Abstract 907: Evaluation of the anti-GPC1 scFv-Fc fusion protein as a detection tool of GPC1 expression in human cancers

Abstract Background: We have developed a glypican-1 (GPC1) targeted CAR-T cell with strong anti-tumor effects against solid tumors in vivo (Kato et al 2020 eLife). In general, an antibody is used for detection of target protein expression in the tumor to identify appropriate targets for the CAR-T cell therapy in solid tumors. However, the local conjunction between CAR-T cells and the target is not exactly the same in the antibody and the target because CAR-T cell recognizes the target by single-chain variable fragment (scFv). In this study, we evaluated the anti-GPC1 scFv-Fc fusion protein as a detection tool of GPC1 expression in human cancer cell lines. Methods: The sequences encoding the anti-GPC1 scFv are based on the sequence of anti-GPC1 mAb (clone: 1-12) that recognizes human and mouse GPC1 (Harada et al., 2017). The sequences of anti GPC-1 scFv were cloned into pCAG-Neo mIgG2a-Fc plasmid for the generation of anti GPC1_scFv-mIgG2a_Fc fusion proteins. The plasmids were transfected into 293 T cells and the fusion proteins were purified from their culture supernatants using Antibody TCS Purification Kit. LK2-GPC1 (a GPC1 overexpressing lung carcinoma cell line), TE8 (an esophageal carcinoma cell line with endogenous GPC1) and LK2-mock (a GPC1-negative lung carcinoma cell line) were used for the evaluation of the anti-GPC1 scFv-Fc fusion protein. Results: In FACS assays, fluorescence microscopy analysis and immunohistochemistry staining, GPC1 expression was successfully detected by both anti-GPC1 scFv of VL-VH and VH-VL in LK2-GPC1 and TE-8, and not detected in LK2-mock. Localization of GPC1 expression on the cell membrane was confirmed by fluorescence microscopy. Perspective: Further comparative evaluation between anti-GPC1 antibody and anti-GPC1 scFv-Fc fusion proteins are needed especially in human tumor tissues. We plan to evaluate a relationship between GPC1 positivity in various human tumor tissues detected by anti-GPC1 scFv-Fc fusion protein and CAR-T cell cytotoxicity against the tissue. Citation Format: Takeshi Sawada, Tomonori Yaguchi, Yuko Sakai, Chinatsu Suzuki, Kenji Morii, Masahiko Aoki, Masaru Fukahori, Satoshi Serada, Tetsuji Naka, Jun K. Yamashita, Yutaka Kawakami, Takako E. Nakajima. Evaluation of the anti-GPC1 scFv-Fc fusion protein as a detection tool of GPC1 expression in human cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 907.