TCR Fingerprinting and Off-Target Peptide Identification

Area of Science:

• Immunology
• Oncology
• Biochemistry

Background:

• Adoptive T cell therapy (TCT) utilizing genetically engineered T-cell receptors (TCRs) offers potential for treating melanoma and solid tumors.
• Clinical applications of TCR-engineered T cells have revealed significant concerns regarding off-target toxicities, necessitating methods to predict and mitigate these adverse effects.

Purpose of the Study:

• To develop a robust method for identifying potential off-target peptides recognized by TCRs, thereby enhancing the safety of TCR-based immunotherapies.
• To characterize the recognition specificity of the NY-ESO-1-specific TCR C259 to understand its potential for cross-reactivity with other peptides.

Main Methods:

• A comprehensive replacement scan assay was employed, systematically altering amino acids in the NY-ESO-1 epitope peptide (SLLMWITQC) to generate 133 variants.
• Three in vitro assays (TCR binding, T-cell activation, and target cell killing) were used to evaluate the interaction between the TCR C259 and each peptide variant.
• Position Weight Matrices (PWMs) were generated to define the TCR recognition kernel, which was then used in a novel algorithm to predict off-target peptide recognition across the human proteome.

Main Results:

• The study identified 7 novel off-target peptides, including variants with up to 7 amino acid differences from the original epitope, that strongly activate NY-ESO-1-specific T cells.
• These findings demonstrate that alanine scans are insufficient for predicting the full spectrum of TCR cross-reactivity.
• The developed platform successfully predicted and validated previously unrecognized TCR-binding peptides, highlighting potential safety risks.

Conclusions:

• The developed replacement scan assay and predictive algorithm provide a powerful platform for defining TCR specificity and identifying potential off-target antigens.
• This methodology is crucial for screening TCR candidates for clinical development and for understanding TCR-specific cross-reactive peptide recognition, ultimately improving the safety of adoptive T cell therapies.