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Strategic addition of an N-linked glycan to a monoclonal antibody improves its HIV-1–neutralizing activity

Abstract

Ibalizumab is a humanized monoclonal antibody that binds human CD4—a key receptor for HIV—and blocks HIV-1 infection. However, HIV-1 strains with mutations resulting in loss of an N-linked glycan from the V5 loop of the envelope glycoprotein gp120 are resistant to ibalizumab. Previous structural analysis suggests that this glycan fills a void between the gp120 V5 loop and the ibalizumab light chain, perhaps causing steric hindrance that disrupts viral entry. If this void contributes to HIV-1 resistance to ibalizumab, we reasoned that 'refilling' it by engineering an N-linked glycan into the ibalizumab light chain at a position spatially proximal to gp120 V5 may restore susceptibility to ibalizumab. Indeed, one such ibalizumab variant neutralized 100% of 118 diverse HIV-1 strains tested in vitro, including 10 strains resistant to parental ibalizumab. These findings demonstrate that the strategic placement of a glycan in the variable region of a monoclonal antibody can substantially enhance its activity.

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Figure 1: Model of glycosylation in V5 of HIV-1 gp120, in the context of both CD4 and ibalizumab (using PyMOL).
Figure 2: N-linked glycosylation in the L chain of ibalizumab.
Figure 3: Neutralization activities of WT ibalizumab and its L-chain mutants.
Figure 4: The influence of glycan size on the HIV-1 neutralization activity of LM52.
Figure 5: Neutralization of a panel of 118 HIV-1 Env pseudoviruses.
Figure 6: HIV-1 strain coverage of LM52.

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References

  1. Baeten, J.M. et al. Antiretroviral prophylaxis for HIV prevention in heterosexual men and women. N. Engl. J. Med. 367, 399–410 (2012).

    Article  CAS  Google Scholar 

  2. Thigpen, M.C. et al. Antiretroviral preexposure prophylaxis for heterosexual HIV transmission in Botswana. N. Engl. J. Med. 367, 423–434 (2012).

    Article  CAS  Google Scholar 

  3. Van Damme, L. et al. Preexposure prophylaxis for HIV infection among African women. N. Engl. J. Med. 367, 411–422 (2012).

    Article  CAS  Google Scholar 

  4. Huber, M., Olson, W.C. & Trkola, A. Antibodies for HIV treatment and prevention: window of opportunity? Curr. Top. Microbiol. Immunol. 317, 39–66 (2008).

    CAS  PubMed  Google Scholar 

  5. Baba, T.W. et al. Human neutralizing monoclonal antibodies of the IgG1 subtype protect against mucosal simian-human immunodeficiency virus infection. Nat. Med. 6, 200–206 (2000).

    Article  CAS  Google Scholar 

  6. Mascola, J.R. et al. Protection of Macaques against pathogenic simian/human immunodeficiency virus 89.6PD by passive transfer of neutralizing antibodies. J. Virol. 73, 4009–4018 (1999).

    Article  CAS  Google Scholar 

  7. Wu, X. et al. Rational design of envelope identifies broadly neutralizing human monoclonal antibodies to HIV-1. Science 329, 856–861 (2010).

    Article  CAS  Google Scholar 

  8. Walker, L.M. et al. Broad and potent neutralizing antibodies from an African donor reveal a new HIV-1 vaccine target. Science 326, 285–289 (2009).

    Article  CAS  Google Scholar 

  9. Scheid, J.F. et al. Sequence and structural convergence of broad and potent HIV antibodies that mimic CD4 binding. Science 333, 1633–1637 (2011).

    Article  CAS  Google Scholar 

  10. Walker, L.M. et al. Broad neutralization coverage of HIV by multiple highly potent antibodies. Nature 477, 466–470 (2011).

    Article  CAS  Google Scholar 

  11. Moldt, B. et al. Highly potent HIV-specific antibody neutralization in vitro translates into effective protection against mucosal SHIV challenge in vivo. Proc. Natl. Acad. Sci. USA 109, 18921–18925 (2012).

    Article  CAS  Google Scholar 

  12. Diskin, R. et al. Increasing the potency and breadth of an HIV antibody by using structure-based rational design. Science 334, 1289–1293 (2011).

    Article  CAS  Google Scholar 

  13. Huang, J. et al. Broad and potent neutralization of HIV-1 by a gp41-specific human antibody. Nature 491, 406–412 (2012).

    Article  CAS  Google Scholar 

  14. Balazs, A.B. et al. Antibody-based protection against HIV infection by vectored immunoprophylaxis. Nature 481, 81–84 (2011).

    Article  Google Scholar 

  15. Jacobson, J.M. et al. Antiviral activity of single-dose PRO 140, a CCR5 monoclonal antibody, in HIV-infected adults. J. Infect. Dis. 198, 1345–1352 (2008).

    Article  Google Scholar 

  16. Burkly, L.C. et al. Inhibition of HIV infection by a novel CD4 domain 2-specific monoclonal antibody. Dissecting the basis for its inhibitory effect on HIV-induced cell fusion. J. Immunol. 149, 1779–1787 (1992).

    CAS  PubMed  Google Scholar 

  17. Jacobson, J.M. et al. Safety, pharmacokinetics, and antiretroviral activity of multiple doses of ibalizumab (formerly TNX-355), an anti-CD4 monoclonal antibody, in human immunodeficiency virus type 1-infected adults. Antimicrob. Agents Chemother. 53, 450–457 (2009).

    Article  CAS  Google Scholar 

  18. Dimitrov, A. Ibalizumab, a CD4-specific mAb to inhibit HIV-1 infection. Curr. Opin. Investig. Drugs 8, 653–661 (2007).

    CAS  PubMed  Google Scholar 

  19. Kuritzkes, D.R. et al. Antiretroviral activity of the anti-CD4 monoclonal antibody TNX-355 in patients infected with HIV type 1. J. Infect. Dis. 189, 286–291 (2004).

    Article  CAS  Google Scholar 

  20. Zhang, X.Q., Sorensen, M., Fung, M. & Schooley, R.T. Synergistic in vitro antiretroviral activity of a humanized monoclonal anti-CD4 antibody (TNX-355) and enfuvirtide (T-20). Antimicrob. Agents Chemother. 50, 2231–2233 (2006).

    Article  CAS  Google Scholar 

  21. Boon, L. et al. Development of anti-CD4 MAb hu5A8 for treatment of HIV-1 infection: preclinical assessment in non-human primates. Toxicology 172, 191–203 (2002).

    Article  CAS  Google Scholar 

  22. Song, R. et al. Epitope mapping of ibalizumab, a humanized anti-CD4 monoclonal antibody with anti-HIV-1 activity in infected patients. J. Virol. 84, 6935–6942 (2010).

    Article  CAS  Google Scholar 

  23. Freeman, M.M. et al. Crystal structure of HIV-1 primary receptor CD4 in complex with a potent antiviral antibody. Structure 18, 1632–1641 (2010).

    Article  CAS  Google Scholar 

  24. Toma, J. et al. Loss of asparagine-linked glycosylation sites in variable region 5 of human immunodeficiency virus type 1 envelope is associated with resistance to CD4 antibody ibalizumab. J. Virol. 85, 3872–3880 (2011).

    Article  CAS  Google Scholar 

  25. Pace, C.S. et al. Anti-CD4 monoclonal antibody ibalizumab exhibits breadth and potency against HIV-1, with natural resistance mediated by the loss of a V5 glycan in envelope. J. Acquir. Immune Defic. Syndr. 62, 1–9 (2013).

    Article  CAS  Google Scholar 

  26. Olden, K., Pratt, R.M. & Yamada, K.M. Role of carbohydrates in protein secretion and turnover: effects of tunicamycin on the major cell surface glycoprotein of chick embryo fibroblasts. Cell 13, 461–473 (1978).

    Article  CAS  Google Scholar 

  27. Vallee, F., Karaveg, K., Herscovics, A., Moremen, K.W. & Howell, P.L. Structural basis for catalysis and inhibition of N-glycan processing class I alpha 1,2-mannosidases. J. Biol. Chem. 275, 41287–41298 (2000).

    Article  CAS  Google Scholar 

  28. Reeves, P.J., Callewaert, N., Contreras, R. & Khorana, H.G. Structure and function in rhodopsin: high-level expression of rhodopsin with restricted and homogeneous N-glycosylation by a tetracycline-inducible N-acetylglucosaminyltransferase I-negative HEK293S stable mammalian cell line. Proc. Natl. Acad. Sci. USA 99, 13419–13424 (2002).

    Article  CAS  Google Scholar 

  29. Haynes, B.F. et al. Cardiolipin polyspecific autoreactivity in two broadly neutralizing HIV-1 antibodies. Science 308, 1906–1908 (2005).

    Article  CAS  Google Scholar 

  30. Mouquet, H. et al. Polyreactivity increases the apparent affinity of anti-HIV antibodies by heteroligation. Nature 467, 591–595 (2010).

    Article  CAS  Google Scholar 

  31. Pace, C.S. et al. Bispecific antibodies directed to CD4 domain 2 and HIV envelope exhibit exceptional breadth and picomolar potency against HIV-1. Proc. Natl. Acad. Sci. USA 110, 13540–13545 (2013).

    Article  CAS  Google Scholar 

  32. Arnold, J.N., Wormald, M.R., Sim, R.B., Rudd, P.M. & Dwek, R.A. The impact of glycosylation on the biological function and structure of human immunoglobulins. Annu. Rev. Immunol. 25, 21–50 (2007).

    Article  CAS  Google Scholar 

  33. Anthony, R.M. & Ravetch, J.V. A novel role for the IgG Fc glycan: the anti-inflammatory activity of sialylated IgG Fcs. J. Clin. Immunol. 30 (suppl. 1), S9–S14 (2010).

    Article  CAS  Google Scholar 

  34. Nimmerjahn, F. & Ravetch, J.V. Divergent immunoglobulin g subclass activity through selective Fc receptor binding. Science 310, 1510–1512 (2005).

    Article  CAS  Google Scholar 

  35. Pepinsky, R.B. et al. Improving the solubility of anti-LINGO-1 monoclonal antibody Li33 by isotype switching and targeted mutagenesis. Protein Sci. 19, 954–966 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Wu, S.J. et al. Structure-based engineering of a monoclonal antibody for improved solubility. Protein Eng. Des. Sel. 23, 643–651 (2010).

    Article  CAS  Google Scholar 

  37. Wei, X. et al. Antibody neutralization and escape by HIV-1. Nature 422, 307–312 (2003).

    Article  CAS  Google Scholar 

  38. Seaman, M.S. et al. Standardized assessment of NAb responses elicited in rhesus monkeys immunized with single- or multi-clade HIV-1 envelope immunogens. Virology 367, 175–186 (2007).

    Article  CAS  Google Scholar 

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Acknowledgements

The authors thank N. Padte for project management; Y. Huang, M. Tsuji, M.-W. Chen, C. Andrews, M. Sun and J. Yu for helpful discussions; and M.C. Nussenzweig for reagents. We also thank T.-L. Hsu, P.-C. Chen and the MS Core Facility at the Genomics Research Center, Academia Sinica (Taiwan), for glycoform profiling. Funding support for this study was provided by the Bill and Melinda Gates Foundation (OPP50714 and OPP1040731) via the Collaboration for AIDS Vaccine Discovery. Additional funding support was provided by the US National Institutes of Health grant number 1DP1DA033263-01.

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Authors

Contributions

R.S. and D.D.H. conceived the study and designed the experiments. R.S., D.F. and M.S.S. performed the experiments. D.A.O., D.F. and R.S. carried out the structural analyses. R.S. and D.D.H. analyzed the data and wrote the manuscript.

Corresponding author

Correspondence to David D Ho.

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Competing interests

D.D.H. is the scientific founder of TaiMed Biologics, Inc., which owns the commercial rights to ibalizumab. In this capacity, D.D.H. has equity in the company. R.S. and D.D.H. are inventors on a patent describing glycan-modified anti-CD4 antibodies for HIV prevention and therapy.

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Supplementary Figures 1–5 and Supplementary Tables 1, 2 and 4 (PDF 720 kb)

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Supplementary Table 3 (PDF 145 kb)

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Song, R., Oren, D., Franco, D. et al. Strategic addition of an N-linked glycan to a monoclonal antibody improves its HIV-1–neutralizing activity. Nat Biotechnol 31, 1047–1052 (2013). https://doi.org/10.1038/nbt.2677

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