IC2 Flashcards
List 2 types of immunotherapies and how they work
- Activation type of immunotherapies: involve use of agents that augment and/or reestablish the immune system’s ability to prevent and fight the disease.
- Suppression type of immunotherapies: involve use of agents that reduce or suppress an immune response.
What is an immunotherapy?
Treatment of a disease by intervening the immune system.
Examples of therapeutic strategies in immunotherapy
- Cytokines (humoral component of innate & adaptive immune response)
- Antibodies (humoral component of adaptive immune response)
- T cells (include cell- based immunotherapy and checkpoint inhibitors)
- Cancer vaccines and many more due to advances in molecular cloning techniques and biotechnology!
Properties/ facts of cytokine
• Though belonging to diverse groups, are usually small proteins of < 20 kDa.
• Are usually glycoproteins (glycosylated). Glycosylation sometimes not essential for cytokine’s biological activity, sometimes contributes to half-life of cytokine.
• Usually have short half-lives.
• Are secreted out of the cell producing it. Usually act at short range in an autocrine (i.e. on the cells that produce them) or paracrine (i.e. on cells nearby) manner.
• Binds specifically to its corresponding cytokine receptor expressed on surface of effector cell -> trigger biological effects.
Two types of IFN and example
- Type I (IFN alpha, beta)
- Type II (IFN gamma)
Which cell produces IFN-alpha?
leukocytes
what cell produces IFN-beta?
somatic cells, including fibroblasts and epithelial cells.
what condition is IFN-beta effective against?
multiple sclerosis
what cell produces IFN-gamma?
T lymphotcytes
production of antiserum
- whole blood from immunized animal collected → left to clot or add coagulant → clotting factors removed → serum obtained as supernatant after centrifugation to separate cellular components.
- Raw serum further purified by eliminating serum proteins and enriching the fraction of Ig that reacts with the target Ag (by protein A/G purification or immunoaffinity column chromatography)
what is an antiserum?
Animal sera containing polyclonal antibodies raised by immunizing the animal with a particular antigen.
drawback of antiserum
immunogenicity issues
what is a monoclonal antibody?
Ab produced from 1 B-cell clone, recognize just 1 epitope of the antigen.
characteristic of monoclonal Ab and its usefulness
• Possess high specificity → Useful for:
– Recombination protein purification work (used in immunoaffinity chromatographic purification of proteins).
• Possess high homogeneity → Effects obtained highly reproducible → Useful for:
– Commercial development into target-specific therapeutic molecules for treatment of diseases with less side effects.
– Commercial development into diagnostic test kits.
– Many experimental research techniques.
characteristic of monoclonal Ab and its usefulness
• Possess high specificity → Useful for:
– Recombination protein purification work (used in immunoaffinity chromatographic purification of proteins).
• Possess high homogeneity → Effects obtained highly reproducible → Useful for:
– Commercial development into target-specific therapeutic molecules for treatment of diseases with less side effects.
– Commercial development into diagnostic test kits.
– Many experimental research techniques.
Limitations of murine Mabs
• human-anti-mouse antibody response (HAMA) characterized by joint swelling, rashes and kidney failure
• Fail to trigger a number of effector functions.
• Shorter half-lives (30 - 40 h)
% of human sequences in chimeric Mab and how is this achieved?
• To replace amino acid sequences on the CH and CL of murine Mab that are not essential for antigen binding with human sequences. Antigen-binding sequences in VH and VL fragments conserved.
• Chimeric Mab retains antigen selectivity and affinity similar to parent murine Mab.
• ~ 75% human
% of human sequences in humanised Mab and how is this achieved?
• Replacing all mouse amino acid sequences except the hypervariable CDR domains of Ig (reside within the VH and VL fragments) in chimeric Mabs → Humanized Mabs.
• > 90% human (reduced immunogenicity as compared to chimeric Mabs).
remaining immunogenicity concerns for recombinant human monoclonal antibodies
impurity proteins from mammalian host cells not removed
Nomenclature of 4 types of Monoclonal Antibody
– Mice (infix, -o-)
– Chimeric (infix, -xi-)
– Humanized (infix, -zu-)
– Antibodies originating in humans (infix, -u-)
Role of pepsin & papain
proteases that cleave peptide bonds to yield F(ab’)2 and Fab respectively.
what is ScFv
single chain variable fragment; contains amino acid sequences of antigen- binding CDRs in the VH and VL in the Fab arm of an Ig in a single polypeptide chain.
what is BiTEs?
Bispecific T cell engagers (BiTEs) - have 2 distinct Fab regions and are capable to bind to two distinct epitopes. Lack a Fc domain.
what is a triomab?
Trifunctional mAbs (triomabs) - bind to 2 different antigens while maintaining the capacity to mediate Fc-dependent effector functions. Possess a Fc domain.
In naturally occurring IgG, is the Fc domain fucosylated? (Y/N)
Yes
Fucosylated Fc domain of IgG causes _____
reduced affinity of the Fc domain to bind to an subtype of activating Fc receptor (FcyRIII) found on effector cells -> reduced ADCC induction by effector cells -> reduced efficacy of fucosylated antibodies
what is TIL therapy
TIL (tumour infiltrating lymphocytes) therapy:
- Isolated TILs from patient’s tumor masses consist of T and NK cells. Isolated T cells are polyclonal, have diverse antigen specificity
- Isolated TILs, mainly T cells are expanded in vitro by a rapid expansion process (REP) - exposed to high dose IL2 + anti-CD3 antibody (to activate CD3 in TCR) + irradiated feeder cells [usually autologous PBMCs (peripheral blood mononuclear cells) obtained from patient]
- Large quantities (~10-150 billion) of lymphocytes produced and infused into patient
what are TCR-T and CAR-T therapies (in general)
T cell receptor engineered T cell (TCR-T) therapy and Chimeric antigen receptor T cell (CAR-T) therapy:
1. T cell isolation from patient’s peripheral blood
2. Genetic modification with CAR/ TCR
3. Genetically engineered T cells (TCR or CAR) expanded in vitro, then infused back into the
patient -> recognize tumor antigens and attack cancer cells.
Advantages & disadvantages of TIL therapy
Advantages: Generally safe compared to CAR-T/ TCR-T
Limitations:
- For some patients, excised tumor masses are devoid of or contain very low quantities of TILs.
- Expanded naturally occurring tumor-specific T cells are heterogeneous possessing varying antigen specificities -> TIL reinfusion may not be lethal enough to attack and eradicate cancer cells.
- Limited or none of the expanded naturally occurring tumor antigen- specific T cells possess high affinity.
Principle of TCR-T therapy
- Genes encoding Va and Vb within the a and b chains that make up a T cell receptor (TCR) are tumor antigen-specific.
- Genes are cloned into retro- or lentiviral vectors, which in turn are used to transduce T cells isolated from patient’s peripheral blood
- Genetically modified T cells less heterogeneous and high transduction efficiency of retro- or lentiviral vectors ensures that reinfused T cells possess high tumor antigen specificity
Advantages of TCR-T therapy
Advantages:
- Engineered TCR-T cells possess full TCR complex -> can recognize antigens expressed at both cell surface/tumor surface and within tumor cell/tumor mass -> can penetrate tumors -> effective against solid and hematological tumors.
- Compared to CAR-T cells, TCR-T cells use full TCR complex for antigen recognition and signal transduction (CD3 adaptor proteins)
-> 1. can be fully activated at low target cell antigen densities,
2. onset of signaling slow but of longer duration and
3. execute more extended killing -> more effective than CAR-T therapy for cancer treatment.
Disadvantages of of TCR-T therapy
Disadvantages:
- Engineered TCR-T cells expressing a particular tumor-specific TCR only limited for use in a patient subpopulation carrying a specific MHC/HLA allele recognized by TCR. (recall: MHC is polygenic and polymorphic)
- Less safe than TIL therapy –
1. On-target off-tumor toxicity (TCR-T cells target normal tissue expressing same antigen. E.g.
both normal melanocytes and melanoma cells express gp100 and MART-1)
2. Off-target toxicity (TCR-T cells not specific and cross-react with other antigenic fragments)
3. Cytokine-release syndrome (CRS) (infusion of TCR-T cells induce a cytokine storm).
Where do CDR 1,2,3 in Va and Vb of TCR bind to?
CDR1: bind to each terminus of the peptide in peptide-MHC
CDR2: recognize and bind to MHC in peptide-MHC
CDR3: recognize and bind to peptide in peptide-MHC
Recall: Strength of interaction between TCR and peptide-MHC contributes to extent of T cell activation and development of antigen-specific T cells as memory T cells
Principle of CAR-T therapy
- Construction of chimeric antigen receptor (CAR): genetic sequence encoding for specific antigen-binding sites within VH and VL in the Fab domain of an identified antibody (known to be targeting specifically a cell surface molecule of interest) is cloned into a retro- or lentiviral vector.
- T cells isolated from patient’s peripheral blood are transduced by vectors carrying CAR gene -> Transduced T cells express CAR on surface (now called CAR-T cells) -> In vitro expansion of CAR-T cells and cells are infused into the patient to bind to antigens on cancer cells to kill them.
Similarities and differences btw the 4 generations of CAR-T cells
Similarity: All contains only ONE extracellular domain -> scFv -> R&B to antigen
Differences:
- 1st gen: 1 intracellular domain CD3z
- 2nd gen: 2 intracellular domain CD3z + CD28/ 4-IBB
- 3nd gen: 3 intracellular domain CD3z + CD28 + 4-IBB
- 4th gen: 3 intracellular domain CD3z + CD28 + 4-IBB + 1 transgene (upon target antigen binding, activated to express cytokines -> activate more T cells to kill cancer cells)
Advantages of CAR-T therapy
- CAR-T cells recognize and bind to unprocessed tumor surface antigens without MHC processing. possess full TCR complex -> can recognize antigens without MHC proteins.
- scFv domain only binds to cell surface antigens -> effective against hematological tumors (patients with acute lymphoid leukemia respond very well), but not effective against solid tumors
Disadvantages of CAR-T therapy
- scFv may guide CAR-T cells into an antigen- independent mechanism -> failed therapy
- Less efficient than TCR-T therapy
- Activated only at higher target cell surface antigen densities
- Only one subunit (scFv) binding to target cell surface antigen -> weaker CAR signaling and activation -> execute faster killing function but lack extended killing. - Adverse effects:
- On-target off-tumor toxicity mainly limited to B cell aplasia (due to CD19-specific CAR-T cells attacking and killing normal B cells also expressing CD19 like the malignant CD19+ B cells)
- Off-target toxicity (not highly reported)
- Cytokine-release syndrome (CRS) (induce CRS more than TCR-T therapy)
Which cell expresses checkpoint molecules proteins on their surface?
T cell
Native function of checkpoint proteins
to counter overstimulation of T cell activity and to prevent autoimmune responses.
Examples of checkpoint molecules identified as targets for development of therapeutics
- Cytotoxic T-lymphocyte associated protein 4 (CTLA-4)
- Programmed death 1 (PD-1)
Principle of anti-CTLA4 Mab
CTLA-4 and CD28 expressed on activated T cells. During antigen presentation by APC, costimulatory molecule CD80/C86 expressed on APC binds simultaneously to CD28 on
T cells to increase T cell activation -> activated T cell executes cell killing effect.
Expressed CTLA-4 competes with CD28 for binding with CD80/C86 -> reduces CD80/C86 - CD28 binding -> T cell activation inhibited
anti-CTLA4 Mab used to promote T cell activation
Principle of PD-1 inhibitors/ PD-L1 inhibitor
PD-1 expressed on activated T cells.
In the intratumor microenvironment, a number of cells including DCs, tumor- associated macrophage (subset of macrophage), fibroblasts and even tumor cells themselves express PD- L1 (PD-1 ligand) and/or PD-L2
PD- L1/PD-L2 binds to PD-1 and suppress T cell activity.
PD-1 inhibitors/ PD-L1 inhibitor used to promote T cell activity.
3 categories of cancer vaccines
– Cell vaccines
– Protein/peptide vaccines
– Nucleic acid (DNA, RNA) vaccines
Designs for Cell Vaccines
- Tumor cell vaccine -> antigens expressed by tumor cells to induce T cells in patient.
- DC vaccine -> tumor antigenic proteins/peptides or tumor cells loaded on DCs, DCs administered into cancer patient for tumor antigens to induce T cells
Principle of Protein/Peptide Vaccines
Vaccine formulations contain antigenic peptide fragments derived from tumor-associated antigens -> upon administration, recognized by T cells and activate T cells.
Why are peptide/ protein vaccines not found to produce strong immune responses?
- Neoantigens (arising from specific gene mutations in a cancer patient) are not included.
- Antigenic peptides of short chain restricted to MHC class I binding -> MHC class II binding not engaged and hence subsequent activation of CD4+ helper T cells followed by CTL induction is absent.
- Antigenic peptides of short chain may bind to any cell without triggering further processing -> may induce anergy (lack of immune response to an antigen) and cause immune tolerance.
Principle of Nucleic acid Vaccines
Vaccine formulations contain DNA or RNA that code for tumor associated-antigenic proteins/peptides. Can be delivered using a viral vector (efficient delivery of DNA/RNA into cells). Non vector- based delivery system also used (e.g. liposomal formulation).
Concern of DNA vaccine
Nucleic acid gets incorporated into host cell genome -> more lasting expression of antigenic proteins/peptides, but risk of inducing carcinogenicity if insertion of gene causes insertional mutagenesis and switch on oncogenes.
Concern for RNA vaccine
No risk of carcinogenicity caused by insertional mutagenesis since RNA not incorporated into host cell genome. Main concern is RNA stability in formulation and upon administration.
