A First-In-Class, Humanized Antibody Targeting Alternatively Spliced Tissue Factor: Preclinical Evaluation in an Orthotopic Model of Pancreatic Ductal Adenocarcinoma

Clayton S Lewis,Henri H Versteeg,Aniruddha Karve,Syed A Ahmad,Bruce J Aronow,Vladimir Y Bogdanov,Timothy Stone,Kateryna Matiash,Jingxing Li,Jordon K Wang,Pankaj B Desai

doi:10.3389/fonc.2021.691685

Abstract

In 2021, pancreatic ductal adenocarcinoma (PDAC) is the 3rd leading cause of cancer deaths in the United States. This is largely due to a lack of symptoms and limited treatment options, which extend survival by only a few weeks. There is thus an urgent need to develop new therapies effective against PDAC. Previously, we have shown that the growth of PDAC cells is suppressed when they are co-implanted with RabMab1, a rabbit monoclonal antibody specific for human alternatively spliced tissue factor (asTF). Here, we report on humanization of RabMab1, evaluation of its binding characteristics, and assessment of its in vivo properties. hRabMab1 binds asTF with a KD in the picomolar range; suppresses the migration of high-grade Pt45.P1 cells in Boyden chamber assays; has a long half-life in circulation (~ 5 weeks); and significantly slows the growth of pre-formed orthotopic Pt45.P1 tumors in athymic nude mice when administered intravenously. Immunohistochemical analysis of tumor tissue demonstrates the suppression of i) PDAC cell proliferation, ii) macrophage infiltration, and iii) neovascularization, whereas RNAseq analysis of tumor tissue reveals the suppression of pathways that promote cell division and focal adhesion. This is the first proof-of-concept study whereby a novel biologic targeting asTF has been investigated as a systemically administered single agent, with encouraging results. Given that hRabMab1 has a favorable PK profile and is able to suppress the growth of human PDAC cells in vivo, it comprises a promising candidate for further clinical development.

Highlights

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy with most patients presenting at stage IV [1]
Chimeric antibodies comprised of the RabMab1 Fab region and a human Fc region characteristic of either an IgG1 or IgG4 isotype were assessed by ELISA for their ability to bind recombinant alternatively spliced isoform of human tissue factor (asTF)
The chimeric and parental RabMab1 antibodies were further analyzed for asTF binding affinity using anti-penta-HIS (HIS1K) biosensors. cRabMab1-hIgG1 and cRabMab1-hIgG4 had asTF equilibrium dissociation constants (KD) of 430 pM and 1.64 nM, respectively; by comparison, rRabMab1 did not dissociate (Figure 1B, SPR sensograms can be found in Supplementary Figure 1)

Summary

Introduction

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy with most patients presenting at stage IV [1]. We reported on the anti-tumor effects of RabMab, our monoclonal antibody that binds the alternatively spliced isoform of human tissue factor (asTF), in a setting of orthotopic co-implantation with TF-expressing human PDAC cells [3]. One of the most extensively studied solid cancers with elevated TF expression comprises PDAC, where it was first identified as the protein responsible for PDAC cell-triggered coagulation [7]. Earlier studies of TF protein in human tissues were done using antibodies that could not discriminate between flTF and asTF; more recently, we reported that flTF and asTF are both overexpressed in breast cancer and PDAC [11, 12]. AsTF fuels cancer cell growth via integrin ligation [13]. This has been seen in PDAC, where asTF promotes PDAC cell growth and spread in an orthotopic mouse model via b1 integrin-linked mechanisms [3]

Methods

Results

Conclusion