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Glycan-modified Anti-cd4 antibodies for HIV prevention and therapy

a technology of cd4 and glycan, which is applied in the field of glycan modification of antibodies and anticd4 antibodies, can solve the problems that no previous study provides the effect of glycan, and achieve the effects of improving activity, preventing infection of target cells, and inhibiting or/and treating infection

Active Publication Date: 2016-12-15
THE ROCKEFELLER UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0008]In one embodiment of the invention, the glycan-modified anti-CD4 antibody is a modified form of an anti-CD4 antibody having an engineered N-linked glycosylation site in its variable region. In some embodiments, the engineered N-linked glycosylation site is located in the variable region of the light chain of an anti-CD4 antibody, such as wild type (WT) or modified ibalizumab, ibalizumab mutant or modified anti-CD4 antibody. In some specific embodiments, the engineered N-linked glycosylation site is located at an amino acid position of the light chain of ibalizumab or corresponding positions thereof, which is selected from the group consisting of residues 30E, 52, 53, 54, 60, 65 and 67 and 76, and combination thereof. In the present invention, the glycosylation site is at the amino acid position selected from the group consisting of positions 30E Gln, 52Ser, 53Thr, 54Arg, 60Asp, 65Ser, 67Ser, and 76Ser. In some examples, the glycosylation site at 30E Gln, 52Ser, 53Thr, 54Arg, 65Ser, or 67Ser provides improved activity. In one particular example of the invention, the glycosylation site is at position 52Ser.
[0012]In another particular example of the invention, a glycan-modified anti-CD4 antibody with improved recycling of the antibody is provided, which has the amino acid sequence of the light chain as set forth in SEQ ID NO: 4, and the heavy chain having the amino acid sequence as set forth in SEQ ID NO: 5.
[0015]In a further aspect, the present invention provides a glycan-modified monoclonal antibody having improved activity, in which the glycan-modified anti-CD4 antibody has N-linked glycans attached to the variable region as defined above. The antibody or antigen binding fragment of the invention is effective for inhibiting, treating or / and preventing infection of target cells by human immunodeficiency virus type 1 (“HIV-1”).

Problems solved by technology

However, none of previous studies provides the effect of a glycan strategically placed in the variable region of an antibody.

Method used

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  • Glycan-modified Anti-cd4 antibodies for HIV prevention and therapy
  • Glycan-modified Anti-cd4 antibodies for HIV prevention and therapy
  • Glycan-modified Anti-cd4 antibodies for HIV prevention and therapy

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0118]Sequence analysis of a panel of 118 viral isolates suggests that ibalizumab resistance was associated with the number of potential N-linked glycosylation sites (PNGS) in the V5 loop of gp120 (Pace et al., J. Acquir. Immune Defic. Syndr. Epub ahead of print: September 2012). As shown in FIG. 1, viruses having two N-linked glycosylation sites in V5 were sensitive to ibalizumab, whereas viruses having no N-linked glycosylation site in V5 were resistant. Bar indicates median. Interestingly, clinical viral isolates that have developed resistance to ibalizumab monotherapy also display a loss in a potential V5 glycosylation site (Toma et al., J. Virology 85(8): 3872-2880, 2011).

[0119]One wild-type virus (AC10.0.29) in the panel has two N-linked glycosylation sites in V5 and is naturally sensitive to ibalizumab. The V5 N-linked glycosylation sites in this virus were systematically deleted using site-directed mutagenesis, and the resulting mutant viruses became resistant to ibalizumab ...

example 2

Design of Ibalizumab Variants

[0123]Studies of ibalizumab-resistant HIV-1 strains revealed that resistance is mainly conferred by the loss of glycan(s) from the V5 loop of HIV-1 Env gp120. To further explore the role of V5 glycosylation in ibalizumab susceptibility, we modeled the interactions between gp120, CD4 and ibalizumab using the structures reported to the Protein Data Bank (accession number 2NXY and 302D). The V5 N-terminal glycan is situated closest to the ibalizumab L chain (FIG. 5A), while the V5 C-terminal glycan is further away from ibalizumab (FIG. 5B). This model raises the possibility that the fit of the N-terminal glycan into the space between gp120 and ibalizumab exerts a mass effect on gp120, thereby disrupting its conformational changes (twists and turns) that are essential for HIV-1 entry into the target cell. This model also suggests that introduction of a similarly sized glycan into the ibalizumab L chain may boost its ability to inhibit entry of HIV-1 strains ...

example 3

CD4 Binding and HIV-1 Neutralization by LMs

[0125]We next evaluated the kinetics of binding of these ibalizumab LMs to human soluble CD4 (sCD4) by surface plasmon resonance. In a Biacore assay using sCD4 as the analyte, WT ibalizumab and LMs bound sCD4 with similar binding kinetics. The KD of six of these LMs bound to sCD4 was in the range of 0.19 nM to 0.8 nM (Table 1), and these numbers were within 2-fold of the KD of wild-type ibalizumab (0.43 nM). These data showed that the addition of an N-linked glycan at these select locations in the L chain of ibalizumab did not markedly affect its ability to bind CD4.

TABLE 1The binding kinetics of ibalizumab and its LMs to human CD4AntibodyKon (105 / Ms)Koff (10−5 / s)KD (0.1 nM)ibalizumab2.8124.3LM30E1.11514LM524.6163.5LM532.9134.5LM543.7277.3LM604.48.31.9LM653.34915LM672.5208LM765.0142.8

[0126]We next explored the HIV-1 neutralizing capacity of the LMs compared to WT ibalizumab. To this end, we tested the LMs against a panel of HIV-1 viruses th...

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Abstract

Disclosed herein are glycan-modified anti-CD4 monoclonal antibodies with N-linked glycans attached to the variable region. Expression vectors and cell lines useful for the production of such antibodies, and use of such antibodies for HIV prevention and therapy are also disclosed.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application claims benefit of U.S. provisional application No. 61 / 738,777, filed Dec. 18, 2012 and U.S. provisional application No. 61 / 864,942, filed Aug. 12, 2013, the disclosures of which are hereby incorporated by reference.FIELD OF THE DISCLOSURE[0002]This disclosure generally relates to HIV prevention and treatment. This disclosure also generally relates to glycan modification of antibodies. In particular, this disclosure relates to glycan-modified anti-CD4 antibodies useful for HIV prevention and therapy.BACKGROUND ART[0003]HIV-1 entry is triggered by interaction of the viral envelope (Env) glycoprotein gp120 with domain 1 (D1) of the T-cell receptor CD4. Ibalizumab (iMab) is a potent and broadly HIV-1 neutralizing Ab (Jacobson et al., Antimicrob. Agents Chemother. 53:450-457, 2009; Kuritzkes et al., J. Infect. Dis. 189:286-291, 2004), which neutralizes HIV by binding mainly to domain 2 (D2) of the CD4 receptor on host T-cells,...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07K16/28
CPCC07K16/2812C07K2317/41A61K2039/505C07K2317/24C07K2317/76A61P31/18C07K2317/33
Inventor SONG, RUIJIANGHO, DAVID D.
Owner THE ROCKEFELLER UNIV
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