Pharmaceutical Potential of Antibody-Drug Conjugate Doublets for Targeted Treatment of Cancers Heterogeneously Expressing RON Receptor Tyrosine Kinase

Vollständiger Abstract

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Introduction/Background: Limited RON expression by cancer cells is detri-mental for the therapeutic efficacy of Antibody-Drug Conjugates (ADCs). The present study aimed to validate a pharmaceutical strategy of using ADCs in vitro to target cancer cells with RON expression below the therapeutic threshold. Materials and Methods: Anti-RON ADCs Zt/g4-MMAE and H5B14-DCM, with distinct epitope-binding properties and different payload-effect mechanisms, were formulated to form anti-RON ADC doublets. Hydrophobic interaction chromatography was used to deter-mine ADC conjugation profile and stability. Flow cytometry was used to study RON inter-nalization. Liquid chromatography-mass spectrometry was used to detect intracellular pay-load molecules. Various biochemical and biological methods were used to determine cell viability, cell cycle, and cell death. results: Results: Anti-RON ADC doublets at a 50:50 ratio were effective with increased efficacy in inducing RON internalization by cancer cells with variable RON expression. This effect increased intracellular payload accumulation, particularly in cells with limited RON expression. Treatment of ADC doublets also caused significant cell cycle arrest, dramatic reductions in cell viability, and increased cell apoptosis. Moreover, cancer cells with limited RON expression responded well to the ADC doublets, exhibiting a dramatic decrease in cell viability and an increase in apoptotic death. A payload synergism within the ADC doublets was responsible for these activities. Finally, the ADC doublets effectively kill cancer cells displaying acquired-payload resistance. This activity was particularly prominent in cancer cells insensitive to ADCs with a single mechanism of action. Results: Anti-RON ADC doublets at a 50:50 ratio were effective, with increased efficacy in inducing RON internalization by cancer cells with variable RON expression. This effect increased intracellular payload accumulation, particularly in cells with limited RON expres-sion. Treatment of ADC doublets also caused significant cell cycle arrest, dramatic reduc-tions in cell viability, and increased cell apoptosis. Moreover, cancer cells with limited RON expression responded well to the ADC doublets, exhibiting a dramatic decrease in cell via-bility and an increase in apoptotic death. Payload synergy within the ADC doublets was responsible for these activities. Finally, the ADC doublets effectively kill cancer cells dis-playing acquired-payload resistance. This activity was particularly prominent in cancer cells insensitive to ADCs with a single mechanism of action. Discussion: By binding to 2 different antigenic epitopes to deliver 2 payloads possessing different effect mechanisms, anti-RON ADC doublets overcome several shortcomings, such as limited target antigen expression, insufficient target internalization, and acquired payload resistance, observed in anticancer studies following a single ADC treatment. Conclusion: Anti-RON ADC doublets with unique pharmacological features demonstrate enhanced in vitro activity compared to single ADCs, delivering sufficient payloads to achieve cytotoxicity against cancer cells with RON expression below the therapeutic thresh-old. These findings provide an opportunity to optimize ADC treatment regimens in vivo for cancers expressing low levels of RON.