Cancer Immunology / Immunotherapy

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Primary neoplasm Metastasis

Tumor evolution & Progression Metastasis

Transformation
 Cancer Immunology / Immunotherapy
Immunotherapy of cancer is the 4th modality approaches in cancer
1st approach – Surgery
2nd approach – Radiation
3rd approach - Chemotherapy

Immunotherapy is treatment that uses certain parts of a person’s immune system to fight
diseases such as cancer. This can be done in a couple of ways:
a) Stimulating your own immune system to work harder or smarter to attack cancer cells
b) Giving you immune system components, such as man-made immune system proteins

Do cancer specific antigens exist and, if so, are they recognized by the
autologous host?
How these possible cancer antigens could be discovered for immunotherapy?
 Host Immune response to tumor

Circumstantial evidences
•Spontaneous regression
•Regression of tumor due to sub lethal dose of chemotherapy
•Regression of metastasis after resection of primary tumor
•Mononuclear cell infiltration into the tumor
•High incidence of tumor after clinical immunosuppression
•High incidence of tumor in immunodeficiency diseases
•High incidence of tumor on aging

Experimental evidences
►Colony inhibition of tumor by sensitive lymphocytes
►Lymphocyte blast formation in presence of tumor extracts
►Lymphocyte enhanced cytotoxicity in patients with tumor
►Macrophage enhanced phagocytosis in patients with tumor
Cellular effectors that mediate Immunity

• Macrophage activated exhibit selective cytotoxicity against tumor cells

T cell
NK cell collaborate in anti-tumor reactivity
Macrophage

(e.g. IFN- secreted by T and NK cell, activator of macrophage) kill by production

of reactive oxygen or secretion of TNF-)

• Humoral mechanism Activation of complement

Induction of ADCC by NK cell
 Antitumor effector arms of immune response: The immune system can impact on tumor growth in
several ways
•Most effective way in vivo is through combined action of CD8 + and IFN-γ-secreting Th1 CD4+ T cells.
•Tumor specific CD8+ T cells activated by DCs presenting tumor antigens can kill tumor cells directly.
•The survival and persistence of CD8 + T cells as memory cells is regulated by tumor specific CD4 + T cells.
•Both CD8+ and , especially CD4+ T cells secrete IFN- γ, which can further sensitize tumor cells to CD8 + T
cells by up regulating MHC-I and other components of antigen-processing machinery promoting the
recruitment NK cells, granulocytes, macrophages, and interfering with crucial function of tumor stroma e.g.
angiogenesis
•Tumor can also be controlled by Th2 type immune response, whereby DCs activate IL-5-secreting Th2
CD4+T cells, which induce the accumulation of eosinophils in the tumor bed and provide ‘T help’ for the
generation of a humoral, antibody-based antitumor response
 Cancer Immunoediting Process:

TNF-related apoptosis-inducing
ligand (TRAIL)

Normal cells ( gray) subject to common oncogenic stimuli ultimately undergo transformation and become tumor cell (red).

At early stage of tumorigenesis, these cells may express distinct tumor specific markers and generate proinflammatory
‘danger’ signals that initiate the cancer immunoediting process.

In the 1st phase of elimination, cells and molecules of innate and adaptive immunity, which comprise the
immunosurveillance network, may eradicate the developing tumor and protect the host from tumor formation.

However, if this process is not successful, the tumor cell may enter equilibrium phase whether they may be either
maintained chronically or immunologically sculpted by immune ‘editors’ to produce new populations of tumor variants.
These variants may eventually evade the immune system by a variety of mechanism and become clinically detectable in the
escape phase.
 Known Intrinsic Mechanisms of Resistance to Immunosurveillance

A) Intrinsic factors lead

to primary or adaptive B) Intrinsic factors
resistance associated with
i) loss of tumor antigen acquired resistance
expression, of cancer,
ii) loss of HLA i) loss of target
expression, antigen, mutation
iii) alterations in antigen in HLA,
processing machinery, ii) loss of T cell
iv) alterations of several functionality.
signalling pathways
(MAPK, PI3K, WNT,
IFN),
v) constitutive PD-
L1( Programme Death
Ligand-1 binding with its
receptor PD-1 on T cells ,
inhibiting TCR-mediated
activation of IL-
2 production and T cell
proliferation) expression.
vi)

PD-L1:Programme Death Ligand-1 binding with its receptor PD-1 on T cells , inhibiting TCR-mediated activation of IL-2 production and T cell proliferation.
 Known Extrinsic Mechanisms of
Resistance to Immunosurveillance:
i) Activation of CTLA-4, PD1, and
other immune checkpoints ( inhibiting
T cell activation),
ii) T cell exhaustion and phenotype
change,
iii) Appearance of immune
suppressive cell populations (Tregs,
MDSC(Myeloid derived suppressor
cells), type II macrophages),
iv) Secretion of tumor growth
promoting cytokine and metabolite
release in the tumor
microenvironment(CSF-1,
tryptophan metabolites, TGF-b,
adenosine)

Abbreviations are as follows:

APC, antigen-presenting cells; MHC, major histocompatibility complex; TCR, T cell receptor; Treg, regulatory T cell;
MDSC, myeloid-derived suppressor cell; Mɸ II, type II macrophage.
TIM3, T cell Ig mucin domain-containing 3; Lymphocyte-activation gene 3, LAG-3;( Inhibitory Signal)
The inducible co-stimulatory molecule (ICOS); GITR, glucocorticoid-induced TNFR family-related protein;
OX40 (CD134) members of the TNFR/TNF superfamily ; 4-1BB ( CD137) ( Stimulatory Signal)
Mechanism of tumor-mediated immune evasion:
►Tumor secrete various factors [vascular endothelial growth factor (VEGF), macrophage colony
stimulating factor (MCSF), IL-6] that promote the accumulation of heterologous population of myeloid
cells with immune-suppressive properties (known as immature myeloid cells) as a consequence of
continuous JAK2-STAT3 signaling.
►These myeloid cells suppress T-cell immunity by various mechanism e.g. depletion of arginine and
elaboration of reactive oxygen species ( ROS) and nitric oxide ( NO).
►The tumor microenvironment also promotes the accumulation of regulatory T cells that suppress
T-cell function through a host mechanisms, some of which include secretion of IL-10 or TGF-β which
are often directly secreted by tumor cells to the same effect.
►Tumors by virtue of their genetic instability, can escape immune elimination by down regulating the
tumor antigens or antigen processing machinery.
 Six key steps for specific CD8+ T cell mediated antitumor immunity
Effective destruction of tumor cells by antigen specific CD8 + T cell is multistep process.
a) Tumor antigen must be present,
b) these antigens must reach and load APC in draining lymph node,
c) specific T cells must respond by proliferation,
d) the circulating T cells must enter the tumor
e) once in the tumor the T cell must be able to overcome local immune suppressive molecules to recognize and kill
targets,
f) memory cells should be generated

Cancer immunotherapy can fail at any of these steps, so the development of assay to analyze of these steps in vivo has
been the key to understand how chemotherapy and immunotherapy interact.
 Assay for Six key steps for specific CD8 mediated
antitumor immunity
Steps In vivo assay system for measuring response

Antigen threshold Quantification of levels of marker antigens by ELISA or western blot

Antigen presentation CFSE ( carboxyfluorescein diacetate succimidyl ester) dye dilution assay

T-cell response a) In vivo CTL assay using CFSE and peptide - loaded target cells
b) Tetramer analysis of tumor specific T-cell numbers; ELISPOT or
intracellular cytokine staining for cytokine production-can be combined
with other assay

T-cell traffic Staining for T-cell infiltration; flow cytometry of extracted cells; trafficking
of dye labeled cells

Target destruction Reduction in size of tumor, cytokine staining in tumor, function of cells
extracted from tumor

Generation of memory Flow cytometry of extracted cells, adoptive transfer of extracted cells,
tumor growth after re-challenge
Tumor Antigens
Tumor-associated transplantation antigen (TATA)

•Re-expressed embryonic antigens, the Oncofetal proteins

•Alpha-fetoprotein (AFP)
•Ex. MAGE-1 Melanoma associated
Antigen in breast cancer cells and
brain tumor cells
• Carcino-embryonic antigen (CEA)
•Differentiation antigens
• Over-expressed normal proteins
•Viral Antigens (TATA on viral tumors)
•Tumors induced by oncogenic viruses

 Tumor-specific antigens (TSA)

• An antigen that is unique to tumor cells only
• Constitute mostly of mutated self antigens
 Identification of tumor antigens
Antigens analysis of the T-cell repertoire against tumors done by using three strategies:

1st: The genetic approach which makes use of antigen-loss tumor cell variants and cytotoxic CD8+ anti-
tumor T-cell clones (cytotoxic T lymphocytes). The discovery of IL-2 permitted the isolation of stable
lines of cytotoxic T cells with specificity for autologous melanoma cells , and this, in turn, led to the
identification of T cell–recognized epitopes on human tumor cells. MAGE (melanoma associated
antigen) and tyrosinase that had originally been defined as T cell-recognized epitopes.

2nd: The biochemical strategy , which is based on the acid elution of antigenic peptides bound to major
histocompatibility complex class I molecules from tumor cells .
Using these approaches, several additional human tumor antigens have been defined at the molecular
level, most notably in malignant melanoma.
Main task is to discover tumor antigens which elicit immune response in autologous host.

3rd approach : Serological screening methods

Other approaches : Approaches that apply cDNA-expression cloning, reverse immunology and
mass spectroscopy, computer based algorithms

Ultimate goal is to Search for Universal Tumor-Associated T Cell Epitopes (Th and Tc, Treg) as
because cellular immunity is more important in tumor immunotherapy
 SEREX (serological analysis of recombinant cDNA expression libraries)

SEREX was developed to combine serological analysis with antigen cloning techniques to
identify human tumor antigens eliciting high-titer immunoglobulin G (IgG) antibodies.
It is based on autologous typing and is able to rapidly analyze thousands of proteins in a tumor
specimen to identify cancer antigen.

*SEREX analysis has now identified a series of provocative cancer antigens that have
relevance to the etiology, diagnosis, and therapy of cancer.

*SEREX provides a way to analyze the humoral immune response to intracellular cancer
antigens, a generally impenetrable for cancer serologists in the past.

* SEREX repertoire can be considered to be a reflection of the CD4+ T cell repertoire.

This approach involves immunoscreening cDNA libraries extracted from fresh tumor tissues
with sera from cancer patients to identify gene products recognized by IgG antibody

SEREX-defined clones can be directly sequenced and their expression profiles can be readily
determined, allowing for immediate structural definition of the antigenic target and subsequent
identification of TAAs
Serology plays a central role in three phases of SEREX analysis.
1. In the initial identification of reactive clones
2. In screening small panels of sera from normal individuals and cancer patients for antibody
3. In large-scale surveys of human sera (grand serology)
 Cancer Neoantigen Screening
Discovery Whole genome/exome sequencing
(arise due to somatic
mutations e.g.
missense, frameshift Selection
mutation in TSA)
Patient HLA typing Epitope Prediction by
computational algorithm

Validation
Synthesizing mutation-derived peptides
In vitro T-cell assay/in vivo immunization in
mouse model

The flowchart shows the process of identifying cancer neoantigens for target of immunotherapy, consisting of three steps:
a) screening, b) selection, and c) validation of the candidate immunogen.
First, the whole genome/exome sequence profile is comprehensively screened to identify tumor-specific somatic mutations
(cancer neoantigens) by massive parallel sequencing of tumor and normal tissues, respectively.
Second, computational algorithms are used for predicting the affinity of the mutation-derived peptides with the patient’s
own HLA and/or TCR.
Third, synthetic mutated peptides and wild-type peptides are used to validate the immunogenicity and specificity of the
identified antigens by in vitro T-cell assay or in vivo immunization.
Validated antigens could be applied to immunotherapy as either biomarkers of responses to immunotherapy, or as targets of
cancer immunotherapy, including cancer vaccines and adoptive T-cell therapy.
 Modalities of Cancer Immunotherapy

a) Monoclonal antibodies to treat cancer

b) Non-specific cancer immunotherapies and adjuvants

c) Immune checkpoint inhibitors

d) Adoptive cell therapy [T cell (Chimeric antigen receptor (CAR) T-

cell therapy and tumor-antigen-specific T cell receptor-engineered
therapy( TCR-T cell), tumor-infiltrating lymphocytes (TILs)]

e) Cancer Vaccine (Tumor cell vaccines, Antigen vaccines, Dendritic

cell vaccines, Vector-based vaccines (Oncolytic virus)
Anti-tumor monoclonal Antibodies:
Antibodies specific or tumor associated antigens may serve as “ magic bullet”
that target cancer cells for destruction while sparing most normal tissues
Mechanism
- Fc receptor mediated engagement of phagocytes
- Complement mediated injury or clearance
-Target toxins or drugs linked to antibodies
-Inhibitory signals induced by antibody

1.Only Antibody
ADCC- Antibody dependent cytotoxicity,
CDC-Complement dependent cytotoxicity,

2. Immunoconjugates
ADEPT- Antibody directed prodrug therapy
Immunotoxin
Immunocytokines
Radioimmunoconjugate

3. Multistep targeting
Biotinylated radioactive ligand
Bispecific antobody
 Macrophage Infiltration
xx
High oxygen region Necrotic core

chemoattractant

macrophages

Hypoxic (low oxygen) region

z
z

Hypoxic tumor cells release chemoattractant, causing

macrophages to localize to hypoxic regions.

•Activated macrophages
- activity demonstrated mainly in vitro
-tumor cells may be more susceptible to macrophages mediated killing
than normal cell
Polarization of macrophage function
IL-1ra: IL-1 receptor
antagonist protein, an acute
phase protein

Macrophages constitute an extremely heterogeneous population, which could be divided schematically into two main
classes: M1 and M2.
Blood monocytes differentiating in the presence of LPS/IFN- mature into M1-polarized cells (classically activated
macrophages). They produce high levels of IL-12, IL-1, IL-23, TNF-α, and CXCL10 and are characterized by cytotoxic
activity against microorganisms and neoplastic cells, expression of high levels of ROI, and capability as APCs.

On the other hand, when monocytes differentiate in the presence of IL-4, IL-13, IL-10, or corticosteroids, they mature
into M2 macrophages (alternatively activated, which secrete IL-10, CCL17, CCL22, CCL18, IL-1ra, and IL-1R decoy.
M2 cells are active workers of the host, promoting scavenging of debris, angiogenesis, remodeling, and repair of
wounded/damaged tissues. Within the tumor mass, they exert the same functions favoring tumor promotion. In addition,
M2 macrophages control the inflammatory response by down-regulating M1-mediated functions and adaptive immunity.
Repolarization of macrophages - a way to treat cancer?
The strong infiltration of human tumors by activated CD8+ cytotoxic lymphocytes (CTL) and CD4+ helper T
(Th) cells but not immune suppressive cells such as regulatory T cells, M2 macrophages and myeloid derived
suppressor cells is a hallmark for improved survival after therapy.

The treatment of cancer may take advantage of therapies that interfere with M2 macrophages, if
combined with standard or immunotherapeutic regimens.
Therapeutic modalities may attack at several levels; the attraction, the differentiation or the activation
of macrophages.

Strategies to inhibit the induction of M2 macrophage

One therapeutic option is to interfere at the level of macrophage attraction and differentiation by
abrogation of the PGE2, IL-6 and STAT3 activation loop.
A second strategy to interfere with immune cells in the tumor micro milieu is the blockade of
cytokines secreted by the tumor or immune cells .

Reprogramming M2 macrophages once they exist:

 Reprogramming of M2 to M1 type macrophages not only requires receptor-mediated activation signals
but the presence of polarizing cytokines (e.g. IFN-γ).
 In order to reprogram macrophages directly in the tumor-microenvironment, it is essential that CD4+
Th1 cells are locally present.
The capacity of tumor-specific Th1 cells to directly alter the tumor microenvironment has also been
recognized in studies on tissue-infiltrating CD8+ T cells in mice models.
 In order to obtain sufficient numbers of tumor-specific CD4+ Th1 cells one may make use of
adoptive T-cell transfer protocols or apply strong vaccines.
T cell based Immunotherapy:
Immune checkpoint inhibitors for CTLA-4, PD-1
Overview of coinhibitory pathways.

Upon T cell activation (mediated by TCR

recognition of antigens presented on APCs and
costimulation through the B7/CD28 interaction),
many different pathways may inhibit the T cell
response.
·Reminiscent of CD28 and CTLA-4, several
receptors can have multiple binding partners. Some
receptors are also expressed on NK cells (LAG-3,
PD-1, TIM-3, CD96, TIGIT), and certain ligands are
expressed on APCs and nonhematopoietic cells and
in tumor tissues (PD-L1, PD-L2, B7-H3, VISTA,
CD155, CD112).

Abbreviations: APC, antigen-presenting cell; TIM-3,

T cell–immunoglobulin–mucin domain 3; VISTA, V-
domain immunoglobulin-containing suppressor of T
cell activation.
Mechanisms of the CTLA-4 pathway and effect of
CTLA-4 blockade.
a) CTLA-4 is induced by T cell activation and can
inhibit the immune response in a T cell–intrinsic
fashion by intracellular signals that inhibit TCR and
CD28 signaling or in a T cell–extrinsic fashion by
reducing expression of B7 (CD80 and CD86) on APCs.
Anti-CTLA-4 monoclonal antibody prevents the
interaction between CTLA-4 and B7 and may deplete
Tregs in the tumor microenvironment, thereby allowing
CD28 signaling without CTLA-4 opposition and
promoting an ongoing immune response.

Mechanisms of the PD-1 pathway and effect of

PD-1/PD-L1 blockade.
(a) PD-1 is inducibly expressed on T cells.
Engagement of PD-1 by PD-L1 or PD-L2 results in
reduced phosphorylation of TCR signaling
molecules, thereby limiting T cell effector functions.
Anti-PD-1 or anti PDL1 or both Anti-PD-1 and
anti PDL1 monoclonal antibody allow T cell to
grow.
 Immune checkpoint inhibitors for CTLA-4, PD-1

T cell activation in the tumor milieu. a Programmed cell death protein 1 (PD1)
receptor is an inhibitory receptor expressed by antigen stimulated T cells.

Interactions between PD1 and its ligand, PD-L1, expressed in many tumors activate
signaling pathways that inhibit T-cell activity and thus block the antitumor immune
response.
Antibodies targeting PD1 or PD-L1 block the PD1 pathway and reactivate T cell activity.
MCH, Major histocompatibility complex; TCR, T cell receptor
Immune checkpoint blockade approach.
This approach to immunotherapy is exemplified by antibodies directed against CTLA ‑4
(ipilimumab, tremilimumab), which block the immunosupression mediated by the interaction
between B7 family members (on antigen-presenting cells) and CTLA ‑4 (on CD8+ and CD4+ T
cells). A second major checkpoint, mediated by the interaction between PD ‑1 on T cells and its
ligand PD‑L1 on either antigen-presenting cells or tumour cells, has been the subject of several
recent clinical trials, and has shown evidence of efficacy in both non-small-cell lung cancer and
renal cell carcinoma.
 Adoptive T cell therapy (ATCT)

With ATCT, T cells are trained or genetically engineered ex vivo to react to tumor antigens then
expanded prior to reinfusion to the patient. The ex vivo manipulation of T cells ensures that they are
properly stimulated and differentiated into armed effector cells prior to their adoptive transfer.

Different ways of ATCT :

Tumor Infiltrating lymphocytes ( TIL) : Ex vivo TIL expansion with IL-2 enhances the number of
cytotoxic lymphocytes available for adoptive cell transfer. Although IL-2 facilitates the large-scale
expansion of TIL, this process requires 14-100 days and cytolytic activity declines with time .

Allogeneic T lymphocytes: Peripheral blood mononuclear cells (PBMNC) from healthy donors are
mixed with lethally irradiated glioma patient PBMNC. The mixed lymphocyte reactions allow for the
expansion of alloreactive cytotoxic T lymphocytes (aCTL) that recognize patient HLA antigens in
bioreactors.

Autologous tumor antigen sensitized T lymphocytes: Glioma patient PBMNC were activated and
expanded by co-culturing lethally irradiated glioma cells with PBMNC in the presence of IL-2. The
lymphocytes were infused in tumor beds.
Chimeric antigen receptor T cells ( CAR T Cell)

Genetic engineering of T cell for the

improvement and broadening of tumor-
infiltrating lymphocyte (TIL) therapy.

Chimeric antigen receptors:

(CARs) consist of an Ig variable extracellular

domain fused to a T cell receptor (TCR) constant
domain.
The engineered T cells obtain the antigen
recognition properties of antibodies and thus are
targeted against any potential cell surface target
antigen. The expression of the TCR confers the
engineered T cell with the antigen specificity of
the transferred TCR.
TIL therapy with TCRs is feasible for patients
whose tumor harbors the human leukocyte
antigen (HLA) allele and expresses the target
antigen recognized by the TCR
 Chimeric antigen receptor (CAR) T-cell therapy
This method involves reconstruction of the CAR receptor found on T cells to recognize specific antigens
on cells that are to be destroyed. In principle and practice, this can include ex vivo growth of T cells
before they are genetically modified by typically a retrotransposon introduced to the T cells by the
various methods including viral vectors.

There are three primary components of CAR receptor including the a) extracellular domain, b)
transmembrane, and c) intracellular domain. The primary feature of the extracellular domain is the scFv
{single-chain variable antibody fragment, (fusion protein of the variable regions of the heavy (VH) and light chain} region which is
similar to the light chain region of an antibody. The transmembrane domain connects the scFv region
costimulatory molecules and can influence the immunogenicity depending on its length.

The four generations of CAR T cells are depicted in a manner that emphasizes the differences in the
intracellular domain region of the CAR. It should be noted that OX-40 is also known as CD134 and that
4-1BB is CD137 for the third generation. Two copies of the same costimulatory molecules are not used
in third generation CAR T cell design. Each generation increases in complexity concerning the addition
of costimulatory molecules or NFAT (Nuclear factor of activated T-cells ) induced promoter for IL-12
transcription and later translation. T cell redirected universal cytokine killing (TRUCK)
 Tumor-antigen-specific T cell receptor-engineered ( TCR-T cell) therapy

*Identifcation of Cancer (leukemia)‑specifc TCRs

* Anti cancer (leukemia) TCR‑T cell construction and establishing high-afnity tumor antigen-
specifc TCR gene modifed T cells
Characteristics of TCR- and CAR-engineered T cells

Cancer cell International 2019

Categories of T-Cell Based cancer Immunotherapy