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Malignancies

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For Iyer and Jay
Two Awesome, Amazing, Loving Brothers
Preface

This volume was born out of an idea I had thought of through an email from
the publisher Springer Nature calling for Editors for the Cancer Treatment and
Research series. Having had experience in both industry and publishing on cancer
immunotherapies, in particular immune checkpoint inhibitors and chimeric antigen
T cell therapies, I thought I would approach the Editor at the time about my idea
to produce an edited collection on Cancer Immunotherapies. It was well received
by the Editor, and the proposal was passed along to the Series Editor for the
Cancer Treatment and Research series, who ultimately approved. After a series
of requests for chapters and further communication, the volume started to take
shape, and finally we had ten contributors established. These contributors are emi-
nent researchers and scholars from a variety of national and international locales
and preeminent cancer centers, including the Fred Hutchinson Cancer Research
Center at University of Washington, Duke University, Memorial Sloan Kettering
Cancer Center, Oxford University, Indiana University School of Medicine, Uni-
versity of Calgary, The University of Hong Kong, Winship Cancer Institute at
Emory University, and the National Yang-Ming Chiao Tung University in Taipei,
Taiwan. The volume begins with an in-depth discussion on the development of can-
cer immunotherapies and covers immune checkpoint inhibitor therapies for solid
tumors such as melanoma. Other contributions focus on chimeric antigen receptor
T cell therapies, CAR-T cell therapy for glioblastoma multiforme, and comprehen-
sive accounts on genitourinary malignancies and acute myeloid leukemia, as well
as molecules in the tumor microenvironment such as Lag3, along with single-cell
sequencing for enhancing cancer immunotherapy. We aim to provide state-of-art
knowledge and expertise on the fast-paced field of cancer immunotherapies for the
clinician, scientist, and interested stakeholders in this collection that the audience
will find enlightening.

San Mateo, CA, USA Priya Hays

vii
Acknowledgements

This edited collection, Cancer Immunotherapies: Solid Tumors and Hematologic

Malignancies in the Cancer Treatment and Research Series published by Springer
Nature, was the result of the copious efforts of many people and institutions. First,
I would like to thank my editor Sydney Keen at Springer Nature who first pro-
posed it to the Cancer Treatment and Research Editor, Corinna Hauser, who then
submitted it to the Series Editor Steven Rosen, who approved it. I have to extend
my utmost gratitude to the Society for Immunotherapy of Cancer who permitted
me to advertise the call for chapter contributors on their website. A most gra-
cious thanks go especially to the brilliant set of scholars and researchers from all
across the globe with whom I collaborated to write and publish this edited col-
lection. They grace this publication with their generous insight and vision for the
state of current advances in the cancer immunotherapy field. Some are established
Springer Nature contributors, while others are more junior in the field, but on their
way to a wonderful future. I also thank Banu Dhayalan for her outstanding efforts
as Production Editor in organizing this volume. They were all a delight to work
with.

ix
Introduction

Cancer Immunotherapies: Solid Tumors and Hematologic Malignancies contains

the following chapters from national and international contributors, spanning the
spectrum of the wide applications of immunotherapies for cancer malignancies of
all types. The first chapter, “Development of Cancer Immunotherapies,” by Diana
DeLucia and John Lee fittingly discusses how cancer immunotherapies evolved as
a “concept of harnessing the immune system” for the purposes of cancer therapy
from its origins in “Coley’s Toxins” to the production of cytokines and mono-
clonal antibodies to vaccines, immune checkpoint inhibitors and chimeric antigen
receptor T cell therapy. The chapter “focuses on recent advances, current strategies
and future outlook” for cancer immunotherapies. The second chapter “Melanoma”
by Vishal Navani and colleagues offers a fascinating window into the role of
immune therapies in non-cutaneous subtypes and a “review of the impact of under-
lying genomic, transcriptomic, epigenetic, proteomic and immunological correlates
alongside their interaction with patient phenotypes” in understanding immune
checkpoint inhibitor therapies for melanoma and the impact of immunotherapy
response and resistance. Michael Brown in the third chapter “Engaging pattern
recognition receptors in solid tumors to generate systemic antitumor immunity”
reflects with great insight on how “malignant frequently exploit innate immu-
nity to evade immune surveillance” and how these contexts are “determined in
large part by pathogen recognition receptors” whose “activation induces the deliv-
ery of T cell priming cues from antigen-presenting cells.” Brown discusses how
this phenomenon influences the tumor microenvironment, “ultimately providing a
personalized antitumor response against relevant tumor-associated antigens.” Zaki
Molvi and Richard O’Reilly provide their contribution for the fourth chapter, “Al-
logeneic tumor antigen-specific T cells for broadly applicable adoptive cell therapy
of cancer” and explain how “tumor antigen-specific, donor-derived T cells are
expected to be the mainstay in the cancer immunotherapy armamentarium” and
“analyze clinical evidence that tumor antigen-specific donor-derived T cells can
induce tumor regressions”. They conclude on the applicability of this technology
in pre-clinical and clinical settings. An excellent, innovative read. Sarwish Rafiq
and Amitesh Verma provide “Chimeric Antigen Receptor (CAR) T Cell Ther-
apy for Glioblastoma,” a well-researched chapter on a topic that focuses on a

xi
xii Introduction

malignancy that has great urgency for novel and efficacious treatments, glioblas-
toma multiforme. They remark that the remarkable clinical outcomes have been
observed in hematologic malignancies and that similar outcomes in solid tumors
such as glioblastoma have been challenging. They note that “recent data sup-
port the clinical efficacy and safety of CAR-T cell therapy” in glioblastoma,
and conclude on “emerging techniques of optimizing CAR-T cell therapy for
GBM.” Francesca Aroldi and colleagues, in “Lag-3: From Bench to Bedside” dis-
cuss Lymphocyte-activation gene 3, a transmembrane protein involved in cytokine
release and inhibitory signaling in T cells, as a target to “overcome the resistance,
improve the activity and reduce the toxicity of checkpoint inhibitor therapy.” The
explain in excellent detail how LAG-3 “is a negative regulator of both CD4+ T
cell and CD8+ T cell and the activity on CD8+ T cell is independent of CD4+
activation” and how the “blockade of LAG-3 has been tested in several com-
bination therapies.” In another valuable contribution to this volume. Kevin Lu
and colleagues provide an in-depth analysis of immunotherapies in genitourinary
malignancies in their chapter, “Immunotherapy in genitourinary malignancy: evo-
lution in revolution or revolution in evolution.” They discuss how immunotherapies
for this tumor type have evolved from IL-2 for metastatic renal cell carcinoma
to immune checkpoint inhibitors, which “demonstrate meaningful survival ben-
efit and durable clinical response.” They cite common hurdles that arise from
“benefits limited to a minority of unselected patients due to the complexities of
biomarker development” and “figuring out which patients best respond to immune
checkpoint inhibitors and which patients won’t respond to immune checkpoint
inhibitors?” They conclude on common therapeutic strategies for genitourinary
cancers for achieving health-related quality of life and efficacy. Fabiana Perna
and colleagues provide a comprehensive, outstanding account of immunotherapies
for acute myeloid leukemia, spanning from allogeneic stem cell transplantation,
immune checkpoint inhibitors, chimeric antigen T cell therapies, and antibody-
drug conjugates. They state that immune checkpoint inhibitors “have been used
with limited success in relapsed/refractory acute myeloid leukemia” since “AML
mutational burden is low” and that “identification of cell surface targets is crit-
ical for the development of other antibody-drug conjugates potentially useful in
the induction and maintenance regimens.” They “highlight active areas of research
investigations toward fulfillment of the great promise of immunotherapy to AML.”
The two penultimate chapters, written by Ryohichi Sugimura and colleagues, “Off-
the-shelf chimeric antigen receptor immune cells from human pluripotent stem
cells” and “The single-cell level perspective of the tumor microenvironment and
its remodeling by CAR-T cells” are meant to be read in sequence. They write that
“autologous donors” for autologous CAR-T therapy face “technical challenges”
and provide evidence for “the development of safe and efficient allogeneic CAR-T
therapy.” “Since the advent of the generation of immune cells from pluripo-
tent stems cells, numerous studies focus on the off-the-shelf generation of CAR
immune cells derived from PSCs,” they write, and conclude that the “combination
of PSCs-derived immune cells and CAR engineering pave the way for develop-
ing next-generation cancer immunotherapy.” The second of the two-series chapters
Introduction xiii

discusses “delineating the tumor microenvironment at a single-cell level”, a very

timely topic, that “will provide useful information for cancer treatment.” They
discuss the “cellular and molecular features that curb response to CAR-T cells,
for example, high expression of immune checkpoint molecules (PD-1, LAG3) and
anti-inflammatory cytokines (IL-4, TGFb) that block CAR-T cell function” They
discuss how newly invented single-cell technologies would benefit the understand-
ing of cancer immunotherapy. The final chapter is written by the Guest Editor
(Priya Hays) upon invitation from the Editor, Corinna Hauser, and Series Edi-
tor, Steven Rosen, entitled “Clinical Development and Therapeutic Applications
of Bispecific Antibodies for Hematologic Malignancies,” focusing on the canoni-
cal bispecific antibody blinatumomab for acute lymphocytic leukemia. Bispecific
antibodies (also discussed by the Perna and colleagues for AML) are “composed
of two monoclonal antibodies that are designed to target tumor cells by directing
T cells to the antigens on these cells. They recognize and bind to two distinct anti-
gens. The majority of bispecific antigens fall into the category of bispecific T cell
engagers or BiTEs.” Blinatumomab is the FDA-approved agent in the BiTE class
with CD19 and CD3 epitopes. This chapter discusses the mechanism of action of
BiTEs, their clinical development and efficacy, and adverse events associated with
their use in treating hematologic malignancies.
In total, these chapters provide profound insight into our understanding of
cancer immunotherapies. I speak for all of the chapter contributors and editors
who worked diligently to produce an edited collection that advances the field
forward and promotes a greater awareness of immunotherapies for all interested
stakeholders for solid tumors and hematologic malignancies and beyond.

San Mateo, CA, USA Priya Hays

Contents

1 Development of Cancer Immunotherapies . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Diana C. DeLucia and John K. Lee
2 Melanoma: An immunotherapy journey from bench to bedside . . . . 49
Vishal Navani, Moira C. Graves, Hiren Mandaliya,
Martin Hong, Andre van der Westhuizen, Jennifer Martin,
and Nikola A. Bowden
3 Engaging Pattern Recognition Receptors in Solid Tumors
to Generate Systemic Antitumor Immunity . . . . . . . . . . . . . . . . . . . . . . . . . 91
Michael Brown
4 Allogeneic Tumor Antigen-Specific T Cells for Broadly
Applicable Adoptive Cell Therapy of Cancer . . . . . . . . . . . . . . . . . . . . . . . 131
Zaki Molvi and Richard J. O’Reilly
5 Chimeric Antigen Receptor (CAR) T Cell Therapy
for Glioblastoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
Amitesh Verma and Sarwish Rafiq
6 Lag3: From Bench to Bedside . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185
Francesca Aroldi, Reem Saleh, Insiya Jafferji, Carmelia Barreto,
Chantal Saberian, and Mark R. Middleton
7 Immunotherapy in Genitourinary Malignancy: Evolution
in Revolution or Revolution in Evolution . . . . . . . . . . . . . . . . . . . . . . . . . . . 201
Kevin Lu, Kun-Yuan Chiu, and Chen-Li Cheng
8 Immune-Based Therapeutic Interventions for Acute Myeloid
Leukemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225
Fabiana Perna, Manuel R. Espinoza-Gutarra, Giuseppe Bombaci,
Sherif S. Farag, and Jennifer E. Schwartz
9 Off-the-Shelf Chimeric Antigen Receptor Immune Cells
from Human Pluripotent Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255
Handi Cao and Ryohichi Sugimura

xv
xvi Contents

10 The Single-Cell Level Perspective of the Tumor

Microenvironment and Its Remodeling by CAR-T Cells . . . . . . . . . . . . 275
Sanxing Gao and Ryohichi Sugimura
11 Clinical Development and Therapeutic Applications
of Bispecific Antibodies for Hematologic Malignancies . . . . . . . . . . . . . 287
Priya Hays
Development of Cancer
Immunotherapies 1
Diana C. DeLucia and John K. Lee

1.1 Cancer Immunotherapy—Precision Medicine

Cancer is a leading cause of death globally that is second only to cardiovascu-

lar diseases such as ischemic heart disease and stroke [1]. It is likely cancer will
become the primary cause of death worldwide as treatments for cardiovascular
disease and infectious diseases continue to improve with modern medicine and
the continued extension of average life expectancy. The development and imple-
mentation of both localized and systemic therapeutic strategies, such as radiation
therapy and chemotherapy, for the treatment of cancer have significantly improved
the quality of life and survival rates for many individuals with cancer. Although
radiation and systemic chemotherapy can successfully eliminate early disease and
help manage disease, improving patient prognosis and overall survival, incomplete
elimination of all cancerous tissue can occur alongside unwanted side effects due
to off-target damage to healthy tissue [2, 3]. Targeted cancer treatment strategies
utilize agents that block the progression of cancer by targeting molecules in the
body that specifically promote the growth and spread of tumor cells. Variation in
tumor-associated genetic alterations is responsible for a high level of molecular
heterogeneity among targetable tumor molecules within patients as well as among
different patients with the same or differing cancers. Such intra and interindivid-
ual heterogeneity have formed the basis of “precision medicine” through which
known characteristics of a patient’s tumor are used to develop a personalized
treatment regime. Despite the success of such strategies, millions of people con-
tinue to die of cancer every year. Cancer immunotherapy is a form of precision

D. C. DeLucia · J. K. Lee (B)

Fred Hutchinson Cancer Research Center and University of Washington, 1100 Fairview Avenue,
Seattle, WA 98109, USA
e-mail: [email protected]

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 1

P. Hays (ed.), Cancer Immunotherapies, Cancer Treatment and Research 183,
https://doi.org/10.1007/978-3-030-96376-7_1
2 D. C. DeLucia and J. K. Lee

Coley’s toxins - Dr. Coley treats cancer Identification of

patients with live, attenuated bacteria cytotoxic T cells Identification of IL-2
allowing for culturing and
First mouse experiments leading to the study of T cell in vitro
discovery of anti-tumor immune response

1891 1909 1940s 1950s 1960s 1980s 1990s –Present

Ehrlich proposes “Immune “Immune surveillance

of cancer hypothesis” Explosion in interest, development and use of
Surveillance Hypothesis”
(Thomas and Burnet) immune-based therapies for cancer treatment

Fig. 1.1 Pioneering events that shaped the historical evolution of immune-oncology and
immunotherapy

medicine whereby the genetic status or protein expression profile of an individ-

ual’s tumor is used to formulate immune-based treatment agents that induce an
anti-tumor immune response in patients. The goal of immunotherapy is to harness
the antigen-specific nature and cytotoxicity of the human immune system to mount
a potent anti-tumor immune response capable of eliminating all tumor cells with-
out harming healthy tissue. Over the past century, an improved understanding of
how the human immune system interacts with cancerous cells in the body has pro-
vided a foundation upon which immunotherapies are developed and studied today
(Fig. 1.1).

1.2 Immune Surveillance and Recognition of Cancer

Efforts to treat cancer with immune-based therapies originated over a century ago,
prior to the identification of tumor antigens and without an appreciation of the
many components of the human immune system and anti-tumor immune response
that we have today. Some of the first reported associations of immunity and cancer
occurred in the late nineteenth century by German physicians Wilhelm Busch and
Friedrich Fehleisen who observed that some of their cancer patients with concur-
rent erysipelas, a common streptococcal infection of the skin, experienced tumor
regression or complete remission [4]. Similar findings and strategies were also
observed and implemented by the American physician, Dr. William Coley, in the
1890s. Upon a vast review of patient medical records in search of information that
might aid in the treatment of his own patients, Coley identified a number of cases
where patients with otherwise incurable tumors entered into remission following
a diagnosis of erysipelas. Coley subsequently used intratumoral inoculations of a
cocktail of heat-killed strains of Streptococcus to treat his patients with soft tissue
sarcomas with unforeseeable success [5, 6]. The streptococcal cocktail was later
coined “Coley’s Toxins”. Despite the unrivaled success of Coley’s toxins at the
time, Coley’s colleagues heavily debated and were hesitant to adapt the therapeutic
technique due to a lack of understanding of the mechanism by which the bacte-
rial inoculation induced tumor regression [5]. Consequently, Coley’s achievements
1 Development of Cancer Immunotherapies 3

went unnoticed for the better part of the following 50 years while surgery and
radiotherapy became the standard of care for cancer patients in the early twentieth
century.

1.3 Cancer Immunoediting

In 1909, Paul Ehrlich was the first to propose the “immune surveillance hypoth-
esis” which postulated that it was not unreasonable to think that the immune
system can identify transformed cells, eliminate them from the body, and pre-
vent primary tumor formation [7]. At the time, Ehrlich lacked the technical means
to generate experimental evidence in support of his hypothesis. In the 1950s,
the conversation of immune surveillance was resurrected by two scientists Lewis
Thomas and Frank McFarlane Burnet. Soon after, the existence and immune
recognition of tumor-specific antigens was empirically demonstrated [8] thereby
providing evidenced-based support for Erlich’s immune surveillance hypothesis.
Despite these groundbreaking findings, it remained clear that many people con-
tinued to develop tumors and succumb to cancer, suggesting anti-tumor immune
response did not always effectively prevent tumor formation. In the 2000s, Robert
Schreiber proposed the “three E’s theory” to describe three stages of cancer immu-
noediting: (1) Elimination phase, previously the immune surveillance hypothesis,
(2) Equilibrium phase, and (3) Escape phase [9]. During the elimination phase,
immune cells effectively identify and eliminate tumor cells in otherwise healthy
tissues. The immune pressure applied to the tumor during the elimination phase
drives some cancerous cells to undergo changes that mediate immune evasion
resulting in a subset of variant tumor cells that are resistant to immune recogni-
tion and/or killing resulting in an equilibrium phase of immune-tumor interaction.
Lastly, tumors enter the escape phase once all immunogenic tumor cells have been
eliminated and only variant tumor cells remain to spread unhindered due to their
acquired immune resistance. Additionally, tumor cells that have undergone Epithe-
lial Mesenchymal Transition (EMT) often acquire improved metastatic potential
through enhanced mobility and resistance to both apoptosis and immune-mediated
killing [10, 11]. Therapeutic strategies targeting EMT have potential to improve
the efficacy of immune-based therapies.
Acquisition of evidence directly demonstrating cancer immunoediting has his-
torically been challenging due to the need for large patient numbers, the heavy
demand for long-term study follow-up, and significant biological sample collec-
tion. The first collective body of data in support of human immune surveillance
of cancer consisted primarily of studies comparing cancer incidence and progres-
sion rates in individuals with robust versus compromised immune systems. By
the late 1980s, it was well documented that immunocompromised individuals,
such as transplant patients, non-transplant patients receiving immunosuppressive
treatment, established cancer patients receiving immunosuppressive chemother-
apy, individuals with genetic immunodeficiencies, and individuals with acquired
immunodeficiency syndrome, have a higher incidence of developing certain types
4 D. C. DeLucia and J. K. Lee

of primary or secondary cancer [12]. A recent study of 907 immunocompromised

heart and/or lung transplant patients between 1989 and 2004 found that the trans-
plant patients were seven times more likely to develop cancer compared to the
non-transplant population [13]. While the clear association between immune defi-
ciency and cancer incidence supported an important role for the immune system
in cancer development, mechanisms of cancer immune surveillance and the use-
fulness of immunotherapies became more evident as the scientific community’s
discovery and understanding of human tumor antigens advanced.

1.4 Tumor Antigens

Throughout much of the twentieth century, it was unclear whether the immune sys-
tem could detect malignant cells or develop a protective immune response against
tumors. The development of inbred mice strains and the discovery that tumors
could be induced with carcinogens and transplanted between mice provided the
tools necessary for the first essential studies that demonstrated immune recogni-
tion and elimination of tumors [14–17]. Mice injected with tumor cells rendered
replication-incompetent through irradiation, were often protected against subse-
quent injection of growth competent cells of the same tumor type, but not of a
different tumor type [18]. These experiments helped validate immune recognition
and protective immunity against tumors and established the concept that differ-
ent tumor types have unique immunogenic antigens, known as tumor recognition
antigens.

1.4.1 Tumor-Specific Antigens

Tumor recognition antigens are categorized into two subsets: Tumor-specific

antigens (TSAs) and tumor-associated antigens (TAAs). TSAs are “self” protein-
derived peptides strictly found in tumor cells and not in nonmalignant cells that
are presented on major histocompatibility complexes (MHC). TSAs are typi-
cally caused by genomic mutations or gene rearrangements leading to amino acid
changes in proteins of cancer cells from which unique peptides can be generated,
known as neoantigens or neopeptides [19]. Neoantigens form when the coding
sequence of a peptide that is normally presented is altered leading to the de novo
presentation of a novel peptide on MHC. For example, specific mutations in CDK4
in melanoma cells result in presentation of highly immunogenic CDK4-derived
neopeptides [20, 21]. Common mutations in tumor-suppressor genes such as TP53
in cancer also represent immune-reactive TSAs [22, 23].
1 Development of Cancer Immunotherapies 5

1.4.2 Tumor-Associated Antigens

TAAs are proteins or peptides that are expressed at a higher level in cancer
cells while also being expressed in healthy cells but at a much lower level.
Cancer-testis antigens, such as melanoma-associate antigens (MAGE) and New
York esophageal squamous cell carcinoma 1 (NY-ESO-1), are well-studied exam-
ples of TAAs because they are only expressed in healthy male germ cells which
lack MHC expression. TAAs also include differentiation antigens which are only
expressed in select tissues such as CD19 on B lymphocytes and B cell lymphomas
and MART-1 in melanoma [24–26]. Expression of TAAs can be upregulated in
malignant cells through abnormal gene expression as well as post-transcriptional
and post-translational modifications [26, 27]. Oncoviral proteins, foreign proteins
expressed by cancer cells following infection and transformation by cancer-causing
viruses, also serve as TAAs. The E6 and E7 proteins encoded by human papillo-
mavirus type 16, one of the most common etiological agents of cervical cancer,
and the K8.1 glycoproteins expressed by Kaposi’s sarcoma virus are examples of
immunogenic oncoviral proteins [28, 29].

1.5 Methods of Tumor Antigen Identification

The identification of cytotoxic T lymphocytes (T cells) in the 1960s [30] and the
development of methods to propagate and study antigen-specific T cells in vitro
in the 1970s and 80s [31–33] provided the foundational knowledge and technical
methods required to identify tumor antigens. Additional technological advance-
ments over the past 30 years have led to a sharp increase in the number of known
tumor antigens, many of which are now utilized for cancer diagnostics and thera-
peutic targeting. A large range of molecular strategies continues to be explored to
improve the identification of tumor antigens.

1.5.1 Gene Expression Profiling

Safety concerns when selecting tumor antigen targets are a primary focus for
preclinical and clinical studies evaluating precision cancer therapies. To avoid
off-tumor toxicity and undesired side effects, ideal tumor antigens for therapeutic
targeting should be expressed exclusively or preferentially by tumor cells com-
pared to healthy cells in the body. Tumor antigen expression should also be present
across all or most malignant cells within tumors to maximize efficacy of the anti-
gen targeting agent or drug and to minimize therapeutic resistance due to antigen
escape. A common strategy to identify tumor-restricted antigens involves com-
paring the gene expression profiles of malignant and healthy cell lines or tissues
using a variety of technologies such as microarrays, RNA-sequencing, direct digi-
tal mRNA counting, and real-time PCR [34–39]. Proteins encoded by genes widely
6 D. C. DeLucia and J. K. Lee

expressed or overexpressed by tumor cells compared to healthy tissue can be inves-

tigated as potential tumor rejection antigens. However, analysis of gene expression
alone does not always mirror protein expression profiles and is less effective for
the discrimination of intracellular versus cell surface protein expression. An alter-
native approach is to integrate the analysis of transcriptomics and proteomics. We
have recently used this method to specifically study differential cell surface protein
expression to identify candidate tumor antigen targets for CAR T cell therapy in
molecular subsets of prostate cancer [40, 41].

1.5.2 Serological Screening for Antibody-Targeting Antigens

Studies demonstrating humoral immune responses against tumors as well as a

continued increase in the use of antibodies for immunological studies sparked
interest in the identification of human antibody-binding tumor antigens. The first
antigen discovery methodology of this type, serological screening of recombinant
cDNA expression libraries (SEREX) was developed in 1995 [42]. SEREX uti-
lized reverse proteomics whereby cancer patient sera were used to screen a phage
cDNA library derived from various human malignancies for potential tumor anti-
gens recognized by patient tumor-specific antibodies. Several novel tumor antigens
have been identified using traditional SEREX strategies, including the cancer-testis
antigen NY-ESO-1 [43]. The use of traditional, modified, and high-throughput
optimized SEREX technology has aided in the identification of thousands of
additional tumor antigens. The serological and proteomic evaluation of antibody
responses (SPEAR) method combines the use of 2-D polyacrylamide gel elec-
trophoresis of patient tumor samples, immunoblotting with patient sera, and mass
spectrometric protein analysis to rapidly identify tumor antigens. SPEAR, as well
as similar methods, has been used successfully in the setting of renal cell carci-
noma for which identification of tumor antigens has been historically challenging
[44, 45].

1.5.3 Recognition by Reactive Patient Lymphocytes

The use of tumor-reactive lymphocytes, specifically T cells, from patients as a

screening tool has been one of the most widely used strategies for tumor antigen
identification and continues to be used today. Tumor-infiltrating lymphocytes or
lymphocytes isolated from patient peripheral blood are co-cultured with human
leukocyte antigen (HLA)-matched cells expressing cDNA libraries corresponding
to protein expression profiles derived from specific tumor types. Reactivity of the T
cells is used to screen for immunogenic tumor antigens. The first tumor-associated
antigen, melanoma-associated antigen MAGE-1 [46], was identified using this
method and many others have followed [47–54]. While advancements in tumor
1 Development of Cancer Immunotherapies 7

antigen identification have led to the discovery of an abundance of potential tar-

gets for immunotherapy, antigen selection remains a challenging and essential step
in the development of all targeted immunotherapeutic modalities.

1.6 Tumor Immune Evasion

Tumors employ various mechanisms to avoid activation of and detection by

the host immune system such as decreasing overall immunogenicity through
modulation of antigen expression, inducing T cell tolerance of tumor antigens,
and creating a suppressive tumor microenvironment (TME). Some cancer types
referred to as “cold tumors”, inherently have poor immunogenicity whereby the
majority of the tumor cells, as well as nonmalignant cells found in the TME, do
not express tumor rejection antigens or other molecules that mediate immune cell
infiltration and activation. Such tumors can (1) alter adhesion molecule expression
that would otherwise mediate contact growth inhibition as well as proinflammatory
signaling, (2) express few neoantigens due to a low tumor mutation burden, or (3)
exhibit aberrant MHC expression due to somatic mutations in antigen-presenting
genes or downregulation of MHC gene expression [55–57].
Tumor cells can also undergo antigen escape due to genetic instability resulting
in altered amino acid sequences of tumor antigens or downregulation of tumor
antigen expression to evade immune detection. Some tumor cells have been found
to downregulate expression of T cell costimulatory molecules such as CD80 and
CD86 which are essential for antigen-specific T cell activation and anti-tumor
cytotoxicity [58, 59]. T cells that interact with tumor antigen presented on MHC
on the tumor cell surface in the absence of these costimulatory signals are more
likely to become tolerized rather than mediate killing of the tumor cell [60]. Addi-
tionally, cancer cells can induce immune suppression and promote a “pro-tumor”
microenvironment by acting directly on cytotoxic T cells through the secretion of
factors that directly suppress T cells such as tumor growth factor-beta (TGF-β) and
indoleamine 2,3-dioxygenase (IDO) or induce tumor cell surface expression of T
cell inhibitory molecules such as PD-L1 [61]. Some of the same factors can attract
or drive the differentiation of suppressive immune cells such as regulatory T cells
(Treg) and myeloid-derived suppressor cells (MDSC) to and within the tumor [61].
The production of collagen by tumor cells often functions to create an extracellu-
lar matrix through which immune cells struggle to pass. This physical barrier can
create a tumor-induced privileged site that effectively blocks the entrance of any
component of the cellular immune response.
Despite the multitude of mechanisms used by tumors to evade elimination by
the immune system, a large body of research conducted over the past several
decades has improved our understanding of the ways in which the immune system
and cancer cells interact, thereby better positioning scientists to develop strategies
for effective immunotherapy against cancer.
8 D. C. DeLucia and J. K. Lee

1.7 Passive Immunotherapy

The overall goal of cancer immunotherapy is to harness the antigen-specific and

cytotoxic potency of the immune system to eliminate cancer cells from the body
at both local and metastatic disease sites. An extensive range of modalities uti-
lizing modified components of both the humoral and cellular immune response is
under investigation for the generalized treatment of a broad spectrum of cancers.
Immunotherapies are grouped into two main categories: passive immunotherapies
and active immunotherapies. Passive forms of immunotherapy involve the intro-
duction of donated or ex vivo-derived immune system components into a cancer
patient to specifically target that patient’s tumor. The most common forms of pas-
sive immunotherapy currently under investigation are tumor-specific antibodies,
immune checkpoint inhibitors, and adoptive T cell therapy.

1.7.1 Tumor-Specific Antibodies

1.7.1.1 Monoclonal Antibodies

Monoclonal antibodies (mAb) have been routinely used for the clinical treatment
of cancer for the past decade and represent some of the earliest immunotherapies
tested in clinical trials and approved for clinical use due to their improved effi-
cacy over current standard of care and versatility. Unlike polyclonal antibodies,
which are a collection of antibodies that recognize different epitopes within the
same antigen due to their isolation from a pool of B cells in an antigen immunized
organism, mAb are isolated from clonal selection of antigen-specific hybridomas
and are therefore identical to one another [62]. Hybridomas generated from the
fusion of B cell clones from antigen immunized mice with immortalized myeloma
cells lacking antibody production were first used to generate mAb [63]. The ability
to produce large amounts of antibodies targeting a single epitope made monoclonal
antibodies ideal for clinical use. Strong human anti-mouse antibody responses in
patients have been demonstrated against some mouse monoclonal antibodies lead-
ing to decreased efficacy of the therapeutic antibody [64]. Molecular techniques
have since been developed to humanize or generate chimeric versions of mouse
mAb in order to eliminate patient immune reactivity without sacrificing the desired
antigen specificity of the original antibody [65]. The variable regions of therapeu-
tic mAb recognize and bind to protein antigens expressed on the surface of tumor
cells effectively tagging the tumor cells for recognition by other immune cells
which bind the constant region (Fc) of the antibody. Fc receptor binding facili-
tates direct killing of tumor cells through complement-dependent cytotoxicity, or
indirectly through antibody-dependent cellular cytotoxicity (ADCC), usually medi-
ated by natural killer (NK) cells [66] or phagocytosis of the tumor cell-mediated
by myeloid cells such as macrophages [67]. The CD20-targeting chimeric mAb,
Rituximab, was shown in a phase II clinical trial of 37 patients with relapsed
non-Hodgkin’s lymphoma following chemotherapy to provide improved patients