CONTRIBUTION OF HEAVY-CHAIN JUNCTIONAL AMINO-ACID DIVERSITY TO ANTIBODY-AFFINITY AMONG P-AZOPHENYLARSONATE-SPECIFIC ANTIBODIES

We showed previously that heavy chain gene junctional amino acid diffe rences among unmutated p-azophenylarsonate (Ars) Abs that share a uniq ue gene segment combination encoding these V regions, termed ''canonic al,'' alter affinity. To determine the contribution of junctional amin o acid differences to binding, we introduced, by site-directed mutagen esis, various amino acids at position 100 and/or 107 (sequential numbe ring) into the unmutated Ab 36-65. Among 22 mutant Abs, 15 preserved o r showed increased Ars binding (1- to 12.9-fold increase) relative to Ab 36-65, while 7 Abs exhibited lower affinity (less than or equal to 0.5-fold). As much as a 150-fold difference in Ars binding was observe d between 2 Abs with different sets of junctions (Asn100/Tyr107 and Va l100/Lys107). Thus, amino acid replacements at D gene junctions ran pr oduce changes in affinity greater than those for any V region somatic mutation observed thus far in vivo among anti-Ars Abs and, potentially ,can result in preferential selection of Abs containing certain juncti ons during affinity maturation. We combined five different junctional residue pairs with mutations at H chain positions 58 and 59 that are k nown to be recurrent in vivo and are associated with increased Ars aff inity. The mutant Abs all showed increased affinity, indicating that d espite variation in D gene junctions of Ars-binding canonical Abs, the combined mutations are additive for enhancement of Ars affinity. Thes e additive effects reflect the ''adaptability'' of the canonical gene segment combination in sustaining somatic mutations leading to affinit y maturation.