Abstract 4587: A human hybrid immuno-oncology construct targeting the TWEAK receptor Fn14 and containing the serine protease granzyme B
Authors: Cienfuegos Ana Alvarez de , Cheung Lawrence H. , Mohamedali Khalid A. A. , Winkles Jeffrey A. , Rosenblum Michael G. 
Published: July 2017

Targeting immune effector cells to tumors has recently emerged as a clinically-relevant approach for therapy. The underlying mechanism for immune effector killing involves T or B-cell-mediated delivery of the serine protease granzyme B to the target cells. The cell-surface receptor Fn14 for the TNF-related cytokine TWEAK has emerged as a potentially valuable target for cancer therapy because its expression is low in most normal tissues but significantly elevated in a variety of solid tumor types. The serine protease Granzyme B (GrB) is a highly cytotoxic component of human immune effector cells and induces multiple, intense pro-apoptotic signals when delivered to the cytoplasm of target cells. This protein has been well-studied, operating through multimodal pathways which are both caspase-dependent and caspase-independent. Our laboratory has designed series of new Fn14-targeted fusion constructs containing an engineered GrB payload. The GrB-Fc-IT4 construct is a completely human homodimer (~200kDa) containing an IgG Fc domain for prolonged serum half-life. This construct displays high-level protein production in an HEK293E expression system. It exhibits high affinity and selective cytotoxicity within the nanomolar range (IC50 ranged from 4 to 284 nM) when tested against a panel of 25 Fn14-positive human cancer cell lines. We are currently assessing a panel of 100 annotated NSCLC cell lines to try to define a molecular fingerprint related to cellular sensitivity or resistance to the GrB payload. Pharmacokinetic studies in mice revealed that GrB-Fc-IT4 exhibited a bi-exponential clearance from plasma with a rapid initial clearance (t ½α = 0.36 hours) followed by a prolonged terminal-phase plasma half-life (t 1/2β = 35 hours). Mice bearing MDA-MB-231 orthotopic tumor xenografts were intravenously treated with saline, or GrB-Fc-IT4 construct (QODX5). On average, tumors from saline-treated mice grew about 10-fold over 40 days. In contrast, tumors from mice treated with GrB-FC-IT4 did not grow during this period and remained the same size as at the onset of treatment. Moreover, 3/5 mice treated with GrB-Fc-IT4 showed complete tumor regression lasting beyond day 80 (end of the study). Overall, treatment with GrB-FC-IT4 was well-tolerated by mice (no loss of body weight) and resulted in significant anti-tumor efficacy. Toxicology and bio-distribution studies are ongoing and will be presented. Research conducted, in part, by the Clayton Foundation for Research.

Citation Format: Ana Alvarez de Cienfuegos, Lawrence H. Cheung, Khalid A. A. Mohamedali, Jeffrey A. Winkles, Michael G. Rosenblum. A human hybrid immuno-oncology construct targeting the TWEAK receptor Fn14 and containing the serine protease granzyme B [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4587. doi:10.1158/1538-7445.AM2017-4587