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Stable human single CD4 domain and fusion proteins

A technology of fusion protein and immunoglobulin, which is used to target specific cell fusion, fusion polypeptide, immunoglobulin, etc., and can solve problems such as misfolding and low affinity

Active Publication Date: 2020-03-27
UNITED STATES OF AMERICA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the purified protein is stable only at low pH (4.0), is partially misfolded, and has an affinity several times lower than that possessed by D1D2 or T4

Method used

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  • Stable human single CD4 domain and fusion proteins
  • Stable human single CD4 domain and fusion proteins
  • Stable human single CD4 domain and fusion proteins

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0092] This example describes the identification of a single domain CD4 (D1) with improved soluble expression, stability and specificity.

[0093] Structure-guided cavity filling and library screening were used to identify D1 mutants with improved soluble expression, stability and specificity. The screen yielded two mutants: mD1.22 (SEQ ID NO:1) and mD1.23 (SEQ ID NO:2) (see figure 1 ).

[0094] As shown in Table 1, the solubility of the above mutants in E. coli periplasm and PBS was significantly higher than that of the previously identified D1 mutant (mD1.2; Chen et al., J. Virol., 85:9395-9405 (2011) ).

[0095] Table 1. Properties of single domain CD4 mutants.

[0096]

[0097] ND = not determined

Embodiment 2

[0099] This example shows the characterization of mD1.22 and mD1.23.

[0100] The binding properties of mD1.2 (Chen et al., 2011, supra), mD1.22 (SEQ ID NO: 1 ) and mD1.23 (SEQ ID NO: 2) to HIV-1 gp140 were evaluated at different D1 concentrations.

[0101] use gp140 Con-s and gp140 CH12.0544.2 ELISA was performed, the gp140 Con-s A consensus gp140 was designed by aligning more than 1,000 sequences of the M group (see Liao et al., Virology, 353:268-282 (2006)). Bound mD1.2 and mD1.22 and mD1.23 mutants were detected by HRP-conjugated anti-hexahistidine tag antibodies (Sigma-Aldrich, St. Louis, MO).

[0102] Such as image 3 As shown in A and 3B, the two mutants are cross-reactive against the gp140 tested.

[0103] Binding of genetically diverse HIV-1 Envs was also analyzed by surface plasmon resonance (SPR) on a Biacore X100 device (GE Healthcare) using a single-cycle approach. Briefly, purified HIV-1 gp140 was diluted in sodium acetate (pH 5.0) and immobilized directly ...

Embodiment 3

[0106] This example shows that the D1 mutant maintains the functional activity of full-length CD4.

[0107]CD4 induces a conformational change in gp120, resulting in the exposure of CD4-inducible (CD4i) epitopes. To determine whether D1 mutants induce such conformational changes, in the absence or presence of mD1.2 (SEQ ID NO:3), mD1.22 (SEQ ID NO:1) or mD1.23 (SEQ ID NO:2) In this case a CD4i antibody based fusion protein, m36h1Fc (see Chen et al., Proc. Natl. Acad. Sci. USA, 105:17121-17126 (2008)) was tested for binding to gp140Con-s. Two-domain human CD4 (D1D2; SEQ ID NO:5) was used as a positive control and as a negative control was Fab b12, an HIV-1 ubiquitous protein targeting the CD4-binding site on gp120. and antibodies (see Roben et al., J. Virol., 68:4821-4828 (1994)).

[0108] Relative to controls, m36h1Fc interacts with gp140 in the presence of the D1 mutant Con-s increased binding (see Figure 4 ). In addition, m36h1Fc and gp140 Con-s The binding of D1D2 an...

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Abstract

The present invention provides polypeptides comprising single domain CD4 and fusion proteins comprising said single domain CD4 and one or more fusion partners. Also provided are nucleic acids encoding the polypeptides or fusion proteins, as well as compositions or cells comprising the polypeptides, fusion proteins or nucleic acids.

Description

[0001] Cross References to Related Applications [0002] This patent application claims the benefit of US Provisional Patent Application Serial No. 61 / 791,885, filed March 15, 2013, which is incorporated by reference. [0003] sequence listing [0004] The Nucleotide / Amino Acid Sequence Listing filed concurrently with this document is hereby incorporated by reference in its entirety. [0005] Background of the invention [0006] CD4 is present in most thymocytes and mature T cell subsets (CD4 + non-polymorphic transmembrane glycoprotein expressed on the surface of T cells) (see Germain, Curr. Biol., 7:R640-R644 (1997)). It consists of four immunoglobulin-like extracellular domains, a transmembrane segment and a cytoplasmic tail non-covalently associated with the src-family tyrosine kinase Lck. As an important component of the immune system, CD4 plays a role in helping CD4 + Role of co-receptors for stronger association of T cell receptor (TCR) on T cells with major histocom...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K38/17
CPCA61K38/162A61K38/1774C07K2319/00A61K38/00A61K47/6425C07K14/70514C07K16/1063C07K2319/30C07K2319/33
Inventor 迪米特尔·S·蒂米特鲁夫陈维灶普拉巴卡兰·邦拉吉
Owner UNITED STATES OF AMERICA
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