Abstract 632: Tumor-targeted fusion constructs containing engineered granzyme B variants with optimized stability and potency
Authors: Mohamedali Khalid A. , Cheung Lawrence H. , Rosenblum Michael G. 
Published: August 2015

Abstract
The serine protease Granzyme B (GrB) is highly cytotoxic generating multiple, intense pro-apoptotic signals when delivered to the cytoplasm of cells and operates through both caspase-dependent and caspase-independent mechanisms. Our laboratory has generated fusion constructs of this payload with VEGF, scFvMEL, BlyS, ML3-9, 4D5 and scFvIT4 targeting molecules thus far. The inhibitor serpin B9 (PI-9) is a 42 kDa protein found at varying levels in cells and plasma binding irreversibly to Granzyme B at the latter's catalytic domain serine (195Serine) resulting in inactive Granzyme B. We undertook modification studies of the GrB primary sequence to improve protein production, serum stability and biological activity. Granzyme B variants were engineered using PCR-based mutagenesis and fused to VEGF121 for direct comparision to the unmodified GrB/VEGF121 fusion protein. Variants designed to prevent binding to PI-9 took into account the impact of the mutation on substrate specificity, and the ability of the mutated GrB to cleave its various cellular substrates. As expected, mutation of 195serine to alanine (S195A) resulted in a complete loss of GrB enzymatic activity as well as in vitro cytotoxicity against VEGFR-1+ and VEGFR-2+ cells. The enzymatic activity of the K27E, R28A (“EA”) double mutant was identical to GrB/VEGF121 when the samples were incubated in PBS. In the presence of 50% human serum, however, the enzymatic activity of GrB/VEGF121 steadily declined to less than 10% over 24 hours while the enzymatic activity of EA-GrB/VEGF121 remained over 40% over that time period. The somewhat similar K27L, R28A (“LA”) double mutant appeared to behave intermediate to the wild-type protein and the EA construct. Both EA and LA GrB mutants retain higher cytotoxic activity after pre-incubation in serum for 4h, compared to unmodified GrB/VEGF121. Mutation of a putative self-cleaving domain and a glycosylation site in GrB appeared to improve enzymatic activity in serum compared to GrB/VEGF121. C210A-GrB/VEGF121, constructed to remove additional thiol sites which may cause aggregation, resulted in a vastly improved yield with otherwise similar serum stability and in vitro cytotoxicity as unmodified GrB/VEGF121. Constructs with modification of the 82PKN84 loop region alone or in combination with the EA mutation had either low activity or had no impact on PI-9 inactivation. Finally, a construct with a caspase-3-cleavable amino acid sequence at the N-terminus showed similar cytotoxic activity to the EA and LA constructs after pre-incubation in serum for 4h. Our studies suggest that these novel GrB variants may significantly improve in vivo half-life. Further studies are underway to determine whether these modifications impact production of soluble material and in vivo therapeutic efficacy. Research conducted, in part, by the Clayton Foundation for Research.
Citation Format: Khalid A. Mohamedali, Lawrence H. Cheung, Michael G. Rosenblum. Tumor-targeted fusion constructs containing engineered granzyme B variants with optimized stability and potency. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 632. doi:10.1158/1538-7445.AM2015-632