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

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.

Picomolar Binding Selective Human Ron Inhibitory Antibodies Directed to Surface Displayed Conformational Epitopes for Imaging and Therapy  

“…They react strongly to intracellular forms of RON and can be used in ow cytometry of xed cells. Their reaction to the RON protein expressed at the surface of cancer cells is weak however and is induced by treatment with tunicamycin [2]. The new antibodies described here show that this weak surface staining is not a feature of all antibodies to RON as the new antibodies in response to the mammalian protein immunogen this new panel of antibodies show strong cell surface binding and are highly effective in inhibiting MSP driven signalling and in the activation of ADCC with human donor NK cells.…”

“…The RON receptor tyrosine kinase (MST1R) is expressed at high levels at the surface of many tumor cells of epithelial origin antibodies to RON have shown therapeutic potential in preclinical models both as intact antibodies [1,2] and as antibody drug conjugates [3]. RON is a member of the MET receptor family and its sole ligand is the macrophage stimulating protein (MSP) [1].…”

“…We have previously reported a panel of mouse monoclonal antibodies to the unglycosylated extra cellular domain of RON and determined that they recognize cryptic epitopes which are present on intracellular forms of RON but are not exposed at the cell surface of cancer cells grown in tissue culture [2].…”

MET and RON receptor tyrosine kinases in colorectal adenocarcinoma: molecular features as drug targets and antibody-drug conjugates for therapy

“…The lead TMABs targeting MET include ARGX-111 [ 64 ], telisotuzumab (ABT-700) [ 65 ], onartuzumab (MetMab) [ 66 ], emibetuzumab (LY2875358) [ 67 ], SAIT-301 [ 68 ], Sym015 [ 69 ], and others. TMABs targeting RON include IMC-41A10 [7-], narnatumab (NCT01119456) [ 71 ], Zt/f2 [ 72 ], and recently produced 6E6, 6D4, 7G8 [ 73 ]. In addition, bispecific TMABs targeting both MET/EGFR [ 74 ], MET/epithelial cell adhesion molecule (EpCAM) [ 75 ], MET/program cell death-1 (PD-1) [ 76 ], MET/VEGFR2 [ 77 ], and MET/RON [ 78 ] have also been reported.…”

“…In contrast, the efficacy of emibetuzumab in xenograft tumor models is relatively weak, but exhibits a strong synergistic effect with chemotherapeutics [ 67 ]. The effect of anti-RON TMABs on tumor growth, on average, is relatively weak as evident from several preclinical studies [ 70 – 73 ]. Complete inhibition by a single anti-RON TMAB has not been observed.…”