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Cancer Immunotherapy 2020
Cancer Immunotherapy 2020
Cancer Immunotherapy 2020
TCR signaling is initiated by two tyrosine kinases: the Src family kinase Lck and ZAP-70.
Activated Lck phosphorylates ITAMs in the intracellular tails of chains of the TCR complex. Doubly
phosphorylated ITAMs recruit ZAP-70 from the cytosol to the plasma membrane. Tyr 315, Tyr 319,
and Tyr 493 of ZAP-70 are phosphorylated by Lck to fully activate ZAP-70, enabling ZAP-70 to
phosphorylate two scaffold proteins, LAT and SLP-76, leading to the recruitment of effector proteins
that stimulate T cell activation.
CD28 engagement also prevents apoptosis and acts synergistically with CD3-dependent
signals to ensure correct T cell proliferation, differentiation and growth
Mechanisms of B-cell tolerance in bone marrow and periphery
Both clonal deletion and anergy are mechanisms utilized in the primary as well as
peripheral lymphoid organs. Strength of BCR signaling affects the fate of the B cells
undergoing tolerance.
Receptor editing occurs primarily in the bone marrow.
Transitional B cells are found in the bone marrow and spleen.
Peripheral tolerance is primarily found in splenic transitional B cells, which depends
on tonic BCR signaling thresholds and survival signals from BAFF.
Tumor expansion
Interaction between PD-1/PD-L1 and T cell immune response. T cells are primed
and activated through the interaction with antigen-presenting cells. T cells recognizes
tumor via MHC-antigen interaction. Tumor PD-L1 and PD-L2 is upregulated by
interferon γ released from activated CD8 T cells. PD-L1 in turn inhibits T cells via
PD-1.
History of T-cell therapy in cancer
Strategies for use of T-cell therapies for cancer
Regulation of T-cell activation.
C) Feasibility
The tumor needs to be resected for isolation and expansion of TILs
Although TILs can be isolated from many cancers, to date, only those
from melanomas consistently possess specific cytolysis against the
tumors from which they were generated
Not so feasible for solid tumors
Approach advantageous for tumor susceptible to checkpoint blockade
Advantageous for tumors arising from EBV or papilloma virus infections
Genetically modified T cells
Approaches on the basis of genetic modification of normal peripheral-blood T cells
c) viral antigens (such as EBV in Hodgkin disease and HPV in cervical cancer).
The presence of epitope-responsive T cells in the peripheral repertoire is
dependent on:
(A) the generation of appropriate TCR-α/β chains upon somatic recombination of V(D)J
gene segments during T-cell development
(B) survival of developing T cells during positive and negative selection in the thymus
Step 1: Direct TCR cloning system that would allow the unbiased analysis of the TCR
repertoire
If a tetramer stain specific for a pathogenic peptide results in a positive signal, this
may indicate that the person’s immune system has encountered and built a
response to that pathogen.
MHC tetramer production
In the presence
of streptavidin which has
four biotin binding sites per
molecule, four MHC
monomers are joined
together to form a tetramer.
Whilst single MHC: peptide complexes and TCR bind weakly, MHC tetramers can bind up to
four TCRs simultaneously, creating a much stronger interaction.
MHC tetramers only bind to the relevant TCR and tetramers specific for any MHC heavy
chain (e.g. HLA A2, HLA B7) can be produced, coupled with any peptide epitope of choice
(e.g. viral, bacterial, tumor and autoimmune antigens).
Genetically modified T cells (TCRs or CARs) are transfected using virus vectors
(retroviruses or lentiviruses) or a transposon system (Sleeping Beauty). Following
transfection, genetically modified T cells are expanded and transferred into patients
treated with preconditioning lymphodepletion similar to that used with TIL protocols
TCR gene therapy against MART1 and gp100 resulted in skin rash,
uveitis, and hearing loss
off-target toxicity
This process is
antigen dependent
Effects of OX40 ligation on effector versus regulatory T cells
CAR-T therapy starts with accumulating the patient’s white blood cells by leukapheresis. The apheresis products are enriched
or deleted for a specific cell subset and then activated by one of following methods, including interleukins (IL-2, IL-7, and IL-
15), anti-CD3/CD28 antibody-coated magnetic beads, soluble CD3 antibody, artificial antigen-presenting cells, plate-bound
antibodies, and adhesion molecules.
The activated T cells are introduced with the CAR transgene through lentiviral or retroviral and non-viral methods
(electroporation of naked DNA and plasmid-based transposon/transposase).
Afterwards, the engineered-T cells undergo an expansion process in static culture bags or dynamic culture vessels or rotating
bioreactors. Eventually, cell numbers are calculated based on the patient’s disease burden, weight, and another formulation.
The CAR modified-T cells transfer to either a container for infusion purposes or cryopreserved for storage.
CAR-T cells for autoimmune disease
Multiple Sclerosis
Transduced CD4+ T cells using a lentiviral vector system encoding the myelin oligodendrocyte glycoprotein
(MOG) CAR gene and Foxp3 gene. The main role of MOG CAR-Tregs is to localize and bind to
MOG+ oligodendrocytes in CNS, providing a protective shield for these cells against immune attacks
T cell expressing CAAR specific for B cell receptors targets aberrant B cells.
Further, it inhibits B cell development from secreting autoreactive antibodies and prevents B cells from
presenting autoantigens to autoreactive T cells, which leads to suppression of T cell-mediated
autoimmune diseases.
CAR T cells
CAR T cells are constructed by fusing an antibody-derived single-chain variable
fragment (scFv) to T-cell intracellular signaling domains
Advantages:
a) T cells recognize cell surface antigens in a non–MHC-restricted manner
d) can target tumors that have downregulated HLA class I molecules or fail to process or
present proteins.
e) rapidly generate tumor-targeted T cells, bypassing the obstacles that preclude the
induction and execution of effective immune responses in vivo
f) Can be used allogenic and do not have genetic barriers (as off the shelf therapy)
Single chain variable fragment (scFv) is derived from a monoclonal antibody or an antigen-
binding region isolated from an immunoglobulin (Ig) heavy and light chain library
The identification of appropriate tumor antigens (TAA) is the greatest challenge
now facing researchers in the field of ACT. Identifying TAAs not present on
vital organs is critical.
The antigen recognition of T cells and CARs
(A) Antigens inside the cell are delivered to the cell surface as peptide fragments by the
MHC and T cells interact with antigens through the TCR. The subsequent costimulatory
signal is also necessary for optimal T-cell proliferation, differentiation, and survival. CD28
and 4-1BB interact with CD80 and 4-1BBL, respectively.
(B) CARs react directly with native tumor antigens expressed on the surface of a tumor cell
in a non-MHC-restricted manner. CARs are composed of antibody-binding domains
fused to T-cell signaling domains. The target binding module of the single-chain
antibody-variable fragment (scFv) are originated from a tumor-specific antibody
Limitations of CAR-T cells:
tumor escape by loss of target expression
on-target/off-tumor toxicity
another theoretical concern for the use of mature T cells as targets for anti-cancer TCRs is the
potential for mis-pairing: that is, the pairing of endogenous cellular TCR chains with
introduced transgenic chains.
Immunosuppressive state of effector CD8þ T cells in the tumor microenvironment
TGFb and IL-10 drive the differentiation of CD4+ T cells toward the regulatory phenotype which results in
T-cell inhibition.
The engagement of PD-1 with its ligand programmed cell death protein ligand 1 (PD-L1) inhibits the
proliferation and effector function of T cells through the recruitment of SHP2, which inactivates TCR
mediated signaling
T-cell exhaustion during chronic antigen exposure
The presentation of antigenic peptides in the context of MHC class I triggers the priming of naive CD8 + T cells and
their differentiation into effector CD8+ T cells. After antigen clearance, effector CD8+ T cells differentiate into
memory CD8 T cells. In contrast, during continuous exposure to antigen, T cells gradually lose effector functions
and proliferation capacity and finally are eliminated via apoptosis. The gain of cell surface inhibitory receptors
such as PD-1 correlated with T-cell exhaustion reduces antitumor immune function.
Different germ line recombination possibilities results in a huge TCR a and b chain repertoire
T cells undergo positive and negative selection in the thymic cortex and medulla,
respectively
mTECs: Medullary
Thymic Epithelial cells
A promiscuous
expression of a wide
array of self-antigens
in the thymus is
essential for the
negative selection of
self-reactive T cells
and the
establishment of
central tolerance.
Autoreactive T cells are negatively selected in the medulla through mTEC expression of self-antigens
such as tissue-restricted antigens (TRAs)
TRAs are promiscuously expressed under the control of the autoimmune regulator Aire and by the transcription factor Fezf2 in mTECs
POSITIVE and Negative SELECTION OF T CELLS
Double positive abTCRlow cells must successfully undergo positive and negative selection before they can
leave the thymus
Cells which have successfully rearranged abTCR will die in the thymus cortex if they do not bind self
MHC within 3-4 days
Positive selection occurs when double positive T cells bind cortical epithelial cells expressing Class I or
Class II MHC plus self peptides with a high enough affinity to get the survival signal.
Positive selection also determines whether the T cell will become a helper or a cytotoxic T cell. Positive
selection on Class I MHC will produce a CD8 Tc cell, while positive selection on Class II MHC will yield a
CD4 Th cell.
Expression of co-receptor (CD4 or CD8) and its binding to MHC is also required for positive selection; mice
expressing a defective CD4 that cannot bind Class II also fail to produce mature Th cells.
Negative selection occurs when double positive T cells bind to bone-marrow derived APC (macrophages
and dendritic cells) expressing Class I or Class II MHC plus self peptides with a high enough affinity to
receive an apoptosis signal. Note that selection occurs on self peptides in the thymus; MHC presents self
peptides in the absence of pathogen.
Development of T cells (milestones):
ETPs progress to a pre-T cell state expressing CD1a and CD5. At this stage,
recombination of the variable (V) region of the TCR β locus to form a complete
rearranged VDJ TCR β locus occurs almost simultaneously with the rearrangement of
the gene segments encoding the γδ TCR.
Depending on the outcome of the TCR segment rearrangements, the cells can then
follow an αβ or a γδ differentiation path.
A successful TCR β rearrangement leads to the process of β-selection and
emergence of a CD4+ immature single positive (ISP) T cell.
The CD4 ISP cell then develops into a double-positive (DP) cell that expresses
both CD4 and CD8 and has begun to rearrange the V and J regions of the TCRα
locus.
The newly generated T cell clones, known as naive T cells, initially circulate throughout
the body at low frequency.
Upon encountering antigen, T cells expand and acquire effector and/or memory functions
This T cell priming requires
a) TCR engagement by HLA-peptide complexes on APCs
b) Concomitant ligation by co-stimulatory ligands on APCs
Human T cell development
Memory T cell subtypes:
Express CD45RO, C-C chemokine receptor type 7 (CCR7), and L-selectin (CD62L)
Central memory T cells also have intermediate to high expression of CD44
Commonly found in the lymph nodes and in the peripheral circulation
Reprogramming via iPSC technology generates T-iPSCs from antigen-specific CTLs. These T-iPSCs can
redifferentiate into CTLs that retain ancestral antigen specificity and cytotoxicity. Rejuvenated CTLs have higher
proliferative capacity and longer telomeres than do the original exhausted CTLs
TCR genes from original CTLs are retained in T-iPSCs
The TCR a- and b-chain genes (TCRA and TCRB) are rearranged during normal ab T-cell
development in the thymus.
Germline-configuration TCR a and b loci are present in iPSCs derived from CTLs, which
retain the same rearranged sequences of TCR a- and b-chain genes as those in the original
antigen-specific CTLs.
CDR3, the most diversifiable antigen-recognition site of the TCR, spans the
V(D)J-junction region, where several random nucleotides are inserted.
CDR3 sequences from T-iPSCs and their original CTL clones were completely identical at
both the TCRA and the TCRB loci, evidence that antigen specificity encoded in the genomic
DNA of original CTLs is retained throughout cloning, reprogramming, and expansion.
TCR genes from original CTLs are retained in T-iPSCs
The TCR a- and b-chain genes (TCRA and TCRB) are rearranged during normal ab T-cell
development in the thymus.
Germline-configuration TCR a and b loci are present in iPSCs derived from CTLs, which
retain the same rearranged sequences of TCR a- and b-chain genes as those in the original
antigen-specific CTLs.
CDR3, the most diversifiable antigen-recognition site of the TCR, spans the
V(D)J-junction region, where several random nucleotides are inserted.
CDR3 sequences from T-iPSCs and their original CTL clones were completely identical at
both the TCRA and the TCRB loci, evidence that antigen specificity encoded in the genomic
DNA of original CTLs is retained throughout cloning, reprogramming, and expansion.
TCR rearrangements in iPSC-derived CTLs during
lymphoid differentiation
Concern
Such TCR rearrangements of TCR may give rise to nonspecific polyclonal T cells that
could occasion ‘‘autoimmune’’ reactions if administered
Successful interaction of the TCR with MHC molecules during thymic selection
downregulates the expression of the recombination activating genes 1 and 2 (RAG-1 and
RAG-2) and thereby prevents further endogenous TCRA rearrangements
a) T-iPSCs are cultured on C3H10T1/2 feeder cells for 2 weeks, yielding iPSC-derived sacs that contain
many hematopoietic progenitor cells
b) Hematopoietic progenitor cells extracted from sacs are transferred onto Notch-ligand-expressing
CH310T1/2 feeder cells
c) After 28 days of culture, T-lineage cells are harvested and stimulated, yielding huge numbers of
rejuvenated antigen-specific CTLs that were generated from T-iPSCs.
Generation of antigen-specific CTLs from T-iPSCs: methods
Step III: antigen specificity of these iPSC-derived rejCTLs is determined by multimer staining
and interferon-g enzyme-linked immunospot assays used to confirm that the specificity of
the original CTL clone is maintained.
b) can quickly expand to large numbers of effector T cells upon re-exposure to their
cognate antigen. By this mechanism they provide the immune system with
"memory" against previously encountered pathogens.
c) Memory T cells may be either CD4+ or CD8+ and usually express CD45RO
Despite having the functional capacity of memory cells, T SCM cells retain a core
of genes expressed by TN cells and share the recirculation patterns and
distribution of TN cells in vivo
Specific characteristics of iPSC-derived T cells
Once the tumor burden is relived these cells can be induced to undergo suicide
In the intrinsic cell death pathway, activated BAX or BID translocates to the mitochondria,
forming pores that allow cytochrome C to move from mitochondrial intermembrane space
to cytosol. There, cytochrome C activates caspase-9, which in turn activates caspase-3 and
leads to apoptosis.
Hot tumours have immune infiltration whereas cold tumours' are
not infiltrated by immune cells in particular T cells
T cells
OV: Oncolytic virus; NK cell: Natural killer cell; PD-1: Programmed death-1; PD-L1:
Programmed death ligand-1; CTLA-4: Cytotoxic T-lymphocyte-associated protein 4; IFN-
a/b: Interferon-alpha or beta; CCL3/4: Chemokine (C-C motif) ligand 3 or 4.
Mechanism of converting a cold tumour to hot tumour by
Oncolytic Viruses
Type-1 interferons promote the expression of PD-L1 on the surface of cancer cells, while
chemokines like CCL3 and CCL4 attract immune cells which often express PD-1 or
CTLA-4.
##Thus, antiviral immunological events inflame the tumor and make it 'hot'. When
checkpoint inhibitors are administered subsequently, they can bind to their respective
targets on either cancer or immune cells. As a final result, oncolytic viruses sensitize
tumors to the therapeutic effects of immune checkpoint inhibitors
Schematic showing the engineering of Talimogene laherparepvec (T-VEC)
The backbone is the JS17 strain of herpes simplex virus, type 1 in which the two viral ICP34.5 genes have bee
deleted and replaced with copies of the hGM-CSF genes under control of a CMV promoter. In addition, th
viral ICP47 gene is deleted transitioning the viral US11 gene to an immediate-early promoter.
Converting Cold into Hot Tumors by Combining Immunotherapies
Schematic Course of Events following Oncolytic Virus and Anti-PD-1 Treatment in Metastatic Melanoma