Melanotransferrin as a Potential Target for the Selective Killing of Melanoma and Triple-Negative Breast Cancer Cells In Vitro Using Novel SNAP-Tag-Based Antibody–AURIF Conjugate

Background: Melanoma and triple-negative breast cancer (TNBC) are the most aggressive skin and breast cancers, often diagnosed at late stages with limited treatment options. The melanoma-associated antigen melanotransferrin (MTf) is overexpressed in these solid tumors, where it drives tumorigenesis, progression, and chemoresistance. Its inhibition correlates with tumor regression, making MTf a promising therapeutic target. This study aimed to develop a novel, selectively targeted antibody–drug conjugate (ADC) against MTf-expressing melanoma and TNBC cancer cells using SNAP-tag fusion protein conjugation technology. Methods: We generated an L49(scFv)-SNAP-tag antibody fusion protein engineered through the genetic fusion of a humanized anti-MTf single-chain variable fragment (scFv) with a SNAP-tag fusion protein capable of site-specific self-labelling with O6-benzylguanine (BG) modified substrates in 1:1 stoichiometry. Binding and internalization of the conjugate labeled with BG-Alexa 488 (L49(scFv)-SNAP-Alexa488) were assessed by confocal microscopy and flow cytometry in MTf-overexpressing cell lines. Cytotoxicity was evaluated using the cell viability XTT assay after conjugating the SNAP-fusion protein to the potent monomethyl auristatin-F (BG-AURIF). Results: The L49(scFv)-SNAP-Alexa488 conjugate demonstrated specific binding and internalization into MTf-positive melanoma and TNBC cells. The corresponding ADC, L49(scFv)-SNAP-Linker-AURIF, exerted potent, antigen and dose-dependent cytotoxicity, with IC50 values in the nanomolar range (4.77–34.43 nM). Conclusions: We successfully generated a novel SNAP-tag-based ADC that selectively eliminates MTf-overexpressing tumor cells. This proof-of-concept highlights MTF’s value as a therapeutic target and demonstrates that a smaller-format, non-cleavable linker SNAP-tag-based ADC can achieve potent nanomolar cytotoxicity, supporting further development of MTF-targeted immunotherapies for melanoma and TNBC.