Balancing the Affinity and Tumor Cell Binding of a Two-in-One Antibody Simultaneously Targeting EGFR and PD-L1.

The optimization of the affinity of monoclonal antibodies is crucial for the development of drug candidates, as it can impact the efficacy of the drug and, thus, the dose and dosing regimen, limit adverse effects, and reduce therapy costs. Here, we present the affinity maturation of an EGFR×PD-L1 Two-in-One antibody for EGFR binding utilizing site-directed mutagenesis and yeast surface display. The isolated antibody variants target EGFR with a 60-fold-improved affinity due to the replacement of a single amino acid in the CDR3 region of the light chain. The binding properties of the Two-in-One variants were confirmed using various methods, including BLI measurements, real-time antigen binding measurements on surfaces with a mixture of both recombinant proteins and cellular binding experiments using flow cytometry as well as real-time interaction cytometry. An AlphaFold-based model predicted that the amino acid exchange of tyrosine to glutamic acid enables the formation of a salt bridge to an arginine at EGFR position 165. This easily adaptable approach provides a strategy for the affinity maturation of bispecific antibodies with respect to the binding of one of the two antigens.

Structural Basis of Interleukin-5 Inhibition by the Small Cyclic Peptide AF17121.

Interleukin-5 (IL-5) is a T-helper cell of subtype 2 cytokine involved in many aspects of eosinophil life. Eosinophilic granulocytes play a pathogenic role in the progression of atopic diseases, such as allergy, asthma and atopic dermatitis and hypereosinophilic syndromes. Here, eosinophils upon activation degranulate leading to the release of proinflammatory proteins and mediators stored in intracellular vesicles termed granula thereby causing local inflammation, which when persisting leads to tissue damage and organ failure. As a key regulator of eosinophil function, IL-5 therefore presents a major pharmaceutical target and approaches to interfere with IL-5 receptor activation are of great interest. Here we present the structure of the IL-5 inhibiting peptide AF17121 bound to the extracellular domain of the IL-5 receptor IL-5Rα. The small 18mer cyclic peptide snugly fits into the wrench-like cleft of the IL-5 receptor, thereby blocking access of key residues for IL-5 binding. While AF17121 and IL-5 seemingly bind to a similar epitope at IL-5Rα, functional studies show that recognition and binding of both ligands differ. Using the structure data, peptide variants with improved IL-5 inhibition have been generated, which might present valuable starting points for superior peptide-based IL-5 antagonists.