Preclinical Screening of T-Cell Therapies

T-cell therapy, has evolved as personalized immunotherapy that engineer patient’s T-cells receptors (TCR) to specifically identify the target cancer cells, autoimmune cells and other chronic infectious viruses. When these T cells bind to the targeted malignant antigen, the downstream signals within them get activated leading to targeted cell killing. These genetically engineered T-cells have enhanced affinity for specific tumor peptide-MHC complexes While this improves therapeutic potency, it also increases the risk of weak TCR cross-reactivity with antigens expressed on healthy tissues, potentially resulting in serious clinical toxicities. In the pre-clinical settings, interspecies differences in proteomic composition reduces the predictive value of in vivo toxicological models for assessing on-target and off-target TCR toxicity.

To mitigate these risks, Sanderson and colleagues at the (Adaptimmune (ADAPY) developed an extensive in vitro pre-clinical testing protocol to evaluate the safety and efficacy of T-cell therapies like CAR-T and specific peptide enhanced affinity receptor (SPEAR) T cells. The strategy involves potency testing of engineered T-cells using 2D and 3D human cell cultures and primary tumor material, in addition to safety testing using human primary cell and cell-line cross reactivity screening and molecular analysis to predict peptides recognized by the affinity-enhanced TCR.

Pre-clinical Screening of T-cell Therapy

This approach follows 3 steps guided by screening and evaluation of T-cell therapy

Step 1: Potency & Efficacy Assessment

The process begins by confirming the anti-tumor activity of engineered T-cell therapy.

• 2D cell line assays are used to measure cytokine release and cytotoxicity.
• Antigen-driven proliferation assays assess T-cell expansion upon target recognition.
• Primary tumor samples are tested to confirm activity against clinically relevant material.
• In vivo efficacy studies evaluate tumor control in xenograft models.
• 3D cell-line models provide a more physiologically relevant assessment of tumor killing.

Only T-cell therapy that show strong and consistent efficacy progress further.

Step 2: Human Cell Testing for Safety

At this step, on-target and off-target toxicities are assessed using human-relevant systems.

• A standard human cell screen covering more than 100 normal cell types is performed to detect unintended reactivity.
• Fresh human tissues, organotypic cultures, and iPSC-derived models are used to improve biological relevance.
• Alloreactivity assays test whether the TCR reacts with alternative HLA alleles, identifying patient populations that must be excluded.

This step filters out T-cell therapy with unacceptable safety risks.

Step 3: Molecular Analysis for Specificity

Molecular studies are then used to understand and predict peptide cross-reactivity of TCRs.

• An X-scan systematically mutates each position of the target peptide to define tolerated substitutions.
• In silico searches use the resulting motif to scan the human proteome for potential mimetic peptides.
• Homolog identification examines related proteins, particularly within antigen families.
• Peptide screening tests candidate peptides for functional T-cell activation.
• Overexpression studies confirm whether candidate off-target peptides are naturally processed and presented.

This step provides mechanistic confidence that there is no clinically relevant off-target recognition is likely.

Step 4: Selection of a Clinical Candidate

Only T-cell therapy that demonstrate:

• Robust anti-tumor efficacy
• Minimal off-target and alloreactive responses
• No functionally relevant molecular cross-reactivity

are advanced to clinical trial as a potent anti-cancer candidate.