Improving TCR affinity on 293T cells.

Area of Science:

• Immunology
• Molecular Biology
• Biotechnology

Background:

• T-cell receptors (TCRs) are crucial for adaptive immunity, recognizing peptide-MHC complexes.
• Improving TCR affinity is essential for developing more effective immunotherapies.
• Current methods for TCR engineering can be complex and time-consuming.

Purpose of the Study:

• To develop and validate an efficient method for enhancing TCR affinity.
• To identify high-affinity mutants of an HLA-A*24:02-restricted, hTERT-specific TCR.
• To establish a functional assay for evaluating affinity-enhanced TCRs.

Main Methods:

• CDR-directed saturation mutagenesis of TCR cDNA.
• Transient TCR display on CD3-expressing HEK293T cells.
• Flow cytometry sorting using HLA-tetramer binding.
• Development of a CD8-independent T cell line (CD8I-J2) for functional assays.

Main Results:

• Identified two high-affinity mutant TCRs (2A7A and 2D162) with mutations in CDR1 and CDR2 of TCRβ.
• Mutant TCRs exhibited enhanced binding to HLA-A24/hTERT tetramers compared to wild-type.
• Affinity-enhanced TCRs recognized the hTERT peptide at lower concentrations in the CD8I-J2 cell line.
• No cross-reactivity with irrelevant peptides was observed for the mutant TCRs.

Conclusions:

• The developed method is efficient for improving TCR affinity.
• The identified mutant TCRs show promise for targeting hTERT-expressing cancers.
• This methodology can be applied to engineer other TCRs for therapeutic purposes.