: Platelets are increasingly recognized as mediators of tumor growth and metastasis. Hypothesizing that activated platelets in the tumor microenvironment provide a targeting epitope for tumor-directed chemotherapy, we developed an antibody-drug conjugate (ADC), comprised of a single-chain antibody (scFv) against the platelet integrin GPIIb/IIIa (scFv) linked to the potent chemotherapeutic microtubule inhibitor, monomethyl auristatin E (MMAE). : We developed an ADC comprised of three components: 1) A scFv which specifically binds to the high affinity, activated integrin GPIIb/IIIa on activated platelets. 2) A highly potent microtubule inhibitor, monomethyl auristatin E. 3) A drug activation/release mechanism using a linker cleavable by cathepsin B, which we demonstrate to be abundant in the tumor microenvironment. The scFv-MMAE was first conjugated with Cyanine7 for imaging. The therapeutic efficacy of the scFv-MMAE was then tested in a mouse metastasis model of triple negative breast cancer. : studies confirmed that this ADC specifically binds to activated GPIIb/IIIa, and cathepsin B-mediated drug release/activation resulted in tumor cytotoxicity. fluorescence imaging demonstrated that the newly generated ADC localized to primary tumors and metastases in a mouse xenograft model of triple negative breast cancer, a difficult to treat tumor for which a selective tumor-targeting therapy remains to be clinically established. Importantly, we demonstrated that the scFv-MMAE displays marked efficacy as an anti-cancer agent, reducing tumor growth and preventing metastatic disease, without any discernible toxic effects. : Here, we demonstrate the utility of a novel ADC that targets a potent cytotoxic drug to activated platelets and specifically releases the cytotoxic agent within the confines of the tumor. This unique targeting mechanism, specific to the tumor microenvironment, holds promise as a novel therapeutic approach for the treatment of a broad range of primary tumors and metastatic disease, particularly for tumors that lack specific molecular epitopes for drug targeting.Rationale