TECHNICAL FIELD

The invention relates to the field of biomedicine. In particular, the invention relates to a polypeptide fragment of pro-BDNF and a protein binding to the polypeptide fragment, and also relates to the use of the polypeptide fragment of pro-BDNF.

BACKGROUND

Autoimmune disease (AID) is a type of disease, in which autoimmune tolerance is broken, and the immune system is activated to attack autoantigens, so as to cause damage to tissues and organs. The etiology and pathogenesis of autoimmune disease are unclear, and it is currently considered to be a hypersensitive reactive disease against autoantigen caused by autoantibodies, autoreactive T lymphocytes, or both. There are no curative medicines in clinical practice. The timely application of traditional glucocorticoids and immunosuppressants can control the disease and improve the survival rate of patients. However, long-term use of them will lead to a series of side effects. In severe cases, the quality of life of patients will be affected, or even their lives will be threatened. Moreover, some patients are not sensitive to glucocorticoid and immunosuppressive therapy.

In recent years, new treatment strategies have been proposed with the deep understanding of the molecular mechanism of AID, including gene therapy, epigenetic intervention, small molecule Toll-like receptor inhibitors, anti-inflammatory factor antibodies, backfusion of stem cells and regulatory T cell autolog, dendritic cell vaccine, etc. Some of these therapeutic drugs or methods have been used in clinic, and some are still in the clinical research stage (such as stem cell autotransfusion therapy, etc.) and even animal experimental stages (such as epigenetic regulation, etc.).

However, these drugs cannot replace glucocorticoids as first-line drugs, therefore, there is an urgent need for new, safe and effective therapeutic drugs and methods for clinical application.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a polypeptide, by which an antibody that specifically binds to the polypeptide can be obtained; and the antibody can be effectively used for the treatment of autoimmune diseases.

In the first aspect, the present invention provides a polypeptide comprising an amino acid sequence as shown in SEQ ID NO: 41.

In a preferred embodiment, the amino acid sequence of the polypeptide is shown in SEQ ID NO: 41.

In the second aspect, the present invention provides a polypeptide comprising an amino acid sequence shown in SEQ ID NO: 44.

In a preferred embodiment, the amino acid sequence of the polypeptide is shown in SEQ ID NO: 44.

In the third aspect, the present invention provides a polypeptide comprising an amino acid sequence as shown in SEQ ID NO: 47.

In a preferred embodiment, the amino acid sequence of the polypeptide is shown in SEQ ID NO: 47.

In a specific embodiment, the present invention provides a polynucleotide encoding a polypeptide according to any one of the first to third aspects of the present invention.

In a specific embodiment, the present invention provides an expression vector comprising the encoding polynucleotide of the present invention.

In a specific embodiment, the present invention provides the use of the polypeptide according to any one of the first to third aspects of the present invention in screening or preparing a specific antibody thereof.

In the fourth aspect, the invention provides a protein that specifically binds to the polypeptide of any one of the first to third aspects of the invention.

In a specific embodiment, the binding affinity of the protein to human pro-BDNF is <10 nM.

In a specific embodiment, the protein is an antibody.

In a preferred embodiment, the antibody is a monoclonal antibody, a polyclonal antibody, or an antibody fragment.

In a preferred embodiment, the antibody may be selected from a chimeric antibody, a humanized antibody, or a fully human antibody.

In a specific embodiment, the light chain variable region of the antibody has:

CDR1 as shown in SEQ ID NO: 4 or SEQ ID NO: 14;

CDR2 as shown in SEQ ID NO: 5 or SEQ ID NO: 15; and

CDR3 as shown in SEQ ID NO: 6 or SEQ ID NO: 16.

In a specific embodiment, the light chain variable region of the antibody has:

CDR1 as shown in SEQ ID NO: 4; CDR2 as shown in SEQ ID NO: 5; and CDR3 as shown in SEQ ID NO: 6; or

CDR1 as shown in SEQ ID NO: 14, CDR2 as shown in SEQ ID NO: 15 and CDR3 as shown in SEQ ID NO: 16.

In a specific embodiment, the heavy chain variable region of the antibody has:

CDR1 as shown in SEQ ID NO: 1 or SEQ ID NO: 11;

CDR2 as shown in SEQ ID NO: 2 or SEQ ID NO: 12; and

CDR3 as shown in SEQ ID NO: 3 or SEQ ID NO: 13.

In a specific embodiment, the heavy chain variable region of the antibody has:

CDR1 as shown in SEQ ID NO: 1, CDR2 as shown in SEQ ID NO: 2 and CDR3 as shown in SEQ ID NO: 3; or

CDR1 as shown in SEQ ID NO: 11, CDR2 as shown in SEQ ID NO: 12 and CDR3 as shown in SEQ ID NO: 13.

In a specific embodiment, the antibody is selected from:

Antibody (a): the heavy chain variable region comprises the sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, and the light chain variable region comprises the sequence shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6;

Antibody (b): the heavy chain variable region comprises the sequences as shown in SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13, and the light chain variable region comprises the sequence as shown in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16;

Antibody (c): a variant of antibody (a) or antibody (b), which has or substantially has the activity of antibody (a) or antibody (b).

In a preferred embodiment, the antibody (c) is obtained by addition, deletion or substitution (preferably substitution) of one or more, for example, 1-3, preferably 1 amino acid in the sequence as shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 of the antibody (a), and has or substantially has the activity of the antibody (a); or

the antibody (c) is obtained by the addition, deletion, or substitution (preferably substitution) of one or more, for example, 1-3, preferably 1 amino acid in the sequence as shown in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 of antibody (b), and has or substantially has the activity of antibody (b).

In a preferred embodiment, the amino acid substitution is a conservative amino acid substitution.

In a specific embodiment, the antibody is selected from:

Antibody (d): having a heavy chain variable region as shown in SEQ ID NO: 7 and a light chain variable region as shown in SEQ ID NO: 8;

Antibody (e): having a heavy chain variable region as shown in SEQ ID NO: 17 and a light chain variable region as shown in SEQ ID NO: 18;

Antibody (f): a variant of antibody (d) or antibody (e), which has or substantially has the activity of antibody (d) or antibody (e).

In a preferred embodiment, the antibody (f) is obtained by the addition, deletion, or substitution (preferably substitution) of one or more, for example, 1-10, preferably 1-6, more preferably 1-3, most preferably 1 amino acid in the sequence as shown in SEQ ID NO: 7 or SEQ ID NO: 8 of antibody (f), and has or substantially has the activity of antibody (f); or

the antibody (f) is obtained by the addition, deletion, or substitution (preferably substitution) of one or more, for example, 1-10, preferably 1-6, more preferably 1-3, most preferably 1 amino acid in the sequence as shown in SEQ ID NO: 17 or SEQ ID NO: 18 of antibody (e), and has or substantially has the activity of antibody (e).

In a preferred embodiment, the antibody has one or more of the following characteristics:

i) binding pro-BDNF polypeptide with a KDof 10 nM or less;

ii) competitively inhibiting the binding of pro-BDNF to p75; and

iii) Pro-BDNF-dependent activity.

In a specific embodiment, the protein is obtained by immunizing an animal with the polypeptide according to any one of the first to third aspects of the present invention or is obtained by screening a phage library using the polypeptide according to any one of the first to third aspects of the present invention.

In the fifth aspect, the present invention provides a pharmaceutical composition comprising the protein according to the fourth aspect of the present invention and optionally one or more pharmaceutically acceptable excipients, diluents or carrier.

In the sixth aspect, the present invention provides the use of the protein according to the fourth aspect of the present invention for preparing a medicament for the prevention, treatment or diagnosis of an autoimmune disease.

In a preferred embodiment, the autoimmune disease is a pro-BDNF-mediated autoimmune disease.

In the seventh aspect, the present invention provides the use of the protein according to the fourth aspect of the present invention for preparing a medicament for the prevention, treatment or diagnosis of a disease mediated by pro-BDNF.

In a specific embodiment, the disease is arthritis, rheumatoid arthritis, ankylosing spondylitis, aplastic anemia, psoriasis, insulin-dependent diabetes mellitus, multiple sclerosis, cryoglobulinemia, Chronic obstructive pulmonary disease, systemic lupus erythematosus (SLE), lupus nephritis, asthma, multiple sclerosis, or cystic fibrosis.

In the eighth aspect, the present invention provides the use of the protein according to the fourth aspect of the present invention for preparing a medicament for the prevention, treatment or diagnosis of a disease by inhibiting the secretion of TNF-α, IL-2, IL-6 or IL-4.

In the ninth aspect, the present invention provides the use of the protein according to the fourth aspect of the present invention for preparing a medicine for relieving pain.

In the tenth aspect, the present invention provides a nucleic acid encoding a protein according to the fourth aspect of the present invention.

In the eleventh aspect, the present invention provides a method for screening antibodies, using the polypeptide according to any one of the first to third aspects of the present invention to immunize animals or screening a phage library by using the polypeptide according to any one of the first to third aspects of the present invention.

It should be understood that, within the scope of the present invention, the technical features specifically mentioned above and below (such as in the Examples) can be combined with each other, thereby constituting a new or preferred technical solution which needs not be individually described.

