CAR T Cellular Therapy

Question 1

how do tumor cells evade immune system?

Answer 1

• show decreased expression of immunological markers
• different MHC gene mutations
• expression of negative costim molecules = dampening immune system
• creating immunosuppressive environments

Question 2

immunotherapy strategies

Answer 2

• oncolytic viruses
• monoclonal ABs
• checkpt inhibitors
• cancer vaccines
• CAR T cells

Question 3

three domains of CAR T cells

Answer 3

intracellular signalling domain
transmembrane domain
extracell target binding domain

= all necessary for sustaining immune response

Question 4

CD3zeta

Answer 4

• primary signalling domain
• necessary to initiate phosphorylation signalling fpr T cell activation

Question 5

transmembrane costim domain

Answer 5

• anchors CAR to membbrane
• contains CD18 or CD137 receptors
• essential for signal transduction that sustains T cell activation
• mediates cytokine release that controls T cell prolife and in-vivo survival for prolonged anti-tumor effect

Question 6

hinge region of CAR T

Answer 6

flexible peptide linker
- important to bind transmembrane domain to extracel binding domain (antigen binding domain)

Question 7

antigen binding domain of CAR T

Answer 7

derived from light and heavy chain portion of single chain variable fragment from antigen binding site of a monoclonal Ab
= affords tumor specificity/ targeting

Question 8

this gives T cells ability to recognize any cell surface molecule to which an Ab can be made

Answer 8

the antigen binding domain of CAR T

Question 9

advantages of CAR T (2)

Answer 9

proteins, glycoproteins, glycolipids can serve as potential targets

recognition is not MHC-restricted (no requirements for antigen processing and presentation)

reprograms regular T cells to T HUNTER cells = specificity of Ab with cytotoxicity and memory of T cells

Question 10

what does CAR T cell manufacturing involve?

Answer 10

CAR protein
engineered into CAR DNA
- built inside a carrier vector

vector inserted inside virus; infects T cells but can not replicate itself (modified retrovirus or lenti-virus) = replicaiton-deficient virus with CAR DNA

infect T cells = CAR DNA built in the genome and translatred into CAR protein = CAR T cell

Question 11

five key steps of CAR T cell therapy

Answer 11

• leukopheresis = pt T cells harvested through apheresis machine
• T cell activation = Ab=coatred beads serving as artiifcl dendritic cels = used to activate isolated T cells
• transduction = genetically reprogramed ex-vivo using lenti-viral mediated transduction to constructing coating of CAR-receptor on the T cell
• expansion = significant increase in number until sufficient dose for infusion (dependent on individual clinical situation)
• lympho-depleting and then CAR T cell infusion; CART ‘s bind to cancer cells to kill them

Question 12

how do CAR T cells kill?

Answer 12

similar to non-gentically modified T cels
BUT…
selectively kills tumor cells, proliferate, migrate in/out lymph nodes, regulated identically to non-genetic T cells, persist for multiple years for anti-tumor cells

Question 13

what is the ideal CAR-T target receptor?

Answer 13

• want target to be tumor-specific
• want it to be expressed only on tumor cells
• not intracell but cell-surface molecule
  = CD19 most frequent; B-cell leukemias; almost always only found on malignant cells; broader and higher expression; has confinement to B-cell lineage in healthy tissue

Question 14

what do we expect when targeting Bcell leukemias with CAR T therapy?

Answer 14

some B cell aplasia
- can alleviate by replacement therapy with IVIg

Question 15

Kymriah

Answer 15

for chidren with relapsed/refractpru B cell ALL
Novartis

Question 16

Yescarta

Answer 16

for adults with relapsed/frefractory large B cell lymphoma

Kitepharma

Question 17

major difference between Kymriah and Yescarta

Answer 17

vector used
lentivirus = Kymriah/Novartis
retroviral = Yescarta/Kite

Question 18

drawbacks of CAR T cell therapy

Answer 18

(most serious & common)
1. cytokine release syndrome
2. neurological toxicity
3. on-target, off-tumor toxicity
4. (theoretical) insertional oncogenesis = not been reported in clinical studies
5. (theoretical) = allergy/anaphylaxis = host reaction due to foreign monoclonal Ab chain and receptors

Question 19

CRS

Answer 19

cytokine release syndrome
- systemic inflamm response
- high IL-10, IL-6, elevated inflam responses
- fever, fatigues, nausea, tachycardia,hypotension, etc. = DIC; can lead to death
- expansion and progressive immune activation of transfer CAR T cells
- degree directly associated with disease burden at time of CAR T cell infusion; high tumor burden (lots of receptors) = immune bomb of CAR Ts infused = these pts will experience the most severe toxicity

Question 20

pros of CAR T cells compared to regular T cells

Answer 20

non-HLA restricted

targets non-peptide antigens

better targeting due to high affinity of Ig binding

can avoid tumor escape through HLA downregulation

increase of invoivo activation due to build up cstimulation

Question 21

cons of CAR T cell therapy vs T cells

Answer 21

cannot target intracellular pathogens

does not deal well with tumor escape mechanisms (ex: soluble antigen shedding, antigen downreg, TSA/TAA epitope mutation… escapes the effectiveness of CAR T Cells)

Question 22

pros of CAR T vs IVIg

Answer 22

CAR T more potent due to T cell cytotoxic activity

long-term effect bc of memory associated with CAR Ts

CAR T = considered curative bc one shot deal whereas IVIg = need multiple

Question 23

cons of CAR Ts vs. IVIG

Answer 23

T cell infusion toxicity significant cost
lengthy process

Question 24

T or F. Patients do not need to be well enough for CAR T cell therapy

Answer 24

F! need to be well enough for infusion

Question 25

allogenieic/off-the-shelf CAR T cell therapy

Answer 25

re-dosing
combination therapy
can pool from donors
less expensive

Question 26

need to do this before doing allogeneneic CAR T

Answer 26

eliminate T Cell HLA expression (knockout genes)