STRUCTURE-SENSITIVE PEPTIDE ANTIGEN OF VON WILLEBRAND FACTOR

Abstract

The present invention discloses a polypeptide antigen sequence, its spatially sensitive position in von Willebrand factor (VWF), and use thereof in the production of preparations for the diagnosis and/or treatment of VWF-related blood diseases. The polypeptide antigen comprises the amino acid sequence as shown in SEQ ID NO: 1, or a biologically active fragment or a variant thereof. The spatial position of the polypeptide transits from non-solvent exposure to solvent exposure when VWF is in activation process upon exposing to blood shear stress, so as to be recognized by an antibody. The antibody that recognizes the active state of VWF prepared by using the polypeptide antigen is useful in diagnosing and treating a variety of VWF-related diseases.

Claims

1. Use of a peptide antigen in the production of preparations for diagnosis and/or treatment of blood diseases associated with von Willebrand factor (VWF), wherein the peptide antigen comprises the amino acid sequence as shown in SEQ ID NO: 1, or a biologically active fragment or variant thereof, and the peptide antigen is derived from the A1 domain of human VWF.

2. The use according to claim 1, wherein the peptide antigen is located in the loop region between the β3 fold and α2 helix in the A1 domain of VWF.

3. The use according to claim 1, wherein when VWF is in an active state, the peptide antigen is recognized by a specific antibody thereof.

4. The use according to claim 1, wherein the peptide antigen is derived from human.

5. The use according to claim 1, wherein the preparation is a vaccine composition, an antibody drug or a diagnostic kit.

6. An antibody, wherein the antibody specifically recognizes a peptide antigen comprising the amino acid sequence as shown in SEQ ID NO: 1, or a biologically active fragment or a variant thereof.

7. The antibody according to claim 6, wherein the antibody recognizes VWF in an active state; and the peptide antigen is derived from the A1 domain of human VWF.

8. A diagnostic preparation, for determining whether a peptide antigen having the amino acid sequence as shown in SEQ ID NO: 1 is present in a sample, comprising the antibody according to claim 6.

9. A therapeutic preparation, for recognizing and inhibiting VWF in an active state, comprising the antibody according to claim 1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIG. 1 shows the test results of the monoclonal antibody prepared in the present invention for the recognition of active and inactive VWF;

[0022] FIG. 2 illustrates the spatial position of the peptide antigens in VWF; and

[0023] FIG. 3 shows the test results of the monoclonal antibody in the comparative example for the recognition of active and inactive VWF.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] The specific embodiments of the present invention will be described in further detail with reference to the accompanying drawings and specific embodiments. The following embodiments are intended to illustrate the present invention, instead of limiting the scope of the present invention.

Example 1

[0025] VWF consists of 2050 amino acids which are divided into 14 domains. The A1 domain is related to the coagulation function, and the A1 domain has 205 amino acids. The positions of these amino acids in the structure of the protein are known. From a region in the A1 domain of human VWF that interacts with the short peptides flanking the A1 domain, a 10-amino acid long peptide was selected as the antigen (GLKDRKRPSE, SEQ ID NO: 1). The peptide antigen is located in the loop region between the β3 fold and α2 helix in the A1 domain of human VWF (FIG. 2). A murine monoclonal antibody was prepared by a standard antibody preparation method using the peptide antigen as follows:

[0026] (1) Animal immunization: Mice were immunized with the peptide antigen of the above sequence to allow the mice produce B lymphocytes.

[0027] (2) Cell fusion: Syngeneic myeloma cells were mixed with mouse spleen cells. In the presence of polyethylene glycol, the lymphocytes and myeloma cells were fused to form hybridoma cells.

[0028] (3) Selective culture: The fused hybridoma cells were screened with HAT selective medium.

[0029] (4) Screening and clonal expansion of positive clones:

[0030] Positive hybridoma cells that can produce the required monoclonal antibodies were screened by an immunofluorescence method (ELISA), followed by clonal expansion.

[0031] FIG. 1 shows the test results of various concentrations of the monoclonal antibody prepared in the present invention for the recognition of active and inactive VWF. The results show that as the antibody concentration increases, the UV absorption of active VWF at 450 nm is enhanced, and the UV absorption of inactive VWF at 450 nm is basically unchanged, indicating that the monoclonal antibody prepared from the peptide antigen of the present invention can specifically recognize active VWF. This is because the peptide antigen is high sensitive to the active state of VWF. VWF is protected by the short peptide regions flanking the A1 domain when it is in an inactive state, and the monoclonal antibody cannot recognize it. When VWF is activated, the peptide antigen region is exposed, and the monoclonal antibody can recognize it.

[0032] The antibody can be used to further prepare antibody drugs, which can recognize only a few active proteins, and thus will not cause the overall protein level to drop, so the risk of bleeding is very low; also, the protein will not be consumed in a large amount, and the durability is good and thus the dosage is low.

COMPARATIVE EXAMPLES

[0033] Detection of the active state of VWF with the monoclonal antibody produced by using the peptide antigen pVWF 1346-1355: QVKYAGSQVD (SEQ ID NO: 2) shows that, the antibody has a similar binding to the active and inactive states of VWF (FIG. 3), indicating that the antibody is not sensitive to the active state of VWF.

[0034] While preferred embodiments of the present invention have been described above, the present invention is not limited thereto. It should be appreciated that various improvements and variations can be made by those skilled in the art without departing from the technical principles of the present invention, which are also contemplated to be within the scope of the present invention.