Abstract 5194: Development of a cytotoxic fusion protein targeting VEGF receptors with improved cytotoxicity, stability and pharmacokinetics
Authors: Mohamedali Khalid A. , Cheung Lawrence H. , Alvarez-Cienfuegos Ana A. , Rosenblum Michael G. 
Published: September 2020

Abstract
The serine protease granzyme B (GrB), a key effector in immune mediated cell killing is capable of inducing apoptosis through both caspase-dependent and caspase-independent multiple-cascade mechanisms. VEGF121 is a naturally-occurring isoform of the VEGF-A family and binds to VEGF receptors R-1 and R-2, which are over-expressed on tumor cells as well as the endothelium of tumor vasculature but not normal vasculature. We have previously reported the expression of a fusion protein composed of Granzyme B and VEGF121 (GrB/VEGF121) and its subsequent in vitro and in vivo characterization. We hypothesized that incorporation of an IgG heavy chain Fc-fragment, resulting in a relatively higher molecular weight (133 kDa vs 80 kDa), would improve in vivo circulation and efficacy. Accordingly, the VEGF inter-chain dimerization domains were mutated so that dimerization occurred only through the Fc domain. Transient expression of GrB-Fc-VEGF121 in HEK-293E cells under serum-free conditions, followed by purification by immobilized metal affinity chromatography, resulted in yields in the range of 20-25 mg/L. Comparison of GrB-Fc-VEGF121 with its expected molecular weight suggests significant glycosylation when produced in HEK293E cells. The enzymatic activity of GrB in GrB-Fc-VEGF121 was comparable to that of commercially available human GrB. Cytotoxicity studies against a panel of tumor and endothelial cells indicated improved cytotoxicity against cell lines expressing high levels of VEGFR-1 or VEGFR-2, unlike GrB/VEGF121, which targeted VEGFR-2+ cells more efficiently. Control (receptor-negative) cells demonstrated IC50 levels in the high micromolar range. Comparison of ex vivo stability of GrB-Fc-VEGF121 to GrB/VEGF121 over 96 hours indicated GrB/VEGF121 signal reduced by approximately 50% over the first 24 hours of incubation, but minimal loss of protein was observed thereafter for the duration of the study. On the other hand, the protein loss of GrB-Fc-VEGF121 was more gradual, while the overall loss of protein over 96 hours was similar to that of GrB/VEGF121. Thus, GrB-Fc-VEGF121 is very stable in mouse serum and further pharmacokinetic analysis is warranted to determine its stability in vivo. In vivo pharmacokinetics and efficacy against various tumor models are currently ongoing and will be presented. In summary, the GrB-Fc-VEGF121 produced in HEK293E cells is active and has significant potential as a targeted therapeutic. Research conducted, in part, by the Clayton Foundation for Research.
Citation Format: Khalid A. Mohamedali, Lawrence H. Cheung, Ana A. Alvarez-Cienfuegos, Michael G. Rosenblum. Development of a cytotoxic fusion protein targeting VEGF receptors with improved cytotoxicity, stability and pharmacokinetics [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5194.