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

Journal name:
Nature Biotechnology
Year published:
Published online


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


  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

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Author information


  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


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.

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