Abstract 6315: Human granzyme B fusions targeting VEGFR on tumor cells and activated vasculature
Authors: Mohamedali Khalid A. , Cheung Lawrence H. , Alvarez-Cienfuegos Ana , Hittelman Walter N. , Rosenblum Michael G. 
Published: June 2023

Abstract
VEGF121 is a naturally-occurring splice variant that binds to VEGF receptors R-1 and R-2, which are over-expressed on the endothelium of tumor vasculature but not normal vasculature, as well as several tumor cells. While anti-VEGF therapy is a key therapeutic in several cancer treatment regimens, none of the current therapeutics (bevacizumab, VEGF-trap, tyrosine kinase inhibitors) are directly cytotoxic against tumor cells. The serine protease Granzyme B (GrB) is a key effector in immune-mediated cell killing by inducing apoptosis through both caspase-dependent and -independent mechanisms. We previously reported the development and characterization of a fusion protein composed of GrB and VEGF121 (GrB/VEGF121). Here, we report on GrB-Fc-VEGF121, a fusion protein that incorporates a human IgG heavy chain Fc fragment for dimerization which increases the molecular weight and improves in vivo circulation and targeting potential. GrB-Fc-VEGF121 was expressed in HEK-293E cells under serum-free conditions and purified from the conditioned medium with a final yield of approximately 40 mg/L. The enzymatic activity of GrB in GrB-Fc-VEGF121 was comparable to that of commercially available human GrB. In vitro studies of GrB-Fc-VEGF121 showed cytotoxicity in the nanomolar range against tumor and endothelial cell lines expressing high levels of VEGFR-1 or VEGFR-2. Receptor-negative cells demonstrated IC50 levels in the high micromolar range. Immunofluorescence and confocal microscopy demonstrated that GrB-Fc-VEGF121 readily internalized into VEGFR-2+ tumor cells and endothelial cells within 2 hours of treatment while untargeted GrB did not internalize, suggesting internalization was receptor-mediated. Treatment of VEGFR-2+ endothelial and tumor cells at the IC50 dose resulted in &gt;50% cell death via apoptosis and/or necrosis within 48 h. Ex vivo serum stability of GrB-Fc-VEGF121 indicated a gradual protein loss of GrB-Fc-VEGF121, with an overall stability of about 50% over 96 hours. No toxicity was observed in BALB/c mice treated with a total 475 mg/kg given IP every other day over 5 doses, indicating that the maximum tolerated dose of GrB-Fc-VEGF121 exceeds this dose level. In vivo efficacy studies in an OVCAR8 tumor xenograft model with GrB-Fc-VEGF121 (100 mg/kg over 5 doses) resulted in significant growth inhibition of established tumors compared to vehicle controls. We observed a significant decrease in the number of CD31+ blood vessels and Ki-67+ proliferating tumor cells in treated tumors compared to controls. Thus, 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. This tumor- and vascular-targeting 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. Human granzyme B fusions targeting VEGFR on tumor cells and activated vasculature [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 6315.