新型 KRAS(G12D) 新抗原特异性 TCR 的鉴定及消除脱靶识别的策略

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.

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).

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 .

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.