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 REGION

We previously showed that a single mutation at heavy (H) position 35 o f Abs specific for p-azophenylarsonate (Ars) resulted in acquisition o f binding to the structurally related hapten p-azophenylsulfonate (Sul f), To explore the sequence and structural diversity of the H chain fi rst 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 germl ine encoded, lacking somatic mutations. Following affinity selection o n Sulf resins, 55 mutant Fab were isolated, revealing seven unique HCD R1 sequences containing different amino acids at position EI:35, All F ab bound Sulf, but not Ars, Site-directed mutagenesis in a variety of HCDR1 sequence contexts indicates that H:35 is critical for hapten spe cificity, independent of the sequence of the remainder of HCDR1, At H: 35, Asn is required for Ars specificity, consistent with the x-ray cry stal structure of the somatically mutated anti-Ars Ab 36-71, while Sul f binding occurs with at least seven different H:35 residues. All Sulf -binding clones selected following phage display contained H:Gly33, ob served previously for Ars-binding Abs that use the same germline V-H s equence. Site directed mutagenesis at H:33 indicates that Gly plays an essential structural role in HCDR1 for both Sulf- and Ars-specific Ab s.