CN115028722A - anti-TSLP antibody, preparation method and application thereof - Google Patents

anti-TSLP antibody, preparation method and application thereof Download PDF

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CN115028722A
CN115028722A CN202110244601.7A CN202110244601A CN115028722A CN 115028722 A CN115028722 A CN 115028722A CN 202110244601 A CN202110244601 A CN 202110244601A CN 115028722 A CN115028722 A CN 115028722A
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韩化敏
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Biocells Beijing Biotech Co Ltd
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Abstract

The present application provides antibodies against Thymic Stromal Lymphopoietin (TSLP), nucleic acid molecules encoding the heavy chain variable region and the light chain variable region in said antibodies, expression vectors comprising said nucleic acid molecules, host cells comprising said nucleic acid molecules or said expression vectors, compositions comprising said antibodies, kits comprising said antibodies, and uses of said antibodies, said nucleic acid molecules, said expression vectors, said host cells, or said compositions.

Description

anti-TSLP antibody, preparation method and application thereof
Technical Field
The present application relates generally to the fields of genetic engineering and antibodies; in particular, antibodies against Thymic Stromal Lymphopoietin (TSLP) and uses thereof. The present application develops novel anti-TSLP antibodies and provides for the use of such antibodies in the prevention, diagnosis, treatment, follow-up and/or prognostic screening of TSLP-mediated diseases.
Background
Thymic Stromal Lymphopoietin (TSLP), also known as IL-7-like cytokine, is a member of the IL-2 cytokine family. TSLP is the induction of dendritic cell-mediated CD4 + Immune cytokines of T cell response, among which the preallophenotypic Dendritic Cells (DCs) activated by TSLP play an important role in the induction and maintenance of allergic inflammatory Th2 and mast cell responses, through the production of pro-allergen cytokines, chemokines and costimulatory molecules, which direct the naive T cells to become Th2 cells, producing the IL-4, IL-5 and IL-13 key mediators of allergic inflammation. Overexpression of TSLP in atopic dermatitis (AtD), netherton syndrome and asthma indicates an important role for this cytokine in the pathogenesis of these allergic inflammatory diseases. This is supported by animal models in which transgenic overexpression of TSLP in the skin or lung and removal of gene targeting effects by negative regulators of TSLP leads to allergic inflammatory diseases very similar to human atopic dermatitis or asthma.
The TSLP receptor (TSLPR) belongs to a member of hematopoietic factor receptor family, and is a type I cytokine receptor protein. TSLPR includes the IL-7 receptor alpha chain (IL-7R alpha) and the TSLP receptor alpha chain (TSLPR alpha), and high affinity is only available when the receptor complexes interact. TSLPR (a co-receptor for TSLP and IL-7) is expressed predominantly on the surface of mature DC cells, mast cells, and some activated T cells.
A large number of studies prove that about 2/3 severe asthma is expressed by over-expression of Th2 cytokines, and TSLP is an important factor causing over-expression of Th2 cytokines. The TSLP-TSLPR action is accomplished primarily through the JAK-STAT signaling pathway. It is thought that upregulation of TSLP, in combination with TSLPR on DC cells, causes JAK activation, recruits the transcription factor STAT5, causes downstream signaling, and ultimately leads to activation of DC cells. DC cell activation shows up-regulation of expression of co-stimulatory molecules (e.g., CD80, CD40, CD86) and secretion of chemokines (TARC/CCL17, MDC/CCL22 and I-309/CCL1), thereby providing a favorable microenvironment for Th0 to Th2 cell differentiation, directing Th2 cell-dominated inflammatory responses, and concomitant factor (IL-4, IL-13, IL-5) release. TSLP transgenic mice are susceptible to specific antigen-induced asthma, while symptoms are significantly reduced in TSLP receptor knockout mice. From the analysis of the mechanism of asthma and inflammation, anti-cytokine (IL-4, IL-13, IL-5) drugs are only targeted to specific inflammatory molecules that drive asthma inflammation and are only suitable for certain types of severe asthma patients, i.e., a subset of patients, such as eosinophilic asthma. TSLP is obviously different from targets such as IL4, IL5 and the like, and the TSLP moves in the early upstream of the inflammatory cascade reaction and is possibly suitable for a wide range of severe uncontrolled asthma patients.
Researchers have conducted extensive research and study on drugs targeting TSLP. The anti-TSLP monoclonal antibody can effectively block the effect of TSLP/TSLPR, reverse airway inflammation, prevent the change of tissue structure and reduce the Airway Hyperresponsiveness (AHR) and TGF-beta 1 level in a mite dust induced mouse asthma model. In the serum protein-induced mouse asthma model, the anti-TSLP monoclonal antibody effectively reduced the expression of Th 2-like factors (IL-4, IL-5, etc.). The safety of anti-TSLP monoclonal antibodies is also well documented in monkeys. Moreover, the only clinically under investigation anti-TSLP monoclonal antibody shows good objective response rate in early clinical stage on a global scale, effectively alleviating the symptoms of the patients.
Therefore, the preparation of monoclonal antibodies that specifically recognize and bind TSLP is of great biological and medical significance in the prevention, diagnosis, treatment, prognosis, etc. of diseases involving TSLP.
Summary of The Invention
In a first aspect, the present application provides an anti-Thymic Stromal Lymphopoietin (TSLP) antibody comprising a heavy chain variable region comprising the amino acid sequences HCDR1, HCDR2 and HCDR3 and a light chain variable region comprising the amino acid sequences LCDR1, LCDR2 and LCDR3, wherein
The amino acid sequence of the HCDR1 is shown as SEQ ID NO.1, the amino acid sequence of the HCDR2 is shown as SEQ ID NO. 2, the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 3, the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 4, the amino acid sequence of the LCDR2 is shown as SEQ ID NO. 5, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 6; or,
the amino acid sequence of the HCDR1 is shown as SEQ ID NO.1, the amino acid sequence of the HCDR2 is shown as SEQ ID NO.7, the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 3, the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 4, the amino acid sequence of the LCDR2 is shown as SEQ ID NO. 5, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 6; or,
the amino acid sequence of the HCDR1 is shown as SEQ ID NO. 8, the amino acid sequence of the HCDR2 is shown as SEQ ID NO. 9, the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 10, the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 11, the amino acid sequence of the LCDR2 is shown as SEQ ID NO.12, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 6; or,
the amino acid sequence of the HCDR1 is shown as SEQ ID NO. 8, the amino acid sequence of the HCDR2 is shown as SEQ ID NO. 9, the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 10, the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 4, the amino acid sequence of the LCDR2 is shown as SEQ ID NO. 5, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 6;
wherein the HCDR and LCDR amino acid sequences are defined according to Kabat.
In some embodiments of the first aspect, the amino acid sequence of the heavy chain variable region of the antibody is at least 90% homologous to any one of SEQ ID NOs 21, 23, 25, 27, 29 and 31.
In some embodiments of the first aspect, the amino acid sequence of the light chain variable region of the antibody has at least 90% homology to any one of SEQ ID NOs 33, 35 and 37.
In some embodiments of the first aspect, the antibody is a whole antibody, a Fab fragment, a Fab 'fragment, a F (ab') 2 A fragment, an Fv fragment, or a single chain Fv fragment (scFv).
In some embodiments of the first aspect, the antibody is a fully human antibody.
In some embodiments of the first aspect, the antibody is a humanized antibody.
In some embodiments of the first aspect, the antibody is a monoclonal antibody.
In some embodiments of the first aspect, the antibody comprises a framework region.
In some embodiments of the first aspect, the antibody comprises a heavy chain constant region selected from the group consisting of the IgG1 subtype, the IgG2 subtype, and the IgG4 subtype.
In some embodiments of the first aspect, the antibody comprises a light chain constant region selected from the kappa subtype or the lambda subtype.
In some embodiments of the first aspect, the antibody is capable of binding TSLP.
In some embodiments of the first aspect, the antibody is capable of inhibiting TSLP binding to the TSLP receptor.
In some embodiments of the first aspect, the antibody is capable of blocking TSLP-mediated activity.
In a second aspect, the present application provides a nucleic acid molecule encoding the heavy chain variable region and the light chain variable region of the antibody of the first aspect.
In a third aspect, the present application provides an expression vector comprising the nucleic acid molecule of the second aspect.
In a fourth aspect, the present application provides a host cell comprising the nucleic acid molecule of the second aspect or the expression vector of the third aspect.
In a fifth aspect, the present application provides a composition comprising an antibody according to the first aspect together with a pharmaceutically acceptable excipient, diluent or carrier.
In a sixth aspect, the present application provides a kit for detecting TSLP in a sample from a subject, comprising an antibody according to the first aspect.
In a seventh aspect, the present application provides the use of an antibody of the first aspect, a nucleic acid molecule of the second aspect, an expression vector of the third aspect, a host cell of the fourth aspect, or a composition of the fifth aspect, in the manufacture of a kit for the diagnostic, therapeutic, tracking, and/or prognostic check of a TSLP-mediated disease.
In an eighth aspect, the present application provides use of an antibody of the first aspect, a nucleic acid molecule of the second aspect, an expression vector of the third aspect, a host cell of the fourth aspect, or a composition of the fifth aspect, in the manufacture of a medicament for treating and/or preventing a TSLP-mediated disease.
Brief Description of Drawings
FIG. 1 is the result of identifying hTSLPR positive cell clones using flow cytometry.
FIG. 2 shows the results of the determination of the binding of rhTSLP-His protein to hTSLPR/hIL-7R α -293T/17 cells.
FIG. 3 shows the results of the measurement of the binding of the murine anti-hTSLP monoclonal antibody to rhTSLP-His recombinant protein.
FIG. 4 shows the results of the activity assay of the murine anti-hTSLP monoclonal antibody in inhibiting the binding of rhTSLP-His recombinant protein to hTSPR/hIL-7 Ra-293T/17 cells.
FIG. 5 shows the results of the measurement of the binding of the anti-hTSLP humanized monoclonal antibody to rhTSLP-His recombinant protein.
FIG. 6 shows the results of the determination that the anti-hTSLP humanized monoclonal antibody inhibits the binding of rhTSLP-His recombinant protein to hTSPR/hIL-7 Ra-293T/17 cells.
DESCRIPTION OF THE SEQUENCES
SEQ ID NO.1 shows the HCDR1 amino acid sequence of anti-TSLP monoclonal antibodies 9F8-1-25, 9F8-46H13L, 10D9-M-432, and M432-69H13L, as defined by Kabat.
SEQ ID NO. 2 shows the HCDR2 amino acid sequence of anti-TSLP monoclonal antibodies 9F8-1-25 and 9F8-46H13L according to the Kabat definition, or a combination of the Kabat and Chothia definitions.
SEQ ID NO 3 shows the HCDR3 amino acid sequence of anti-TSLP monoclonal antibodies 9F8-1-25, 9F8-46H13L, 10D9-M-432, and M432-69H13L according to the Kabat definition, the Chothia definition, or a combination of the Kabat and Chothia definitions.
SEQ ID NO. 4 shows the LCDR1 amino acid sequence of anti-TSLP monoclonal antibodies 9F8-1-25, 9F8-46H13L, 10D9-M-432, M432-69H13L and 5F10-69H13L, according to the Kabat definition, the Chothia definition or a combination of the Kabat and Chothia definitions.
SEQ ID NO 5 shows the LCDR2 amino acid sequence of anti-TSLP monoclonal antibodies 9F8-1-25, 9F8-46H13L, 10D9-M-432, M432-69H13L and 5F10-69H13L according to the Kabat definition, the Chothia definition, or a combination of the Kabat and Chothia definitions.
SEQ ID NO 6 shows the LCDR3 amino acid sequence of anti-TSLP monoclonal antibodies 9F8-1-25, 9F8-46H13L, 10D9-M-432, M432-69H13L, 5F10-2-25, and 5F10-69H13L according to the Kabat definition, the Chothia definition, or a combination of the Kabat and Chothia definitions.
SEQ ID NO 7 shows the HCDR2 amino acid sequence of anti-TSLP monoclonal antibodies 10D9-M-432 and M432-69H13L according to the Kabat definition, or a combination of the Kabat and Chothia definitions.
SEQ ID NO 8 shows the HCDR1 amino acid sequences of anti-TSLP monoclonal antibodies 5F10-2-25 and 5F10-69H13L, according to the Kabat definition.
SEQ ID NO 9 shows the HCDR2 amino acid sequence of anti-TSLP monoclonal antibodies 5F10-2-25 and 5F10-69H13L according to the Kabat definition, or a combination of the Kabat and Chothia definitions.
SEQ ID NO 10 shows the HCDR3 amino acid sequences of anti-TSLP monoclonal antibodies 5F10-2-25 and 5F10-69H13L according to the Kabat definition, the Chothia definition, or a combination of the Kabat and Chothia definitions.
SEQ ID NO 11 shows the LCDR1 amino acid sequence of anti-TSLP monoclonal antibody 5F10-2-25, which will be according to the Kabat definition, the Chothia definition, or a combination of the Kabat and Chothia definitions.
SEQ ID NO.12 shows the LCDR2 amino acid sequence of anti-TSLP monoclonal antibody 5F10-2-25, which will be according to the Kabat definition, the Chothia definition, or a combination of the Kabat and Chothia definitions.
13 shows the HCDR1 amino acid sequence of anti-TSLP monoclonal antibodies 9F8-1-25 and 9F8-46H13L as defined by Chothia.
SEQ ID NO. 14 shows the HCDR2 amino acid sequence of anti-TSLP monoclonal antibodies 9F8-1-25, 9F8-46H13L, 10D9-M-432, and M432-69H13L as defined by Chothia.
15 shows the HCDR1 amino acid sequence of anti-TSLP monoclonal antibodies 10D9-M-432 and M432-69H13L as defined by Chothia.
16 shows the HCDR1 amino acid sequence of anti-TSLP monoclonal antibodies 5F10-2-25 and 5F10-69H13L, defined according to Chothia.
SEQ ID NO. 17 shows the HCDR2 amino acid sequence of anti-TSLP monoclonal antibodies 5F10-2-25 and 5F10-69H13L, as defined by Chothia.
18 shows the HCDR1 amino acid sequence of anti-TSLP monoclonal antibodies 9F8-1-25 and 9F8-46H13L in combination according to the Kabat and Chothia definitions.
SEQ ID NO 19 shows the HCDR1 amino acid sequence of anti-TSLP monoclonal antibodies 10D9-M-432 and M432-69H13L in combination according to the Kabat and Chothia definitions.
SEQ ID NO. 20 shows the HCDR1 amino acid sequences of anti-TSLP monoclonal antibodies 5F10-2-25 and 5F10-69H13L in combination according to the Kabat and Chothia definitions.
SEQ ID NO 21 shows the amino acid sequence of the heavy chain variable region of anti-TSLP monoclonal antibody 9F 8-1-25.
SEQ ID NO. 22 shows the nucleotide sequence encoding the heavy chain variable region of anti-TSLP monoclonal antibody 9F 8-1-25.
SEQ ID NO. 23 shows the amino acid sequence of the heavy chain variable region of anti-TSLP monoclonal antibody 10D 9-M-432.
SEQ ID NO. 24 shows the nucleotide sequence encoding the heavy chain variable region of anti-TSLP monoclonal antibody 10D 9-M-432.
SEQ ID NO. 25 shows the amino acid sequence of the heavy chain variable region of anti-TSLP monoclonal antibody 5F 10-2-25.
SEQ ID NO. 26 shows the nucleotide sequence encoding the heavy chain variable region of anti-TSLP monoclonal antibody 5F 10-2-25.
27 shows the amino acid sequence of the heavy chain variable region of anti-TSLP humanized monoclonal antibody 9F8-46H 13L.
28 shows the nucleotide sequence of the heavy chain variable region encoding anti-TSLP humanized monoclonal antibody 9F8-46H 13L.
29 shows the amino acid sequence of the heavy chain variable region of anti-TSLP humanized monoclonal antibody M432-69H 13L.
SEQ ID NO. 30 shows the nucleotide sequence encoding the heavy chain variable region of anti-TSLP humanized monoclonal antibody M432-69H 13L.
31 shows the amino acid sequence of the heavy chain variable region of anti-TSLP humanized monoclonal antibody 5F10-69H 13L.
SEQ ID NO. 32 shows the nucleotide sequence of the heavy chain variable region encoding anti-TSLP humanized monoclonal antibody 5F10-69H 13L.
33 shows the amino acid sequence of the light chain variable region of anti-TSLP monoclonal antibodies 9F8-1-25 and 10D 9-M-432.
SEQ ID NO. 34 shows the nucleotide sequence of the light chain variable region encoding anti-TSLP monoclonal antibodies 9F8-1-25 and 10D 9-M-432.
SEQ ID NO. 35 shows the amino acid sequence of the light chain variable region of anti-TSLP monoclonal antibody 5F 10-2-25.
SEQ ID NO 36 shows the nucleotide sequence encoding the light chain variable region of anti-TSLP monoclonal antibody 5F 10-2-25.
37 shows the amino acid sequence of the light chain variable region of the anti-TSLP humanized monoclonal antibodies 9F8-46H13L, M432-69H13L and 5F10-69H 13L.
38 shows the nucleotide sequence of the light chain variable region encoding the anti-TSLP humanized monoclonal antibodies 9F8-46H13L, M432-69H13L and 5F10-69H 13L.
SEQ ID NO 39 shows the amino acid sequence of the antibody heavy chain constant region.
SEQ ID NO 40 shows the nucleotide sequence encoding the constant region of the antibody heavy chain.
SEQ ID NO 41 shows the amino acid sequence of the constant region of the antibody light chain.
42 shows the nucleotide sequence encoding the constant region of the antibody light chain.
SEQ ID NO 43 shows the amino acid sequence of the recombinant protein rhTSLP-His.
44 shows the nucleotide sequence of recombinant protein rhTSLP-His.
SEQ ID NO 45 shows the amino acid sequence of the recombinant protein rhTSLP-His leader peptide.
SEQ ID NO 46 shows the amino acid sequence of hTSLPR.
SEQ ID NO 47 shows the nucleotide sequence encoding hTSLPR.
SEQ ID NO 48 shows the amino acid sequence of hIL-7R α.
SEQ ID NO. 49 shows a nucleotide sequence encoding hIL-7R α.
Detailed Description
The inventors of the present application have obtained novel anti-TSLP antibodies by antibody engineering techniques. In various aspects of the present application, novel anti-TSLP antibodies, nucleic acid molecules encoding the heavy chain variable region and the light chain variable region of the antibodies, expression vectors comprising the nucleic acid molecules, host cells comprising the nucleic acid molecules or expression vectors, compositions comprising the antibodies, methods of making and purifying the antibodies, kits comprising the antibodies, and biological and medical uses of the antibodies, the nucleic acid molecules, the expression vectors, the host cells, or the compositions are provided. Based on the sequences of the variable regions of the antibodies provided herein, full-length antibody molecules can be constructed as components of kits for the diagnosis, treatment, tracking, and/or prognostic examination of TSLP-mediated diseases.
The practice of the present application employs, unless otherwise indicated, conventional molecular biology, microbiology, cell biology, biochemistry, and immunology techniques.
Unless otherwise indicated, terms used in the present application have meanings commonly understood by those skilled in the art.
Definition of
The term "antibody", as used herein, refers to an immunoglobulin molecule capable of specifically binding to a target via at least one antigen recognition site located in the variable region of the immunoglobulin molecule. Targets include, but are not limited to, carbohydrates, polynucleotides, lipids, polypeptides, and the like. As used herein, "antibody" includes not only intact (i.e., full-length) antibodies, but also antigen-binding fragments thereof (e.g., Fab fragments, Fab 'fragments, F (ab') 2 Fragment or Fv fragment), variants thereof, fusion proteins comprising an antibody portion, humanized antibodies, chimeric antibodies, diabodies, linear antibodies, single chain antibodies, multispecific antibodies (e.g., bispecific antibodies), and any other modified configuration of an immunoglobulin molecule comprising an antigen recognition site of a desired specificity, including glycosylation variants of an antibody, amino acid sequence variants of an antibody, and covalently modified antibodies.
Typically, a complete or full-length antibody comprises two heavy chains and two light chains. Each heavy chain comprises a heavy chain variable region (VH) and first, second and third constant regions (CH1, CH2 and CH 3). Each light chain contains a light chain variable region (VL) and a constant region (CL). Full-length antibodies can be of any class, such as IgD, IgE, IgG, IgA, or IgM (or subclasses thereof), but the antibodies need not belong to any particular class. Depending on the antibody amino acid sequence of the constant domain of the heavy chain, immunoglobulins can be assigned to different classes. Generally, there are five main classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these classes can be further classified into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA 2. The heavy chain constant domains corresponding to different immunoglobulin classes are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional structures of different classes of immunoglobulins are well known.
The term "antigen-binding fragment or antigen-binding portion" as used herein refers to a portion or region of an intact antibody molecule that is responsible for binding an antigen. The antigen binding domain may comprise a heavy chain variable region (VH), a light chain variable region (VL), or both. Each of VH and VL typically contains three complementarity determining regions CDR1, CDR2, and CDR 3.
The CDR sequences in the variable region sequences can be analyzed in a variety of ways for the variable region sequences of a given antibody, as can be determined using the online software Abysis (http:// www.abysis.org /).
Examples of antigen-binding fragments include, but are not limited to: (1) a Fab fragment, which can be a monovalent fragment having a VL-CL chain and a VH-CH1 chain; (2) f (ab') 2 A fragment, which may be a bivalent fragment having two Fab 'fragments linked by a disulfide bridge of the hinge region (i.e., a dimer of Fab'); (3) fv fragments of VL and VH with one arm of the antibody; (4) single chain fv (scfv), which may be a single polypeptide chain consisting of VH and VL via a peptide linker; and (5) (scFv) 2 It may comprise two VH connected by peptide linkers and two VL combined with the two VH via disulfide bridges.
The term "specific binding" as used herein refers to a non-random binding reaction between two molecules, e.g. binding of an antibody to an epitope of an antigen.
The term "monoclonal antibody" as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for the possible presence of naturally occurring mutations in a small number of individuals. The monoclonal antibodies described herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the heavy and/or light chain is identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, and also include fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al, Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984).
The term "diabody", as used herein, refers to a small antibody fragment having two antigen binding sites, which fragment comprises a heavy chain variable domain (VH) linked to a light chain variable domain (VL) (VH-VL or VL-VH) in the same polypeptide chain. By using a short linker that does not allow pairing between the two domains on the same chain, each domain is forced to pair with the complementary domain of the other chain, thereby creating two antigen binding sites.
The term "humanized antibody" as used herein refers to a form of an antibody that contains the sequences of a non-human (e.g., murine) antibody as well as the sequences of a human antibody. Such antibodies contain minimal sequences derived from non-human immunoglobulins. Generally, a humanized antibody comprises at least one (typically two variable domains) in which the hypervariable region corresponds to a non-human immunoglobulin and the FR region is a human immunoglobulin sequence. The humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (e.g., an Fc region), which is typically a human immunoglobulin constant region. Humanized versions of rodent antibodies will typically comprise the CDR sequences of the parent rodent antibody, although certain amino acid substitutions may be included to improve affinity or increase stability of the humanized antibody.
The term "homology", as used herein, is defined as the percentage of residues in an amino acid or nucleotide sequence variant that are identical, if necessary to the maximum percentage, after alignment and the introduction of gaps in the sequence. Methods and computer programs for alignment are well known in the art.
The term "autoimmune disease" as used herein refers to a disease caused by damage to self-tissues resulting from an immune response from the body to self-antigens, including but not limited to asthma, scleroderma, systemic lupus erythematosus, rheumatoid arthritis, Churg-Strauss syndrome, Wegener's granulomatosis, goodpasture's syndrome, allergic pneumonia, atopic dermatitis, rhinitis, ankylosing spondylitis, rheumatic fever, fibromyalgia, psoriatic arthritis, chronic nephritis, sjogren's syndrome, as well as multiple sclerosis, allergic dermatitis, allergic conjunctivitis, atopic dermatitis fibrosis, inflammatory bowel disease, and the like.
The term "inflammatory disease" as used herein refers to the general term for diseases in which inflammation is the main damaging factor. Inflammation is the biological response of tissue to noxious stimuli, a condition that is accompanied by three events, tissue degeneration, circulatory disturbance and fluid exudation, and hypertrophy. Examples of inflammatory diseases include acute and chronic diseases including, but not limited to, asthma, scleroderma, systemic lupus erythematosus, rheumatoid arthritis, Churg-Strauss syndrome, Wegener's granulomatosis, goodpasture's syndrome, pulmonary hemorrhage-nephritis syndrome, allergic pneumonia, atopic dermatitis, rhinitis, ankylosing spondylitis, rheumatic fever, fibromyalgia, psoriatic arthritis, chronic nephritis, sjogren's syndrome, as well as multiple sclerosis, allergic dermatitis, allergic conjunctivitis, atopic dermatitis fibrosis, inflammatory bowel disease, and the like.
The term "tumor" as used herein refers to a neoplasm or solid lesion formed by abnormal cell growth. Tumors can be benign, premalignant, or malignant, including but not limited to hodgkin's lymphoma, breast cancer, pancreatic cancer, melanoma, B-cell acute lymphocytic leukemia, cervical cancer, cutaneous T-cell lymphoma, gastric cancer, and lung cancer, among others.
In a first aspect, the present application provides an anti-Thymic Stromal Lymphopoietin (TSLP) antibody comprising a heavy chain variable region comprising the amino acid sequences HCDR1, HCDR2 and HCDR3 and a light chain variable region comprising the amino acid sequences LCDR1, LCDR2 and LCDR3, wherein
The amino acid sequence of the HCDR1 is shown as SEQ ID NO.1, the amino acid sequence of the HCDR2 is shown as SEQ ID NO. 2, the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 3, the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 4, the amino acid sequence of the LCDR2 is shown as SEQ ID NO. 5, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 6; or,
the amino acid sequence of the HCDR1 is shown as SEQ ID NO.1, the amino acid sequence of the HCDR2 is shown as SEQ ID NO.7, the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 3, the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 4, the amino acid sequence of the LCDR2 is shown as SEQ ID NO. 5, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 6; or,
the amino acid sequence of the HCDR1 is shown as SEQ ID NO. 8, the amino acid sequence of the HCDR2 is shown as SEQ ID NO. 9, the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 10, the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 11, the amino acid sequence of the LCDR2 is shown as SEQ ID NO.12, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 6; or,
the amino acid sequence of the HCDR1 is shown as SEQ ID NO. 8, the amino acid sequence of the HCDR2 is shown as SEQ ID NO. 9, the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 10, the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 4, the amino acid sequence of the LCDR2 is shown as SEQ ID NO. 5, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 6;
wherein the HCDR and LCDR amino acid sequences are defined according to Kabat.
It is well known to those skilled in the art that the Complementarity Determining Regions (CDRs), usually CDR1, CDR2 and CDR3, are the regions of the variable region that have the greatest impact on the affinity and specificity of an antibody. There are two common definitions of CDR sequences for VH or VL, namely the Chothia definition and the Kabat definition. (see, e.g., Kabat, "Sequences of Proteins of Immunological Interest", National Institutes of Health, Bethesda, Md. (1991); A1-Lazikani et al, J.mol.biol.273: 927-.
As defined in accordance with Chothia, the anti-Thymic Stromal Lymphopoietin (TSLP) antibodies provided herein are as follows: comprising a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 amino acid sequences and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 amino acid sequences, wherein
The amino acid sequence of the HCDR1 is shown as SEQ ID NO. 13, the amino acid sequence of the HCDR2 is shown as SEQ ID NO. 14, the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 3, the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 4, the amino acid sequence of the LCDR2 is shown as SEQ ID NO. 5, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 6; or,
the amino acid sequence of the HCDR1 is shown as SEQ ID NO. 15, the amino acid sequence of the HCDR2 is shown as SEQ ID NO. 14, the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 3, the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 4, the amino acid sequence of the LCDR2 is shown as SEQ ID NO. 5, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 6; or,
the amino acid sequence of the HCDR1 is shown as SEQ ID NO. 16, the amino acid sequence of the HCDR2 is shown as SEQ ID NO. 17, the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 10, the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 11, the amino acid sequence of the LCDR2 is shown as SEQ ID NO.12, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 6; or,
the amino acid sequence of the HCDR1 is shown as SEQ ID NO. 16, the amino acid sequence of the HCDR2 is shown as SEQ ID NO. 17, the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 10, the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 4, the amino acid sequence of the LCDR2 is shown as SEQ ID NO. 5, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 6.
The anti-Thymic Stromal Lymphopoietin (TSLP) antibodies provided herein, when combined according to Kabat and Chothia definitions, are shown below: comprising a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 amino acid sequences and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 amino acid sequences, wherein
The amino acid sequence of the HCDR1 is shown as SEQ ID NO. 18, the amino acid sequence of the HCDR2 is shown as SEQ ID NO. 2, the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 3, the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 4, the amino acid sequence of the LCDR2 is shown as SEQ ID NO. 5, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 6; or,
the amino acid sequence of the HCDR1 is shown as SEQ ID NO. 19, the amino acid sequence of the HCDR2 is shown as SEQ ID NO.7, the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 3, the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 4, the amino acid sequence of the LCDR2 is shown as SEQ ID NO. 5, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 6; or,
the amino acid sequence of the HCDR1 is shown as SEQ ID NO. 20, the amino acid sequence of the HCDR2 is shown as SEQ ID NO. 9, the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 10, the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 11, the amino acid sequence of the LCDR2 is shown as SEQ ID NO.12, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 6; or,
the amino acid sequence of the HCDR1 is shown as SEQ ID NO. 20, the amino acid sequence of the HCDR2 is shown as SEQ ID NO. 9, the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 10, the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 4, the amino acid sequence of the LCDR2 is shown as SEQ ID NO. 5, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 6.
In some embodiments of the first aspect, the amino acid sequence of the heavy chain variable region of the antibody has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology to any of SEQ ID NOs 21, 23, 25, 27, 29 and 31.
In some embodiments of the first aspect, the amino acid sequence of the light chain variable region of the antibody has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology to any of SEQ ID NOs 33, 35 and 37.
In some embodiments of the first aspect, the heavy chain variable region of the antibody has the amino acid sequence set forth in any one of SEQ ID NOs 21, 23, 25, 27, 29, and 31.
In some embodiments of the first aspect, the amino acid sequence of the variable region of the light chain of the antibody is as set forth in any one of SEQ ID NOs 33, 35 and 37.
In some particular embodiments of the first aspect,
the amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO. 21, and the amino acid sequence of the light chain variable region of the antibody is shown as SEQ ID NO. 33; or
The amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO. 23, and the amino acid sequence of the light chain variable region of the antibody is shown as SEQ ID NO. 33; or
The amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO. 25, and the amino acid sequence of the light chain variable region of the antibody is shown as SEQ ID NO. 35; or
The amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO. 27, and the amino acid sequence of the light chain variable region of the antibody is shown as SEQ ID NO. 37; or
The amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO. 29, and the amino acid sequence of the light chain variable region of the antibody is shown as SEQ ID NO. 37; or
The amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO. 31, and the amino acid sequence of the light chain variable region of the antibody is shown as SEQ ID NO. 37.
In some specific embodiments of the first aspect, the antibody comprises a heavy chain variable region as set forth in SEQ ID NO 27, a light chain variable region as set forth in SEQ ID NO 37, a heavy chain constant region as set forth in SEQ ID NO 39, and a light chain constant region as set forth in SEQ ID NO 41; or alternatively
The antibody comprises a heavy chain variable region as shown in SEQ ID NO. 29, a light chain variable region as shown in SEQ ID NO. 37, a heavy chain constant region as shown in SEQ ID NO. 39, and a light chain constant region as shown in SEQ ID NO. 41; or alternatively
The antibody comprises a heavy chain variable region as set forth in SEQ ID NO 31, a light chain variable region as set forth in SEQ ID NO 37, a heavy chain constant region as set forth in SEQ ID NO 39, and a light chain constant region as set forth in SEQ ID NO 41.
In some embodiments of the first aspect, the antibody is a whole antibody, an Fab fragment, an Fab 'fragment, an F (ab')2 fragment, an Fv fragment, or a single chain Fv fragment (scFv).
In some embodiments of the first aspect, the antibody is a fully human antibody.
In some embodiments of the first aspect, the antibody is a humanized antibody.
In some embodiments of the first aspect, the antibody is a monoclonal antibody.
In some embodiments of the first aspect, the antibody comprises a framework region.
In some embodiments of the first aspect, the framework regions are humanized.
In some embodiments of the first aspect, the antibody comprises a heavy chain constant region selected from the IgG1 subtype, the IgG2 subtype, or the IgG4 subtype.
In some embodiments of the first aspect, the heavy chain constant region comprises a heavy chain constant region of the human IgG2 subtype.
In some embodiments of the first aspect, the antibody comprises a light chain constant region selected from the kappa subtype or the lambda subtype.
In some embodiments of the first aspect, the antibody is capable of binding TSLP.
In some embodiments of the first aspect, the antibody is capable of inhibiting TSLP binding to the TSLP receptor.
In some embodiments of the first aspect, the antibody is capable of blocking TSLP-mediated activity.
In a second aspect, the present application provides a nucleic acid molecule encoding the heavy chain variable region and the light chain variable region of the antibody of the first aspect.
In some embodiments of the second aspect, the nucleic acid molecule is operably linked to a control sequence that is recognized by a host cell transformed with the vector.
In a third aspect, the present application provides an expression vector comprising the nucleic acid molecule of the second aspect.
In a fourth aspect, the present application provides a host cell comprising the nucleic acid molecule of the second aspect or the expression vector of the third aspect.
In some embodiments of the fourth aspect, the host cell may be a mammalian or insect cell, such as a Chinese Hamster Ovary (CHO) cell, a Human Embryonic Kidney (HEK)293T cell, a mouse myeloma NSO cell, a Baby Hamster Kidney (BHK) cell. In some specific embodiments, the host cell is a Human Embryonic Kidney (HEK)293T cell.
In a fifth aspect, the present application provides a composition comprising an antibody according to the first aspect together with a pharmaceutically acceptable excipient, diluent or carrier.
In some embodiments of the fifth aspect, compositions for diagnostic, therapeutic, tracking and/or prognostic applications can be formulated for storage in lyophilized formulations or aqueous solutions by mixing the reagents of the desired purity with, optionally, pharmaceutically acceptable carriers, diluents, excipients, and the like.
In a sixth aspect, the present application provides a kit for detecting TSLP in a sample from a subject, comprising an antibody of the first aspect.
In some embodiments of the sixth aspect, the subject is a mammal.
In some embodiments of the sixth aspect, the subject is a human.
In a seventh aspect, the present application provides the use of an antibody of the first aspect, a nucleic acid molecule of the second aspect, an expression vector of the third aspect, a host cell of the fourth aspect, or a composition of the fifth aspect, in the manufacture of a kit for the diagnostic, therapeutic, tracking, and/or prognostic check of a TSLP-mediated disease.
In some embodiments of the seventh aspect, the TSLP-mediated disease is an immune system-related disease or tumor.
In some embodiments of the seventh aspect, the immune system-related disease is selected from autoimmune diseases and inflammatory diseases.
In some embodiments of the seventh aspect, the autoimmune disease and inflammatory disease are selected from the group consisting of: asthma, scleroderma, systemic lupus erythematosus, rheumatoid arthritis, Churg-Strauss syndrome, Wegener's granulomatosis, goodpasture's syndrome, allergic pneumonia, atopic dermatitis, rhinitis, ankylosing spondylitis, rheumatic fever, fibromyalgia, psoriatic arthritis, chronic nephritis, sjogren's syndrome, as well as multiple sclerosis, allergic dermatitis, allergic conjunctivitis, atopic dermatitis fibrosis, inflammatory bowel disease, and the like.
In some embodiments of the seventh aspect, the tumor is selected from: hodgkin's lymphoma, breast cancer, pancreatic cancer, melanoma, B-cell acute lymphocytic leukemia, cervical cancer, cutaneous T-cell lymphoma, gastric cancer and lung cancer.
In an eighth aspect, the present application provides the use of an antibody of the first aspect, a nucleic acid molecule of the second aspect, an expression vector of the third aspect, a host cell of the fourth aspect, or a composition of the fifth aspect, in the manufacture of a medicament for the treatment and/or prevention of a TSLP-mediated disease.
In some embodiments of the eighth aspect, the TSLP-mediated disease is an immune system-related disease or a tumor.
In some embodiments of the eighth aspect, the immune system-related disease is selected from autoimmune diseases and inflammatory diseases.
In some embodiments of the eighth aspect, the autoimmune and inflammatory diseases are selected from: asthma, scleroderma, systemic lupus erythematosus, rheumatoid arthritis, Churg-Strauss syndrome, Wegener's granulomatosis, goodpasture's syndrome, allergic pneumonia, atopic dermatitis, rhinitis, ankylosing spondylitis, rheumatic fever, fibromyalgia, psoriatic arthritis, chronic nephritis, sjogren's syndrome, as well as multiple sclerosis, allergic dermatitis, allergic conjunctivitis, atopic dermatitis fibrosis, inflammatory bowel disease, and the like.
In some embodiments of the eighth aspect, the tumor is selected from: hodgkin's lymphoma, breast cancer, pancreatic cancer, melanoma, B-cell acute lymphocytic leukemia, cervical cancer, cutaneous T-cell lymphoma, gastric cancer and lung cancer.
In addition, the present application provides a method of producing a polypeptide, the method comprising:
culturing the host cell of the fourth aspect in a culture medium under conditions in which the nucleic acid sequence is expressed, thereby producing a polypeptide comprising a light chain variable region and a heavy chain variable region; and
recovering the polypeptide from the host cell or culture medium.
For recombinant production of the antibodies herein, the nucleic acids encoding the heavy and light chains, respectively, are isolated and inserted into one or more replicable vectors for amplification or expression of further cloned DNA. DNA encoding the monoclonal antibody is readily isolated and sequenced using conventional protocols (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody). Many vectors are available. Vector components generally include, but are not limited to, a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. In one embodiment, both the light and heavy chains of an anti-TSLP antibody herein are expressed from the same vector (e.g., a plasmid or adenoviral vector).
The antibodies of the present application can be produced by any method known in the art. In some embodiments, the antibody is expressed in cultured mammalian or insect cells, such as Chinese Hamster Ovary (CHO) cells, Human Embryonic Kidney (HEK)293T cells, mouse myeloma NSO cells, Baby Hamster Kidney (BHK) cells. In some specific embodiments, the host cell is a Human Embryonic Kidney (HEK)293T cell.
The present application also provides a method of detecting the presence or amount of TSLP in a sample from a subject comprising providing an antibody as described in the first aspect.
In some embodiments, the subject is a mammal. In some specific embodiments, the subject is a human.
Antibody-based detection methods are well known in the art and include, for example, ELISA, immunoblotting, radioimmunoassay, immunofluorescence, immunoprecipitation, and other related techniques.
It should be understood that the above detailed description is only for the purpose of making the content of the present application more clearly understood by those skilled in the art, and is not intended to be limiting in any way. Various modifications and changes to the described embodiments will be apparent to those skilled in the art.
Examples
The following examples are for the purpose of illustration only and are not intended to limit the scope of the present application.
Example 1 preparation of recombinant protein
1.1 Synthesis of hTSLP Gene and construction of expression vector
The process of making monoclonal antibodies against TSLP requires the use of a variety of different recombinant proteins, including full-length human TSLP, which have a number of post-translational modifications (e.g., glycosylation or disulfide bonding, etc.), and thus the use of mammalian cell expression systems would be more advantageous in maintaining the structure and function of the recombinant protein. His labels are added at the C end of the recombinant proteins, which is beneficial to the purification of the recombinant proteins and the identification of the functions of the monoclonal antibodies. The amino acid and nucleotide sequences of the recombinant human TSLP protein (rhTSLP-His) containing the His tag are respectively shown in SEQ ID NO 43 and SEQ ID NO 44, and SEQ ID NO 45 is a guide peptide for cracking the recombinant protein rhTSLP-His mature product.
Genes (including His tags) of various recombinant proteins were designed and synthesized based on the amino acid sequences of various recombinant proteins of interest in the Uniprot database. The various recombinant protein genes synthesized are cloned into a suitable eukaryotic expression vector (e.g., pcDNA3.1(-) from invitrogen) using conventional molecular biology techniques.
1.2 expression of the rhTSLP-His fusion Gene in 293T/17 cells
1) Cell preparation
The day before transfection, 293T/17 cells were digested and passaged to 10cm dishes. The culture medium is DMEM medium containing 10% FBS, the culture temperature is 37 ℃, the cell fusion degree on the next day is about 80%, and the DMEM medium without serum is changed 1h before transfection.
2) Preparation of transfection reagents
Test tube a: plasmid 25 μ g/dish, adding transfection water 250 μ L, mixing well, adding 250 μ L of 0.5M CaCl 2 Mixing the solution with CaCl 2 The solution was mixed well before use.
Test tube B: 500. mu.L of a2 XHEPES solution was added
The solution in tube A was added drop by drop to tube B and mixed well. Standing at room temperature for 15-20min, and if the solution precipitates, it cannot be used.
3) Sample application
Adding the transfection reagent in the step 2) into 293T/17 cells along the wall of the dish, and rotating the dish to uniformly distribute the transfection reagent.
4) Liquid changing device
The expression medium was changed 6h after transfection (Gibco, 12338-018).
5) Culturing
The transfected cells were incubated at 32 ℃ with 5% CO 2 And (5) incubation in an incubator. Observing cell state and culture medium color every day, and timely replenishing liquid. The supernatant was collected on day 7 after transfection, centrifuged at 4500rpm at 4 ℃ for 15min in a refrigerated centrifuge, collected and filtered using a3 μm filter for the next protein purification step.
1.3 purification of rhTSLP-His
The supernatant of 293T/17 cells of the recombinant protein rhTSLP-His prepared in section 1.2 was captured using a Ni column (GE, 17-3712-02), and the eluate was collected by washing the Ni column with 50mL of 10mM imidazole. Eluting with 500mM imidazole at an elution flow rate of 3mL/min, and collecting 12-15mL of eluate. Protein content was determined using a 3KD 15mL ultrafiltration tube (Millipore, UFC900396) concentrated by ultrafiltration in PBS buffer at 280 nm. The concentrated protein is frozen in liquid nitrogen and stored at-80 deg.C.
Example 2 construction of hTSLPR/hIL-7 Ra-293T Stable cell line
2.1 selection of hTSLPR/hIL-7R alpha-293T Stable cell line
Respectively synthesizing full-length genes of human TSLP receptor (hTSPR, the amino acid sequence is shown as SEQ ID NO:46, the nucleotide sequence is shown as SEQ ID NO: 47) and human IL-7 Ra (hIL-7 Ra, the amino acid sequence is shown as SEQ ID NO:48, and the nucleotide sequence is shown as SEQ ID NO: 49), and respectively inserting the full-length genes into proper eukaryotic expression vectors (such as pCDH-CMV-MCS-EF-GFP-PURO) by utilizing a conventional molecular cloning method. The plasmid is transferred into 293T/17 cells by an electric shock transfection method, 5 mu g/mL puromycin is added, and a stable clone can be obtained after one week. The cells of each clone were transferred to a 24-well plate, and the puromycin concentration was adjusted to 2. mu.g/mL. After the cells were confluent, clones were selected based on fluorescence intensity and bound to TSLPR flow antibody PE-TSLPR (Biolegend, 322805), and then further characterized by flow cytometry, and clones with high TSLPR receptor expression levels in flow screening were selected for further amplification, with the results shown as Q1-UR in fig. 1.
2.2 binding assay of hTSSLPR/hIL-7 Ra-293T/17 Stable cells with rhTSLP-His protein
hTSLPR/hIL-7R α -293T/17 cells were harvested and 5% milk blocked (on ice) for 1 hour. Spreading in v-bottom 96-well plate (nai Si, 701201) at 5X 10/well 5 And (4) cells. rhTSLP-His protein was serially diluted from 1. mu.g/ml for 6 concentration gradients, 100. mu.L was added per well and bound for 1h at 4 ℃ with shaking. The cells were harvested by centrifugation, washed with PBS, resuspended in 100. mu.L of anti-His-HRP (Abcam No.1269, 1:3000), and incubated on ice for 1 h. After PBS washing, TMB was added for color development, and 100. mu.L of the mixture was centrifuged after signal termination to determine the OD450 value. The results are shown in FIG. 2, where hTSLPR/hIL-7 Ra-293T/17 stable cells were able to bind rhTSLP-His protein.
Example 3 immunization of mice and determination of antibody titers in serum
Balb/c mice (purchased from Beijing Spbefu laboratory animal technology Co., Ltd.) were immunized using the rhTSLP-His protein prepared in example 1 as an immunogen. A total of 3 mice were immunized by dorsal subcutaneous multi-point injection with Freund's complete adjuvant at an immunization dose of 50. mu.g/200. mu.L/mouse. The first immunization was performed by mixing 50. mu.g of rhTSLP-His protein with 100. mu.L of Freund's complete adjuvant (Sigma, F5881), and the second, third and fourth immunizations were performed by mixing 50. mu.g of rhTSLP-His with 100. mu.L of Freund's incomplete adjuvant (Sigma, F5506), for the four immunizations, at days 0, 14, 28 and 42, respectively. After the fourth immunization, 3 mice were blood-sampled by eye, and the titer of anti-hTSLP antibody in the serum of the immunized mice was determined by ELISA method.
The serum titer is measured by adopting an ELISA method, and the measuring method is as follows: first, rhTSLP-His protein was diluted to 1. mu.g/mL with 50mM carbonate coating buffer (pH 9.6), 100. mu.L/well was added to a 96-well ELISA plate (West, 504201), and incubated overnight at 4 ℃. The following day, the plates were washed 3 times with PBST (phosphate buffer containing 0.5% Tween 20) and blocked for 2 hours at 37 ℃ by adding blocking solution (3% BSA phosphate buffer). The plate was washed 1 time with a blank well of PBS solution, 100. mu.L/well was added to the ELISA plate and incubated at room temperature for 1 hour, the plate was washed 3 times, goat anti-mouse IgG (H + L) -HRP antibody (ProteinTech, SA00001-1) was added to a final concentration of 1. mu.g/mL and incubated at room temperature for 1 hour. The plate was washed 4 times, and then TMB (Solebao, PR1200) developing solution was added for development at room temperature, and then stop solution was added, and the absorbance was read on a microplate reader (Thermo, multiscan GO) at a wavelength of 450nm, and the results are shown in Table 1.
TABLE 1 measurement of serum titers of immunized mice
Figure BDA0002963610810000221
Example 4 preparation of hybridomas
The mice of example 3 were shock immunized within 2 weeks after the fourth immunization, spleens of mice were harvested within 4 days, milled in saline and then taken as a B cell and pre-B cell rich suspension, and cell fused with myeloma cells SP2/0 under the action of the fusion agent PEG4000(Sigma, 95904). The fused cells were divided into 30 96-well cell culture plates and replete in 20% fetal bovine serum (Gibco,1099-141) RPMI-1640 containing HAT (Sigma, H0262)In nutrient medium (Thermo, 31800089), in 5% CO 2 After one week of incubation at 37 ℃ the culture was continued in HT medium (Sigma, H0137).
The screening method for hybridomas and subclones was as follows:
1) screening hybridoma positive clones with strong binding activity with antigen rhTSLP-His by enzyme-linked immunosorbent assay (ELISA);
adding a confining liquid into a 96-well ELISA plate coated by 100 ng/well rhTSLP-His protein, sealing for 2 hours at 37 ℃, adding 100 mu L of hybridoma supernatant liquid for combination for 1 hour, then adding anti-His-HRP, incubating for 1 hour at 37 ℃, adding TMB developing liquid for developing for 1-3 minutes at 37 ℃, adding a stop solution, and reading a light absorption value on an enzyme-linked immunosorbent assay (ELISA) instrument at a wavelength of 450 nm.
2) Screening hybridoma positive clones with blocking activity by flow cytometry or ELISA method
hTSLPR/hIL-7R alpha-293T/17 cells at 5X 10 5 Density of individual/well V-bottom 96-well plates were added and 100ng of rhTSLP-His and 100 μ L of anti-TSLP hybridoma supernatant were incubated at room temperature for 1h, then mixed with cells, incubated on ice for 1 hour and washed three times with PBS buffer. Anti-6 × His antibody (iFluor 647, Kisrey, A01802-100) or APC anti-His tag antibody (Biolegend, J095G46) were raised at a rate of 1: 400 diluted with PBS buffer, 100 u L/hole, protected from light incubation for 30min, PBS buffer washing two times after 100 u L/hole heavy suspension cells, using the flow meter (Beckman, Cytoflex-AR46042) to detect FL-1 fluorescence path.
When screening hybridoma cells by ELISA, anti-His-HRP (Abcam No.1269, 1:3000) is added, incubated at 37 ℃ for 1 hour, then TMB developing solution is added to develop color at 37 ℃, and after termination, the absorbance is read on an enzyme-linked immunosorbent assay at a wavelength of 450 nm. Since the neutralizing antibody can prevent the receptor hTSPR from being combined with rhTSLP, the rhTSLP-His combination quantity is reduced, and the OD450 value is reduced. Clones with blocking efficiency as high as more than 90% are selected for subcloning. Cloning cells in the holes by adopting a limiting dilution method, and establishing a monoclonal hybridoma cell strain for stably secreting a human TSLP neutralizing antibody after three times of subcloning.
Example 5 obtaining of murine TSLP monoclonal antibodies
Examples of the invention4 to a total number of 1X 10 5 The cells were harvested by centrifugation at 1000rpm for 10 minutes and total RNA was extracted (Qiagen, No. 74104). First strand cDNA (Takara, PrimeScript) was synthesized using the extracted total RNA as a template TM II first strand cDNA Synthesis kit), the variable region DNA sequence corresponding to the hybridoma cells is amplified using the first strand cDNA as a subsequent template. The primer sequences used in the amplification reaction are complementary to the first framework and constant regions of the antibody variable region, reference (Larrick JW, et al (1990) and Coloma MJ, et al (1991)). The DNA polymerase used for PCR amplification was a fidelity PCR amplification enzyme (Takara, R045Q).
3 monoclonal antibodies (9F8-1-25, 10D9-M-432 and 5F10-2-25) having neutralizing activity were obtained together. Meanwhile, hybridoma cells having neutralizing activity were cultured to logarithmic growth phase, and the cell concentration was adjusted to 2X 10 with PBS 6 Individual cells/mL. 6-8 week old Balb/c mice were injected intraperitoneally with 1mL of a mixture of Freund's incomplete adjuvant (Sigma, F5506) and hybridoma cells (1:1) to grow hybridomas in the abdominal cavities of the mice and produce ascites. The abdominal cavity of the mouse can be enlarged 7-10 days after the hybridoma cells are injected, ascites of the mouse is collected, and the ascites is purified by protein A column affinity. The ascites fluid obtained is collected and captured using either a ProteinA 5mL prepacked column (GE, 17-5080-02) or a ProteinA/G1 mL prepacked column (Biyunnan, P2028) and eluted with 0.1M citric acid pH 3.0. After completion of collection, the collected solutions were pooled, concentrated by ultrafiltration in PBS buffer via a 30K 4mL ultrafiltration tube (Millipore, UFC803096), and the protein content was determined by BCA method. The obtained protein is frozen by liquid nitrogen and then stored at-80 ℃.
Example 6 Performance validation of murine monoclonal antibodies
The affinity of murine monoclonal antibodies to human TSLP and their inhibition of the binding activity of TSLP to TSLPR cells were determined by ELISA techniques, in which the control antibody was Tezepelumab expressed by eukaryotic cells (antibody developed by Anin and Aslicon). The specific operation is as follows: blocking solution was added to a 96-well ELISA plate coated with 100 ng/well rhTSLP-His recombinant protein, and after blocking for 2 hours at 37 ℃, 100 μ L of hybridoma supernatant (hybridoma supernatant of 9F8-1-25, 10D9-M-432, or 5F10-2-25) and control antibody (Tezepelumab) were added to bind for 1h, respectively, followed by addition of anti-His-HRP, incubation for 1 hour at 37 ℃, addition of TMB developing solution for development for 1-3 minutes at 37 ℃, addition of stop solution, and reading of absorbance at 450nm wavelength on a microplate reader, with the results shown in fig. 3 and table 2: antibodies 9F8-1-25, 10D9-M-432, 5F10-2-25 and Tezepelumab were all capable of binding to rhTSLP-His recombinant protein.
The blocking activity of the antibody was evaluated mainly by blocking the binding of rhTSLP-His recombinant protein to hTSPR/hIL-7 Ra-293T/17 cells by the antibody. The specific operation is as follows: hTSLPR/hIL-7R α -293T/17 cells were harvested and 5% milk blocked (on ice) for 1 hour. Spreading on v-bottom 96-well plate with 5 × 10 wells 5 And (4) cells. After 2-fold serial dilution, the antibody was combined with 100ng rhTSLP-His protein for 30min at room temperature, a v-bottom 96-well plate was added, the cells were resuspended, and the binding was performed for 1h with shaking on ice. Cells were harvested by centrifugation, washed with PBS, resuspended in 100. mu.L of anti-His-HRP, and incubated on ice for 1 h. After washing with PBS, TMB was added for color development. After termination of the signal, 100. mu.L of the supernatant was centrifuged to determine the OD450 value. Taking the OD450 of the cell binding hole added with only 100ng of rhTSLP-His recombinant protein as an unblocked control,
Figure BDA0002963610810000241
the results are shown in fig. 4 and table 2: antibodies 9F8-1-25, 10D9-M-432, 5F10-2-25 and Tezepelumab were all able to inhibit the binding of rhTSLP-His recombinant protein to hTSPR/hIL-7 Ra-293T/17 cells, and the inhibitory effect of antibodies 9F8-1-25, 10D9-M-432 and 5F10-2-25 was comparable to that of antibody Tezepelumab.
TABLE 2 murine TSLP monoclonal antibody binding and neutralizing Activity
10D9-M-432 9F8-1-25 5F10-2-25 Tezepelumab
EC 50 (nM) 8.16 11.38 12.36 2.929
IC 50 (μg/mL) 12.03 10.71 13.45 6.216
Example 7 humanization and Performance validation of murine anti-TSLP monoclonal antibodies
1. Humanization of murine anti-TSLP monoclonal antibodies
Respectively carrying out anti-mouse TSLP monoclonal antibody 9F8-1-25 (heavy chain variable region amino acid sequence is SEQ ID NO:21, nucleotide sequence is SEQ ID NO:22, light chain variable region amino acid sequence is SEQ ID NO:33, nucleotide sequence is SEQ ID NO:34), 10D9-M-432 (heavy chain variable region amino acid sequence of SEQ ID NO:23, nucleotide sequence of SEQ ID NO: 24; light chain variable region amino acid sequence of SEQ ID NO:33, nucleotide sequence of SEQ ID NO:34) and 5F10-2-25 (heavy chain variable region amino acid sequence of SEQ ID NO:25, nucleotide sequence of SEQ ID NO: 26; light chain variable region amino acid sequence of SEQ ID NO:35, nucleotide sequence of SEQ ID NO:36) were humanized to reduce immunogenicity. The nucleic acid molecules encoding the heavy chain variable region (VH) and light chain variable region (VL) were compared with human antibody germline gene sequences in the IMGT database, respectively, and appropriate germline gene sequences were selected to provide the gene sequences encoding framework regions 1 to 3(FR1+ FR2+ FR3) of the antibody, and appropriate J region gene sequences were selected to provide the gene sequence encoding framework region 4(FR 4). This template may be selected based on a number of factors, such as: the relative overall length of the antibody, the size of the CDR regions, the amino acid residues located at the junction between the Framework (FR) and hypervariable (CDR) regions of the antibody, the homology of the sequence as a whole, etc. The template selected may be a chimeric version of multiple sequences or may be a consensus template, in order to maintain as appropriate a conformation of the parental Complementarity Determining Regions (CDRs) as possible. CDR grafting of murine anti-TSLP monoclonal antibodies 10D9-M-432, 9F8-1-25 and 5F10-2-25 was performed based on the selected templates to obtain three humanized versions of M432-69H13L, 9F8-46H13L and 5F10-69H 13L. Designing and synthesizing antibody variable region genes according to the amino acid sequence of a humanized antibody, wherein the amino acid sequences of a heavy chain variable region and a light chain variable region of the antibody 9F8-46H13L after the humanized antibody are respectively SEQ ID NO:27 and SEQ ID NO:37, and the nucleotide sequences are respectively SEQ ID NO:28 and SEQ ID NO: 38; the amino acid sequences of the heavy chain variable region and the light chain variable region of M432-69H13L are SEQ ID NO:29 and SEQ ID NO:37 respectively, and the nucleotide sequences are SEQ ID NO:30 and SEQ ID NO:38 respectively; the amino acid sequences of the heavy chain variable region and the light chain variable region of 5F10-69H13L are SEQ ID NO 31 and SEQ ID NO 37, respectively, and the nucleotide sequences are SEQ ID NO 32 and SEQ ID NO 38, respectively. The recombinant expression vector is constructed by connecting and cloning an IgG2 subtype antibody heavy chain constant region (with an amino acid sequence of SEQ ID NO:39 and a nucleotide sequence of SEQ ID NO:40) and a light chain constant region (with an amino acid sequence of SEQ ID NO:41 and a nucleotide sequence of SEQ ID NO:42) to a eukaryotic expression vector pBUDce4.1 plasmid by using a conventional molecular cloning technology. The recombinant expression plasmids carrying the various humanized and modified light chain and heavy chain combinations are transferred into 293T cells, and the expression and purification steps in the 293T cells are the same as the previous sections 1.2 and 1.3 of the example 1 and the example 5, so as to prepare the humanized TSLP monoclonal antibody.
ELISA (enzyme Linked immunosorbent assay) determination of binding activity of anti-hTSLP humanized monoclonal antibody to rhTSLP-His recombinant protein and blocking of binding of rhTSLP-His recombinant protein and hTSPR/hIL-7 Ralpha-293T/17 cells
The binding activity of the resulting humanized antibodies (9F8-46H13L, M432-69H13L and 5F10-69H13L) (results are shown in FIG. 5 and Table 3) and the ability to block the binding of rhTSLP-His recombinant protein/hTSLPPR/hIL-7 Ra-293T/17 cells (results are shown in FIG. 6 and Table 3) were determined in the same ELISA technique as in the binding and neutralization experiment in example 6. The results show that the anti-hTSLP humanized monoclonal antibodies (9F8-46H13L, M432-69H13L and 5F10-69H13L) can be combined with rhTSLP-His protein, and can obviously inhibit the combination of the rhTSLP-His recombinant protein and the hTSPR/hIL-7 Ra-293T/17 cells, and the inhibition capacity is stronger or equivalent to that of a human TSLP monoclonal antibody Tezepelumab (Amgen).
TABLE 3 binding and neutralizing Activity of humanized monoclonal antibodies against hTSLP
M432-69H13L 9F8-46H13L 5F10-69H13L Tezepelumab
EC 50 (nM) 4.046 9.522 31.56 1.796
IC 50 (μg/mL) 4.353 4.048 7.565 8.928
Example 8 anti-oxidative stability test of antibodies
Proteins undergo varying degrees of degradation during purification and storage. Oxidation is one of the major degradation pathways for proteins and has a devastating effect on protein stability and potency. The oxidative degradation rate of antibodies can be determined by 2,2 '-azobis (2-amidinopropane) dihydrochloride (2, 2' -azobis (2-amidinopropane) dihydrochloride, AAPH) stress experiments and liquid phase analysis (Ji JA, et al (2009)).
AAPH stress experiments:
first, a 1mM AAPH solution was prepared using a 20mM ammonium acetate buffer solution. Then, after the concentration of the antibody sample is subjected to primary concentration detection, 1 mu L of sample is taken and HPLC is used for recording the corresponding peak-appearing distribution condition, retention time and main peak area. Data were counted at 0h time point. Next, according to n antibody: the mixture was mixed at a ratio of 1:50 nAAPH, and oxidation test was carried out by adding 10. mu.L of AAPH to 50. mu.L of the antibody solution at a concentration of 1mg/L after concentration. At this ratio, the oxidation reaction solution was mixed. The main peak retention time and the main peak area corresponding to 0h are respectively observed at 2h, 4h, 6h and 24 h. And finally, carrying out data statistics, recording the peak area change conditions of the main peak at different times, and calculating the oxidative degradation rate.
Liquid phase analysis: the liquid chromatography separation method of this experiment requires liquid chromatography conditions of liquid a: 0.065% aqueous TFA; and B, liquid B: 0.05% TFA + 100% ACN, ZORBAX-300SB-C18 chromatographic column, flow rate of 1mL/min, column temperature of 60 ℃, detection wavelength of 220nm, gradient method of 0-30min, B liquid of 5-65%. The results show that different antibodies have different rates of oxidative degradation and show different peak areas in high performance liquid chromatography. The slower decrease in peak area of the HPLC at various time points over 24H compared to Tezepelumab and 9F8-46H13L also means that it has better stability against oxidation (table 4).
TABLE 4 antioxidant stability results of antibodies characterized by peak area
Figure BDA0002963610810000271
Although the invention has been described in detail hereinabove by way of general illustration and specific embodiments, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Reference to the literature
1.Larrick JW,Coloma MJ,del Valle J,Fernandez ME,Fry KE,Gavilondo-Cowley JV.Immunoglobulin V regions of a bactericidal anti-Neisseria meningitidis outer membrane protein monoclonal antibody.Scand J Immunol,1990,32:121-128；
2.Coloma MJ,Larrick JW,Ayala M,Gavilondo-Cowley JV.Primer design for the cloning of immunoglobulin heavy-chain leader-variable regions from mouse hybridoma cells using the PCR.Biotechniques,1991,11(2):152-4,156；
3.Ji JA，Zhang B，Cheng W，Wang YJ.Methionine,Tryptophan,and Histidine Oxidation in a Model Protein,PTH:Mechanisms and Stabilization.J Pharm Sci,2009,98:4485-4500.
Sequence listing
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115
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Val Thr Val Ser Ser
115
<210> 32
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<210> 33
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Asp Ile Asp Leu Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly
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20 25 30
Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val
35 40 45
Tyr Asn Ala Lys Ile Leu Ala Glu Ser Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Gly Ile Tyr Ser Cys Gln His His Tyr Asp Thr Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu
100 105
<210> 34
<211> 315
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
gacattgacc tcacccagtc tccagcctcc ctatctgcat ctgtgggaga aactgtcacc 60
atcacatgtc gagcaggtga gaatatttac agttatttag catggtatca gcagaaacag 120
ggaaaatctc ctcagctcct ggtctataat gcaaaaattt tagcagaaag tgtgccctca 180
aggttcagtg gcagtggatc aggcacacag ttttctctga agatcaacag cctgcagcct 240
gaagattttg ggatttattc ctgtcaacat cattatgata ctccgtggac gttcggtgga 300
ggcaccaagc tggaa 315
<210> 35
<211> 108
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 35
Asp Ile Asp Leu Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Glu Thr Val Thr Ile Thr Cys Arg Thr Ser Glu Asn Ile Tyr Ser Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val
35 40 45
Tyr Asn Ala Lys Ser Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His His Tyr Asp Thr Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Asn Lys Thr
100 105
<210> 36
<211> 324
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
gacattgacc tcacccagtc tccagcctcc ctatctgcat ctgtgggaga aactgtcacc 60
atcacatgtc gaacaagtga aaatatttac agttatttag catggtatca gcagaaacag 120
ggaaaatctc ctcagctcct ggtctataat gcaaaaagtt tagcagaagg tgtgccatca 180
aggttcagtg gcagtggatc aggcacccag ttttctctga agatcaacag cctgcagcct 240
gaagattttg ggagttatta ctgtcaacat cattatgata ctccgtggac gttcggtgga 300
ggcaccaagc tggaaaataa aacg 324
<210> 37
<211> 107
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 37
Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Gly Glu Asn Ile Tyr Ser Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Val
35 40 45
Tyr Asn Ala Lys Ile Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His His Tyr Asp Thr Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105
<210> 38
<211> 321
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
gacatccagc tcacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60
atcacttgcc gggcaggtga gaatatttac agttatttag cctggtatca gcagaaacca 120
gggaaagctc ctaagctcct ggtgtataat gcaaaaattt tagcagaagg ggtcccatca 180
aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240
gaagattttg caacttatta ctgtcaacat cattatgata ctccttggac gttcggcgga 300
ggcaccaagc tgaccgtgct g 321
<210> 39
<211> 326
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 39
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg
1 5 10 15
Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr
65 70 75 80
Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro
100 105 110
Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
115 120 125
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
130 135 140
Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly
145 150 155 160
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn
165 170 175
Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp
180 185 190
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro
195 200 205
Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu
210 215 220
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn
225 230 235 240
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
245 250 255
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
260 265 270
Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
275 280 285
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
290 295 300
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
305 310 315 320
Ser Leu Ser Pro Gly Lys
325
<210> 40
<211> 978
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 40
gccagcacca agggaccctc cgtgttcccc ctggctccct gcagcaggtc caccagcgag 60
tccaccgctg ccctgggctg cctggtgaag gactacttcc ccgagcccgt gaccgtgagc 120
tggaactccg gagccctgac cagcggcgtg cacaccttcc cagccgtgct gcagtccagc 180
ggcctgtact ccctgagctc cgtggtgacc gtgcccagct ccaacttcgg cacccagacc 240
tacacctgca acgtggacca caagcccagc aacaccaagg tggacaagac cgtggagagg 300
aagtgctgcg tggagtgccc tccctgccct gccccacccg tggctggccc ctccgtgttc 360
ctgttccctc ccaagcccaa ggacaccctg atgatcagca gaacccccga ggtgacctgc 420
gtggtggtgg acgtgtccca cgaggacccc gaggtgcagt tcaactggta cgtggacggc 480
gtggaggtgc acaacgccaa gaccaagccc agggaggagc agttcaacag caccttcaga 540
gtggtgtccg tgctgaccgt ggtgcaccag gactggctga acggcaagga gtacaagtgc 600
aaggtgagca acaagggcct gcctgctccc atcgagaaga ccatctccaa gaccaagggc 660
cagcccagag agccccaggt gtacaccctg cctcccagca gagaggagat gaccaagaac 720
caggtgtccc tgacctgcct ggtgaagggc ttctacccca gcgacatcgc cgtggagtgg 780
gagtccaacg gccagcccga gaacaactac aagaccaccc ctcccatgct ggacagcgac 840
ggctccttct tcctgtacag caagctgacc gtggacaagt ccagatggca gcagggcaac 900
gtgttcagct gctccgtgat gcacgaggcc ctgcacaacc actacaccca gaagagcctg 960
tccctgagcc ctggcaag 978
<210> 41
<211> 106
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 41
Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser
1 5 10 15
Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp
20 25 30
Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro
35 40 45
Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn
50 55 60
Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys
65 70 75 80
Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val
85 90 95
Glu Lys Thr Val Ala Pro Thr Glu Cys Ser
100 105
<210> 42
<211> 321
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 42
ggccagccca aggctgcccc cagcgtgacc ctgttccctc cctccagcga ggagctgcag 60
gccaacaagg ccaccctggt gtgcctgatc agcgacttct accctggagc cgtgaccgtg 120
gcctggaagg ccgactccag ccccgtgaag gctggcgtgg agaccaccac ccccagcaag 180
cagtccaaca acaagtacgc cgccagctcc tacctgagcc tgacccctga gcagtggaag 240
agccaccgct cctacagctg ccaggtgacc cacgagggca gcaccgtgga gaagaccgtg 300
gctcccaccg agtgcagctg a 321
<210> 43
<211> 175
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 43
Met Val Pro Phe Ala Leu Leu Tyr Val Leu Ser Val Ser Phe Arg Lys
1 5 10 15
Ile Phe Ile Leu Gln Leu Val Gly Leu Val Leu Thr Tyr Asp Phe Thr
20 25 30
Asn Cys Asp Phe Glu Lys Ile Lys Ala Ala Tyr Leu Ser Thr Ile Ser
35 40 45
Lys Asp Leu Ile Thr Tyr Met Ser Gly Thr Lys Ser Thr Glu Phe Asn
50 55 60
Asn Thr Val Ser Cys Ser Asn Arg Pro His Cys Leu Thr Glu Ile Gln
65 70 75 80
Ser Leu Thr Phe Asn Pro Thr Ala Gly Cys Ala Ser Leu Ala Lys Glu
85 90 95
Met Phe Ala Met Lys Thr Lys Ala Ala Leu Ala Ile Trp Cys Pro Gly
100 105 110
Tyr Ser Glu Thr Gln Ile Asn Ala Thr Gln Ala Met Lys Lys Arg Arg
115 120 125
Lys Arg Lys Val Thr Thr Asn Lys Cys Leu Glu Gln Val Ser Gln Leu
130 135 140
Gln Gly Leu Trp Arg Arg Phe Asn Arg Pro Leu Leu Lys Gln Gln Gly
145 150 155 160
Gly His His His His His His Asp Tyr Lys Asp Asp Asp Asp Lys
165 170 175
<210> 44
<211> 525
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 44
atggtgccct tcgccctgct gtacgtgctg agcgtgagct tccgcaagat cttcatcctg 60
cagctggtgg gcctggtgct gacctacgac ttcaccaact gcgacttcga gaagatcaag 120
gccgcctacc tgagcaccat cagcaaggac ctgatcacct acatgagcgg caccaagagc 180
accgagttca acaacaccgt gagctgcagc aaccgccccc actgcctgac cgagatccag 240
agcctgacct tcaaccccac cgccggctgc gccagcctgg ccaaggagat gttcgccatg 300
aagaccaagg ccgccctggc catctggtgc cccggctaca gcgagaccca gatcaacgcc 360
acccaggcca tgaagaagcg ccgcaagcgc aaggtgacca ccaacaagtg cctggagcag 420
gtgagccagc tgcagggcct gtggcgccgc ttcaaccgcc ccctgctgaa gcagcaggga 480
ggccatcacc atcaccatca cgactacaaa gacgatgacg acaaa 525
<210> 45
<211> 28
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 45
Met Phe Pro Phe Ala Leu Leu Tyr Val Leu Ser Val Ser Phe Arg Lys
1 5 10 15
Ile Phe Ile Leu Gln Leu Val Gly Leu Val Leu Thr
20 25
<210> 46
<211> 371
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 46
Met Gly Arg Leu Val Leu Leu Trp Gly Ala Ala Val Phe Leu Leu Gly
1 5 10 15
Gly Trp Met Ala Leu Gly Gln Gly Gly Ala Ala Glu Gly Val Gln Ile
20 25 30
Gln Ile Ile Tyr Phe Asn Leu Glu Thr Val Gln Val Thr Trp Asn Ala
35 40 45
Ser Lys Tyr Ser Arg Thr Asn Leu Thr Phe His Tyr Arg Phe Asn Gly
50 55 60
Asp Glu Ala Tyr Asp Gln Cys Thr Asn Tyr Leu Leu Gln Glu Gly His
65 70 75 80
Thr Ser Gly Cys Leu Leu Asp Ala Glu Gln Arg Asp Asp Ile Leu Tyr
85 90 95
Phe Ser Ile Arg Asn Gly Thr His Pro Val Phe Thr Ala Ser Arg Trp
100 105 110
Met Val Tyr Tyr Leu Lys Pro Ser Ser Pro Lys His Val Arg Phe Ser
115 120 125
Trp His Gln Asp Ala Val Thr Val Thr Cys Ser Asp Leu Ser Tyr Gly
130 135 140
Asp Leu Leu Tyr Glu Val Gln Tyr Arg Ser Pro Phe Asp Thr Glu Trp
145 150 155 160
Gln Ser Lys Gln Glu Asn Thr Cys Asn Val Thr Ile Glu Gly Leu Asp
165 170 175
Ala Glu Lys Cys Tyr Ser Phe Trp Val Arg Val Lys Ala Met Glu Asp
180 185 190
Val Tyr Gly Pro Asp Thr Tyr Pro Ser Asp Trp Ser Glu Val Thr Cys
195 200 205
Trp Gln Arg Gly Glu Ile Arg Asp Ala Cys Ala Glu Thr Pro Thr Pro
210 215 220
Pro Lys Pro Lys Leu Ser Lys Phe Ile Leu Ile Ser Ser Leu Ala Ile
225 230 235 240
Leu Leu Met Val Ser Leu Leu Leu Leu Ser Leu Trp Lys Leu Trp Arg
245 250 255
Val Lys Lys Phe Leu Ile Pro Ser Val Pro Asp Pro Lys Ser Ile Phe
260 265 270
Pro Gly Leu Phe Glu Ile His Gln Gly Asn Phe Gln Glu Trp Ile Thr
275 280 285
Asp Thr Gln Asn Val Ala His Leu His Lys Met Ala Gly Ala Glu Gln
290 295 300
Glu Ser Gly Pro Glu Glu Pro Leu Val Val Gln Leu Ala Lys Thr Glu
305 310 315 320
Ala Glu Ser Pro Arg Met Leu Asp Pro Gln Thr Glu Glu Lys Glu Ala
325 330 335
Ser Gly Gly Ser Leu Gln Leu Pro His Gln Pro Leu Gln Gly Gly Asp
340 345 350
Val Val Thr Ile Gly Gly Phe Thr Phe Val Met Asn Asp Arg Ser Tyr
355 360 365
Val Ala Leu
370
<210> 47
<211> 1116
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 47
atggggcggc tggttctgct gtggggagct gccgtctttc tgctgggagg ctggatggct 60
ttggggcaag gaggagcagc agaaggagta cagattcaga tcatctactt caatttagaa 120
accgtgcagg tgacatggaa tgccagcaaa tactccagga ccaacctgac tttccactac 180
agattcaacg gtgatgaggc ctatgaccag tgcaccaact accttctcca ggaaggtcac 240
acttcggggt gcctcctaga cgcagagcag cgagacgaca ttctctattt ctccatcagg 300
aatgggacgc accccgtttt caccgcaagt cgctggatgg tttattacct gaaacccagt 360
tccccgaagc acgtgagatt ttcgtggcat caggatgcag tgacggtgac gtgttctgac 420
ctgtcctacg gggatctcct ctatgaggtt cagtaccgga gccccttcga caccgagtgg 480
cagtccaaac aggaaaatac ctgcaacgtc accatagaag gcttggatgc cgagaagtgt 540
tactctttct gggtcagggt gaaggctatg gaggatgtat atgggccaga cacataccca 600
agcgactggt cagaggtgac atgctggcag agaggcgaga ttcgggatgc ctgtgcagag 660
acaccaacgc ctcccaaacc aaagctgtcc aaatttattt taatttccag cctggccatc 720
cttctgatgg tgtctctcct ccttctgtct ttatggaaat tatggagagt gaagaagttt 780
ctcattccca gcgtgccaga cccgaaatcc atcttccccg ggctctttga gatacaccaa 840
gggaacttcc aggagtggat cacagacacc cagaacgtgg cccacctcca caagatggca 900
ggtgcagagc aagaaagtgg ccccgaggag cccctggtag tccagttggc caagactgaa 960
gccgagtctc ccaggatgct ggacccacag accgaggaga aagaggcctc tgggggatcc 1020
ctccagcttc cccaccagcc cctccaaggc ggtgatgtgg tcacaatcgg gggcttcacc 1080
tttgtgatga atgaccgctc ctacgtggcg ttgtga 1116
<210> 48
<211> 459
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 48
Met Thr Ile Leu Gly Thr Thr Phe Gly Met Val Phe Ser Leu Leu Gln
1 5 10 15
Val Val Ser Gly Glu Ser Gly Tyr Ala Gln Asn Gly Asp Leu Glu Asp
20 25 30
Ala Glu Leu Asp Asp Tyr Ser Phe Ser Cys Tyr Ser Gln Leu Glu Val
35 40 45
Asn Gly Ser Gln His Ser Leu Thr Cys Ala Phe Glu Asp Pro Asp Val
50 55 60
Asn Ile Thr Asn Leu Glu Phe Glu Ile Cys Gly Ala Leu Val Glu Val
65 70 75 80
Lys Cys Leu Asn Phe Arg Lys Leu Gln Glu Ile Tyr Phe Ile Glu Thr
85 90 95
Lys Lys Phe Leu Leu Ile Gly Lys Ser Asn Ile Cys Val Lys Val Gly
100 105 110
Glu Lys Ser Leu Thr Cys Lys Lys Ile Asp Leu Thr Thr Ile Val Lys
115 120 125
Pro Glu Ala Pro Phe Asp Leu Ser Val Val Tyr Arg Glu Gly Ala Asn
130 135 140
Asp Phe Val Val Thr Phe Asn Thr Ser His Leu Gln Lys Lys Tyr Val
145 150 155 160
Lys Val Leu Met His Asp Val Ala Tyr Arg Gln Glu Lys Asp Glu Asn
165 170 175
Lys Trp Thr His Val Asn Leu Ser Ser Thr Lys Leu Thr Leu Leu Gln
180 185 190
Arg Lys Leu Gln Pro Ala Ala Met Tyr Glu Ile Lys Val Arg Ser Ile
195 200 205
Pro Asp His Tyr Phe Lys Gly Phe Trp Ser Glu Trp Ser Pro Ser Tyr
210 215 220
Tyr Phe Arg Thr Pro Glu Ile Asn Asn Ser Ser Gly Glu Met Asp Pro
225 230 235 240
Ile Leu Leu Thr Ile Ser Ile Leu Ser Phe Phe Ser Val Ala Leu Leu
245 250 255
Val Ile Leu Ala Cys Val Leu Trp Lys Lys Arg Ile Lys Pro Ile Val
260 265 270
Trp Pro Ser Leu Pro Asp His Lys Lys Thr Leu Glu His Leu Cys Lys
275 280 285
Lys Pro Arg Lys Asn Leu Asn Val Ser Phe Asn Pro Glu Ser Phe Leu
290 295 300
Asp Cys Gln Ile His Arg Val Asp Asp Ile Gln Ala Arg Asp Glu Val
305 310 315 320
Glu Gly Phe Leu Gln Asp Thr Phe Pro Gln Gln Leu Glu Glu Ser Glu
325 330 335
Lys Gln Arg Leu Gly Gly Asp Val Gln Ser Pro Asn Cys Pro Ser Glu
340 345 350
Asp Val Val Ile Thr Pro Glu Ser Phe Gly Arg Asp Ser Ser Leu Thr
355 360 365
Cys Leu Ala Gly Asn Val Ser Ala Cys Asp Ala Pro Ile Leu Ser Ser
370 375 380
Ser Arg Ser Leu Asp Cys Arg Glu Ser Gly Lys Asn Gly Pro His Val
385 390 395 400
Tyr Gln Asp Leu Leu Leu Ser Leu Gly Thr Thr Asn Ser Thr Leu Pro
405 410 415
Pro Pro Phe Ser Leu Gln Ser Gly Ile Leu Thr Leu Asn Pro Val Ala
420 425 430
Gln Gly Gln Pro Ile Leu Thr Ser Leu Gly Ser Asn Gln Glu Glu Ala
435 440 445
Tyr Val Thr Met Ser Ser Phe Tyr Gln Asn Gln
450 455
<210> 49
<211> 1380
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 49
atgacaattc taggtacaac ttttggcatg gttttttctt tacttcaagt cgtttctgga 60
gaaagtggct atgctcaaaa tggagacttg gaagatgcag aactggatga ctactcattc 120
tcatgctata gccagttgga agtgaatgga tcgcagcact cactgacctg tgcttttgag 180
gacccagatg tcaacatcac caatctggaa tttgaaatat gtggggccct cgtggaggta 240
aagtgcctga atttcaggaa actacaagag atatatttca tcgagacaaa gaaattctta 300
ctgattggaa agagcaatat atgtgtgaag gttggagaaa agagtctaac ctgcaaaaaa 360
atagacctaa ccactatagt taaacctgag gctccttttg acctgagtgt cgtctatcgg 420
gaaggagcca atgactttgt ggtgacattt aatacatcac acttgcaaaa gaagtatgta 480
aaagttttaa tgcacgatgt agcttaccgc caggaaaagg atgaaaacaa atggacgcat 540
gtgaatttat ccagcacaaa gctgacactc ctgcagagaa agctccaacc ggcagcaatg 600
tatgagatta aagttcgatc catccctgat cactatttta aaggcttctg gagtgaatgg 660
agtccaagtt attacttcag aactccagag atcaataata gctcagggga gatggatcct 720
atcttactaa ccatcagcat tttgagtttt ttctctgtcg ctctgttggt catcttggcc 780
tgtgtgttat ggaaaaaaag gattaagcct atcgtatggc ccagtctccc cgatcataag 840
aagactctgg aacatctttg taagaaacca agaaaaaatt taaatgtgag tttcaatcct 900
gaaagtttcc tggactgcca gattcatagg gtggatgaca ttcaagctag agatgaagtg 960
gaaggttttc tgcaagatac gtttcctcag caactagaag aatctgagaa gcagaggctt 1020
ggaggggatg tgcagagccc caactgccca tctgaggatg tagtcatcac tccagaaagc 1080
tttggaagag attcatccct cacatgcctg gctgggaatg tcagtgcatg tgacgcccct 1140
attctctcct cttccaggtc cctagactgc agggagagtg gcaagaatgg gcctcatgtg 1200
taccaggacc tcctgcttag ccttgggact acaaacagca cgctgccccc tccattttct 1260
ctccaatctg gaatcctgac attgaaccca gttgctcagg gtcagcccat tcttacttcc 1320
ctgggatcaa atcaagaaga agcatatgtc accatgtcca gcttctacca aaaccagtga 1380

Claims (10)

1. An anti-Thymic Stromal Lymphopoietin (TSLP) antibody comprising a heavy chain variable region comprising the amino acid sequences of HCDR1, HCDR2, and HCDR3 and a light chain variable region comprising the amino acid sequences of LCDR1, LCDR2, and LCDR3, wherein
The amino acid sequence of the HCDR1 is shown as SEQ ID NO.1, the amino acid sequence of the HCDR2 is shown as SEQ ID NO. 2, the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 3, the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 4, the amino acid sequence of the LCDR2 is shown as SEQ ID NO. 5, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 6; or,
the amino acid sequence of the HCDR1 is shown as SEQ ID NO.1, the amino acid sequence of the HCDR2 is shown as SEQ ID NO.7, the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 3, the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 4, the amino acid sequence of the LCDR2 is shown as SEQ ID NO. 5, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 6; or,
the amino acid sequence of the HCDR1 is shown as SEQ ID NO. 8, the amino acid sequence of the HCDR2 is shown as SEQ ID NO. 9, the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 10, the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 11, the amino acid sequence of the LCDR2 is shown as SEQ ID NO.12, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 6; or,
the amino acid sequence of the HCDR1 is shown as SEQ ID NO. 8, the amino acid sequence of the HCDR2 is shown as SEQ ID NO. 9, the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 10, the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 4, the amino acid sequence of the LCDR2 is shown as SEQ ID NO. 5, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 6;
wherein the HCDR and LCDR amino acid sequences are defined according to Kabat.
2. The antibody of claim 1, wherein
The amino acid sequence of the heavy chain variable region of the antibody has at least 90% homology with any one of SEQ ID NOs 21, 23, 25, 27, 29 and 31; and/or
The amino acid sequence of the variable region of the light chain of the antibody has at least 90% homology with any one of SEQ ID NOs 33, 35 and 37;
preferably, the amino acid sequence of the heavy chain variable region of the antibody is as shown in any one of SEQ ID NOs: 21, 23, 25, 27, 29 and 31; and/or
The amino acid sequence of the variable region of the light chain of the antibody is shown in any one of SEQ ID NO 33, 35 and 37;
more preferably, the amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO. 21, and the amino acid sequence of the light chain variable region of the antibody is shown as SEQ ID NO. 33; or
The amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO. 23, and the amino acid sequence of the light chain variable region of the antibody is shown as SEQ ID NO. 33; or
The amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO. 25, and the amino acid sequence of the light chain variable region of the antibody is shown as SEQ ID NO. 35; or
The amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO. 27, and the amino acid sequence of the light chain variable region of the antibody is shown as SEQ ID NO. 37; or
The amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO. 29, and the amino acid sequence of the light chain variable region of the antibody is shown as SEQ ID NO. 37; or
The amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO. 31, and the amino acid sequence of the light chain variable region of the antibody is shown as SEQ ID NO. 37;
optionally, the antibody comprises a heavy chain variable region as set forth in SEQ ID NO 27, a light chain variable region as set forth in SEQ ID NO 37, a heavy chain constant region as set forth in SEQ ID NO 39, and a light chain constant region as set forth in SEQ ID NO 41; or
The antibody comprises a heavy chain variable region as shown in SEQ ID NO. 29, a light chain variable region as shown in SEQ ID NO. 37, a heavy chain constant region as shown in SEQ ID NO. 39, and a light chain constant region as shown in SEQ ID NO. 41; or alternatively
The antibody comprises a heavy chain variable region as shown in SEQ ID NO. 31, a light chain variable region as shown in SEQ ID NO. 37, a heavy chain constant region as shown in SEQ ID NO. 39, and a light chain constant region as shown in SEQ ID NO. 41.
3. The antibody of any one of claims 1-2, wherein
The antibody is a whole antibody, a Fab fragment, a Fab 'fragment, or a F (ab') 2 A fragment, Fv fragment, or single chain Fv fragment (scFv), preferably, the antibody is a fully human antibody; and/or
The antibody is a humanized antibody; and/or
The antibody is a monoclonal antibody; and/or
The antibody comprises a framework region, preferably the framework region is humanized; and/or
The antibody comprises a heavy chain constant region selected from the group consisting of an IgG1 subtype, an IgG2 subtype, or an IgG4 subtype; preferably, the heavy chain constant region comprises a heavy chain constant region of the human IgG2 subtype; and/or
The antibody comprises a light chain constant region selected from the kappa subtype or the lambda subtype; and/or
The antibody is capable of binding to TSLP; and/or
The antibody is capable of inhibiting binding of TSLP to the TSLP receptor; and/or
The antibodies are capable of blocking TSLP-mediated activity.
4. A nucleic acid molecule encoding the heavy chain variable region and/or the light chain variable region in the antibody of any one of claims 1-3.
5. An expression vector comprising the nucleic acid molecule of claim 4.
6. A host cell comprising the nucleic acid molecule of claim 4 or the expression vector of claim 5.
7. A composition comprising the antibody of any one of claims 1-3 and a pharmaceutically acceptable excipient, diluent, or carrier.
8. A kit for detecting TSLP in a sample from a subject comprising the antibody of any one of claims 1-3;
preferably, the subject is a mammal; more preferably, the subject is a human.
9. Use of the antibody of any one of claims 1-3, the nucleic acid molecule of claim 4, the expression vector of claim 5, the host cell of claim 6, or the composition of claim 7 in the preparation of a kit for the diagnostic, therapeutic, tracking, and/or prognostic check of a TSLP-mediated disease;
optionally, the TSLP-mediated disease is an immune system-related disease or tumor;
preferably, the immune system-related disease is selected from autoimmune diseases as well as inflammatory diseases, such as asthma, scleroderma, systemic lupus erythematosus, rheumatoid arthritis, Churg-Strauss syndrome, Wegener's granulomatosis, goodpasture's syndrome, hypersensitivity pneumonitis, atopic dermatitis, rhinitis, ankylosing spondylitis, rheumatic fever, fibromyalgia, psoriatic arthritis, chronic nephritis, sjogren's syndrome, as well as multiple sclerosis, allergic dermatitis, allergic conjunctivitis, atopic dermatitis fibrosis, inflammatory bowel disease, and the like;
preferably, the tumor is selected from: hodgkin's lymphoma, breast cancer, pancreatic cancer, melanoma, B-cell acute lymphocytic leukemia, cervical cancer, cutaneous T-cell lymphoma, gastric cancer and lung cancer.
10. Use of the antibody of any one of claims 1-3, the nucleic acid molecule of claim 4, the expression vector of claim 5, the host cell of claim 6, or the composition of claim 7 in the manufacture of a medicament for treating and/or preventing a TSLP-mediated disease;
optionally, the TSLP-mediated disease is an immune system-related disease or tumor;
preferably, the immune system-related disease is selected from autoimmune diseases as well as inflammatory diseases, such as asthma, scleroderma, systemic lupus erythematosus, rheumatoid arthritis, Churg-Strauss syndrome, Wegener's granulomatosis, goodpasture's syndrome, hypersensitivity pneumonitis, atopic dermatitis, rhinitis, ankylosing spondylitis, rheumatic fever, fibromyalgia, psoriatic arthritis, chronic nephritis, sjogren's syndrome, as well as multiple sclerosis, allergic dermatitis, allergic conjunctivitis, atopic dermatitis fibrosis, inflammatory bowel disease, and the like;
preferably, the tumor is selected from: hodgkin's lymphoma, breast cancer, pancreatic cancer, melanoma, B-cell acute lymphocytic leukemia, cervical cancer, cutaneous T-cell lymphoma, gastric cancer and lung cancer.
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