Fc Antigen-Binding Fragment Drug Conjugates as Novel Antibody-Based Format for Targeted Drug Delivery

The fundamental prerequisite of therapeutic efficacy of antibody drug conjugates (ADCs) in solid tumors is the exposition of cancer cells to a therapeutically active dose of drug. One prominent strategy to maximize drug exposure, is to increase tissue penetration by smaller sized drug conjugates that extravasate faster and distribute deeper into the tumor. Fc antigen binding fragments (Fcabs) are engineered homodimeric 50 kDa Fc regions that incorporate all essential immunoglobulin G (IgG) functionalities such as neonatal Fc receptor (FcRn)-mediated recycling and antigen binding at only one-third of the size. The combination of reduced size and favorable pharmacokinetics makes Fcabs an attractive alternative to the conventional IgG scaffold employed in ADCs, with potentially improved tissue penetration capabilities. In the present study, Fcabs were explored for the first time as a novel scaffold for ADCs. For proof-of-principle, HER2- and EGFR-targeting Fcabs were selected, coupled to a pH sensitive dye, and subsequently tested for receptor-mediated endocytosis on tumor cells. Target-dependent cellular binding and uptake confirmed the suitability of the selected Fcabs for targeted intracellular drug delivery. Moreover, Fcabs were conjugated to cytotoxic monomethyl auristatin E (MMAE) employing site-specific enzymatic or cysteine coupling. The feasibility of Fcab-drug conjugates could be demonstrated by retained binding to target receptor and half-life prolonging FcRn, conjugation site dependent serum stability and target-mediated cell killing at sub-nanomolar to double digit nanomolar concentrations. Moreover, HER2-targeting Fcab-dye constructs were tested in a tumor cell spheroid model and showed elevated penetration compared to a 150 kDa Fcab derivative with unrelated Fab arms or to the clinically validated HER2-targeting antibody Trastuzumab. Overall, this study demonstrates the applicability of the Fcab format as targeting scaffold for the generation of drug conjugates and emphasizes the potential of Fcab drug conjugates for improved tumor penetration in solid cancers