(T1030-03-20) Therapeutic Potential of Dual-Action Antibody-Drug Conjugates as a Novel Strategy for Cancers Heterogeneously Expressing RON Receptor Tyrosine Kinase

Purpose: Antibody–Drug Conjugates (ADCs) are a class of targeted Immunotherapeutics designed to selectively eliminate malignant cells by recognizing tumor-associated antigens overexpressed on tumor cells relative to normal tissues. Among emerging targets, the receptor tyrosine kinase RON (Recepteur d’Origine Nantais), a member of the MET proto-oncogene family, has been implicated in advanced tumor staging, poor prognosis, and increased metastatic potential. While RON regulates physiological processes such as immune modulation and tissue repair, its aberrant expression drives oncogenic progression by activating intracellular signaling pathways, promoting proliferation, survival, and metastasis.¹ To exploit RON as a therapeutic target, our laboratory developed two monoclonal antibodies, Zt/G4 and H5B14, which bind distinct, non-overlapping epitopes on the extracellular domain of RON while sparing the Macrophage-Stimulating Protein (MSP) binding site. These antibodies induce transient RON phosphorylation, triggering receptor internalization—a critical mechanism for ADC-mediated cytotoxic payload delivery. However, effective monotherapy requires high antigen density (~8,000 receptors per cell) to achieve a 95% reduction in viability, limiting use in tumors with low RON expression.² To address this limitation, we proposed a combinatorial ADC strategy involving co-administration of Zt/G4 and H5B14, each conjugated to a distinct cytotoxic payload—MMAE and Duocarmycin, respectively. This approach aims to enhance tumor selectivity, cytotoxicity, and efficacy by leveraging complementary epitope targeting and potentially synergistic mechanisms of action.

Methods: A competitive binding assay was initially conducted to evaluate potential interference in binding affinity when the two antibodies were co-administered. In this assay, one antibody was maintained at a fixed concentration of 2 µg/mL, while the other was serially diluted from 10 µg/mL to 0.015 µg/mL. Following a one-hour incubation at 4°C to allow binding, cells were washed and subsequently stained with a fluorescently labeled secondary antibody specific to the fixed antibody. The binding efficiency was then quantified using flow cytometry to assess any competitive displacement. Three cancer cell lines expressing RON levels ranging from 5,000 to 19,000 receptors per cell were used. Subsequently, time-dependent internalization and downregulation studies were conducted using flow cytometry, immunocytochemistry (ICC), and western blotting to assess changes in surface and total RON levels following antibody co-treatment. Lysosomal trafficking was analyzed via ICC by assessing colocalization with the lysosomal marker LAMP1. Both antibodies were then conjugated to MMAE and Duocarmycin at drug-to-antibody ratios (DARs) of 4.7 and 4.5, respectively, with conjugation quality confirmed via hydrophobic interaction chromatography (HIC). Cytotoxic potential was evaluated using MTT assays, and cell cycle progression was assessed by propidium iodide staining and flow cytometry.

Results: The competitive binding assay demonstrated no interference in antibody binding when Zt/G4 and H5B14 were co-administered, confirming their compatibility for combination therapy. Co-treatment induced a rapid, time-dependent reduction in surface RON expression across all three cell lines, as detected by flow cytometry. Western blot analysis corroborated these findings, showing decreased total RON levels with combination treatment compared to monotherapy. While ICC did not show significant differences in receptor internalization between single and combination treatments, lysosomal uptake (LAMP1 colocalization) was significantly higher when the antibody combination was used, supporting enhanced receptor trafficking and degradation through the lysosomal pathway. Cytotoxicity assays revealed a notable decrease in IC₅₀ values for the combination treatment in both high and moderate RON-expressing cell lines. In contrast, no significant reduction in IC₅₀ was observed in the low RON-expressing line. However, cell cycle analysis demonstrated a complete G₂–M phase arrest in the low antigen cell line with combination treatment, suggesting a cell growth arrest. In high and moderate RON-expressing cell lines, combination treatment led to substantial G₀–G₁ depletion and marked G₂–M phase arrest as early as 6 hours post-treatment, particularly at a 1:1 ADC ratio.

Conclusion: This study highlights the therapeutic potential of dual-epitope ADC combination therapy targeting RON-expressing tumors. Co-administration of Zt/G4 and H5B14 enhanced RON internalization and downregulation relative to monotherapy. Cytotoxicity studies confirmed reduced IC₅₀ values and G₂–M phase arrest, especially in cell lines with high and moderate RON expression. Notably, even in low RON-expressing cells, combination treatment induced pronounced cell cycle effects, supporting the feasibility of this strategy in tumors traditionally considered suboptimal for ADC monotherapy. Further studies investigating cell death and in vivo efficacy are ongoing. These findings underscore the promise of combinatorial ADC regimens as a means to overcome antigen threshold limitations, broaden therapeutic applicability, and improve outcomes in solid tumors, paving the way for future clinical translation.

References: 1. Wang MH, Zhang R, Zhou YQ, Yao HP. Pathogenesis of RON receptor tyrosine kinase in cancer cells: activation mechanism, functional crosstalk, and signaling addiction. J Biomed Res. 2013 Sep;27(5):345-56. doi: 10.7555/JBR.27.20130038. Epub 2013 Jul 30.
2. Yao HP, Suthe SR, Tong XM, Wang MH. Targeting RON receptor tyrosine kinase for treatment of advanced solid cancers: antibody-drug conjugates as lead drug candidates for clinical trials. Ther Adv Med Oncol. 2020 May 12;12:1758835920920069. doi: 10.1177/1758835920920069


Downregulation of surface RON β-chain analyzed by Western blot.


Downregulation of surface RON analyzed by Flow cytometry.


Cytotoxicity of the ADCs and the combinations using MTT Assay.