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

Journal name:
Nature Biotechnology
Volume:
31,
Pages:
1047–1052
Year published:
DOI:
doi:10.1038/nbt.2677
Received
Accepted
Published online

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.

At a glance

Figures

  1. Model of glycosylation in V5 of HIV-1 gp120, in the context of both CD4 and ibalizumab (using PyMOL).
    Figure 1: Model of glycosylation in V5 of HIV-1 gp120, in the context of both CD4 and ibalizumab (using PyMOL).

    The complex was modeled by superimposing the structure of D1 and D2 of CD4 in complex with gp120 (Protein Data Bank (PDB) accession number 2NXY) onto the same domains of CD4 in complex with ibalizumab (PDB 3O2D). The glycan (dark blue) was introduced at the relevant asparagine by superimposing the asparagine with that of a glycan-bound asparagine from PDB 3TYG. The H and L chains of ibalizumab are shown as cyan and magenta ribbons, respectively. The first two domains of human CD4 are green, whereas HIV-1 gp120 is orange/brown. (a) Man5GlcNac2 at the position 459 of gp120 in the V5 loop (N terminus). (b) Man5GlcNac2 at the position of 463 of gp120 in the V5 loop (C terminus).

  2. N-linked glycosylation in the L chain of ibalizumab.
    Figure 2: N-linked glycosylation in the L chain of ibalizumab.

    (a) Ibalizumab L-chain mutants (LMs) were constructed, co-transfected into 293A cells with the WT ibalizumab H chain plasmid, purified on a protein-A agarose column and analyzed by SDS-PAGE. WT ibalizumab was analyzed the same way. (b) Purified WT, LM30E, LM53 and LM52 antibodies were treated with or without PNGase F at denaturing conditions and analyzed by SDS-PAGE. (c) N-linked glycoforms on the L chain of LM52 produced in 293A cells were analyzed by mass spectrometry. Percentage of each glycoform among the total is plotted. Error bars, mean ± s.e.m.

  3. Neutralization activities of WT ibalizumab and its L-chain mutants.
    Figure 3: Neutralization activities of WT ibalizumab and its L-chain mutants.

    Neutralization against a panel of ibalizumab-resistant or partially ibalizumab-resistant pseudovirus or replication-competent HIV-1 strains was measured by TZM-bl assay. 96USHIPs9, BK132/GS009 and 96USHIPs7 are replication-competent. CAAN5342.A2-dd and AC10.0.29-dd are site-directed Env mutants without any potential N-linked glycosylation sites in V5 and are resistant or partially resistant to neutralization by WT ibalizumab. 9015-07 A1 and 1051-D927 are clade B transmitted founder viruses. The data represent three independent experiments.

  4. The influence of glycan size on the HIV-1 neutralization activity of LM52.
    Figure 4: The influence of glycan size on the HIV-1 neutralization activity of LM52.

    (a) LM52 was produced in HEK293A cells with tunicamycin (LM52-T) or kifunensine (LM52-K). Alternatively, LM52 was produced in the N-acetylglucosaminyltransferase I-negative GnT1(−) HEK293S cells (LM52-G). The purified LM52 proteins, together with unmodified LM52 and WT ibalizumab, were analyzed by SDS-PAGE. (b) Neutralization activities of ibalizumab and different glycan variants of LM52 against three ibalizumab-resistant pseudoviruses, as measured in TZM-bl cells. (c) Depiction (using PyMOL) of the space filled by glycans of representative conformations and sizes, when tagged on residue 52 of ibalizumab. Depiction is based on the model generated in Figure 1 and colors are the same. The 7-ring N-glycan, Man5GlcNac2, was extracted from PDB entry 3TYG. An 11-ring N-glycan, Man3GlcNac5Fuc, was extracted from PDB entry 3QUM. These results represent three independent experiments.

  5. Neutralization of a panel of 118 HIV-1 Env pseudoviruses.
    Figure 5: Neutralization of a panel of 118 HIV-1 Env pseudoviruses.

    Neutralization by LM52 and WT ibalizumab was measured in a TZM-bl assay. For each virus, black bars indicate MPI when tested at antibody concentrations up to 10 μg/ml, and the corresponding IC50 (μg/ml) or IC80 (μg/ml). Viruses are ordered by descending MPI for ibalizumab. Given the large number of viruses being tested, this experiment was done only once.

  6. HIV-1 strain coverage of LM52.
    Figure 6: HIV-1 strain coverage of LM52.

    Viral coverage of WT ibalizumab, LM52 and PG9, 10E8, VRC01 and NIH45-46G54W HIV-1 broadly neutralizing antibodies. LM52 and ibalizumab were tested at up to 10 μg/ml, whereas the other mAbs were tested at up to 50 μg/ml. The data for PG9, 10E8, VRC01 and NIH45-46G54W were obtained from the published literature7, 8, 12, 13 or newly generated by M.S.S.

Accession codes

Referenced accessions

References

  1. Baeten, J.M. et al. Antiretroviral prophylaxis for HIV prevention in heterosexual men and women. N. Engl. J. Med. 367, 399410 (2012).
  2. Thigpen, M.C. et al. Antiretroviral preexposure prophylaxis for heterosexual HIV transmission in Botswana. N. Engl. J. Med. 367, 423434 (2012).
  3. Van Damme, L. et al. Preexposure prophylaxis for HIV infection among African women. N. Engl. J. Med. 367, 411422 (2012).
  4. Huber, M., Olson, W.C. & Trkola, A. Antibodies for HIV treatment and prevention: window of opportunity? Curr. Top. Microbiol. Immunol. 317, 3966 (2008).
  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, 200206 (2000).
  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, 40094018 (1999).
  7. Wu, X. et al. Rational design of envelope identifies broadly neutralizing human monoclonal antibodies to HIV-1. Science 329, 856861 (2010).
  8. Walker, L.M. et al. Broad and potent neutralizing antibodies from an African donor reveal a new HIV-1 vaccine target. Science 326, 285289 (2009).
  9. Scheid, J.F. et al. Sequence and structural convergence of broad and potent HIV antibodies that mimic CD4 binding. Science 333, 16331637 (2011).
  10. Walker, L.M. et al. Broad neutralization coverage of HIV by multiple highly potent antibodies. Nature 477, 466470 (2011).
  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, 1892118925 (2012).
  12. Diskin, R. et al. Increasing the potency and breadth of an HIV antibody by using structure-based rational design. Science 334, 12891293 (2011).
  13. Huang, J. et al. Broad and potent neutralization of HIV-1 by a gp41-specific human antibody. Nature 491, 406412 (2012).
  14. Balazs, A.B. et al. Antibody-based protection against HIV infection by vectored immunoprophylaxis. Nature 481, 8184 (2011).
  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, 13451352 (2008).
  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, 17791787 (1992).
  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, 450457 (2009).
  18. Dimitrov, A. Ibalizumab, a CD4-specific mAb to inhibit HIV-1 infection. Curr. Opin. Investig. Drugs 8, 653661 (2007).
  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, 286291 (2004).
  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, 22312233 (2006).
  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, 191203 (2002).
  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, 69356942 (2010).
  23. Freeman, M.M. et al. Crystal structure of HIV-1 primary receptor CD4 in complex with a potent antiviral antibody. Structure 18, 16321641 (2010).
  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, 38723880 (2011).
  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, 19 (2013).
  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, 461473 (1978).
  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, 4128741298 (2000).
  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, 1341913424 (2002).
  29. Haynes, B.F. et al. Cardiolipin polyspecific autoreactivity in two broadly neutralizing HIV-1 antibodies. Science 308, 19061908 (2005).
  30. Mouquet, H. et al. Polyreactivity increases the apparent affinity of anti-HIV antibodies by heteroligation. Nature 467, 591595 (2010).
  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, 1354013545 (2013).
  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, 2150 (2007).
  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), S9S14 (2010).
  34. Nimmerjahn, F. & Ravetch, J.V. Divergent immunoglobulin g subclass activity through selective Fc receptor binding. Science 310, 15101512 (2005).
  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, 954966 (2010).
  36. Wu, S.J. et al. Structure-based engineering of a monoclonal antibody for improved solubility. Protein Eng. Des. Sel. 23, 643651 (2010).
  37. Wei, X. et al. Antibody neutralization and escape by HIV-1. Nature 422, 307312 (2003).
  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, 175186 (2007).

Download references

Author information

Affiliations

  1. Aaron Diamond AIDS Research Center, The Rockefeller University, New York, New York, USA.

    • Ruijiang Song,
    • David Franco &
    • David D Ho
  2. Structural Biology Resource Center, The Rockefeller University, New York, New York, USA.

    • Deena A Oren
  3. Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA.

    • Michael S Seaman

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.

Competing financial 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.

Corresponding author

Correspondence to:

Author details

Supplementary information

PDF files

  1. Supplementary Text and Figures (738 KB)

    Supplementary Figures 1–5 and Supplementary Tables 1, 2 and 4

  2. Supplementary Table 3 (148 KB)

    Supplementary Table 3

Additional data