Abstract
Abstract Anti-VEGF therapy is a key therapeutic in several cancer treatment regimens. However, none of the current therapeutics are directly cytotoxic against tumor cells. VEGF121 is the smallest isoform in the VEGF-A family of cytokines that binds to VEGF receptors R-1 and R-2. Both receptors are over-expressed on several tumor cells and the endothelium of tumor vasculature but not normal vasculature. We hypothesized that delivery of a cytotoxic payload through VEGFR-2 into tumor cells and tumor neovasculature would significantly enhance cancer therapy. Granzyme B is a key effector in immune-mediated cell killing through both caspase-dependent and -independent mechanisms. We previously reported the development of a fusion protein composed of granzyme B (GrB) and VEGF121 (GrB/VEGF121). Here, we report on GrB-Fc-VEGF121, a fusion protein incorporating a human IgG heavy chain Fc fragment for dimerization to increase the molecular weight and improve in vivo circulation and targeting potential. We expressed and purified GrB-Fc-VEGF121 from HEK-293E cells under serum-free conditions 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. We characterized this construct against both human and mouse VEGFR+ cell lines as this ligand cross-reacts to both species. GrB-Fc-VEGF121 showed in vitro cytotoxicity in the nanomolar range against tumor and endothelial cell lines expressing high levels of VEGFR-1 or VEGFR-2, while receptor-negative cells demonstrated IC50 levels in the high micromolar range. GrB-Fc-VEGF121 internalized into VEGFR-2+ tumor and endothelial cells within 2 hours of treatment while untargeted GrB did not internalize, suggesting internalization was receptor-mediated. Treatment of VEGFR2+ cells at the respective IC50 dose resulted in >50% cell death via apoptosis and/or necrosis within 48 h. Ex vivo serum stability studies indicated a gradual protein loss of GrB-Fc-VEGF121 with an overall stability of about 50% over 96 hours. No toxicity was observed in mice treated with a total dose of 475 mg/kg (IP, QOD x 5), indicating that the maximum tolerated dose of GrB-Fc-VEGF121 exceeds this dose level. In vivo efficacy studies in an OVCAR8 tumor xenograft model (20 mg/kg GrB-Fc-VEGF121 x 5) resulted in significant growth inhibition of established tumors compared to vehicle controls. The number of CD31+ blood vessels and Ki-67+ proliferating tumor cells decreased significantly in treated tumors compared to controls. Pharmacokinetic studies are underway and will be reported. Thus, GrB-Fc-VEGF121 significantly reduces tumor growth in vivo with no observed toxicity against normal tissues. 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. Targeting VEGFR on tumor cells and activated vasculature through delivery of granzyme B [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5974.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have