Structural correlates of high antibody affinity: three engineered amino acid substitutions can increase the affinity of an anti-p-azophenylarsonate antibody 200-fold.

Abstract

Communicated by Alfred Nisonoff, April 9, 1990 (received for review February 22, 1990 ABSTRACTThe basis for the 200-fold difference in affinity between two hybridoma antibodies specific for the hapten p-azophenylarsonate (Ars) that have diversified by somatic hypermutation was examined. Oligonucleotide-directed mutagenesis was used to sequentially convert the nucleotide sequence ofthe lower-affinity antibody into that ofthe higher-affinity one, and the mutant antibodies generated by transfection of hybridoma cells were analyzed for affinity to Ars-tyrosine. The data

The Primary Antibody Repertoire Represents a Linked Network of Degenerate Antigen Specificities

“…A significant, perhaps unexpected, consequence of receptor plasticity was that it also allowed for a convergence of Ab reactivities, with binding affinities being well within the range described for primary immune responses (12,(42)(43)(44). Notably, this was predominantly supported by entropy-controlled binding pathways and required varying degrees of adaptability by the individual germline Abs.…”

Structural Analysis of Mutants of High-Affinity and Low-Affinityp-Azophenylarsonate-Specific Antibodies Generated by Alanine Scanning of Heavy Chain Complementarity-Determining Region 2

“…It was also shown that, taken together, the 11 L chain mutations in 36 -71 Ab do not affect Ab affinity, as expression of 36 -71 H chain with either 36 -71L or 36 -65L resulted in Abs with indistinguishable affinity, despite the fact that several L chain mutations in 36 -71 are in CDRs ( Fig. 1) (11). Thus, high-affinity binding in 36 -71 was associated with mutations in the H chain.…”

“…The production of the murine A/J hybridoma cell lines 36 -65 and 36 -71 (␥1, ), and 36 -65 and 36 -71 transfectomas (IgG2b (␥ 2 b), ) and their H chain loss variants 36 -65L and 36 -71L were previously described (4,11,15). …”

“…It was shown by Sharon et al (9,11) that when two contiguous residues in H chain complementarity-determining region (HCDR2) of the germline-encoded 36 -65 Ab were mutated from Thr 58 to Ile and Lys 59 to Thr (similar to 36 -71) ( Fig. 1), Ab affinity increased 8-fold.…”

“…However, the affinity of 36 -71 is 100-to 200-fold higher than 36 -65 (9,10). Two mechanisms have been shown to contribute to the higher affinity of 36 -71 Ab: D gene junctional rearrangement and accumulation and selection of favorable somatic mutations in the H chain (11).…”