WO2021155861A1 - Human thymus stromal lymphopoietin monoclonal antibody and application thereof - Google Patents

Abstract

The present invention provides a human thymus stromal lymphopoietin (TSLP) monoclonal antibody and an application thereof. The TSLP antibody provided in the present invention can specifically bind to hTSLP, has high affinity, and can effectively inhibit the combination of TSLP and a receptor thereof so as to inhibit activation of a downstream signal path and activation of immune cells by TSLP. The TSLP antibody has great significance in content measurement of hTSLP and diagnosis, treatment and prognosis of inflammatory or allergic inflammatory diseases such as allergic asthma, chronic obstructive pulmonary disease, allergic dermatitis, allergic rhinitis, allergic nasosinusitis, eosinophilic granulocytic esophagitis, allergic conjunctivitis, inflammatory bowel disease or atopic dermatitis.

Description

Human thymic stromal lymphopoietin monoclonal antibody and its application

cross reference

This application claims the priority of the Chinese patent application No. 202010082636.0 filed on February 7, 2020 entitled "Human Thymic Stromal Lymphogenin Monoclonal Antibody and Its Application", the entire disclosure of which is incorporated herein by reference in its entirety.

Technical field

The present invention relates to the technical field of antibodies, in particular to human thymic stromal lymphopoietin monoclonal antibodies and applications thereof.

Background technique

Thymic stromal lymphopoietin (TSLP) consists of epithelial cells, airway smooth muscle cells, lung fibroblasts and stromal cells in the thymus, lung, skin, intestine, and tonsils when exposed to allergens, viruses, bacteria, cigarettes and the environment. The particles are induced to produce behind the lead. Human thymic stromal lymphopoietin (hTSLP) is an interleukin 7 (IL-7)-like cytokine. Mature hTSLP is composed of 131 amino acid residues and has a typical 4-strand α-helix bundle structure.

The TSLP receptor complex is a heterodimer composed of TSLP receptor (TSLPR) and IL-7 receptor α (IL-7Ra), mainly in dendritic cells, CD4 ⁺ T cells, eosinophils, and eosinophils. It is expressed on basic granulocytes, mast cells and type 2 innate lymphocytes (ILC2). The cells with the highest expression of TSLPR and IL-7Rα are myeloid dendritic cells. After TSLP binds to the surface receptors of myeloid dendritic cells, the dendritic cells secrete IL-8 and eotaxin-2 to recruit neutrophils and eosinophils, and secrete TARC and MDC to recruit Th2 cells. In addition, DC cells activated by TSLP induce CD4 ⁺ T cells to differentiate into Th2 cells. Th2 cells can produce IL4, IL-5, IL-13 and TNF. These cytokines promote the production of IgE, eosinophils and mucus to initiate metamorphosis. Reactions cause diseases such as asthma and atopic dermatitis. In addition, TSLP induces the aggregation of fibroblasts and the deposition of collagen in animals, confirming its role in promoting fibrotic disorders.

The role of TSLP in the development and maintenance of allergic diseases has been confirmed in animal models. Mice deficient in TSLP signaling resist the development of asthma. Neutralizing TSLP or its receptor with antibodies is effective in murine or primate asthma or rhinitis models. For example, blocking TSLP with anti-TSLPR mAb in a primate asthma model (cynomolgus monkeys that are naturally sensitive to Ascaris suum antigens) reduced eosinophil airway resistance and IL 13 levels.

Asthma is a common chronic disease that affects approximately 300 million people worldwide. In many patients, bronchodilators, inhaled or oral corticosteroids can be used to control symptoms. However, most patients with moderate and severe asthma still have symptoms or improper control, which affects their quality of life and has a significant burden of health care. In particular, many patients with severe asthma may not respond or respond poorly to high doses of steroids. TSLP is overexpressed in the epithelium and lamina propria of the lungs of asthmatic patients, even in patients who use high-dose inhaled corticosteroids. Strong supporting data for the importance of TSLP in asthma comes from the anti-TSLP monoclonal antibody (AMG 157 /MEDI9929) in the allergen challenge study of mild asthma patients: after 6 or 12 weeks of treatment with AMG 157 (monthly administration) Significant utility was observed in early and late reactions measured by FEV1 and changes in blood and sputum eosinophil count and FeNO levels.

Therefore, the preparation of monoclonal antibodies that can specifically recognize and bind TSLP is of great significance in the diagnosis, treatment, and prognosis of diseases involving TSLP.

Summary of the invention

In order to solve the technical problems in the prior art, the purpose of the present invention is to provide an antibody that can specifically recognize and bind human thymic stromal lymphopoietin. The present invention also provides a preparation method, application and related products of the antibody.

To achieve the above purpose, the present invention uses the C-terminal fusion mouse Fc fragment (mFc) hTSLP as the antigen to immunize mice, prepare hybridoma cells, screen and obtain monoclonal antibodies that can specifically bind to hTSLP. Affinity, and can effectively inhibit the binding of hTSLP and TSLPR. On the basis of this murine monoclonal antibody, in order to reduce its heterogeneity to the human body, a humanized antibody was constructed: on the one hand, by fusing the variable region of the murine antibody with the constant region of the human antibody, Human-mouse chimeric antibody; on the other hand, the FR region of the murine monoclonal antibody is specifically humanized to obtain a humanized antibody modified in the FR region. The humanized antibody has high affinity to hTSLP and can effectively inhibit the binding of hTSLP and TSLPR.

Specifically, the technical solution of the present invention is as follows:

In the first aspect, the present invention provides a thymic stromal lymphopoietin antibody, an antigen-binding fragment thereof, a polypeptide thereof, or a fusion protein thereof, wherein the thymic stromal lymphopoietin antibody comprises a light chain CDR and a heavy chain CDR; Chain CDR contains:

(1) HCDR1: Contains the sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2, or contains one or more of the sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2 deleted, replaced, or inserted The amino acid sequence of a protein or polypeptide that can specifically bind to thymic stromal lymphopoietin derived from three amino acids, or an amino acid sequence that has at least 90% homology with the sequence shown in SEQ ID NO.1 or SEQ ID NO.2 ；

(2) HCDR2: Contains the sequence shown in SEQ ID NO. 3 or SEQ ID NO. 4, or contains the sequence shown in SEQ ID NO. 3 or SEQ ID NO. 4 with one or more deletions, substitutions, or insertions The amino acid sequence of a protein or polypeptide that can specifically bind to thymic stromal lymphopoietin derived from three amino acids, or an amino acid sequence that has at least 90% homology with the sequence shown in SEQ ID NO. 3 or SEQ ID NO. 4 ；

(3) HCDR3: contains the sequence shown in SEQ ID NO. 5 or SEQ ID NO. 6, or contains the sequence shown in SEQ ID NO. 5 or SEQ ID NO. 6 with one or more deletions, substitutions, or insertions The amino acid sequence of a protein or polypeptide that can specifically bind to thymic stromal lymphopoietin derived from three amino acids, or an amino acid sequence that has at least 90% homology with the sequence shown in SEQ ID NO.5 or SEQ ID NO.6 ；

The light chain CDR includes:

(1) LCDR1: contains the sequence shown in SEQ ID NO. 7 or SEQ ID NO. 8, or contains the sequence shown in SEQ ID NO. 7 or SEQ ID NO. 8 with one or more deletions, substitutions, or insertions The amino acid sequence of a protein or polypeptide that can specifically bind to thymic stromal lymphopoietin derived from three amino acids, or an amino acid sequence that has at least 90% homology with the sequence shown in SEQ ID NO.7 or SEQ ID NO.8 ；

(2) LCDR2: contains the sequence shown in SEQ ID NO. 9 or SEQ ID NO. 10, or contains the sequence shown in SEQ ID NO. 9 or SEQ ID NO. 10 with one or more deletions, substitutions, or insertions The amino acid sequence of a protein or polypeptide that can specifically bind to thymic stromal lymphopoietin obtained from three amino acids, or an amino acid sequence that has at least 90% homology with the sequence shown in SEQ ID NO.9 or SEQ ID NO.10 ；

(3) LCDR3: Contains the sequence shown in SEQ ID NO. 11 or SEQ ID NO. 12, or contains the sequence shown in SEQ ID NO. 11 or SEQ ID NO. 12 with one or more deletions, substitutions, or insertions The amino acid sequence of a protein or polypeptide that can specifically bind to thymic stromal lymphopoietin derived from three amino acids, or an amino acid sequence that has at least 90% homology with the sequence shown in SEQ ID NO.11 or SEQ ID NO.12 .

The aforementioned homology is preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

In the present invention, "deleted, replaced, or inserted one or more amino acids" refers to a sequence that differs from the sequence shown in the present invention at one or more amino acid residues but retains the biological activity of the resulting molecule, It can be a "conservatively modified variant" or modified by "conservative amino acid substitution". "Conservatively modified variants" or "conservative amino acid substitutions" refer to amino acid substitutions known to those skilled in the art, and making such substitutions generally does not change the biological activity of the resulting molecule. Generally speaking, those skilled in the art recognize that a single amino acid substitution in a non-essential region of a monoclonal antibody does not substantially change the biological activity.

In the present invention, "a plurality of" in the "amino acid sequence of a protein with the same function obtained by deleting, replacing, or inserting one or more amino acids" is ≥2 and ≤20.

The above heavy chain CDRs and light chain CDRs can be combined to obtain different thymic stromal lymphopoietin antibodies. Among the above different combinations, antibodies using the following CDR combinations (1) or (2) have better The affinity of thymus stromal lymphopoietin and receptor binding inhibition:

(1) HCDR1: Contains the sequence shown in SEQ ID NO.1, or contains the sequence shown in SEQ ID NO.1 by deleting, replacing or inserting one or more amino acids and can specifically bind to thymic stromal lymphocyte production The amino acid sequence of the protein or polypeptide of the protein, or the amino acid sequence that has at least 90% homology with the sequence shown in SEQ ID NO.1;

HCDR2: contains the sequence shown in SEQ ID NO. 3, or contains the sequence shown in SEQ ID NO. 3, a protein that can specifically bind to thymic stromal lymphopoietin, obtained by deleting, replacing or inserting one or more amino acids Or the amino acid sequence of the polypeptide, or an amino acid sequence that has at least 90% homology with the sequence shown in SEQ ID NO. 3;

HCDR3: Contains the sequence shown in SEQ ID NO. 5, or contains the sequence shown in SEQ ID NO. 5, a protein that can specifically bind to thymic stromal lymphopoietin, obtained by deleting, replacing or inserting one or more amino acids Or the amino acid sequence of the polypeptide, or an amino acid sequence that has at least 90% homology with the sequence shown in SEQ ID NO.5;

LCDR1: Contains the sequence shown in SEQ ID NO. 7, or contains the sequence shown in SEQ ID NO. 7, a protein that can specifically bind to thymic stromal lymphopoietin obtained by deleting, replacing or inserting one or more amino acids Or the amino acid sequence of the polypeptide, or an amino acid sequence that has at least 90% homology with the sequence shown in SEQ ID NO. 7;

LCDR2: Contains the sequence shown in SEQ ID NO.9, or contains the sequence shown in SEQ ID NO.9, a protein that can specifically bind to thymic stromal lymphopoietin, obtained by deleting, replacing or inserting one or more amino acids Or the amino acid sequence of the polypeptide, or an amino acid sequence that has at least 90% homology with the sequence shown in SEQ ID NO. 9;

LCDR3: Contains the sequence shown in SEQ ID NO.11, or contains the sequence shown in SEQ ID NO.11, a protein that can specifically bind to thymic stromal lymphopoietin obtained by deleting, replacing or inserting one or more amino acids Or the amino acid sequence of the polypeptide, or an amino acid sequence that has at least 90% homology with the sequence shown in SEQ ID NO.11;

(2) HCDR1: Contains the sequence shown in SEQ ID NO. 2, or contains the sequence shown in SEQ ID NO. 2, which is obtained by deleting, replacing or inserting one or more amino acids and can specifically bind to thymic stromal lymphocyte production The amino acid sequence of the protein or polypeptide of the protein, or an amino acid sequence that has at least 90% homology with the sequence shown in SEQ ID NO. 2;

HCDR2: Contains the sequence shown in SEQ ID NO. 4, or contains the sequence shown in SEQ ID NO. 4, a protein that can specifically bind to thymic stromal lymphopoietin obtained by deleting, replacing or inserting one or more amino acids Or the amino acid sequence of the polypeptide, or an amino acid sequence that has at least 90% homology with the sequence shown in SEQ ID NO. 4;

HCDR3: contains the sequence shown in SEQ ID NO. 6, or contains the sequence shown in SEQ ID NO. 6, a protein that can specifically bind to thymic stromal lymphopoietin, obtained by deleting, replacing or inserting one or more amino acids Or the amino acid sequence of the polypeptide, or an amino acid sequence that has at least 90% homology with the sequence shown in SEQ ID NO. 6;

LCDR1: Contains the sequence shown in SEQ ID NO. 8, or contains the sequence shown in SEQ ID NO. 8, a protein that can specifically bind to thymic stromal lymphopoietin obtained by deleting, replacing or inserting one or more amino acids Or the amino acid sequence of the polypeptide, or an amino acid sequence that has at least 90% homology with the sequence shown in SEQ ID NO. 8;

LCDR2: Contains the sequence shown in SEQ ID NO. 10, or contains the sequence shown in SEQ ID NO. 10, a protein that can specifically bind to thymic stromal lymphopoietin, obtained by deleting, replacing or inserting one or more amino acids Or the amino acid sequence of the polypeptide, or an amino acid sequence that has at least 90% homology with the sequence shown in SEQ ID NO. 10;

LCDR3: Contains the sequence shown in SEQ ID NO. 12, or contains the sequence shown in SEQ ID NO. 12, a protein that can specifically bind to thymic stromal lymphopoietin obtained by deleting, replacing or inserting one or more amino acids Or the amino acid sequence of the polypeptide, or an amino acid sequence that has at least 90% homology with the sequence shown in SEQ ID NO.12.

The aforementioned homology is preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

The thymic stromal lymphopoietin antibody or antigen-binding fragment thereof of the present invention can specifically bind to human TSLP (sequence shown in SEQ ID NO. 102) or conservative mutants thereof.

The above-mentioned thymic stromal lymphopoietin antibody also includes a heavy chain FR and a light chain FR; specifically, the heavy chain FR includes:

(1) HFR1: contains the sequence shown in any one of SEQ ID NO. 13-20, or contains the sequence shown in any one of SEQ ID NO. 13-20 obtained by deleting, replacing or inserting one or more amino acids The amino acid sequence of a protein or polypeptide having the same function, or an amino acid sequence that has at least 90% homology with the sequence shown in SEQ ID NO. 13-20;

(2) HFR2: contains the sequence shown in any one of SEQ ID NO. 21-27, or contains the sequence shown in any one of SEQ ID NO. 21-27 obtained by deleting, replacing or inserting one or more amino acids The amino acid sequence of a protein or polypeptide having the same function, or an amino acid sequence that has at least 90% homology with the sequence shown in any one of SEQ ID NOs. 21-27;

(3) HFR3: contains the sequence shown in any one of SEQ ID NO. 28-35, or contains the sequence shown in any one of SEQ ID NO. 28-35 by deleting, replacing or inserting one or more amino acids The amino acid sequence of a protein or polypeptide having the same function, or an amino acid sequence that has at least 90% homology with the sequence shown in SEQ ID NO. 28-35;

(4) HFR4: contains the sequence shown in any one of SEQ ID NO. 36-39, or contains the sequence shown in any one of SEQ ID NO. 36-39 by deleting, replacing or inserting one or more amino acids The amino acid sequence of a protein or polypeptide having the same function, or an amino acid sequence that has at least 90% homology with the sequence shown in any one of SEQ ID NOs. 36-39;

The light chain FR includes:

(1) LFR1: contains the sequence shown in any one of SEQ ID NO. 40-47, or contains the sequence shown in any one of SEQ ID NO. 40-47 by deleting, replacing or inserting one or more amino acids The amino acid sequence of a protein or polypeptide having the same function, or an amino acid sequence that has at least 90% homology with the sequence shown in any one of SEQ ID NO. 40-47;

(2) LFR2: contains the sequence shown in any one of SEQ ID NO.48-53, or contains the sequence shown in any one of SEQ ID NO.48-53 obtained by deleting, replacing or inserting one or more amino acids The amino acid sequence of a protein or polypeptide with the same function, or an amino acid sequence that has at least 90% homology with the sequence shown in any one of SEQ ID NO. 48-53;

(3) LFR3: contains the sequence shown in any one of SEQ ID NO. 54-61, or contains the sequence shown in any one of SEQ ID NO. 54-61 by deleting, replacing or inserting one or more amino acids The amino acid sequence of a protein or polypeptide with the same function, or an amino acid sequence that has at least 90% homology with the sequence shown in any one of SEQ ID NO. 54-61;

(4) LFR4: contains the sequence shown in any one of SEQ ID NO. 62-65, or contains the sequence shown in any one of SEQ ID NO. 62-65 obtained by deleting, replacing or inserting one or more amino acids The amino acid sequence of a protein or polypeptide having the same function, or an amino acid sequence having at least 90% homology with the sequence shown in SEQ ID NO. 62-65.

The aforementioned homology is preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

The present invention develops humanized antibodies based on the obtained murine antibodies. In the above FRs, such as SEQ ID NO. 13, 21, 28, 36, 40, 48, 54, 62, 14, 22, 29 The FRs shown in, 37, 41, 49, 55 and 63 are mouse-derived FRs; the remaining FRs are humanized FRs.

The above heavy chain FRs, CDRs and light chain FRs, CDRs can be combined to obtain different thymic stromal lymphopoietin antibodies. In the above different combinations, there are the following (1) to (8) variable regions The antibody has better affinity for thymic stromal lymphopoietin and receptor binding inhibition:

(1) Heavy chain variable region: contains the sequence shown in SEQ ID NO.66, or contains the sequence shown in SEQ ID NO.66, which is a protein with the same function obtained by deleting, replacing or inserting one or more amino acids Or the amino acid sequence of the polypeptide, or an amino acid sequence that has at least 90% homology with the sequence shown in SEQ ID NO. 66;

Light chain variable region: comprising the sequence shown in SEQ ID NO. 67, or a protein or polypeptide with the same function obtained by deleting, replacing or inserting one or more amino acids in the sequence shown in SEQ ID NO. 67 An amino acid sequence, or an amino acid sequence that has at least 90% homology with the sequence shown in SEQ ID NO. 67;

(2) Heavy chain variable region: contains the sequence shown in SEQ ID NO. 68, or contains the sequence shown in SEQ ID NO. 68, which has the same functional protein obtained by deleting, replacing or inserting one or more amino acids Or the amino acid sequence of the polypeptide, or an amino acid sequence that has at least 90% homology with the sequence shown in SEQ ID NO. 68;

Light chain variable region: comprising the sequence shown in SEQ ID NO. 69, or a protein or polypeptide with the same function obtained by deleting, replacing or inserting one or more amino acids in the sequence shown in SEQ ID NO. 69 An amino acid sequence, or an amino acid sequence that has at least 90% homology with the sequence shown in SEQ ID NO. 69;

(3) Light chain variable region: contains the sequence shown in SEQ ID NO. 70, or contains the sequence shown in SEQ ID NO. 70, which is obtained by deleting, replacing or inserting one or more amino acids with the same function Or the amino acid sequence of the polypeptide, or an amino acid sequence that has at least 90% homology with the sequence shown in SEQ ID NO. 70;

Heavy chain variable region: contains the sequence shown in any one of SEQ ID NO. 73-75, or contains the sequence shown in any one of SEQ ID NO. 73-75 obtained by deleting, replacing or inserting one or more amino acids The amino acid sequence of the protein or polypeptide with the same function, or the amino acid sequence that has at least 90% homology with the sequence shown in SEQ ID NO. 73-75;

(4) Light chain variable region: contains the sequence shown in SEQ ID NO.71, or contains the sequence shown in SEQ ID NO.71 by deleting, replacing or inserting one or more amino acids to obtain a protein with the same function Or the amino acid sequence of the polypeptide, or an amino acid sequence that has at least 90% homology with the sequence shown in SEQ ID NO. 71;

Heavy chain variable region: contains the sequence shown in any one of SEQ ID NO. 73-75, or contains the sequence shown in any one of SEQ ID NO. 73-75 obtained by deleting, replacing or inserting one or more amino acids The amino acid sequence of the protein or polypeptide with the same function, or the amino acid sequence that has at least 90% homology with the sequence shown in SEQ ID NO. 73-75;

(5) Light chain variable region: comprising the sequence shown in SEQ ID NO.72, or a protein with the same function obtained by deleting, replacing or inserting one or more amino acids in the sequence shown in SEQ ID NO.72 Or the amino acid sequence of the polypeptide, or an amino acid sequence that has at least 90% homology with the sequence shown in SEQ ID NO. 72;

Heavy chain variable region: contains the sequence shown in any one of SEQ ID NO. 73-75, or contains the sequence shown in any one of SEQ ID NO. 73-75 obtained by deleting, replacing or inserting one or more amino acids The amino acid sequence of the protein or polypeptide with the same function, or the amino acid sequence that has at least 90% homology with the sequence shown in SEQ ID NO. 73-75;

(6) Light chain variable region: comprising the sequence shown in SEQ ID NO.76, or a protein with the same function obtained by deleting, replacing or inserting one or more amino acids in the sequence shown in SEQ ID NO.76 Or the amino acid sequence of the polypeptide, or an amino acid sequence that has at least 90% homology with the sequence shown in SEQ ID NO. 76;

Heavy chain variable region: contains the sequence shown in any one of SEQ ID NO. 79-81, or contains the sequence shown in any one of SEQ ID NO. 79-81 obtained by deleting, replacing or inserting one or more amino acids The amino acid sequence of the protein or polypeptide with the same function, or the amino acid sequence that has at least 90% homology with the sequence shown in SEQ ID NO. 79-81;

(7) Light chain variable region: contains the sequence shown in SEQ ID NO.77, or contains the sequence shown in SEQ ID NO.77 by deleting, replacing or inserting one or more amino acids to obtain a protein with the same function Or the amino acid sequence of the polypeptide, or an amino acid sequence that has at least 90% homology with the sequence shown in SEQ ID NO. 77;

Heavy chain variable region: contains the sequence shown in any one of SEQ ID NO. 79-81, or contains the sequence shown in any one of SEQ ID NO. 79-81 obtained by deleting, replacing or inserting one or more amino acids The amino acid sequence of the protein or polypeptide with the same function, or the amino acid sequence that has at least 90% homology with the sequence shown in SEQ ID NO. 79-81;

(8) Light chain variable region: contains the sequence shown in SEQ ID NO.78, or contains the sequence shown in SEQ ID NO.78 by deleting, replacing or inserting one or more amino acids to obtain a protein with the same function Or the amino acid sequence of the polypeptide, or an amino acid sequence that has at least 90% homology with the sequence shown in SEQ ID NO. 78;

Heavy chain variable region: contains the sequence shown in any one of SEQ ID NO. 79-81, or contains the sequence shown in any one of SEQ ID NO. 79-81, obtained by deleting, replacing or inserting one or more amino acids The amino acid sequence of the protein or polypeptide with the same function, or the amino acid sequence with at least 90% homology with the sequence shown in SEQ ID NO. 79-81.

In the present invention, the thymic stromal lymphopoietin antibody is a murine antibody, a humanized antibody, a chimeric antibody or a multispecific antibody. The antigen binding fragment is Fab, Fab', F(ab') ₂ , Fd, Fv, scFv, SdAb or Fab/c.

On the basis of the above-mentioned light chain variable region and heavy chain variable region, those skilled in the art can construct monoclonal antibodies, multispecific antibodies (such as bispecific antibodies) or Fab, Fab using conventional technical means in the art as needed. ', F(ab') ₂ , Fd, Fv, scFv, SdAb or Fab/c antibody.

Preferably, the heavy chain of the thymic stromal lymphopoietin antibody is of IgG2 type or IgG1 type, and the light chain is of κ type.

As a preferred solution of the present invention, the thymic stromal lymphopoietin antibody is any one of the following (1) to (8):

(1) The heavy chain sequence is shown in SEQ ID NO.82, and the light chain sequence is shown in SEQ ID NO.83;

(2) The heavy chain sequence is shown in SEQ ID NO.84, and the light chain sequence is shown in SEQ ID NO.85;

(3) The light chain sequence is shown in SEQ ID NO. 86, and the heavy chain sequence is shown in any one of SEQ ID NO. 89-91;

(4) The light chain sequence is shown in SEQ ID NO. 87, and the heavy chain sequence is shown in any one of SEQ ID NO. 89-91;

(5) The light chain sequence is shown in SEQ ID NO.88, and the heavy chain sequence is shown in any one of SEQ ID NO.89-91;

(6) The light chain sequence is shown in SEQ ID NO. 92, and the heavy chain sequence is shown in any one of SEQ ID NO. 95-97;

(7) The light chain sequence is shown in SEQ ID NO.93, and the heavy chain sequence is shown in any one of SEQ ID NO.95-97;

(8) The light chain sequence is shown in SEQ ID NO.94, and the heavy chain sequence is shown in any one of SEQ ID NO.95-97;

(9) The light chain sequence is shown in SEQ ID NO. 98, and the heavy chain sequence is shown in SEQ ID NO. 99;

(10) The light chain sequence is shown in SEQ ID NO.100, and the heavy chain sequence is shown in SEQ ID NO.101.

In the second aspect, the present invention provides a hybridoma cell capable of producing the thymic stromal lymphopoietin antibody, its antigen-binding fragment, its polypeptide or its fusion protein.

In the third aspect, the present invention provides a nucleic acid that encodes the thymic stromal lymphopoietin antibody, its antigen-binding fragment, its polypeptide or its fusion protein.

In a fourth aspect, the present invention provides a biological material comprising a nucleic acid encoding the thymic stromal lymphopoietin antibody, its antigen-binding fragment, its polypeptide, or its fusion protein. Specifically, the biological material is selected from one of an expression cassette, a vector, a transposon, a host cell, or a cell line.

The vectors include, but are not limited to, cloning vectors, expression vectors, and plasmid vectors. All vectors containing the nucleic acid encoding the thymic stromal lymphopoietin antibody, its antigen-binding fragment or its polypeptide or fusion protein are within the protection scope of the present invention. Inside.

The host cell or cell line may be a cell or cell line derived from a microorganism, plant or animal, all containing nucleic acid or its vector encoding the thymic stromal lymphopoietin antibody, its antigen-binding fragment or its polypeptide or fusion protein The host cells or cell lines of are all within the protection scope of the present invention.

In the fifth aspect, the present invention provides a method for preparing thymic stromal lymphopoietin antibody, its antigen-binding fragment, its polypeptide or its fusion protein, which is to prepare the thymic stromal lymphopoietin antibody by using the hybridoma cells , Its antigen-binding fragment, its polypeptide or its fusion protein; or, using host cells to express the nucleic acid encoding the thymic stromal lymphopoietin antibody, its antigen-binding fragment, its polypeptide or its fusion protein.

In the sixth aspect, the present invention provides a complex or conjugate comprising the thymic stromal lymphopoietin antibody, its antigen-binding fragment, its polypeptide or its fusion protein.

As an embodiment of the present invention, the complex is obtained by chemically labeling or biomarking the thymic stromal lymphopoietin antibody, its antigen-binding fragment, its polypeptide or its fusion protein.

The chemical markers include, but are not limited to, isotopes, immunotoxins, and/or chemical drugs.

The biomarkers include, but are not limited to, biotin, avidin, or enzyme markers.

As an embodiment of the present invention, the conjugate is obtained by coupling the thymic stromal lymphopoietin antibody, its antigen-binding fragment, its polypeptide or its fusion protein, or the complex and a carrier.

The carrier may be a solid phase carrier, including but not limited to polystyrene plates and the like.

In the seventh aspect, the present invention provides the thymic stromal lymphopoietin antibody, its antigen-binding fragment, its polypeptide or its fusion protein, or the hybridoma cell or the nucleic acid or the biological material or the complex or couple Any of the following applications of Lianwu:

(1) Application in the preparation of reagents or kits for diagnosing or assisting in the diagnosis of diseases related to thymic stromal lymphopoietin or its receptor;

(2) Application in the preparation of drugs for the treatment or adjuvant treatment of diseases related to thymic stromal lymphopoietin or its receptor;

(3) Application in the preparation of reagents or kits for tracking or prognostic inspection of thymic stromal lymphopoietin or its receptor-related diseases;

(4) Application in detecting the content of thymic stromal lymphopoietin;

(5) Application in inhibiting the binding of thymic stromal lymphopoietin to its receptor.

Preferably, the disease related to thymic stromal lymphopoietin or its receptor is an immune system disease or a tumor disease; the immune system disease includes an inflammatory or allergic inflammatory disease; the tumor disease includes a breast tumor, Huo Chikin lymphoma, pancreatic cancer, melanoma and lung cancer;

More preferably, the inflammatory or allergic inflammatory disease is selected from allergic asthma, chronic obstructive pulmonary disease, allergic dermatitis, allergic rhinitis, allergic sinusitis, eosinophilic esophagitis, allergic conjunctivitis , Inflammatory bowel disease or atopic dermatitis.

In an eighth aspect, the present invention provides a pharmaceutical composition comprising the thymic stromal lymphopoietin antibody, its antigen-binding fragment, its polypeptide or its fusion protein, or the complex or conjugate.

The pharmaceutical composition may also include a pharmaceutically acceptable carrier or excipient. The type of carrier or excipient used particularly depends on the mode of administration of the pharmaceutical composition of the present invention (such as oral, nasal, intradermal, subcutaneous, intramuscular or intravenous, etc.).

The pharmaceutical composition may also contain antibodies or active ingredients with other functions.

The pharmaceutical composition can be prepared into different dosage forms as required, such as a dry powder formulation.

The pharmaceutical composition can be used to prevent or treat diseases related to thymic stromal lymphopoietin or its receptor.

In a ninth aspect, the present invention provides a kit comprising the thymic stromal lymphopoietin antibody, its antigen-binding fragment, its polypeptide or its fusion protein, or the complex or conjugate.

The kit may also contain other reagents required for immunological detection. For example, the kit also includes one or more of coating buffer, washing solution, blocking solution, and color developing solution.

The kit can be used for detecting the content of thymic stromal lymphopoietin, or for the diagnosis, tracking and prognosis of diseases related to thymic stromal lymphopoietin or its receptor.

In a ninth aspect, the present invention provides a method for the prevention, treatment or adjuvant treatment of diseases related to thymus stromal lymphopoietin or its receptors, comprising: administering the thymic stromal lymphopoietin antibody or antigen-binding fragment thereof to a subject , Its polypeptide or its fusion protein or the pharmaceutical composition.

Preferably, the disease related to thymic stromal lymphopoietin or its receptor is an immune system disease or a tumor disease; the immune system disease includes an inflammatory or allergic inflammatory disease; the tumor disease includes a breast tumor, Hodgkin's lymphoma, pancreatic cancer, melanoma and lung cancer.

More preferably, the inflammatory or allergic inflammatory disease is selected from allergic asthma, chronic obstructive pulmonary disease, allergic dermatitis, allergic rhinitis, allergic sinusitis, eosinophilic esophagitis, allergic conjunctivitis , Inflammatory bowel disease or atopic dermatitis.

In a tenth aspect, the present invention provides a method for detecting the content of thymic stromal lymphopoietin, using the thymic stromal lymphopoietin antibody, its antigen-binding fragment, its polypeptide or its fusion protein, or using the complex or coupling Or use the kit to detect the content of thymic stromal lymphopoietin.

The beneficial effects of the present invention are as follows: The TSLP antibody provided by the present invention can specifically bind to hTSLP, has high affinity, and can effectively inhibit the binding of TSLP to its receptor, thereby effectively inhibiting TSLP from activating downstream signaling pathways and the activation of immune cells. Content detection and inflammation such as allergic asthma, chronic obstructive pulmonary disease, allergic dermatitis, allergic rhinitis, allergic sinusitis, eosinophilic esophagitis, allergic conjunctivitis, inflammatory bowel disease or atopic dermatitis Or the diagnosis, treatment and prognosis of allergic inflammatory diseases are of great significance.

Description of the drawings

Figure 1 shows the results of the assay results of the murine TSLP monoclonal antibody inhibiting the binding of TSLP to TSLPR in Example 6 of the present invention.

Fig. 2 is the result of the determination of the activity of the chimeric antibody in inhibiting the combination of TSLP and TSLPR in Example 7 of the present invention.

Fig. 3 is the result of determination of STAT5-luciferase activity of 293F-transfected cells stimulated by the chimeric antibody in Example 7 of the present invention.

Fig. 4 shows the detection result of the binding capacity of the human TSLP humanized monoclonal antibody to the human recombinant TSLP protein in Example 8 of the present invention.

Fig. 5 is the detection result of the human TSLP humanized monoclonal antibody inhibiting the combination of TSLP and TSLPR in Example 8 of the present invention.

Figure 6 shows the detection result of the human TSLP humanized monoclonal antibody inhibiting the STAT5-luciferase activity of TSLP-stimulated 293F transfected cells in Example 8 of the present invention.

Detailed ways

The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are given for illustrative purposes only, and are not intended to limit the scope of the present invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from the spirit and spirit of the present invention.

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

The amino acid or nucleotide sequence involved in the present invention is specifically as follows:

Mouse monoclonal antibody 1K/1H HCDR1: SEQ ID NO. 1; HCDR2: SEQ ID NO. 3; HCDR3: SEQ ID NO. 5; LCDR1: SEQ ID NO. 7; LCDR2: SEQ ID NO. 9; LCDR3 ：SEQ ID NO.11;

Mouse monoclonal antibody 1K/1H HFR1: SEQ ID NO. 13; HFR2: SEQ ID NO. 21; HFR3: SEQ ID NO. 28; HFR4: SEQ ID NO. 36; LFR1: SEQ ID NO. 40; LFR2 : SEQ ID NO. 48; LFR3: SEQ ID NO. 54; LFR4: SEQ ID NO. 62;

The variable region of the heavy chain of the murine monoclonal antibody 1K/1H: SEQ ID NO.66; the variable region of the light chain: SEQ ID NO.67; the heavy chain: SEQ ID NO.82; the light chain: SEQ ID NO.83 .

Mouse monoclonal antibody 6K/3H HCDR1: SEQ ID NO. 2; HCDR2: SEQ ID NO. 4; HCDR3: SEQ ID NO. 6; LCDR1: SEQ ID NO. 8; LCDR2: SEQ ID NO. 10; LCDR3 ：SEQ ID NO.12;

Mouse monoclonal antibody 6K/3H HFR1: SEQ ID NO. 14; HFR2: SEQ ID NO. 22; HFR3: SEQ ID NO. 29; HFR4: SEQ ID NO. 37; LFR1: SEQ ID NO. 41; LFR2 : SEQ ID NO. 49; LFR3: SEQ ID NO. 55; LFR4: SEQ ID NO. 63;

The variable region of the heavy chain of the murine monoclonal antibody 6K/3H: SEQ ID NO.68; the variable region of the light chain: SEQ ID NO.69; the heavy chain: SEQ ID NO.84; the light chain: SEQ ID NO.85 .

HFR1 of humanized heavy chain 1H1: SEQ ID NO.15; HFR2: SEQ ID NO.23; HFR3: SEQ ID NO.30; HFR4: SEQ ID NO.38;

HFR1 of humanized heavy chain 1H2: SEQ ID NO.16; HFR2: SEQ ID NO.24; HFR3: SEQ ID NO.31; HFR4: SEQ ID NO.39;

HFR1 of humanized heavy chain 1H3: SEQ ID NO.17; HFR2: SEQ ID NO.25; HFR3: SEQ ID NO.32; HFR4: SEQ ID NO.38;

LFR1 of the humanized light chain 1K1: SEQ ID NO. 42; LFR2: SEQ ID NO. 48; LFR3: SEQ ID NO. 56; LFR4: SEQ ID NO. 64;

LFR1 of the humanized light chain 1K2: SEQ ID NO. 43; LFR2: SEQ ID NO. 50; LFR3: SEQ ID NO. 57; LFR4: SEQ ID NO. 64;

LFR1 of the humanized light chain 1K3: SEQ ID NO. 44; LFR2: SEQ ID NO. 48; LFR3: SEQ ID NO. 58; LFR4: SEQ ID NO. 64;

HFR1 of humanized heavy chain 3H1: SEQ ID NO.18; HFR2: SEQ ID NO.26; HFR3: SEQ ID NO.33; HFR4: SEQ ID NO.38;

HFR1 of humanized heavy chain 3H2: SEQ ID NO.19; HFR2: SEQ ID NO.27; HFR3: SEQ ID NO.34; HFR4: SEQ ID NO.38;

HFR1 of humanized heavy chain 3H3: SEQ ID NO.20; HFR2: SEQ ID NO.26; HFR3: SEQ ID NO.35; HFR4: SEQ ID NO.38;

LFR1 of the humanized light chain 6K1: SEQ ID NO.45; LFR2: SEQ ID NO.51; LFR3: SEQ ID NO.59; LFR4: SEQ ID NO.64;

LFR1 of the humanized light chain 6K2: SEQ ID NO. 46; LFR2: SEQ ID NO. 52; LFR3: SEQ ID NO. 60; LFR4: SEQ ID NO. 65;

LFR1 of the humanized light chain 6K3: SEQ ID NO.47; LFR2: SEQ ID NO.53; LFR3: SEQ ID NO.61; LFR4: SEQ ID NO.62;

The variable region of the light chain of the humanized monoclonal antibody 1K1/1H1: SEQ ID NO. 70; the variable region of the heavy chain: SEQ ID NO. 73; the light chain: SEQ ID NO. 86; the heavy chain: SEQ ID NO. 89;

The variable region of the light chain of the humanized monoclonal antibody 1K1/1H2: SEQ ID NO. 70; the variable region of the heavy chain: SEQ ID NO. 74; the light chain: SEQ ID NO. 86; the heavy chain: SEQ ID NO. 90;

The variable region of the light chain of the humanized monoclonal antibody 1K1/1H3: SEQ ID NO. 70; the variable region of the heavy chain: SEQ ID NO. 75; the light chain: SEQ ID NO. 86; the heavy chain: SEQ ID NO. 91;

The variable region of the light chain of the humanized monoclonal antibody 1K2/1H1: SEQ ID NO. 71; the variable region of the heavy chain: SEQ ID NO. 73; the light chain: SEQ ID NO. 87; the heavy chain: SEQ ID NO. 89;

The variable region of the light chain of the humanized monoclonal antibody 1K2/1H2: SEQ ID NO. 71; the variable region of the heavy chain: SEQ ID NO. 74; the light chain: SEQ ID NO. 87; the heavy chain: SEQ ID NO. 90;

The variable region of the light chain of the humanized monoclonal antibody 1K2/1H3: SEQ ID NO. 71; the variable region of the heavy chain: SEQ ID NO. 75; the light chain: SEQ ID NO. 87; the heavy chain: SEQ ID NO. 91;

The variable region of the light chain of the humanized monoclonal antibody 1K3/1H1: SEQ ID NO. 72; the variable region of the heavy chain: SEQ ID NO. 73; the light chain: SEQ ID NO. 88; the heavy chain: SEQ ID NO. 89;

The variable region of the light chain of the humanized monoclonal antibody 1K3/1H2: SEQ ID NO. 72; the variable region of the heavy chain: SEQ ID NO. 74; the light chain: SEQ ID NO. 88; the heavy chain: SEQ ID NO. 90;

The variable region of the light chain of the humanized monoclonal antibody 1K3/1H3: SEQ ID NO. 72; the variable region of the heavy chain: SEQ ID NO. 75; the light chain: SEQ ID NO. 88; the heavy chain: SEQ ID NO. 91;

The light chain variable region of the humanized monoclonal antibody 6K1/3H1: SEQ ID NO.76; the heavy chain variable region: SEQ ID NO.79; the light chain: SEQ ID NO.92; the heavy chain: SEQ ID NO. 95;

The variable region of the light chain of the humanized monoclonal antibody 6K1/3H2: SEQ ID NO. 76; the variable region of the heavy chain: SEQ ID NO. 80; the light chain: SEQ ID NO. 92; the heavy chain: SEQ ID NO. 96;

The variable region of the light chain of the humanized monoclonal antibody 6K1/3H3: SEQ ID NO. 76; the variable region of the heavy chain: SEQ ID NO. 81; the light chain: SEQ ID NO. 92; the heavy chain: SEQ ID NO. 97;

The variable region of the light chain of the humanized monoclonal antibody 6K2/3H1: SEQ ID NO.77; the variable region of the heavy chain: SEQ ID NO.79; the light chain: SEQ ID NO.93; the heavy chain: SEQ ID NO. 95;

The variable region of the light chain of the humanized monoclonal antibody 6K2/3H2: SEQ ID NO.77; the variable region of the heavy chain: SEQ ID NO.80; the light chain: SEQ ID NO.93; the heavy chain: SEQ ID NO. 96;

The variable region of the light chain of the humanized monoclonal antibody 6K2/3H3: SEQ ID NO.77; the variable region of the heavy chain: SEQ ID NO.81; the light chain: SEQ ID NO.93; the heavy chain: SEQ ID NO. 97;

The light chain variable region of the humanized monoclonal antibody 6K3/3H1: SEQ ID NO.78; the heavy chain variable region: SEQ ID NO.79; the light chain: SEQ ID NO.94; the heavy chain: SEQ ID NO. 95;

The variable region of the light chain of the humanized monoclonal antibody 6K3/3H2: SEQ ID NO. 78; the variable region of the heavy chain: SEQ ID NO. 80; the light chain: SEQ ID NO. 94; the heavy chain: SEQ ID NO. 96;

The variable region of the light chain of the humanized monoclonal antibody 6K3/3H3: SEQ ID NO.78; the variable region of the heavy chain: SEQ ID NO.81; the light chain: SEQ ID NO.94; the heavy chain: SEQ ID NO. 97.

Chimeric antibody 1K-1H: the light chain sequence is shown in SEQ ID NO.98, and the heavy chain sequence is shown in SEQ ID NO.99;

Chimeric antibody 6K-3H: The light chain sequence is shown in SEQ ID NO.100, and the heavy chain sequence is shown in SEQ ID NO.101.

Example 1 Preparation of immunogen

Construct a human TSLP eukaryotic expression vector, transfect 293F cells, and purify the culture supernatant through a Protein A column to prepare human TSLP protein for mouse immunization, clone screening and functional identification.

(1) Construction of human TSLP eukaryotic expression vector

The sequence of human TSLP starts from Tyr 29 of natural human TSLP to Gln 159, with a total of 131 amino acids (sequence shown in SEQ ID NO.102). The mouse Fc fragment (mFc) (Accession number: AJ487681, Allele or Gene:IGHG1*01), can bind to the recombinant protein A in the protein A column, which can be purified by affinity chromatography. Therefore, the rhTSLP-mFc fusion protein (amino acid sequence such as SEQ ID NO.103 Shown).

PCR amplifies the above rhTSLP-mFc fusion gene, and constructs a recombinant plasmid by homologous recombination with pcDNA3.1(+) (V790-20, Thermo). The positive clone is identified by PCR and restriction enzyme digestion, and finally the expression vector is verified by sequencing The correctness is named pcDNA3.1(+)-rhTSLP-mFc. Use plasmid extraction kit to extract plasmid for transformation.

(2) Expression of rhTSLP-mFc fusion gene in 293F cells

Twenty-four hours before transfection, 293F (Kerry Zhuhai) was diluted with 293 medium (Kerry Zhuhai, K03252) to a density of 3.0×10 ⁶ cells/ml. Incubate in a constant temperature shaker at 130 rpm, 37°C, and 5% CO ₂ so that the cell density (hemocytometer) on the day of transfection is 4.0-6.0×10 ⁶ cells/ml. To ensure the best transfection effect, cell viability (trypan blue staining method) should be greater than 97%.

Take the transfection of 100ml cell suspension as an example, prepare two 15ml sterile centrifuge tubes, add 5ml KPM (Zhuhai Kairui, K03125L) and 100μg sterile plasmid pcDNA3.1(+)-rhTSLP-mFc to one of them, Gently pipette to mix; add another 5ml KPM and 500μl TA-293 transfection reagent (Kerry Zhuhai, K20001), gently pipette to mix; transfer all the liquid in the centrifuge tube containing the transfection reagent to the plasmid In the centrifuge tube, gently pipette to mix; let stand at room temperature for 10 minutes to prepare the plasmid-vector complex; remove the cells from the constant temperature shaker, add the prepared plasmid-vector complex while shaking, and return to the CO ₂ Incubate in a constant temperature shaker. After 24 hours of transfection, 600μl 293 cell protein expression enhancer (KE-293) (Kerry Zhuhai, K30001) and transient transfection nutrient additives (KT-Feed 50×) (Kerry Zhuhai, K40001) can be added to increase product expression Amount; collect the supernatant about 5 days after transfection, freeze the centrifuge at 9000 rpm / separation for 20 minutes, collect the supernatant for the next step of protein purification.

(3) Purification of rhTSLP-mFc

The above-mentioned 293F cell supernatant containing the antigen rhTSLP-mFc was centrifuged and captured using a Protein A column (GE Healthcare Bio-Sciences, 17-5080-02), and then captured with 50 mM citric acid-sodium citrate buffer ( pH 3.0) elution, collect the eluate (1.0ml/tube), add 50μl 1M Tris-HCl buffer (pH 8.0) to neutralize to neutrality, pass the 10K dialysis membrane (Generay, M1915) in phosphate buffer PBS After dialysis, the protein content was measured at 280nm. Store at -80°C after filtration and sterilization.

Example 2 Mouse Immunization and Determination of Antibody Titer in Serum

Use rhTSLP-His (rhTSLP coupled with His tag, the sequence is shown in SEQ ID NO.104) or rhTSLP-mFc prepared in Example 1 as the immunogen to Balb/c mice (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) for immunization. A total of 10 mice were immunized, 6 of which were rhTSLP-His and were immunized with Freund's complete adjuvant; the other 4 mice were rhTSLP-mFc and were quickly immunized with a water-soluble adjuvant.

When using Freund's complete adjuvant immunization, multiple subcutaneous injections were used on the back, and the immunization dose was 50μg/200μl/mouse. For the first immunization, mix 50μg rhTSLP-His with 100μl Freund’s complete adjuvant (Sigma, F5881), and mix 50μg rhTSLP-His with 100μl Freund’s adjuvant for the second, third, fourth and fifth immunizations. Complete adjuvant (Sigma, F5506) was mixed, and the five immunization times were 0, 14, 28, 42, and 56 days. After the fifth immunization (day 63) of the 6 mice, blood was collected from the eyeballs, and the titer of anti-human TSLP antibody in the serum of the immunized mice was determined by ELISA. After the fifth immunization, No. 1 and No. 6 mice with the highest titers of mouse serum were selected for subsequent antigen shock immunization and preparation of hybridoma cells.

When using the rapid immunization method, the immunization method is intramuscular injection, and the immunization dose is 20μg/100μl/mouse. For the first immunization, mix 20μg/50μL rhTSLP-mFc antigen and immune adjuvant (QuickAntibody-Mouse5W, Boolon, KX0210041, a new water-soluble adjuvant) at a volume ratio of 1:1. Three weeks after the first immunization, the second booster immunization was performed in the same manner. Two weeks after the second immunization, blood was collected for titer determination. The mice No. 2 and No. 4 with the highest titers of mouse serum were selected for the subsequent antigen shock immunization and the preparation of hybridoma cells.

Serum obtained by the above two methods are immunized by ELISA for titer determination, assay methods are briefly described below: First, a coating buffer _{(0.05M Na 2 CO 3 -NaHCO 3} , pH 9.6) (CB) was diluted rhTSLP- hFc (amino acid sequence shown in SEQ ID NO. 105) to 1 μg/ml, 100 μl/well was added to a 96-well ELISA plate (Costar, 2592), overnight at 4°C. The next day, the plate was washed 3 times with PBST (0.5‰), and blocking solution (3% BSA in1×PBS) was added for blocking at 37°C for 2 hours. Wash the plate once, dilute the mouse serum and negative mouse serum obtained above with PBS starting from 1:100 in a 2-3 times gradient. The blank wells are PBS, and 100μl/well is added to the ELISA plate. Incubate at room temperature for 1 hour. Wash the plate Three times, goat anti-mouse IgG (H+L)-HRP (ProteinTech, SA00001-1) with a final concentration of 1 μg/ml was added, and incubated for 1 hour at room temperature. Wash the plate 4 times, add TMB (Zuman Bio, ZD311) color developing solution at room temperature to develop color for 10-20 minutes, add stop solution, and read the absorbance at 450nm wavelength on a microplate reader (BioTek, ELx808). A _{positive clone is defined as a positive clone with an OD 450} value greater than 2 times the negative well. At the highest dilution of the serum, the _{higher the OD 450} value, the stronger the immunoreactivity to human TSLP. The results of potency determination are shown in Table 1 and Table 2.

Table 1 Determination of serum titer of immunized mice

Table 2 Determination of serum titer of immunized mice

Example 3 Preparation of Hybridoma

After the last immunization in Example 2, the spleens of the 2 mice with the highest titer obtained by each immunization method (Freund's complete adjuvant immunization No. 1 and No. 6; rapid immunization No. 2 and No. 4) were mixed with each other. After milling in normal saline, a suspension rich in B cells was taken and fused with the myeloma cell SP2/0 under the action of the fusion agent PEG (Sigma, P7181). Divide the fused cells into 15 96-well cell culture plates, and place them in 20% fetal bovine serum RPMI-1640 complete medium (Thermo, 31800089) containing HAT (Sigma, H0262) and place them in 5% CO ₂ at 37°C. After cultivating for one week, switch to HT medium (Sigma, H0137) to continue culturing.

Example 4 Screening and subcloning of hybridomas

The positive clones were screened in the hybridoma cells prepared in Example 3. The specific method is as follows:

(1) Enzyme-linked immunosorbent assay (ELISA) to screen positive hybridoma clones with strong binding activity to the antigen hTSLP

The ELISA plate was coated with rhTSLP-hFc for the first round of screening of positive cell lines. After the first round of screening, select the positive hybridoma monoclonal with OD value>1.0.

(2) Enzyme-linked immunosorbent assay to screen positive clones of hybridomas with rhTSLP neutralizing activity

Dilute rhTSLP-hFc to 1 μg/ml with coating buffer (0.05M Na ₂ CO ₃ -NaHCO ₃ , pH 9.6) (CB), add 100 μl/well to a 96-well ELISA plate (Costar, 2592), and overnight at 4°C. The next day, the plate was washed 3 times with PBST (0.5‰), and blocking solution (3% BSA in 1×PBS) was added to block for 2 hours at 37°C. Wash the plate once. 50μl of hybridoma supernatant and rhTSLPR-His (sequence shown in SEQ ID NO.106, concentration of 2μg/ml) 50μl after incubation for 2h, bind to the blocking plate for 1h, wash the plate 3 times, add 1:10000 diluted anti-His tag- HRP (ProteinTech, HRP-66005), incubated at 37°C for 1 hour. Wash the plate 4 times, add TMB (Zuman Bio, ZD311) color developing solution at 37°C for 10-20 minutes, add stop solution, and read the absorbance at 450nm wavelength on a microplate reader (BioTek, ELx808). The neutralizing antibody can prevent the receptor TSLPR from binding to TSLP, resulting in a decrease in the amount of rhTSLPR-His binding and a decrease in the OD ₄₅₀ value.

(3) Obtaining of subclones of hybridomas with neutralizing activity

Select _{wells with a low OD 450} value, and use the limiting dilution method to clone the cells in the wells. After three times of subcloning, a monoclonal hybridoma cell line that stably secretes human TSLP neutralizing antibodies is established.

Example 5 Obtaining Murine TSLP Monoclonal Antibody

Example 4 The culture of hybridoma cells having the total amount obtained in the activity to ¹⁰⁷ cells, centrifuged at 1000 rpm for 10 minutes to collect cells, and the cartridge using Trizol (CWBio, CW0580S) extraction of total RNA. After the first-strand cDNA (CWBio, CW0744M) is synthesized using the extracted total RNA as a template, the first-strand cDNA is used as a subsequent template to amplify the variable region DNA sequence corresponding to the hybridoma cell. The primer sequence used in the amplification reaction is complementary to the first framework region and the constant region of the antibody variable region, refer to (Larrick, JW, et al. (1990), Scand. J. Immunol., 32: 121-128 and Coloma , J Jet al., (1991) BioTechniques, 11, 152-156). The DNA polymerase used for PCR amplification is Taq enzyme (NEB, M0491S).

Two groups of mouse TSLP monoclonal antibody sequences (1K/1H and 6K/3H) with neutralizing activity were obtained, and they were named as monoclonal antibodies 3A1H2B2E1 (1K/1H) and 7A9E3G10 (6K/3H), respectively.

At the same time, 6-8 weeks old Balb/c mice were injected intraperitoneally with 0.5ml of Freund’s incomplete adjuvant (Sigma, F5506). Seven days after the injection, the hybridoma cells with neutralizing activity obtained in Example 4 were cultured to In the logarithmic growth phase, the cell concentration was adjusted to 1×10 ⁶ ～2×10 ⁶ cells/ml with PBS, and 0.5 ml/mouse was injected into the abdominal cavity to grow hybridomas in the abdominal cavity of mice and produce ascites. 7-10 days after injection of hybridoma cells, mouse abdominal cavity enlargement can be seen. Collect mouse ascites and affinity-purify ascites with protein A column to obtain corresponding mouse TSLP monoclonal antibodies 3A1H2B2E (1K/1H) and 7A9E3G10 (6K/3H) ).

Example 6 Performance verification of mouse-derived antibodies

1. Determination of affinity constant

ForteBio Blitz Biomolecular Interaction Analysis (ForteBio) instrument was used to detect the affinity of the murine monoclonal antibodies 3A1H2B2E1 and 7A9E3G10E5 prepared in Example 5 for human TSLP, and the affinity constant detection results are shown in Table 3.

Table 3 Affinity constant determination results of mouse monoclonal antibodies

name	KD(M)	Ka(1/Ms)	Kd(1/s)
3A1H2B2E1	1.751e -9	4.177e 5	7.315e -4
7A9E3G10E5	2.009e -8	2.519e 5	5.059e -3

2. ELISA to determine the activity of murine TSLP monoclonal antibody to inhibit the binding of TSLP to TSLPR

Dilute rhTSLP-hFc to 1 μg/ml with coating buffer (0.05M Na ₂ CO ₃ -NaHCO ₃ , pH 9.6) (CB), add 100 μl/well to a 96-well ELISA plate (Costar, 2592), and overnight at 4°C. The next day, the plate was washed 3 times with PBST (0.5‰), and blocking solution (3% BSA in 1×PBS) was added to block for 2 hours at 37°C. Wash the plate once with PBST. Different concentrations of humanized TSLP monoclonal antibody and Tezepelumab 50μl (starting at 50μg/ml, 4-fold dilution to 0.012μg/ml) and 2μg/ml rhTSLPR-His 50μl were mixed and incubated for 2h and then blocked. Plate, combine at 37°C for 1h. Wash the plate 3 times with PBST, add 1:10000 diluted anti-His tag-HRP (ProteinTech, HRP-66005), and incubate at 37°C for 1 hour. Wash the plate 4 times with PBST, add TMB (Zuman Bio, ZD311) color developing solution at 37°C for 10-20 minutes, add stop solution, and read the absorbance at 450nm wavelength on a microplate reader (BioTek, ELx808).

The results are shown in Figure 1 and Table 4. The results show that the murine monoclonal antibodies 3A1H2B2E1 and 7A9E3G10E5 can significantly inhibit the binding of TSLP to TSLPR, and their inhibitory ability is better than that of human TSLP monoclonal antibody Tezepelumab/MEDI9929 (Amgen, hereinafter referred to as Tezepelumab, sequence The information is as follows: IMGT: Chain ID: INN 10172_H, 20172-L) is significantly stronger.

Table 4 Determination of murine monoclonal antibody inhibiting the binding activity of TSLP and TSLPR

Antibody	3A1H2B2E1	7A9E3G10E5	Tezepelumab
IC 50 (μg/mL)	0.3235	0.4279	0.6918

Example 7 Construction and performance verification of chimeric antibodies

1. Construction of chimeric antibody

The variable regions of murine monoclonal antibodies 3A1H2B2E1 and 7A9E3G10E5 were fused with the constant region of human IgG1 to construct chimeric antibodies 1K-1H and 6K-3H. The construction methods are as follows:

PCR amplified light chain variable regions 1K, 6K, heavy chain variable regions 1H, 3H, human light chain κ chain constant region Cκ, and heavy chain IgG1 constant region CH1-3. 1K, Cκ, 6K, Cκ, 1H, CH1-3, 3H, CH1-3 were constructed by homologous recombination with pcDNA3.1(+) (V790-20, Thermo), respectively, and the expression was verified by sequencing. The correctness of the vector is named pcDNA3.1(+)-1K, pcDNA3.1(+)-1H, pcDNA3.1(+)-6K, pcDNA3.1(+)-3H, and transfer the above recombinant expression plasmid into 293F cells, the chimeric antibody was expressed and purified in 293F cells.

2. ELISA measures the activity of chimeric antibody to inhibit the combination of TSLP and TSLPR

The method is the same as in Example 6. The results are shown in Figure 2 and Table 5. The results show that both chimeric antibodies 1K-1H and 6K-3H can significantly inhibit the binding of TSLP and TSLPR, and their inhibitory ability is significantly stronger than that of human TSLP monoclonal antibody Tezepelumab/MEDI9929.

Table 5 Determination of the activity of chimeric antibodies 1K-1H and 6K-3H in inhibiting the binding of TSLP to TSLPR

Antibody	1K-1H	6K-3H	Tezepelumab
IC 50 (μg/mL)	0.3882	0.6089	0.8316

3. Chimeric antibody inhibits STAT5-luciferase activity in 293F transfected cells stimulated by TSLP

Take 293F cells in logarithmic growth phase, adjust the cell density to 0.8×10 ⁶ /ml, inoculate a 6-well plate with 2ml/well, and co-transfect hTSLPR(Origene RC221190)/hIL7Rα(Sino Biological HG10975-CM)/pGL4 after 30 minutes. 52[luc2P/STAT5 RE/Hygro] (Promega E4651), the amount of plasmid is 231ng/231ng/1538ng. Inoculated into 96-well plates (black board, 5×10 ⁴ ～10×10 ⁴ /50 μl/well) 24 hours after transfection. Mix 8μg/ml TSLP-hFc and human TSLP humanized monoclonal antibody prepared in Example 6 of different concentrations (300μg/ml starting, 3-fold dilution to 0.046μg/ml) in equal volume and then incubate at 37°C 2h. Take 50 μl of the above-mentioned mixed sample and add it to the 96-well light-proof plate with added cells, and incubate at 37°C. After 24 hours, 100 μl/well of luminescent substrate was added, and the fluorescence intensity was measured by a chemiluminescence instrument (Promega Glomax) after 3 minutes.

The results are shown in Figure 3 and Table 6. The results show that both chimeric antibodies 1K-1H and 6K-3H can significantly inhibit TSLP-stimulated STAT5-luciferase activity, and the inhibitory capacity is significantly greater than that of human TSLP monoclonal antibody Tezepelumab/MEDI9929 powerful.

Table 6 Chimeric antibodies 1K-1H and 6K-3H inhibit STAT5-luciferase activity

Antibody	1K-1H	6K-3H	Tezepelumab
IC 50 (μg/mL)	3.482	0.2598	5.719

Example 8 Humanized modification and performance verification of mouse TSLP monoclonal antibody

1. Humanization of mouse TSLP monoclonal antibody

The sequences of the variable regions of the murine TSLP monoclonal antibodies 3A1H2B2E1 (1K/1H) and 7A9E3G10 (6K/3H) prepared in Example 5 were humanized, and the obtained humanized sequences were as follows:

1K/1H humanized transformation: light chain: 1K1, 1K2, 1K3; heavy chain: 1H1, 1H2, 1H3;

6K/3H humanized transformation: light chain: 6K1, 6K2, 6K3; heavy chain: 3H1, 3H2, 3H3.

The nucleotide sequences of the light chain and the heavy chain are connected into the modified pcDNA3.1(+) vector plasmid based on seamless cloning to construct a recombinant expression vector. The combination of light chain and heavy chain is as follows: 1K1/1H1, 1K1/1H2, 1K1/1H3, 1K2/1H1, 1K2/1H2, 1K2/1H3, 1K3/1H1, 1K3/1H2, 1K3/1H3; 6K1/3H1 6K1/3H2, 6K1/3H3, 6K2/3H1, 6K2/3H2, 6K2/3H3, 6K3/3H1, 6K3/3H2, 6K3/3H3.

The recombinant expression plasmids carrying the above-mentioned various humanized modified light chain and heavy chain combinations were transferred into 293F cells, and the expression and purification steps in the 293F cells were the same as those in (2) and (3) of the previous example 1. Sourced TSLP monoclonal antibody 1K1/1H1, 1K1/1H2, 1K1/1H3, 1K2/1H1, 1K2/1H2, 1K2/1H3, 1K3/1H1, 1K3/1H2, 1K3/1H3; 6K1/3H1, 6K1/3H2 6K1/3H3, 6K2/3H1, 6K2/3H2, 6K2/3H3, 6K3/3H1, 6K3/3H2, 6K3/3H3.

2. ELISA to determine the binding activity of humanized TSLP monoclonal antibody to TSLP

The rhTSLP-mFc antigen was _{diluted to 1μg/ml with coating buffer (0.05M Na 2} CO ₃ -NaHCO ₃ , pH 9.6) CB (pH=9.6), added to the microtiter plate at 100μl/well, and coated overnight at 4°C . After washing the plate twice with PBST, 200μl/well of 3% BSA was added, and the plate was blocked at 37°C for 2 hours. Wash the plate again with PBST, and add 100μl/well of the humanized TSLP monoclonal antibody prepared in Example 6 and Tezepelumab (Amgen) (starting at 50μg/ml, 4-fold dilution to 0.012μg/ml). , Incubate at 37°C for 2 hours. The plate was washed 3 times with PBST, HRP (horseradish peroxidase) labeled goat anti-human IgG antibody (ProteinTech, SA00001-1) was added, and incubated at 37°C for 1 hour. Wash the plate 4 times with PBST, add 100μl/well TMB color developing solution (Zuman Bio, ZD311), incubate at 37°C for 15 minutes, add 50μl/well stop solution (1M sulfuric acid), on the microplate reader (BioTek, ELx808) Read the absorbance at 450nm wavelength.

The results are shown in Figure 4, and the results indicate that the human TSLP humanized monoclonal antibody specifically binds to the human recombinant TSLP protein.

3. ELISA assay humanized TSLP monoclonal antibody inhibits the binding of TSLP and TSLPR

The measurement method is the same as that in Example 6. The results of the assay are shown in Figure 5. The results show that human TSLP humanized monoclonal antibodies can significantly inhibit the combination of TSLP and TSLPR, and the inhibitory ability is stronger or equivalent than that of human TSLP monoclonal antibody Tezepelumab (Amgen). _{The IC 50} value of each humanized antibody is shown in Table 7.

Table 7 Determination of the activity of humanized antibodies to inhibit the binding of TSLP and TSLPR

4. Humanized TSLP monoclonal antibody inhibits STAT5-luciferase activity in 293F transfected cells stimulated by TSLP

The measurement method is the same as that of 3 in Example 7. The results of the determination are shown in Figure 6, and the results show that the human TSLP humanized monoclonal antibodies can significantly inhibit TSLP-stimulated STAT5-luciferase activity. The IC ₅₀ values of each humanized antibody are shown in Table 8.

Table 8 Humanized antibodies inhibit STAT5-luciferase activity

Antibody	1K3/1H2	6K3/3H2	Tezepelumab
IC 50 (μg/mL)	3.217	0.1821	7.037

Although the present invention has been described in detail above with general descriptions and specific implementations, some modifications or improvements can be made on the basis of the present invention, which is obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention belong to the scope of the present invention.

Industrial applicability

The present invention provides human thymic stromal lymphopoietin monoclonal antibodies and applications thereof. The TSLP antibody provided by the present invention can specifically bind to hTSLP, has high affinity, and can effectively inhibit the binding of TSLP to its receptor, thereby inhibiting TSLP from activating downstream signaling pathways and the activation of immune cells. The TSLP antibody provided by the present invention detects the content of hTSLP as well as allergic asthma, chronic obstructive pulmonary disease, allergic dermatitis, allergic rhinitis, allergic sinusitis, eosinophilic esophagitis, allergic conjunctivitis, inflammatory bowel Diagnosis, treatment and prognosis of inflammatory or allergic inflammatory diseases such as atopic dermatitis are of great significance and have good economic value and application prospects.

Claims (15)

1. Thymus stromal lymphopoietin antibody, its antigen binding fragment, its polypeptide or its fusion protein, characterized in that said thymus stromal lymphopoietin antibody comprises heavy chain CDR and light chain CDR; said heavy chain CDR comprises:

   (1) HCDR1: Contains the sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2, or contains one or more of the sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2 deleted, replaced, or inserted The amino acid sequence of a protein or polypeptide that can specifically bind to thymic stromal lymphopoietin derived from three amino acids, or an amino acid sequence that has at least 90% homology with the sequence shown in SEQ ID NO.1 or SEQ ID NO.2 ；

   (2) HCDR2: Contains the sequence shown in SEQ ID NO. 3 or SEQ ID NO. 4, or contains the sequence shown in SEQ ID NO. 3 or SEQ ID NO. 4 with one or more deletions, substitutions, or insertions The amino acid sequence of a protein or polypeptide that can specifically bind to thymic stromal lymphopoietin derived from three amino acids, or an amino acid sequence that has at least 90% homology with the sequence shown in SEQ ID NO. 3 or SEQ ID NO. 4 ；

   (3) HCDR3: contains the sequence shown in SEQ ID NO. 5 or SEQ ID NO. 6, or contains the sequence shown in SEQ ID NO. 5 or SEQ ID NO. 6 with one or more deletions, substitutions, or insertions The amino acid sequence of a protein or polypeptide that can specifically bind to thymic stromal lymphopoietin derived from three amino acids, or an amino acid sequence that has at least 90% homology with the sequence shown in SEQ ID NO.5 or SEQ ID NO.6 ；

   The light chain CDR includes:

   (1) LCDR1: contains the sequence shown in SEQ ID NO. 7 or SEQ ID NO. 8, or contains one or more of the sequence shown in SEQ ID NO. 7 or SEQ ID NO. 8 deleted, replaced, or inserted The amino acid sequence of a protein or polypeptide that can specifically bind to thymic stromal lymphopoietin derived from three amino acids, or an amino acid sequence that has at least 90% homology with the sequence shown in SEQ ID NO.7 or SEQ ID NO.8 ；

   (2) LCDR2: contains the sequence shown in SEQ ID NO. 9 or SEQ ID NO. 10, or contains the sequence shown in SEQ ID NO. 9 or SEQ ID NO. 10 with one or more deletions, substitutions, or insertions The amino acid sequence of a protein or polypeptide that can specifically bind to thymic stromal lymphopoietin obtained from three amino acids, or an amino acid sequence that has at least 90% homology with the sequence shown in SEQ ID NO.9 or SEQ ID NO.10 ；

   (3) LCDR3: Contains the sequence shown in SEQ ID NO. 11 or SEQ ID NO. 12, or contains the sequence shown in SEQ ID NO. 11 or SEQ ID NO. 12 with one or more deletions, substitutions, or insertions The amino acid sequence of a protein or polypeptide that can specifically bind to thymic stromal lymphopoietin derived from three amino acids, or an amino acid sequence that has at least 90% homology with the sequence shown in SEQ ID NO.11 or SEQ ID NO.12 .
2. The thymic stromal lymphopoietin antibody, its antigen-binding fragment, its polypeptide, or its fusion protein according to claim 1, wherein the thymic stromal lymphopoietin antibody comprises the following (1) or (2):

   (1) HCDR1: Contains the sequence shown in SEQ ID NO.1, or contains the sequence shown in SEQ ID NO.1 by deleting, replacing or inserting one or more amino acids and can specifically bind to thymic stromal lymphocyte production The amino acid sequence of the protein or polypeptide of the protein, or the amino acid sequence that has at least 90% homology with the sequence shown in SEQ ID NO.1;

   HCDR2: contains the sequence shown in SEQ ID NO. 3, or contains the sequence shown in SEQ ID NO. 3, a protein that can specifically bind to thymic stromal lymphopoietin, obtained by deleting, replacing or inserting one or more amino acids Or the amino acid sequence of the polypeptide, or an amino acid sequence that has at least 90% homology with the sequence shown in SEQ ID NO. 3;

   HCDR3: Contains the sequence shown in SEQ ID NO. 5, or contains the sequence shown in SEQ ID NO. 5, a protein that can specifically bind to thymic stromal lymphopoietin, obtained by deleting, replacing or inserting one or more amino acids Or the amino acid sequence of the polypeptide, or an amino acid sequence that has at least 90% homology with the sequence shown in SEQ ID NO.5;

   LCDR1: Contains the sequence shown in SEQ ID NO. 7, or contains the sequence shown in SEQ ID NO. 7, a protein that can specifically bind to thymic stromal lymphopoietin obtained by deleting, replacing or inserting one or more amino acids Or the amino acid sequence of the polypeptide, or an amino acid sequence that has at least 90% homology with the sequence shown in SEQ ID NO. 7;

   LCDR2: Contains the sequence shown in SEQ ID NO.9, or contains the sequence shown in SEQ ID NO.9, a protein that can specifically bind to thymic stromal lymphopoietin, obtained by deleting, replacing or inserting one or more amino acids Or the amino acid sequence of the polypeptide, or an amino acid sequence that has at least 90% homology with the sequence shown in SEQ ID NO. 9;

   LCDR3: Contains the sequence shown in SEQ ID NO.11, or contains the sequence shown in SEQ ID NO.11, a protein that can specifically bind to thymic stromal lymphopoietin obtained by deleting, replacing or inserting one or more amino acids Or the amino acid sequence of the polypeptide, or an amino acid sequence that has at least 90% homology with the sequence shown in SEQ ID NO.11;

   (2) HCDR1: Contains the sequence shown in SEQ ID NO. 2, or contains the sequence shown in SEQ ID NO. 2, which is obtained by deleting, replacing or inserting one or more amino acids and can specifically bind to thymic stromal lymphocyte production The amino acid sequence of the protein or polypeptide of the protein, or an amino acid sequence that has at least 90% homology with the sequence shown in SEQ ID NO. 2;

   HCDR2: Contains the sequence shown in SEQ ID NO. 4, or contains the sequence shown in SEQ ID NO. 4, a protein that can specifically bind to thymic stromal lymphopoietin obtained by deleting, replacing or inserting one or more amino acids Or the amino acid sequence of the polypeptide, or an amino acid sequence that has at least 90% homology with the sequence shown in SEQ ID NO. 4;

   HCDR3: contains the sequence shown in SEQ ID NO. 6, or contains the sequence shown in SEQ ID NO. 6, a protein that can specifically bind to thymic stromal lymphopoietin, obtained by deleting, replacing or inserting one or more amino acids Or the amino acid sequence of the polypeptide, or an amino acid sequence that has at least 90% homology with the sequence shown in SEQ ID NO. 6;

   LCDR1: Contains the sequence shown in SEQ ID NO. 8, or contains the sequence shown in SEQ ID NO. 8, a protein that can specifically bind to thymic stromal lymphopoietin obtained by deleting, replacing or inserting one or more amino acids Or the amino acid sequence of the polypeptide, or an amino acid sequence that has at least 90% homology with the sequence shown in SEQ ID NO. 8;

   LCDR2: Contains the sequence shown in SEQ ID NO. 10, or contains the sequence shown in SEQ ID NO. 10, a protein that can specifically bind to thymic stromal lymphopoietin, obtained by deleting, replacing or inserting one or more amino acids Or the amino acid sequence of the polypeptide, or an amino acid sequence that has at least 90% homology with the sequence shown in SEQ ID NO. 10;

   LCDR3: Contains the sequence shown in SEQ ID NO. 12, or contains the sequence shown in SEQ ID NO. 12, a protein that can specifically bind to thymic stromal lymphopoietin obtained by deleting, replacing or inserting one or more amino acids Or the amino acid sequence of the polypeptide, or an amino acid sequence that has at least 90% homology with the sequence shown in SEQ ID NO.12.
3. The thymic stromal lymphopoietin antibody, its antigen-binding fragment, its polypeptide, or its fusion protein according to claim 1 or 2, wherein the thymus stromal lymphopoietin antibody further comprises a heavy chain FR and a light chain FR;

   The heavy chain FR includes:

   (1) HFR1: contains the sequence shown in any one of SEQ ID NO. 13-20, or contains the sequence shown in any one of SEQ ID NO. 13-20 obtained by deleting, replacing or inserting one or more amino acids The amino acid sequence of a protein or polypeptide having the same function, or an amino acid sequence that has at least 90% homology with the sequence shown in SEQ ID NO. 13-20;

   (2) HFR2: contains the sequence shown in any one of SEQ ID NO. 21-27, or contains the sequence shown in any one of SEQ ID NO. 21-27 obtained by deleting, replacing or inserting one or more amino acids The amino acid sequence of a protein or polypeptide having the same function, or an amino acid sequence that has at least 90% homology with the sequence shown in any one of SEQ ID NOs. 21-27;

   (3) HFR3: contains the sequence shown in any one of SEQ ID NO. 28-35, or contains the sequence shown in any one of SEQ ID NO. 28-35 by deleting, replacing or inserting one or more amino acids The amino acid sequence of a protein or polypeptide having the same function, or an amino acid sequence that has at least 90% homology with the sequence shown in SEQ ID NO. 28-35;

   (4) HFR4: contains the sequence shown in any one of SEQ ID NO. 36-39, or contains the sequence shown in any one of SEQ ID NO. 36-39 by deleting, replacing or inserting one or more amino acids The amino acid sequence of a protein or polypeptide having the same function, or an amino acid sequence that has at least 90% homology with the sequence shown in any one of SEQ ID NOs. 36-39;

   The light chain FR includes:

   (1) LFR1: contains the sequence shown in any one of SEQ ID NO. 40-47, or contains the sequence shown in any one of SEQ ID NO. 40-47 by deleting, replacing or inserting one or more amino acids The amino acid sequence of a protein or polypeptide having the same function, or an amino acid sequence that has at least 90% homology with the sequence shown in any one of SEQ ID NO. 40-47;

   (2) LFR2: contains the sequence shown in any one of SEQ ID NO.48-53, or contains the sequence shown in any one of SEQ ID NO.48-53 obtained by deleting, replacing or inserting one or more amino acids The amino acid sequence of a protein or polypeptide with the same function, or an amino acid sequence that has at least 90% homology with the sequence shown in any one of SEQ ID NO. 48-53;

   (3) LFR3: contains the sequence shown in any one of SEQ ID NO. 54-61, or contains the sequence shown in any one of SEQ ID NO. 54-61 by deleting, replacing or inserting one or more amino acids The amino acid sequence of a protein or polypeptide with the same function, or an amino acid sequence that has at least 90% homology with the sequence shown in any one of SEQ ID NO. 54-61;

   (4) LFR4: contains the sequence shown in any one of SEQ ID NO. 62-65, or contains the sequence shown in any one of SEQ ID NO. 62-65 obtained by deleting, replacing or inserting one or more amino acids The amino acid sequence of a protein or polypeptide having the same function, or an amino acid sequence having at least 90% homology with the sequence shown in SEQ ID NO. 62-65.
4. The thymic stromal lymphopoietin antibody, its antigen-binding fragment, its polypeptide, or its fusion protein according to any one of claims 1 to 3, wherein the thymus stromal lymphopoietin antibody comprises the following (1) ~(8) Any one:

   (1) Heavy chain variable region: contains the sequence shown in SEQ ID NO.66, or contains the sequence shown in SEQ ID NO.66, which is a protein with the same function obtained by deleting, replacing or inserting one or more amino acids Or the amino acid sequence of the polypeptide, or an amino acid sequence that has at least 90% homology with the sequence shown in SEQ ID NO. 66;

   Light chain variable region: comprising the sequence shown in SEQ ID NO. 67, or a protein or polypeptide with the same function obtained by deleting, replacing or inserting one or more amino acids in the sequence shown in SEQ ID NO. 67 An amino acid sequence, or an amino acid sequence that has at least 90% homology with the sequence shown in SEQ ID NO. 67;

   (2) Heavy chain variable region: contains the sequence shown in SEQ ID NO. 68, or contains the sequence shown in SEQ ID NO. 68, which has the same functional protein obtained by deleting, replacing or inserting one or more amino acids Or the amino acid sequence of the polypeptide, or an amino acid sequence that has at least 90% homology with the sequence shown in SEQ ID NO. 68;

   Light chain variable region: comprising the sequence shown in SEQ ID NO. 69, or a protein or polypeptide with the same function obtained by deleting, replacing or inserting one or more amino acids in the sequence shown in SEQ ID NO. 69 An amino acid sequence, or an amino acid sequence that has at least 90% homology with the sequence shown in SEQ ID NO. 69;

   (3) Light chain variable region: contains the sequence shown in SEQ ID NO. 70, or contains the sequence shown in SEQ ID NO. 70, which is obtained by deleting, replacing or inserting one or more amino acids with the same function Or the amino acid sequence of the polypeptide, or an amino acid sequence that has at least 90% homology with the sequence shown in SEQ ID NO. 70;

   Heavy chain variable region: comprising the sequence shown in SEQ ID NO. 73-75, or comprising the sequence shown in SEQ ID NO. 73-75 by deleting, replacing or inserting one or more amino acids. The amino acid sequence of the same functional protein or polypeptide, or an amino acid sequence that has at least 90% homology with the sequence shown in SEQ ID NO. 73-75;

   (4) Light chain variable region: contains the sequence shown in SEQ ID NO.71, or contains the sequence shown in SEQ ID NO.71 by deleting, replacing or inserting one or more amino acids to obtain a protein with the same function Or the amino acid sequence of the polypeptide, or an amino acid sequence that has at least 90% homology with the sequence shown in SEQ ID NO. 71;

   Heavy chain variable region: contains the sequence shown in any one of SEQ ID NO. 73-75, or contains the sequence shown in any one of SEQ ID NO. 73-75 obtained by deleting, replacing or inserting one or more amino acids The amino acid sequence of the protein or polypeptide with the same function, or the amino acid sequence that has at least 90% homology with the sequence shown in SEQ ID NO. 73-75;

   (5) Light chain variable region: comprising the sequence shown in SEQ ID NO.72, or a protein with the same function obtained by deleting, replacing or inserting one or more amino acids in the sequence shown in SEQ ID NO.72 Or the amino acid sequence of the polypeptide, or an amino acid sequence that has at least 90% homology with the sequence shown in SEQ ID NO. 72;

   Heavy chain variable region: contains the sequence shown in any one of SEQ ID NO. 73-75, or contains the sequence shown in any one of SEQ ID NO. 73-75 obtained by deleting, replacing or inserting one or more amino acids The amino acid sequence of the protein or polypeptide with the same function, or the amino acid sequence that has at least 90% homology with the sequence shown in SEQ ID NO. 73-75;

   (6) Light chain variable region: comprising the sequence shown in SEQ ID NO.76, or a protein with the same function obtained by deleting, replacing or inserting one or more amino acids in the sequence shown in SEQ ID NO.76 Or the amino acid sequence of the polypeptide, or an amino acid sequence that has at least 90% homology with the sequence shown in SEQ ID NO. 76;

   Heavy chain variable region: contains the sequence shown in any one of SEQ ID NO. 79-81, or contains the sequence shown in any one of SEQ ID NO. 79-81 obtained by deleting, replacing or inserting one or more amino acids The amino acid sequence of the protein or polypeptide with the same function, or the amino acid sequence that has at least 90% homology with the sequence shown in SEQ ID NO. 79-81;

   (7) Light chain variable region: contains the sequence shown in SEQ ID NO.77, or contains the sequence shown in SEQ ID NO.77 by deleting, replacing or inserting one or more amino acids to obtain a protein with the same function Or the amino acid sequence of the polypeptide, or an amino acid sequence that has at least 90% homology with the sequence shown in SEQ ID NO. 77;

   Heavy chain variable region: contains the sequence shown in any one of SEQ ID NO. 79-81, or contains the sequence shown in any one of SEQ ID NO. 79-81 obtained by deleting, replacing or inserting one or more amino acids The amino acid sequence of the protein or polypeptide with the same function, or the amino acid sequence that has at least 90% homology with the sequence shown in SEQ ID NO. 79-81;

   (8) Light chain variable region: contains the sequence shown in SEQ ID NO.78, or contains the sequence shown in SEQ ID NO.78 by deleting, replacing or inserting one or more amino acids to obtain a protein with the same function Or the amino acid sequence of the polypeptide, or an amino acid sequence that has at least 90% homology with the sequence shown in SEQ ID NO. 78;

   Heavy chain variable region: contains the sequence shown in any one of SEQ ID NO. 79-81, or contains the sequence shown in any one of SEQ ID NO. 79-81 obtained by deleting, replacing or inserting one or more amino acids The amino acid sequence of the protein or polypeptide with the same function, or the amino acid sequence with at least 90% homology with the sequence shown in SEQ ID NO. 79-81.
5. The thymic stromal lymphopoietin antibody, its antigen-binding fragment, its polypeptide, or its fusion protein according to any one of claims 1 to 4, wherein the thymic stromal lymphopoietin antibody is a murine antibody, Humanized antibody, chimeric antibody or multispecific antibody; the antigen-binding fragment is Fab, Fab', F(ab') ₂ , Fd, Fv, scFv, SdAb or Fab/c;

   Preferably, the heavy chain of the thymic stromal lymphopoietin antibody is of type IgG2 or IgG1, and the light chain is of type κ;

   More preferably, the thymic stromal lymphopoietin antibody is any one of the following (1) to (8):

   (1) The heavy chain sequence is shown in SEQ ID NO.82, and the light chain sequence is shown in SEQ ID NO.83;

   (2) The heavy chain sequence is shown in SEQ ID NO.84, and the light chain sequence is shown in SEQ ID NO.85;

   (3) The light chain sequence is shown in SEQ ID NO. 86, and the heavy chain sequence is shown in any one of SEQ ID NO. 89-91;

   (4) The light chain sequence is shown in SEQ ID NO. 87, and the heavy chain sequence is shown in any one of SEQ ID NO. 89-91;

   (5) The light chain sequence is shown in SEQ ID NO.88, and the heavy chain sequence is shown in any one of SEQ ID NO.89-91;

   (6) The light chain sequence is shown in SEQ ID NO. 92, and the heavy chain sequence is shown in any one of SEQ ID NO. 95-97;

   (7) The light chain sequence is shown in SEQ ID NO.93, and the heavy chain sequence is shown in any one of SEQ ID NO.95-97;

   (8) The light chain sequence is shown in SEQ ID NO.94, and the heavy chain sequence is shown in any one of SEQ ID NO.95-97;

   (9) The light chain sequence is shown in SEQ ID NO. 98, and the heavy chain sequence is shown in SEQ ID NO. 99;

   (10) The light chain sequence is shown in SEQ ID NO.100, and the heavy chain sequence is shown in SEQ ID NO.101.
6. Hybridoma cells are characterized in that they are capable of producing the thymic stromal lymphopoietin antibody, its antigen-binding fragment, its polypeptide, or its fusion protein according to any one of claims 1 to 5.
7. A nucleic acid, characterized in that it encodes the thymic stromal lymphopoietin antibody, its antigen-binding fragment, its polypeptide, or its fusion protein according to any one of claims 1 to 5.
8. The biological material is characterized by comprising the nucleic acid of claim 7; the biological material is one selected from the group consisting of expression cassettes, vectors, transposons, host cells, or cell lines.
9. A method for preparing thymic stromal lymphopoietin antibody, its antigen-binding fragment, its polypeptide or its fusion protein, characterized in that the hybridoma cell of claim 6 is used to prepare the thymic stromal lymphopoietin antibody, Its antigen-binding fragment, its polypeptide, or its fusion protein; or, using a host cell to express the nucleic acid of claim 7.
10. A complex or conjugate, characterized by comprising the thymic stromal lymphopoietin antibody, its antigen-binding fragment, its polypeptide or its fusion protein according to any one of claims 1 to 5;

    Preferably, the complex is the thymic stromal lymphopoietin antibody, its antigen-binding fragment, its polypeptide, or its fusion protein according to any one of claims 1 to 5, which is obtained by chemical labeling or biomarking;

    The conjugate is obtained by coupling the thymic stromal lymphopoietin antibody, its antigen-binding fragment, its polypeptide or its fusion protein according to any one of claims 1 to 5, or the complex and a carrier.
11. The thymic stromal lymphopoietin antibody, its antigen-binding fragment, its polypeptide or its fusion protein according to any one of claims 1 to 5 or the hybridoma cell according to claim 6 or the nucleic acid according to claim 7 or claim Any one of the following applications of the biological material according to claim 8 or the complex or conjugate according to claim 10:

    (1) Application in the preparation of reagents or kits for diagnosing or assisting in the diagnosis of diseases related to thymic stromal lymphopoietin or its receptor;

    (2) Application in the preparation of drugs for the treatment or adjuvant treatment of diseases related to thymic stromal lymphopoietin or its receptor;

    (3) Application in the preparation of reagents or kits for tracking or prognostic inspection of thymic stromal lymphopoietin or its receptor-related diseases;

    (4) Application in detecting the content of thymic stromal lymphopoietin;

    (5) Application in inhibiting the binding of thymic stromal lymphopoietin to its receptor;

    Preferably, the disease related to thymic stromal lymphopoietin or its receptor is an immune system disease or a tumor disease; the immune system disease includes an inflammatory or allergic inflammatory disease; the tumor disease includes a breast tumor, Huo Chikin lymphoma, pancreatic cancer, melanoma and lung cancer;

    More preferably, the inflammatory or allergic inflammatory disease is selected from allergic asthma, chronic obstructive pulmonary disease, allergic dermatitis, allergic rhinitis, allergic sinusitis, eosinophilic esophagitis, allergic conjunctivitis , Inflammatory bowel disease or atopic dermatitis.
12. A pharmaceutical composition, characterized in that it comprises the thymic stromal lymphopoietin antibody, its antigen-binding fragment, its polypeptide or its fusion protein according to any one of claims 1 to 5, or the composition of claim 10 Complex or conjugate.
13. A kit, characterized in that it comprises the thymic stromal lymphopoietin antibody, its antigen-binding fragment, its polypeptide or its fusion protein according to any one of claims 1 to 5, or the complex according to claim 10物 or conjugate.
14. A method for the prevention, treatment or adjuvant treatment of diseases related to thymic stromal lymphopoietin or its receptor, characterized by comprising: administering the thymic stromal lymphopoietin according to any one of claims 1 to 5 to a subject Antibody, its antigen-binding fragment, its polypeptide or its fusion protein or the pharmaceutical composition of claim 12;

    Preferably, the disease related to thymic stromal lymphopoietin or its receptor is an immune system disease or a tumor disease; the immune system disease includes an inflammatory or allergic inflammatory disease; the tumor disease includes a breast tumor, Hodgkin's lymphoma, pancreatic cancer, melanoma and lung cancer;

    More preferably, the inflammatory or allergic inflammatory disease is selected from allergic asthma, chronic obstructive pulmonary disease, allergic dermatitis, allergic rhinitis, allergic sinusitis, eosinophilic esophagitis, allergic conjunctivitis , Inflammatory bowel disease or atopic dermatitis.
15. The method for detecting the content of thymic stromal lymphopoietin is characterized by using the thymic stromal lymphopoietin antibody, its antigen-binding fragment, its polypeptide or its fusion protein according to any one of claims 1 to 5, or the right to use The complex or conjugate according to claim 10, or the kit according to claim 13 for detecting the content of thymic stromal lymphopoietin.

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