title: 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.
  
  
  
  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.
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Young D
authors.initials: YD
authors.lastname: Kwon
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Ivelin S
authors.initials: IS
authors.lastname: Georgiev
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Gilad
authors.initials: G
authors.lastname: Ofek
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Baoshan
authors.initials: B
authors.lastname: Zhang
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Mangaiarkarasi
authors.initials: M
authors.lastname: Asokan
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Robert T
authors.initials: RT
authors.lastname: Bailer
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Amy
authors.initials: A
authors.lastname: Bao
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: William
authors.initials: W
authors.lastname: Caruso
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Xuejun
authors.initials: X
authors.lastname: Chen
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Misook
authors.initials: M
authors.lastname: Choe
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Aliaksandr
authors.initials: A
authors.lastname: Druz
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Sung-Youl
authors.initials: SY
authors.lastname: Ko
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Mark K
authors.initials: MK
authors.lastname: Louder
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Krisha
authors.initials: K
authors.lastname: McKee
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Sijy
authors.initials: S
authors.lastname: O'Dell
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Amarendra
authors.initials: A
authors.lastname: Pegu
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Rebecca S
authors.initials: RS
authors.lastname: Rudicell
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Wei
authors.initials: W
authors.lastname: Shi
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Keyun
authors.initials: K
authors.lastname: Wang
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Yongping
authors.initials: Y
authors.lastname: Yang
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Mandy
authors.initials: M
authors.lastname: Alger
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Michael F
authors.initials: MF
authors.lastname: Bender
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Kevin
authors.initials: K
authors.lastname: Carlton
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Jonathan W
authors.initials: JW
authors.lastname: Cooper
authors.affiliation: Duke Human Vaccine Institute, Departments of Medicine, Surgery, Pediatrics and Immunology, Duke University School of Medicine, Durham, North Carolina, USA.|Center for HIV/AIDS Vaccine Immunology-Immunogen Discovery, Duke University, Durham, North Carolina, USA.
authors.forename: Julie
authors.initials: J
authors.lastname: Blinn
authors.affiliation: Duke Human Vaccine Institute, Departments of Medicine, Surgery, Pediatrics and Immunology, Duke University School of Medicine, Durham, North Carolina, USA.|Center for HIV/AIDS Vaccine Immunology-Immunogen Discovery, Duke University, Durham, North Carolina, USA.
authors.forename: Joshua
authors.initials: J
authors.lastname: Eudailey
authors.affiliation: Duke Human Vaccine Institute, Departments of Medicine, Surgery, Pediatrics and Immunology, Duke University School of Medicine, Durham, North Carolina, USA.|Center for HIV/AIDS Vaccine Immunology-Immunogen Discovery, Duke University, Durham, North Carolina, USA.
authors.forename: Krissey
authors.initials: K
authors.lastname: Lloyd
authors.affiliation: Duke Human Vaccine Institute, Departments of Medicine, Surgery, Pediatrics and Immunology, Duke University School of Medicine, Durham, North Carolina, USA.|Center for HIV/AIDS Vaccine Immunology-Immunogen Discovery, Duke University, Durham, North Carolina, USA.
authors.forename: Robert
authors.initials: R
authors.lastname: Parks
authors.affiliation: Duke Human Vaccine Institute, Departments of Medicine, Surgery, Pediatrics and Immunology, Duke University School of Medicine, Durham, North Carolina, USA.|Center for HIV/AIDS Vaccine Immunology-Immunogen Discovery, Duke University, Durham, North Carolina, USA.
authors.forename: S Munir
authors.initials: SM
authors.lastname: Alam
authors.affiliation: Duke Human Vaccine Institute, Departments of Medicine, Surgery, Pediatrics and Immunology, Duke University School of Medicine, Durham, North Carolina, USA.|Center for HIV/AIDS Vaccine Immunology-Immunogen Discovery, Duke University, Durham, North Carolina, USA.
authors.forename: Barton F
authors.initials: BF
authors.lastname: Haynes
authors.affiliation: The Aaron Diamond AIDS Research Center, Rockefeller University, New York, New York, USA.
authors.forename: Neal N
authors.initials: NN
authors.lastname: Padte
authors.affiliation: The Aaron Diamond AIDS Research Center, Rockefeller University, New York, New York, USA.
authors.forename: Jian
authors.initials: J
authors.lastname: Yu
authors.affiliation: The Aaron Diamond AIDS Research Center, Rockefeller University, New York, New York, USA.
authors.forename: David D
authors.initials: DD
authors.lastname: Ho
authors.affiliation: HIV-Specific Immunity Section, Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Jinghe
authors.initials: J
authors.lastname: Huang
authors.affiliation: HIV-Specific Immunity Section, Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Mark
authors.initials: M
authors.lastname: Connors
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Richard M
authors.initials: RM
authors.lastname: Schwartz
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA jmascola@mail.nih.gov pdkwong@nih.gov.
authors.forename: John R
authors.initials: JR
authors.lastname: Mascola
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA jmascola@mail.nih.gov pdkwong@nih.gov.
authors.forename: Peter D
authors.initials: PD
authors.lastname: Kwong
mesh_terms: D000818:Animals; D057134:Antibodies, Neutralizing / Q000737:chemistry* / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D002623:Chemistry Techniques, Analytical; D018360:Crystallography, X-Ray; D004220:Disulfides; D015483:HIV Antibodies / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D015497:HIV-1 / Q000276:immunology*; D006207:Half-Life; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008253:Macaca mulatta; D008958:Models, Molecular; D012995:Solubility
chemical_list: D057134:Antibodies, Neutralizing; D004220:Disulfides; D015483:HIV Antibodies