Abstract 2167: A protease-mediated regulatory chimeric antigen receptor (CAR): “Scissors” CAR improves target-specificity through regulation of T-cell activity
Authors: Aoyama Satoru , Yasuda Shunichiro , Watanabe Daisuke , Akiyama Hiroki , Okada Keigo , Umezawa Yoshihiro , Nogami Ayako , Miura Osamu , Kawamata Norihiko 
Published: September 2020

Abstract
[Introduction] CAR-T cell therapy is an attractive methodology in the field of cancer immunotherapy. The anti-CD19 CAR-T cell therapy shows excellent therapeutic effects against refractory B-cell malignancies. However, the CAR-T cells attack not only target-tumor-cells, but also normal tissues expressing the target molecule, for example, normal B-cells with CD19. For the CAR-T cell therapy, such “on-target / off-tumor” effect is one of major obstacles to be overcome. Here, we show the protease-mediated regulatory CAR-T cell system, which improved the target-specificity by recognizing two distinct antigens. We designed two types of CAR: "effector CAR" and "scissors CAR". The “effector CAR” is constituted of a single chain Fv fragment (scFv) targeting an antigen (protein X) on tumor cells, Human Immunodeficiency Virus protease (HIVPR) recognition site, and a functional domain of CD3-ζ. The “scissors CAR” is constituted of a scFv targeting another antigen (protein Y) and HIVPR. The “scissors CAR” induces cleavage of the “effector CAR” leading to inactivation when the CAR-T cells contact with cells expressing both proteins X and Y.
[Material and Methods] For proof of principle, we first constructed the anti-CD19 “mCherry CAR” harboring mCherry fluorescence protein in the cytoplasmic region under the HIVPR recognition site. Also, we constructed the anti-HER2 “scissors CAR”. To analyze the target-cell dependent cleavage of “mCherry CAR”, 293T cells expressing the CARs were co-cultured with target-cells, including Raji (CD19+, HER2-) and engineered SK-BR-3 (CD19+, HER2+). Locations of mCherry were analyzed under the microscopy and by Western blotting. Next, to assess T-cell activation, we established Jurkat cells expressing both the anti-CD19 “effector CAR” and the anti-HER2 “scissors CAR”. These cells were co-cultured with the target-cells described above. T-cell activation was analyzed by both flowcytometric analysis and measurement of IL-2 mRNA expression.
[Results] Transduced anti-CD19 “mCherry CAR” was detected as a membranous protein in 293T cell. Co-cultivation of 293T cell expressing both the anti-CD19 “mCherry CAR” and the anti-HER2 “scissors CAR” with Raji (CD19+, HER2-) did not change the localization of mCherry. However, engineered SK-BR-3 (CD19+, HER2+) induced cleavage of the recognition site and translocation of the mCherry from the membrane to cytoplasm. Furthermore, the anti-HER2 “scissors CAR” attenuated T-cell activation driven by the anti-CD19 “effector CAR” when Jurkat cells expressing both the CARs contacted with the target-cells expressing both CD19 and HER2.
[Discussion] This novel protease-mediated CAR-T system recognized expression pattern of target molecules and regulated T-cell activity. Our CAR-T system would attenuate the adverse effects and contribute to expansion in application of CAR-T cell therapy.
Citation Format: Satoru Aoyama, Shunichiro Yasuda, Daisuke Watanabe, Hiroki Akiyama, Keigo Okada, Yoshihiro Umezawa, Ayako Nogami, Osamu Miura, Norihiko Kawamata. A protease-mediated regulatory chimeric antigen receptor (CAR): “Scissors” CAR improves target-specificity through regulation of T-cell activity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2167.