Abstract 6316: In vitro and in vivo studies of human granzyme B fusion constructs targeting mesothelin

Abstract

Abstract Mesothelin is highly expressed on tumor cells and promotes tumor growth and drug resistance. Pancreatic, ovarian, mesothelioma, and lung cancers highly express mesothelin and are cancers of unmet needs with ≤16% relative 5-year survival using standard of care. When delivered to tumor cells by immune effector cells, granzyme B (GrB) is a highly cytotoxic serine protease which induces apoptosis through both caspase-dependent and -independent mechanisms. We developed a fusion protein (GrB-Fc-SD1) composed of human GrB and containing SD1, a novel human single-domain antibody that uniquely targets Region III of the glycoprotein, mesothelin. The SD1 and GrB domains are connected by a human IgG heavy chain fragment which enables dimerization. Thus, the increase in total construct molecular weight leads to an increased in vivo circulation time and anti-tumor efficacy. GrB-Fc-SD1 was transiently expressed in HEK-293E cells under serum-free conditions and purified to homogeneity to a final yield of approximately 5 mg/L. Anti-GrB western blot analysis confirmed the expression of GrB-Fc-SD1 exclusively as a dimer. Comparison of GrB-Fc-SD1 with its expected molecular weight suggests significant glycosylation when produced in HEK-293E cells. The enzymatic activity of granzyme B in GrB-Fc-SD1 was comparable to that of commercially available human granzyme B. In vitro cytotoxicity against a limited panel of lung, pancreatic and ovarian cancer cell lines expressing surface mesothelin indicate cytotoxicity in the nanomolar range, while cytotoxicity against mesothelin-negative cells was at micromolar levels. GrB-Fc-SD1 bound specifically to mesothelin extra-cellular domain compared to a non-specific control, as determined by ELISA. Biacore analysis confirmed binding with a dissociation constant between 10−7 to 10−8 M. These data warrant continued investigation of the protein as a therapeutic candidate. Accordingly, ex vivo serum stability studies are underway. In addition, screening of an expanded panel of tumor cell lines for in vitro cytotoxicity is currently in progress and will be reported. In vivo efficacy studies against nude mice bearing Capan-2 xenograft models are currently underway and will also be reported. Research supported by H2Biologics. Research conducted, in part, by the Clayton Foundation for Research. Citation Format: Khalid A. Mohamedali, Lawrence H. Cheung, Louis R. DePalatis, Claire Thuning-Roberson, Michael G. Rosenblum. In vitro and in vivo studies of human granzyme B fusion constructs targeting mesothelin [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 6316.