Abstract 5216: Optimizing antitumor efficacy of granzyme B human fusion constructs targeting the Fn14 antigen in solid tumors. Pre IND pharmacokinetics, schedule, dose optimization and MTD studies
Authors: De Cienfuegos Ana Alvarez , Cheung Lawrence H. , Mohamedali Khalid A. , Gagea Mihai , Rosenblum Michael G. 
Published: June 2022

Abstract
We previously demonstrated that a human fusion protein construct targeting the Fn14 receptor and containing the cytotoxic granzyme B (GrB) payload (GrB-Fc-IT4) displays impressive in vitro and in vivo cytotoxic effects. Comprehensive mechanism of action studies show that GrB-Fc-IT4 effectively induces cell death in vitro against a broad selection of tumor types with high specificity. The intracellular pathway includes irreversible activation of the caspase cascade and independent mitochondrial depolarization leading to an intense apoptotic cellular damage. This activation occurs quickly and efficiently. Pharmacokinetic studies in mice showed that GrB-Fc-IT4 was cleared bi-exponentially from plasma with a rapid initial clearance (t ½alpha = 0.36 hours) followed by a prolonged terminal-phase plasma half-life (t 1/2beta = 35 hours) similar to that of IgGs. Based on our pharmacokinetic study, we employed a QODX5 therapy schedule and demonstrated impressive antitumor activity against several tumor models including PDX-lung, A459 (NSCLC) and MDA-MB231(TNBC) tumor models. In the present study we compared the in vivo antitumor efficacy of GrB-Fc-IT4 against A549 (NSCLC) tumor cell line, using two different dosing schedules (QODx5 vs QWx5). There was a clear dose and schedule dependence and that treating mice bearing A549 tumors with 32mg/kg/dose using a QOWx5 schedule induced a complete tumor growth inhibition (7/10 tumors regressed) with 3/10 tumors showing no growth up to 50 days after the last dose. Preliminary results suggest that GrB-Fc-IT4 may reduce the formation of spontaneous A549 metastasis into lungs. Further studies are planned to examine the potential of the fusion construct to prevent spontaneous metastatic spread in lung tumor models. Although the fusion construct cross-reacts with the murine Fn14 antigen, maximum tolerated dose studies have confirmed that GrB-Fc-IT4 is a safe product, showing no evidence of toxicity (weight loss) in mice treated with up to 500mg/kg. This suggests that the therapeutic index (TI) for this class of agents is &gt;4. Our data suggest that GrB-Fc-IT4 is a novel class of antitumor agents with a unique mechanism of action. This agent seems to be an exceptionally safe and effective drug and appears to be an ideal candidate for advancing to clinical trials. Research conducted, in part, by Clayton Foundation for Research.
Citation Format: Ana Alvarez De Cienfuegos, Lawrence H. Cheung, Khalid A. Mohamedali, Mihai Gagea, Michael G. Rosenblum. Optimizing antitumor efficacy of granzyme B human fusion constructs targeting the Fn14 antigen in solid tumors. Pre IND pharmacokinetics, schedule, dose optimization and MTD studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5216.