Identification and validation of a T cell receptor targeting KRAS G12V in HLA-A*11:01 pancreatic cancer patients
Xiongfei Xu, Shiwei Guo, Haihui Gu, Zhanshan Cha, Xiaohan Shi, Xiaoyi Yin, Huan Wang, Suizhi Gao, Bo Li, Lingyu Zhu, Wei Jing, Kailian Zheng, Zhuo Shao, Peng Cheng, Chunhong Zheng, Yi-Ping Shih, Yunguang Li, Baohua Qian, Dong Gao, Eric Tran, Gang Jin
Xiongfei Xu, Shiwei Guo, Haihui Gu, Zhanshan Cha, Xiaohan Shi, Xiaoyi Yin, Huan Wang, Suizhi Gao, Bo Li, Lingyu Zhu, Wei Jing, Kailian Zheng, Zhuo Shao, Peng Cheng, Chunhong Zheng, Yi-Ping Shih, Yunguang Li, Baohua Qian, Dong Gao, Eric Tran, Gang Jin
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Research Article Immunology Oncology

Identification and validation of a T cell receptor targeting KRAS G12V in HLA-A*11:01 pancreatic cancer patients

Abstract

T cells targeting a KRAS mutation can induce durable tumor regression in some patients with metastatic epithelial cancer. It is unknown whether T cells targeting mutant KRAS that are capable of killing tumor cells can be identified from peripheral blood of patients with pancreatic cancer. We developed an in vitro stimulation approach and identified HLA-A*11:01–restricted KRAS G12V–reactive CD8+ T cells and HLA-DRB1*15:01–restricted KRAS G12V–reactive CD4+ T cells from peripheral blood of 2 out of 6 HLA-A*11:01–positive patients with pancreatic cancer whose tumors expressed KRAS G12V. The HLA-A*11:01–restricted KRAS G12V–reactive T cell receptor (TCR) was isolated and validated to specifically recognize the KRAS G12V8–16 neoepitope. While T cells engineered to express this TCR specifically recognized all 5 tested human HLA-A*11:01+ and KRAS G12V+ pancreatic cancer organoids, the recognition was often modest, and tumor cell killing was observed in only 2 out of 5 organoids. IFN-γ priming of the organoids enhanced the recognition and killing by the TCR-engineered T cells. The TCR-engineered T cells could significantly slow the growth of an established organoid-derived xenograft in immunodeficient mice. Our data suggest that this TCR has potential for use in TCR-gene therapy, but additional strategies that enhance tumor recognition by the TCR-engineered T cells likely will be required to increase clinical activity.

Authors

Xiongfei Xu, Shiwei Guo, Haihui Gu, Zhanshan Cha, Xiaohan Shi, Xiaoyi Yin, Huan Wang, Suizhi Gao, Bo Li, Lingyu Zhu, Wei Jing, Kailian Zheng, Zhuo Shao, Peng Cheng, Chunhong Zheng, Yi-Ping Shih, Yunguang Li, Baohua Qian, Dong Gao, Eric Tran, Gang Jin

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Figure 4

TCR-001–transduced T cells specifically recognized endogenously processed and presented antigen and human pancreatic cancer cell lines.

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TCR-001–transduced T cells specifically recognized endogenously processe...
(A) TCR-001–transduced allogeneic T cells were cocultured overnight with Cos7 cells transiently transfected with plasmids encoding HLA-A*11:01 and the KRAS G12V or WT full-length gene. 4-1BB/OX-40 upregulation was assayed by flow cytometry. Error bars represent SEM of 4 biological replicates. Dots indicate biological replicates. **P < 0.01. (B) KRAS mutation and HLA profiles of human pancreatic cancer cell lines. Information is from the TRON Cell Line portal (https://github.com/TRON-Bioinformatics/TCLP). (C) TCR-001–transduced allogeneic T cells were cocultured overnight with human pancreatic cancer cell lines naturally expressing KRAS G12 mutations with and without HLA-A*11:01 transfection. 4-1BB/OX-40 upregulation was assayed by flow cytometry. Anti–human HLA-ABC antibody (20 μg/mL) was used in 1 group. Error bars represent SEM of at least 3 biological replicates. Dots indicate biological replicates. **P < 0.01. (D) TCR-001–transduced allogeneic T cells were cocultured overnight with human pancreatic cancer cell lines with and without HLA-A*11:01 transfection pulsed with 1 μg/mL KRAS WT or G12V 9-mer peptides. 4-1BB/OX-40 upregulation was assayed by flow cytometry. Error bars represent SEM of at least 3 biological replicates. Dots indicate biological replicates. **P < 0.01. (E and F) TCR-001–transduced allogeneic T cells were cocultured with naturally HLA-A*11:01–expressing Panc-1 cells pulsed with various concentrations of KRAS WT or G12V 9-mer peptides overnight or for 5 hours. 4-1BB/OX-40 upregulation was assayed by flow cytometry. CD107a upregulation and IFN-γ/TNF-α secretion were assayed by intracellular cytokine staining. The results of mTCRβ+ CD8+ T cells (D) and mTCRβ+ CD4+ T cells (E) are shown. Statistical differences were determined with 1-way ANOVA followed by Tukey’s multiple-comparison test (A and D) or mixed-effects analysis with Tukey’s multiple-comparison test (C).