Identification of novel KRAS(G12D) neoantigen specific TCRs and a strategy to eliminate off-target recognition.

BACKGROUND: T cell receptor (TCR)-engineered T cells targeting neoantigens originated from mutations in KRAS gene have demonstrated promising outcomes in clinical trials against solid tumors. However, the challenge lies in developing tumor-specific TCRs that avoid cross-reactivity with self-antigens to minimize the possibility of severe clinical toxicities. Current research efforts have been put towards strategies to eliminate TCR off-target recognition. METHODS: Naive T cell repertoire was used for screening KRAS(G12D)-reactive TCRs. Specific TCRs were subsequently identified and their functionality was assessed using TCR Jurkat cells and TCR T cells. Peptide specificity was evaluated using the X-scan assay. To enhance TCR specificity for KRAS(G12D) and reduce their reactivity to self-peptide SMC1A(29-38), mammalian TCR display libraries were employed for the design of modification in the complementarity-determining region (CDR). RESULTS: HLA-A*11:01-restricted TCRs targeting the KRAS(G12D) epitope were isolated, and TCR1 was characterized with superior functional avidity and specificity. Alongside a robust recognition of endogenous KRAS(G12D) epitope, this TCR displayed cross-reactivity with the SMC1A(29-38) epitope. With an approach utilizing structural-guided mutations in the CDR-1A region of TCR1, we obtained an engineered TCR variant (TCR1a7). Functional characterization of TCR1a7 showed that this TCR not only exhibited enhanced specificity towards KRAS(G12D), but also demonstrated successful elimination of the off-target recognition of SMC1A(29-38). CONCLUSIONS: TCRs targeting the KRAS(G12D) peptide could be isolated from naive T cell repertoires. Integrating the TCR-peptide-HLA complex structure with a mammalian TCR library system could serve as a functional strategy to reduce potential TCR cross-reactivity with self-antigens, such as SMC1A(29-38). Our findings evidenced an operable method to enhance TCR peptide specificity, while maintaining advanced functional avidity and potent anti-tumor activity.