Biopharm pdts: T cell therapy + Cancer Vaccines

Question 1

Tumor Infiltrating Lymphocyte (TIL) therapy involves autologous lymphocytes

True or False

Answer 1

True, it involves autologous TILs isolated from excised tumor masses

Question 2

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)?

Answer 2

Exposed to a high dose of IL2 + anti-CD3 antibody + irradiated feeder cells

1. IL2: stimulate growth + differentiation + activation of T cells, B cells, NK cells
2. Anti-CD3 Ab: activate CD3 in TCR, stimulate T cell activation
3. Irradiated feeder cells: autologous peripheral blood mononuclear cells (PBMC) to aid in proliferation (enter cell cycle division for activation, can also produce cytokines)

Question 3

Even though TIL therapy is generally safe, what are 3 disadvantages associated with it?

Answer 3

1. Excised tumor mass may be devoid of or contain low quantities of TILs (aka cold tumor)
2. Heterogenous/polyclonal T cells with different antigen specificies, thus not lethal enough
3. Limited or none of the naturally occurring T cells possess high affinity for the antigen (don’t bind strongly to the antigen)

Question 4

In TCR-T and CAR-T therapy, T cells are isolated from excised tumor mass

TRUE or FALSE

Elaborate

Answer 4

FALSE, they are isolated from patient’s peripheral blood, and subsequently genetically modified to tumor antigen-specific T cells

Question 5

What do the genes transduced into TCR-T cells encode for?

*T cell receptor engineered T cells

Answer 5

Genes encode for Va and VB regions within the alpha and beta chains

Question 6

With respect to TCR-T therapy, how are the genes cloned before transduction into TCR-T cells? Explain the choice of the vectors.

Answer 6

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

Question 7

Both TCR-T and CAR-T are effective against solid and hematological tumors (leukemia)

TRUE or FALSE

Answer 7

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)

Question 8

What are the 3 advantage that TCR-T therapy have over CAR-T therapy?

(Hint: density, onset, duration of action)

Answer 8

TCR-T cells use full TCR complex (6CDRs) for antigen recognition and signal transduction. Hence,

1. Can be fully activated at low target cell antigen densities
2. 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
3. Execute more extended killing (stronger binding and signal transduction since 6CDRs available)
   *CAR-T lack extended killing due to weaker binding, signaling, and activation

Question 9

MHC is polygenic and polymorphic. How does this affect the efficacy of TCR-T therapy?

Answer 9

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

Question 10

Name 3 disadvantages that both TCR-T and CAR-T therapy have in common.

Answer 10

1. On-target off-tumor toxicity (target normal tissue/cell expressing same antigen)
2. Off-target toxicity (T cell is not specific, cross-reacts) *not much reported w CAR-T
3. Cytokine-release syndrome CRS (infusion causes cytokine storm) *more so in CAR-T

Question 11

What does the gene transduced into CAR-T cells encode for?

*Chimeric Antigen Receptor T cell therapy

Answer 11

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

Question 12

Explain why CAR-T cells have faster onset than TCR-T cells (2 reasons)

*Think about their structure (ScFv VS TCR complex)

Answer 12

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)

Question 13

Name a disadvantage of CAR-T therapy due to the use of scFv.

Answer 13

scFv may guide CAR-T cells into antigen independent mechanism (if not specific, recognize other antigens)

Question 14

How do the 4 generations of CAR-T cells differ in structure?

Answer 14

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

Question 15

What improves from the modifications of 1st gen to 4th gen CAR T cells?
Explain the action of transgene in 4th gen.

Answer 15

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

Question 16

Can antigen-negative cancer cells be eliminated by CAR T cells?

Answer 16

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)

Question 17

Why do T cells express checkpoint molecule proteins on their surface? (what is the function)

Answer 17

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

Question 18

How do anti-CTLA-4 mAbs work?

Answer 18

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

Question 19

How do PD1 inhibitors work?

Answer 19

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

Question 20

Name 2 checkpoint molecule proteins that can be expressed on activated T cells

Answer 20

1. CTLA-4
2. PD-1

Question 21

What are the 3 current limitations of checkpoint inhibitors?

Answer 21

1. Not all cancer pt show response
2. Efficacy can be transient
3. Development of immune-related adverse events (autoimmune reactions, since remove safeguard of body’s immune system)

Question 22

Name 2 types of cell vaccines for cancer, and explain how they work

Answer 22

1. Tumor cell vaccine
   - antigen expressed on autologous/non-autologous tumor cells to induce T cells in pt
2. Dendritic cell vaccine
   - tumor antigenic peptides loaded on DCs, DCs express p-MHC molecules on cell surface, induce T cells in pt

Question 23

For dendritic cell vaccine, how are DCs extracted and cultured before re-infusion into pt?

Answer 23

DCs extracted from pt peripheral blood

DCs cultured with fusion protein consisting: antigen + immune signalling factor GM-CSF for APC maturation

Question 24

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

Answer 24

1. Neoantigens can arise as cancer cells mutate differently (diff tumor antigens in diff pt)
2. 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)
3. May induce aenergy (whereby peptide binds to cell but does not trigger any immune response) ==> this can cause immune tolerance

Question 25

What do nucleic acid vaccines contain?

Answer 25

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

Question 26

What is the risk of using DNA vaccines?

Answer 26

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

Question 27

Does RNA vaccines have risk of carcinogenicity?

Answer 27

No. It is not incorporated into host cell genome, it will not cause insertional mutagenesis. RNA lies in the cytoplasm.

Question 28

What is the main concern of using RNA vaccines?

Answer 28

RNA stability in formulation and upon administration

Other concerns:
- encode for too short peptide, only bind to MHC class I
- evoke immune response