W14 Cancer therapy (December 1st)

What are the three phases of tumor–immune interaction, and what do they mean?

• Elimination: immune surveillance detects and destroys emerging tumor cells.

• Equilibrium: immune system controls but does not fully eradicate tumor; selection pressure shapes tumor variants.

• Escape: tumor variants that avoid recognition or actively suppress immunity outgrow and become clinically apparent.

How do innate and adaptive immunity differ, and why does each matter in cancer?

• Innate: fast, generic, same each time; includes NK cells, macrophages, DCs, complement. It provides early tumor killing and crucial activation signals

• Adaptive: slower initially, antigen-specific, forms memory; includes T and B cells. It provides high-precision, long-lasting tumor-specific responses that we try to harness in immunotherapy

What is cross-presentation, and why is it essential for anti-tumor CD8+ T cell responses?

Cross-presentation occurs when DCs take up exogenous tumor material and present it on MHC I

• This is essential because naïve CD8+ T cells cannot be primed by tumor cells directly; they rely on DCs to receive antigen plus co-stimulation and danger signals.

What happens if dendritic cells present antigen without danger signals?

They remain immature and lack co-stimulation, leading naïve T cells to become anergic (unresponsive) rather than activated

• This contributes to tolerance instead of anti-tumor immunity

Why is CD4+ T cell help critical in cancer vaccination?

CD4+ T cells:

• Provide cytokines (e.g. IL-2) and co-stimulation that enhance CD8+ T-cell activation and proliferation

• Are necessary for generating memory CD8+ T cells

  • Without including CD4 epitopes, vaccines may induce initial CD8 responses but fail to create durable memory and protection upon tumor re-challenge

Define self-antigen, TAA, and TSA/ neoantigen.

• Self-antigen: normal body protein, expressed in healthy tissue

• TAA (tumor-associated antigen): self-antigen that is over-expressed or restricted to tumors or specific lineages

• TSA/ neoantigen: antigen created by tumor-specific mutations or viral proteins; absent from normal cells and usually highly immunogenic

Why are neoantigen-rich tumors often more responsive to immunotherapy?

High mutational/neoantigen load increases the chance that tumor cells display novel peptides recognized as foreign, driving strong T-cell responses and making checkpoint blockade and vaccines more effective

How do tumors use the immune system to their advantage?

• They express checkpoint ligands (e.g. PD-L1) to inhibit T cells.

• They recruit Tregs via TGFβ and other factors.

• They polarize myeloid cells into TAMs and MDSCs that secrete suppressive mediators.
  Net effect: an immunosuppressive TME that protects the tumor.

What is the immunoscore, and what does a high immunoscore mean?

The immunoscore quantifies CD3+/CD8+ T cells in the tumor core (CT) and invasive margin (IM). A high immunoscore indicates many T cells in and around the tumor, correlating with improved survival and lower risk of recurrence.

What’s the difference between hot, excluded, and cold tumors, and why does it matter?

• Hot: T cells infiltrate tumor core → more likely to respond to immunotherapy.

• Excluded: T cells accumulate at margins but don’t enter.

• Cold: few T cells present at all.
  Therapies may need to first recruit and activate T cells (e.g. oncolytic viruses) before checkpoint blockade can work, especially in cold tumors.

How do anti-CTLA-4 and anti-PD-1 antibodies restore T-cell function?

They block inhibitory receptors on T cells or their ligands on APC/tumor cells:

• Anti-CTLA-4 enhances T-cell priming in lymph nodes.

• Anti-PD-1/PD-L1 restores T-cell activity in the TME.
  Together, they “release the brakes,” enabling T cells to kill tumor cells.

What is a major downside of immune checkpoint blockade?

Because checkpoints are global brakes on T cells, systemic blockade can induce immune-related adverse events resembling autoimmunity (e.g. colitis, dermatitis, endocrinopathies), requiring close clinical monitoring and sometimes immunosuppression.

Why are combination therapies necessary in cancer immunotherapy?

Tumors evade at multiple levels: antigenicity, TME suppression, checkpoints, physical barriers. Single interventions rarely fix all steps. Combining vaccines/neoantigen targeting, checkpoint blockade, TME-modulating strategies (oncolytic viruses, TAM/MDSC targeting), and sometimes chemo/radiotherapy increases the chance of re-establishing the full cancer immunity cycle.

What is the rationale behind CAR-T cell therapy?

CAR-T cells are T cells engineered to express synthetic receptors that recognize tumor antigens independently of MHC. This bypasses issues like low MHC expression and allows strong, targeted killing, especially effective in some hematologic malignancies.

Why is “personalized immunotherapy” often needed?

Neoantigens, immunoscore, TME composition, checkpoint expression, and mutational load differ:

• Between tumor types

• Between patients with the same tumor type
  Hence, the optimal combination of interventions (vaccine, ICB, cell therapy, TME-modulation) must be tailored to each patient’s tumor characteristics.

What is the core limitation of checkpoint inhibitors in many solid tumors?

They rely on pre-existing tumor-resident T cells

• Many tumors either have too few T cells or mostly bystander, non-tumor-specific T cells

• In those cases, releasing checkpoints cannot generate effective anti-tumor immunity, so patients fail to respond.

How does TIL therapy conceptually differ from checkpoint inhibition?

Checkpoint inhibitors re-activate T cells already in the tumor

TIL therapy removes those tumor-infiltrating T cells, massively expands and reinvigorates them ex vivo with IL-2 and stimulation, then reinfuses billions of tumor-reactive T cells after lymphodepletion

• It is an adoptive transfer rather than in situ re-activation

Compare the antigen types targeted by CAR-T vs TCR-T cells

• CAR-T: recognizes surface antigens only, via antibody-like chimeric receptors; MHC independent.

• TCR-T: recognizes peptide–HLA complexes, derived from both intracellular and extracellular proteins after processing; MHC restricted. This allows TCR-T to access a much broader antigen repertoire

Why is finding a suitable CAR target harder in solid tumors than in B-cell malignancies?

• In B-cell malignancies, CD19 is acceptable even though it’s not tumor-specific because patients can survive with B-cell depletion

• In solid tumors, many candidate antigens are also expressed in vital normal tissues (e.g., bowel), so targeting them would cause unacceptable organ damage

  • Truly tumor-specific surface antigens are rare in solid cancers

What are the main problems with standard viral TCR transduction?

1. Random integration into the genome.

2. Co-expression with endogenous TCR leads to competition for surface expression.

3. Mispairing between transgenic and endogenous α/β chains can produce new, unpredictable TCRs with unknown specificity, posing safety risks.

4. Each TCR requires a separate viral vector, which is expensive and patient-specific

How does CRISPR/ Cas9 editing solve TCR mispairing and allow multiple TCRs?

CRISPR/ Cas9 uses guide RNAs to cut TRBC and TRAC loci:

• Endogenous β chains are knocked out; endogenous α is replaced by a transgenic αβ cassette inserted into TRAC via HDR.

• Each edited cell expresses only the transgenic TCR, no endogenous TCR, eliminating mispairing.

• By splitting T cells into pools and using different HDR templates with the same gRNAs, we can generate a multi-TCR product made of separate T-cell sub-populations, each with one defined TCR

Outline the key steps in manufacturing a TIL product

1. Surgical resection of a metastatic lesion (~2 cm³).

2. Mechanical/enzymatic tumor digestion to single-cell suspension.

3. Initial outgrowth phase with very high-dose IL-2 (≈6000 IU/mL) for 2–3 weeks.

4. Rapid expansion protocol with anti-CD3, IL-2 (~3000 IU/mL), and irradiated feeder PBMCs in flasks then bioreactors.

5. Lymphodepleting chemotherapy (cyclophosphamide + fludarabine).

6. Infuse 10⁹–10¹¹ TILs, followed by systemic high-dose IL-2

What are ATMPs, and why is TIL therapy classified as one?

ATMP = Advanced Therapy Medicinal Product, a regulatory category for cutting-edge therapies like gene and cell therapies

• TILs are a living cell-based medicinal product made specifically for each patient, so they fall under ATMP regulation and must be produced under strict GMP conditions with defined release criteria

What were the main clinical results of the phase III melanoma TIL trial vs ipilimumab?

In unresectable stage IIIC–IV melanoma:

• Objective response rate (ORR): 50% with TIL vs 21% with ipilimumab.

• Complete response (CR): 20% vs 7%.

• Progression-free survival significantly improved in the TIL arm. This established TIL therapy as a superior option for this population.

What is meant by TIL products being a “white bag but black box”?

The final infusion bag looks uniform (white cell suspension), but its internal composition is complex and initially unknown: it contains mixtures of tumor-reactive T cells (neoantigen and shared antigen-specific) and bystander viral-specific T cells

Without mapping, you don’t know which T cells drive clinical responses or toxicity

How is the antigen landscape in TIL products mapped experimentally?

1. Identify patient-specific mutations via tumor exome and RNA sequencing.

2. Combine these neoantigens with known shared TAAs and viral antigens to form a library.

3. Express each antigen as a minigene in autologous immortalized B cells.

4. Co-culture B-cell library with TILs and perform an antigen drop-out screen: antigens whose B-cell clones disappear are those recognized and killed by TILs.

5. Use single antigen-expressing B cells and TCR sequencing to identify specific TCR clonotypes

How do autoimmunity and tumor control relate in some TIL-treated patients?

• Some complete responders developed autoimmune toxicity (e.g., uveitis, vitiligo, hearing loss)

• Mapping showed broad T-cell recognition of melanocyte-associated antigens—present on both melanoma cells and normal melanocytes—explaining why powerful tumor immunity came with off-target attack on normal pigment cells

Why is presence of tumor-reactive TCRs not enough to guarantee clinical success?

Tumor-reactive T cells must also:

• Be present at sufficient numbers in the product.

• Have a favourable phenotype (less exhausted, more stem-like or memory).

• Persist and function in vivo within the immunosuppressive tumor microenvironment.

Without these, even good TCRs cannot translate into meaningful tumor control

What are major future improvements planned for TIL therapy?

• Use TILs for more tumor types (NSCLC, penile, endometrial, etc.).

• Develop fully closed bioreactor systems.

• Replace IL-2 with better cytokines to reduce toxicity and IL-2 dependence.

• Replace feeder PBMCs with synthetic factor mixes.

• Genetically engineer TILs for improved homing, persistence, and resistance to tumor microenvironmental suppression