Therapeutic anti-cancer activity of antibodies targeting sulfhydryl bond constrained epitopes on unglycosylated RON receptor tyrosine kinase

Xin Yu Koh,Siti Aishah Binte Rahmat,Fernando Jose Ferrer,David P Lane,Xiao Hui Koh,Le-Ann Hwang,Dilraj Lama

doi:10.1038/s41388-019-0946-8

Abstract

Recepteur d’origine nantais (RON) receptor tyrosine kinase (RTK) and its ligand, serum macrophage-stimulating protein (MSP), are well-established oncogenic drivers for tumorigenesis and metastasis. RON is often found to be alternatively spliced resulting in various isoforms that are constitutively active. RON is therefore an attractive target for cancer therapeutics, including small molecular inhibitors and monoclonal antibodies. While small molecule inhibitors of RON may inhibit other protein kinases including the highly similar MET kinase, monoclonal antibodies targeting RON are more specific, potentially inducing fewer side effects. Although anti-RON monoclonal antibody therapies have been developed and tested in clinical trials, they were met with limited success. Cancer cells have been associated with aberrant glycosylation mechanisms. Notably for RON, the loss of N-bisected glycosylation is a direct cause for tumorigenesis and poorer prognosis in cancer patients. Particularly in gastric cancer, aberrant RON glycosylation augments RON activation. Here, we present a novel panel of monoclonal antibodies which potentially widens the specific targeting of not only the glycosylated RON, but also unglycosylated and aberrantly glycosylated RON. Our antibodies can bind strongly to deglycosylated RON from tunicamycin treated cells, recognise RON in IHC/IF and possess superior therapeutic efficacy in RON expressing xenograft tumours. Our most potent antibody in xenograft assays, is directed to the RON alpha chain and targets a sulfhydryl bond constrained epitope that appears to be cryptic in the crystal structure. This establishes the paradigm that such constrained and cryptic epitopes represent good targets for therapeutic antibodies.

Highlights

Recepteur d’origine nantais (RON) is a tyrosine kinase receptor belonging to the MET family of receptors [1]
A total of 2112 hybridoma clones were screened against RON antigens in enzyme-linked immunosorbent assay (ELISA) and high throughput immunofluorescence screening
From our ELISA screening data (Fig. 1a), 18 hybridoma clones were identified as positive binders for RON1 and RON3 antigens that were not cross reactive to the negative antigen GST

Summary

Introduction

Recepteur d’origine nantais (RON) is a tyrosine kinase receptor belonging to the MET family of receptors [1]. Like MET, RON is expressed as an 185 kDa glycosylated pro-RON chain, which is subsequently processed by protease mediated cleavage into a functional 35 kDa alpha RON chain and a 150 kDa beta RON chain, linked by disulphide bridges [3]. Activation of the MSP/RON pathway leads to downstream activation of multiple signalling pathways including the PI3K/Akt pathways, resulting in regulation of important cellular processes like cell survival and proliferation, angiogenesis and transformation [3]. Aberrant RON activities in cancer are mostly caused by receptor overexpression and alternative splicing, which results in constitutively active RON isoforms [6, 7]. Therapeutic anti-cancer activity of antibodies targeting sulfhydryl bond constrained epitopes on

Methods

Results

Conclusion