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Updated: Jul 21, 2025
Measurement of T Cell Alloreactivity Using Imaging Flow Cytometry
Published on: April 19, 2017
Multi-tiered approach to detect autoimmune cross-reactivity of therapeutic T cell receptors
Kazusa Ishii1, John S Davies1,2, Andrew L Sinkoe1
1Center for Immuno-Oncology, Center for Cancer Research (CCR), National Cancer Institute (NCI), National Institutes of Health (NIH), Bethesda, MD, USA.
Abstract:
T cell receptor (TCR)-engineered T cell therapy using high-affinity TCRs is a promising treatment modality for cancer. Discovery of high-affinity TCRs especially against self-antigens can require approaches that circumvent central tolerance, which may increase the risk of cross-reactivity. Despite the potential for toxicity, no standardized approach to screen cross-reactivity has been established in the context of preclinical safety evaluation. Here, we describe a practical framework to prospectively detect clinically prohibitive cross-reactivity of therapeutic TCR candidates. Cross-reactivity screening consisted of multifaceted series of assays including assessment of p-MHC tetramer binding, cell line recognition, and reactivity against candidate peptide libraries. Peptide libraries were generated using conventional contact residue motif-guided search, amino acid substitution matrix-based search unguided by motif information, and combinatorial peptide library scan-guided search. We demonstrate the additive nature of a layered approach, which efficiently identifies unsafe cross-reactivity including one undetected by conventional motif-guided search. These findings have important implications for the safe development of TCR-based therapies.
Insights
Developing T cell receptor (TCR)-engineered T cell therapies requires robust cross-reactivity screening. A new framework effectively identifies potentially harmful cross-reactivity for safer cancer treatments.
Area of Science:
- Immunology
- Oncology
- Biotechnology
Background:
- T cell receptor (TCR)-engineered T cell therapy shows promise for cancer treatment.
- High-affinity TCR discovery, especially against self-antigens, may risk T cell receptor cross-reactivity and toxicity.
- No standardized preclinical safety evaluation exists for TCR cross-reactivity.
Purpose of the Study:
- To establish a practical framework for prospectively detecting clinically prohibitive cross-reactivity in therapeutic TCR candidates.
- To ensure the safety and efficacy of TCR-based therapies.
Main Methods:
- Multifaceted assays including p-MHC tetramer binding and cell line recognition.
- Screening against peptide libraries generated through motif-guided, unguided matrix-based, and combinatorial scan-guided searches.
- Utilizing a layered approach to identify cross-reactivity.
Main Results:
- The layered screening approach effectively detected clinically prohibitive cross-reactivity.
- Identified cross-reactivity missed by conventional motif-guided searches.
- Demonstrated the additive benefit of a multifaceted screening strategy.
Conclusions:
- A practical framework for TCR cross-reactivity screening has been established.
- This approach enhances the safety evaluation of TCR-engineered T cell therapies.
- Findings are crucial for the safe development of TCR-based cancer treatments.
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