The crystallizable fragment (Fc) of the immunoglobulin class G (IgG) is an attractive scaffold for the design of novel therapeutics. Upon engineering the C-terminal loops in the CH3 domains, Fcabs (Fc domain with antigen-binding sites) can be designed. We present the first crystal structures of Fcabs, i.e., of the HER2-binding clone H10-03-6 having the AB and EF loop engineered and the stabilized version STAB19 derived by directed evolution. Comparison with the crystal structure of the Fc wild-type protein reveals conservation of the overall domain structures but significant differences in EF-loop conformations. Furthermore, we present the first Fcab-antigen complex structures demonstrating the interaction between the engineered Fcab loops with domain IV of HER2. The crystal structures of the STAB19-HER2 and H10-03-6-HER2 complexes together with analyses by isothermal titration calorimetry, SEC-MALS, and fluorescence correlation spectroscopy show that one homodimeric Fcab binds two HER2 molecules following a negative cooperative binding behavior.
Keywords: ErbB2; Fcab; Fcab-HER2 structure; HER2; IgG1-Fc; X-ray crystallography; binding stoichiometry; fluorescence correlation spectroscopy.
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