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
View: Text | PDF
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

×

Figure 7

IFN-γ priming enhanced the recognition and killing of human pancreatic cancer organoids by TCR-001–transduced T cells.

Options: View larger image (or click on image) Download as PowerPoint
IFN-γ priming enhanced the recognition and killing of human pancreatic c...
(A–C) TCR-001–transduced allogeneic T cells were cocultured with human pancreatic cancer organoid cells naturally expressing KRAS G12 mutations or HLA-A*11:01. In some groups, 10 μg/mL anti–human PD-1 antibody (Beigene) or human IgG (Invitrogen) was added. 4-1BB/OX-40 upregulation (A), CD107a upregulation (B), and IFN-γ/TNF-α secretion (C) were assayed by flow cytometry and intracellular cytokine staining. (D–F) TCR-001–transduced allogeneic T cells were cocultured with human pancreatic cancer organoid cells naturally expressing KRAS G12 mutations and HLA-A*11:01. In some groups, organoid cells were primed with 100 ng/mL IFN-α2b or 125 ng/ml IFN-γ for 24 hours before the coculture. 4-1BB/OX-40 upregulation (D), CD107a upregulation (E), and IFN-γ/TNF-α secretion (F) were assayed by flow cytometry and intracellular cytokine staining. (G) TCR-001–transduced allogeneic T cells or mock T cells were cocultured with human pancreatic cancer organoid cells naturally expressing the KRAS G12V mutation and HLA-A*11:01 at an effector/target ratio of 10:1. Human pancreatic cancer organoid cells were labeled with CellTrace Yellow before the coculture. In some groups, PDAC-59 organoid cells were primed with 125 ng/mL IFN-γ before the coculture. After 24 hours of coculture, tumor cell apoptosis was detected by green fluorescent caspase 3/7 probe. Representative images with caspase 3/7 green fluorescence and merged figures between caspase 3/7 green fluorescence and corresponding bright-field and CellTrace Yellow are shown. Scale bar: 100 μm. (H and I) Apoptosis in Yellow+ organoid cells was assayed by green fluorescent caspase 3/7 probe using flow cytometry. Representative overlay plots (H) and summarized data (I) are shown. Numbers in overlay plots indicate the percentage of caspase 3/7+ cells among gated Yellow+ organoid cells. Error bars represent SEM of at least 3 biological replicates. Dots indicate biological replicates. *P < 0.05; **P < 0.01 vs. “T alone” group or indicated group. Statistical differences were determined with 1-way ANOVA followed by Tukey’s multiple-comparison test (A–F and I).