Abstract 337: Development of an immune-oncology agent containing granzyme B to target VEGFR+ ovarian cancer cells and activated tumor vasculature

Abstract

Abstract Granzyme B is a key effector in immune-mediated cell killing by inducing apoptosis through both caspase-dependent and -independent mechanisms. We developed a fusion protein (GrB-Fc-VEGF121) composed of granzyme B, VEGF121 (a naturally-occurring isoform of the VEGF-A family that binds to VEGF receptors R-1 and R-2), and a human IgG heavy chain fragment (for dimerization and to augment in vivo circulation and anti-tumor efficacy). GrB-Fc-VEGF121 was transiently expressed in HEK-293E cells under serum-free conditions purified with a final yield of approximately 40 mg/L. Comparison of GrB-Fc-VEGF121 with its expected molecular weight suggests significant glycosylation when produced in HEK-293E cells. The enzymatic activity of granzyme B in GrB-Fc-VEGF121 was comparable to that of commercially available human granzyme B. GrB-Fc-VEGF121 readily internalized into VEGFR-2+ cells within 2 hours of treatment while untargeted granzyme B was not internalized, demonstrating that internalization was receptor-mediated. Cytotoxicity studies against a panel of tumor and endothelial cells indicated cytotoxicity in the nanomolar range against cell lines expressing high levels of VEGFR-1 or VEGFR-2 while control (receptor-negative) cells demonstrated IC50 levels in the high micromolar range. Treatment of VEGFR-2+ endothelial and tumor cells with GrB-Fc-VEGF121 resulted in robust cell death via apoptosis and/or necrosis. Ex vivo serum stability of GrB-Fc-VEGF121 indicated a gradual protein loss of GrB-Fc-VEGF121, with an overall loss of about 50% over 96 hours. The maximum tolerated dose of GrB-Fc-VEGF121 was not reached when BALB/c mice were treated with a total 475 mg/kg given every other day over 5 doses. Treatment of mice bearing OVCAR8 tumor xenografts with GrB-Fc-VEGF121 (100 mg/kg over 5 doses) resulted in significant growth inhibition of established tumors compared to vehicle controls. Treated tumors showed a significant decrease in the number of CD31+ blood vessels and Ki-67+ proliferating tumor cells compared to controls, when assessed at the end of the study. These studies clearly indicate that GrB-Fc-VEGF121 has significant anti-tumor effects in vivo with virtually no toxicity against normal tissues at doses 4-5 times higher than therapeutic levels. The tumor- and vascular-targeted agent appears to have significant potential as a new class of targeted therapeutic agents with a unique mechanism of action. Research conducted, in part, by the Clayton Foundation for Research. Citation Format: Khalid A. Mohamedali, Lawrence H. Cheung, Ana Alvarez-Cienfuegos, Walter N. Hittelman, Michael G. Rosenblum. Development of an immune-oncology agent containing granzyme B to target VEGFR+ ovarian cancer cells and activated tumor vasculature [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 337.