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 ∼10-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 ∼10 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.

Importance: Antibody 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.

FIG 1

Epitope recognition, HIV-1 neutralization, polyreactivity, and turbidity of 10E8 variants with alterations of coplanar paratope residues. (A) Fab 10E8 is shown in ribbon representation, with the MPER epitope in red and coplanar residues with the epitope highlighted. (B) Ninety-degree view of coplanar residues selected for alteration to tryptophan. (C) Effect of single and double tryptophan/phenylalanine substitutions on neutralization. Numbers represent the ratio of the IC₅₀ or IC₈₀ of variants to that of 10E8. (D) Cardiolipin binding assay with 4E10 and palivizumab (Synagis) as positive and negative controls, respectively; polyreactive binding based on an ELISA criterion of being three times the background (0.18 OD at 450 nm) is indicated by yellow highlighting. (E) Breadth and potency assessed on a panel of 20 HIV-1 isolates. (F) Turbidity of antibody 10E8 and HC6-S74Y/L3 variant.

FIG 2

Cocrystal structure of 10E8 S74W in complex with HIV-1 MPER. The variable domain is shown in C_α-backbone representation in light blue. All residues were superimposed with the parent 10E8 within a Cα RMSD of 1Å, except for the Cα of residue Ile75 (in red) in the heavy chain. Cα distances of all residues between the parent 10E8 in the constant domain were greater than 1Å and are colored in red. Residues that are different from the 10E8v4 structure described in Fig. 10 are labeled.

FIG 3

Reversion to germ line of hydrophobic residues in the framework region 3 of the 10E8 heavy chain variant (HC6-S74Y) enhances solubility. (A) Fab 10E8 in surface representation, with colors indicating electrostatic potential: red, electronegative; blue, electropositive; white, hydrophobic. A hydrophobic patch is formed by Leu72, Ile75, and Phe77. (B) Alteration of four hydrophobic residues to their germ line counterparts to create heavy chain variant 10E8v1. Sequences are shown based on the Kabat numbering scheme. (C) Turbidity of 10E8v1. (D) Neutralization potency of 10E8v1 versus the wild type.

FIG 4

Somatic 10E8 variants showed increased solubility but reduced potency versus 10E8. (A) Sequences of 10E8 and somatic variants (residues in red indicate reversion to germ line); (B) turbidity; (C) neutralization potency.

FIG 5

Swaps of variant heavy chain residues proximal to epitope and pairing with L3 light chain enhanced neutralization potency. (A) Structure of 10E8 highlighting location of residues altered to improve potency; (B) sequence of 10E8 heavy chain variants and alterations to improve potency; (C) turbidity of the variants in PBS; (D) neutralization potency.

FIG 6

Swaps of variant light chain residues further improved solubility. (A) Structure of 10E8 highlighting location of residues altered to improve potency; (B) sequence of 10E8 light chain variants and alterations to improve solubility; (C and D) turbidity of 10E8 light chain variants; (E) neutralization potency.

FIG 7

10E8v1, 10E8v4, and 10E8v5 retain extraordinary breadth and potency of the parent 10E8. (A) Breadth-potency curve for a panel of 200 HIV-1 isolates; (B) aggregate IC₅₀s (the percentage of viruses resistant to neutralization [IC₅₀ > 50 μg/ml] is shown at the top of each column).

FIG 8

10E8v1, 10E8v4, and 10E8v5 are soluble and monodisperse. (A) Solubility of 10E8 and 10E8 variants after dialysis in PBS, pH 7.4; (B) turbidity in PBS; (C) kinetic concentration assay, with the volume after centrifugation (left) and the absorbance at 280 nm (right); (D) dynamic light scattering indicating 10E8 to be polydispersed, whereas 10E8v1, 10E8v4, and 10E8v5 were monodispersed.

FIG 9

10E8v1, 10E8v4, and 10E8v5 showed no polyreactivity, with increased bioavailability in mice and macaques for 10E8v4 and 10E8v4-LS. (A) HEp2 cell staining assays on 10E8 variants. (B) Anti-cardiolipin ELISA. (C) BALB/c mice were divided into groups of three, and the mice in each group were administered intraperitoneally (IP) 100 μg of 10E8, 10E8v4, or 10E8v5 antibody on day 0. The serum antibody levels shown are the mean values for each group of mice, and error bars indicate standard deviations. (D) Serum antibody level assessment for rhesus macaques. IV, intravenous. “LS” indicates alteration of the heavy chain to increase affinity for neonatal Fc receptor.

FIG 10

Cocrystal structure of 10E8v4 in complex with HIV-1 MPER. Residues that differ between 10E8v4 and the parent 10E8 are highlighted in stick representation, labeled, and colored cyan.

FIG 11

Size exclusion chromatography anomaly resulted from slow conformational isomerization, which an engineered disulfide resolves. (A) Size exclusion chromatography showed 10E8v4 to elute as three peaks (top chromatogram); when each fraction was immediately reinjected, it eluted as a mostly single peak (left chromatograms). However, if each of the separated peaks was allowed to equilibrate for 22 h, each now isomerized into three peaks (right chromatograms). (B) Multiangle light scattering (SEC-MALS) analysis. MW, molecular weight in thousands. (C) Neutralization potency of 10E8v4 and 10E8v4-DS. (D) An engineered disulfide between residue 100e on the heavy chain and residue 30 on the light chain to prevent isomerization. (E) 10E8v4 with an interchain disulfide, 100eC-30C (DS), eluted as a single peak in size exclusion chromatography.