Identification of novel KRASG12D neoantigen specific TCRs and a strategy to eliminate off-target recognition

Abstract

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.

Keywords: Cross-reactivity; KRAS mutation; Off-target; T cell receptor.

Fig. 1

Discovery and functional characterization of HLA-A*11:01-restricted KRAS^G12D specific TCR clones. a KRAS^G12D-specific T cells were assessed using KRAS^G12D tetramer, showing live⁺ CD3⁺ CD8⁺ T cells. b TCR Jurkat cells were evaluated for TCR expression and KRAS^G12D tetramer binding. c, d TCR Jurkat cells were co-cultured with HLA-A11:01⁺ K562 cells loaded with KRAS^G12D peptide and functional avidity (EC50) determined by CD69 expression. Wild-type KRAS peptide served as negative control. Peptide specificity of TCR1 e and TCR2 f was assessed by X-scan assay. Normalized data are displayed as heatmaps. g TCR1 expression on CD8⁺ T cells was measured; TCR1 T cells were co-cultured with HLA-A11:01⁺ K562 cells and analyzed for CD137 (h) and IFN-γ release (i). j TCR1 T cells were co-cultured with HLA-A*11:01⁺ SW480 cells expressing KRAS^WT or KRAS^G12D and the cytotoxicity were assessed after 24 h. Data are presented as mean ± SD and all panels represent data from two independent experiments. HD healthy donor. N.T. non-transduced

Fig. 2

Peptide specificity profiling of TCR1 T cells for identifying potential off-target peptides. a TCR1 peptide specificity was assessed by X-scan assay and shown as a heatmap. b TCR T cells were co-cultured with HLA-A11:01⁺ K562 cells with Minigene-cluster constructs; CD137 expression was analyzed after 24 h. E7 TCR and P64 TCR served as negative and positive controls, respectively. c Peptides from Minigene-cluster 1 were loaded onto HLA-A11:01⁺ K562 cells and co-cultured with TCR1 T cells; CD137 expression was measured. d TCR1 T cells were co-cultured with HLA-A11:01⁺ K562 cells loaded with SMC1A_29–38 peptide. CD137 expression was analyzed. e TCR1 T cells were co-cultured with HLA-A11:01⁺ COS-7 cells expressing full-length SMC1A; CD137 expression was measured. Data are presented as mean ± SD and all panels represent data from two independent experiments. N.T. non-transduced T cells, P.C. positive control, N.C. negtive control, N.P. Non-peptide

Fig. 3

Diminishing TCR1 recognition of SMC1A29-38 peptide through Q112.1 residue mutation in the CDR-3B region. a Analysis of the TCR and peptide interaction region of the JDI/pHLA complex. The AA sequences of KRAS^G12D and SMC1A29-38 are shown. b Q112.1 residue was mutated into 19 other AAs. These mutant TCR clones were stained with KRAS^G12D Tetramer and SMC1A_29–38 Tetramer. Functional avidity of TCR1^Q112.1A clone and TCR1^Q112.1E clone towards KRAS^G12D peptide c and SMC1A_29–38 peptide d were assessed. All panels represent data from two independent experiments. AA amino acid

Fig. 4

Functional evaluation and peptide specificity profiling of TCR clones derived from TCR1 libraries. a Functional avidity (EC50) of 24 TCR clones was assessed by co-culturing with HLA-A11:01⁺ K562 cells loaded with KRAS^G12D peptide, measuring CD69 expression after 6 h. Dashed line represents TCR1 EC50. b TCR clones were stained with SMC1A29-38 and KRAS^G12D tetramers. c TCR clones were co-cultured with HLA-A11:01⁺ K562 cells loaded with 0.1 µM and 1 µM SMC1A_29–38 peptides, and CD69 expression was analyzed. HLA-A11:01⁺ K562 cells loaded with DMSO as negative control. d TCR activation was assessed with K562 cells under three conditions: unmodified, HLA-A11:01⁺ without peptide, and HLA-A11:01⁺ with KRAS^G12D peptide. e Peptide specificity of TCR clones was profiled using X-scan assay and results are shown as heatmaps. Data are presented as mean ± SD and all panels represent data from two independent experiments. AA amino acid

Fig. 5

Functional characterization and specificity profiling of engineered TCR1a7 T cells. hTCR-KO TCRs T cells were co-cultured with HLA-A11:01⁺ K562 cells loaded with KRAS^G12D peptide. After 24 h, CD137 expression a and IFN-γ release b were analyzed. c hTCR-KO TCR T cells were co-cultured with HLA-A11:01⁺ K562 cells loaded with SMC1A29-38 peptide and CD137 expression was measured. E7 TCR-T cells served as N.C. d–f TCR1a7 T cells were co-cultured with HLA-A11:01⁺ AGS, AsPC-1, or KRAS^G12D SW480 cells, and cytotoxicity was assessed. g CD137 expression on TCR1a7 T cells co-cultured with HLA-A11:01⁺ cancer cell lines was analyzed. h, i Peptide specificity profiling of TCR1a7 was performed by X-scan assay. j TCR1a7 T cells were co-cultured with HLA-A*11:01⁺ K562 cells expressing Minigene-cluster constructs, and CD137 expression was analyzed. E7 TCR-T cells served as N.C. and P64 TCR-T cells as P.C. HLA⁺ K562 cells without constructs were N.T., and those with antigen peptide were P.C. Data are presented as mean ± SD and all panels represent data from two independent experiments. N.T. non-transduced, N.C. negative control, P.C. positive control