Comparison of an Fcab-Drug Conjugate with Other Antibody-Drug Conjugate Formats towards Exposure and Antitumor Activity

Abstract Antibody-drug conjugates (ADCs) based on Fc antigen-binding fragments (Fcabs) promise improved tumor penetration while retaining the FcRn-mediated long half-life. Building on prior spheroid studies, we directly compared a 50 kDa HER2-targeted Fcab-MMAE ADC with a matched 150 kDa control molecule (aDIG-Fcab-MMAE) consisting of the same HER2-targeting Fcab ADC equipped with additional non-targeting anti-digoxigenin (aDIG) Fab arms and trastuzumab Fab- and IgG-based ADCs (50 kDa T-Fab-MMAE and 150 kDa T-IgG-MMAE) using the same linker-payloads. Site-specific conjugation yielded high-purity DAR2 species with excellent serum stability. In vitro cytotoxicity assays revealed that Fcab-MMAE and aDIG-Fcab-MMAE exhibited lower potency compared to T-Fab-MMAE and T-IgG-MMAE, correlating with their approximately 10-fold lower binding affinity for HER2. A pharmacokinetic study in mice showed similar exposure for the Fcab-MMAE, its 150 kDa control, and a higher exposure for T-IgG-MMAE ADC, while the T-Fab-MMAE ADC cleared rapidly. In an NCI-N87 gastric cancer SCID mouse xenograft model, T-IgG-MMAE demonstrated superior efficacy, while Fcab-MMAE and aDIG-Fcab-MMAE showed similar efficacy despite their size difference, with T-Fab-MMAE showing lower efficacy. These data indicate that at similar exposure, reduced scaffold size alone may not improve in vivo efficacy.