Adoptive T cell immunotherapy Tumor Infiltrating Lymphocytes (TILs) and Engineered Lymphocytes

what cells are depleted with lymphodepletion? Why is it advisable to prepare before TIL

1.Eliminates Regulatory T cells

2.Eliminates cellular sinks (= normal cells that compete for

T cell supporting cytokines IL-2, IL-7,IL-15)

The infused T cells need an environment in which they can expand and function, rather than immediately competing with the patient's existing immune cells.

What are the challenges for TIL therapy

Increase success rate ( 1/3 of the cultures fail)

Reduce time for generation ( ~ 6 weeks for generation of TILs)

Improve in vivo survival and persistence

Abrogate immune escape in the TME

Difference between TIL and TCR engineered T cells

TIL therapy expands tumour-reactive cells that already exist,

TCR engineering gives T cells a chosen receptor specificity for a specific antigen

Principle/Workflow behind TCR engineered T cells

• Obtain tumour tissue or peripheral blood T cells.

• Identify and isolate a tumour-reactive TCR.

• Sequence the TCR genes.

• Introduce the genes into recipient T cells using a vector.

• Expand the engineered cells and infuse them into the patient.

What is TCR mispairing?

Introduced TCR chains accidentally pair with the patient's original TCR chains, potentially creating receptors with unintended specificity.

How to produce tumor-specific T cells faster? What is the disadvantage of this?

TCR Gene Transfer.

TCR mispairing

Challenges for TCR gene Transfer Therapy

• expensive.

• TCR mispairing:

• Poor infiltration and persistence: engineered cells may fail to reach or survive in tumours.

• Adverse effects: cytokine release syndrome (CRS) and autoimmune reactions are potential concerns.

What is the key recognition difference between TCR and CAR-T cells?

TCRs recognise peptides presented by HLA; CARs generally bind accessible antigens directly on the cell surface (EXTRACELLULARLY ACCESSIBLE), independent of HLA BUT STILL ANTIGEN DEPENDENT

What are the four generation of CAR T cells

First = Antigen recognition and activation; second = one costimulatory domain; third = two; fourth = additional functional equipment.

CAR-T versus TCR-engineered T cells

What causes on-target, off-tumour toxicity?

Occurs when a CAR-T cell correctly recognises its intended antigen, but that antigen is also present on healthy tissue.

CEA targeting: damage to normal intestinal tissue can cause severe colitis.

How can lowering CAR affinity reduce off-tumour toxicity?

a lower-affinity CAR may still recognise the tumour while responding less strongly to healthy cells with low CD38 expression.

What are the four logic gates used in engineered T cells, and how do they work? / Boolean logic gating / How to make CAR cells better functionally

1. OR gate (A OR B): The T cell activates when it recognises either antigen A or antigen B. This broadens tumour recognition and helps target cancer cells that may express only one of the two antigens.

2. AND gate (A AND B): The T cell requires recognition of both antigen A and antigen B to become fully activated. This improves tumour specificity by reducing activation against cells expressing only one antigen.

3. AND NOT gate (A AND NOT B): The T cell activates when it recognises tumour-associated antigen A, but an inhibitory receptor suppresses activation when it recognises antigen B, which may be present on healthy cells. This helps reduce damage to healthy tissue.

4. IF–THEN gate: Recognition of antigen A triggers a genetic programme that enables the T cell to respond to antigen B. This creates a conditional response, allowing the T cell to attack cells expressing B only after it has first detected A.

Tumour recognition is necessary but not sufficient; CAR-T cells must also overcome the suppressive environment. True or false

True

Why should we engineer donor cells to make off the shelf car t cells

Donor-derived cells create two important immunological problems:

1. Graft-versus-host disease: donor TCRs may recognise recipient tissues as foreign.

2. Rejection: the recipient's immune system may recognise donor cells as foreign .

How do you overcome the barriers for off the shelf CAR cells

• CRISPR/Cas9 gene editing: Knock out endogenous TCR genes to reduce GvHD; modify HLA-related pathways to reduce recipient-mediated rejection, while considering NK-cell recognition.

• CAR-NK therapy: Use CAR-engineered NK cells, which have a lower risk of conventional TCR-mediated GvHD and can kill tumour cells through both CAR-dependent and natural cytotoxic mechanisms.