CN119798431A - Anti-TSLP antibodies and their applications - Google Patents

Abstract

The invention provides an anti-TSLP antibody and application thereof. The anti-TSLP antibody of the invention can specifically bind to human/monkey TSLP protein with high affinity, and can block the binding of TSLP and its receptor TSLPR, thereby inhibiting downstream STAT5 signaling pathway induced by TSLP, and has the function of treating TSLP related diseases.

Description

Anti-TSLP antibodies and uses thereof

Technical Field

The invention relates to the field of biological medicine. In particular, the invention relates to an anti-TSLP antibody and uses thereof.

Background

Thymic stromal lymphopoietin (Thymic stromal lymphopoietin, TSLP) is a potential target for allergic diseases, tumors, immunological diseases. TSLP promotes B cell differentiation and also co-stimulates thymocytes and mature T cells. TSLP binds to a specific heterodimeric receptor on human CD11c⁺ Dendritic Cells (DCs). The receptor heterodimer consists of a heterodimer of a common gamma-like receptor chain (TSLP receptor; TSLPR) and an IL7Rα chain. Ligands that bind to the receptor induce DC secretion of chemokines that attract Th2, TARC (thymus and activation regulated chemokines) and MDC (macrophage derived chemokines). TSLP signaling also leads to activation of STAT5 and STAT3 transcription factors and initiates expression of downstream genes.

TSLP promotes the differentiation of Th0 cells into Th2 type cells by activating myeloid dendritic cells (mDC), and generates a large amount of Th2 type cytokines such as IL-4, IL-13, IL-5, and TNF-alpha, thereby causing allergic inflammation. TSLP also acts on CD4⁺ T cells and mast cells, enhancing Th 2-type inflammatory responses. Acute and chronic atopic dermatitis patients have been reported to overexpress TSLP at skin wounds, indicating that TSLP expression is associated with allergic inflammation in vivo. In addition to skin keratinocytes, high levels of TSLP expression have also been found in bronchial epithelial cells, smooth muscle and lung fibroblasts, supporting the possible role of TSLP in respiratory allergy indications.

With the deep research mechanism of TSLP/TSLPR, the immunoregulatory function of TSLP antibodies is becoming clear, and the use of TSLP antibodies for treating allergic diseases, neoplastic diseases and immune diseases has been verified. The TSLP is used as a target point, so that the development of antibody medicaments not only has theoretical feasibility, but also has practical significance. Based on current theory and basic research results, it is expected that TSLP antibodies will have application prospects for a variety of indications.

Disclosure of Invention

The invention aims to provide an anti-TSLP antibody and application thereof.

In a first aspect of the invention there is provided an anti-TSLP antibody comprising a heavy chain and a light chain, wherein the heavy chain variable region has a Complementarity Determining Region (CDR) selected from the group consisting of:

1) VH-CDR1 shown in SEQ ID NO 72 or 73,

VH-CDR2 as shown in SEQ ID NO 74, 75 or 76, and

The VH-CDR3 shown in any one of SEQ ID NOs 77 to 87;

2) VH-CDR1 shown in SEQ ID No. 91, VH-CDR2 shown in SEQ ID No. 92, and VH-CDR3 shown in SEQ ID No. 93;

And, the light chain variable region has a Complementarity Determining Region (CDR) selected from the group consisting of:

1) VL-CDR1 shown in SEQ ID NO. 88, VL-CDR2 shown in SEQ ID NO. 89, and VL-CDR3 shown in SEQ ID NO. 90, and

2) VL-CDR1 shown in SEQ ID NO. 94, VL-CDR2 shown in SEQ ID NO. 95, and VL-CDR3 shown in SEQ ID NO. 96.

In another preferred embodiment, the heavy chain variable region has a Complementarity Determining Region (CDR) selected from the group consisting of:

1) VL-CDR1 shown in SEQ ID NO. 72, VL-CDR2 shown in SEQ ID NO. 74, and VL-CDR3 shown in SEQ ID NO. 77;

2) VL-CDR1 shown in SEQ ID NO. 72, VL-CDR2 shown in SEQ ID NO. 75, and VL-CDR3 shown in SEQ ID NO. 77;

3) VL-CDR1 shown in SEQ ID NO. 72, VL-CDR2 shown in SEQ ID NO. 76, and VL-CDR3 shown in SEQ ID NO. 77;

4) VL-CDR1 shown in SEQ ID NO. 73, VL-CDR2 shown in SEQ ID NO. 76, and VL-CDR3 shown in SEQ ID NO. 77;

5) VL-CDR1 shown in SEQ ID NO. 72, VL-CDR2 shown in SEQ ID NO. 76, and VL-CDR3 shown in SEQ ID NO. 78;

6) VL-CDR1 shown in SEQ ID NO. 72, VL-CDR2 shown in SEQ ID NO. 76, and VL-CDR3 shown in SEQ ID NO. 79;

7) VL-CDR1 shown in SEQ ID NO. 72, VL-CDR2 shown in SEQ ID NO. 76, and VL-CDR3 shown in SEQ ID NO. 80;

8) VL-CDR1 shown in SEQ ID NO. 72, VL-CDR2 shown in SEQ ID NO. 76, and VL-CDR3 shown in SEQ ID NO. 81;

9) VL-CDR1 shown in SEQ ID NO. 72, VL-CDR2 shown in SEQ ID NO. 76, and VL-CDR3 shown in SEQ ID NO. 82;

10 VL-CDR1 shown in SEQ ID NO. 72, VL-CDR2 shown in SEQ ID NO. 76, and VL-CDR3 shown in SEQ ID NO. 83;

11 VL-CDR1 shown in SEQ ID NO. 72, VL-CDR2 shown in SEQ ID NO. 76, and VL-CDR3 shown in SEQ ID NO. 84;

12 VL-CDR1 shown in SEQ ID NO. 72, VL-CDR2 shown in SEQ ID NO. 76, and VL-CDR3 shown in SEQ ID NO. 85;

13 VL-CDR1 shown in SEQ ID NO. 72, VL-CDR2 shown in SEQ ID NO. 76, and VL-CDR3 shown in SEQ ID NO. 86, and

14 VL-CDR1 shown in SEQ ID NO. 72, VL-CDR2 shown in SEQ ID NO. 76, and VL-CDR3 shown in SEQ ID NO. 87;

And the light chain variable region has a Complementarity Determining Region (CDR) of VL-CDR1 shown in SEQ ID NO. 88, VL-CDR2 shown in SEQ ID NO. 89, and VL-CDR3 shown in SEQ ID NO. 90.

In another preferred embodiment, the heavy chain variable region has a Complementarity Determining Region (CDR) of VH-CDR1 shown in SEQ ID NO. 91, VH-CDR2 shown in SEQ ID NO. 92, and VH-CDR3 shown in SEQ ID NO. 93;

and the light chain variable region has a Complementarity Determining Region (CDR) of VL-CDR1 shown in SEQ ID NO. 94, VL-CDR2 shown in SEQ ID NO. 95, and VL-CDR3 shown in SEQ ID NO. 96.

Any one of the amino acid sequences described above also includes derivative sequences that are optionally added, deleted, modified and/or substituted for at least one amino acid and that allow a derivative antibody comprising the heavy and light chains of the derivative CDR sequences to retain TSLP binding affinity.

In another preferred embodiment, the antibody specifically binds to a TSLP protein.

In another preferred embodiment, the TSLP protein is a human TSLP protein, a cynomolgus TSLP protein, or a murine TSLP protein.

In another preferred embodiment, the amino acid sequence of the human TSLP protein is set forth in SEQ ID NO. 1.

In another preferred embodiment, the cynomolgus TSLP protein has the amino acid sequence shown in SEQ ID NO. 2.

In another preferred embodiment, the antibody blocks binding of TSLP to the TSLP receptor.

In another preferred embodiment, the TSLP receptor is TSLPR or TSLPR-IL7Rα heterodimer.

In another preferred embodiment, said blocking means that the binding rate of said TSLP receptor to TSLP is reduced by 50%, preferably by 70%, more preferably by 90%.

In another preferred embodiment, the heavy chain variable region of the antibody further comprises a framework region of human origin, and/or the light chain variable region of the antibody further comprises a framework region of human origin.

In another preferred embodiment, the heavy chain variable region of the antibody further comprises a framework region of murine origin, and/or the light chain variable region of the antibody further comprises a framework region of murine origin.

In another preferred embodiment, the amino acid sequence of the heavy chain variable region has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs 6, 8, 12-20, 27-60, 64-67 of the sequence Listing.

In another preferred embodiment, the heavy chain variable region has the amino acid sequence set forth in any one of SEQ ID NOs 6, 8, 12-20, 27-60, 64-67.

In another preferred embodiment, the heavy chain variable region has a mutation with respect to the sequence set forth in SEQ ID NO.19 selected from the group consisting of:

V2Q, K23Q, K23Q, K24Q, K25Q, K25Q, K26Q, K26Q, K27Q, K28Q, K29Q, K30Q, K30Q, K32Q, K74Q, K and Q, K74 39330D and T74Q, K75Q, K T and T74E and S75Q, K77Q, K100Q, K100Q, K101Q, K101Q, K102Q, K102Q, K103Q, K105Q or a combination thereof.

In another preferred embodiment, the amino acid sequence of the light chain variable region has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or sequence identity to the amino acid sequence as set forth in any one of SEQ ID NOs 7, 9, 22-26, 68-71 of the sequence Listing.

In another preferred embodiment, the light chain variable region has the amino acid sequence set forth in any one of SEQ ID NOs 7, 9, 22-26, 68-71.

In another preferred embodiment, the heavy chain variable region of the antibody comprises the amino acid sequence set forth in any one of SEQ ID NOS.6, 8, 12-20, 27-60, 64-67, and the light chain variable region of the antibody comprises the amino acid sequence set forth in any one of SEQ ID NOS.7, 9, 22-26, 68-71.

In another preferred embodiment, the antibody has:

(1) A heavy chain variable region as shown in SEQ ID NO. 19 and a light chain variable region as shown in SEQ ID NO. 23;

(2) A heavy chain variable region as shown in SEQ ID NO. 40 and a light chain variable region as shown in SEQ ID NO. 25;

(3) A heavy chain variable region as set forth in SEQ ID NO. 46 and a light chain variable region as set forth in SEQ ID NO. 25;

(4) A heavy chain variable region as set forth in SEQ ID NO. 64 and a light chain variable region as set forth in SEQ ID NO. 68;

(5) A heavy chain variable region as set forth in SEQ ID NO. 64 and a light chain variable region as set forth in SEQ ID NO. 69;

(6) A heavy chain variable region as set forth in SEQ ID NO. 64 and a light chain variable region as set forth in SEQ ID NO. 70;

(7) A heavy chain variable region as shown in SEQ ID NO. 64 and a light chain variable region as shown in SEQ ID NO. 71;

(8) A heavy chain variable region as set forth in SEQ ID NO. 65 and a light chain variable region as set forth in SEQ ID NO. 68;

(9) A heavy chain variable region as shown in SEQ ID NO. 65 and a light chain variable region as shown in SEQ ID NO. 69;

(10) A heavy chain variable region as set forth in SEQ ID NO. 65 and a light chain variable region as set forth in SEQ ID NO. 70;

(11) A heavy chain variable region as shown in SEQ ID NO. 65 and a light chain variable region as shown in SEQ ID NO. 71;

(12) A heavy chain variable region as set forth in SEQ ID NO. 66 and a light chain variable region as set forth in SEQ ID NO. 68;

(13) A heavy chain variable region as set forth in SEQ ID NO. 66 and a light chain variable region as set forth in SEQ ID NO. 69;

(14) A heavy chain variable region as set forth in SEQ ID NO. 66 and a light chain variable region as set forth in SEQ ID NO. 70;

(15) A heavy chain variable region as set forth in SEQ ID NO. 66 and a light chain variable region as set forth in SEQ ID NO. 71;

(16) A heavy chain variable region as set forth in SEQ ID NO. 67 and a light chain variable region as set forth in SEQ ID NO. 68;

(17) A heavy chain variable region as shown in SEQ ID NO. 67 and a light chain variable region as shown in SEQ ID NO. 69;

(18) A heavy chain variable region as set forth in SEQ ID NO. 67 and a light chain variable region as set forth in SEQ ID NO. 70;

(19) A heavy chain variable region as shown in SEQ ID NO. 67 and a light chain variable region as shown in SEQ ID NO. 71;

(20) A heavy chain variable region as shown in SEQ ID NO. 8 and a light chain variable region as shown in SEQ ID NO. 9, or

(21) A heavy chain variable region as shown in SEQ ID NO. 6 and a light chain variable region as shown in SEQ ID NO. 7.

In another preferred embodiment, the antibody is selected from the group consisting of a murine antibody, a chimeric antibody, a humanized antibody, a fully human antibody, or a combination thereof.

In another preferred embodiment, the light chain and/or heavy chain of the antibody further comprises a constant region.

In another preferred embodiment, the constant region is of human or murine origin.

In another preferred embodiment, the constant region of the heavy chain is derived from an IgG, preferably from a human IgG, more preferably a human IgG4 heavy chain constant region.

In another preferred embodiment, the constant region of the light chain is derived from an IgG, preferably from a human IgG, more preferably a human IgG4 kappa chain.

In another preferred embodiment, the constant region sequence of the heavy chain is shown in SEQ ID NO. 10.

In another preferred embodiment, the constant region sequence of the light chain is shown in SEQ ID NO. 11.

In a second aspect of the present invention, there is provided a recombinant protein comprising:

(i) An antibody according to the first aspect of the invention, and

(Ii) Optionally a tag sequence to assist expression and/or purification.

In another preferred embodiment, the tag sequence comprises a 6His tag, a GGGS sequence, a FLAG tag.

In another preferred embodiment, the recombinant protein (or polypeptide) comprises a fusion protein.

In another preferred embodiment, the recombinant protein is a monomer, dimer, or multimer.

In another preferred embodiment, the recombinant protein is specifically anti-TSLP.

In another preferred embodiment, the recombinant protein is a fusion protein.

In another preferred embodiment, the fusion protein is a monospecific antibody (i.e., a monospecific antibody against TSLP), a bispecific antibody, or a multispecific antibody (e.g., a trispecific antibody).

In another preferred embodiment, the bispecific or multispecific antibody not only is anti-TSLP, but also specifically binds to an additional target antigen (e.g., other tumor antigen).

In a third aspect of the invention there is provided a Chimeric Antigen Receptor (CAR) construct, the antigen binding region of which comprises a single chain variable fragment (scFv) that specifically binds to TSLP, and which scFv has the heavy chain variable region and the light chain variable region of an antibody according to the first aspect of the invention.

In a fourth aspect of the invention, there is provided a recombinant immune cell expressing an exogenous CAR construct according to the third aspect of the invention.

In another preferred embodiment, the immune cells are selected from the group consisting of NK cells, T cells.

In another preferred embodiment, the immune cells are derived from a human or non-human mammal (e.g., a mouse).

In a fifth aspect of the invention there is provided the use of an active ingredient selected from the group consisting of an antibody according to the first aspect of the invention, a recombinant protein according to the second aspect of the invention, an immune cell according to the fourth aspect of the invention or a combination thereof, wherein the active ingredient is used to:

(a) Preparing a diagnostic reagent, a test plate or a kit, and/or

(B) Preparing a medicament for preventing and/or treating diseases related to TSLP expression or dysfunction.

In another preferred embodiment, the reagent is used to detect TSLP.

In another preferred embodiment, the agent, assay plate or kit is used to detect a disease associated with TSLP expression or dysfunction.

In another preferred embodiment, the agent is for the prevention and/or treatment of allergic diseases, tumors or cancers, or immunological diseases.

In another preferred embodiment, the agent is used for preventing and/or treating a disease selected from the group consisting of asthma (e.g., moderately severe asthma), atopic dermatitis, urticaria, chronic Obstructive Pulmonary Disease (COPD), chronic rhinitis nasal polyp, eosinophilic esophagitis, or a combination thereof.

In a sixth aspect of the present invention, there is provided a pharmaceutical composition comprising:

(i) An active ingredient selected from the group consisting of an antibody according to the first aspect of the invention, a recombinant protein according to the second aspect of the invention, an immune cell according to the fourth aspect of the invention, or a combination thereof, and

(Ii) A pharmaceutically acceptable carrier.

In another preferred embodiment, the pharmaceutical composition is a liquid formulation.

In another preferred embodiment, the pharmaceutical composition is an injection.

In another preferred embodiment, the pharmaceutical composition comprises 0.01-99.99% of the antibody according to the first aspect of the invention, the recombinant protein according to the second aspect of the invention, the immune cell according to the fourth aspect of the invention, or a combination thereof, and 0.01-99.99% of a pharmaceutically acceptable carrier, wherein the percentages are mass percentages of the pharmaceutical composition.

In another preferred embodiment, the pharmaceutical composition is used for preventing and/or treating allergic diseases, tumors or cancers, or immunological diseases.

In a seventh aspect of the invention, there is provided a polynucleotide encoding a polypeptide selected from the group consisting of:

(1) An antibody according to the first aspect of the invention;

(2) Recombinant proteins according to the second aspect of the invention, or

(3) A Chimeric Antigen Receptor (CAR) construct according to the third aspect of the invention.

In an eighth aspect of the invention there is provided a vector comprising a polynucleotide according to the seventh aspect of the invention.

In another preferred embodiment, the vector comprises a bacterial plasmid, a phage, a yeast plasmid, a plant cell virus, a mammalian cell virus.

In another preferred embodiment, the vector is a plasmid, adenovirus, or retrovirus, or other vector.

In a ninth aspect of the invention there is provided an engineered host cell comprising a vector or genome according to the eighth aspect of the invention incorporating a polynucleotide according to the seventh aspect of the invention.

In a tenth aspect of the present invention, there is provided a method of detecting TSLP in a sample, the method comprising the steps of:

(1) Contacting the sample with an antibody according to the first aspect of the invention;

(2) Detecting whether an antigen-antibody complex is formed, wherein the formation of a complex indicates the presence of TSLP in the sample.

In another preferred embodiment, the detection is for non-therapeutic non-diagnostic purposes in vitro.

In an eleventh aspect of the invention, there is provided a composition for in vitro detection of TSLP in a sample comprising as an active ingredient an antibody according to the first aspect of the invention, a recombinant protein according to the second aspect of the invention, an immune cell according to the fourth aspect of the invention or a combination thereof.

In a twelfth aspect of the invention there is provided a test plate comprising a substrate (support plate) and a test strip comprising an antibody according to the first aspect of the invention, a recombinant protein according to the second aspect of the invention, an immune cell according to the fourth aspect of the invention or a combination thereof.

In a thirteenth aspect of the present invention, there is provided a kit comprising:

(1) A first container comprising an antibody according to the first aspect of the invention, and/or

(2) A second container comprising a second antibody against the antibody of the first aspect of the invention;

or alternatively

The kit contains a detection plate according to the twelfth aspect of the invention.

In a fourteenth aspect of the present invention, there is provided a method for producing a recombinant polypeptide, the method comprising:

(a) Culturing a host cell according to the ninth aspect of the invention under conditions suitable for expression;

(b) Isolating the recombinant polypeptide from the culture, said recombinant polypeptide being an antibody according to the first aspect of the invention or a recombinant protein according to the second aspect of the invention.

In a fifteenth aspect of the invention there is provided the use of an antibody according to the first aspect of the invention, or a recombinant protein according to the second aspect of the invention, or an immune cell according to the fourth aspect of the invention and/or a pharmaceutical composition according to the sixth aspect of the invention in combination with a medicament for the manufacture of a medicament for the treatment of a disease associated with TSLP expression or dysfunction.

In another preferred embodiment, the abnormal expression of TSLP is TSLP overexpression.

In another preferred embodiment, the overexpression is the ratio of the expression level (F1) of TSLP to the expression level (F0) under physiological conditions (i.e., F1/F0). Gtoreq.1.5, preferably. Gtoreq.2, more preferably. Gtoreq.2.5.

In another preferred embodiment, the medicament is for the prevention and/or treatment of allergic diseases, tumors or cancers, or immunological diseases.

In another preferred embodiment, the medicament is for the prevention and/or treatment of a disease selected from the group consisting of asthma (e.g. moderately severe asthma), atopic dermatitis, urticaria, chronic Obstructive Pulmonary Disease (COPD), chronic rhinitis nasal polyp, eosinophilic esophagitis, or a combination thereof.

In a sixteenth aspect of the invention there is provided a method of treating a disease associated with abnormal TSLP expression or function comprising administering to a subject in need thereof an effective amount of an antibody according to the first aspect of the invention, or a recombinant protein according to the second aspect of the invention, or an immune cell according to the fourth aspect of the invention, or a pharmaceutical composition according to the sixth aspect of the invention, or a combination thereof.

In another preferred embodiment, the disease associated with abnormal TSLP expression or function is an allergic disease, a tumor or cancer, or an immunological disease.

In another preferred embodiment, the disease associated with abnormal TSLP expression or function is a disease selected from the group consisting of asthma (e.g., moderately severe asthma), atopic dermatitis, urticaria, chronic Obstructive Pulmonary Disease (COPD), chronic rhinitis nasal polyp, eosinophilic esophagitis, or a combination thereof.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

The following drawings are illustrative of particular embodiments of the invention and are not intended to limit the scope of the invention as defined by the claims.

FIG. 1 shows ELISA detection results of binding of humanized antibodies to human TSLP.

Figure 2 shows the ELISA assay results of binding of humanized antibodies to cynomolgus TSLP.

Figure 3 shows the results of an ELISA assay for binding of affinity matured antibodies to human TSLP.

Figure 4 shows the ELISA assay results for binding of affinity matured antibodies to cynomolgus TSLP.

FIG. 5 shows ELISA assay results for receptor-ligand (human TSLP-human TSLPR) binding blocking ability of humanized antibodies.

Figure 6 shows the results of ELISA assays for receptor-ligand (human TSLP-human TSLPR) binding blocking capacity of affinity matured antibodies.

Figure 7 shows the results of ELISA assays for receptor-ligand (cynomolgus TSLP-cynomolgus TSLPR) binding blocking capacity of humanized antibodies.

Figure 8 shows the results of ELISA assays for receptor-ligand (cynomolgus TSLP-cynomolgus TSLPR) binding blocking capacity of affinity matured antibodies.

FIG. 9 shows the results of a flow-through assay for the blocking ability of humanized antibodies to human TSLP binding to cell surface receptors.

FIG. 10 shows the results of a flow-through assay for the blocking ability of affinity matured antibodies to human TSLP binding to cell surface receptors.

FIG. 11 shows the effect of humanized antibodies on BaF3-TSLPR/IL7Rα cell proliferation.

FIG. 12 shows the effect of affinity matured antibodies on BaF3-TSLPR/IL7Rα cell proliferation.

FIG. 13 shows the results of reporter gene assay detection of humanized antibody function.

FIG. 14 shows the results of reporter gene assay detection of affinity matured antibody function.

Detailed Description

The inventors have conducted extensive and intensive studies to obtain a class of anti-TSLP monoclonal antibodies. The invention also provides humanized antibodies and affinity matured antibodies to TSLP. The anti-TSLP antibody of the invention can specifically bind with high affinity to human/cynomolgus monkey TSLP protein, can block the binding of TSLP and cell surface receptor TSLPR, inhibit downstream STAT5 signal path induced by TSLP, and achieve the function of treating TSLP related diseases. On this basis, the present invention has been completed.

Terminology

In order that the invention may be more readily understood, certain technical and scientific terms are defined below. Unless defined otherwise herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Before describing the present invention, it is to be understood that this invention is not limited to the particular methodology and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, as the scope of the present invention will be limited only by the appended claims.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, when used in reference to a specifically recited value, the term "about" means that the value can vary no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

The three-letter and one-letter codes for amino acids used in the present invention are as described in J.biol. Chem,243, p3558 (1968).

As used herein, the term "treatment" refers to the administration of an internal or external therapeutic agent, including antibodies to TSLP of the invention and compositions thereof, to a patient having one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Typically, the patient is administered an amount of the therapeutic agent (therapeutically effective amount) effective to alleviate one or more symptoms of the disease.

As used herein, the term "optional" or "optionally" means that the subsequently described event or circumstance may, but need not, occur.

"Sequence identity" as used herein refers to the degree of identity between two nucleic acid or two amino acid sequences when optimally aligned and compared with appropriate substitutions, insertions, or deletions of mutations. The sequence identity between the sequences described in the present invention and sequences with which it has identity may be at least 85%, 90% or 95%, preferably at least 95%. Non-limiting examples include 85%,86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98%,99%,100%.

TSLP

Thymic stromal lymphopoietin (Thymic stromal lymphopoietin, TSLP) is a short-chain cytokine with a four-helix bundle folding structure, similar to IL-7, belonging to the IL-2 cytokine family of members. It is named after Firend et al, which was first isolated from the culture supernatant of thymic stromal cells. The TSLP protein is a short chain type I cytokine consisting of A, B, C, D helical bundles. Human TSLP exists in 2 isoforms, isoform I, a 159 amino acid protein and isoform II, a 60 amino acid protein. The human TSLP gene is located on chromosome 5q22.1, adjacent to the atopic cytokine gene cluster 5q31, which encodes mainly the Th2 cytokines IL-4, IL-5, IL-9 and IL-13. The highest levels of TSLP are found in lung and skin epithelial cells, and TSLP can also be produced by fibroblasts, ASMC (airway smooth muscle cells), endothelial cells, mast cells, macrophages/monocytes, granulocytes and DCs.

Antibodies to

As used herein, the term "antibody" or "immunoglobulin" is an iso-tetralin protein of about 150000 daltons, consisting of two identical light chains (L) and two identical heavy chains (H), having identical structural features. Each light chain is linked to the heavy chain by a covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end followed by a plurality of constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end, the constant region of the light chain being opposite the first constant region of the heavy chain and the variable region of the light chain being opposite the variable region of the heavy chain. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.

As used herein, the term "variable" means that certain portions of the variable regions in an antibody differ in sequence, which results in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three fragments in the light and heavy chain variable regions called Complementarity Determining Regions (CDRs) or hypervariable regions. The more conserved parts of the variable region are called Framework Regions (FR). The variable regions of the natural heavy and light chains each comprise four FR regions, which are generally in a β -sheet configuration, connected by three CDRs forming the connecting loops, which in some cases may form part of the β -sheet structure. The CDRs in each chain are held closely together by the FR regions and together with the CDRs of the other chain form the antigen binding site of the antibody (see Kabat et al, NIH publication No.91-3242, vol. I, pp. 647-669 (1991)). The constant regions are not directly involved in binding of the antibody to the antigen, but they exhibit different effector functions, such as participation in antibody-dependent cytotoxicity of the antibody.

The "light chain" of a vertebrate antibody (immunoglobulin) can be classified into one of two distinct classes (called kappa and lambda) depending on the amino acid sequence of its constant region. Immunoglobulins can be assigned to different classes based on the amino acid sequence of their heavy chain constant region. There are mainly class 5 immunoglobulins IgA, igD, igE, igG and IgM, some of which can be further divided into subclasses (isotypes) such as IgG1, igG2, igG3, igG4, igA and IgA2. The heavy chain constant regions corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. Subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population, i.e., the individual antibodies contained in the population are identical, except for a few naturally occurring mutations that may be present. Monoclonal antibodies are highly specific for a single antigenic site. Moreover, unlike conventional polyclonal antibody preparations (typically having different antibodies directed against different determinants), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by hybridoma culture and are not contaminated with other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring any particular method for producing the antibody.

In general, the antigen binding properties of antibodies can be described by 3 specific regions located in the heavy and light chain variable regions, called variable regions (CDRs), which are separated into 4 Framework Regions (FRs), the amino acid sequences of the 4 FRs being relatively conserved and not directly involved in the binding reaction. These CDRs form a loop structure, the β -sheets formed by the FR therebetween are spatially close to each other, and the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen binding site of the antibody. It is possible to determine which amino acids constitute the FR or CDR regions by comparing the amino acid sequences of the same type of antibody.

The term "antigen-binding fragment of an antibody" (or simply "antibody fragment") refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen. Fragments of full length antibodies have been shown to be useful for performing the antigen binding function of antibodies. Examples of binding fragments included in the term "antigen-binding fragment of an antibody" include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL and CH1 domains, (ii) F (ab') 2 fragments, divalent fragments comprising two Fab fragments linked by a disulfide bridge over the longer chain region, (iii) Fd fragments consisting of VH and CH1 domains, and (iv) Fv fragments consisting of VH and VL domains of a single arm of an antibody. Fv antibodies contain antibody heavy chain variable regions, light chain variable regions, but no constant regions, and have a minimal antibody fragment of the entire antigen binding site. Generally, fv antibodies also comprise a polypeptide linker between the VH and VL domains, and are capable of forming the structures required for antigen binding.

The invention includes not only intact monoclonal antibodies but also immunologically active antibody fragments or fusion proteins of antibodies with other sequences, such as Fab or (Fab')₂ fragments, antibody heavy chains, antibody light chains. Thus, the invention also includes fragments, derivatives and analogues of said antibodies.

The term "epitope" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. Epitopes typically comprise at least 3,4,5,6,7,8,9,10,11,12,13,14 or 15 contiguous or non-contiguous amino acids in a unique spatial conformation. Epitopes may be discrete on an antigen, three-dimensional spatial sites recognized by an antibody or antigen binding fragment of the invention.

The terms "specific binding," "selective binding," "selectively binding," and "specifically binding" refer to binding of an antibody to an epitope on a predetermined antigen. Typically, the antibody binds with an affinity (KD) of about less than 10^-7 M, e.g., about less than 10^-8M、10^-9 M or l0^-10 M or less. In one embodiment of the invention, the anti-TSLP antibodies of the present invention are capable of binding to TSLP molecules with an affinity (KD) of l0^-11M、l0^-12 M or less.

In the present invention, antibodies include murine, chimeric, humanized or fully human antibodies prepared by techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, including human and non-human portions, can be obtained by standard DNA recombination techniques, all of which are useful antibodies. Chimeric antibodies are a molecule in which different portions are derived from different animal species, e.g., chimeric antibodies having variable regions from murine monoclonal antibodies, and constant regions from human immunoglobulins (see, e.g., U.S. Pat. No. 4,816,567 and U.S. Pat. No. 4,816,397, incorporated herein by reference in their entirety). Humanized antibodies refer to antibody molecules derived from non-human species having one or more Complementarity Determining Regions (CDRs) derived from the non-human species and a framework region derived from a human immunoglobulin molecule (see U.S. Pat. No. 5,585,089, incorporated herein by reference in its entirety). These chimeric and humanized monoclonal antibodies can be prepared using DNA recombination techniques well known in the art.

In the present invention, antibodies may be monospecific, bispecific, trispecific, or more multispecific.

As used herein, the term "heavy chain variable region" is used interchangeably with "VH".

As used herein, the term "light chain variable region" is used interchangeably with "VL".

As used herein, the term "complementarity determining regions (complementarity determining region, CDRs)" refers to hypervariable regions within the variable domains of an antibody that contribute primarily to antigen binding. One of the most commonly used definitions of the CDRs is provided by Kabat E.A et al, (1991) Sequences of proteins of immunological interface. Furthermore, IMGT (Lefranc, 2003), chothia (Al-Lazikani, 1997) all provide CDR definition rules, which are well known to those skilled in the art.

In a preferred embodiment of the invention, the heavy chain of the antibody comprises a heavy chain variable region and a heavy chain constant region, which may be of murine or human origin, e.g. derived from human IgG4.

In a preferred embodiment of the invention, the light chain of the antibody comprises a light chain variable region and a light chain constant region, which may be murine or human in origin, e.g., derived from a human IgG4 kappa chain.

In the present invention, the antibodies of the invention also include conservative variants thereof, meaning that up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids are replaced by amino acids of similar or similar nature to the amino acid sequence of the antibodies of the invention to form a polypeptide. These conservatively variant polypeptides are preferably generated by amino acid substitutions according to Table A.

Table A

Anti-TSLP antibodies

As used herein, the terms "antibody of the invention", "anti-TSLP antibody of the invention" and "TSLP antibody of the invention" are used interchangeably and refer to an antibody of the invention that targets TSLP.

The function of the antibodies of the invention is determined by the antibody light and heavy chain variable region gene-specific gene sequences. The antibody of the present invention can specifically bind to TSLP, has high affinity, and can efficiently block the binding of TSLP to its receptor TSLPR and to the cell surface TSLPR/IL7R surface receptor, and can inhibit proliferation of target cells highly expressing TSLPR/IL7Rα and inhibit expression of STAT5 gene therein. Using the variable region genes or Complementarity Determining Region (CDR) genes of the antibodies of the invention, genetically engineered antibodies of different forms can be engineered and produced in any expression system that utilizes prokaryotic and eukaryotic cells.

The heavy chain variable region of the anti-TSLP antibodies of the invention has a Complementarity Determining Region (CDR) selected from the group consisting of:

1) VH-CDR1 shown in SEQ ID NO 72 or 73,

VH-CDR2 as shown in SEQ ID NO 74, 75 or 76, and

The VH-CDR3 shown in any one of SEQ ID NOs 77 to 87;

2) VH-CDR1 shown in SEQ ID No. 91, VH-CDR2 shown in SEQ ID No. 92, and VH-CDR3 shown in SEQ ID No. 93;

And, the light chain variable region of the antibody has Complementarity Determining Regions (CDRs) selected from the group consisting of:

1) VL-CDR1 shown in SEQ ID NO. 88, VL-CDR2 shown in SEQ ID NO. 89, and VL-CDR3 shown in SEQ ID NO. 90, and

2) VL-CDR1 shown in SEQ ID NO. 94, VL-CDR2 shown in SEQ ID NO. 95, and VL-CDR3 shown in SEQ ID NO. 96.

The amino acid sequence also includes sequences formed by adding, deleting, modifying and/or substituting at least one amino acid sequence, preferably amino acid sequences having homology or sequence identity of at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95%.

The light and/or heavy chain of an antibody of the invention, or the light and/or heavy chain variable region thereof, may also have (e.g., at the N-terminus) a signal peptide that contributes to the expression or secretion of the antibody. An alternative signal peptide is shown, for example, in SEQ ID NO.21 or 63.

Methods of determining Sequence homology or identity known to those of ordinary skill in the art include, but are not limited to, computer molecular biology (Computational Molecular Biology), lesk, a.m. editions, oxford university Press, new york, 1988, biological calculations, informatics and genome projects (Biocomputing: informatics and Genome Projects), smith, d.w. editions, academic Press, new york, 1993, computer analysis of Sequence data (Computer Analysis of Sequence Data), first part, griffin, a.m. and Griffin, h.g. editions, humana Press, new jersey, 1994, sequence analysis in molecular biology (Sequence ANALYSIS IN Molecular Biology), von Heinje, g., academic Press, 1987, and Sequence analysis primers (Sequence ANALYSIS PRIMER), gribskov, m. and Devereux, j.m. stock Press, about 1991 and Carillo, h.and Lipman, d., siam.applied 1073:1988. The preferred method of determining identity is to obtain the greatest match between the sequences tested. Methods for determining identity are compiled in publicly available computer programs. Preferred computer program methods for determining identity between two sequences include, but are not limited to, GCG package (Devereux, J. Et al, 1984), BLASTP, BLASTN, and FASTA (Altschul, S, F. Et al, 1990). BLASTX programs are available to the public from NCBI and other sources (BLAST handbook, altschul, S. Et al, NCBI NLM NIH Bethesda, md.20894; altschul, S. Et al, 1990). The well-known SMITH WATERMAN algorithm can also be used to determine identity.

Preferably, the antibodies described herein are one or more of full length antibodies, antigen-antibody binding domain protein fragments, bispecific antibodies, multispecific antibodies, single chain antibodies (SINGLE CHAIN antibody fragment, scFv), single domain antibodies (singledomain antibody, sdAb) and single domain antibodies (Signle-domain antibodies), and monoclonal or polyclonal antibodies made from the above antibodies. The monoclonal antibodies can be developed by a variety of routes and techniques, including hybridoma technology, phage display technology, single lymphocyte gene cloning technology, etc., and the main stream is to prepare monoclonal antibodies from wild-type or transgenic mice by hybridoma technology.

The antibody full-length protein is a conventional antibody full-length protein in the art, and comprises a heavy chain variable region, a light chain variable region, a heavy chain constant region and a light chain constant region. The heavy chain variable region and the light chain variable region of the protein, the human heavy chain constant region and the human light chain constant region form the full-length protein of the fully human antibody. Preferably, the antibody full-length protein is of the IgG1, igG2, igG3 or IgG4 type.

The antibody of the present invention may be a double-or single-chain antibody, and may be selected from animal-derived antibodies, chimeric antibodies, humanized antibodies, more preferably humanized antibodies, human-animal chimeric antibodies, and even more preferably fully humanized antibodies.

The antibody derivatives of the present invention may be single chain antibodies, and/or antibody fragments such as Fab, fab ', (Fab') 2 or other known antibody derivatives in the art, etc., as well as IgA, igD, igE, igG and any one or more of IgM antibodies or other subclasses of antibodies.

The single-chain antibody is a conventional single-chain antibody in the field, and comprises a heavy chain variable region, a light chain variable region and a short peptide of 15-20 amino acids.

Wherein the animal is preferably a mammal, such as a mouse.

The antibodies of the invention can be chimeric, humanized, CDR grafted and/or modified antibodies that target TSLP (e.g., human, cynomolgus, or murine TSLP).

In the above-described aspect of the present invention, the number of amino acids added, deleted, modified and/or substituted is preferably not more than 40%, more preferably not more than 35%, more preferably 1 to 33%, more preferably 5 to 30%, more preferably 10 to 25%, more preferably 15 to 20% of the total amino acids in the original amino acid sequence.

In the above aspect of the present invention, more preferably, the number of the added, deleted, modified and/or substituted amino acids may be 1 to 7, more preferably 1 to 5, still more preferably 1 to 3, still more preferably 1 to 2.

Recombinant proteins

The invention also provides a recombinant protein comprising one or more of the heavy chain CDR1 (VH-CDR 1), heavy chain CDR2 (VH-CDR 2) and heavy chain CDR3 (VH-CDR 3) of the antibody of the invention, and/or one or more of the light chain CDR1 (VL-CDR 1), light chain CDR2 (VL-CDR 2) and light chain CDR3 (VL-CDR 3) of the antibody of the invention.

Preferably, the recombinant protein further comprises an antibody heavy chain constant region and/or an antibody light chain constant region, wherein the antibody heavy chain constant region is conventional in the art, preferably a rat or human antibody heavy chain constant region, more preferably a human antibody heavy chain constant region. The antibody light chain constant region is conventional in the art, preferably a rat light chain antibody constant region or a human antibody light chain constant region, more preferably a human antibody light chain constant region.

In another preferred embodiment, the recombinant protein comprises an antibody of the invention.

The recombinant protein is a protein conventional in the art, preferably, it is one or more of an antibody full-length protein, an antigen-antibody binding domain protein fragment, a bispecific antibody, a multispecific antibody, a single chain antibody (SINGLE CHAIN antibody fragment, scFv), a single domain antibody (singledomain antibody, sdAb) and a single domain antibody (Signle-domain antibody), and a monoclonal antibody or polyclonal antibody produced by the above antibodies.

The single-chain antibody is a conventional single-chain antibody in the field, and comprises a heavy chain variable region, a light chain variable region and a short peptide of 15-20 amino acids.

The antigen-antibody binding domain protein fragment is a conventional antigen-antibody binding domain protein fragment in the art, which comprises the Fd segment of the light chain variable region, the light chain constant region and the heavy chain constant region. Preferably, the antigen-antibody binding domain protein fragments are Fab and F (ab') 2.

The single domain antibody is a conventional single domain antibody in the art, which comprises a heavy chain variable region and a heavy chain constant region.

The single region antibody is a conventional single region antibody in the art, which comprises only the heavy chain variable region.

Wherein, the preparation method of the recombinant protein is a preparation method conventional in the field. The preparation method is preferably obtained by isolation from an expression transformant in which the protein is expressed recombinantly or by artificially synthesizing the protein sequence. The preferred method for isolation from the recombinant expression transformant for expressing the protein comprises cloning the polynucleotide molecule encoding the protein and carrying point mutation into a recombinant vector, transforming the recombinant vector into the transformant to obtain a recombinant expression transformant, and culturing the recombinant expression transformant to obtain the recombinant protein.

Polynucleotide

The invention also provides a polynucleotide encoding an antibody or recombinant protein of the invention or Chimeric Antigen Receptor (CAR) construct of an antibody of the invention as described above.

The preparation method of the polynucleotide is a preparation method conventional in the art, and preferably comprises the step of obtaining a nucleic acid molecule encoding the above protein by a gene cloning technique or obtaining a nucleic acid molecule encoding the above protein by a method of artificial total sequence synthesis.

It is known to those skilled in the art that a nucleotide sequence encoding the amino acid sequence of the above protein may be appropriately introduced into a substitution, deletion, alteration, insertion or addition to provide a homolog of a polynucleotide. Homologs of the polynucleotides of the invention may be obtained by substitution, deletion or addition of one or more bases of the gene encoding the protein sequence within a range that retains antibody activity.

Carrier body

The invention also provides a recombinant expression vector comprising the polynucleotide.

Wherein the recombinant expression vector can be obtained by a method conventional in the art, namely, by connecting the polynucleotide molecule of the invention to various expression vectors. The expression vector is a variety of vectors conventional in the art, as long as it is capable of harboring the aforementioned polynucleotide molecule. The vector preferably includes various plasmids, cosmids, phage or viral vectors, and the like.

The invention also provides a recombinant expression transformant containing the recombinant expression vector.

The recombinant expression transformant is prepared by a conventional preparation method in the field, preferably by transforming the recombinant expression vector into a host cell. The host cell is a variety of host cells conventional in the art, so long as the recombinant expression vector can stably replicate itself and the polynucleotide carried can be expressed effectively. Preferably, the host cell is an E.coli TG1 or E.coli BL21 cell (expressing a single chain antibody or Fab antibody), or HEK293 or CHO cell (expressing a full length IgG antibody). The recombinant expression plasmid is transformed into a host cell, so that the preferred recombinant expression transformant of the invention can be obtained. Wherein the conversion process is conventional in the art, preferably chemical, heat shock or electrotransformation.

Preparation of antibodies

The sequence of the DNA molecule of the antibody or fragment thereof of the present invention can be obtained by a conventional technique such as amplification by PCR or screening of a genomic library. In addition, the coding sequences for the light and heavy chains may be fused together to form a single chain antibody.

Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.

Furthermore, the sequences concerned, in particular fragments of short length, can also be synthesized by artificial synthesis. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them.

At present, it is already possible to obtain the DNA sequences encoding the antibodies of the invention (or fragments or derivatives thereof) described, entirely by chemical synthesis. The DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors, for example) and cells known in the art. In addition, mutations can be introduced into the protein sequences of the invention by chemical synthesis.

The invention also relates to vectors comprising the above-described suitable DNA sequences and suitable promoter or control sequences. These vectors may be used to transform an appropriate host cell to enable expression of the protein.

The host cell may be a prokaryotic cell, or a lower eukaryotic cell, or a higher eukaryotic cell, such as a mammalian cell.

Typically, the transformed host cell is cultured under conditions suitable for expression of the antibodies of the invention. The antibodies of the invention are then purified by conventional isolation and purification means well known to those skilled in the art.

The resulting monoclonal antibodies can be identified by conventional means. For example, the binding specificity of a monoclonal antibody can be determined using immunoprecipitation or in vitro binding assays, such as Radioimmunoassays (RIA) or enzyme-linked immunosorbent assays (ELISA). The binding affinity of monoclonal antibodies can be determined, for example, by Scatchard analysis by Munson et al, anal. Biochem.,107:220 (1980).

The antibodies of the invention may be expressed intracellularly, or on the cell membrane, or secreted extracellularly. If desired, the recombinant proteins can be isolated and purified by various separation methods using their physical, chemical and other properties. Such methods are well known to those skilled in the art.

Application of

The invention also provides for the use of an antibody, recombinant protein, chimeric Antigen Receptor (CAR) construct and/or immune cell of the invention, e.g., for the preparation of a diagnostic formulation or for the preparation of a medicament.

Preferably, the medicament is a medicament for preventing and/or treating a disease associated with abnormal TSLP expression or function.

In the present invention, the disease associated with TSLP expression or dysfunction is a disease associated with TSLP expression or dysfunction that is conventional in the art. Preferably, the disease associated with abnormal TSLP expression or function is an allergic disease, a tumor, an immune disease, or a combination thereof. Preferably, the disease associated with abnormal TSLP expression or function is selected from the group consisting of asthma (e.g., moderately severe asthma), atopic dermatitis, urticaria, chronic Obstructive Pulmonary Disease (COPD), chronic rhinitis nasal polyp, eosinophilic esophagitis, or a combination thereof.

Detection application and kit

The antibodies of the invention may be used in detection applications, for example for detecting samples, thereby providing diagnostic information.

In the present invention, the samples (specimens) used include cells, tissue samples and biopsy specimens. The term "biopsy" as used herein shall include all kinds of biopsies known to a person skilled in the art. Thus biopsies used in the present invention may include, for example, resected samples of tumors, tissue samples prepared by endoscopic methods or puncture of organs or needle biopsies.

Samples for use in the present invention include fixed or preserved cell or tissue samples.

The invention also provides a kit comprising an antibody (or fragment thereof) of the invention, which in a preferred embodiment of the invention further comprises a container, instructions for use, buffers, etc. In a preferred embodiment, the antibody of the present invention may be immobilized on a detection plate.

Pharmaceutical composition

The invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition comprising an antibody or active fragment thereof or fusion protein thereof or corresponding immune cell as described above, and a pharmaceutically acceptable carrier.

The antibodies of the invention may also be expressed intracellularly from the nucleotide sequence for use in cell therapy.

The pharmaceutical composition is used for preventing and/or treating diseases related to TSLP expression or dysfunction.

The pharmaceutical composition of the present invention contains a safe and effective amount of the monoclonal antibody of the present invention as described above and a pharmaceutically acceptable carrier or excipient. The pharmaceutical formulation should be compatible with the mode of administration. The amount of active ingredient administered is a therapeutically effective amount. In addition, the polypeptides of the invention may also be used with other therapeutic agents.

In one embodiment of the invention, the polypeptides of the invention may be used in combination with other therapeutic agents for the treatment and/or prevention of diseases, for example therapeutic agents for the treatment and/or prevention of allergic diseases, tumors, or immunological diseases.

In the present invention, the pharmaceutical composition of the present invention preferably further comprises one or more pharmaceutically acceptable carriers. The pharmaceutical carrier is a conventional pharmaceutical carrier in the field, and can be any suitable physiologically or pharmaceutically acceptable pharmaceutical excipients. The pharmaceutical excipients are conventional pharmaceutical excipients in the field, and preferably comprise pharmaceutically acceptable excipients, fillers or diluents and the like.

In the present invention, the pharmaceutical composition is preferably administered in an amount effective to reduce or delay the progression of the disease, degenerative or damaging condition. The effective amount can be determined on an individual basis and will be based in part on the symptoms to be treated and the consideration of the results sought. The skilled artisan can determine the effective amount by using the factors described above on an individual basis and the like and using no more than routine experimentation.

The invention provides application of the pharmaceutical composition in preparing medicines for preventing and/or treating diseases related to TSLP expression or dysfunction. Preferably, the disease associated with abnormal TSLP expression or function is an allergic disease, a tumor, an immune disease, or a combination thereof.

Methods, compositions for detecting TSLP in a sample

The invention also provides a method for detecting TSLP in a sample (e.g., detecting cells that overexpress TSLP), comprising the step of contacting the antibody with a sample to be detected in vitro, and detecting whether the antibody binds to the sample to be detected to form an antigen-antibody complex.

The meaning of overexpression is conventional in the art and refers to overexpression of RNA or protein in the sample to be tested (due to increased transcription, post-transcriptional processing, translation, post-translational processing and altered protein degradation), as well as to local overexpression and increased functional activity due to altered protein transport patterns (increased cell membrane localization), as in the case of increased enzymatic hydrolysis of the substrate.

In the present invention, the above-mentioned detection mode of whether or not an antigen-antibody complex is formed by binding is a conventional detection mode in the art, preferably a flow cytometry (FACS) detection.

The present invention provides a composition for detecting TSLP in a sample comprising as an active ingredient an antibody, recombinant protein, immune cell, or combination thereof as described above. Preferably, it further comprises a compound composed of the functional fragment of the above antibody as an active ingredient.

The main advantages of the invention include:

1) The anti-TSLP antibodies of the present invention are capable of binding TSLP proteins of different species, with high affinity for both human and cynomolgus TSLP proteins.

2) The anti-TSLP antibodies of the present invention are capable of efficiently blocking the binding of TSLP to its receptor TSLPR molecules, as well as to cell surface TSLPR/IL7Rα receptors.

3) The anti-TSLP antibodies of the present invention are capable of inhibiting proliferation of target cells that highly express TSLPR/IL7Rα and inhibit expression of the TSLP downstream signaling pathway STAT5 gene therein.

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedures, which do not address the specific conditions in the examples below, are generally followed by conventional conditions, such as those described in Sambrook et al, molecular cloning, a laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989), or by the manufacturer's recommendations. Percentages and parts are weight percentages and parts unless otherwise indicated.

EXAMPLE 1 preparation of TSLP and TSLPR

For animal immunization and antibody sequence screening, sequences encoding His-tagged and Fc-tagged human, cynomolgus monkey sequences were synthesized onto the pt 5 vector, constructed into expression plasmids for subsequent transfection of Expi293F (Thermo).

The specific transfection procedure was to inoculate the expression medium with 0.8X10⁶/ml of Expi293F cells with a viability of >95% and to place them in 6% CO₂, 130rpm,37℃for a further 24 hours. After 24 hours, the sterile plasmid to be transfected was sterilized with a 0.22 μm filter and the concentration was measured, and 1mg/ml of the transfection reagent Polyethylenimine (PEI) was sterilized with a 0.22 μm filter for use, the mass ratio of PEI to plasmid being 2:1. Taking 200ml of Expi293F cells as an example, 10ml of Opti-MEM and 200 μg of plasmid are mixed uniformly, and the mixture is kept stand for 5min, and 10ml of Opti-MEM and 400 μl of PEI are mixed uniformly and the mixture is kept stand for 5min. Mixing the plasmid mixed solution and PEI mixed solution uniformly, and standing at room temperature for 15min. The plasmid and PEI mixture was slowly added to 200ml of Expi293F cells and incubated in a shaker at 6% CO₂, 130rpm,37 ℃. Transfection 18-24h 250mM sodium valproate solution was added at 0.8% of the transfection volume, and 5% of the volume of the feed medium was supplemented on days 1 and 3 of transfection, respectively. On day 5 of transfection, the cell supernatant was collected by centrifugation at 4000rpm for 15min, filtered and the collected cell supernatant was purified.

The relevant amino acid sequences are shown below:

Human TSLP:

MFPFALLYVLSVSFRKIFILQLVGLVLTYDFTNCDFEKIKAAYLSTISKDLITYMSGTKSTEFNNTVSCSNRPHCLTEIQSLTFNPTAGCASLAKEMFAMKTKAALAIWCPGYSETQINATQAMKKARKSKVTTNKCLEQVSQLQGLWRRFNRPLLKQQ(SEQ ID NO.1)

Cynomolgus monkey TSLP:

MKSLGQSKKEEVSFRKFFIFQFVGLVLTYDFTNCDFQKIEADYLRTISKDLITYMSGTKSTDFNNTVSCSNRPHCLTEIQSLTFNPTPRCASLAKEMFARKTKATLALWCPGYSETQINATQAMKKARKSKVTTNKCLEQVSQLLGLWRRFIRTLLKKQ(SEQ ID NO.2)

Annotation underlined amino acids are mutated amino acid sites, avoiding Furin protease cleavage

Human TSLPR (extracellular domain):

MGRLVLLWGAAVFLLGGWMALGQGGAAEGVQIQIIYFNLETVQVTWNASKYSRTNLTFHYRFNGDEAYDQCTNYLLQEGHTSGCLLDAEQRDDILYFSIRNGTHPVFTASRWMVYYLKPSSPKHVRFSWHQDAVTVTCSDLSYGDLLYEVQYRSPFDTEWQSKQENTCNVTIEGLDAEKCYSFWVRVKAMEDVYGPDTYPSDWSEVTCWQRGEIRDACAETPTPPKPKLSKFILISSLAILLMVSLLLLSLWKLWRVKKFLIPSVPDPKSIFPGLFEIHQGNFQEWITDTQNVAHLHKMAGAEQESGPEEPLVVQLAKTEAESPRMLDPQTEEKEASGGSLQLPHQPLQGGDVVTIGGFTFVMNDRSYVAL(SEQ ID NO.3)

Cynomolgus TSLPR (extracellular domain):

MGRLVLLWGAAVFLLGSWMALGQVATGEGLQIQIIYFNLETVQVTWNASHYPRSNLSFHYKFSRDEAYDQCTVYILQEGHTSGCLLDAEQQDDILYFSIRNGTHPVFTASRWIFYYLKPSSPKQVSFSWHQDAVTVTCSDLSYRGLLYEVQYRSPFDTEWQSKQENTCNVTIEDLDAEKCYAFRARVKAMEDAYGPDTYPSDWSEVTCWQRGKTRDSCPEPRTPPKPKLSKFMLVSSLAILLMVCLLLLSLRKLWR(SEQ ID NO.4)

EXAMPLE 2 purification of TSLP and TSLPR recombinant proteins

Purification of His tag fusion protein, namely, high-speed centrifugation of a cell expression supernatant sample to remove impurities, filtration, 20mM PB+0.5M NaCl solution balancing of a nickel column and flushing for 10 times of column volume. The filtered supernatant samples were loaded onto a column. The column was washed with 20mM PB+0.5M NaCl solution containing 20mM imidazole until the A280 reading dropped to baseline. The target protein was eluted with 20mM PB+0.5M NaCl solution containing 250mM imidazole and the elution peaks were collected. And replacing buffer with PBS, and subpackaging for standby after SDS-PAGE identification is correct.

And (3) purifying the Fc tag recombinant Protein, namely centrifuging a cell expression supernatant sample at a high speed to remove impurities, and purifying the recombinant Protein expression supernatant by using a Protein A column. The column was washed with PBS until the a280 reading dropped to baseline. The target protein was eluted with 100mM glycine pH3.0 and neutralized with 1M Tris-HCl, pH 8.0. And replacing buffer with PBS, and subpackaging the obtained protein for standby after SDS-PAGE identification is correct.

EXAMPLE 3 construction of human TSLPR/IL7Rα receptor Co-expressing cells

To screen for antibodies that block TSLP binding to the TSLP receptor CHO-K1 and BaF3 cell lines expressing both human TSLP receptor and human IL7 ra (TSLPR/IL 7 ra) were constructed. And (3) packaging the target gene TSLPR/IL7 Ralpha by using slow viruses, and cloning the target gene TSLPR/IL7 Ralpha into a target cell strain to form a stable high-expression cell strain. The human TSLPR and human IL7Rα genes were first cloned into pCDH-CMV-MCS-EF1-puro and pCDH-CMV-MCS-EF1-Neo (SBI, CD 500B-1) plasmids, respectively, and then cloned into CHO-K1 and BaF3 cell lines by lentiviral infection, and selected for culture under a screening pressure of 10. Mu.g/ml puromycin (puromycin, gibco, US) for three weeks. A second round of infection was performed on this basis, and the human IL7Rα gene was cloned into cells and screened with 1mg/ml G418 (Gibco, US) and 10. Mu.g/ml puromycin for two to three weeks. Finally screening out CHO-K1 and BaF3 monoclonal cell strains which simultaneously express TSLPR and IL7RR in high expression by a flow separation method.

The sequence information for both receptors is as follows:

human TSLPR is shown as SEQ ID NO. 3.

Human IL7rα:

MTILGTTFGMVFSLLQVVSGESGYAQNGDLEDAELDDYSFSCYSQLEVNGSQHSLTCAFEDPDVNITNLEFEICGALVEVKCLNFRKLQEIYFIETKKFLLIGKSNICVKVGEKSLTCKKIDLTTIVKPEAPFDLSVVYREGANDFVVTFNTSHLQKKYVKVLMHDVAYRQEKDENKWTHVNLSSTKLTLLQRKLQPAAMYEIKVRSIPDHYFKGFWSEWSPSYYFRTPEINNSSGEMDPILLTISILSFFSVALLVILACVLWKKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMSSFYQNQ(SEQ ID NO.5)

EXAMPLE 4 discovery and screening of anti-human TSLP antibodies

Anti-human TSLP monoclonal antibodies were generated by immunizing mice, laboratory SJL white mice, females, 6-8 weeks old (Beijing Veitz Lihua laboratory animal technologies Co., ltd., animal production license number SCXK (Beijing) 2012-0001). The feeding environment is SPF grade. After the mice are purchased, the mice are kept in a laboratory environment for 1 week, the light/dark period is regulated for 12/12 hours, the temperature is 20-25 ℃, and the humidity is 40-60%. The acclimatized mice were immunized with recombinant proteins huTSLP-Fc (25. Mu.g), huTSLP-his (12.5. Mu.g) and cyno TSLP-his (12.5. Mu.g) with TiterMax, alum or CpG adjuvant. After 4-5 immunizations, mice with high and plateau titers in serum were selected, and after sacrifice, spleen cells were taken and fused with myeloma cells. The spleen lymphocytes were fused with myeloma cells Sp2/0 (CRL-8287. TM.) using an optimized PEG-mediated fusion procedure to give hybridoma cells.

Primary screening was performed using ELISA binding assays against human and monkey TSLP, assays that block binding of human TSLP to its receptor TSLPR, and the like. After transferring the hybridoma cells to a 24-well plate, the supernatant was rescreened. In addition to the above experiments, positive clones screened by increasing a reporter gene signal up-regulation experiment for inhibiting TSLP induction, a proliferation experiment for inhibiting BaF3 cells induced by TSLP, and the like are subjected to two rounds of subcloning to obtain hybridoma clones which are used for antibody production and purified by an affinity method.

The monoclonal hybridoma cell strains 64B8F8 and 217H11C5 with good activity are obtained by screening, hybridoma cells in logarithmic growth phase are respectively collected, RNA is extracted by using NucleoZol (MN), and reverse transcription is carried out. The cDNA obtained by reverse transcription is amplified by PCR and then sent to sequencing company for sequencing. The murine anti-TSLP antibody 64B8F8, 217H11C5 sequences were obtained by sequencing, and the amino acid sequences were as follows:

64B8F8 murine antibody sequence information:

64B8F8-mouse-VH:

EVQLQQSGAELVRSGASVKLSCTASGFNIKDNYMHWVKQRPEQGLEWI GWIDPENGNTEYVPKFQAKATMTADTSSNTVYLHLSSLTSEDTAVYYCNAR FYGSGYRNYFDYWGQGTTLTVSS(SEQ ID NO.6)

64B8F8-mouse-VL:

QIVLTQSPAIMSTSPGEKVTMTCSATSSVTYMFWYQQKPGSSPRLLIYDT SNLASGVPFRFSGSGSGTSSSLTISRMEAEDAATYYCQQWNTYPPTFGAGTK LELK(SEQ ID NO.7)

217H11C5 murine antibody sequence information:

217H11C5-mouse-VH:

QIQLVQSGPELKKPGETVKISCKASGYNFITSGMSWVKQAPGKGLKWM GWINTYSGVATYADDLKGRFAFSLETSASTASLQINNLKIEDTATYFCARGEP PAYWGQGTLVTVSA(SEQ ID NO.8)

217H11C5-mouse-VL:

DILLTQSPAILSVSPGERVSFSCRASQSIGTSIHWYQQRTNGSPRLLIKYA SESISGIPSRFSGSGSGTDFTLSINSVESEDIADYFCQESNSWPTWTFGGGTKLE IK(SEQ ID NO.9)

wherein single underline indicates CDR regions.

Example 5 humanized design of anti-human TSLP antibodies

In this example, antibodies were engineered, non-human antibodies were engineered to be humanized antibodies, thereby improving their safety and efficacy in clinical applications. The structure of the antibody is regulated by genetic engineering technology and protein engineering technology, so that the antibody has better immune tolerance in human body and enhances the affinity and specificity of the antibody. The antibodies involved were the 64B8F8 and 217H11C5 antibodies. The humanized antibody engineering method is as follows:

selecting a human antibody skeleton, namely screening and selecting a proper human antibody skeleton through the comparison of protein primary sequences and the comparison of antibody structures;

CDR replacement, namely, grafting and replacing CDR regions of a selected human antibody skeleton by using CDR regions of 64B8F8 and 217H11C5 murine antibodies to obtain framework region grafted antibody variable region sequences;

referring to the simulated three-dimensional structures of the 64B8F8 and 217H11C5 murine antibodies, carrying out back mutation on partial key residues of the replaced VH and VL, and obtaining humanized antibody molecules through cross screening of VH and VL back mutation sequences;

The constant region of the non-human antibody was replaced with the constant region of human, the heavy chain was constructed as a chimeric antibody of IgG4, and the light chain was constructed as an IgG4 kappa chain constant region.

The heavy and light chain constant region sequences of control antibody AMG157 were as follows:

AMG157-VH:

QMQLVESGGGVVQPGRSLRLSCAASGFTFRTYGMHWVRQAPGKGLEW VAVIWYDGSNKHYADSVKGRFTITRDNSKNTLNLQMNSLRAEDTAVYYCA RAPQWELVHEAFDIWGQGTMVTVSS**(SEQ ID NO.61)

AMG157-VL:

SYVLTQPPSVSVAPGQTARITCGGNNLGSKSVHWYQQKPGQAPVLVVY DDSDRPSWIPERFSGSNSGNTATLTISRGEAGDEADYYCQVWDSSSDHVVFG GGTKLTVL**(SEQ ID NO.62)

the humanized antibody and the control antibody were selected as IgG4, unless otherwise specified.

The IgG4 heavy chain constant region sequence is as follows:

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG

VHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESK

YGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQF

NWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVS

NKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDI

AVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVM

HEALHNHYTQKSLSLSLGK**(SEQ ID NO.10)

the IgG4 light chain constant region sequence is as follows:

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC**(SEQ ID NO.11)

wherein, represents a stop codon.

5.1 Selection and back-mutation of the human FR region of a 64B8F8 murine antibody

(1) Selection and back-mutation of human FR regions

The FR of 64B8F8 humanized VH selects IGHV1-46 x 01+IGHJ4 x 01 and the FR of humanized VL selects IGKV3-11 x 01+IGKJ2 x 01. The CDR regions of the 64B8F8 murine antibody were grafted onto the human template described above, and the sequence obtained was as follows:

>64B8F8-VH0

QVQLVQSGAEVKKPGASVKVSCKASGFNIKDNYMHWVRQAPGQGLE WMGIIDPENGNTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCN ARFYGSGYRNYFDYWGQGTLVTVSS(SEQ ID NO.12)

>64B8F8-VL0

EIVLTQSPATLSLSPGERATLSCSATSSVTYMFWYQQKPGQAPRLLIYDT SNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWNTYPPTFGQGTKL EIK(SEQ ID NO.22)

The structure of the parent antibody was modeled by the MOE homology modeling program. Humanized antibodies were designed using CDR grafting, the CDRs of the parent antibodies were grafted into a human framework to obtain humanized light and heavy chains for each parent antibody. Affinity ranking experiments were performed with 8 heavy chains (VH 1, VH2, VH3, VH4, VH5, VH6, VH7, VH 8) paired with 4 light chains (VL 1, VL2, VL3 and VL 4). The combination is detailed in tables 1 and 2

The constant region heavy chain was constructed as a chimeric antibody to IgG4 and the light chain was constructed as an IgG4 kappa chain constant region.

Wherein the IgG4 heavy chain constant region sequence is (SEQ ID NO. 10)

IgG4 light chain constant region sequence (SEQ ID NO. 11)

The humanized light and heavy chain variable region amino acid sequences are as follows:

>64B8F8-VH1

>64B8F8-VH2

>64B8F8-VH3

>64B8F8-VH4

>64B8F8-VH5

>64B8F8-VH6

QVQLVQSGAEVKKPGASVKVSCKASGFNIKDNYMHWVKQRPGQGLEWMGIIDPENGNTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCNARFYGSGYRNYFDYWGQGTLVTVSS(SEQ ID NO.18)

>64B8F8-VH7

>64B8F8-VH8

The N-terminal of the heavy chain variable region sequence may be linked to a signal peptide METDTLLLWVLLLWVPGSTG (SEQ ID NO. 21).

Light chain

>64B8F8-VL1

EIVLTQSPATLSLSPGERATLSCSATSSVTYMFWYQQKPGQAPRLLIYDT SNLASGIPARFSGSGSGTDSTLTISSLEPEDFAVYYCQQWNTYPPTFGQGTKL EIK(SEQ ID NO.23)

>64B8F8-VL2

>64B8F8-VL3

>64B8F8-VL4

The N-terminal of the light chain variable region sequence may be linked to a signal peptide METDTLLLWVLLLWVPGS TG (SEQ ID NO. 21).

Note that the CDR regions are underlined, bold and italics are back mutations.

The VL and VH described above were combined with the light and heavy chain constant regions of the human germline to obtain full-length sequence antibodies.

Further obtaining antibody molecules with different combinations through genetic engineering technology, protein engineering technology, cell culture technology and protein purification technology. The above two sets of sequences were run in two batches for antibody production as shown in tables 1 and 2 below.

Table 1 first lot combinations

	64B8F8-VH1	64B8F8-VH2	64B8F8-VH3	64B8F8-VH4
					64B8F8-VL1	SZ213	SZ214	SZ215	SZ216
64B8F8-VL2	SZ217	SZ218	SZ219	SZ220
					64B8F8-VL3	SZ221	SZ222	SZ223	SZ224
64B8F8-VL4	SZ225	SZ226	SZ227	SZ228

Table 2 second batch combinations

	64B8F8-VH5	64B8F8-VH6	64B8F8-VH7	64B8F8-VH8
					64B8F8-VL1	SZ247	SZ248	SZ249	SZ250

The different antibody molecules are detected by ELISA and cell function experiment, and the binding activity and the functional activity of the different antibody molecules are verified.

(2) Affinity maturation of 64B8F8 humanized antibodies

Antibody drugs are important drugs and diagnostic reagents in the biomedical field, and their affinities are key factors affecting biological functions and therapeutic effects. Thus, increasing the affinity of an antibody to a target antigen is an important goal to improve the quality and efficacy of antibodies. In this example, optimized antibody molecules capable of blocking TSLP/TSLPR binding are screened. The affinity maturation method comprises the following steps:

the antigen-antibody complex structure analysis, namely determining the complex structure of the antigen-antibody by means of cyno-EM, computer molecule docking and the like;

point mutation design, namely designing a point mutation scheme according to structural information of an antibody antigen complex, and pertinently replacing amino acid residues of a binding site of an antibody;

The implementation of point mutation, namely introducing the designed point mutation into the gene sequence of the antibody by utilizing a genetic engineering technology, and synthesizing the mutated antibody;

Affinity evaluation, namely evaluating the binding affinity of the mutated antibody and a target antigen through an SPR technology or a BLI technology, and screening antibody candidates with higher affinity;

Functional verification, namely performing biological functional verification on the antibody after affinity maturation, including antigen neutralization activity, intracellular signal transduction and the like, and verifying the effectiveness of the antibody in a biological process.

TABLE 3 affinity maturation mutations

The constant region heavy chain was constructed as a chimeric antibody to IgG4 and the light chain was constructed as an IgG4 kappa chain constant region. Wherein the IgG4 heavy chain constant region sequence is (SEQ ID NO. 10)

IgG4 light chain constant region sequence (SEQ ID NO. 11)

The affinity maturation variable region sequence is as follows:

>64B8F8-VH7-M1

>64B8F8-VH7-M2

>64B8F8-VH7-M3

>64B8F8-VH7-M4

>64B8F8-VH7-M5

>64B8F8-VH7-M6

>64B8F8-VH7-M7

>64B8F8-VH7-M8

>64B8F8-VH7-M9

>64B8F8-VH7-M10

>64B8F8-VH7-M11

>64B8F8-VH7-M12

>64B8F8-VH7-M13

>64B8F8-VH7-M14

>64B8F8-VH7-M15

>64B8F8-VH7-M16

>64B8F8-VH7-M17

>64B8F8-VH7-M18

>64B8F8-VH7-M19

QVQLVQSGAEVKKPGASVKVSCKASGFNIKDNYMHWVKQRPGQGLEWMGWIDPENGNTEYVPKFQGKVTMTADNSTNTVYMELSSLRSEDTAVYYCNARFYGSGYRNYFDYWGQGTLVTVSS(SEQ ID NO.45)

>64B8F8-VH7-M20

>64B8F8-VH7-M21

>64B8F8-VH7-M22

>64B8F8-VH7-M23

>64B8F8-VH7-M24

>64B8F8-VH7-M25

>64B8F8-VH7-M26

>64B8F8-VH7-M27

>64B8F8-VH7-M28

>64B8F8-VH7-M29

>64B8F8-VH7-M30

>64B8F8-VH7-M31

>64B8F8-VH7-M32

>64B8F8-VH7-M33

>64B8F8-VH7-M34

Note that the CDR regions are shown in single underline and that the bold + italics indicate back mutations.

The N-terminal of the affinity maturation variable region sequence can be linked to a signal peptide METDTLLLWVLLLW VPGSTG (SEQ ID NO. 21).

64B8F8 and humanized and affinity matured antibody variable region CDRs are shown in Table 4.

Table 4 64B8F8 and humanized and affinity matured antibody variable region CDRs thereof

(3) Production of chimeric and humanized antibodies

DNA sequences encoding the heavy and light chains of the antibodies were synthesized and expression plasmids for full length IgG antibodies were constructed. Antibody expression was performed in 4mL CHO cell culture and the supernatant was purified using a protein a affinity column. Purified antibody buffer was exchanged to PBS using a PD-10 desalting column.

(4) Multi-concentration affinity assay of chimeric and selected humanized antibodies

The affinity to Human TSLP-his was determined using the Surface Plasmon Resonance (SPR) biosensor Biacore T200 (GE HEALTHCARE) to select chimeric antibodies and the first 3 antibodies (with smaller KD as the preferred antibody, with the smaller KD being taken into account comprehensively in the case of comparable KD). Antibodies were captured on the sensor chip by Fc capture method. Human TSLP-his as analyte. Data for dissociation (kd) and association (ka) rate constants were obtained using Biacore T200 evaluation software. The equilibrium dissociation constant (KD) is calculated from the ratio of KD to ka.

TABLE 5 antibody affinity

The affinity assay results showed that the 64B8F8 antibody had an increased affinity for binding to Cyno TSLP after affinity maturation compared to the chimeric antibody.

5.2 Selection and back-mutation of the human FR region of a 217H11C5 murine antibody

(1) Selection and back-mutation of human FR regions

The FR of the 217H11C5 humanized VH selected IGHV 9-3+IGH-J3.times.01 and the FR of the humanized VL selected IGKV 5-48.times.01+IGKJ1.times.01. CDR regions of the 217H11C5 murine antibody were grafted onto the human template described above.

The structure of the parent antibody was modeled by the MOE homology modeling program. Humanized antibodies were designed using CDR grafting, the CDRs of the parent antibodies were grafted into a human framework to obtain humanized light and heavy chains for each parent antibody. Affinity ranking experiments were performed with 4 heavy chains (VH 1, VH2, VH3, VH 4) paired with 4 light chains (VL 1, VL2, VL3, and VL 4).

The constant region heavy chain was constructed as a chimeric antibody to IgG4 and the light chain was constructed as an IgG4 kappa chain constant region.

Wherein the IgG4 heavy chain constant region sequence is (SEQ ID NO. 10)

IgG4 light chain constant region sequence (SEQ ID NO. 11)

The humanized light and heavy chain variable region amino acid sequences are as follows:

>217H11C5-VH1

QVQLVQSGSELKKPGASVKVSCKASGYNFITSGMSWVRQAPGQGLEW MGWINTYSGVATYADDLKGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCAR GEPPAYWGQGTLVTVSS(SEQ ID NO.64)

>217H11C5-VH2

QVQLVQSGSELKKPGASVKVSCKASGYNFITSGMSWVRQAPGQGLKW MGWINTYSGVATYADDLKGRFVFSLDTSVSTAYLQISSLKAEDTAVYFCAR GEPPAYWGQGTLVTVSS(SEQ ID NO.65)

>217H11C5-VH3

QVQLVQSGSELKKPGASVKVSCKASGYNFITSGMSWVRQAPGQGLKW MGWINTYSGVATYADDLKGRFVFSLDTSVSTASLQISSLKAEDTAVYFCARG EPPAYWGQGTLVTVSS(SEQ ID NO.66)

>217H11C5-VH4

QVQLVQSGSELKKPGASVKVSCKASGYNFITSGMSWVKQAPGQGLKW MGWINTYSGVATYADDLKGRFVFSLDTSVSTASLQISSLKAEDTAVYFCARG EPPAYWGQGTLVTVSS(SEQ ID NO.67)

>217H11C5-VL1

EIVLTQSPDFQSVTPKEKVTITCRASQSIGTSIHWYQQKPDQSPKLLIKYA SESISGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCQESNSWPTWTFGGGTK LEIK(SEQ ID NO.68)

>217H11C5-VL2

EIVLTQSPDFQSVTPKEKVTITCRASQSIGTSIHWYQQKPDQSPKLLIKYA SESISGIPSRFSGSGSGTDFTLTINSLEAEDAATYFCQESNSWPTWTFGGGTKL EIK(SEQ ID NO.69)

>217H11C5-VL3

EIVLTQSPDFQSVTPKEKVTITCRASQSIGTSIHWYQQKPDQSPRLLIKYA SESISGVPSRFSGSGSGTDFTLTINSLEAEDAADYFCQESNSWPTWTFGGGTK LEIK(SEQ ID NO.70)

>217H11C5-VL4

EIVLTQSPDFQSVTPKEKVTFTCRASQSIGTSIHWYQQKPDQSPRLLIKY ASESISGIPSRFSGSGSGTDFTLTINSLEAEDAADYFCQESNSWPTWTFGGGTK LEIK(SEQ ID NO.71)

The N-terminal of the above humanized variable region sequence may be linked to a signal peptide MGWSCIILFLVATATGVH S (SEQ ID NO. 63).

The CDRs of the 217H11C5 and humanized and affinity matured antibody variable regions are shown in table 6.

Table 6 217H11C5 and humanized and affinity matured antibody variable region CDRs thereof

(2) Production of chimeric and humanized antibodies

DNA sequences encoding the heavy and light chains of the antibodies were synthesized and expression plasmids containing full-length IgG were constructed. Antibody expression was performed in 4mL CHO cell (ATCC) cultures and the supernatant was purified using protein a affinity columns. Purified antibody buffer was exchanged to PBS using a PD-10 desalting column.

(3) Affinity assay for chimeric and humanized antibodies

The affinity of the purified antibodies to Human TSLP-his binding was determined separately using the Surface Plasmon Resonance (SPR) biosensor Biacore T200 (GE HEALTHCARE). Antibodies were immobilized on the sensor chip by Fc capture. Human TSLP-his was used as the analyte. Data for dissociation (kd) and association (ka) rate constants were obtained using Biacore T200 evaluation software. The equilibrium dissociation constant (KD) is calculated from the ratio of KD to ka. Antibodies are ordered by their dissociation rate constant (dissociation rate, kd).

Table 7 affinity of 217H11C5 humanized antibodies

And (3) carrying out single-point affinity sequencing on the humanized antibody, and taking the antibody with higher affinity in the table for further research.

(4) Multi-concentration affinity assay of chimeric and selected humanized antibodies

The affinity to Human TSLP-his was determined using the Surface Plasmon Resonance (SPR) biosensor Biacore T200 (GE HEALTHCARE) to select chimeric antibodies and the first 3 antibodies (with smaller KD as the preferred antibody, with the smaller KD being taken into account comprehensively in the case of comparable KD). Antibodies were captured on the sensor chip by Fc capture method. Human TSLP-his as analyte. Data for dissociation (kd) and association (ka) rate constants were obtained using Biacore T200 evaluation software. The equilibrium dissociation constant (KD) is calculated from the ratio of KD to ka.

Table 8 antibody affinity

The preferred sequences were subjected to multi-concentration affinity assay, and the antibodies with higher affinities in the above table were further studied.

Example 6 ELISA detection of anti-human TSLP antibodies binding to human TSLP and cynomolgus monkey TSLP

The wells were coated with hTSLP-His and cynoTSLP-His at a concentration of 2. Mu.g/ml diluted in PBS, 100. Mu.L per well. Sealing by a sealing plate film, and standing overnight at 2-8 ℃. The sealing plate membrane was removed and 200 μl of PBST wash plate was added to each well and repeated 3 times. 200 μl of 3% BSA was added to each well, sealed with a plate membrane, and allowed to stand at 37deg.C for 2h. The sealing plate membrane was removed and 200 μl of PBST wash plate was added to each well and repeated 3 times. The initial concentration of the sample was 20000ng/mL, 8 dilutions were made at 4-fold dilution and 100. Mu.L/well, the dilutions were PBS+1% BSA.

Detection of antigen-antibody binding the antibody to be detected was incubated with antigen 37 ℃ for 1h, amg157 as positive control. The sealing plate membrane was removed and 200 μl of PBST wash plate was added to each well and repeated 3 times. HRP-IgG antibody (diluted 1:5000 with PBS+1% BSA) was added and allowed to stand at 37℃for 1h at 100. Mu.L per well. The sealing plate membrane was removed and 200 μl of wash plate was added to each well and repeated 4 times. 100 mu L of single-component TMB color development liquid is added into each hole, the sealing plate film is sealed, and the mixture is kept stand for 10min at room temperature in a dark place. The plate was removed and 100. Mu.L of ELISA stop solution was added to each well. OD450 was detected using a microplate reader. Using GRAPHPAD PRISM to process the data, the average value of OD450 absorbance values of samples at each concentration is taken as a Y axis, the concentration value of the samples is taken as an X axis, and four-parameter fitting is performed to obtain EC50 and fitting constant R².

The results are shown in FIGS. 1-4. Humanized antibodies and affinity matured antibodies can specifically bind to human/cynomolgus TSLP protein with high affinity.

Example 7 BIACORE assay of anti-TSLP humanized antibodies binding to human/cynomolgus monkey TSLP

The affinity of the purified antibodies to Human TSLP-his binding was determined separately using the Surface Plasmon Resonance (SPR) biosensor Biacore T200 (GE HEALTHCARE). Antibodies were immobilized on the sensor chip by Fc capture. Human TSLP-his was used as the analyte. Data for dissociation (kd) and association (ka) rate constants were obtained using Biacore T200 evaluation software. The equilibrium dissociation constant (KD) is calculated from the ratio of KD to ka. The results are shown in tables 5, 7 and 8.

Example 8 ELSA-based anti-TSLP antibodies block TSLP binding to TSLP receptor experiments

The wells were coated with hTSLPR-Fc and cynoTSLPR-Fc diluted in PBS at a concentration of 2. Mu.g/mL, 100. Mu.L per well. Sealing by a sealing plate film, and standing overnight at 2-8 ℃. The sealing plate membrane was removed and 200 μl of PBST wash plate was added to each well and repeated 3 times. 200. Mu.L of 1% BSA was added to each well, the wells were sealed with a plate membrane, and allowed to stand at 37℃for 2 hours. The sealing plate membrane was removed and 200 μl of PBST wash plate was added to each well and repeated 3 times. The initial concentration of the sample was 10000ng/mL (prepared concentration 20000 ng/mL), and 8 dilutions were prepared at a 4-fold ratio dilution of 50. Mu.L/well, and the dilution was 1% BSA-PBS.

The blocking effect of the antibody on TSLP binding to TSLPR was detected by adding TSLP-His at a final concentration of 100ng/mL (configured concentration of 200 ng/mL), 50. Mu.L/well and cynoTSLP-His at a final concentration of 20ng/mL (configured concentration of 40 ng/mL), 50. Mu.L/well. AMG157 antibody served as positive control. The reaction was incubated at 37℃for 1h. The sealing plate membrane was removed and 200 μl of PBST wash plate was added to each well and repeated 3 times. HRP-his antibody (diluted 1:5000 with PBS+1% BSA) was added and allowed to stand at 37℃for 1h at 100. Mu.L per well. The sealing plate membrane was removed and 200 μl of wash plate was added to each well and repeated 4 times. 100 mu L of single-component TMB color development liquid is added into each hole, the sealing plate film is sealed, and the mixture is kept stand for 10min at room temperature in a dark place. The plate was removed and 100. Mu.L of ELISA stop solution was added to each well. The microplate reader detects OD450.GRAPHPAD PRISM processing data, namely taking the average value of OD450 absorbance values of samples with various concentrations as a Y axis, taking the concentration value of the samples as an X axis, and performing four-parameter fitting to obtain an IC50 and a fitting constant R².

The results are shown in FIGS. 5-8. Both humanized and affinity matured antibodies can efficiently block ligand-receptor binding between the human/cynomolgus TSLP protein and its receptor (TSLPR).

Example 9 FACS-based TSLP antibody blocking TSLP binding to TSLP receptor experiments

CHOK1-tslpr+il7r cells prepared in example 3 were collected, exchange medium pbs+1% fbs, cell density was adjusted to 4×10⁶/mL, seeded at 50 μl/well into 96 well V-plates, and the supernatant was discarded by centrifugation. The TSLP-His ligand was diluted to a concentration of 0.4. Mu.g/ml (final concentration of 0.1. Mu.g/ml), 25. Mu.l/well, the antibody was diluted in a gradient to an initial concentration of 200. Mu.g/ml (final concentration of 50. Mu.g/ml), and 8 dilutions were made at 5-fold ratio, 25. Mu.l/well. The ligand and antibody were added to the 96-well V-plate at a 1:1 ratio, 25. Mu.l: 25. Mu.l, and incubated at 37℃for 1h. Centrifuging at 1500rpm for 5min, discarding supernatant, washing cells twice, diluting APC-His secondary antibody (1:100), adding 96-well V-shaped plate, 100 μl/well, incubating at 37deg.C for 30min, and washing cells twice. Flow cytometer detection GRAPHDPAD PRISM processes the data.

The results are shown in FIGS. 9-10. Both humanized and affinity matured antibodies can efficiently block ligand-receptor binding between the human TSLP protein and the TSLP receptor expressed on the cell surface.

Example 10 anti-TSLP antibodies inhibit TSLP-induced proliferation of BaF3-TLSPR/IL7R cells

Cells prepared in example 3 were collected, baF3-TSLPR/IL7R:1000R/min was centrifuged for 5min, cells in the logarithmic growth phase were collected, the cells were washed twice with 1640+10% FBS medium, the cells were resuspended with 1640+10% FBS and the cell concentration was adjusted to 5X 10⁴/mL. Cells were seeded at 2.5×10³/well, 50 μl/well in 96-well plates with a dilution of 1640+10% fbs. Cell viability was greater than 80%. The TSLP antibody was diluted in a gradient to 25. Mu.L/well in 96-well plates inoculated with cells, the initial concentration of TSLP antibody was 40000ng/mL (final concentration 10000 ng/mL), 8 dilutions were made by 4-fold dilution, TSLP-His was diluted to 0.4ng/mL (final concentration 0.1 ng/mL), 25. Mu.L/well in 96-well plates inoculated with cells, and two wells were used for each concentration. The plates were incubated at 37℃for 72h.

Detection reagent use2.0Luminescent Cell Viability Assay (Northenzan). The test cell plates were removed from the incubator and left at room temperature for 30min to equilibrate the plate temperature to room temperature. 100. Mu. L CellCounting-Lite 2.0 was pipetted into 100. Mu.L of the test cell culture. Shaking and mixing for 2-5min to allow the cells to be completely lysed, standing at room temperature for 10min to stabilize luminescence signal, and detecting. And detecting the full wavelength Luminesecnce (RLU) in a chemiluminescent mode of the enzyme-labeled instrument. And analyzing test data by utilizing Graphpad software, taking TSLP antibody concentration as an X axis, and correspondingly Luminesecnce (RLU) as a Y axis, selecting a four-parameter equation regression model, and fitting an effect curve.

The results are shown in FIGS. 11-12. Humanized antibodies and affinity matured antibodies all inhibit human TSLP protein-induced BaF3 cell proliferation.

Example 11 anti-TSLP antibodies inhibit TSLP-induced BaF3-TLSPR/IL7R/STAT5-Luc reporter Gene signalling

Plasmid pGL4.52[ luc2P/STAT5 RE/Hygro ] was transfected into the receptor overexpressing cells of example 3 on the basis of the TSLPR/IL-7R overexpressing BaF3 cells prepared as described in example 3. Cells were seeded the day before transfection to achieve a confluence of 50% -80% cells on the day of transfection. On the day of transfection, the serum-free culture medium opti-MEM and the transfection reagent FUGENE are equilibrated to room temperature, the transfection reagent and DNA complex is prepared, during the preparation process, FUGENE transfection reagent is firstly added into the MEM culture medium, the mixture is incubated for 5min at room temperature, then plasmid DNA is added, the mixture is uniformly mixed, and the mixture is incubated for 15min at room temperature. After 48h of transfection, antibiotics are added for pressure screening, and FACS and reporter gene detection are performed after the cells grow up. Cells were centrifuged one day in advance of reporter gene detection, washed once with 1640+10% fbs medium, and starved overnight with 1640+10% fbs (without ml 3) in exchange medium. Cells BaF3TSLPR+IL7R were harvested, centrifuged at 1000R/min for 5min, cells in the logarithmic growth phase were collected, 1640+10% FBS resuspended and cell concentration adjusted to 4X 10⁵ cells/mL. Cells were seeded at 2x10⁴/well, 50 μl/well into 96-well plates, dilutions 1640+10% fbs. The TSLP antibody was diluted in a gradient to 25. Mu.L/well in 96-well plates inoculated with cells, the initial concentration of TSLP antibody was 40000ng/mL (final concentration: 10000 ng/mL), 8 dilutions were made by 10-fold dilution, TSLP-His was diluted to 0.4ng/mL (final concentration: 0.1 ng/mL), 25. Mu.L/well in 96-well plates inoculated with cells, and two wells were used for each concentration. The plates were incubated at 37℃for 5h.

Detection reagent useLuciferase ASSAY SYSTEM (cat.#E6120). The test cell plates were removed from the incubator and left at room temperature for 10min to equilibrate the plate temperature to room temperature. Mu.l of the detection reagent is pipetted into 100. Mu.l of the test cell culture. Shaking and mixing for 2-5min to allow the cells to be completely lysed, standing at room temperature for 10min to stabilize luminescence signal, and detecting. And detecting the full wavelength Luminesecnce (RLU) in a chemiluminescent mode of the enzyme-labeled instrument. And (3) analyzing data by utilizing Graphpad software, namely analyzing test data, taking TSLP antibody concentration as an X axis, and correspondingly Luminesecnce (RLU) as a Y axis, selecting a four-parameter equation regression model, and fitting an effect curve.

The results are shown in FIGS. 13-14. Both humanized and affinity matured antibodies can inhibit TSLP-induced downstream STAT5 signaling.

All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.

Claims (17)

1. An anti-TSLP antibody comprising a heavy chain and a light chain, wherein the heavy chain variable region of the heavy chain has a Complementarity Determining Region (CDR) selected from the group consisting of:

1) VH-CDR1 shown in SEQ ID NO 72 or 73,

VH-CDR2 as shown in SEQ ID NO 74, 75 or 76, and

The VH-CDR3 shown in any one of SEQ ID NOs 77 to 87;

2) VH-CDR1 shown in SEQ ID No. 91, VH-CDR2 shown in SEQ ID No. 92, and VH-CDR3 shown in SEQ ID No. 93;

and, the light chain variable region of the light chain has a Complementarity Determining Region (CDR) selected from the group consisting of:

1) VL-CDR1 shown in SEQ ID NO. 88, VL-CDR2 shown in SEQ ID NO. 89, and VL-CDR3 shown in SEQ ID NO. 90, and

2) VL-CDR1 shown in SEQ ID NO. 94, VL-CDR2 shown in SEQ ID NO. 95, and VL-CDR3 shown in SEQ ID NO. 96.

2. The antibody of claim 1, wherein the heavy chain variable region has a Complementarity Determining Region (CDR) selected from the group consisting of:

1) VL-CDR1 shown in SEQ ID NO. 72, VL-CDR2 shown in SEQ ID NO. 74, and VL-CDR3 shown in SEQ ID NO. 77;

2) VL-CDR1 shown in SEQ ID NO. 72, VL-CDR2 shown in SEQ ID NO. 75, and VL-CDR3 shown in SEQ ID NO. 77;

3) VL-CDR1 shown in SEQ ID NO. 72, VL-CDR2 shown in SEQ ID NO. 76, and VL-CDR3 shown in SEQ ID NO. 77;

4) VL-CDR1 shown in SEQ ID NO. 73, VL-CDR2 shown in SEQ ID NO. 76, and VL-CDR3 shown in SEQ ID NO. 77;

5) VL-CDR1 shown in SEQ ID NO. 72, VL-CDR2 shown in SEQ ID NO. 76, and VL-CDR3 shown in SEQ ID NO. 78;

6) VL-CDR1 shown in SEQ ID NO. 72, VL-CDR2 shown in SEQ ID NO. 76, and VL-CDR3 shown in SEQ ID NO. 79;

7) VL-CDR1 shown in SEQ ID NO. 72, VL-CDR2 shown in SEQ ID NO. 76, and VL-CDR3 shown in SEQ ID NO. 80;

8) VL-CDR1 shown in SEQ ID NO. 72, VL-CDR2 shown in SEQ ID NO. 76, and VL-CDR3 shown in SEQ ID NO. 81;

9) VL-CDR1 shown in SEQ ID NO. 72, VL-CDR2 shown in SEQ ID NO. 76, and VL-CDR3 shown in SEQ ID NO. 82;

10 VL-CDR1 shown in SEQ ID NO. 72, VL-CDR2 shown in SEQ ID NO. 76, and VL-CDR3 shown in SEQ ID NO. 83;

11 VL-CDR1 shown in SEQ ID NO. 72, VL-CDR2 shown in SEQ ID NO. 76, and VL-CDR3 shown in SEQ ID NO. 84;

12 VL-CDR1 shown in SEQ ID NO. 72, VL-CDR2 shown in SEQ ID NO. 76, and VL-CDR3 shown in SEQ ID NO. 85;

13 VL-CDR1 shown in SEQ ID NO. 72, VL-CDR2 shown in SEQ ID NO. 76, and VL-CDR3 shown in SEQ ID NO. 86, and

14 VL-CDR1 shown in SEQ ID NO. 72, VL-CDR2 shown in SEQ ID NO. 76, and VL-CDR3 shown in SEQ ID NO. 87;

And the light chain variable region has a Complementarity Determining Region (CDR) of VL-CDR1 shown in SEQ ID NO. 88, VL-CDR2 shown in SEQ ID NO. 89, and VL-CDR3 shown in SEQ ID NO. 90.

3. The antibody of claim 1, wherein the heavy chain variable region has a Complementarity Determining Region (CDR) of VH-CDR1 of SEQ ID NO. 91, VH-CDR2 of SEQ ID NO. 92, and VH-CDR3 of SEQ ID NO. 93;

And the light chain variable region has a Complementarity Determining Region (CDR) of VL-CDR1 shown in SEQ ID NO. 94, VL-CDR2 shown in SEQ ID NO. 95, and VL-CDR3 shown in SEQ ID NO. 96.

4. The antibody of claim 1, wherein the heavy chain variable region has the amino acid sequence set forth in any one of SEQ ID NOs 6, 8, 12-20, 27-60, 64-67, and/or

The light chain variable region has an amino acid sequence shown in any one of SEQ ID NOs 7, 9, 22-26 and 68-71.

5. The antibody of claim 1, wherein the antibody has:

(1) A heavy chain variable region as shown in SEQ ID NO. 19 and a light chain variable region as shown in SEQ ID NO. 23;

(2) A heavy chain variable region as shown in SEQ ID NO. 40 and a light chain variable region as shown in SEQ ID NO. 25;

(3) A heavy chain variable region as set forth in SEQ ID NO. 46 and a light chain variable region as set forth in SEQ ID NO. 25;

(4) A heavy chain variable region as set forth in SEQ ID NO. 64 and a light chain variable region as set forth in SEQ ID NO. 68;

(5) A heavy chain variable region as set forth in SEQ ID NO. 64 and a light chain variable region as set forth in SEQ ID NO. 69;

(6) A heavy chain variable region as set forth in SEQ ID NO. 64 and a light chain variable region as set forth in SEQ ID NO. 70;

(7) A heavy chain variable region as shown in SEQ ID NO. 64 and a light chain variable region as shown in SEQ ID NO. 71;

(8) A heavy chain variable region as set forth in SEQ ID NO. 65 and a light chain variable region as set forth in SEQ ID NO. 68;

(9) A heavy chain variable region as shown in SEQ ID NO. 65 and a light chain variable region as shown in SEQ ID NO. 69;

(10) A heavy chain variable region as set forth in SEQ ID NO. 65 and a light chain variable region as set forth in SEQ ID NO. 70;

(11) A heavy chain variable region as shown in SEQ ID NO. 65 and a light chain variable region as shown in SEQ ID NO. 71;

(12) A heavy chain variable region as set forth in SEQ ID NO. 66 and a light chain variable region as set forth in SEQ ID NO. 68;

(13) A heavy chain variable region as set forth in SEQ ID NO. 66 and a light chain variable region as set forth in SEQ ID NO. 69;

(14) A heavy chain variable region as set forth in SEQ ID NO. 66 and a light chain variable region as set forth in SEQ ID NO. 70;

(15) A heavy chain variable region as set forth in SEQ ID NO. 66 and a light chain variable region as set forth in SEQ ID NO. 71;

(16) A heavy chain variable region as set forth in SEQ ID NO. 67 and a light chain variable region as set forth in SEQ ID NO. 68;

(17) A heavy chain variable region as shown in SEQ ID NO. 67 and a light chain variable region as shown in SEQ ID NO. 69;

(18) A heavy chain variable region as set forth in SEQ ID NO. 67 and a light chain variable region as set forth in SEQ ID NO. 70;

(19) A heavy chain variable region as shown in SEQ ID NO. 67 and a light chain variable region as shown in SEQ ID NO. 71;

(20) A heavy chain variable region as shown in SEQ ID NO. 8 and a light chain variable region as shown in SEQ ID NO. 9, or

(21) A heavy chain variable region as shown in SEQ ID NO. 6 and a light chain variable region as shown in SEQ ID NO. 7.

6. A recombinant protein, wherein said recombinant protein comprises:

(i) The antibody of claim 1, and

(Ii) Optionally a tag sequence to assist expression and/or purification.

7. A Chimeric Antigen Receptor (CAR) construct, the antigen binding region of which comprises a single chain variable fragment (scFv) that specifically binds to TSLP, and which scFv has the heavy chain variable region and the light chain variable region of the antibody of claim 1.

8. A recombinant immunocyte, characterized in that, the immune cell expresses an exogenous CAR construct according to claim 7.

9. A pharmaceutical composition, in the form of a capsule, the pharmaceutical composition comprises:

(i) An active ingredient selected from the group consisting of an antibody according to claim 1, a recombinant protein according to claim 6, an immune cell according to claim 8, or a combination thereof, and

(Ii) A pharmaceutically acceptable carrier.

10. Use of an antibody according to claim 1, or a recombinant protein according to claim 6, or an immune cell according to claim 8 and/or a pharmaceutical composition according to claim 9 in the manufacture of a medicament for the treatment of a disease associated with TSLP expression or dysfunction.

11. A polynucleotide encoding a polypeptide selected from the group consisting of:

(1) The antibody of claim 1;

(2) The recombinant protein according to claim 6, or

(3) The Chimeric Antigen Receptor (CAR) construct of claim 7.

12. A carrier, characterized in that, the vector comprising the polynucleotide of claim 11.

13. An engineered host cell containing the vector or genome of claim 12 having the polynucleotide of claim 11 integrated therein.

14. A method of detecting TSLP in a sample, the method comprising the steps of:

(1) Contacting the sample with the antibody of claim 1;

(2) Detecting whether an antigen-antibody complex is formed, wherein the formation of a complex indicates the presence of TSLP in the sample.

15. A test plate comprising a substrate and a test strip comprising the antibody of claim 1, the recombinant protein of claim 6, the immune cell of claim 8, or a combination thereof.

16. A kit of parts for the manufacture of a kit, the kit comprises the following components:

(1) A first container comprising the antibody according to claim 1, and/or

(2) A second container comprising a second antibody against the antibody of claim 1;

or alternatively

The kit comprising the assay plate of claim 15.

17. A method of producing a recombinant polypeptide, the method comprising:

(a) Culturing the host cell of claim 13 under conditions suitable for expression;

(b) Isolating the recombinant polypeptide from the culture, wherein the recombinant polypeptide is the antibody of claim 1 or the recombinant protein of claim 6.