CTLA-4: IMMUNE CHECKPOINT BLOCKADE THERAPY,,,, THE 2018 NOBEL PRIZE WINNING STUDY IN THE FIELD OF PHYSIOLOGY/ MEDICINE,,, JOINTLY AWARDED TO JAMES P. ALLISON OF USA and TASAKU HONJU OF JAPAN .

1. CURRENT TRENDS IN IMMUNOCHEMISTRY
PRESENTATION
Anti-CTLA-4: IMMUNE CHECK POINT BLOCKADE THERAPY
Presented by: Saima Barki
DEPARTMENT OF BIOCHEMISTRY
QUAID-E-AZAM UNIVERISTY, ISLAMABAD

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3. Cytotoxic T-lymphocyte antigen 4(CTLA-4)
HISTORY:
• 1st identified by Pierre Golstein and colleagues In 1987.
• November 1995, the labs of Tak Wah Mak and Arlene H. Sharpe independent discovery→ function of CTLA-
4 as a negative regulator of T-cell activation, by knocking out gene in mice.
• In the early 1990s, Jim Allison→CTLA-4 acts as an inhibitory molecule to restrict T cell responses.
• In 1996, Allison was the first to show that antibody blockade of a T-cell inhibitory molecule (known as CTLA-
4) could lead to enhanced anti-tumor immune responses and tumor rejection.
GENOMIC LOCATION IN HUMAN: 2q33.2
STRUCTURE:
• 3 domained structure, a) extracellualar, b). TM, 3) cytoplasmic tail (homology to CD28)→No intrinsic
catalytic activity and contian 2 motifs, one YVKM motif able to bind PI3K, PP2A and SHP-2 and 2nd one
proline-rich motif .
• The first role of CTLA-4 in inhibiting T cell responses seem to be directly via SHP-2 and PP2A
dephosphorylation of TCR-proximal signalling proteins as CD3 and LAT.
• Membrane boundform dimeric, monomers bound toghether by disulphide bond
• Free form as monomer
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4. BASICS OF IMMUNE SYSTEM
• To distinguish self from non self.
• Complex system.
• Main compenent:
• Recognition and binding of TCR to antigen displayed in connection with MHC of APC.
• Binding leads to→ Activation or Anergy
• T cell development:
• Progenitor cell→ T cell in bone marrow.
• Commitment to lineage in thymus, by exposing to Ag on the surface of APCs.
• In start T cell double negative(DN) i.e, CD4-CD8-TCR-.
• Then gene rearrangment unique TCR.
• Then selection or check for tolerance.
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5. T CELL TOLERANCE
Two Types Of Tolerance Mechanism:
• central tolerance
• peripheral tolerance
• CENTRAL TOLERANCE/NEGATIVE SELECTION:
• Location→ primary lymphoid organ.
• Process of eliminating any developing T or B lymphocyte reactive to self Ag.
• Ensure immune system not reactive to self molecules.
• Not a perfect system.
• SIGNALS/AFFINITY FOR SELECTION:
• Strong signal→ apoptotic death
• Intermediate signal→ T regulatory cells
• Weak signal→ T effector cells
• All cells either Treg or Teff depending on affinity towards self peptide.
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6. • PERIPHERAL TOLERANCE:
• Secondary mechanism
• Weak affinity bearing or Teff cells released as naive T cells to circulate in
blood, spleen, lymphatic organs.
• Its main purpose is to ensure that self-reactive T and B cells which escaped
central tolerance do not cause autoimmune disease.
• Then exposure to proffesional APCs bearing self mutated/altered antigen or
foreign antigen.
• Some self reactive so autoimmunity, prevented by immune checkpoint, being
regulated at multiple steps.
• IMMUNE CHECKPOINT PATHWAY MOLECULES: CTLA-4 AND PD-1
• Cytotoxic T lymphocyte like antigen-4(CTLA-4)→ leader→stop autoreactive
T cell at priming stage of the naive cell activation
• Programmed death protein 1 (PD-1)→later working→ in pheripheral tissues
• Key similarites and differences between both immune checkpoint pathways
• WHY A HOT TOPIC IN CANCER RESEARCH?
Tumors cell developed pathway to evade host immune system taking
advantage of peripheral tolerance.
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8. Normal T Cell Positve Costimulatory Signal
• T cell activation→complex→>1 stimualtory signal.
• TCR+MHC+Ag→T cell activation specificity but not complete signal.
• APC bearing B7-1(CD80) or B7-2(CD86) and T cell CD28→ +ve
costimualtory signal.
• Sufficient level of CD28:B7-1/2 →+ve signal.
• Proliferation,
• Increased T cell survival,
• Differentiation through growth cytokines(IL-2),
• Increased energy metabolism,
• Upregulation of cell survival genes, etc.,
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9. IMMUNE CHECKPOINT BLOCKADE
• Removes inhibitory signals of T cell activation.
• Enables tumor reactive cell to overcome regulatory mechanism and mounting
of anti tumor response.
• Inhibition result in increased activation of immune system.
• Induce durable response across multiple types of cancers.
• Discovery of immune checkpoint blockade and durable response, a
transformative event.
• Different inhibitors approval from FDA since 2011.
• Inhibitors approved being anti-CTLA-4 i.e. Ipilimumab and Tremilimumab for
metastatic melanoma.
• Combination therapy of ipilimumab and novolumab(anti PD-1)→synergistic
effect in advance melanoma patients.
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10. CTLA-4 PATHWAY: NEGATIVE COSTIMUALTORY SIGNAL
• CTLA-4 : CD28 HOMOLOG also known as CD125
• Regulate at T Cell priming in secnodary lymphoid organs,
• Higher avidity and affinity for B7 as compared to CD28,
• Rapid binding kinetics withB7-1,
• Competative binding,
• Relative amount of CD28 :B7 binding verses CTLA-4:B7 →T cell Activation or
Anergy respectively
• Attenuates positive signalling by CD28 and limits downstream signalling
mediated by PI3K and AKT,
• CTLA-4:B7 binding→inhibitory signals
• INHIBITORY MECHANISM:
• Direct inhibiton at TCR immune synapse
• Inhibition of CD28 or its signalling pathway
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11. CTLA-4: Intrinsic Control and Role In +Ve And –Ve Signalling
• Localization within the cell, intracellular
compartment.
• CD28:B7 and TCR Stimualtory
signal→upregualtion of CTLA-4 on surface by
increased exocytosis of vesicels.
• Graded feedback loop→strong signal so more
CTLA-4 translocation
• Net negative signal through CTLA-4:B7,
→inhibition of T cell activation by IL-2
production and cell cycle progression.
• Treg control Teff so improtant regulator of
pheripheral tolerance
• Treg→ constitutitutive expression of CTLA-4
genetic CTLA-4 deficiency in Treg→loss of
supressive functions.
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T CELL: ACTIVATION, INHIBITION, POTENTIATION

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NEGATIVE IMMUNE CHECKPOIINT REGULATION

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MOLECULAR MECHANISM OF CTLA-4
ATTENUATION OF T-CELL ACTIVATION

15. Mechanism of Tregcontrolling Teff
by:
• Downregulation of B7 ligands
on APCs→reduced CD28
costimualtion
MECHANISM OF NEGATIVE
COSTIMULATION VERSES MECHANISM
OF CHECKPOINT BLOCKADE:
• Cancer highly analogous to
infectious disease so principles of
virus system applicabe here where
chronic antigen stimulation results
in Tcell exhaustion
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16. MECHANISM OF ACTION OF CTLA-4 BLOCKADE INDUCED
TUMOR REJECTION
CTLA-4 blockade→tumor rejection
Exact Mechanisms not clear yet,, possibly these 3 mechanisms:
1. Primarily through direct blockade of CTLA-4 competition for B7-1/2 costimulatory
signal→unrestrained CD28 +ve signalling sp. activation.
Tumor cell→ No B7 ligands expression , so occur in tumor draining lymph nodes and tumor
microenvironment, require tumor cell death→ tumor antigen(neoantigens, TAA) cross
represented by APC to prime tumor reactive T cells.
Tumor presentation possibily in the tertiarry lymphoid structure TLS (presence associated
with improved survival), Context dependent antitumor functionality,Treg presence suppress
antitumor response
• CTLA-4 blockade specific response, expansion of specifically tumor neoantigen-CD28
(TIL) in tumor microenvironment not in lymphoid organ. TIL population include exhauseted
CD8 T cells and PD-1+ICOS+TBET+Th1 like CD4 effector cells.
• ICOS+CD4 Teff cells→pharmacodynamic marker of anti CTLA-4 therapy.
• CD4 Teff cells →Anti Ctla-4 affect differentiation along the activation.
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17. 2. DEPLETION of Treg population: mechanism of anti-CTLA-4 therapy
• Treg depletion partial contribution towards efficacy
• Differential efficiancy, as context dependent
• Blockade of both effector and reg t cell compaartment derived ctla-4 is required for effective tumor
rejection
• Ipilimumab: purely human IgG1,blocking antibody not depleting so no treg depletion evidence with
treatment, induce antibody dependent cellular cytotoxicity mediated killiing of Treg
• High affinity Fc receptor→improved response with iplilimumab so efficacy dependend on effector
funciton not depletion
• Tremelimumab:human IgG2→less effective ADCC than ipilumumab
3. Modulation of TCR repertoir:
3. Ipilimumab remodelling of peripheral TCR repertoir i.e. CD8 T cells but associated with irAEs.
4. Loss of CTLA-4, lowering of threshold for T cell activation, effective recognition of tumor
specific antigen→major driver of tumor immunogenicity.
• Survival associated with the maintainess of clones present in high frequency , more the frequency of clones
more the survival chances
• Effective anti CTLA-4 depend on the retention of pre-existing high avidtity cell with relevance to antitumor
response
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19. Clinical Efficacy and Characteristics of Response
with Immunecheckpoint Inhibitors
• Ipilimumab,1st anti CTLA-4 , prolong overall survival in melanoma.
• Phase ll/lll→survival curve plateau about 3 years
• Immune checkpoint blockade response efficacy vary with:
a.) Variation in kinetics and efficacy of ind. patient immune system.
b.) Interplay with tumor and metastasis
• Initial increase in tumor volume before generation of effective
tumor response so faster activation lead to inflammation and
influx of immune cells to tumor site.
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21. BIOMARKERS and IMMUNOLOGIC TOXICITIES
Retrospective studies reveal biomarkers including:
1. Absoulte lynphocyte count
2. Upregulation of T cell inducable costimulator(ICOS)-activation marker
3. Development of polyfunctional T cell response
IMMUNOLOGIC TOXICITIES:
• Immune checkpoint inhibitors →most promising drugs→toxicities
• Effect→skin,colon, endocrine glands,liver,lungs(common) and neurological
and myocardial disorders(rare)
• Ipilimumab irAEs: anti-ctla-4 3mg/kg in 60-85% paitent of grade 1 and 2 but
27% develop grade 3 to 4 toxicities, 2.1% deaath in 1st phase l trials
• Onset varies, 8 to 12 weeks of treatment initiation,
• Toxicity dose dependent
COMBINATION THERAPY ASSOCIATED TOXICITIES:
95% of patients→adverse effects→55%pf grade 3 to 4
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22. Summary Of Biological And Molecular
Functions Of T-cell
MOLECULE LIGANDS RECEPTOR
EXPRESSION
PATTERN
BIOLOGICAL
FUNCTION
MOLECULAR
FUNCTION
CTLA-4 B7-1(CD80),
B7-2(CD86)
Activated T cells,
Treg
Naïve T-cell
costimulation
(primarily at
priming): prevent
tonic signalling
and/or attenuate
high affinity clones
Competative
inhibitors of CD28
costimualtion
(binding of B7-
1(CD80),
B7-2(CD86)
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23. CLTA-4 Pathway Inhibitors Approved Or In Phase ll
and/or Phase lll Clinical Trail Stage Of Development
TARGET NAME STATUS COMPANY
CTLA-4 Ipilimumab •Approved for metastatic
melanoma
•Phase lll: lung, kidney
and prostate cancer
•Phase ll: cervical,
colorectal, gastric,
pancreatic, ovarian and
urothelial cancers
•Bristol-myers
•Squibb
CTLA-4 Tremelimumab •Phase lll studies in lung
cancer
•Medimmune/
•AstraaZenecca
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25. CONTINUED....
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