Abstract 2779: Molecular mechanistic and in vivo efficacy studies of Fn14-targeted fusion constructs containing human granzyme B

Abstract

Abstract The past 15 years have seen impressive preclinical and clinical efforts to develop smart therapeutics with enhanced specificity for tumor cells and with much reduced “off-target” or nonspecific toxicity to normal tissues. For the antibody-drug conjugate approach, these constructs are composed of a targeting antigen binding domain, which delivers the cytotoxic domain into the cell. The majority of the cytotoxic payloads used in these constructs are categorized as either microtubule inhibitors or DNA-damaging drugs. We have developed a fusion protein, GrB-Fc-IT4 (MRT-101), that contains a humanized scFv binding domain targeting the cell surface receptor Fn14, an antigen highly expressed in a variety of solid tumors, and containing the human serine protease granzyme B (GrB) as the cytotoxic payload. The construct includes the IgG hinge Fc domain linker for efficient dimerization and an overall high molecular weight thereby designed to provide a prolonged serum half-life (~40 h). This unique format mimics human immune effector cell function and induces target cell death through activation of a variety of well described pro-apoptotic cascade signals. Western blot studies on human TNBC cells (MDA-MD-231) have shown that intact MRT101 is translocated into the cytosol in less than 1 h after exposure and is detectable in the cytosol for at least 8 h. The free GrB component is also detected by Western blot 4 h after treatment and persists for up to 8 h. Both of these agents trigger apoptotic cascades through activation of various caspases and induction of mitochondrial damage. Studies demonstrating cytochrome C release and mitochondrial depolarization are ongoing and will be reported. Incubation with the lysosomotropic agent chloroquine did not alter the IC50 of MRT-101, suggesting that the fusion protein is not appreciably held in the endosomal compartment. In vivo studies have shown that MRT-101 is well tolerated in BALB/c mice after intravenous administration of 5 doses at 20 mg/kg/dose. This dose level showed no evidence of toxicity in any of the major organs such as liver and kidneys. In vivo efficacy studies conducted on NSG-NOD scid mice demonstrated significant tumor growth inhibition of established orthotopic breast tumors (MDA-MB-231), with no tumor growth for up to 30 days after implantation. Treatment of nude mice bearing lung PDX tumors showed a 60% tumor growth inhibition when compared to the vehicle control group. These results, in combination with previous in vitro and in vivo studies, demonstrate that the completely human MRT-101 construct is a selective, highly potent, non-toxic and effective antigen-driven drug with significant potential for the treatment of Fn-14 positive tumors that acts through a new and unique mechanism of action. Research supported by Mirata Biopharma LLC and conducted, in part, by the Clayton Foundation for Research. Citation Format: Ana Alvarez de Cienfuegos, Lawrence A. Cheung, Khalid A. Mohamedali, Landon J. Inge, Timothy Whitsett, Jeffrey A. Winkles, Louis DePalatis, Linda Paradiso, Michael G. Rosenblum. Molecular mechanistic and in vivo efficacy studies of Fn14-targeted fusion constructs containing human granzyme B [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 2779.