Mutational Analysis of 48G7 Reveals that Somatic Hypermutation Affects Both Antibody Stability and Binding Affinity

Abstract

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.

A Combination of Structural and Empirical Analyses Delineates the Key Contacts Mediating Stability and Affinity Increases in an Optimized Biotherapeutic Single-chain Fv (scFv)

“…Importantly, it has been shown that although in vivo-generated somatic mutations can improve the potency of antibodies, they can also destabilize the v-domains (30). In the case of 3B4, however, the unique V H -CDR3 interaction with CXCL13 was resistant to mutagenesis (13), and so the interface instability had to be repaired by mutation of a germline residue in the VL-CDR3.…”

“…5E shows that R93L removes a positively charged patch in the paratope, which may dilute the nearby attractive electrostatic interactions described above and introduce a repulsive interaction with Lys 46 of CXCL13. Leu 93 also makes new intra-domain interactions with both Trp 32 and Tyr 30 in V L -CDR1, leading to increased scFv stability (Fig. 5F).…”

A general approach to antibody thermostabilization

“…This may be especially true of antibodies generated and matured via in vitro selection systems frequently used to optimize antibody affinity in which there is no inherent selection for thermal stability. 7,8 The thermostabilized scaffold was then applied to improve the stability of 11 antibodies, including a number of FDA-approved therapeutics, resulting in as much as a 10 C improvement in T m with only minimal loss in antigen binding affinity. Stabilized antibodies were of either mouse or human origin, and originated from in vitro-or in vivo-based antibody generation methodologies.…”

“…In all cases but one, K D measurements for the stable-grafted antibodies were within 3-fold of the starting antibody. Cetuximab displayed an 8-fold loss in antigen binding affinity despite its 7 C increase in T m . Omalizumab, the only antibody in this group for which a T m improvement was not observed, had high HC homology to the stable framework (92.4%), yet the LC sequence was among the lowest homology to the stabilized framework of all antibodies tested (65%).…”

“…6 It has been suggested that these methods lack an inherent selection for thermostability that occurs in vivo. 7,8 To take advantage of the full breadth of specificity existing in both native and synthetic antibody repertoires, a general approach is needed for antibody stabilization that can be applied across multiple antigen specificities and antibody formats, without compromising antigen binding affinity. The previously reported 9 stabilization and affinity maturation of an anti-MS2 antibody was achieved by a combination of grafting complementarity-determining regions (CDRs) onto known stable frameworks, 10 consensus design, 11 disulfide bond engineering, 12,13 and computational design.…”