title: Eliminating antibody polyreactivity through addition of N-linked glycosylation.
abstract: Antibody polyreactivity can be an obstacle to translating a candidate antibody into a clinical product. Standard tests such as antibody binding to cardiolipin, HEp-2 cells, or nuclear antigens provide measures of polyreactivity, but its causes and the means to resolve are often unclear. Here we present a method for eliminating antibody polyreactivity through the computational design and genetic addition of N-linked glycosylation near known sites of polyreactivity. We used the HIV-1-neutralizing antibody, VRC07, as a test case, since efforts to increase VRC07 potency at three spatially distinct sites resulted in enhanced polyreactivity. The addition of N-linked glycans proximal to the polyreactivity-enhancing mutations at each of the spatially distinct sites resulted in reduced antibody polyreactivity as measured by (i) anti-cardiolipin ELISA, (ii) Luminex AtheNA Multi-Lyte ANA binding, and (iii) HEp-2 cell staining. The reduced polyreactivity trended with increased antibody concentration over time in mice, but not with improved overall protein stability as measured by differential scanning calorimetry. Moreover, glycan proximity to the site of polyreactivity appeared to be a critical factor. The results provide evidence that antibody polyreactivity can result from local, rather than global, features of an antibody and that addition of N-linked glycosylation can be an effective approach to reducing antibody polyreactivity.
authors: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, 20892.
authors: Gwo-Yu
authors: GY
authors: Chuang
authors: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, 20892.
authors: Baoshan
authors: B
authors: Zhang
authors: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, 20892.
authors: Krisha
authors: K
authors: McKee
authors: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, 20892.
authors: Sijy
authors: S
authors: O'Dell
authors: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, 20892.
authors: Young Do
authors: YD
authors: Kwon
authors: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, 20892.
authors: Tongqing
authors: T
authors: Zhou
authors: Duke Human Vaccine Institute, Duke University Medical Center, Durham, North Carolina, 103020.
authors: Julie
authors: J
authors: Blinn
authors: Duke Human Vaccine Institute, Duke University Medical Center, Durham, North Carolina, 103020.
authors: Krissey
authors: K
authors: Lloyd
authors: Duke Human Vaccine Institute, Duke University Medical Center, Durham, North Carolina, 103020.
authors: Robert
authors: R
authors: Parks
authors: Duke Human Vaccine Institute, Duke University Medical Center, Durham, North Carolina, 103020.
authors: Tarra
authors: T
authors: Von Holle
authors: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, 20892.
authors: Sung-Youl
authors: SY
authors: Ko
authors: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, 20892.
authors: Wing-Pui
authors: WP
authors: Kong
authors: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, 20892.
authors: Amarendra
authors: A
authors: Pegu
authors: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, 20892.
authors: Keyun
authors: K
authors: Wang
authors: Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, Oxford, OX1, 3QU, United Kingdom.
authors: Kavitha
authors: K
authors: Baruah
authors: Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, Oxford, OX1, 3QU, United Kingdom.
authors: Max
authors: M
authors: Crispin
authors: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, 20892.
authors: John R
authors: JR
authors: Mascola
authors: Duke Human Vaccine Institute, Duke University Medical Center, Durham, North Carolina, 103020.
authors: M Anthony
authors: MA
authors: Moody
authors: Duke Human Vaccine Institute, Duke University Medical Center, Durham, North Carolina, 103020.
authors: Barton F
authors: BF
authors: Haynes
authors: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, 20892.
authors: Ivelin S
authors: IS
authors: Georgiev
authors: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, 20892.
authors: Peter D
authors: PD
authors: Kwong
mesh_terms: D000818:Animals; D057134:Antibodies, Neutralizing / Q000737:chemistry* / Q000276:immunology*; D000918:Antibody Specificity / Q000235:genetics / Q000276:immunology*; D006031:Glycosylation; D015483:HIV Antibodies / Q000737:chemistry* / Q000276:immunology*; D056945:Hep G2 Cells; D006801:Humans; D051379:Mice; D009154:Mutation; D015202:Protein Engineering / Q000379:methods*
chemical_list: D057134:Antibodies, Neutralizing; D015483:HIV Antibodies
keywords: antibody engineering; bioinformatics; broadly neutralizing antibody; glycan engineering; polyreactivity