Somatic hypermutation and clonal selection lead to B cells expressing high-affinity antibodies. Here we show that somatic mutations not only play a critical role in antigen binding, they also affect the thermodynamic stability of the antibody molecule. Somatic mutations directly involved in antigen recognition by antibody 93F3, which binds a relatively small hapten, reduce the melting temperature compared with its germ-line precursor by up to 9 °C. The destabilizing effects of these mutations are compensated by additional somatic mutations located on surface loops distal to the antigen binding site. Similarly, somatic mutations enhance both the affinity and thermodynamic stability of antibody OKT3, which binds the large protein antigen CD3. Analysis of the crystal structures of 93F3 and OKT3 indicates that these somatic mutations modulate antibody stability primarily through the interface of the heavy and light chain variable domains. The historical view of antibody maturation has been that somatic hypermutation and subsequent clonal selection increase antigen–antibody specificity and binding energy. Our results suggest that this process also optimizes protein stability, and that many peripheral mutations that were considered to be neutral are required to offset deleterious effects of mutations that increase affinity. Thus, the immunological evolution of antibodies recapitulates on a much shorter timescale the natural evolution of enzymes in which function and thermodynamic stability are simultaneously enhanced through mutation and selection.

The monoclonal antibody 48G7 differs from its germline precursor by ten somatic mutations, a number of which appear to be functionally silent. We analyzed the effects of individual somatic mutations and combinations thereof on both antibody binding affinity and thermal stability. Individual somatic mutations that enhance binding affinity to hapten decrease the stability of the germline antibody; combining these binding mutations produced a mutant with high affinity for hapten but exceptionally low stability. Adding back each of the remaining somatic mutations restored thermal stability. These results, in conjunction with recently published studies, suggest an expanded role for somatic hypermutation in which both binding affinity and stability are optimized during clonal selection.

The X‐ray crystal structure of a bovine antibody (BLV1H12) revealed a unique structure in its ultralong heavy chain complementarity determining region 3 (CDR3H) that folds into a solvent‐exposed β‐strand “stalk” fused to a disulfide crosslinked “knob” domain. We have substituted an antiparallel heterodimeric coiled‐coil motif for the β‐strand stalk in this antibody. The resulting antibody (Ab‐coil) expresses in mammalian cells and has a stability similar to that of the parent bovine antibody. MS analysis of H–D exchange supports the coiled‐coil structure of the substituted peptides. Substitution of the knob‐domain of Ab‐coil with bovine granulocyte colony‐stimulating factor (bGCSF) results in a stably expressed chimeric antibody, which proliferates mouse NFS‐60 cells with a potency comparable to that of bGCSF. This work demonstrates the utility of this novel coiled‐coil CDR3 motif as a means for generating stable, potent antibody fusion proteins with useful pharmacological properties.

The X-ray crystal structure of a bovine antibody (BLV1H12) revealed a unique structure in its ultralong heavy chain complementarity determining region 3 (CDR3H) that folds into a solvent-exposed b-strand "stalk" fused to a disulfide crosslinked "knob" domain. We have substituted an antiparallel heterodimeric coiled-coil motif for the b-strand stalk in this antibody. The resulting antibody (Ab-coil) expresses in mammalian cells and has a stability similar to that of the parent bovine antibody. MS analysis of H-D exchange supports the coiled-coil structure of the substituted peptides. Substitution of the knob-domain of Ab-coil with bovine granulocyte colonystimulating factor (bGCSF) results in a stably expressed chimeric antibody, which proliferates mouse NFS-60 cells with a potency comparable to that of bGCSF. This work demonstrates the utility of this novel coiled-coil CDR3 motif as a means for generating stable, potent antibody fusion proteins with useful pharmacological properties.

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