Random mutagenesis of two complementarity determining region amino acids yields an unexpectedly high frequency of antibodies with increased affinity for both cognate antigen and autoantigen.

Abstract

SummaryTo gain insight into the mechanism and limitations of antibody affinity maturation leading to memory B cell formation, we generated a phage display library of random mutants at heavy chain variable (V) complementarity determining region 2 positions 58 and 59 of an anti-pazophenylarsonate (Ars) Fab. Single amino acid substitutions at these positions resulting from somatic hypermutation are recurrent products of affinity maturation in vivo. Most of the ex vivo mutants retained specificity for Ars. Among the many mutants displaying high Ars-binding activity, only one contained a position 58 and 59 amino acid combination that has been previously observed among monoclonal antibodies (mAbs) derived from Ars-immunized mice. Affinity measurements on 14 of the ex vivo mutants with high Ars-binding activity showed that 11 had higher intrinsic affinities for Ars than the wild-type V region. However, nine of these Fabs also bound strongly to denatured DNA, a property neither displayed by the wild-type V region nor observed among the mutants characteristic of in vivo affinity maturation. These data suggest that ex vivo enhancement of mAb affinity via site-directed and random mutagenesis approaches may often lead to a reduction in antibody specificity that could complicate the use of the resulting mAbs for diagnostic and therapeutic applications. Moreover, the data are compatible with a hypothesis proposing that increased specificity for antigen, rather than affinity per se, is the driving force for formation of the memory B cell compartment.D uring the course of a T cell-dependent antibody response, the V regions of antibodies expressed by B cells differentiating to memory are structurally altered because of hypermutation of the genes encoding them. Working in concert with hypermutation, a stringent process of antigen selection results in the affinity maturation of the antibody response. Frequent end products of the mutation-selection process are V regions containing recurrent amino acid changes at particular positions (1-3). Site-directed mutagenesis studies have shown that many of these recurrent mutations individually result in increased affinity for the eliciting antigen (2-4).Affinity for antigen is only one of the factors that could influence the composition of the somatically mutated antibody repertoire expressed by the memory B cell compartment, however. Somatic mutation takes place throughout the length of V region genes, but is not a random process; hotspots and hot areas for mutation clearly exist (1, 5-7). In some responses, mutants with altered idiotypy (8) or antigen binding on-rate (9) may be differentially selected. It has also been suggested that somatic mutation will create autoantibodies de novo (10-13), and that peripheral tolerance mechanisms must eliminate B cells expressing these autoreactive antibodies (14).Secondary anti-p-azophenylarsonate (Ars) 1 antibodies from A/J mice are largely encoded by a single combination of V gene segments (termed "canonical"). Recurrent amino acid substitutions du...

Differential regulation of self-reactivity discriminates between IgG+human circulating memory B cells and bone marrow plasma cells

“…Selection in the germinal center is based on high affinity of the B cell antigen receptor that is necessary to capture and present sufficient amounts of antigen to T follicular helper cells, presumably the limiting factor in the selection process (21-23). The low level of self-and polyreactivity that we measured in IgG-positive bone marrow plasma cells may be a by-product of somatic mutations and does not exclude the possibility that the antibodies show high affinity for foreign antigens that were not tested (24). Antibody self-and polyreactivity may be tolerated during germinal center reactions if affinity for the activating antigen is high enough and antigen presentation is sufficient to receive proper help from cognate T follicular helper cells.…”

Quantitatively Reduced Participation of Anti-Nuclear Antigen B Cells That Down-Regulate B Cell Receptor during Primary Development in the Germinal Center/Memory B Cell Response to Foreign Antigen

“…In anamnestic HKIR AFCs, this ratio in CDRs was somewhat lower than in GCs, but this ratio in FRs was ϳ1. In addition, in the V H sequences obtained from anamnestic AFC foci, two mutations in CDR codons 58 and 59 known to increase affinity for Ars (27), were often observed. From the six independent AFC foci analyzed from HKIR3 B6 chimeras, five gave rise to multiple V H sequences that had either one, or both, of these position 58 and 59 mutations (data not shown).…”

“…Ag (anti-Ars), DNA, and clonotype-specific (E4) serum Igs were measured by solid-phase ELISA on 96-well plates (Immulon-4; Thermo Electron) as previously described (27).…”

Primary Development and Participation in a Foreign Antigen-Driven Immune Response of a Chromatin-Reactive B Cell Clonotype Are Not Influenced by TLR9 or Other MyD88-Dependent TLRs

“…Ars-specific total serum IgM or IgG and E4 was measured by ELISA on 96-well plates (Immunolon-4; Thermo Electron) as previously described (34).…”