Cancer Immunology

Question 1

Explain the three steps of immune surveillance

Answer 1

1) Elimination
- Innate and adaptive immunity work together to detect presence of developing tumor and destroy it before it becomes clinically apparent
2) Equilibrium
- Tumor is formed but functionally dormant because immune system is controlling it
3) Escape
- Functional dormancy is lost, tumor begins to grow in immunologically unrestricted manner and is clinically apparent disease
- Due to active immunoediting process (mutations to escape immune system), aging and senescence

Question 2

What is the difference between a tumor-associated-antigen (TAA) and a neo-antigen? What type of cellular therapy would you use to target either?

Answer 2

Tumor-associated-antigens:
- Naturally-occurring proteins in the body that have here overexpressed on tumor cell surface.
- Difficult to distinguish from self-cells so endogenous T cells are not a good therapy as they undergo negative selection and will not recognize self antigen
- use mAbs and engineered T cells

Neo-Antigens:
- Mutations in cancer cell genome that have led to mutations in the surface proteins/antigens expressed on the cancer
- Not expressed on normal self-cells.
- Good targets for endogenous T cell therapy

Question 3

What is the cancer context you would use for the antibody Pembrolizumab (Keytruda)?

Answer 3

Keytruda is for non-small-cell lung cancer.
- When T cells are recruited to tumor cells, a PD1-PDL1 interaction occurs that exhausts T cell activity/proliferation.
- Some cancers overexpress PDL1. to try to downregulate T cell activity.
- Immune checkpoint inhibitor drug: Pembrolizumab Ab binds preferentially to PD1 and allows T cells to stay active and proliferating.

Question 4

Which 3 different approaches can be taken to target tumors with immunotherapy?/Three different ways to target tumors with mAbs?

Answer 4

1) mAb-mediated cell signaling
- Blocking the binding of an activating ligand responsible for the survival of the cancer cell -> checkpoint blockade
- Inhibiting dimerization of a receptor and thus blocking signal activation/inducing apoptosis

2) Ab-drug conjugates
- Uses a toxin linked to the Ab. When Ab binds to a target, it is internalized through the receptor, bringing in the drug that drives a toxic response killing cancer cells

3) Angiogenesis Inhibition
- If an Ab targets vascular endothelial growth factor receptors (VEGF-R) or VEGF, blood vessel growth in the tumors can be slowed/inhibited, preventing angiogenesis

Additional:
4) Via the Fc region
- ADCP (target overexpressed receptor in cancer cell, Fc region drives macrophage attack)
- ADCC (IgG Fc drives NKs to attack cancer cell
- CDC (complement proteins bind to Fc, drives tumor cell lysis)

Question 5

Why are certain tumors highly immunogenic? How could you use that to develop a mAb based therapy?

Answer 5

Some tumors are highly immunogenic because cancer cells are genomically unstable due to mutations occurring during mismatch repair, leading to a higher number of mutations. This increases the probability of the tumor cells expressing mutation-associated neo-antigen on the surface, which can be recognized by endogenous TCRs and thus trigger a strong anti-tumor response.

A mAb-based therapy could be developed if the tumor cell is expressing PDL1 immune checkpoint ligand, by making an Ab that preferentially binds to PD1 on the T cell and keeps T cell response active.

Question 6

A patient has cancer with a very high mutation rate and high T cell infiltration. Design a comprehensive workflow for developing a personalized cellular therapy for this patient. Please describe what is done in each step.

Answer 6

1) Single Cell Sequencing of T cells
- The TCR region is sequenced to get information on the VDJ and transcriptome.
- Since the patient has cancer with a high mutation rate, can assume they have many neo-antigens expressed -> it is likely targeted by endogenous T cells (immunogenic tumor)
- Sequencing is necessary to get effector state and functional profile information on T cell.

2) TCR and Tumor Antigen ID
- Sequencing data is used to identify TCRs that are clonally expanded or are part of a possible tumor-specific phenotype
- Tumor cells can also be sequenced (NGS) to discover possible neo-antigens or TAAs

3) Cellular Engineering
- Patient’s autologous T cells are genetically engineered (via lentivirus or CRISPR-Cas9) to express a synthetic receptor (CAR or TCR) designed with the TCR that was identified to exhibit a high specificity to a tumor target.
- Expanded and re-injected into patient

Question 7

Draw a CAR and label its major domains and regions, and briefly explain their function.

Answer 7

CAR:
- Variable domains of Ab are linked to intracellular signaling and co-stimulatory domains that are highly involved in T cell activation.

Extracellular Domain:
- Binds the target
- Composed of scFv Ab fragment variable heavy and light chain
- Also nanobodies, cytokines, ligands

Linker & Transmembrane Domain:
- Not functionally relevant but important for the architecture of the molecule

Intracellular Signaling Domain:
- Co-stimulation via:
- CD28: faster expansion, less long term persistence of T cell
- 4-1BB: delayed expansion, more persistence
- Activation via:
- CD3-zeta domain

Armour:
- Another gene that causes expression of other proteins (cytokines, chemokines, ligands or receptors)