Protease-Cleavable Linkers Modulate the Anticancer Activity of Noninternalizing Antibody–Drug Conjugates

Antibody–drug conjugates (ADCs) represent an attractive class of biopharmaceutical agents, with the potential to selectively deliver potent cytotoxic agents to tumors. It is generally assumed that ADC products should preferably bind and internalize into cancer cells in order to liberate their toxic payload, but a growing body of evidence indicates that also ADCs based on noninternalizing antibodies may be potently active. In this Communication, we investigated dipeptide-based linkers (frequently used for internalizing ADC products) in the context of the noninternalizing F16 antibody, specific to a splice isoform of tenascin-C. Using monomethyl auristatin E (MMAE) as potent cytotoxic drug, we observed that a single amino acid substitution of the Val–Cit dipeptide linker can substantially modulate the in vivo stability of the corresponding ADC products, as well as the anticancer activity in mice bearing the human epidermoid A431 carcinoma. In these settings, the linker based on the Val–Ala dipeptide exhibited better performances, compared to Val–Cit, Val–Lys, and Val–Arg analogues. Mass spectrometric analysis revealed that the four linkers displayed not only different stability in vivo but also differences in cleavage sites. Moreover, the absence of anticancer activity for a F16–MMAE conjugate featuring a noncleavable linker indicated that drug release modalities, based on proteolytic degradation of the immunoglobulin moiety, cannot be exploited with noninternalizing antibodies. ADC products based on the noninternalizing F16 antibody may be useful for the treatment of several human malignancies, as the cognate antigen is abundantly expressed in the extracellular matrix of several tumors, while being virtually undetectable in most normal adult tissues.