IMMUNE Flashcards
innate cellular component
Phagocytes (neut, macrophage)
Natural killer (NK cells)
Dendritic cells
Mast cells
innate humoural
Cytokines (IFN, IL)
Complement proteins
adaptive cellular
T cell
B cell
adaptive humoural
Cytokines
Antibodies (from B cell
composition of Ab
4 pp
2 light chain
2 heavy chain
Ab binding sites
• 2 antigen-binding fragments (Fab)
○ Bind to antigen
○ DIFFER FOR EACH AB
• 1 constant fragment ( Fc)
○ Immune triggering module
○ Biological effector
® Binds to (macro, NK, neut via Fc receptors)
○ Bind to complement proteins
○ SAME FOR ALL Ig
components of Fab
Fab: light chain (VL), heavy chain (VH) – variable domain
• s-s disulfide bonds
• Cysteine interchain and intrachain
• 3D conformation
•Recognise epitope in antigen for binding
components of Fc
Fc: CL, CH
• Glycosylation
• Add carbohydrate chain to aa
• Post-translation modification
○ Bind to effector cells
○Bind to complement proteins
Complementarity-determining region
CDR purpose
responsible for diversity of antigen specificities of AB produced by mature B cells
CDR structure
1) 10-20 bases, sparsely arranged/ folded
2) Each Fab arm, acid sequence in variable domain of both VH and VL chains
a. Arranged to form 3 CDRs
how mancy CDR in 1 Ab
1 AB = 2 identical Fab =
3 CDRs x2 (light, heavy) x 2Fab = 12CDRs per AB
paratope definition
a) Part of Fab region
b) Antigen-binding site. Binds to epitope
c) Tip of Fab arm
how many CDR in 1 paratope
Consist of 6CDRs (3- light chain/ 3- heavy chain)
a. CDR1
b. CDR2
c. CDR3
antigen affinity measures
strength of interaction between antibody (paratope) and antigen (epitope)
• High affinity binds strongly
• At single antigenic site
Ab specificity
GOODNESS of fit between paratope and antigen
• Ability of paratope in AB to distinguish similar and dissimilar antigens
• Low specificity = cross reactivity = paratope react >1 epitope
avidity vs affinity
• strength which AB binds to target
• if target has Multiple antigenic sites (multiple epitope on antigen) – IgM binds
T cells possess TCR for
T cells possess TCR for
antigen recognition and activation
TCR responsible for binding to peptide antigens presented on MHC class II
structure of TCR
Found on surface of T cells
α chain and β chain (each chain encoded by specific gene)
- 2 extracellular domains (glycosylated)
Variable (V) region - bind to antigen
Constant region
- transmembrane region (hydrophobic region)
- short cytoplasmic tail (hydrophilic region)
TCR V and C region structures
2 extracellular domains (glycosylated)
Variable (V) region
Constant region – cysteine residue for disulfide bond to link a and b chain
TCR 3 domains
A) 2 extracellular domains (glycosylated)
• Variable (V) region - bind to antigen
• Constant region (cys residue disulfide bond to link α chain and β chain)
○ 3D conformation
B) Transmembrane region (hydrophobic, cuts phospholipid region)
C) short cytoplasmic tail (hydrophilic)
α chain and β chain arranged to ensure
• antigen recognition/ binding
• Docking onto the plasma mem
• Cytoplasmic tail too short to mediate signal transduction for T cell activation
Upon TCR-antigen binding
tyrosine residues in ITAMs is phosphorylated
1) Post-translational modification
2) Initiate downstream T cell signaling event
3) T cell activation
α chain and β chain has invariant CD3 dimers (adapter proteins)
CD3e, Cd3y, CD38, CD3z
=CD3ey, CD3e8, CD3zz
Form octameric complex in plasma mem
6 monomers = 3 dimers
CD3 has ITAMS =
immunoreceptor tyrosine-based activation motif
(mediates signal since tail too short)
CD3 has ITAMS =
immunoreceptor tyrosine-based activation motif
1 TCR =
10 ITAMs
i) CD3e = 1 ITAM monomer x 2 = 2
ii) CD3y = 1 ITAM monomer x 1 = 1
iii) CD38 = 1 ITAM monomer x1 = 1
iv) CD3 z = 3 ITAMs monomer x2 = 6
Ab vs TCR differences
• CDR is AB responsible for antigen recognition and binding
• AB binds to diverse antigens
• 12 CDRs per AB (3x 4 chains)
• Activate effector cells
• TCR forms complex with CD3 adaptor proteins but TCR responsible for binding to peptide antigens presented on MHC class II
Only those presented on MHC
6 CDRs per TCR
similarity between TCR and Ab
• Have variable regions (Fab, extracellular TCR region)
• Variable regions (Va, Vb of TCR — VL, VH of AB)
• Variable regions are antigen binding sites
• Constant regions
• Disulfide bonds
• Glycosylation at constant region
• Member of immunoglobin superfamily
B cells origin
Virgin B cells express B cell receptor
Leave bone marrow (development: progenitor B cells rearrange Ig genes.
Clones of immature B cells express B cell antigen receptor)
• Found in circulation
• found in 2nd peripheral lymphoid tissues
activation of B cell
1) Virgin B cell encounter pathogenic antigens
2) Activated, mature
3) Produce, secrete IgM (first response, pentameric)
B cell variability by:
1) somatic recombination
2) class switching
3) affinity maturation
CLASS SWITCH
gene rearrangement of constant regions in Fc domain in IgM
• Mature B cells switch production to IgG
• 1st response = IgM (produced by naïve B cell)
• After exposure to antigen, 2nd response = higher affinity AB
AFFINITY MATURATION (gene shuffling)
each B cell clone continue to undergo gene rearrangement, shuffling under low dose of antigen
• VL, VH regions of Fab domain of IgG genes
• Each B cell clone ends up a diff hypervariable CDR
○ Aa seq diff
○ Diff antigen specificity: hypervariable CDR
○ IgG produced by diff mature B cell clones have diff CDRs (diff aa seq, diff antigen specificity) recog diff epitope of antigen
§ Each B cell prod AB to 1 epitope
T lymphocutes types
• CD4+: T helper
• CD8+: cytotoxic T cells
T cell develop
T lymphocyte progenitor travel from bone marrow to thymus (lymphoid tissue) develop into T lymphocytes
• Localized in:
• Primary lymphoid tissue
• 2nd peripheral lymphoid tissue (lymph nodes, spleen
T cell activation
1) Virgin T cell encounter pathogenic antigens
2) Activated, mature
3) Release cytokines, activate other effector cells
genetic recombination of T CELL
DNA segment in genes encoding Va and Vb regions of TCR
• Unique combination of segments for each recombined TCR expressed by individual somatic T cell
○ Va:
§ VJ recombine
§ CDR1a, CDR3a
○ Vb:
§ VJ recombine
§ VDJ recombination
□ CDR1b, CDR2b, CDR3b
Va (in a chain of TRCR)
○ Va:
§ VJ recombine
§ CDR1a, CDR3a
Vb (in b chain of TRC)
§ VJ recombine
§ VDJ recombination
CDR1b, CDR2b, CDR3b
why TCR diversity
allows diff T cells to recognise and bind to diff antigens presented by MHC molecules
lymphoid tissues
1) thymus, bone marrow
2) lymph node, spleen, tonsils
T cell memory (like B cell) develop
• After infection
• Antigen-driven EXPANSION of antigen-specific T cells
○ Clear antigen
○ Effector T cells die after combat with pathogens
• Remaining antigen-specific T cells differentiate to memory T cells
memory cells found in
○ Located in lymphoid tissues, bone marrow, specific organs (intestines, lungs, skin
purpose of memory cells
○ Respond more quickly, bind to antigens (shorter lag time)
§ Faster, more potent T cell-mediated immune response
§ When encounter same antigen
MHC proteins purpose
Major Histocompatibility complex (MHC)/ Human Leukocyte Antigen (HLA)
• Cell surface proteins needed for adaptive immune system to function. IMMUNE FUNCTION:
MHC 2 immune functions
○ Bind peptide fragments (antigen) and display for recognition by T cells
○ Aid immune system distinguish self from non-self/ foreign antigens
MHC 2 immune functions
1) Bind peptide fragments (antigen) and display for recognition by T cells
2) Aid immune system distinguish self from non-self/ foreign antigens
ADAPTIVE immunity by cell-mediated
§ Executed by cytotoxic T lymph (CTLs)
□ Secrete perforin to promote form holes/ apoptosis
§ Lyse infected cells
§ Pathogen/ antigen clearance
adaptive humoural response
§ AB secreted by plasma cells
§ AB recognise and bind to specific epitopes expressed by pathogens
§ Followed by Fc domain in AB binding to Fc receptor (on effector cells)
□ NK, macro, neutrophils
□ Activated effector cells engulf/ lyse pathogens
□ Clear pathogen/ antigens
MHC genes
• Chrom 6 (contains MHC region: MHC class I, II, III), family of genes (>200 genes)
○ APC contains both MHC I, II
• ~50% of genes in MHC region are encoding proteins having immune functions
MHC subtypes coded from MHC region
1) class I – apc
2) class II – apc
3) clas III
what class to activate adaptive (APC)
• Class II
• In APC (maco, dendritic, B cells)
how activation is done with MHC
1) Invading pathogen recognised by APC
2) Engulfed by phagocytosis
3) Bacterial proteins broken up into peptide fragments (antigenic fragment)
4) Each MHC class II protein binds to antigenic fragment
5) Peptide-MHC II (on APC) presented to helper T cell (CD4+)
i. IL2, other cytokines released
ii. Cell-mediated immune
iii. Humoural immunity (AB)
MHC II presents__ to CD4 T cell
Bind to peptide fragment of EXOGENOUS antigens
Foreign antigens from invading pathogens
peptide-MHC II
how is peptide-MHC II formed?
- Exogenous antigens taken into APC (phagocytosis) by endosome
- Degraded by proteases to peptide fragment
- Process of peptide-MHC II involve ER
Present onto surface for antigen presentation to CD4
how CD4 recognise MHC II
• CD4 recognise/ dock to MHC II molecule present in peptide-MHC II proteins
• Proximity so that respective TCR expressed on cell surface binds to antigenic peptide and MHC II molecules
• Trigger signaling event by activated TCR
MHC II vs I enzymes
II: proteases
I: proteasome, enzyme peptidases
MHC I function
Present antigen (peptide-MHC I) expressed on cell surface to CD8+ cytotoxic T cell
1) NK cell recognise lack of inhibitory MHC I (healthy cells) / incr in activating ligands on cell surface 2) NK cell activated to release cytotoxic granules, destroy abnormal by LYSIS, APOPTOSIS
where is MHC I found
• In all nucleated cells and platelets
Not in RBC (no nucleus)
what do MHC I bind to
Binds to peptide fragment of endogenous antigens
1) Normal self antigen
2) Viral components from virus-infected cell
3) Neoantigens (exclusively expressed by cancer cell)
how is peptide-MHC I formed?
- Endogenous antigen. Cellular proteins
- Proteasome:
a. Protein complex ubiquitously present in cells to degrade unwanted/ damaged proteins by proteolysis
b. Cleavage of peptide bonds
c. Proteins degrade to peptide frag ~15 aa - Enzyme peptidases
- Transport to ER by TAP transporter, helper proteins
- Processing and expression of peptide-MHC I molecule involves ER and GA
Fuse vesicle onto surface of cell mem (exocytosis) – CD8
how CD8 recognise peptide-MHC I
• CD8 recognise/ dock to MHC I molecule present in peptide-MHC I proteins
• Proximity so that respective TCR expressed on cell
• surface binds to antigenic peptide and MHC I molecules
Trigger signaling event by activated TCR
how to regulate MHC expression
○ Cytokines regulate expression of MHC I, II
○ Interferons (IFNa, IFNy) incr expression of MHC I, II
§ Activate appropriate T cells in times of infection □ IFNa: early resp for infection, incr transcription & expression of MHC □ IFNy: immunomodulatory cytokine
why need regulate MHC expression
level of MHC molecule expression affects extent of T cell activation
MHC genes is ___ (2 features)
POLYGENIC: several genes encode family related proteins
POLYMORPHIC: multiple alleles of each gene (freq>1%)
MHC is polymorphic which helps __
§ Human genome –> diff MHC class I, II genes (>20) –> sets of class I/ II molecules possess diff peptide binding specificities expressed
§ Diff set of MHC class I/ II molecules present diff antigens to T cells
***** Ensure that adaptive immune response mediated by activated T, B cells
- Broad coverage against diff antigens present in invading pathogen
polymorphic MHC helps
§ Presence of diff alleles of in EACH MHC gene
§ Pdt of each MHC allele differ from one another by around 20 aa
□ Difference found in peptide binding domains of MHC molecules
□ –> varied antigen recognition by T cells in diff individuals
****Incr diversity of MHC molecules expressed by indiv (add-on to polygeny)
- So that adaptive immune response mediated by activated T, B cells provide broad coverage against diff antigens present in an invading pathogen
immunotherapy definition
2 types
• Treatment of disease by intervening the immune system
- activation
- suppresion
activation immunotherapy
involve use of agents that augment/ reestablish immune system ability to prevent and fight disease
suppression immunotherapy
involve use of agents that reduce or suppress immune response
eg of immunotherapies
○ Eg: cytokines - humoural of innate/ adaptive response
○ AB - adaptive immune response
○ T cells - cell based immunotherapy/ checkpt inhibitors
○ Cancer vaccines
CYTOKINE THERAPY
○ Key roles in innate and adaptive immune response - immunity, inflammation, hematopoiesis
§ Recombinant/ anti-cytokine use to treat, manage conditions
§ Infectious disease, cancer, wound heal, inflammatory disease
2 types of cytokines therapy
1) anti-cytokine (MAB binds to receptor, inhibits
2) recombinant cytokine (MAB bind to receptor, incr activaiton)
purpose of cytokines
a) Diverse group of small proteins secreted by immune cells (macrophage, T cells – both myeloid & lymphoid lineage)
- Diff immune cells can secrete diff cytokines
b) Mediate and regulate immunity, inflammation, hematopoiesis
- Hematopoiesis (erythropoietin included as well)
c) Glycoproteins, short half-lives
types of cytokines
1) INTERFERONS
2) INTERLEUKINS
3) COLONY STIMULATING FACTORS
4) CHEMOKINES
5) OTHERS (tumour necrosis factor)
INTERFERONS uses
a) Produced by cells response to viral + tumours + biological inducers.
b) Promote antiviral state
· Induce cellular resistance to viral attack · Regulate immune function · Regulate growth and differentiation
action of cytokines
- autocrine (short range, on cells that produce them)
- paracrine (cells nearby)
binds specifically to cytokine receptor expressed on surface of effector cells (trigger biological effects)
2 types of IFN
- type 1 = IFN a,b (antiviral// regulate interferon activity)
- type II = IFN y (phago)
IFN a
leukocytes
14 subtypes, diff profile of antiviral and antiproliferative activity,
upregulates immune system for antiviral/ anticancer therapy
IFN b
expressed by somatic cells (fibroblast, epithelial). GLYCOSYLATED 80/166 (but may not be essential).
1) Inhibits IFN-Y activity,
2) slow growth of self-attacking cells
3) stop production of myelin destroying (multiple sclerosis)
IFN y
T-lymph, glycosylated.
Immunomodulatory cytokine (activate phagocytes)
1) Activate resident macrophage and monocytes (to differentiate)
2) increase phagocytic activity
3) Induce macrophage express MHC/ Fc/ cytokines (IL2, TNF)
INTERLEUKINS
produced by leukocytes (WBC).
Affect growth, diff of hematopoietic cells. Immunity, inflammation, hematopoiesis
· Mostly glycosylated · Involved in regulation of immune cell growth, differentiation and maturation · Short circulation time, regulated by +, - loops (die) · Biological effects varies
IL 2
· T cell growth factor, secreted by T cells - autocrine
· Immunomodulatory prop:
- Stimulates growth
- differentiation
- activation of T,B, NK cells [IL2 receptors]
Proleukin: recombinant IL2 = stimulate body activate T cells kill own cancer cells
IL 11
· Thrombopoietic growth factor produced by fibroblasts and bone marrow stromal cells
· Stimulate proliferation of hematopoietic stem cells · · ·
· induce megakaryocyte maturation (more platelets form)
(recombinant) Neumega – downstream processed to be PEGylated: travel to bone marrow, slow increase platelet count.
incr half life >8-10 days
hematopoietic GF
i) Single chain pp: generally glycoproteins
ii) Proliferation and diff of pluripotent stem cells —> functional immunologically active cells
[promote hematopoiesis]
clinical use, recombinant: Help restore severe deficiency of hematopoietic cells from chemotherapy/ radiation
- CSF, EPO (Erythropoietin), IL11 (platelets), Thrombopoietin
eg of myeloid growth factors recombinant
1) G-CSF (grastim): WBC—> neutrophil incr
□ Chemo-induced neutrocytopenia
2) GM-CSF (gramostim): incr neutrophils, eosinophil, monocytes counts
□ Accelerate myeloid cell recovery after bone marrow transplant
□ After antiviral therapy (AIDS)
(antifungal, CD, antibiral)
3) glycosylation (not for activity), incr half life
- lenograstin > filgrastin (potent, stable)
types of Colony stimulating factors
CSF
EPO (Erythropoietin)
IL11 (platelets)
Thrombopoietin ( megakaryocytes – platelets)
polyclonal vs monoclonal Ab
poly: mixed pop of Ab, bind diff target molecule
mono: single species, only bind to single specific site
antiserum
○ contain polyclonal AB raised by immunising the animal with particular antigen
- Generated by variety of animal species
○ eg: Inject into animal antigen of another species. Polyclonal AB raised in host animal
(antimouse Ab raised in a rabbit)
antiserum production
1) whole blood collected from immunised animal
2) . Left to clot/ add coagulant
3) remove clotting factors
4) Serum obtained - supernatant of centrifugation
antiserum purification
serum proteins and fraction Ig that react Ag
a. Protein A/G purification
i. Staphylococcus aureus protein A & G have affinity for Fc of Ig
ii. pulls all types of Ig down, not specific
b. IMMUNOAffinity purification
i. antigen covalent bond to column: specific antigen-AB binding.
ii. Ig of desired specificity obtained
uses of antiserum
passive immunisation: snake venom, tetanus
○ host has genetic defect
○ cannot wait for immunity to develop
monoclonal Ab produced
Produced from 1 B cell clone, recognise 1 epitope of antigen
features of MAB
○ High specificity: useful for immunoaffinity chromatographic purification
○ High homogenity: effects obtained highly reproducible
· Target-specific therapeutics, less SE
· Diagnostic test kits
· Experimental research technique
Hybridoma technology - to prepare monoclonal AB
murine MABS
- induce immunogenicity
- fail to trigger a number of effector functions
- shorter half live, did not bind to effector cells Fc domain
~0% human
Hybridoma technology - to prepare monoclonal AB
murine MABS
- induce immunogenicity
human anti-mous Ab response HAMA = joint swell, rash, kidney failure - fail to trigger a number of effector functions
- shorter half live (30-40h), did not bind to effector cells Fc domain
~0% human
Chimeric MABS
- CH and CL constant regions in Fc and Fab domains in chimeric MABs contribute to immunogenicity○ replace aa seq on CH and CL of MAB that are not essential for antigen binding with human seq○ antigen-binding sequences in VH and VL fragments conserved○ chimeric MAB retains antigen selectivity and affinity similar to parent murine MAB
~75% human = 25% is VARIABLE REGION
Humanised MABS
- replace all mouse aa seq except HYPERVARIABLE CDR domains of Ig (VH and VL fragments)
○ CDR: hypervariable region of Ig - bind epitope
○ responsible for antigen specificity and affinity of AB
~90% human (reduced immunogenicity) = 10% is HYPERVARIABLE CDR domains
Recombinant human MAB
- use of recombinant DNA technology to genetically engineer mammalian host cells: human MABs
- aa seq of heavy, light chain entirely human
- 100% human = no immunogenicity
○ CHO cells used: impurity proteins from mammalian host cells not removed
○high cost
Ab derivative no need Fc because:
- may not need Fc domain in these applications:
○ antagonism of enzyme actions by binding to enzyme active site (achieve enzyme inhibition)○ neutralise receptor ligands (hormones/ cytokines), counter overproduction of cytokines § Fc may be needed not for effector function but to extend half-life in circulation
types of Ab derivative
1) Ig conjugate
2) F(ab’)2 // Fab
3) ScFv
4) biospecific/ triomabs
Ig conjugate
Conjugate with: radioisotope, cytotoxic cytokine or toxin
To full-length AB
1. When AB bind to surface molecule of cell —> surface molecule-Ig conjugate complex
2. Phagocytose by cell
3.Conjugate exerts lethal effect on cell
Ig conjugate
Conjugate with: radioisotope, cytotoxic cytokine or toxin
To full-length AB
1. When AB bind to surface molecule of cell —> surface molecule-Ig conjugate complex
2. Phagocytose by cell
Conjugate exerts lethal effect on cell
F(ab’)2 and Fab
Pepsin and papain proteases
Cleave peptide bonds
ScFv
single chain variable fragment
Contain aa sequence of antigen binding CDR in VH and VL n Fab arm in single pp chain
biospecific
Recognise 2 diff antigens/ epitopes
Lack Fc domai
triomabs
Resemble full length AB but have diff Fab
Bind 2 diff antigens, maintain capacity to mediate Fc dependent effector functions
(Fc domain included)
- complement-dependent cytotoxicity (CDC) - AB-dependent cellular cytotoxicity (ADCC)
- targets surface bound antigens on cancer cells & T cell CD 3 receptor
1) bring cytotoxic T cell closer to CANCER cells
2) stimulate cytotoxic T cells to kill cancer cells
fucosylated Ab is formed by:
- naturally in IgG
○ Fc domain is N-linked (Asn) glycosylated = 2 N-link oligosaccharide chains bound to Fc region
○ N-glycans attach to Asn 297 in Fc domain link to FUCOSE
why fucosylated is not good
- reduce affinity of Fc domain to bind to subtype of activating Fc receptor on effector cells
- fucosylated COMPETES with therapeutic Ab (recombinants) for binding to effector cells
- cannot elicit ADCC (less therapeutic)
defucosylated AB benefits
no fucose at Asn 297
- enhanced affinity of defucosylated AB to Fc - preferred interaction than IgG
- greater ADCC induction by effector cells, more effective AB recombinant
T cellls as therapeutics
mainly for cancer treatment
Adoptive cell transfer (ACT)
Tumour infiltrating lymphocyte (TIL) therapy
retrieve T cells:
- Autologous tumour-infiltrating lymphocytes isolated from excised tumour masses (from pt)
a. Isolated TIL from pt tumour masses consists of T and NK cells
b. Isolated T cells are polyclonal
c. Diverse antigen specificity
TIL expansion
- Primed and expanded (in vivo)
a. Rapid expansion process
b. Exposed to high dose
i. IL2
§ Produced by CD4 T cell when exposed to antigen
§ T cell growth factor, secreted by T cells - autocrine
§ Immunomodulatory prop: Stimulates growth, diff, activation of T,B, NK cells [IL2 recep]ii. Anti-CD3 antibody § AB bind to CD3 § Activates T cell iii. Irradiated feeder cells § Incr ppt, T cells to proliferate § More cytokine release from irradiation• Peripheral Blood Mononuclear Cells
○ Obtained from pt
TIL infusion back to pt
a. Therapeutic TIL re-infiltrate tumour
i. Large quantity ~10-150 billion of lymph produced and infused into patient
ii. T lymph > NK cell preferred
§ T cell memory cell
§ NK cell die first
b. Recognise tumour antigens
c. Attack cancer cells
T cell receptor engineered T cell
(TCR-T) therapy
retrieve T cells
- T cells isolated from pt peripheral blood
(and expanded if quantities are not high enough genetic manipulation)
genetic modification of TCR cells
- genetically modified under laboratory conditions –> development of tumour antigen-specific T cellsa. Modify with TCR
○ Genes encoding Va, Vb within TCR a, b chains are tumour antigen-specific
○ Genes are cloned into retro/ lentiviral vectors
○ Transduce T cells isolated from pt peripheral blood
§ Genetically modified T cells
□ less heterogenous,
□ high transduction
b. Or with CAR
TCR-T
T cell expansion
a. Recognise tumour antigens
b. Attack cancer cells
- Efficiency of vectors (genes cloned into retro/ lentiviral vectors) ensures that reinfused T cells possess high tumour antigen specificity
TCR-T reinfused to pt
Eg TCR-antigen targets:
- carcinoembryonic antigen CEA (colorectal cancer)
- glycoprotein gp100, melanoma antigen (T cells 1 (MART-1)
TCR added can only link with MHC proteins
- target naturally occuring antigens
- MHC mark cancer cells with recognisable antigens
Chimeric antigen receptor T cell (CAR-T)
genetic modification
1) Construct chimeric antigen receptor (CAR)
a) Genetic sequence encoding for specific antigen-binding sites within VH and VL in Fab domain of an identified AB
b) That targets specifically a cell surface molecule of interest
2) Cloned into a retro- / lentiviral vector
3) T cells isolated from pt peripheral blood transduced by vectors carrying CAR gene Transduced T cells express CAR on surface
generation of CAR-T cells
• Design of generations of CAR-T cells
• Based on structure of intracellular domain
• Extracellular domain is only the scFV found in Fab arm of an Ig resp for antigen recognition and binding
○ 1-3rd focus on intracellular binding
ScFv to recognise specific tumour antigens
1st —–> 3rd gen of CART
1) intracellular binding only
a) 1 signaling domain (CD3z)
b) Not strong enough to sustain CAR-T cell expansion (in vivo)
c) Fail to execute potent antitumour activity
2) 2 signaling domain (CD3z + costimulatory CD28/ 4-1BB domain) a) 2nd activation signal upon target antigen recognition b) Stronger signaling, longer in vitro proliferation c) Potent antitumour activity 3) 3 signaling domain (CD3z + costimulatory CD28 + 4-1BB domain) a) Stronger activation
1st —–> 3rd gen of CART
1) intracellular binding only
a) 1 signaling domain (CD3z)
b) Not strong enough to sustain CAR-T cell expansion (in vivo)
c) Fail to execute potent antitumour activity
2) 2 signaling domain (CD3z + costimulatory CD28/ 4-1BB domain) a) 2nd activation signal upon target antigen recognition b) Stronger signaling, longer in vitro proliferation c) Potent antitumour activity 3) 3 signaling domain (CD3z + costimulatory CD28 + 4-1BB domain) a) Stronger activation
4th gen CART
4) 3 signaling domain (CD3z + costimulatory CD28 + 4-1BB domain) + TRANSGENE
a) Upon target antigen binding, strong CAR signaling +
b) Transgene activated to express cytokine (IL12) — broad-acting, activate proliferation of leuk
i. Exerts autocrine &/ paracrine effect on T cells at target site
ii. More T cell activated to eliminate cancer cells
** target antigen-negative cancer cells
TIL advantage
TIL therapy generally safe among different ACT techniques
TIL disadvantage
1) Excised tumour masses are devoid of or contain very low quantities of TILs
• COLD: poor infiltrating lymphocytes
• HOT: high load of lymph into tumour
2) Expanded naturally occurring tumour-specific T cells are heterogenous possessing varying antigen specificities
• TIL reinfusion may not be lethal enough to attack and eradicate cancer
• Polyclonal T cell (wide diverse spectrum of antigen specificity)
○ Expand T cell growth but cannot control that those with higher specificity grow slower instead
3) Limited or none of the expanded naturally occurring tumour antigen-specific T cells possess high affinity
• Dependent on natural supply of patients
Not genetically modified
advantage of TCR-T cells
1) Engineered TCR T cells possess full TCR complex
• Can recognise antigens expressed at both:
○ cell surface/ tumour surface
○ Within tumour cell/ mass
• Can penetrate tumours
○ Effective against solid and hematological tumours
§ Leukemias
2) TCR T cells use full TCR complex for antigen recognition and signal transduction
• CD3 adaptor proteins
○ Can be fully activated at low target cell antigen densities
○ Onset of signalling slow but of longer duration
○ Execute more extended killing
§ Further elimination of cancer cells
More effective than CAR-T therapy for cancer treatment
TCR T cell disadvantage
1) Engineered TCR T cells express a particular tumour-specific TCR only limited for use in a pt subpopulation carrying specific MHC/ HLA allele recognised by TCR
• But MHC is polygenic, polymorphic
2) Less safe than TIL therapy
• On target off tumour toxicity
○ TCR T cells target normal tissue expressing same antigen
○ Both normal melanocytes and melanoma cells express gp100 and MART-1
• Off-target toxicity
○ TCR T cells not specific and cross-react with other antigenic fragment
• Cytokine-release syndrome
○ Infusion of TCR T cells induce cytokine storm
○ Upset balance of T cells
advantage of CART cells
1) CAR T cells recognise and bind to unprocessed tumour surface antigens without MHC processing
• Possess full TCR complex
• Recognise antigens without MHC proteins
• Through scFV on CAR-T (not reliant on TCR)
2) scF domain binds to cell surface antigens
• Effective against hematological tumours
• Acute lymphoid leukemia (blood type cancers)
• Not for solid tumours
CAR-T cells diasadvantage
1) scFV may guide CART cells into antigen independent mechanism
• Failed therapy
2) Less efficient than TCR T therapy
• Activated only at higher target cell surface antigen densities
○ Require tumour cells to have high conc of antigens on surface
• Only 1 subunit (scFv) binding to target cell surface antigen
○ Weaker CAR signally and activation
○ Execute killing function but lack EXTENDED killing
3) ADR:
• On-target off-tumour toxicity limited to B cell aplasia
○ Due to CD19 specific CAR T cells attacking and kill normal B cells – also have CD19
• Off-target toxicity
• Cytokine-release syndrome
• Autoimmune response
immune checkpoint inhibition
• T cells express checkpoint molecule proteins on surface
○ Ligands binding to these checkpoint molecules trigger suppression of T cell activity
• Function of checkpt proteins: counter overstimulation of T cell activity.
Prevent autoimmune response
CTLA-4 (NORMAL FUNCTION)
Cytotoxic T-lymphocyte associated protein 4
1) CTLA-4 and CD28 expressed on activated T cell
2) During APC: co-stimulatory molecule CD80/ CD86 expressed on APC binds to CD28 on T cells
a. Incr T cell activation
b. Execute cell killing effect
3) Expressed CTLA-4 competes with CD28 to bind with CD80/ CD86 a. Reduce CD80/ CD86 - CD28 binding
T cell activation inhibited
Therapeutic: anti-CTLA4 mab
1) Ipilimumab bind to CTA-4 (INHIBITS)
2) So that CD80/ CD86 - CD28 binding
Programme death 1 (PD-1)
normal function
• PD1 expressed on activated T cells in intratumour microenvironment
• DC, tumour-associated macrophage, fibroblasts, some tumour cells
• PD-L1 (ligand)/ L2 binds to PD-1 and suppress T cell activity
- cancer cells develop to hide
PD1 inhibitor
a. Pembrolizumab, nivolumab
b. Prevent PDL1 bind to PD1 on T cell
c. So T cell activity continued
PD-L1 inhibitor
a. Atezolizumab, avelmab, durvalumab
b. Blocks PDL1, not able to bind to PD1 of T cell
limitation of checkpoint inhibitors
1) not all cancer pt response to checkpoint inhibitors (tumour microenvironment is complex)
2) efficacy can be transient (resistance)
3) development of immune-related ADR
- reduced clinical outcomes
- dose interruption/ discontinuation
- death
cancer vaccines
why?
types?
strategy in cancer immunotherapy, acquisition of immune memory
• Given to cancer pt for cancer management by potentiating or reactivating pt own immune system
1) cell – tumour/ dendritic
2) protein/ peptide
3) nucleic acid (RNA, DNA)
cancer vaccines: cell vaccines
(tumour, dendritic)
1) tumour cell vaccine: Contains autologous/ non-autologous tumour cells (may or may not be genetically modified)
□ Antigens expressed by tumour cells to induce T cells in pt
2) Dendritic cell vaccine: tumour antigenic proteins/ peptides or tumour cells loaded on DC
a) DC administered into cancer pt for tumour antigens to induce T cells
b) Eg: provenge 1) Leukocyte fraction extracted from pt peripheral blood (DC main APC in pdt) 2) Dc cultured ex vivo with fusion protein i) Fusion proteins = (antigen prostatic acid phosphatase + immune signaling factor GM-CSF for APC maturation) 3) Activated APC reinfuse into pt i) Evoke immune response against cancer cells carrying antigen
cancer vaccines — nucleic acid vaccines
(RNA, DNA)
Vaccine formulation contain DNA, RNA code for tumour associated-antigenic proteins/ peptides
Delivered using:
□ viral vector
-Efficient delivery of DNA/ RNA into cells)
□ Non-viral vector
- Liposomal formulation
Considerations:
1) DNA vaccines: nucleic acid incorporated into host cell genome
a) Risk induce carcinogenicity if insert gene cause mutagenesis, switch on ONCOGENEs
i) Esp insert into exons
2) RNA vaccines
a) No risk of carcinogenicity since RNA not incorporated into host cell genome
b) But RNA stability in formulation and administration needs to be maintained
