Chemical Basis for the Affinity Maturation of a Camel Single Domain Antibody

Abstract

Affinity maturation of classic antibodies supposedly proceeds through the pre-organization of the reactive germ line conformational isomer. It is less evident to foresee how this can be accomplished by camelid heavychain antibodies lacking light chains. Although these antibodies are subjected to somatic hypermutation, their antigen-binding fragment consists of a single domain with restricted flexibility in favor of binding energy. An antigen-binding domain derived from a dromedary heavy-chain antibody, cAb-Lys3, accumulated five amino acid substitutions in CDR1 and CDR2 upon maturation against lysozyme. Three of these residues have hydrophobic side chains, replacing serines, and participate in the hydrophobic core of the CDR1 in the mature antibody, suggesting that conformational rearrangements might occur in this loop during maturation. However, transition state analysis of the binding kinetics of mature cAb-Lys3 and germ line variants show that the maturation of this antibody relies on events late in the reaction pathway. This is reflected by a limited perturbation of k a and a significantly decreased k d upon maturation. In addition, binding reactions and the maturation event are predominantly enthalpically driven. Therefore, maturation proceeds through the increase of favorable binding interactions, or by the reduction of the enthalpic penalty for desolvation, as opposed to large entropic penalties associated with conformational changes and structural plasticity. Furthermore, the crystal structure of the mutant with a restored germ line CDR2 sequence illustrates that the matured hydrophobic core of CDR1 in cAb-Lys3 might be compensated in the germ line precursor by burying solvent molecules engaged in a stable hydrogen-bonding network with CDR1 and CDR2.The success of the humoral immunity relies heavily on the fast generation of highly specific and tight binding antibodies against virtually any foreign target molecule. These antibodies occur concomitantly as membrane-bound receptors on B-cells or as soluble proteins. The antigen-combining sites of classic antibodies are composed of two variable domains (VH and VL) 1 located at the N-terminal end of the heavy and light chains (1). Both domains display high sequence diversity, mainly concentrated in three complementarity-determining regions (CDRs) in each V domain. Structurally, these regions form loops, clustered at the N-terminal side of the folded domain, and provide the antigen contacts. Upon antigenic challenge, somatic hypermutation or gene conversion mechanisms further diversify the V sequences as well as the affinities of reactive B-cell receptors. A clonal selection system then enriches for those B-lymphocytes that display receptors with a higher affinity for the antigen. This process of somatic hypermutation and clonal selection is known as antibody (or B-cell receptor) affinity maturation (2-4).It has been reported on several occasions that antibody paratopes for haptenic molecules or small peptides lose conformational plasticity upon maturation. We...

Functional Maturation of the Human Antibody Response to Rotavirus

“…In contrast to our findings, a previous detailed study of a virus-neutralizing Ab using engineered mutations selected for affinity suggested that improvements in the on-rate enhanced neutralizing activity (18). Abs directed against some model Ags that have been characterized previously at a kinetic level also show an importance of the off-rate in affinity maturation caused by somatic mutations (14,15,17,37,38).…”

First molecular and biochemical analysis ofin vivoaffinity maturation in an ectothermic vertebrate

“…However, the degree to which different organisms rely on the fine-tuning conferred through somatic hypermutation appears to somewhat of a ''sliding scale.'' According to recent publications, the camel single-domain VHH repertoire seems structurally limited (30), so low-( M) affinity binders are selected from the primary repertoire, and mutation is required to significantly (1,000-fold) improve binding affinity (11). In contrast, the study of an IgNAR V-region family presented here demonstrated that high-affinity (nM) binders could be selected directly from the primary repertoire.…”

“…Furthermore, little is known of how singledomain antibody fragments, which lack one of the V domains, are matured. A study of an anti-hen egg-white lysozyme (HEL) camel heavy-chain antibody variable (VHH) domain in its germ line and mutated form showed that, despite the lack of binding-site plasticity conferred by pairing with a second domain and the presence of a complementarity-determining region (CDR)3-constraining disulphide bond, a 300-fold increase in binding affinity was achieved by increasing VHH-antigen complementarity and apolar buried surface area (11). However, all but one of the clones in the study were generated in vitro and, so, tell little of how the antigen-stimulated repertoire is generated.…”

Structures of P-glycoprotein reveal its conformational flexibility and an epitope on the nucleotide-binding domain

“…Thus, although Nb592 itself might not directly be used as a clinical inhibitor, it provides a template for the future development of this type of molecular scaffold. Several techniques may also be applied to improve the binding affinity of Nb592 (31).…”