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Structural Requirements for a Specificity Switch and for Maintenance of Affinity Using Mutational Analysis of a Phage-Displayed Anti-Arsonate Antibody of Fab Heavy Chain First Complementarity-Determining Region¹

Yee Wah Wong^*, Davinder S. Gill, Behnaz Parhami-Seren, Mary K. Short^§, Seshi Reddy Sompuram^¶ and Michael N. Margolies^2,

^* Department of Obstetrics and Gynecology, The Ohio State University, Columbus, OH 43120; IntraImmune Therapies, Boston, MA 02215; Department of Surgery, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114; ^§ Department of Hematology/Oncology, Children’s Hospital Medical Center, Cincinnati, OH 45229; and ^¶ Hubert Humphrey Cancer Center and Department of Pathology, Boston University School of Medicine, Boston, MA 02118

We previously showed that a single mutation at heavy (H) position 35 of Abs specific for p-azophenylarsonate (Ars) resulted in acquisition of binding to the structurally related hapten p-azophenylsulfonate (Sulf). To explore the sequence and structural diversity of the H chain first complementarity-determining region (HCDR1) in modulating affinity and specificity, positions 30–36 in Ab 36–65 were randomly mutated and expressed as Fab in a bacteriophage display vector. Ab 36–65 is germline encoded, lacking somatic mutations. Following affinity selection on Sulf resins, 55 mutant Fab were isolated, revealing seven unique HCDR1 sequences containing different amino acids at position H:35. All Fab bound Sulf, but not Ars. Site-directed mutagenesis in a variety of HCDR1 sequence contexts indicates that H:35 is critical for hapten specificity, independent of the sequence of the remainder of HCDR1. At H:35, Asn is required for Ars specificity, consistent with the x-ray crystal structure of the somatically mutated anti-Ars Ab 36–71, while Sulf binding occurs with at least seven different H:35 residues. All Sulf-binding clones selected following phage display contained H:Gly33, observed previously for Ars-binding Abs that use the same germline V_H sequence. Site-directed mutagenesis at H:33 indicates that Gly plays an essential structural role in HCDR1 for both Sulf- and Ars-specific Abs.

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