Abstract Upon therapeutic pressure, cancers commonly select for drug-resistant, invasive subpopulations with elevated expression of the receptor tyrosine kinase AXL. Besides the correlation between high AXL expression and induction of epithelial-to-mesenchymal transition, a process known to support metastasis, ample evidence also links AXL to resistance against a variety of targeted therapies, including inhibitors of the MAPK pathway in malignant melanoma and the EGFR pathway in lung cancer. AXL-107-MMAE (HuMax-AXL-ADC) is a therapeutic antibody-drug conjugate specific for AXL, containing the microtubule disrupting agent monomethyl auristatin E as the cytotoxic payload. AXL-107-MMAE was previously shown to induce potent cytotoxicity in vitro and in vivo, which was dependent expression of AXL on the cell surface. We evaluated the capacity of AXL-107-MMAE to target AXL-positive MAPK pathway inhibitor resistant tumor cells, using malignant melanoma as a clinically relevant example. First, AXL-107-MMAE was shown to induce cytotoxicity in BRAF-mutant tumor cell lines that showed AXL expression upon acquired resistance to BRAF-inhibitors. In contrast, no cytotoxicity was observed in the BRAF-inhibitor-sensitive, AXL-negative parental cell lines. In heterogeneous tumor cell cultures, treatment with a BRAF-inhibitor selected for AXL-high, MAPK pathway inhibitor-insensitive melanoma cells, which was prevented by combined BRAF-inhibitor and AXL-107-MMAE treatment. Interestingly, we observed marked AXL upregulation in biopsies obtained from patients after they developed resistance to MAPK pathway inhibitors compared to paired pre-treatment biopsies. The in vivo potential of AXL-107-MMAE in malignant melanoma was demonstrated using BRAFV600E-mutated xenograft model derived from a patient who developed resistance to the BRAF inhibitor vemurafenib in the clinic. This indicates that AXL expression levels in treatment-resistant malignant melanoma are sufficient to induce tumor regression with AXL-107-MMAE, at least in this model system. These findings merit clinical validation of the targeting of both treatment-naïve and drug-resistant cancers with AXL-107-MMAE, either alone or in combination with other targeted therapies. Citation Format: Julia Boshuizen, Louise A. Koopman, Esther C. Breij, David Satijn, Daniel Peeper, Paul W. Parren. Specific elimination of invasive and multidrug-resistant cancer cells by an antibody-drug conjugate targeting AXL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4591. doi:10.1158/1538-7445.AM2017-4591