SGN-CD228A is an investigational antibody-drug conjugate (ADC) directed to melanotransferrin (CD228, MELTF, MFI2, p97), a cell-surface protein first identified in melanoma. SGN-CD228A consists of a humanized antibody, hL49, with high specificity and affinity for CD228 that is stably conjugated to 8 molecules of the clinically validated microtubule-disrupting agent monomethyl auristatin E (MMAE) via a novel glucuronide linker. We performed comprehensive immunohistochemical studies, which corroborated published RNA-seq data and confirmed low CD228 expression in normal tissues and high expression in several cancers, including melanoma, squamous non-small cell lung cancer (NSCLC), triple-negative breast cancer (TNBC), colorectal cancer, and pancreatic cancer. SGN-CD228A was efficiently internalized in various tumor cell types, and its cytotoxic activity was dependent on CD228 expression and internalization and intrinsic sensitivity to the MMAE payload. Compared to the valine-citrulline dipeptide linker, the novel glucuronide linker increased the cellular retention of MMAE in vitro and conferred improved antitumor activity against melanoma cell lines in vitro and in vivo. Additionally, SGN-CD228A was active across melanoma, TNBC, and NSCLC cell line- and patient-derived xenograft models with heterogeneous antigen expression. In vivo, CD228 expression was important for response to SGN-CD228A but was not well-correlated across all tumor types, suggesting that other factors associated with ADC activity are important. Overall, SGN-CD228A is a CD228-directed, investigational ADC that employs innovative technology and has compelling preclinical antitumor activity. SGN-CD228A is being investigated in an ongoing Phase I clinical trial (NCT04042480) in patients with advanced solid tumors.