and 71 months are recommended
protein conjugate).
polyribosyl ribitol
capsule is regarded as a safeguard to provide
to receive routine vaccination of liquid
phosphate (PRP) antigen of
protection against haemophilus influenzae
PedvaxHIB against invasive disease caused
haemophilus B capsule.
type B.
by haemophilus influenzae type b.
ActHIB [180, 181] Haemophilus b conjugate Causes the development
To provide protection against haemophilus
Vaccination for preventing haemophilus
vaccine (tetanus toxoid
of antibodies against the
influenzae type B, an antibody concentration
influenzae type b-induced invasive
conjugate).
haemophilus B capsule’s
of >0.15 to >1.0 g/mL against the PRP of the
diseases.
polyribosyl ribitol phosphate haemophilus B capsule is considered to be a
(PRP) antigen.
safeguard.

— SECTION 46 —
HIBERIX
Haemophilus b conjugate Promotes the production
Haemophilus influenzae type B protection is
For the purpose of actively vaccinating
[182–190]
vaccine (tetanus toxoid
of antibodies against the
provided by antibodies with concentrations
against invasive infection caused by
conjugate).
haemophilus B capsule’s
of >0.15 to >1.0 g/mL against the PRP of the
haemophilus influenzae type b.
polyribosyl ribitol phosphate
(PRP) antigen.
haemophilus B capsule.

— SECTION 47 —
PENTACEL
Tetanus toxoids,
The synthesis of specific

The antibody concentration of ≥1.0 µg/mL
For effective protection against invasive
[191–194]
diphtheria toxoids,
antibodies that are effective against PRP is considered as a safeguard
infections brought on by diphtheria,
acellular pertussis
against the following
against haemophilus influenzae type b.
tetanus, pertussis, poliomyelitis, and
adsorbed, inactivated
diseases is increased:
A concentration of ≥0.1 IU/mL against
haemophilus influenzae type b.
poliovirus and
Diphtheria, tetanus,

diphtheria and tetanus, and ≥10 mIU/mL
haemophilus b conjugate haemophilus influenzae type against HBsAg are considered as safeguards
(tetanus toxoid conjugate) B, hepatitis B, pertussis, and for hepatitis B virus infection. Additionally,
vaccine.
poliovirus.
an antibody concentration of 0.075–0.180 IU/
ml is required to provide protection against
poliovirus.

TABLE 11.2 (Continued)
Cancer Vaccine and Chemotherapeutic Agent: Advances in Cancer Management
331

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PAGE 351

332
TABLE 11.2 (Continued)
FDA Approved
Types of Vaccine
Mechanism
Immunization Potential
Application
Brand of Vaccine
PEDIARIX [195, Diphtheria toxoids,
Boosts the synthesis of
An antibody concentration of 0.075–0.180
PEDIARIX is recommended for active
196]
acellular pertussis
specific antibodies that are
IU/ml is required to provide protection
vaccination against tetanus, poliomyelitis,
adsorbed, tetanus toxoids, effective against diphtheria, against poliovirus. Concentrations of ≥1.0
diphtheria, pertussis, and infections
hepatitis B (recombinant) tetanus, haemophilus
µg/mL against PRP are considered as a
brought on by all recognized subtypes of
and inactivated poliovirus influenzae type B, pertussis, safeguard against haemophilus influenzae
the hepatitis B virus.
vaccine combined.
hepatitis B, and poliovirus.
type b. Concentrations of ≥0.1 IU/mL against
tetanus and ≥10 mIU/mL against HBsAg are
considered as safeguards for hepatitis B virus
infection. Lastly, a concentration of ≥0.1 IU/
mL is required to provide protection against
diphtheria.
Cancer Vaccination and Challenges, Volume 2

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Cancer Vaccine and Chemotherapeutic Agent: Advances in Cancer Management
333
environment, thereby weakening the cytolytic capacity of these cells and
even promoting metastasis [200]. These antigens can be used as diagnostic
and prognostic markers for multiple cancers. CEA and CA 15-3 can be used
to monitor breast cancer treatment efficacy and predict progression to an
advanced stage [201].

— SECTION 50 —
11.5.3 FDA APPROVED CVS
The first experiment on the use of therapeutic vaccines was conducted by
Dr. William Coley in 1890 to treat patients suffering from sarcoma. Due to
the potential failure of existing therapies and the involvement of immune
components in cancer development, numerous attempts have been made to
develop cancer vaccines (CVs). CV is part of Immunotherapy [202]. CVs
encompass various modalities, including peptide-based, whole cell-based,
protein-based, DC-based, mRNA-based, and DNA-based approaches [19].
The world is in need of prophylactic personalized and therapeutic vaccines
to reduce cancer-related morbidity and mortality. The FDA has approved
four preventive CVs. Cervarix is used to prevent cervical, head, and neck
cancer caused by HPV 16 and 18 infections. Gardasil and Gardasil-9 target
other strains of HPV and provide protection against throat cancer and other
cancers. HEPLISAV-B offers protection against hepatitis B virus (HBV)­
induced liver cancer. Sipuleucel-T, approved by the FDA, is a vaccine for
advanced stage prostate cancer based on the presence of prostatic acid phos­
phatase (PAP). Bacillus Calmette-Guérin (BCG) has FDA approval for the
treatment of early-stage bladder cancer by activating general immunity [156].
Numerous CVs are currently in clinical trials, including Mammaglobin-A
(NCT02204098), QUILT-3.013 (NCT02751528) for breast cancer, and
Granulocyte Macrophage Colony-Stimulating Factor (GM-CSF) with Folate
Receptor Alpha Peptide for Triple Negative Breast Cancer (NCT02593227).
These trials are currently in progress [204].

— SECTION 51 —
11.5.4 CHALLENGES AHEAD
Although the success achieved for marketed CVs and many that are under
clinical trials, there is concern regarding the complexity related to antigens
and immunity. Tumors with low immunogenicity, changes in the heterog­
enous population of cancer cells, the presence of glycocalyx covering the
surface antigen, the immunocompromised state of many diseases such as

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Cancer Vaccination and Challenges, Volume 2
acquired immunodeficiency syndrome (AIDS) and tuberculosis, changes
in major histocompatibility complex (MHC) interaction with antigens, the
systemic prevalence of immunosuppression, and the development of large
tumor size at advanced stages of malignancy all pose great challenges that
need to be resolved. There is evidence available that indicates a complex
correlation between vaccine-stimulated immunity and therapeutic benefits
[205]. The ever-changing heterogenous population of tumors and the

— SECTION 52 —
unsteady tumor microenvironment create difficulties in drawing conclusions
about therapeutic vaccines. To overcome all these obstacles of immune
tolerance and evasion, adjuvant therapy is needed with CVs [206]. The
preclinical screening model cannot be exactly extrapolated to the clinical
situation, making it difficult to predict vaccine efficacy [207]. If an antigen
has low binding capacity to MHC, it should be administered at a high dose
to properly stimulate the lymphatic system of defense [208]. Improving the
biopharmaceutical properties of vaccines is difficult due to inadequate data
on vaccine ADME (absorption, distribution, metabolism, and excretion)
[209]. Sometimes, commercialization pressure urges the design of trials with
insufficient dosing schedules [210, 211].

— SECTION 53 —
11.5.5 THERAPEUTIC CV WITH CHEMO AGENTS – CONCEPT OF
SYNERGISM
The concern of multiple hallmarks in cancer development and progression
makes it necessary to suppress it from multiple sites to achieve sustainable
progression-free life with an increase in overall survival (OS). The abscopal
effect induced by radiation was first observed in 1950 during a preclinical
experiment. It demonstrated that non-irradiated tumors shrink after the irra­
diation of the main tumor. The abscopal effects occur due to the awakening of
systemic immunity. The exposed tumor releases certain cytokines that signal
the immune system to recognize and eliminate similar tumors throughout
the body. Thus, the exposed tumor could act as a live vaccine [212]. A more
beneficial synergism can be achieved in the case of chemotherapy and cancer
therapeutic vaccine. Cyclophosphamide (CTX) induces immunogenic
tumor cell death, releasing many tumor-associated antigens and activating
systemic immune response. Previous studies provide clues for the success
of chemotherapy with immunomodulators. Chemotherapy not only causes
cytotoxicity but also modulates host immunity. The positive changes include
exaggerated immunogenicity of cancer cells, activation of tumoricidal cells

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Cancer Vaccine and Chemotherapeutic Agent: Advances in Cancer Management
335
(CD8+ T lymphocytes, NK cells), and secretion of cytokines like TNF-α and
interferon gamma [213]. The combination of granulocyte colony-stimulating
factors (CSFs) with CTX has shown clear clinical benefits for advanced
pancreatic cancer with reduced toxicities [214]. Treatment for hepatocellular
carcinoma (HCC) is challenging with chemotherapy and shows a poor five-
year survival rate. The development of resistance due to an immune suppres­
sive milieu and patients with cirrhosis condition show poor response and
resistance to chemotherapy alone [215]. The combined approaches improve
the therapeutic outcomes due to tumor cell death by antibodies, stimulation
of NK cells and T lymphocytes, while suppressing Treg cells. Chemotherapy
overcomes immunosuppression, enhances the interaction of tumor cells with
immunological components, and alters TME subpopulations [216, 217].
CTX-induced immunomodulation enhances the HER-2 specific monoclonal
antibody vaccine-induced anticancer response in neu mice and breast cancer
patients [218]. The inoculation of CTX-loaded hydrogel with tumor lysate
in CT26 colorectal tumor-bearing mice shows promising outcomes in terms
of tumor growth inhibition, increased survival rate, and sustained immune-
based memory to prevent recurrence [219]. Pancreatic cancer has a low
cure rate with chemotherapy alone, but combination with peptide-based CV
shows great hope for the patients [220].

— SECTION 55 —
11.6 NOVELTY, NEW POSSIBILITIES, AND FUTURE PERSPECTIVES
Currently, various antigens or proteins are being screened for the development
of cancer vaccines (CVs). These include mesothelin (targeting metastasis),
WT1 (for Wilms’ tumor), telomerase, and survivin (targeting the immortality
of tumor cells). Virus-infected tumor cells express virus-derived proteins that
can be used as targets for vaccine-based therapy [221]. Neoantigen-based
vaccines may be available, sparing normal cells and targeting tumor cells
more aggressively. Currently, researchers are developing personalized CVs
based on massive parallel sequencing of epitopes and neoantigens from indi­
vidual patients [222]. The use of preparative chemotherapy with CTX and
fludarabine supports the treatment of autologous tumor-infiltrating lympho­
cytes for melanoma patients by increasing the level of IL-15, which dampens
the activity of Treg cells [223]. Nano-technology-based formulations need
to be developed to gain more advantages in targeted drug delivery to cancer
cells and dose reduction [224]. In the future, the use of multiple antigens
targeting vaccines may achieve a robust response against malignancy from

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— SECTION 56 —

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Cancer Vaccination and Challenges, Volume 2
various immune cells [203]. Resistance development after discontinuation of
chemotherapy can be managed by vaccination at certain time intervals [153].

— SECTION 57 —
11.7 CONCLUSION
The multifactorial nature of cancer must be suppressed with the combined
edge of chemotherapy and vaccines. Monotherapy with either of these
cannot fulfill the goal of cancer treatment. The chances of cure rate,
disease progression-free life, and overall survival are improved when using
combination therapy, while the development of resistance and recurrence is
minimized. The ever-changing nature of cancer may necessitate the use of
specific chemotherapy based on the prevailing cancer, along with the use of
personalized vaccines. Many more studies are indeed required to find hope
for the deadliest cancers, such as lung, colorectal, stomach, and pancreas.
KEYWORDS

• 
  cancer vaccine
• 
  chemotherapeutic agents
• 
  chemotherapy
• 
  combination therapy
• 
  immunomodulation
• 
  immunotherapy
• 
  monotherapy
• 
  recurrence

— SECTION 58 —
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PAGE 372

Index
A
Abscesses, 277
Absorption, 24, 26, 33, 75, 81, 200, 201,
276, 277, 296, 334
Accounting, 55, 134, 235
Accumulation, 32, 33, 54, 80, 81, 137, 247,
282
Acidic pH values, 83
Acids, 97
Acquired immunodeficiency syndrome

— SECTION 77 —
(AIDS), 283, 334
Acquisition, 94, 215
Activation, 3, 4, 13, 14, 26, 27, 30, 31,
82, 98, 102, 104–110, 115, 121, 122,
124, 125, 135, 145, 146, 149, 152, 156,
159, 160, 162, 179, 181, 184, 188, 197,
198, 202, 219, 241, 250, 254, 255, 259,
273–275, 281, 282, 284, 288, 290, 291,
315–318, 322, 325–327, 334
Actuator, 13
Acupuncture, 311
Acute myeloid leukemia, 293
Adaptive immunity, 97, 102, 121, 279, 291,
296
Adaptor, 106
Additives, 7
Adenocarcinoma, 65, 119, 134, 137, 140,
204, 219, 220, 320, 322
Adenosine, 320, 323
triphosphate, 320
Adenovirus, 16, 35, 66, 149, 154, 155
genome, 16
vector, 16
Adherent cultures, 7
Adhesion, 142, 154, 249, 310
Adjuvant, 3, 20, 22, 23, 26, 27, 29–32,
35, 65, 111, 114, 156, 159, 160, 212,
217–219, 221, 237, 238, 251, 253,
267–282, 284–295, 298–301, 321, 322,
334
treatment, 251, 288
Adoptive
cellular therapy, 161
transfer (AT), 248, 282, 313
Adsorption, 277, 280, 292
Aerosols, 63
Air pollution, 137
Aldehyde dehydrogenase, 324
Alginate
chitosan, 83
cyclodextrin, 83
keratin composite, 83
liposome hydrogels, 83
polyamidoamine (PAMAM), 21, 28, 81–83
Algorithms, 115, 213
Allergens, 278
AlO(OH)-polymer nanoparticles, 292
Alum, 157, 160, 267, 276, 277, 279, 280,
284, 300, 301
Amino acids, 62, 67, 151, 279, 323
Amphiphilic peptide vaccines, 32
Anatomical barriers, 97, 98
Anatomy, 260
Anchoring, 294
Androgen-deprivation therapy, 182
Anemia, 236, 270, 312, 321
Anesthetics, 13
Aneuploid, 95
Aneuploidy, 85
Angiogenesis, 16, 18, 53, 58, 84, 101, 135,
141, 220, 253, 288, 311, 314
Animal lifespan, 10
Ankara, 256
Anthracyclines, 256, 311
Antibacterial medicines, 67
Antibiotics, 21, 247
Antibodies, 3, 4, 15, 28, 30, 32, 33, 64, 66,
80, 96, 102, 107–109, 114, 121, 136, 143,
149, 154, 156, 158, 159, 177, 198–200,
218, 241, 255, 278, 284, 288, 289, 315,
324, 325, 329–332, 335
antibody
drug conjugates (ADCs), 326, 327
mediated immunity, 107

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PAGE 373

354
Index
Anticancer
activity, 17, 81, 144, 156, 178, 245
agents, 63, 74
gene therapy, 67
therapy, 125, 126, 156, 180
Antigens, 1, 3–6, 9, 14, 15, 21, 22, 24, 26,
27, 29, 31–35, 56, 57, 59, 60, 64, 68, 69,
73, 75, 95–97, 99, 103, 105, 107–113,
115–119, 121–126, 133, 134, 136, 143,
144, 146, 147, 150–153, 155, 156, 158,
159, 180, 182, 184–187, 193, 196, 199,
201, 202, 204, 205, 211–215, 218, 220,
221, 237–241, 244, 246, 249, 251, 252,
254, 255, 259, 268, 269, 274, 276, 277,
286, 288–293, 295, 298, 299, 301, 315,
324, 325, 328, 333–335
binding domain, 143
neutral, 98
presentation, 21, 22, 33, 66, 116, 118,
150, 160–162, 175, 196, 213, 241, 249,
254, 256–258, 292, 293, 324
presenting cells (APCs), 1, 3, 4, 9, 14, 15,
22, 24, 31, 33, 57, 87, 98, 102, 104,
105, 110, 111, 117, 121, 122, 124, 136,
138, 148, 152, 158, 159, 161, 184, 196,
198, 199, 201, 202, 218, 220, 239–241,
245, 246, 250, 258, 268, 269, 273–276,
278, 279, 283–292, 299, 310, 313, 316,
326
processing and presenting machinery

— SECTION 79 —
(APM), 172, 254, 256
receptors, 102
Antigenic
determinants, 83
stimulation, 104, 126
Anti-infective vaccines, 54
Anti-inflammatory treatment, 82
Anti-neoplastic vaccines, 30
Anti-proliferation action, 82
Antitumor, 10, 26, 65, 95, 102, 110, 121,
124, 141, 144, 145, 150, 161, 163, 179,
200, 204, 212, 242, 268, 278, 283, 284,
290–292, 294, 312, 322, 326, 327
antibodies, 327
effect, 95, 179, 200, 204, 268, 284, 327
effector cells, 95
immunity, 99, 110, 120, 220, 259
Antitumorigenic M1 macrophages, 99
Apoptosis, 16, 53, 94, 135, 158, 160, 177,
180, 205, 251, 258, 309, 311, 313, 314,
316, 318–320, 322, 325, 328
Aqueous
environments, 29
media, 67
Archaeal lipid liposomes, 1, 73
Archaeosomes, 1
Arginine, 22, 99, 323
Aspirin, 83
Asthma, 213
Attenuation, 18, 297
Autoimmune
polyendocrine disorders, 104
regulator, 104
Autoimmunity, 113, 123
Autologous tumor cell-based vaccines, 212,
227
Automation, 67
Autophagy, 85, 285, 313
Autoreactive immune reactions, 106
Auxiliary transport systems, 68
Azurin protein, 18

— SECTION 80 —
B
Bacillus Calmette Guerin (BCG), 154, 155,
171, 237, 287, 329, 333
Bacteria, 17, 18, 54, 97, 148, 218, 243, 269,
295, 328
Bacterial vectors, 17
Barriers, 35, 96, 97, 199, 300
Basal cell carcinoma, 294
B-cell receptors (BCRs), 107, 109, 273
Bifidobacterium
adolescentis, 18
longum, 18
Bilayer, 30, 60, 61, 293, 298, 299
structure, 60
Bioavailability, 24, 79, 223, 293
Biocompatible, 30, 62, 80–82, 277
Biodegradable, 21, 36, 272
drug administration, 30
linear amino polysaccharide, 21
polymers, 21
Bioinspired hydrogels, 83
Biojector 2000, 5
administrations, 8
Biolistic approaches, 5

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— SECTION 81 —

PAGE 374

355
Index
Biological
hypermutation, 108
targets, 82
Biomarkers, 54, 161, 163, 226, 261
Biomaterials, 55, 286
Biomedicine, 277
Biomolecules, 94
Bionics, 84
Biopsy, 135, 215
Bioresorbability, 27
Bioresponsive hydrogels, 83
Bladder cancer, 171, 204, 247, 329, 333
Blockade, 34, 113, 119, 162, 212
Blood, 12, 76, 77, 82, 84, 100, 107, 112,
117, 120, 121, 142, 143, 145, 146, 211,
216, 221, 227, 235, 239, 240, 251, 297,
318, 320, 328
brain barrier, 297
pressure, 227
vessels, 12, 77, 84, 142, 318
Bone, 17, 68, 101, 107, 109, 142, 185, 287,
288, 296, 319–322, 324
marrow (BM), 68, 101–103, 107, 109,
274, 278, 285, 287, 297, 319–322, 324
Booster vaccination, 5, 280
Bordetella, 279
Bortezomib, 58
Bourquin, 26
Bovine, 293, 301
serum albumin (BSA), 284, 293, 294, 301
Breast cancer, 16–19, 26, 32, 82, 101, 115,
134, 200, 203, 239, 242, 252–254, 270,
278, 295, 297, 310–313, 322, 333, 335
Bridges, 85
Bypass, 15, 317

— SECTION 82 —
C
Calcium, 30, 105, 247, 271, 276
carbonate (CaCO3), 23, 27, 276
microspheres, 83
Calreticulin (CRT), 10, 254, 256, 258
Cancer, 2–4, 6, 7, 9–12, 15–20, 23–31,
33–36, 53–60, 62–64, 69, 73–75, 77–87,
93–96, 98, 99, 101, 102, 110–115,
117–126, 133–138, 140–147, 149–163,
171–173, 175–181, 184–188, 193,
194, 196–206, 211–220, 222, 226, 227,
235–239, 241–249, 252–262, 267–273,
276, 278–281, 283–291, 293–295,
297–301, 309–314, 316, 318, 319, 321,
322, 324, 325, 327–330, 333–336
antigens, 35
associated fibroblast, 142, 246, 312, 318
cells, 2–4, 15, 16, 35, 55, 56, 59, 60, 63,
74, 75, 77, 81, 84, 85, 87, 95, 112, 124,
135, 141, 152, 153, 155, 156, 158,
162, 172, 173, 176, 177, 179–181, 205,
211–213, 216, 219, 222, 236, 237, 241,
242, 244–246, 248, 249, 253, 256–260,
314, 319, 325, 333–335
immunology, 93, 95
immunotherapy, 9, 17, 31, 33, 110, 112,
135, 136, 141, 142, 144, 145, 152, 154,
155, 161, 177, 193, 197, 199, 200, 202,
206, 212, 216, 237, 243, 253, 272, 278,
291, 299
microenvironment (CME), 33, 124, 126,
162, 312
research advances, 5
stem cells (CSC), 81, 84, 204, 309, 313,
323–325
testis antigens, 56
therapy, 3, 17, 75, 78, 79, 81, 85, 87, 142,
172, 185, 186, 213, 235, 247, 279, 285
tissue microenvironment, 77
treatment, 2, 16, 19, 27, 29, 35, 74, 75,
77, 78, 80, 81, 86, 96, 102, 112, 113,
123, 135, 145, 159, 172, 173, 176, 177,
179, 180, 186, 188, 194, 198, 205, 212,
213, 226, 237, 243, 248, 260, 286–288,
290, 311, 318, 336
vaccinations, 75, 120, 123, 176, 181, 188,
213, 235, 249, 260
vaccine (CV), 1–3, 6, 9, 11, 20, 22–25,
27, 29, 31–34, 36, 53–55, 59–61, 63,
64, 73, 93, 95, 96, 110, 111, 115, 116,
122–126, 133–136, 145–147, 149–154,
157–163, 171–173, 201, 202, 204, 205,
211–215, 218, 220, 221, 236–240, 245,
250–253, 258–261, 267–269, 272, 273,
278, 279, 282, 284, 295, 298, 300, 310,
328, 333–336
delivery systems, 36
Cancerous
phenotype, 58
tumors, 86

================================================================================

— SECTION 83 —

PAGE 375

356
Index
Capillary network, 76
Capsid, 7, 19, 160, 270, 329, 330
Capsules, 22, 295
Carbohydrate, 268, 279
antigens, 27
polymers, 21
Carbon dioxide (CO2), 11
Carcinoembryonic antigen (CEA), 11, 144,
181, 184, 221, 255, 309, 328, 333
Carcinogenesis, 94, 309, 313
Carcinogens, 313
Carcinoids, 134
Carcinoma, 19, 20, 22, 28–30, 54, 56, 57,
64, 74, 78, 111, 116, 134–138, 140, 143,
144, 178, 180–182, 184, 219, 238, 247,
254, 256, 280, 286, 289, 319–322, 329
in situ (CIS), 238, 329
Cardiac arrest, 145
Cardiovascular disease, 83, 268
Cargo delivery, 81
CART cell therapy, 143
Cascade, 26, 111, 122, 199, 255, 282
Cassettes, 13
Cationic
polyelectrolytes, 22
polymer, 21, 22, 36, 283
CCL2 (C-C motif chemokine ligand 2), 250,
258, 317, 323
CCR7 (C-C chemokine receptor 7), 98, 119,
288
Cell
adhesion molecules, 81
cycle, 18, 58, 82, 86, 139, 172, 258, 311,
313
phase, 86
death, 4, 18, 99, 101, 121, 124, 135, 146,
176–179, 181, 212, 239, 241–243, 248,
256, 310, 324, 335
membrane, 1, 8, 31, 35, 68, 81, 84, 107,
108, 110, 154, 161, 199–201, 256, 278,
293, 294, 297, 298, 301
migration, 118
models, 34
penetrating peptides (CPP), 1, 36, 53, 67,
70, 201
proliferation, 94, 99, 108, 109, 119, 139,
143, 174, 175, 311, 313, 314, 318, 328
therapy, 144
Cellular, 6, 7, 9, 33, 68, 75, 76, 81, 82, 94, 95,
98, 105, 119, 135, 148, 151, 152, 156, 158,
159, 173, 186, 236, 238–240, 251, 256,
274, 277, 282, 285, 286, 288, 293, 295,
296, 300, 311, 313, 316, 320, 322, 325
entrance, 9
machinery, 98
proliferation, 94
Cervical cancer, 26, 30, 156, 178, 281, 291,
300, 311
Cetuximab, 80, 325, 327
Chemoimmunotherapy (CIT), 175–177,
236, 244, 245, 248, 261, 262
Chemokine, 4, 9, 98, 119, 141, 142, 149,
175, 282, 323
receptor, 99, 288
Chemo-physical properties, 85
Chemotactic factors, 327
Chemotherapeutics, 245
agents, 176, 187, 236, 244, 249, 260, 262,
311, 318, 324, 326, 336
drugs, 74, 78, 186, 243, 297
Chemotherapy, 2, 18, 33, 55–58, 74, 84, 86,
100, 112, 115, 124, 133, 135–137, 145,
153–155, 171, 172, 174–178, 180–182,
185, 186, 188, 204, 205, 216, 218–221,
226, 235–237, 243–245, 247–250,
252–256, 258–261, 286, 309–312, 318,
323–325, 327, 334–336
regimens, 78
Chest, 65, 135, 138, 145
wall tumors, 65
Chickenpox, 102
Chimeric antigen receptor (CAR), 17, 22,
31, 110, 121, 135, 141, 143, 144, 159,
161, 162, 198, 202, 206, 325
Chitin, 268, 294
Chitosan, 21, 24, 26, 80, 146, 268, 292, 294
nanoparticles (NPs), 24
Cholesterol, 29, 30, 61, 200, 271
management, 29
Chromatin, 85, 139, 259
Chromosomal
defects, 94
instability (CIN), 11, 85
Chromosomes, 67, 95, 108
Chronic
condition, 12, 227
lymphocytic leukemia, 9

================================================================================

— SECTION 84 —

PAGE 376

357
Index
Cirrhosis, 280, 320, 335
Cisplatin, 33, 186, 247, 256
Clinical
specimens, 100
trials, 3, 7, 11, 15, 19, 24, 29, 34, 53, 64,
67, 96, 115–117, 134, 135, 141, 144,
149–151, 154–157, 161, 162, 185, 201,
202, 205, 213–215, 220, 226, 261, 268,
281, 289, 300, 312, 333
Clonal cell, 94
Clostridium acetobutylicum, 18
Cluster of differentiation (CD), 12, 63, 64,
95, 145, 218, 285
Clustered regularly interspaced short palin­
dromic repeats (CRISPR), 118
Clusters, 117
Codons, 103
Co-encapsulation, 62, 111
Colitis, 98, 185
Colloidal
gold, 27
immunostimulatory delivery systems, 73
vaccine delivery system, 73
Colon
cancer cells, 9, 28, 83, 297
carcinomas, 7
transitory, 85
Colony stimulating factor (CSF), 27, 117,
145, 146, 181, 239, 242, 286–288, 300
Colorectal cancer (CRC), 11, 83, 85, 149,
203, 255, 309, 313, 316, 317
recurrence, 85
Colorectum, 83
Combat cancer cells, 3
Combination therapy, 78, 86, 163, 183, 185,
243, 246, 248, 336
Complementary DNA (CDNA), 195
Computed tomography, 135
Confocal laser scanning microscopy

— SECTION 85 —
(CLSM), 293
Congestive heart failure (CHF), 319
Contract manufacturing, 31
Conventional type 2 DCs (CDC2s), 99,
118–120
Copy number alteration (CNA), 85
Co-stimulatory molecules, 104, 153, 217,
259, 281, 324
Covalent bond, 94
COVID-19, 59, 204, 327
Cuba, 111, 136, 156
Curative immunization, 56
Curcumin, 26, 83
Cutaneous, 5, 15, 239, 286, 287
C-X-C motif chemokine ligand 1 (CXCL1),
258
Cyclic GMP-AMP synthase (CGAS), 258
Cyclophosphamide (CTX), 16, 181, 186,
248, 250–253, 258, 259, 310, 320, 334,
335
Cyto pulse, 11
Cytokines, 4, 8, 9, 14, 15, 18, 77, 95, 98,
99, 103, 106–110, 116, 117, 119, 135,
141–146, 149, 153, 154, 175, 179, 198,
199, 202, 212, 225, 242, 246, 252, 259,
268, 269, 273–276, 282, 284, 285, 287,
288, 291, 294, 312, 314, 315, 321, 326,
334, 335
Cytoplasm, 9, 21, 29, 76, 199, 298
Cytosine-phosphate-guanine (CpG), 8, 23,
25, 26, 29, 30, 32, 109, 148, 161, 268,
270, 276, 277, 281–283, 290–293
Cytosol, 18, 105, 297
Cytosolic domain, 106
Cytostatic drugs, 180
Cytotoxic
agent, 75, 86, 171, 239
cells, 159, 315, 327
effectors, 327
lymphocytes (CTLs), 4, 7, 10, 15, 21, 23,
26, 28–30, 77, 96, 121–124, 136, 146,
152, 153, 158, 175, 177, 184, 211, 214,
215, 219, 241, 244, 250–252, 254, 256,
273, 275, 282, 285, 290, 291, 323, 324,
326, 328
mediators, 103
T-cells, 96, 106, 121, 199, 218, 317
therapies, 78
T-lymphocyte, 4, 15, 67, 77, 96, 123, 136,
158, 177, 227, 249, 251, 252, 254, 273,
323, 328
antigen 4, 324
Cytotoxicity, 4, 22, 54, 70, 81, 100, 175,
247, 249, 254, 257, 278, 283, 284, 309,
314, 315, 321, 323, 324, 334

================================================================================

— SECTION 86 —

PAGE 377

358
Index
D
Damage-associated molecular pattern
(DAMP), 314
Deactivation, 313
Death protein 1 (PD-1), 15, 114, 119, 120,
143, 153, 162, 178, 198, 214, 241–243,
247–249, 315–317, 324, 325
Decision-making, 261, 262
Deep penetration, 84
Degradation, 68, 83, 106, 194, 197, 201,
202, 204, 270–272, 290
Dendrimer, 21, 28, 62, 70, 80–82, 201
architecture, 82
Dendritic
cell (DC), 3, 4, 14, 15, 17, 23, 25–27, 33,
57, 68, 75, 96, 98, 99, 111, 115, 116,
119–124, 136, 145–147, 158–162, 175,
181, 186–188, 198, 202, 204, 218–221,
224, 227, 237, 239, 249, 250, 252–258,
261, 268, 269, 274, 278, 287–289, 291,
293, 298, 300, 301, 310, 314, 318, 326,
333
polymers, 62, 81
Deoxyribonucleic acid (DNA), 5–13, 16,
18, 21, 29, 30, 53, 57–60, 66–68, 85,
94, 109, 116, 147–151, 153, 174, 177,
179, 194, 195, 197, 225, 226, 236–238,
252, 254–256, 258, 259, 267, 269, 278,
284–286, 291, 295, 296–298, 311, 313,
318–322, 328, 333
immunization, 8, 11, 238
methylation, 259
nanospheres, 21
vaccination, 5, 7, 8, 10–12, 53, 254, 284,
285, 291, 296
vaccines, 5, 10, 13, 18, 59, 147, 197, 226,
238, 267, 269, 278, 284, 286, 296
Depletion, 99, 117, 120, 124
Depots, 159, 292
Dermal layer, 14
DermaVax, 11
Destruction, 2, 95, 107, 123, 124, 135, 173,
177, 198, 244, 259, 273, 293, 326
Determinants, 141
Detox, 8, 10
Dextranspermine polycations, 21
Diabetes, 12
Diacylglycerol (DAG), 105, 106
Diagnosis, 74–76, 135, 136, 138, 311
Diameter, 13, 23, 79, 286
Diarrhea, 236, 311, 319, 320
Diethylaminoethyl-dextran (DEAE­
dextran), 21
Difluoro, 32
Diphtheria, 279, 280, 330–332
Diphtheria, pertussis, and tetanus (DPT),
280
Diphtheria, tetanus, pertussis (DTP), 279, 331
Disease-free survival (DFS), 218, 224, 238,
288
Disintegration, 24
Disorder, 75, 161
Docetaxel, 33, 216, 217, 254
Dolomite bio, 86
Dormancy, 84, 85, 314
Dosage, 3, 13, 34, 74, 78, 145, 181, 184,
185, 249, 261
Double-positive
lymphocytes, 103
T-cells, 103, 104
thymocytes, 104
Doxorubicin, 33, 81, 186, 187, 251, 253,
256, 259, 310, 311
silicon NPs, 81
Drive tumor growth, 94
Drug, 18, 21, 33, 54, 55, 58, 61, 62, 67, 68,
73–75, 77–80, 82, 85, 126, 134, 143, 145,
155, 161, 171–174, 176–181, 186, 218,
235, 236, 243, 245, 247, 249, 252, 254,
256, 258, 277, 283, 297, 311
administration, 54
carriers, 34

— SECTION 87 —
delivery methods & systems, 69, 75, 76,
78, 79, 83, 85–87, 298
design, 126
entrapment, 82
hydrodynamic radius, 83
metabolizing enzymes, 94
penetration, 80
resistance, 77, 84, 86, 177, 180, 186, 241,
243, 245, 248
retention, 62
toxicokinetics, 110
Dual-drug delivery system, 83
Dynamic hydrogels, 83
Dysfunctional cells, 95

================================================================================

— SECTION 88 —

PAGE 378

359
Index
E
Easy vax, 11
Egg yolk phosphatidylcholine liposomes, 30
Ehrlich carcinoma, 22
Electron microscope, 19, 80
Electroporation, 296, 299, 301
Embryogenesis, 112
Emulsions, 29
Endocytosis, 55, 108, 110, 122, 199, 292
Endoplasmic reticulum, 160, 258
Endosomal membrane, 66, 283
Endothelial cells, 28, 77, 141, 318
Enhanced permeability and retention (EPR),
75, 79
Environmental factors, 53, 242, 313
Enzymes, 18, 97, 141, 318, 320
Epidemics, 54
Epidermal
growth factor receptor (EGFR), 7, 8, 23,
80, 135, 136, 139, 140, 143, 176, 313,
325
layer, 11
Epigenetics, 75, 135, 313
Epithelial
cells, 53
mesenchymal transition, 85, 237
-to mesenchymal transition (EMT), 85,
317, 327
Epitopes, 19, 21, 32, 34, 68, 109, 113, 151,
212, 213, 220, 324, 335
Epstein-Barr virus (EBV), 19, 20, 147, 328
Equilibrium, 95, 315
Eradication, 77, 106, 136, 294, 315
Erlotinib, 58
Errors, 85, 320
Erysipelas, 17
Escherichia coli, 18
Ethylene glycol, 22
Etiology, 107, 123
Etiopathology, 2
Evolution, 86, 97, 106, 144, 215, 314, 323
Ex vivo, 7, 14, 17, 117, 118, 121, 239, 240,
256
Exfoliative cytology, 135
Exhaustion, 123, 315, 323
Exosome, 80, 81, 84, 242, 292
biomimetic, 81
membranes, 81
Exposure, 24, 53, 55, 97, 100, 108, 133, 134,
137, 196, 247, 250, 251, 274, 289, 297
Extracellular
matrix (ECM), 76, 77, 101, 141, 283
vesicles, 80
Extrusion, 81

— SECTION 89 —
F
Fatal, 79, 218, 236
Fibrillary structures, 32
Fibrin, 77
Fibroblast, 77, 226, 247, 248, 318
growth factor (FGF), 84
Fibrosarcoma, 118
Fluidigm C1, 86
Fluorescent dyes, 68
Food and Drug Administration (FDA), 110,
116, 125, 142, 150, 151, 177, 216, 218,
226, 238, 239, 267, 269–272, 288, 296,
297, 310, 312, 324, 326, 327, 329–333
Forming, 18, 27, 137
Fragmentation, 320
Fragments, 62, 108, 110, 122, 199, 314

— SECTION 90 —
Free radical polymerization methods, 83
Freeze/thaw cycles, 81
G
Galactosidase (LacZ), 12
Galactosyl ceramide (GC), 26, 109, 110,
161, 274
Gamma
herpes, 19
polyglutamic acid, 24
Gastric cancer, 26, 269, 270, 328
Gastroenterology, 10, 83
Gefitinib, 58
Gelatin, 26
Gels, 146
Gene, 18, 35, 66, 85, 86, 94, 102–106, 108,
117, 137, 139, 147, 153, 162, 172, 175,
186, 225, 238, 258, 259, 313, 317, 321,
328
cannon, 5
directed enzyme prodrug therapy

— SECTION 91 —
(GDEPT), 16
expression, 6, 9, 86, 94, 99, 100, 117,
241, 259, 286, 296

================================================================================

PAGE 379

360
Index
gun, 5–8, 10, 296, 299
bombardment, 5, 8
delivery, 5, 6, 299
segment, 103
therapy, 11, 12, 21, 29, 35, 58, 66, 67, 73,
86, 296
Genetic, 313
mutations, 94, 193, 313, 314, 328
resources, 84
variation, 186
Genetically modified tumor vaccines
(GMTVs), 147
Genome, 18, 85, 113, 172, 186, 194, 196,
215
Genomic, 313
instability, 53, 172
Germinal center, 109
Glands, 187, 223
GlaxoSmithKline (GSK), 31, 160, 278
Glioblastoma, 115, 214, 297
Gliomas, 283, 288
Glucans, 294
Glucocorticoids, 175
Glutathione, 247, 248, 322
Glycol, 161
chitosan, 161
Glyconanoparticles, 27
Gold, 27, 283, 288
nanoparticles, 27
standard, 288
Good manufacturing practice (GMP), 159,
299
Gram-positive bacteria, 18
Granulocyte-macrophage colony-stimu­
lating factor (GM-CSF), 7, 28, 63, 117,
146, 149, 150, 157, 160, 183, 184, 198,
212, 216, 217, 219, 239, 240, 250, 253,
268, 286–288, 296, 300, 301, 310, 333
Graphene, 277, 301
oxide (GO), 277, 301
Growth factors, 62, 198, 321

— SECTION 92 —
H
Hallmarks, 53, 94, 135, 312, 314, 324, 334
Healthy tissues, 74, 76, 99
Heat shock protein, 30, 105, 160, 258
Helium, 11, 13
microcylinder, 13
Hemagglutinin, 20, 276, 280
Hemolytic toxicity, 82
Hemorrhage, 312
Hepatitis, 19, 280, 299, 300, 329–333
B virus (HBV), 19, 59, 133, 136, 156,
159, 280, 281, 296, 329–333
Hepatocellular carcinoma (HCC), 136, 156,
280, 291, 297, 335
Herpes, 35, 59, 238, 239, 276
simplex virus, 16, 35, 238, 276
Heterogeneity, 86, 139, 156, 246, 300
High
biodegradability, 33
cell density, 76
metabolic activity, 18
mobility group box 1 (HMGB1), 258
molecular-weight, 22
neutralizing antibody titers, 20
Histological analysis, 100
Histology, 54, 140, 147
Homeostasis, 99, 284, 311, 314
Hormone therapy, 182, 223, 239
Host waste, 98
Human
cyclin, 16
epidermal growth factor receptor 2

— SECTION 93 —
(HER2), 82, 139, 144, 253, 310, 312,
325, 328
granulocyte/macrophage colony-stimu­
lating factor (hGM-CSF), 18
immunodeficiency virus (HIV), 201, 278,
285, 328
immunome project, 115
leukocyte antigen (HLA), 65, 105, 114,
151, 153, 180, 181, 217, 220, 221, 241,
252, 258, 259, 315
papillomavirus (HPV), 7, 8, 10, 11, 25,
32, 59, 133, 136, 147, 156, 159, 160,
171, 268, 270, 278–281, 284, 291, 293,
296, 310, 328–330, 333
type 16 (HPV16), 7
telomerase reverse transcriptase (hTERT),
16, 156, 157, 217, 221, 289
Humoral immunity, 107
Hyaluronic acid (HA), 15, 83, 246, 247
Hybrid
hydrogels, 83
NPs, 86, 87

================================================================================

— SECTION 94 —

PAGE 380

361
Index
Hydrogel, 14, 83, 87
networks, 32
Hydrogen, 82
Hydrophilic, 28, 61, 62, 68, 80, 83
3D porous networks, 83
substances, 68, 80
Hydrophobic, 28, 61, 80, 82, 292
biodegradable polymeric, 82
Hydrophobicity, 278
Hyperplasia, 182
Hypersensitivity, 220, 236, 237, 251
Hypoglycemia, 156
Hypoxia, 16, 76, 77, 99, 156, 248, 249, 314,
318
inducible factor (HIF), 98, 248
response element (HRE), 16
Hypoxic tumor microenvironments, 86, 87

— SECTION 95 —
I
Imaging, 64, 75, 119, 283
Imatinib, 175
Immortality, 156, 314, 335
Immune
cell, 14, 15, 18, 24, 59, 84, 95, 101, 117,
118, 121, 136, 141, 149, 154, 156, 161,
173, 176, 177, 213, 216, 237, 239–241,
247, 257–259, 261, 283, 290, 294, 295,
309, 314, 315, 325, 336
therapy, 58, 59, 173, 176–179
checkpoint inhibitors (ICIs), 100, 112,
121, 123, 134, 141, 146, 155, 156, 159,
174, 178, 198, 216, 219, 226, 242, 244,
248, 249, 300
evasion, 65
mediated adverse events, 206
modulators, 4
response, 1–37, 59–68, 73, 95–99, 103,
104, 110–116, 118, 121–125, 133, 134,
141, 147–150, 152–154, 157, 159–163,
172, 173, 175, 179, 181, 184, 186,
188, 196–201, 204–206, 211–213, 215,
217–219, 222, 223, 225, 226, 236–244,
247–250, 252, 253, 255, 260, 261, 268,
269, 272–274, 276–278, 280–286,
288–296, 298, 301, 316–318, 324–326,
329, 334
stimulating complexes, 1, 36
surveillance, 54, 84, 95, 141, 245, 317
system, 1–4, 7, 14, 16, 21, 24, 27, 29, 33,
36, 54–59, 64, 66, 75, 87, 93, 95–98,
101, 102, 123, 124, 126, 133, 136, 141,
145–147, 150, 152, 153, 160, 171, 173,
177–179, 185, 186, 193, 199, 211–213,
216, 218, 220, 225, 235, 237, 238, 242,
243, 251, 252, 254, 259, 260, 267–269,
274, 278, 283, 287, 295, 309, 314, 316,
324, 334
Immunity, 6, 8, 11, 17, 26, 28–31, 34, 69,
96–99, 102, 103, 110, 115, 118, 119, 121,
122, 124, 151, 159, 160, 173, 175, 177,
179, 181, 182, 186, 188, 196, 218, 221,
240, 244, 245, 252, 254, 259, 260, 268,
269, 272, 274, 275, 277, 279, 285, 292,
294, 295, 298, 300, 312, 326, 330, 333,
334
Immunization, 4, 5, 9, 19, 23, 30, 60, 67–69,
103, 112, 114, 115, 117, 124, 181–183,
205, 218–220, 238, 244, 245, 248, 249,
251, 276, 282, 285, 286, 291, 293, 329,
330
Immunocytes, 145
Immunodetection, 83
Immunogenic
cell death (ICD), 124, 177, 246, 258, 259
neoantigens, 114
substance, 9
tumor cell, 118, 186, 334
vaccination, 115, 294
Immunogens, 30, 68, 181
Immunoglobulins, 109, 317
Immunohistochemistry, 162
Immunologic
dormancy, 85
memory, 102, 196
Immunological
checkpoint, 126, 242, 259, 324
blockade (ICB), 102, 110, 118
dysfunction, 115
effects, 93
memory, 4, 69, 102, 111, 163, 184, 290,
327
milieu, 33
Immunology, 2, 4, 93, 95, 188, 292
Immunomodulation, 186, 283, 335, 336
Immunomodulatory factors, 87
Immuno-stimulating complex, 30

================================================================================

— SECTION 96 —

PAGE 381

362
Index
Immunosuppression, 99, 100, 120, 121, 141,
146, 162, 188, 215, 243, 244, 275, 334, 335
Immunosuppressive
compounds, 77
mouse melanoma, 114

— SECTION 97 —
TME, 101, 144, 246
Immunosurveillance, 95, 142, 153, 155, 255
Immunotherapeutics, 10, 96, 202
Immunotherapy, 1, 3, 24, 33, 34, 53, 57,
58, 64, 65, 73, 84, 87, 93, 95, 96, 110,
112, 113, 118, 121–123, 125, 126, 133,
135, 142, 145, 146, 152, 153, 155, 156,
159–163, 171–175, 177–179, 182,
183, 186, 188, 193, 194, 196, 200, 202,
204, 205, 211–218, 225–227, 235–237,
239–241, 243–245, 248–252, 255,
258–262, 267, 269, 283, 286, 287,
291–295, 309–311, 317, 323, 324, 329,
333, 336
potentiation pathways, 126
resistance, 34, 227, 235, 241
vaccines, 87
In silico, 57
In situ, 26, 124, 283
vaccine (ISVs), 124, 146
In vitro, 7, 9, 11, 19, 22, 28, 81, 96, 110,
116, 143, 144, 195, 197, 199, 204, 215,
278, 283, 285, 290, 292
transcription (IVT), 195, 204
In vivo, 9, 10, 12, 15, 17, 18, 21, 27, 30, 62,
117, 142, 194, 197, 200, 201, 205, 239,
247, 271, 283, 285, 288, 292, 294, 296,
299
Indicators, 26, 53, 116, 179, 186
Indomethacin, 82
Induce apoptosis, 83, 249, 327
Infectious
disease, 10
organisms, 54
Inflammation, 3, 7, 98, 100, 117, 311, 314,
321
Inflammatory, 9, 17, 31, 82, 95, 97, 98, 101,
106, 146, 158, 160, 179, 197, 199, 248,
254, 279, 282, 283, 314, 320, 321, 323, 328
Cytokine (IFN-γ), 4, 14, 15, 17, 100, 101,
157, 199, 241, 248, 254, 250, 258
mediators, 158
reactions, 98, 197
Influenza, 20, 32, 201, 276, 296, 299
matrix peptide, 32
Inhalation, 55, 63
Inhibitors, 119, 133, 135, 141, 142, 146,
149, 153–155, 161, 162, 172, 214, 216,
236, 259, 261, 326
Injections, 5, 23, 216, 290
route, 5
Innate
autoimmune process, 110
immune system, 97, 100, 141, 149, 193,
197, 204, 269
immunity, 14, 97–99, 121, 259, 269, 294
lymphocytes, 101
lymphoid cell (ILCs), 141
Inorganic particles, 27
Integrins, 81
Interferon (IFN), 4, 14, 15, 17, 23, 65, 99,
100, 118–121, 145, 146, 157, 158, 160,
180, 199, 224, 241, 248, 250, 258, 285,
287, 288, 290, 291, 294, 314, 316, 327, 335
producing cells (IPCs), 99
Interleukin (IL), 8, 15, 16, 18, 31, 80, 84,
99–101, 103, 104, 106–108, 110, 112,
113, 117, 119, 120, 145, 146, 150, 157,
158, 160, 175, 187, 188, 197, 198, 200,
202, 246, 249, 250, 254, 256, 257, 268,
270, 276, 277, 282, 284, 285, 288, 291,
294, 298, 300, 312, 314, 316, 317, 323,
327, 335

— SECTION 98 —
21 (IL-21), 327
9 (IL-9), 327
Internalization, 33, 82, 98, 111, 292
Intracellular, 31, 33, 57, 81, 94, 96, 98, 122,
143, 199, 200, 225, 248, 249, 258, 283,
284, 325
Intradermal
injection, 6, 8
layers, 11
Intramuscular
administration, 7
injection, 6, 8
needle injection, 7
Intramuscularly (I.M.), 5–11, 200, 221
Intranasal administration, 5
Intra-nodal vaccination, 5
Intra-tumoral
injection, 9
levels, 120

================================================================================

— SECTION 99 —

PAGE 382

Index
363
Intravenous
administration, 81
injection, 26, 75, 223
Intravenously (I.V.), 9
Intrinsic
immunogenicity, 33
pathways, 98
Invasion, 58, 75, 80, 94, 97, 100, 120, 135,
144, 174, 220, 237, 246, 312, 314, 317
Inverted cytokine receptor, 17
Ions, 293
Iron, 271, 283
Irradiation, 219
Isolated splenocytes, 23

— SECTION 100 —
J
Japan, 279, 280
Jet
impacts, 12
injection, 11, 12
K
Karyotypic mutation, 95
Key element, 96, 112
Keyhole limpet hemocyanin (KLH), 29,
154, 155, 252, 278
Kidney cancers, 28
Kinase, 282
inhibitors, 33, 175

— SECTION 101 —
L
Laser therapy, 5
Latent membrane protein 2 (LMP2), 20
Lead, 4, 7, 24, 56, 85, 87, 115, 126, 172,
180, 193, 204, 227, 236, 247, 252, 290,
312, 313, 328
Leucine-rich repeat (LRR), 283
Leukemia, 22, 150, 313, 319, 320, 322
cells, 22
Ligand, 14, 21, 33, 80, 118, 259, 268, 274,
275, 279, 290, 291, 299, 314, 315
proteins, 81
Lipid, 67, 200, 271
bilayer structure, 67
nanoparticles (LNP), 28, 200, 201
Lipopolyamines, 21
Lipopolysaccharide (LPS), 148, 246, 276,
278, 279, 291, 301
Lipoproteins, 68, 148
Liposomal co-encapsulation, 29
Liposomes, 1, 28–30, 35, 36, 60, 61, 66, 67,
73, 80, 146, 160, 271, 283, 289, 292, 298,
299, 301
Liquid jet injectors, 12
Listeria monocytogenes, 18
Liver, 26, 29, 103, 271, 280, 310, 311, 328,
333
cancer, 271, 280, 310, 328, 333
Loading, 27, 33, 62, 81, 83, 292
Long-term memory, 325
Luminescent PSiNPs, 81
Lung
cancer, 7, 53–55, 57–60, 63, 64, 69, 120,
134–138, 140, 144, 148, 151, 152, 154,
156, 158, 159, 161–163, 178, 187, 242,
270, 280, 288, 296, 311, 328
antigen mechanism, 55, 56
treatment, 53
CV delivery technology, 55
nodule carcinoma, 64
tissues, 53, 60
Lymph
angiogenesis, 101
nodes (LN), 8, 14, 15, 27, 28, 32, 33, 35,
68, 69, 73, 110, 111, 116–119, 122, 142,
158, 162, 260, 273, 286, 294, 301, 314
Lymphatic system, 102, 107, 173, 334
Lymphocytes, 26, 65, 95, 98, 102–104, 106,
107, 114, 119, 121, 122, 152, 179, 180,
197, 214, 242, 250, 254, 257, 260, 268,
269, 276, 282, 291, 296, 315–317, 325,
335
Lymphoid
follicle, 109
organs, 102, 122
tissues, 7, 68, 260, 296
Lymphokines, 96, 188
Lymphomas, 19, 200, 320, 321
Lyse tumor cells, 16
Lysing cells, 16
Lysosomes, 122, 202

— SECTION 102 —
M
Macaques, 11
Macromolecular nanoscopic, 81
Macromolecules, 28, 175, 179, 180, 273

================================================================================

PAGE 383

364
Index
Macrophage, 9, 14, 68, 84, 99, 104, 121,
124, 145, 150, 159, 187, 201, 242, 246,
253, 258, 269, 276, 279, 285, 287, 294,
310, 314, 315, 321, 323
colony, 157, 217, 219, 238, 268, 286, 287
Magic bullet, 74
Magnetic resonance imaging (MRI), 69
Major histocompatibility (MHCII), 104
class I (MHCI), 118, 215
complex (MHC), 10, 21, 23, 34, 57, 60,
66, 98, 103, 104, 107, 108, 110, 112,
122, 154, 158, 171, 172, 177, 188, 196,
199, 213, 218, 239–241, 249, 257, 258,
274, 276, 279, 281, 288, 289, 299, 310,
315, 326, 334
Malaria, 278, 283, 299
Malignancies, 2, 8, 10, 12, 16–20, 30, 57,
58, 73, 76, 85, 86, 94–96, 99, 101, 113,
120, 123, 173, 182, 186, 205, 237, 241,
243, 244, 246, 252, 256, 259–261, 282,
285, 287, 288, 322
Malignant
cell, 3, 17, 18, 22, 74, 95, 112, 113, 173,
178, 193, 196, 235, 240, 252, 318, 320
clones, 95
tissue, 16, 75
transformation, 58
tumors, 114, 120
Mammalian cells, 29, 80, 194
Manufacturing, 60, 67, 99, 116, 205, 212,
241, 268, 300
Mast cell, 101, 141
secreted molecules, 101
tryptase, 101
Matrix
metalloproteases, 101
metalloproteinase (MMP), 246
Maturation, 15, 69, 102–104, 109, 120, 160,
161, 188, 198, 202, 245, 252, 253, 258,
278, 288
Maximum tolerable dose (MTD), 244, 245
Measles, 102, 257
vaccine virus (MeV), 257
Mediastinal lymph nodes, 293, 301
Medical therapy, 79
Medications, 11–14, 16, 53–55, 60–63, 75,
78, 80, 82, 85–87, 98, 112, 115, 123, 126,
177–179, 181, 212, 216, 236, 237, 240,
243, 248–252, 254, 256, 258–261, 282,
283, 322, 324
Melanoma, 7, 12, 14, 15, 18, 23, 25, 26, 32,
63, 64, 111, 112, 114–116, 119, 120, 150,
153, 154, 157, 181, 184, 185, 197, 198,
218, 221, 238, 239, 242, 252, 253, 283,
286–291, 293, 296, 297, 335
associated antigen A3 (MAGE-A), 25, 31,
134, 153, 157
patients, 112, 114, 115, 150, 197, 198,
238, 239, 286, 287, 289, 335
tumors, 14, 15, 32
Membranes, 20, 61, 277, 295
Memory B-cells, 109, 110
Merkel cell carcinoma, 143
Mesenchymal stem cells, 28, 142
Mesoporous silica rods, 27
Mesothelin (MSLN), 144
Mesothelioma, 64, 248, 253
Messenger ribonucleic acid (MRNA), 8, 12,
28, 59, 60, 116, 159, 193–206, 218, 246,
271, 272, 277, 289, 291, 293, 328, 333
Metabolism, 94, 135, 248, 313, 314, 317,
322, 334
Metastasis, 18, 76, 84, 85, 94, 95, 111, 135,
138, 156, 172, 174, 175, 178, 220, 236,
245–248, 312, 314, 333, 335
Metastatic castrate-resistant prostate cancer

— SECTION 103 —
(MCRPC), 216, 222, 240
Methotrexate, 83, 187, 250, 257, 320
Methylcellulose (MC), 83
Mice, 5, 9–11, 15, 18–20, 22, 23, 26, 29, 32,
114, 117, 118, 149, 184, 245, 246, 248,
251, 255, 257, 277, 281, 282, 310, 335
Micelles, 32, 61, 62, 68
Microbes, 84, 98, 134, 163, 292
Microneedle (MN), 13–15
Microorganisms, 68, 97, 123, 136, 269
Microparticles (MPs), 22–24, 36, 66, 179
Micro-RNA (MiRNA), 314
Micro-scale, 22
Microsecond pulses, 9
Migraine, 12
Migration, 7, 58, 77, 99, 100, 116, 120, 219,
288, 296
Minimalistic components, 2
Minor
intragenic mutations, 53
mechanical injuries, 7
Mitochondrial membrane, 82

================================================================================

— SECTION 104 —

PAGE 384

365
Index
Mitomycin, 181, 187
Molecular
agents, 58
biology, 148, 188
mechanisms, 98
Molecules, 4, 10, 14, 18, 33, 35, 57, 60, 62,
67, 75, 76, 84, 93, 94, 96, 98, 103–105,
107, 108, 110, 114, 118, 122–124, 141,
142, 145, 153, 158, 159, 171, 174, 178,
187, 188, 198, 201, 258, 259, 261, 274,
288, 293, 297, 298, 310, 314, 315, 318,
324, 328
Monoclonal antibodies (mAbs), 55, 110,
135, 142, 146, 174, 178, 198–200, 244,
286, 312, 325–327
Monocytes, 99, 101, 113, 117, 160, 178,
248, 258, 293, 321, 325
derived DCs (MoDCs), 99, 116–119
Monodispersed, 81
Mononuclear, 101, 106, 240
phagocyte activation, 106
Mononucleosis, 19
Monophosphoryl lipid A (MPLA), 23, 25,
29, 31, 268, 276–278, 280, 284, 291
Monotherapy, 162, 172, 218, 219, 246, 248,
255, 261, 262, 311, 312, 336
Morphology, 95, 277
Mortality, 2, 23, 54, 116, 172, 217, 310,
311, 333
Motifs, 105
Motion, 13
MP vector (MSV), 23
Multidimensional design, 75
Multidisciplinary scientific field, 73
Multidrug resistance (MDR), 86, 173, 247
associated protein (MRP-1), 247
Multifactorial antigens, 93, 126
Multilamellar vesicles, 61
Multiple microstructured projections, 13
Multipurpose nanoadjuvants (M-NA), 283
Muramyl dipeptide, 29
Murine leukemia, 22
Mutants, 114
Mutational antigen, 147
Mutations, 85, 93, 94, 114, 126, 136, 137,
139, 143, 147, 153, 172, 213, 215, 313,
315, 320, 321, 323, 324, 327
Myeloid
cells, 99, 100, 153, 245, 256
derived suppressor cell (MDSC), 100,
121, 179, 242, 250, 253, 257, 317
progenitor cells, 99
Myeloma, 142, 150, 321, 325
Myungi, 28

— SECTION 105 —
N
Nadia, 86
Nano-curcumin, 26
Nano-drug, 75–77, 79
dimensions, 76
penetration, 75
Nanoformulations, 4
Nanomaterials, 145, 151, 293
Nanomedicines, 34, 77
Nanometers, 14, 76
Nano-micelles, 15
Nanoparticles (NPs), 1, 4, 14, 24–30, 32–36,
54, 55, 62, 70, 73–76, 79–82, 84, 86,
87, 120, 161, 201, 247, 276, 277, 279,
293–296, 300, 301
Nanopolymer, 83
Nano-scale structures, 61
Nanosecond electrical impulses, 9
Nanoshells, 70
Nanospheres, 62, 66
Nanostructures, 295
Nanosystems, 27, 35
Nanotechnology, 86, 135, 163, 200, 295
Nano-vaccines, 28, 87, 135, 161, 163, 295
Nasal
cavity, 294
route, 5
National Cancer Institute (NCI), 182, 183,
185, 328
Natural killer (NK), 18, 101, 102, 107, 124,
141, 145, 150, 158, 175, 187, 199, 250,
254, 255, 259, 273, 278, 286, 290, 294,
314, 316, 321, 325, 327, 335
Necrosis, 7, 320
Needle intramuscular injection, 8
Neoantigen, 34, 113–115, 147, 153, 155,
156, 161, 193, 203, 204, 206, 213–215,
224–226, 240, 258, 293, 294, 312, 328, 335
immunization, 114
peptide, 114

================================================================================

— SECTION 106 —

PAGE 385

366
Index
Neoplasm, 1, 12, 18, 24, 95, 185
Neoplastic growth, 53, 70
Neural tube defect, 85
Neuraminidase, 20
Neuroblastoma (NB), 286, 319, 320
Neuropathy, 236, 237, 311, 319, 322
Neutral pH, 22
Neutrophils, 84, 100, 101, 124, 325
Next-generation sequencing (NGS), 57, 213
Nitric oxide (NO), 101, 297
Nitrogen, 11, 198
Nivolumab, 143, 153, 161, 162
Non-cell small cell lung cancer, 163
Non-immunocytes, 145
Non-infectious vectors, 66
Non-self-antigen, 102, 150
Non-small cell lung cancer (NSCLC), 55, 57,
64, 117, 120, 134–141, 143, 144, 153–157,
162, 163, 203, 256, 270, 271, 299, 322
Nontoxicity, 27
Non-UV active Sendai virus liposomes, 29
Non-viral
gene transfection, 12
vectors, 36, 202
Normal
cells, 17, 58, 74, 75, 77, 135, 144, 173,
174, 186, 213, 313, 317, 318, 327, 335
vasculature, 79
Notch-1, 103
Novel
alkylated dendrimers, 28
method, 114
strategy, 82
Nucleic acid, 19, 54, 59, 147, 288, 294, 295,
319, 322
glycoprotein polysaccharides, 68
Nucleosides, 177
Nucleotide, 215, 281, 318, 322
vaccinations, 115
Nucleus, 9, 66, 106, 193, 194, 319

— SECTION 107 —
O
Oil, 14, 269, 272, 290
Oligodeoxynucleotides (ODN), 268, 270,
277, 281–283, 291
Oligonucleotides, 58, 67, 68, 281
Oncogenes, 98, 139, 172, 313
Oncologists, 1, 53
Oncology, 110, 112, 150, 173, 203, 216
Oncolytic
vectors, 18
viruses, 16, 17, 142, 146
Open reading frame (ORF), 194, 195
Ophthalmology, 83
Opsonization, 80
Oral medicine, 83
Osmosis, 21
Ovalbumin vaccine (OVA), 14, 15, 20, 23,
25–27, 257, 271, 277
Ovarian cancer, 253, 284, 319, 322
Overall survival (OS), 100, 154, 155, 157,
161, 217, 221, 224, 225, 248, 252, 253,
256, 288, 334, 336
Oxygen, 77, 106, 197, 198, 249, 297, 323

— SECTION 108 —
P
Paclitaxel, 78, 181, 187, 246, 250–256
Pancreas, 336
Pancreatic cancer, 100, 133, 201, 212,
217–221, 225–227, 253, 257, 285, 297,
322, 335
Pandemics, 136
Papillomas, 94
Paracellular, 76, 294
Parameters, 79, 110, 276
Paramyxovirus, 16
Parasites, 97, 328
Participants, 183, 217, 224, 225
Parts of the body, 74, 137, 216
Pathogenesis, 137
Pathogens, 21, 31, 54, 97, 98, 102, 103,
105–107, 109, 121, 125, 148, 151, 159,
198, 238, 268, 269, 281, 290, 291, 295, 299
Pathological properties, 76
Pathology, 2
Pattern recognition
molecules, 98, 126
receptor, 98, 292, 300
PEGylation, 83, 202
Pembrolizumab, 114, 143, 161, 203, 326
Peptide, 2, 21, 25, 27, 31, 32, 34, 36, 54, 59,
62, 65, 67, 80, 104, 105, 111, 113, 115,
116, 124, 147, 151, 156, 157, 199, 202,
203, 215, 220, 221, 238, 240, 245, 274,
284, 286, 287, 289, 290, 295, 296, 299,
301, 328, 333
epitopes, 34, 65, 218

================================================================================

— SECTION 109 —

PAGE 386

367
Index
vaccine, 30–34, 113, 136, 151, 217, 221,
238, 268, 281, 289, 296, 300
Peritoneal cavity, 23
Permeability, 11, 82, 321
Peroral, 83
Peroxidase system, 101
P-glycoprotein, 81, 247
pH, 24, 30, 62, 76, 77, 83, 200, 293, 299
Phagocytes, 79
Phagocytosis, 99, 150, 186, 279, 315
Pharmaceutical, 14, 78, 174
action, 77
research, 21
Pharmacokinetics, 86, 173
Pharmacological agents, 58
Phenotype, 75, 100, 117, 120, 153, 171,
245, 249, 314, 315
Phosphatidylinositol-3,4,5-triphosphate

— SECTION 110 —
(PIP3), 104, 105
Phospholipid, 28, 30, 61, 81, 298
bilayer, 21, 28, 61, 80
Photodynamic therapy (PDT), 84, 161
Photothermal therapy (PTT), 77, 84
Phylogeny, 86
Physiological
fluids, 84
growth, 117
Placebo, 217, 256
Plasma
blasts, 109, 110
cells, 109
Plasmacytoid dendritic cell (pDCs), 99, 120
Plasmid, 11, 60, 238
DNA (pDNA), 7, 9, 12, 195, 296, 297
Platelet (PLT), 84, 103, 313
derived growth factor (PDGF), 84, 313,
318
Platinum, 236, 256, 258, 311
Pluronic F127, 14, 15
Polarization, 99, 275
Pollution, 310
Poly(L-lysine) (PLL), 21, 36
Polyacrylate polymers, 5
Polyamidoamine, 21, 81
Polydimethylsiloxane (PDMS), 15
Polyethylene, 14, 80, 200
glycol (PEG), 14, 15, 21, 28, 80, 83, 146,
161, 200, 201, 293
Polyethyleneimine (PEI), 21, 22, 24, 36,
161, 293
Polylactic acid (PLA), 23, 24
Poly-lysine, 21
polycytidylic acid, 247
Polymeric
materials, 62
matrix, 62
microparticles, 36
nanoparticles, 36, 62, 293
systems, 33, 34
Polymers, 13, 21, 23, 24, 28, 33, 201, 292
Polymorphonuclear, 100, 101, 321
Poly-N-L-glutamate (PNLG) polymer, 22
Polypeptide, 103, 104
Polyplexes, 21, 22
Polysaccharides, 68, 83, 109, 273, 295
Polyurethane, 67
Populations, 116, 201, 242, 257, 314
Porous silicon, 23, 295
microparticle, 295
Potency, 34, 65, 87, 149, 198
Powderject method, 13
Preclinical animal models, 100
Primary immune response, 68
Production costs, 3
Prognosis, 53, 76, 85, 99, 101, 118, 137, 138,
153, 155, 216, 220, 226, 237, 248, 260
Programmed
cell death, 119, 143, 177, 179, 243, 319
death-ligand 1 (PD-L1), 121, 143, 144,
160, 162, 178, 214, 240–242, 248, 249,
259, 315, 316
Progressive free survival (PSF), 253
Pro-inflammatory cytokine (PICs), 100,
179, 274, 314
Prophylactic defense, 3
Prostaglandin (PG), 323
Prostate, 10, 19, 116, 133, 150, 151, 157,
182, 183, 201, 202, 212, 215–219,
221–223, 226, 227, 239, 253, 254,
287–289, 296, 298, 310, 312, 322, 328,
333
cancer, 10, 19, 116, 150, 151, 157, 182,
183, 201, 202, 212, 215–219, 222, 223,
226, 227, 239, 253, 254, 296, 310, 333
specific antigen (PSA), 19, 25, 149, 181,
183, 185, 186, 216, 217, 223, 255, 328

================================================================================

— SECTION 111 —

PAGE 387

368
Index
Protein, 9, 11, 19–21, 31, 32, 56–58, 60, 66,
67, 81, 84, 95–98, 103–106, 108, 112, 116,
124, 145–147, 160, 174, 194, 196, 199,
213, 216, 240, 241, 254, 255, 268, 273,
279, 281, 295, 296, 325, 327, 328, 335
conjugated delivery system, 31
Proteinase-activated receptor-2 (PAR-2), 101
Proteo-liposomal carrier systems, 21
Proteolysis, 83
Protumorigenic M2 macrophages, 99
Pseudomonas aeruginosa, 18
Public health, 172, 235
Pulmonary
cancer, 53, 54, 57, 63
route, 53, 70

— SECTION 112 —
Q
Quality control, 86
Quality of life (QoL), 54, 55, 78, 96, 154,
163, 199, 236
Quest, 78
Quillaja saponaria, 31
R
Radiation, 2, 57, 74, 77, 78, 86, 100, 115,
134, 135, 138, 172, 180, 188, 205, 216,
218, 236, 310, 311, 324, 334
Radiotherapeutic alternatives, 77
Radiotherapy, 2, 55, 99, 112, 133, 135, 204,
226, 323
Rapid phase, 83
Ras homolog gene family member C
(RhoC), 217
Reactive oxygen species, 100, 161, 163
Receptor, 17, 27, 54, 57, 94, 102–105, 108,
110, 143, 144, 156, 158, 161, 182, 188,
197, 242, 258, 259, 281, 282, 313, 317,
323–325
ligands, 54
Recombinant proteins, 125, 289
Recruits, 104, 106
Rectal, 29, 32, 83
cancer, 29, 32
distribution, 83
Recurrence, 3, 85, 110, 111, 114, 216, 220,
224, 237, 243, 245, 248, 249, 261, 309,
311, 312, 314, 324, 335, 336
Red blood cells, 84
Refining, 296
Regulators, 273, 313
Relapse, 3, 172, 175, 211, 224, 237, 245
Renal excretion, 83
Residues, 105, 112
Resiquimod, 14
Respiration, 313, 317, 322
Retinoic acid-inducible gene I (RIG-I), 197,
199
Retroviruses, 16, 35, 66
Rhabdovirus, 16
Ribonucleic acid (RNA), 12, 16, 24, 29, 33,
35, 57, 86, 100, 114, 117, 120, 151, 174,
194, 195, 197, 199–203, 215, 258, 286,
289, 294–296, 298, 301, 320, 321
interference, 24, 33
Risk factors, 124, 310
Robust antigen, 4, 65
Rodents, 257
Routes, 125, 239, 272

— SECTION 113 —
S
Salmonella, 18, 278, 291
typhimurium, 18
Sarcoma, 29, 65, 152, 319, 328, 333
Scale-up process, 35
Secondary lymphoid organs (SLO), 109, 260
Secretion, 97, 119, 187, 188, 199, 285, 316,
326, 328, 335
Selenium nanoparticles (SeNPs), 295
Self-assembling peptide, 32, 35, 300
Self-MHC peptide, 104
Self-peptides, 103
Sendai fusion protein, 29
Sepsis, 213, 236, 321
Sequencing, 85, 86, 100, 117, 141, 215, 335
Serum, 19, 62, 319
Sialyl-Tn, 252, 328
Side effects, 54, 55, 74, 78, 82, 122, 155, 173,
211, 216–218, 220, 221, 225, 236, 311
Signal transducer and activator of transcrip­
tion 3 (STAT3), 311, 316
Silicon, 13, 23, 81
dioxide, 13
NPs, 81
Simulated annealing single-cell inference

— SECTION 114 —
(SASC), 86

================================================================================

PAGE 388

369
Index
Single
cell analysis system and isolator, 86
positive morphology & T-lymphocytes,
103, 104
Skin cancer, 30, 294
Small
cartridge, 11
cell lung cancer (SCLC), 55, 64, 134,
135, 137–139, 141, 144, 153–155,
161–163
lymphocytic lymphoma (SLL), 9
Smallpox, 2, 6, 54, 212, 312, 327
Socioeconomic factors, 311
Sodium, 83, 277
Soft tissue sarcomas, 17
Solid
cancers, 16, 144
dose injectors, 13
phase peptide production, 31
Solubility, 145
Spectacular deterioration, 65
Spectrum, 99, 106, 157
Spherical nanovesicles, 28
Spleen, 23
Spontaneous mutation, 95
Spring mechanism, 11
Squamous cell
cancer (SCC), 9, 80
carcinoma, 65, 134, 137, 140, 290
Static hydrogels, 83
Stellate cells, 77
Stem cells, 84, 237
Steric stabilization, 21
Stimulation, 24, 26, 34, 64, 96, 102–105,
123, 126, 146, 150, 175, 193, 240, 249,
252, 257, 273, 277, 278, 282, 284, 285,
316, 321, 335
Stimulator of interferon genes (STING),
160, 258, 293
Stratum corneum, 13
Streptococcus pyogenes, 17
Stroma, 53, 77, 101, 226, 247
Stromal
cells, 141, 142
tissue, 76
Sub-chromosomal
CNAs, 85
modifications, 85
Subcutaneous, 5, 11, 80, 239, 281, 282, 284
Sulfur, 197
Supersonic speeds, 13
Supervision, 226
Supramolecular assembly, 32
Surface-bound, 31
Synergy, 171, 245, 249, 251, 259, 261
Synthesis, 6, 18, 31, 77, 99, 104, 174,
197, 199, 204, 236, 276, 277, 283, 311,
318–322, 331, 332
Synthetic long peptide (SLP), 25, 57, 105,
245, 253, 293

— SECTION 115 —
T
Tattooing, 6, 7
T-cell, 3, 4, 7, 8, 10, 11, 14–18, 23, 27,
29, 30, 34, 56, 57, 64–66, 68, 75, 95,
99, 102–123, 126, 135, 136, 141–145,
149–152, 156–163, 171, 174, 175, 181,
182, 184, 186–188, 193, 196, 198, 199,
203–206, 212–215, 218–221, 223–225,
237–246, 248–258, 261, 273–275, 278,
284, 286, 288, 290–296, 310, 314, 316,
317, 321, 323–325, 327, 329
activity, 99, 104, 177
clones, 104, 106, 184
dependent (TD), 108, 109
development, 102
dysfunction, 114
effectors, 106
independent (TI), 108, 109
receptor (TCR), 34, 103, 104, 106, 108,
110, 158, 175, 193, 198, 225, 274, 316
constant, 198
stimulators, 115
therapy, 31
T-helper type 2 (Th2), 5
T-lymphocytes, 10, 17, 57, 101–104, 107,
112–114, 118, 119, 121, 122, 124, 179,
187, 199, 211, 213, 215, 238, 244, 257,
275, 287, 314, 335
Telomerase, 147, 253, 317, 335
Tetanus toxoid, 32, 279, 280, 331, 332
Therapeutic
agent, 27, 77, 173, 176, 186, 198, 312
anti-tumor, 10
approach, 2, 58, 96, 110, 115, 121, 183,
211, 219, 237, 257

================================================================================

— SECTION 116 —

PAGE 389

370
Index
chemicals, 14
development, 261, 262
drugs, 74, 251
effectiveness, 78, 249
effects, 34, 82
efficacy, 33, 63, 78, 113, 178, 183, 211,
237, 277
environment, 82
fields, 83
indices, 78
nanoparticle delivery system (TLNP), 246
potential, 1, 34, 82, 115, 205
resistance, 327
standpoint, 79
strategy, 80
vaccinations, 57, 237
vaccines, 3, 134, 155, 171
vehicle, 24
Thermo-sensitive
hydrogels, 83
poloxamer 407 hydrogels, 83
Thomsen-Friedenreich antigen, 32
Three-dimensional (3D), 81
Throat cancer, 333
Thymic
cortex, 103
epithelium, 103, 104
Thymocyte proliferation, 103
Thymus, 102, 103, 112, 113, 182, 214, 274
Tissue, 3, 54, 55, 58, 68, 76, 81, 94–97, 101,
103, 112, 145, 154, 171, 172, 201, 214,
237, 239, 260, 319, 328
plasminogen activator, 10
T-lymphocytes, 316
Toll-like receptor (TLR), 4, 23, 24, 26, 27,
69, 109, 116, 145, 148, 159–161, 175,
178, 197, 199, 202, 219, 244, 258, 268,
269, 274, 276, 278–281, 283, 284, 290,
291, 293, 301
Toxicity, 24, 33, 35, 58, 62, 64, 74, 77, 78,
83, 84, 86, 144, 146, 152, 161, 183, 184,
201, 205, 219, 224, 235, 237, 261, 269,
288, 319, 320, 322, 325, 326
Toxins, 97, 148, 178
Transcellular transport, 76
Transcription factors, 105, 282, 321
Transdermal, 13, 82, 83
routes, 82
Transforming growth factor (TGF), 64, 84,
99, 100, 146, 152, 175, 250, 285, 312,
313, 316–318, 323
Transport, 9, 14, 27, 29, 35, 36, 61, 68, 69,
79, 84, 193, 201, 202, 288, 292, 322
proteins, 201
Tuberculosis, 137, 278, 299, 329, 334
Tumor
antigen (TA), 3, 15, 16, 23, 56, 57, 59,
64, 69, 73, 95, 96, 122–124, 133, 136,
144, 146, 147, 154, 155, 157–159, 161,
163, 180, 198, 211, 213, 214, 218, 220,
238–241, 250, 251, 310, 315, 324
associated
antigen (TAA), 7, 26, 56, 57, 147, 156,
157, 214, 217, 255, 326
carbohydrate antigens (TACAs), 278
macrophages (TAMs), 99, 100, 120,
121, 141, 246, 247, 316, 323
neutrophils (TANs), 100, 141
cell, 3, 4, 15–18, 22, 23, 26, 30, 58,
64, 69, 75–78, 81, 84, 85, 95, 100,
122–124, 141, 143, 147, 149, 150, 152,
153, 156–160, 171, 173, 176–179, 181,
186, 188, 193, 198, 211–213, 215, 216,
218, 223, 236, 237, 239–243, 249–252,
254, 255, 257, 258, 261, 284, 286, 287,
296, 309–318, 321, 323–327, 335
migration, 101
draining lymph nodes (TDLN), 99
growth, 3, 17, 22, 23, 26, 30, 33, 74, 77,
85, 96, 101, 121, 144, 149, 152, 156,
177, 198, 201, 205, 245, 249, 292, 312,
313, 318, 323, 335
heterogeneity, 85, 240, 246
immunity, 87, 117, 154
infiltrating lymphocytes (TILs), 95, 119,
126, 153, 173, 226, 227, 247, 318
initiating cell (TIC), 323, 324
lymph node-metastasis (TNM), 260
microenvironment (TME), 3, 17, 65,
83, 87, 96, 101, 122, 123, 134, 138,
141–143, 146, 149, 151, 156, 160, 179,
196, 199, 202, 204, 206, 238, 246, 257,
259, 261, 297, 316, 318, 334, 335
mutational burden (TMB), 153, 162
necrosis factor (TNF), 4, 12, 15, 17, 84, 98,
145, 146, 157, 158, 245, 246, 248, 250,
282, 285, 294, 312, 314, 316, 327, 335

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— SECTION 117 —

PAGE 390

371
Index
alpha (TNF-α), 12, 17, 157, 250, 312,
316, 327, 335
penetration, 81
progression, 12, 95, 123, 145, 147, 219,
318
proportion score (TRS), 162
remission, 327
resistance, 85, 204
shrinkage, 114, 172, 246
suppressor, 139
targeting, 36, 54, 86
tissues, 75–77, 122, 123
Tumorigenesis, 151, 313, 314
Tyrosinase, 25, 32, 113, 147
Tyrosine, 10, 20, 58, 105, 143, 144, 174,
175, 313, 326
kinase inhibitors (TKIs), 58, 175

— SECTION 118 —
U
Ultrasound, 8, 282, 289, 296, 297
microbubbles (USMB), 296, 297
Uniform lipoplex molecules, 67
Unilamellar
lipid membrane vesicles, 20
vesicles, 61
Untranslated region, 195
Uracil/cytosine (U/C), 197
Uric acid, 279
Urinary tract, 213

— SECTION 119 —
V
VacciMax (VM), 29
Vaccination, 1, 2, 4–7, 10, 12, 15–19,
23–27, 29, 30, 32, 34–36, 54, 64–66, 68,
95, 96, 102, 112–116, 120–125, 146, 155,
171, 172, 180–188, 205, 211–220, 224,
237, 238, 245, 249–256, 261, 267, 268,
272–283, 286–289, 293–296, 298–300,
310, 312, 327, 331, 332, 336
vector, 15
Vaccine, 1–5, 7–14, 16–32, 35, 36, 53–57,
59, 60, 63–66, 69, 73–75, 87, 93, 96,
110–117, 120–124, 133, 134, 136, 142,
147–157, 159–163, 171, 181–186, 188,
193–198, 201–206, 211–226, 235–240,
244–256, 261–269, 272–296, 298–301,
309, 310, 312, 327–336
administration system, 35
based immunotherapy regimen (VBIR), 149
delivery carrier & systems, 22, 53
development, 2, 27, 29, 31, 69, 73, 110,
111, 116, 153, 159, 160, 163, 204
Vaccinia, 16
virus, 66, 149, 286
Vaginal intraepithelial neoplasia (VaIN),
281
Vascular endothelial growth factor (VEGF),
64, 77, 84, 101, 126, 246, 250, 311, 318
Vascularization, 94
Vectors, 15–18, 35, 36, 58, 66, 84, 85, 115,
125, 134, 148, 162, 201, 217, 218, 286,
296
Versatility, 27, 108
Vesicles, 60, 61, 80, 84, 316
Vesicular stomatitis virus (VSV), 278
Veterinary medicine, 18, 212
Viral
antigens, 56
protein, 16, 21
strains, 16
vector, 15
Virosomal formulations, 21
Virosomes, 1, 20, 160, 296
Virotherapy, 16
Virus, 15, 16, 19, 35, 54, 57, 59, 66, 97,
149, 156, 201, 211, 255, 295, 296, 328
like particle (VLP), 1, 19, 20, 25, 36, 68,
160, 296
morphology, 19
Void spaces, 82
Vulvar intraepithelial neoplasia (VIN), 281

— SECTION 120 —
W
Warts, 281, 330
Weak immunogenicity, 73
White blood cells, 173, 175
Whole
cell tumor vaccination, 26
chromosomal CNAs, 85
chromosome, 85
Whooping cough, 279
Wisconsin, 226
World Health Organization (WHO), 2, 134,
235
Worm-like micelles, 32

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— SECTION 121 —

PAGE 391

372
Index
X
Z
Zeta potential, 33
Zika, 201
Zinc, 271, 276
X chromosome, 103, 107
Xenografts, 15
Xenotransplantation, 220
X-linked SCID disorder, 103

— SECTION 122 —
Y
Yeast, 19, 237, 279, 281
Yellow fever, 269

— END OF CHAPTER 11 —