Abstract PO-041: Preclinical in vivo characterization of a first-in-class humanized antibody targeting alternatively spliced Tissue Factor
Authors: Lewis Clayton S. , Karve Aniruddha , Matiash Kateryna , Stone Timothy , Desai Pankaj , Bogdanov Vladimir Y. 
Published: November 2020

Abstract
Tissue Factor, the initiator of the extrinsic pathway of coagulation, and its isoform alternatively spliced Tissue Factor (asTF) are often overexpressed in cancer cells. While asTF is only minimally procoagulant, it activates both the extracellular mechanical and intracellular signaling functions of beta-integrins. This leads to activation of multiple MAPK pathways thereby promoting increases in proliferation, migration, and angiogenesis. Previously, we have shown that RabMab1, a rabbit monoclonal antibody specific for human asTF, disrupts the binding of asTF to beta-integrins and thereby inhibits the growth of breast cancer and pancreatic ductal adenocarcinoma (PDAC) cell lines in vitro and in vivo. Here, we report on humanization of RabMab1, assessment of its binding characteristics, and determination of its in vivo properties. The variable regions of the heavy and light chains of hybridoma derived RabMab1 were cloned and used to generate a rabbit/human chimera (cRabMab1). Substitutions of species-specific residues were then carried out to produce humanized RabMab1 (hRabMab1; IgG1 isotype). Antigen binding was assessed via anti-human IgG Fc capture biosensor assays. cRabMab1 was found to have a KD of 4.24 nM whereas hRabMab1 was found to have a KD in the low picomolar range, which could not be precisely measured due to an extremely slow off-rate. The in vivo half-lives of each were determined to be 280 and 908 hours, respectively. Orthotopic co-implantation of cRabMab1 with high-grade, human PDAC cell line Pt45.P1 in athymic nude mice resulted in tumors that were 82% and 92% smaller than tumors in the vehicle and IgG control group groups, respectively, with no statistical difference between vehicle and IgG isotype control groups. Intravenous administration of hRabMab1 slowed the growth of pre-formed orthotopic Pt45.P1 tumors in athymic nude mice by 64% and 61% compared to vehicle and isotype control groups, respectively; again, there was no difference between vehicle and IgG isotype control groups. Immunohistochemical analysis of tumor tissue revealed a statistically significant 68% reduction in neovascularization (CD31), a 58% reduction in M2-polarized macrophages (CD206), and a 24% reduction in proliferating cells (Ki67) in the hRabMab1 cohort. RNAseq analysis of tumor tissue revealed a significant suppression of pathways that promote mitosis and the cell cycle. By qRT-PCR, we validated a greater than 82% reduction in the expression of HBEGF, a gene encoding a cell surface mitogen, and a greater than 68% reduction in the expression of STN1, a gene encoding a component of the CST-complex, a telomere replication and maintenance structure. This is the first proof-of-concept study whereby a novel biologic that targets asTF has been used as a systemically administered single agent, with encouraging results. Because hRabMab1 has a favorable pharmacokinetic profile and is able to suppress PDAC tumor growth in vivo, it comprises an attractive candidate for further clinical development.
Citation Format: Clayton S. Lewis, Aniruddha Karve, Kateryna Matiash, Timothy Stone, Pankaj Desai, Vladimir Y. Bogdanov. Preclinical in vivo characterization of a first-in-class humanized antibody targeting alternatively spliced Tissue Factor [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-041.