Abstract 1203: Targeting activated tumor vasculature with human Granzyme B variants engineered for improved stability and activity
Authors: Mohamedali Khalid A. , Cheung Lawrence H. , Rosenblum Michael G. 
Published: July 2016

Abstract
Angiogenesis is a critical process in numerous diseases, and targeting tumor neovasculature has therapeutic value in controlling tumor progression and metastatic spread. Vascular endothelial growth factor-A (VEGF-A) and its receptors VEGFR-1 and VEGFR-2 have been implicated as central mediators of normal angiogenesis and tumor neovascularization. VEGF121 is a naturally-occurring VEGF-A splice variant that binds to these receptors, which are over-expressed on the endothelium of activated tumor vasculature or neovasculature but not normal vasculature. Granzyme B (GrB) plays a critical role in the body's defense against viral infection and tumor development by initiating the apoptotic cascade through both caspase-dependent and -independent mechanisms. The fusion protein GrB/VEGF121 expressed in mammalian cells and purified to homogeniety was lethal to tumor and endothelial cells in vitro in a manner that correlated closely to total VEGFR-2 expression. IC50 levels were found to be in the nanomolar range. GrB/VEGF121 internalized rapidly into VEGFR-2 expressing cells while the internalization into VEGFR-1 expressing cells was significantly reduced. We engineered GrB variants for improved protein production and for resistance to serpin B9 (PI-9), a natural inhibitor of Granzyme B found at varying levels in cells and plasma, and compared these GrB/VEGF121 constructs directly to unmodified GrB/VEGF121. Variants 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 the active site 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. Enzymatic activity in PBS of the K27E, R28A (“EA-GV”) double mutant was identical to that of GrB/VEGF121. In 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-GV remained over 40% during that time period. This construct retained higher cytotoxic activity after pre-incubation in serum for 4h, compared to GrB/VEGF121. C-GrB/VEGF121, without an unpaired cysteine, resulted in reduced aggregation and a vastly improved yield with otherwise similar serum stability and in vitro cytotoxicity as unmodified GrB/VEGF121. The pharmacokinetic profile for C-GrB/VEGF121 indicated a t1/2α of 6.6 min and t1/2β of 33.3 h. A limited profile for EA-GV indicates a longer terminal-phase half-life for this construct compared to C-GrB/VEGF121. 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 against established tumor xenograft models. Research conducted, in part, by the Clayton Foundation for Research.
Citation Format: Khalid A. Mohamedali, Lawrence H. Cheung, Michael G. Rosenblum. Targeting activated tumor vasculature with human Granzyme B variants engineered for improved stability and activity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1203.