Abstract 2780: Engineering human fusion constructs for targeted delivery of granzyme B to CD33 and CEA antigens

Abstract

Abstract Our laboratory has developed a unique platform for the delivery of the highly pro-apoptotic serine protease granzyme B (GrB). The construct contains enzymatically active GrB and therefore does not require release from the targeting carrier by a fragile linker in contrast to conventional antibody-drug conjugates (ADCs). We employed two scFvs targeting the CD33 antigen found on AML and the juxtamembrane epitope of CEA found on numerous solid tumors. These human scFvs (designated HU33 and CEA respectively) were fused to human GrB through an engineered IgG heavy-chain fragment (hinge to Fc and designated Fc) which contains a dimerization domain. The GrB-Fc-HU33 and GrB-Fc-CEA constructs were cloned into the pSECTag vector containing an upstream 5kDa purification tag with an enterokinase (EK) cleavage site for facile removal. The molecules were expressed in transiently-transfected HEK-293E suspension cells and grown at 37°C for 3 days in 5% CO2. The molecules were isolated from culture media and purified by IMAC followed by EK cleavage and SP chromatography to remove the free tag. The yields were 6-10 mg/L for both molecules. Specific binding of each molecule to the antigen target is undergoing Biacore and/or ELISA assessment. The cytotoxicity of the GrB-Fc-HU33 against HL-60 cells showed a specific cytotoxic effect with an IC50 of 120 nM. Tests of this agent against a panel of cells expressing various levels of CD33 are ongoing. The cytotoxic effects of the GrB-Fc-CEA were assessed against HT29, HT1080-CEA and A431 cells and demonstrated IC50 values of 80, 110 and 95 nM respectively. Based on IC50 values, these data demonstrate that the fusion construct targeting CD33 is efficiently internalized and effectively delivers active serine protease to the cytosol even at relatively low antigen expression levels. In addition, the juxtamembrane epitope of the CEA antibody also allows efficient internalization for delivery of GrB to the cytosol. Immunofluorescence and confocal studies to examine the time course of internalization and the pro-apoptotic events triggered by these agents are under examination and will be presented. Research conducted, in part, by the Clayton Foundation for Research. Citation Format: Lawrence H. Cheung, Khalid A. Mohamedali, Walter N. Hittelman, Michael G. Rosenblum. Engineering human fusion constructs for targeted delivery of granzyme B to CD33 and CEA antigens [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2780.