Crystal structure of rademikibart Fab-IL-4Rα complex reveals molecular basis for next-generation potent IL-4Rα inhibition

Summary: Abstract Body: Rademikibart (CBP-201) is a human monoclonal antibody with higher binding affinity to IL-4Rα compared to dupilumab. Dupilumab was the initial first-generation interleukin-4 receptor alpha (IL-4Rα) inhibitor for treating both type I and type II IL-4Rα-dependent inflammatory disorders, like atopic dermatitis and asthma. Rademikibart, however, demonstrated better inhibition of STAT6 intracellular signaling in vitro and provided similar potency inhibiting both IL-4 induced TARC release and IL-4 induced B cell activation. We determined the crystal structure of rademikibart fragment antigen binding (Fab) bound to IL-4Rα at 2.71 Å. This structure was analyzed and compared to the 2.82 Å resolution structure of dupilumab Fab bound to IL-4Rα. The rotation angle between dupilumab and rademikibart bound to IL-4Rα is 59.17°. This rotation enables the epitope of rademikibart, but not dupilumab, on IL-4Rα to overlap more closely with the conserved binding interface utilized by IL-4 and IL-13 cytokines. Molecular dynamics (MD) studies on rademikibart and dupilumab bound to IL-4Rα examined the stability of the complexes and effects of amino acid mutations on complex formation. MD simulations showed the third interface loop (residues 148 to 152 in domain 2) of IL-4Rα interacts directly with rademikibart, which is absent in dupilumab/IL-4Rα complex. This finding is confirmed by hydrogen bond interactions at the interface between the antibodies and IL-4Rα, demonstrating superior binding energy for rademikibart. Through single amino acid mutation analysis on rademikibart, we identified residue Y50 on rademikibart as the key residue interacting with IL-4Rα’s third interface loop. Our data provide a molecular and structural rationale for the enhanced IL-4Rα inhibition by rademikibart over dupilumab, confirming rademikibart as an optimized second-generation IL-4Rα inhibitor. Yuanjun Shi², Minh Ho¹, Haote Li², Raul Collazo³, Christopher G. Bunick¹ 1. Dermatology, Yale University School of Medicine, New Haven, CT, United States. 2. Chemistry, Yale University, New Haven, CT, United States. 3. Connect Biopharm LLC, San Diego, CA, United States. Translational Studies: Cell and Molecular Biology