"Armed" CEA CAR-T with a SIRPγ-CD28 chimeric co-receptor to exhibit the enhanced antitumor activity in preclinical study of colorectal cancer.

person Yongchun Zhao Chongqing Precision Biotech Co., Ltd., Chongqing, China info_outline Yongchun Zhao, Xiuxiu Zhu, Junjie Shen, Yanmin Xu, Zhi Yang, Yanan Qi, Jun Chen, Juan Hong, Linling Wang, Lei Qin, Ge Liu, Xia Huang, Jingwen Hu, Wenxu Zhao, Yunfan Chen, Xiaorui Jiang, Liurong Jiao, Nan Chen, Cheng Qian

Authors person Yongchun Zhao Chongqing Precision Biotech Co., Ltd., Chongqing, China info_outline Yongchun Zhao, Xiuxiu Zhu, Junjie Shen, Yanmin Xu, Zhi Yang, Yanan Qi, Jun Chen, Juan Hong, Linling Wang, Lei Qin, Ge Liu, Xia Huang, Jingwen Hu, Wenxu Zhao, Yunfan Chen, Xiaorui Jiang, Liurong Jiao, Nan Chen, Cheng Qian Organizations Chongqing Precision Biotech Co., Ltd., Chongqing, China Abstract Disclosures Research Funding Pharmaceutical/Biotech Company ChongQing Precision Biotech Co., Ltd. Background: Nowadays, CAR-T cell therapy still faces limited therapeutic efficacy for solid tumors. SIRPγ is known as a ligand for a well-known immune checkpoint molecule CD47. It is distinguished from other members of its family in that it has a very short intracytoplasmic tail and is incapable of transducing signals on its own. Of note, the role of SIRPγ still largely unknown to date. Preliminary data indicated SIRPγ plays a key role in T-cell transendothelial migration and promotes antigen-specific T-cell proliferation. We thus designed a novel SIRPγ-CD28 chimeric receptor comprising the extracellular part of SIRPγ, the transmembrane and intracellular domains of CD28 and used as the co-receptor for a CEA-targeting CAR. Here, we report on the activity of this "armed" CAR-T with the SIRPγ-CD28 co-receptor in colorectal tumor (CRC) and its anti-tumor efficacy in mice xenograft model. Methods: In vitro luciferase-based cytotoxicity assay were used by coculturing CAR-T and DLD1-CEA CRC cells to assess the activities of CAR-T w/wo the co-receptor. The incubation supernatants were collected for detecting the release of cytokines. In vivo evaluation of the anti-tumor efficacy of CAR-T with the SIRPγ-CD28 co-receptor versus control T cell, and control CAR-T was performed in NOG mice CRC xenograft model. DLD1-CEA cells expressing luciferase reporter were s.c. implanted and T cells were i.v. injected. Anti-tumor efficacy was assessed by in vivo system. Results: CAR-T with the SIRPγ-CD28 co-receptor exhibited specific cytotoxicity only to CEA + CRC cells, and the efficacies of CAR-T w/wo the co-receptor were comparable (88.6% vs 87.8%, p > 0.05). However, cytokines were significantly higher in the CAR-T group with the co-receptor. The levels of IFNγ, IL2 and TNFα in groups of CAR-T w/wo the co-receptor were 72,793.33 vs 19,013.33 pg/ml (p < 0.05), 1834.67 vs 25.15 pg/ml (p < 0.05) and 79.65 vs 0 pg/ml (p < 0.05), respectively. In mice CRC xenografts model, the tumors were totally eliminated in the CAR-T group with the co-receptor at 21 days and lasted until the end of experiment, while the tumor growth was suppressed in the control CAR-T group at first and out-of-control in the end (Table). Conclusions: These preclinical studies demonstrated the potential of SIRPγ-CD28 co-receptor as a novel T cellular activation signals for CAR-T. The "armed" CAR-T exhibits a fabulous anti-tumor efficacy in CRC and has great potential for applications in other solid tumors. In vivo assay of CAR-T in mice CRC xenograft model. Days after cell infusion Control T a CAR-T a CAR-T with SIRP-CD28 a P value b 0 1.32E9 ± 0.63E9 1.92E9 ± 1.16E9 2.88E9 ± 0.77E9 0.021* 21 3.40E10 ± 1.51E10 2.02E9 ± 3.85E9 9.91E5 ± 6.79E5 0.001* 49 6.85E10 ± 3.11E10 3.76E10 ± 3.74E10 3.83E5 ± 0.28E5 0.041* a Bioluminescent signal (photons/sec) values (mean ± SD, n = 5). b. One-way ANOVA test, significant difference (*).

Pre-clinical