Optimization of the Solubility of HIV-1-Neutralizing Antibody 10E8 through Somatic Variation and Structure-Based Design

Abstract

Extraordinary antibodies capable of near pan-neutralization of HIV-1 have been identified. One of the broadest is antibody 10E8, which recognizes the membrane-proximal external region (MPER) of the HIV-1 envelope and neutralizes >95% of circulating HIV-1 strains. If delivered passively, 10E8 might serve to prevent or treat HIV-1 infection. Antibody 10E8, however, is markedly less soluble than other antibodies. Here, we describe the use of both structural biology and somatic variation to develop optimized versions of 10E8 with increased solubility. From the structure of 10E8, we identified a prominent hydrophobic patch; reversion of four hydrophobic residues in this patch to their hydrophilic germ line counterparts resulted in an ϳ10-fold decrease in turbidity. We also used somatic variants of 10E8, identified previously by next-generation sequencing, to optimize heavy and light chains; this process yielded several improved variants. Of these, variant 10E8v4 with 26 changes versus the parent 10E8 was the most soluble, with a paratope we showed crystallographically to be virtually identical to that of 10E8, a potency on a panel of 200 HIV-1 isolates also similar to that of 10E8, and a half-life in rhesus macaques of ϳ10 days. An anomaly in 10E8v4 size exclusion chromatography that appeared to be related to conformational isomerization was resolved by engineering an interchain disulfide. Thus, by combining a structure-based approach with natural variation in potency and solubility from the 10E8 lineage, we successfully created variants of 10E8 which retained the potency and extraordinary neutralization breadth of the parent 10E8 but with substantially increased solubility. IMPORTANCEAntibody 10E8 could be used to prevent HIV-1 infection, if manufactured and delivered economically. It suffers, however, from issues of solubility, which impede manufacturing. We hypothesized that the physical characteristic of 10E8 could be improved through rational design, without compromising breadth and potency. We used structural biology to identify hydrophobic patches on 10E8, which did not appear to be involved in 10E8 function. Reversion of hydrophobic residues in these patches to their hydrophilic germ line counterparts increased solubility. Next, clues from somatic variants of 10E8, identified by next-generation sequencing, were incorporated. A combination of structure-based design and somatic variant optimization led to 10E8v4, with substantially improved solubility and similar potency compared to the parent 10E8. The cocrystal structure of antibody 10E8v4 with its HIV-1 epitope was highly similar to that with the parent 10E8, despite 26 alterations in sequence and substantially improved solubility. Antibody 10E8v4 may be suitable for manufacturing. O ver the last 5 years, extraordinary antibodies capable of effectively neutralizing human immunodeficiency virus type 1 (HIV-1) have been identified (1-9). In addition to serving as potential templates for an antibody-based HIV-1 vaccine, the passive delivery of these ant...

Surface-Matrix Screening Identifies Semi-specific Interactions that Improve Potency of a Near Pan-reactive HIV-1-Neutralizing Antibody

“…(Figure 1F). To understand the impact of Arg5 HC and Phe100c HC mutations on the structure of 10E8, we added these mutations to antibody 10E8v4 (Kwon et al, 2016), a solubility-improved variant with breadth and neutralization potency similar to those of 10E8, and crystallized the antigen-binding fragment (Fab) of 10E8v4-5R+100cF in complex with a 19-mer peptide encompassing its MPER epitope. Diffraction data extended to 3.1 Å resolution, and structure solution by molecular replacement and refinement yielded an R work /R free of 0.24/0.28 (Table S2).…”

Structural basis for broad neutralization of HIV-1 through the molecular recognition of 10E8 helical epitope at the membrane interface

“…Also, consistent with a higher degree of cross-reactivity, 10E8 neutralizes SIVs that infect chimpanzees and gorillas with nanomolar potency8, surpassing the efficacy of other broadly neutralizing antibodies directed against different vulnerability regions on Env. Engineered versions of the 10E8 with improved solubility have been recently reported10, a finding that broadens the therapeutic potential of this broadly neutralizing antibody69.…”

“…It appears that this potency is developed after extensive somatic hypermutation of the heavy-chain complementarity determining regions 2 and 3 (CDRH2 and CDRH3, respectively)3. The exceptionally high degree of conservation of the MPER sequence45 justifies immunotherapeutic approaches based on the 10E8 antibody678910. Supporting its functional activity in vivo , 10E8 confers complete protection to rhesus macaques against infection by a simian immunodeficiency virus-HIV chimera6.…”

Structural Basis of Pan-Ebolavirus Neutralization by an Antibody Targeting the Glycoprotein Fusion Loop

“…These examples indicate a conserved immunological solution for presenting hydrophobic residues on CDRH3 loops (Mian et al, 1991), although antibodies necessarily form highly unique structures to accommodate such a wide range of ligands (Regep et al, 2017), highlighting the importance of structural data to assist therapeutic development. In cases in which these antibodies suffer from solubility, for example, with the HIV antibody 10E8, it has been possible to engineer framework residues to counteract these issues (Kwon etal., 2016). For ADI-15878, however, hydrophobic residues are largely sequestered within the paratope before making contact, possibly enabling greater solubility in the unli- ganded state and generating higher specificity.…”