Biopharm pdts: T cell therapy + Cancer Vaccines Flashcards
Tumor Infiltrating Lymphocyte (TIL) therapy involves autologous lymphocytes
True or False
True, it involves autologous TILs isolated from excised tumor masses
TILs consist of polyclonal T cells and NK cells.
What are the 3 components involved in its expansion in vitro via the rapid expansion process (using cell culture)?
Exposed to a high dose of IL2 + anti-CD3 antibody + irradiated feeder cells
- IL2: stimulate growth + differentiation + activation of T cells, B cells, NK cells
- Anti-CD3 Ab: activate CD3 in TCR, stimulate T cell activation
- Irradiated feeder cells: autologous peripheral blood mononuclear cells (PBMC) to aid in proliferation (enter cell cycle division for activation, can also produce cytokines)
Even though TIL therapy is generally safe, what are 3 disadvantages associated with it?
- Excised tumor mass may be devoid of or contain low quantities of TILs (aka cold tumor)
- Heterogenous/polyclonal T cells with different antigen specificies, thus not lethal enough
- Limited or none of the naturally occurring T cells possess high affinity for the antigen (don’t bind strongly to the antigen)
In TCR-T and CAR-T therapy, T cells are isolated from excised tumor mass
TRUE or FALSE
Elaborate
FALSE, they are isolated from patient’s peripheral blood, and subsequently genetically modified to tumor antigen-specific T cells
What do the genes transduced into TCR-T cells encode for?
*T cell receptor engineered T cells
Genes encode for Va and VB regions within the alpha and beta chains
With respect to TCR-T therapy, how are the genes cloned before transduction into TCR-T cells? Explain the choice of the vectors.
Genes are cloned into retro- or lentiviral vectors that have high transduction efficiency
Ensures that genetically modifed T cells are less heterogenous and possess high tumor antigen specificity
Both TCR-T and CAR-T are effective against solid and hematological tumors (leukemia)
TRUE or FALSE
FALSE
TCR-T: effective against solid and hematological tumors (recognize antigen on tumor cell surface, and within cell)
CAR-T: effective against hematological tumors only (ScFv domain can only bind to cell surface antigens, unable to infiltrate solid tumors)
What are the 3 advantage that TCR-T therapy have over CAR-T therapy?
(Hint: density, onset, duration of action)
TCR-T cells use full TCR complex (6CDRs) for antigen recognition and signal transduction. Hence,
- Can be fully activated at low target cell antigen densities
- Onset of signalling is slow (due to bulkier receptor, time needed to attach to pMHC protein) but of longer duration
*CAR-T have faster onset since single chain can bind more quickly to antigen, MHC-independent as well - Execute more extended killing (stronger binding and signal transduction since 6CDRs available)
*CAR-T lack extended killing due to weaker binding, signaling, and activation
MHC is polygenic and polymorphic. How does this affect the efficacy of TCR-T therapy?
Engineered TCR-T cells express a particular tumor-specific TCR limited for use only in a patient subpopulation carrying that specific MHC allele.
*CDR2 in Va and VB recognizes and binds to MHC in peptide-MHC
Name 3 disadvantages that both TCR-T and CAR-T therapy have in common.
- On-target off-tumor toxicity (target normal tissue/cell expressing same antigen)
- Off-target toxicity (T cell is not specific, cross-reacts) *not much reported w CAR-T
- Cytokine-release syndrome CRS (infusion causes cytokine storm) *more so in CAR-T
What does the gene transduced into CAR-T cells encode for?
*Chimeric Antigen Receptor T cell therapy
Single genetic sequence encoding for scFv (specific antigen-binding sites within VH and VL in Fab domain) of an identified antibody that targets the antigen of interest
Transduced into T cells to express chimeric antigen receptors on their surface
Explain why CAR-T cells have faster onset than TCR-T cells (2 reasons)
*Think about their structure (ScFv VS TCR complex)
CAR-T cells are MHC-independent, able to bind to unprocessed tumor surface antigens without MHC processing (vs TCR-T cells that require time to bind to peptide-MHC)
CAR-T may also have faster onset because single chain can bind more quickly to antigen (vs bulky TCR of TCR-T cells)
Name a disadvantage of CAR-T therapy due to the use of scFv.
scFv may guide CAR-T cells into antigen independent mechanism (if not specific, recognize other antigens)
How do the 4 generations of CAR-T cells differ in structure?
They differ in the intracellular domain that mediates signal transduction.
1st Gen: 1 signalling domain, CD3ζ
2nd Gen: 2 signalling domains, CD3ζ + costimulatory CD28 or 4-1BB domain
3rd Gen: 3 signalling domains, CD3ζ + CD28 + 4-1BB
4th Gen: 3 signalling domains, CD3ζ + CD28 + 4-1BB AND transgene
What improves from the modifications of 1st gen to 4th gen CAR T cells?
Explain the action of transgene in 4th gen.
1st gen lack in vivo survival, fail to execute potent antitumor activity
2nd and 3rd gen have stronger signaling, longer lasting in vitro proliferation, potent antitumor activity
4th gen have stronger signaling + additional transgene that encode for protein cytokine (e.g., able to express cytokine IL12 - broad acting, induce proliferation of leukocytes) *Cytokines can exert autocrine & paracrine effect on T cells - activate more T cells
Can antigen-negative cancer cells be eliminated by CAR T cells?
Yes, if it is 4th gen CAR T cell.
Activation of the transgene that encodes for cytokine, can activate immune cells (without antigen binding to scFv)
Why do T cells express checkpoint molecule proteins on their surface? (what is the function)
Binding of extracellular ligands to these checkpoint molecules trigger SUPPRESSION of T cell activity
Native function: prevent overstimulation of T cell activity, prevent autoimmune response
How do anti-CTLA-4 mAbs work?
They bind to CTLA-4 on T cells, preventing CTLA-4 from competing with CD28 for binding to costimulatory molecule CD80/CD86 expressed on APC.
Thus reduce CD80/CD86-CTLA-4 binding which signals suppression
Increase CD80/CD86-CD28 binding which signals activation
*Both CTLA-4 and CD28 are expressed on activated T cells
How do PD1 inhibitors work?
PD1 inhibitors bind to PD1 expressed on activated T cells, blocks binding of PD1 to PD-L1/PD-L2 expressed on tumor cells/APCs, therefore preventing suppression of T cell activity
*When PD1 binds to PD-L1/PD-L2, T cell does not attack
Name 2 checkpoint molecule proteins that can be expressed on activated T cells
- CTLA-4
- PD-1
What are the 3 current limitations of checkpoint inhibitors?
- Not all cancer pt show response
- Efficacy can be transient
- Development of immune-related adverse events (autoimmune reactions, since remove safeguard of body’s immune system)
Name 2 types of cell vaccines for cancer, and explain how they work
- Tumor cell vaccine
- antigen expressed on autologous/non-autologous tumor cells to induce T cells in pt - Dendritic cell vaccine
- tumor antigenic peptides loaded on DCs, DCs express p-MHC molecules on cell surface, induce T cells in pt
For dendritic cell vaccine, how are DCs extracted and cultured before re-infusion into pt?
DCs extracted from pt peripheral blood
DCs cultured with fusion protein consisting: antigen + immune signalling factor GM-CSF for APC maturation
Why are protein/peptide vaccines not useful? (3 reasons)
*Administration of antigenic peptide fragments derived from tumor-associated antigens in order to evoke immune response from T cells
- Neoantigens can arise as cancer cells mutate differently (diff tumor antigens in diff pt)
- Antigenic peptides of short chain ~15AAs only bind to MHC Class I molecules (only activate CD8+ T cells, no activation of CD4+ which is impt for switching on full adaptive immunity)
- May induce aenergy (whereby peptide binds to cell but does not trigger any immune response) ==> this can cause immune tolerance
What do nucleic acid vaccines contain?
DNA/RNA code for tumor-associated antigenic proteins/peptides, delivered using a viral vector into cells
*Non vector-based delivery system use lipid vesicles in liposomal formulations
What is the risk of using DNA vaccines?
Nucleic acid can get incorporated into host cell genome
Risk of inducing carcinogenicity if insertion of gene causes insertional mutagenesis (e.g., if insert in exon) ==> may switch off tumor suppression genes and switch on oncogenes
Does RNA vaccines have risk of carcinogenicity?
No. It is not incorporated into host cell genome, it will not cause insertional mutagenesis. RNA lies in the cytoplasm.
What is the main concern of using RNA vaccines?
RNA stability in formulation and upon administration
Other concerns:
- encode for too short peptide, only bind to MHC class I
- evoke immune response
