Abstract 540: c-MET mutations sensitize to antibody-drug conjugate telisotuzumab vedotin through efficient internalization and rapid intracellular drug delivery

Background: Telisotuzumab vedotin (Teliso-V) is an antibody-drug conjugate (ADC) composed of the c-MET antibody ABT-700 and the microtubule inhibitor monomethyl auristatin E (MMAE). Patients with EGFR wildtype (WT) nonsquamous non-small cell lung cancer with high c-MET expression had an overall response rate of 54% with Teliso-V. In addition to overexpression and amplification, genetic alterations in c-MET, such as exon 14 deletion (ex14del) and missense mutations in the tyrosine kinase domain (TKD), contribute to its constitutive activation. The efficacy of Teliso-V in context of these genomic backgrounds is currently unknown. Methods: Recombinant retroviruses were used to express WT-c-MET, c-MET-TKD (D1228H/V/N and Y1230C/H mutations), c-MET-Y1003F/N (CBL E3 ubiquitin ligase binding site mutation), and c-MET-ex14del (47 aa juxtamembrane deletion) mutants in NIH3T3 and Ba/F3 cells. c-MET surface expression, intracellular signaling, ADC internalization and endolysosomal (EL) trafficking (using pHrodo Red-labeled Teliso-V), intracellular MMAE by LC/MS, and Teliso-V sensitivity were studied in 2D/3D cell cultures. Full-length c-MET protein structures were created with the ColabFold program and molecular dynamics simulation software (with/without artificial lipid bilayer) was used to predict the mechanistic basis for differential function. Results: c-MET expression was required for Teliso-V cytotoxicity and intracellular payload delivery in all cell lines. Compared with WT-c-MET, various mutants showed oncogene-driven transformation evident by elevated levels of phosphorylated c-MET/AKT, cytokine-independent colony formation, oncogene-driven proliferation, and tyrosine kinase inhibitor (TKI) resistance. Interestingly, c-MET-TKD-expressing cells were the most sensitive to Teliso-V, which was associated with faster ADC internalization and EL trafficking. An AI-generated protein model and associated molecular dynamics simulation demonstrated that all oncogenic TKD variants tested stabilized the c-MET-plexin-semaphorin-integrin domain (R592-L614), known for its ability to position the extracellular ligand-binding (ELB) site for optimal activation. Conclusions: c-MET-TKD mutations induced TKI resistance and enhanced MMAE delivery in association with robust ADC internalization and EL trafficking. Previous studies have shown that the HER2 ADC fam-trastuzumab deruxtecan-nxki was highly effective in cell lines and patients with ERBB2 kinase domain-mutant lung cancer v