Preclinical Strategies to Identify Off-Target Toxicity of High-Affinity TCRs

Area of Science:

• Immunology
• Oncology
• Cell Biology

Background:

• Adoptive T-cell therapy using engineered T-cell receptors (TCRs) offers clinical benefits for cancer treatment.
• A significant concern with TCR-based therapies is the potential for off-target toxicity due to unintended T-cell activation.

Purpose of the Study:

• To investigate the cross-reactive profile of a specific T-cell clone (7B5) targeting the minor histocompatibility antigen HA-2.
• To identify the cause of unexpected off-target recognition observed in T-cell clone 7B5.
• To establish improved methods for validating TCR specificity and detecting potential toxicities before clinical application.

Main Methods:

• Utilized an alanine scanning approach to analyze peptide recognition by the 7B5 T-cell clone.
• Employed a combinatorial peptide library scanning approach to identify novel peptide targets.
• Validated findings by assessing T-cell recognition of CDH13-transduced cell lines and endogenous CDH13-expressing cells.

Main Results:

• The 7B5 T-cell clone effectively recognized HA-2 positive cells but also exhibited off-target recognition of fibroblasts and keratinocytes.
• Alanine scanning indicated a low risk for broad cross-reactivity, suggesting specific peptide interactions.
• A CDH13-derived peptide was identified as an unexpected activator of the 7B5 T-cell clone, confirmed by recognition of CDH13-expressing cells, including proximal tubular epithelial cells.

Conclusions:

• TCR specificity validation requires more than just assessing the primary target antigen.
• Combinatorial peptide library scanning is crucial for uncovering cross-reactivities with unrelated peptides.
• Comprehensive screening, including assessment against diverse cell subsets, is recommended to mitigate off-target toxicity risks in TCR-based therapies.