Anti-TSLP antibody and application thereof

Abstract

The invention provides an anti-TSLP antibody and an application thereof. The anti-TSLP antibody disclosed by the invention can be specifically combined with human / monkey TSLP protein with high affinity, and can block the combination of TSLP and a receptor TSLPR thereof, so that a downstream STAT5 signal channel induced by TSLP is inhibited, and the anti-TSLP antibody has a function of treating TSLP related diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to an anti-TSLP antibody and its application. Background Art

[0002] Thymic stromal lymphopoietin (TSLP) is a potential target for allergic diseases, tumors, and immune diseases. TSLP promotes B cell differentiation and can also co-stimulate thymocytes and mature T cells. TSLP binds to human CD11c + A specific heterodimeric receptor on dendritic cells (DCs). The receptor heterodimer consists of a heterodimer of a common γ-like receptor chain (TSLP receptor; TSLPR) and an IL7Rα chain. Ligand binding to the receptor induces DCs to secrete Th2-attracting chemokines, TARC (thymus and activation-regulated chemokine) and MDC (macrophage-derived chemokine). TSLP signals can also lead to the activation of STAT5 and STAT3 transcription factors and initiate the expression of downstream genes.

[0003] TSLP can promote the differentiation of Th0 cells into Th2 cells by activating myeloid dendritic cells (mDC), producing a large amount of Th2 cytokines such as IL-4, IL-13, IL-5 and TNF-α, thus leading to the occurrence of allergic inflammation. + T cells and mast cells, enhancing Th2-type inflammatory responses. It has been reported that patients with acute and chronic atopic dermatitis overexpress TSLP in 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.

[0004] With the deepening of TSLP / TSLPR research mechanism, the immunomodulatory function of TSLP antibodies has become increasingly clear, and the use of TSLP antibodies in treating allergic diseases, tumor diseases, and immune diseases has been verified. Using TSLP as a target, the development of antibody drugs is not only theoretically feasible, but also has practical significance. Based on the current theoretical and basic research results, it can be expected that TSLP antibodies are expected to have application prospects for a variety of indications. Summary of the invention

[0005] The purpose of the present invention is to provide an anti-TSLP antibody and its application.

[0006] In a first aspect of the present invention, an anti-TSLP antibody is provided, the antibody comprising a heavy chain and a light chain, wherein the heavy chain variable region has a complementary determining region (CDR) selected from the following group:

[0007] 1) VH-CDR1 shown in SEQ ID NO: 72 or 73,

[0008] VH-CDR2 shown in SEQ ID NO:74, 75 or 76, and

[0009] VH-CDR3 shown in any one of SEQ ID NOs: 77-87;

[0010] 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;

[0011] Furthermore, the light chain variable region has a complementarity determining region (CDR) selected from the group consisting of:

[0012] 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

[0013] 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.

[0014] In another preferred embodiment, the heavy chain variable region has a complementarity determining region (CDR) selected from the following group:

[0015] 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;

[0016] 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;

[0017] 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;

[0018] 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;

[0019] 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;

[0020] 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;

[0021] 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;

[0022] 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;

[0023] 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;

[0024] 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;

[0025] 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;

[0026] 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;

[0027] 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

[0028] 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;

[0029] Furthermore, the light chain variable region has the following complementarity determining regions (CDRs): 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.

[0030] In another preferred embodiment, the heavy chain variable region has the following complementarity determining regions (CDRs): 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;

[0031] Furthermore, the light chain variable region has the following complementarity determining regions (CDRs): 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.

[0032] Any of the amino acid sequences in the above CDR sequences also includes a derivative sequence that is optionally supplemented, deleted, modified and / or substituted with at least one amino acid, and enables the derivative antibody composed of the heavy chain and light chain containing the derived CDR sequence to retain TSLP binding affinity.

[0033] In another preferred embodiment, the antibody specifically binds to TSLP protein.

[0034] In another preferred embodiment, the TSLP protein is human TSLP protein, cynomolgus monkey TSLP protein, or mouse TSLP protein.

[0035] In another preferred embodiment, the amino acid sequence of the human TSLP protein is shown in SEQ ID NO.1.

[0036] In another preferred example, the amino acid sequence of the cynomolgus monkey TSLP protein is shown as SEQ ID NO.2.

[0037] In another preferred embodiment, the antibody blocks the binding of TSLP to TSLP receptor.

[0038] In another preferred embodiment, the TSLP receptor is TSLPR or TSLPR-IL7Rα heterodimer.

[0039] In another preferred embodiment, the blocking refers to reducing the binding rate of the TSLP receptor to TSLP by 50%, preferably by 70%, and more preferably by 90%.

[0040] In another preferred example, the heavy chain variable region of the antibody further includes a human framework region, and / or the light chain variable region of the antibody further includes a human framework region.

[0041] In another preferred example, the heavy chain variable region of the antibody further includes a framework region of mouse origin, and / or the light chain variable region of the antibody further includes a framework region of mouse origin.

[0042] In another preferred example, 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 with the amino acid sequence shown in any one of SEQ ID NOs: 6, 8, 12-20, 27-60, 64-67 in the sequence listing.

[0043] In another preferred example, the heavy chain variable region has an amino acid sequence shown in any one of SEQ ID NOs: 6, 8, 12-20, 27-60, 64-67.

[0044] In another preferred embodiment, the heavy chain variable region has a mutation selected from the following group relative to the sequence shown in SEQ ID NO.19:

[0045] V2Q, K23Q, K23W, K23E, A24F, S25Q, S25W, S25D, G26Q, G26E, F27Y, N28T, I29E, K30D, K30T, N32F, T74D, T74E, T74N, K30D and T74E, S75D, K30T and T74E and S75D, N77D, N77E, F100V, F100W, Y101E, Y101V, G102S, G102H, G102K, S103H, S103K, Y105Q or a combination thereof.

[0046] In another preferred example, 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 with the amino acid sequence shown in any one of SEQ ID NOs: 7, 9, 22-26, 68-71 in the sequence listing.

[0047] In another preferred example, the light chain variable region has an amino acid sequence shown in any one of SEQ ID NOs: 7, 9, 22-26, 68-71.

[0048] In another preferred example, the heavy chain variable region of the antibody contains the amino acid sequence shown in any one of SEQ ID NOs: 6, 8, 12-20, 27-60, 64-67; and the light chain variable region of the antibody contains the amino acid sequence shown in any one of SEQ ID NOs: 7, 9, 22-26, 68-71.

[0049] In another preferred embodiment, the antibody has:

[0050] (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;

[0051] (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;

[0052] (3) a heavy chain variable region as shown in SEQ ID NO:46 and a light chain variable region as shown in SEQ ID NO:25;

[0053] (4) a heavy chain variable region as shown in SEQ ID NO:64 and a light chain variable region as shown in SEQ ID NO:68;

[0054] (5) a heavy chain variable region as shown in SEQ ID NO:64 and a light chain variable region as shown in SEQ ID NO:69;

[0055] (6) a heavy chain variable region as shown in SEQ ID NO:64 and a light chain variable region as shown in SEQ ID NO:70;

[0056] (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;

[0057] (8) a heavy chain variable region as shown in SEQ ID NO:65 and a light chain variable region as shown in SEQ ID NO:68;

[0058] (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;

[0059] (10) the heavy chain variable region shown in SEQ ID NO:65 and the light chain variable region shown in SEQ ID NO:70;

[0060] (11) the heavy chain variable region shown in SEQ ID NO:65 and the light chain variable region shown in SEQ ID NO:71;

[0061] (12) a heavy chain variable region as shown in SEQ ID NO:66 and a light chain variable region as shown in SEQ ID NO:68;

[0062] (13) the heavy chain variable region shown in SEQ ID NO:66 and the light chain variable region shown in SEQ ID NO:69;

[0063] (14) the heavy chain variable region shown in SEQ ID NO:66 and the light chain variable region shown in SEQ ID NO:70;

[0064] (15) the heavy chain variable region shown in SEQ ID NO:66 and the light chain variable region shown in SEQ ID NO:71;

[0065] (16) the heavy chain variable region shown in SEQ ID NO:67 and the light chain variable region shown in SEQ ID NO:68;

[0066] (17) the heavy chain variable region shown in SEQ ID NO:67 and the light chain variable region shown in SEQ ID NO:69;

[0067] (18) the heavy chain variable region shown in SEQ ID NO:67 and the light chain variable region shown in SEQ ID NO:70;

[0068] (19) the heavy chain variable region shown in SEQ ID NO:67 and the light chain variable region shown in SEQ ID NO:71;

[0069] (20) the heavy chain variable region shown in SEQ ID NO: 8 and the light chain variable region shown in SEQ ID NO: 9; or

[0070] (21) The heavy chain variable region shown in SEQ ID NO:6 and the light chain variable region shown in SEQ ID NO:7.

[0071] In another preferred embodiment, the antibody is selected from the following group: a murine antibody, a chimeric antibody, a humanized antibody, a fully human antibody, or a combination thereof.

[0072] In another preferred embodiment, the light chain and / or heavy chain of the antibody further includes a constant region.

[0073] In another preferred embodiment, the constant region is of human or mouse origin.

[0074] In another preferred embodiment, the constant region of the heavy chain is derived from IgG, preferably from human IgG, and more preferably from human IgG4 heavy chain constant region.

[0075] In another preferred embodiment, the constant region of the light chain is derived from IgG, preferably from human IgG, more preferably from human IgG4 kappa chain.

[0076] In another preferred example, the constant region sequence of the heavy chain is shown as SEQ ID NO.10.

[0077] In another preferred example, the constant region sequence of the light chain is shown as SEQ ID NO.11.

[0078] In a second aspect of the present invention, a recombinant protein is provided, wherein the recombinant protein comprises:

[0079] (i) the antibody according to the first aspect of the present invention; and

[0080] (ii) optionally a tag sequence to facilitate expression and / or purification.

[0081] In another preferred embodiment, the tag sequence includes a 6His tag, a GGGS sequence, and a FLAG tag.

[0082] In another preferred embodiment, the recombinant protein (or polypeptide) includes a fusion protein.

[0083] In another preferred embodiment, the recombinant protein is a monomer, a dimer, or a polymer.

[0084] In another preferred embodiment, the recombinant protein is specific for anti-TSLP.

[0085] In another preferred embodiment, the recombinant protein is a fusion protein.

[0086] In another preferred embodiment, the fusion protein is a monospecific antibody (ie, a monospecific antibody against TSLP), a bispecific antibody, or a multispecific antibody (eg, a trispecific antibody).

[0087] In another preferred embodiment, the bispecific antibody or multispecific antibody is not only anti-TSLP, but also specifically binds to additional target antigens (such as other tumor antigens).

[0088] In the third aspect of the present invention, a chimeric antigen receptor (CAR) construct is provided, wherein the antigen binding region of the chimeric antigen receptor construct includes a single-chain variable fragment (scFv) that specifically binds to TSLP, and the scFv has the heavy chain variable region and light chain variable region of the antibody as described in the first aspect of the present invention.

[0089] In the fourth aspect of the present invention, a recombinant immune cell is provided, wherein the immune cell expresses an exogenous CAR construct as described in the third aspect of the present invention.

[0090] In another preferred embodiment, the immune cells are selected from the following group: NK cells and T cells.

[0091] In another preferred embodiment, the immune cells are from humans or non-human mammals (such as mice).

[0092] In a fifth aspect of the present invention, a use of an active ingredient is provided, wherein the active ingredient is selected from the group consisting of the antibody according to the first aspect of the present invention, the recombinant protein according to the second aspect of the present invention, the immune cell according to the fourth aspect of the present invention, or a combination thereof, wherein the active ingredient is used for:

[0093] (a) preparing diagnostic reagents, test panels or test kits; and / or

[0094] (b) preparing drugs for preventing and / or treating diseases associated with abnormal TSLP expression or function.

[0095] In another preferred embodiment, the reagent is used to detect TSLP.

[0096] In another preferred embodiment, the reagent, detection plate or kit is used to detect diseases related to abnormal TSLP expression or function.

[0097] In another preferred embodiment, the medicament is used to prevent and / or treat allergic diseases, tumors or cancers, or immune diseases.

[0098] In another preferred embodiment, the agent is used to prevent and / or treat a disease selected from the following group: asthma (such as moderate to severe asthma), atopic dermatitis, urticaria, chronic obstructive pulmonary disease (COPD), chronic rhinitis and nasal polyps, eosinophilic esophagitis, or a combination thereof.

[0099] In a sixth aspect of the present invention, a pharmaceutical composition is provided, comprising:

[0100] (i) an active ingredient, wherein the active ingredient is selected from the group consisting of the antibody according to the first aspect of the present invention, the recombinant protein according to the second aspect of the present invention, the immune cell according to the fourth aspect of the present invention, or a combination thereof; and

[0101] (ii) a pharmaceutically acceptable carrier.

[0102] In another preferred embodiment, the pharmaceutical composition is a liquid preparation.

[0103] In another preferred embodiment, the pharmaceutical composition is an injection.

[0104] In another preferred example, the pharmaceutical composition comprises 0.01 to 99.99% of the antibody as described in the first aspect of the present invention, the recombinant protein as described in the second aspect of the present invention, the immune cell as described in the fourth aspect of the present invention, or a combination thereof and 0.01 to 99.99% of a pharmaceutical carrier, wherein the percentage is the mass percentage of the pharmaceutical composition.

[0105] In another preferred embodiment, the pharmaceutical composition is used to prevent and / or treat allergic diseases, tumors or cancers, or immune diseases.

[0106] In the seventh aspect of the present invention, a polynucleotide is provided, which encodes a polypeptide selected from the following group:

[0107] (1) The antibody according to the first aspect of the present invention;

[0108] (2) the recombinant protein as described in the second aspect of the present invention; or

[0109] (3) The chimeric antigen receptor (CAR) construct as described in the third aspect of the present invention.

[0110] In the eighth aspect of the present invention, a vector is provided, wherein the vector contains the polynucleotide as described in the seventh aspect of the present invention.

[0111] In another preferred embodiment, the vector includes: bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus.

[0112] In another preferred embodiment, the vector is a plasmid, an adenovirus, a retrovirus, or other vectors.

[0113] In the ninth aspect of the present invention, an engineered host cell is provided, wherein the host cell contains the vector as described in the eighth aspect of the present invention or the polynucleotide as described in the seventh aspect of the present invention is integrated into the genome.

[0114] In a tenth aspect of the present invention, a method for detecting TSLP in a sample is provided, the method comprising the steps of:

[0115] (1) contacting the sample with the antibody according to the first aspect of the present invention;

[0116] (2) Detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of TSLP in the sample.

[0117] In another preferred embodiment, the detection is for in vitro non-therapeutic and non-diagnostic purposes.

[0118] In the eleventh aspect of the present invention, a composition for detecting TSLP in a sample in vitro is provided, which comprises the antibody as described in the first aspect of the present invention, the recombinant protein as described in the second aspect of the present invention, the immune cell as described in the fourth aspect of the present invention, or a combination thereof as active ingredients.

[0119] In the twelfth aspect of the present invention, a detection plate is provided, which comprises: a substrate (support plate) and a test strip, wherein the test strip contains the antibody described in the first aspect of the present invention, the recombinant protein described in the second aspect of the present invention, the immune cell described in the fourth aspect of the present invention, or a combination thereof.

[0120] In the thirteenth aspect of the present invention, a kit is provided, comprising:

[0121] (1) a first container, wherein the first container contains the antibody according to the first aspect of the present invention; and / or

[0122] (2) a second container, wherein the second container contains a secondary antibody against the antibody described in the first aspect of the present invention;

[0123] or,

[0124] The kit contains the detection plate as described in the twelfth aspect of the present invention.

[0125] In a fourteenth aspect of the present invention, a method for preparing a recombinant polypeptide is provided, the method comprising:

[0126] (a) culturing the host cell according to the ninth aspect of the present invention under conditions suitable for expression;

[0127] (b) isolating a recombinant polypeptide from the culture, wherein the recombinant polypeptide is the antibody according to the first aspect of the present invention or the recombinant protein according to the second aspect of the present invention.

[0128] In the fifteenth aspect of the present invention, there is provided the use of the antibody as described in the first aspect of the present invention, or the recombinant protein as described in the second aspect of the present invention, or the immune cell as described in the fourth aspect of the present invention and / or the combination of the pharmaceutical composition as described in the sixth aspect of the present invention and a drug in the preparation of a drug for treating diseases related to abnormal TSLP expression or function.

[0129] In another preferred embodiment, the abnormal expression of TSLP refers to overexpression of TSLP.

[0130] In another preferred embodiment, the overexpression refers to the ratio of the expression level of TSLP (F1) to the expression level under physiological conditions (F0) (ie, F1 / F0) being ≥1.5, preferably ≥2, and more preferably ≥2.5.

[0131] In another preferred embodiment, the drug is used to prevent and / or treat allergic diseases, tumors or cancers, or immune diseases.

[0132] In another preferred embodiment, the drug is used to prevent and / or treat a disease selected from the following group: asthma (such as moderate to severe asthma), atopic dermatitis, urticaria, chronic obstructive pulmonary disease (COPD), chronic rhinitis and nasal polyps, eosinophilic esophagitis, or a combination thereof.

[0133] In the sixteenth aspect of the present invention, a method for treating a disease associated with abnormal TSLP expression or function is provided, comprising administering to a subject in need thereof an effective amount of the antibody as described in the first aspect of the present invention, or the recombinant protein as described in the second aspect of the present invention, or the immune cell as described in the fourth aspect of the present invention, or the pharmaceutical composition as described in the sixth aspect of the present invention, or a combination thereof.

[0134] In another preferred embodiment, the disease associated with abnormal TSLP expression or function is an allergic disease, a tumor or cancer, or an immune disease.

[0135] In another preferred embodiment, the disease associated with abnormal TSLP expression or function is a disease selected from the following group: asthma (such as moderate to severe asthma), atopic dermatitis, urticaria, chronic obstructive pulmonary disease (COPD), chronic rhinitis and nasal polyps, eosinophilic esophagitis, or a combination thereof.

[0136] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0137] The following drawings are used to illustrate specific embodiments of the present invention and are not used to limit the scope of the present invention defined by the claims.

[0138] Figure 1 The results of ELISA detection of humanized antibodies binding to human TSLP are shown.

[0139] Figure 2 The ELISA results of the binding of humanized antibodies to cynomolgus monkey TSLP are shown.

[0140] Figure 3 ELISA results showing the binding of affinity matured antibodies to human TSLP.

[0141] Figure 4 ELISA results of affinity matured antibodies binding to cynomolgus monkey TSLP are shown.

[0142] Figure 5 The ELISA test results showing the receptor-ligand (human TSLP-human TSLPR) binding blocking ability of the humanized antibody.

[0143] Figure 6 ELISA test results showing the receptor-ligand (human TSLP-human TSLPR) binding blocking ability of affinity matured antibodies.

[0144] Figure 7 The ELISA test results of the receptor-ligand (cynomolgus monkey TSLP-cynomolgus monkey TSLPR) binding blocking ability of the humanized antibodies are shown.

[0145] Figure 8 ELISA results showing the receptor-ligand (cynomolgus TSLP-cynomolgus TSLPR) binding blocking ability of affinity matured antibodies.

[0146] Figure 9 The results of flow cytometry analysis show the ability of humanized antibodies to block the binding of human TSLP to cell surface receptors.

[0147] Figure 10 The results of flow cytometry analysis show the ability of affinity matured antibodies to block the binding of human TSLP to cell surface receptors.

[0148] Figure 11 The effect of humanized antibodies on the proliferation of BaF3-TSLPR / IL7Rα cells is shown.

[0149] Figure 12 The effect of affinity matured antibodies on the proliferation of BaF3-TSLPR / IL7Rα cells is shown.

[0150] Figure 13 The results of reporter gene assay showing the function of humanized antibodies.

[0151] Figure 14 Reporter gene assay results showing affinity matured antibody functionality. DETAILED DESCRIPTION

[0152] After extensive and in-depth research, the inventors have obtained a class of monoclonal antibodies against TSLP. The present invention also provides humanized antibodies and affinity mature antibodies against TSLP. The anti-TSLP antibodies of the present invention can specifically bind to human / cynomolgus monkey TSLP protein with high affinity, and can block the binding of TSLP to the cell surface receptor TSLPR, inhibit the downstream STAT5 signaling pathway induced by TSLP, and achieve the function of treating TSLP-related diseases. On this basis, the present invention is completed.

[0153] the term

[0154] In order to more easily understand the present invention, some technical and scientific terms are specifically defined below. Unless otherwise clearly defined in this article, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs. Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, because such methods and conditions can be changed. It should also be understood that the terms used herein are intended only to describe specific embodiments, and are not intended to be restrictive, and the scope of the present invention will be limited only by the appended claims.

[0155] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. As used herein, the term "about" when used in reference to a specific recited value means that the value may vary by 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.).

[0156] 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).

[0157] As used herein, the term "treatment" refers to administering an internal or external therapeutic agent, including antibodies to TSLP of the present invention and compositions thereof, to a patient who has one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered to the patient in an amount effective to alleviate one or more symptoms of the disease (therapeutically effective amount).

[0158] As used herein, the terms "optional" or "optionally" mean that the subsequently described event or circumstance may occur but need not occur.

[0159] "Sequence identity" as used herein refers to the degree of identity between two nucleic acid or amino acid sequences when optimally aligned and compared with appropriate mutations such as substitutions, insertions or deletions. The sequence identity between the sequences described herein and the sequences with which they are identical 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%.

[0160] TSLP

[0161] Thymic stromal lymphopoietin (TSLP) is a short-chain cytokine with a four-helix bundle folding structure. Similar to IL-7, it belongs to the IL-2 cytokine family. It was first isolated from the culture supernatant of thymic stromal cells by Firend et al. and was named after it. TSLP protein is a short-chain type I cytokine composed of four helical bundles, A, B, C, and D. There are two isoforms of human TSLP, isoform I and isoform II. The former is a protein of 159 amino acids and the latter is a protein of 60 amino acids. The human TSLP gene is located on chromosome 5q22.1, adjacent to the atopic cytokine gene cluster 5q31, which mainly encodes Th2-type cytokines IL-4, IL-5, IL-9, and IL-13. The highest levels of TSLP are found in epithelial cells of the lung and skin, and fibroblasts, ASMCs (airway smooth muscle cells), endothelial cells, mast cells, macrophages / monocytes, granulocytes, and DCs (dendritic cells) can also produce TSLP.

[0162] Antibody

[0163] As used herein, the term "antibody" or "immunoglobulin" is a heterotetrameric glycoprotein of about 150,000 daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes varies. 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 multiple 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 is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.

[0164] As used herein, the term "variable" means that some parts of the variable region in an antibody are different in sequence, which forms the binding and specificity of various specific antibodies to their specific antigens. However, variability is not evenly distributed throughout the variable region of an antibody. It is concentrated in three fragments called complementary determining regions (CDRs) or hypervariable regions in the variable regions of light and heavy chains. The more conservative part of the variable region is called the framework region (FR). The variable regions of natural heavy and light chains each contain four FR regions, which are roughly in a β-folded configuration, connected by three CDRs forming a connecting loop, and in some cases can form a partial β-folded structure. The CDRs in each chain are closely together through the FR region and together with the CDRs of the other chain form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Volume I, 647-669 pages (1991)). The constant region does not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in the antibody's antibody-dependent cytotoxicity.

[0165] The "light chains" of vertebrate antibodies (immunoglobulins) can be classified into one of two distinct classes (called kappa and lambda) based on the amino acid sequence of their constant regions. Immunoglobulins can be divided into different classes based on the amino acid sequence of their heavy chain constant regions. There are five main classes of 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 the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structure and three-dimensional configuration of the different classes of immunoglobulins are well known in the art.

[0166] As used herein, the term "monoclonal antibody (mAb)" 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 (which typically have different antibodies directed against different determinants), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they are synthesized by hybridoma culture and are not contaminated by other immunoglobulins. The modifier "monoclonal" indicates the characteristic of the antibody, which is obtained from a substantially homogeneous antibody population, and should not be construed as requiring any special method to produce the antibody.

[0167] Generally, the antigen binding properties of an antibody can be described by three specific regions located in the variable regions of the heavy and light chains, called variable regions (CDRs). This segment is divided into four framework regions (FRs). The amino acid sequences of the four FRs are relatively conservative and do not directly participate in the binding reaction. These CDRs form a ring structure, and the β-folds formed by the FRs in between are close to each other in spatial structure. The CDRs on the heavy chain and the CDRs on the corresponding light chains constitute the antigen binding site of the antibody. The amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR region.

[0168] 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 to an antigen. It has been shown that fragments of full-length antibodies can be used to perform the antigen-binding function of an antibody. Examples of binding fragments included in the term "antigen-binding fragment of an antibody" include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments connected by a disulfide bridge on the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VH and VL domains of a single arm of an antibody. The Fv antibody contains the variable region of the antibody heavy chain, the variable region of the light chain, but no constant region, and is the smallest antibody fragment with all antigen binding sites. Generally, the Fv antibody also contains a polypeptide linker between the VH and VL domains, and is capable of forming the structure required for antigen binding.

[0169] The present invention includes not only complete monoclonal antibodies, but also antibody fragments with immunological activity or fusion proteins formed by antibodies and other sequences, such as Fab or (Fab')2 fragments; antibody heavy chains; antibody light chains. Therefore, the present invention also includes fragments, derivatives and analogs of the antibodies.

[0170] The term "epitope" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 consecutive or non-continuous amino acids in a unique spatial conformation. An epitope can be a discontinuous three-dimensional site on an antigen that is recognized by an antibody or antigen-binding fragment of the invention.

[0171] The terms "specific binding", "selective binding", "selectively binds" and "specifically binds" refer to the binding of an antibody to a predetermined epitope on an antigen. -7 M, for example, less than about 10 -8 M, 10 -9 M or l0 -10In one embodiment of the present invention, the anti-TSLP antibody of the present invention can bind with an affinity (KD) of 10 M or less. -11 M, l0 -12 M or less affinity (KD) binds to the TSLP molecule.

[0172] In the present invention, antibodies include murine, chimeric, humanized or fully human antibodies prepared using techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, including human and non-human parts, can be obtained by standard DNA recombinant techniques, and they are all useful antibodies. A chimeric antibody is a molecule in which different parts are from different animal species, such as a chimeric antibody having a variable region from a monoclonal antibody from a mouse, and a constant region from a human immunoglobulin (see, for example, U.S. Pat. No. 4,816,567 and U.S. Pat. No. 4,816,397, which are hereby incorporated by reference in their entirety). A humanized antibody refers to an antibody molecule derived from a non-human species, having one or more complementary determining regions (CDRs) derived from a non-human species and a framework region derived from a human immunoglobulin molecule (see U.S. Pat. No. 5,585,089, which is hereby incorporated by reference in its entirety). These chimeric and humanized monoclonal antibodies can be prepared using DNA recombinant techniques well known in the art.

[0173] In the present invention, the antibodies may be monospecific, bispecific, trispecific, or more multispecific.

[0174] As used herein, the term "heavy chain variable region" is used interchangeably with "VH".

[0175] As used herein, the term "light chain variable region" is used interchangeably with "VL."

[0176] As used herein, the term "complementarity determining region (CDR)" refers to the hypervariable region within the variable domain of an antibody that primarily contributes to antigen binding. One of the most commonly used definitions of the CDR is provided by Kabat E.A et al. (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242). In addition, IMGT (Lefranc, 2003) and Chothia (Al-Lazikani, 1997) both provide CDR definition rules, which are well known to those skilled in the art.

[0177] In a preferred embodiment of the present invention, the heavy chain of the antibody includes a heavy chain variable region and a heavy chain constant region, and the heavy chain constant region may be of murine or human origin, for example, derived from human IgG4.

[0178] In a preferred embodiment of the present invention, the light chain of the antibody comprises a light chain variable region and a light chain constant region, and the light chain constant region may be of murine or human origin, for example, derived from human IgG4κ chain.

[0179] In the present invention, the antibody of the present invention also includes its conservative variants, which refers to a polypeptide formed by replacing at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids with amino acids of similar or similar properties compared to the amino acid sequence of the antibody of the present invention. These conservative variant polypeptides are preferably produced by amino acid substitution according to Table A.

[0180] Table A

[0181]

[0182]

[0183] Anti-TSLP Antibodies

[0184] As used herein, the terms "antibody of the present invention", "anti-TSLP antibody of the present invention" and "TSLP antibody of the present invention" are used interchangeably and all refer to the antibody targeting TSLP as described in the first aspect of the present invention.

[0185] The function of the antibody of the present invention is determined by the specific gene sequence of the light chain and heavy chain variable region genes of the antibody. 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 TSLPR / IL7R surface receptor on the cell surface, and can inhibit the proliferation of target cells that highly express TSLPR / IL7Rα, and inhibit the expression of the STAT5 gene therein. Using the variable region gene or complementary determining region (CDR) gene of the antibody of the present invention, different forms of genetically engineered antibodies can be transformed and produced in any expression system using prokaryotic and eukaryotic cells.

[0186] The heavy chain variable region of the anti-TSLP antibody of the present invention has a complementarity determining region (CDR) selected from the following group:

[0187] 1) VH-CDR1 shown in SEQ ID NO: 72 or 73,

[0188] VH-CDR2 shown in SEQ ID NO:74, 75 or 76, and

[0189] VH-CDR3 shown in any one of SEQ ID NOs: 77-87;

[0190] 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;

[0191] Furthermore, the light chain variable region of the antibody has a complementarity determining region (CDR) selected from the group consisting of:

[0192] 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

[0193] 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.

[0194] The amino acid sequence also includes a sequence formed by adding, deleting, modifying and / or replacing at least one amino acid sequence, preferably an amino acid sequence with a homology or sequence identity of at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95%.

[0195] The light chain and / or heavy chain of the antibody of the present invention, or its light chain variable region and / or heavy chain variable region may also have a signal peptide (e.g. at the N-terminus) that facilitates antibody expression or secretion. The optional signal peptide is shown in, for example, SEQ ID NO. 21 or 63.

[0196] Methods for determining sequence homology or identity known to those of ordinary skill in the art include, but are not limited to: Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM and Griffin, HG, ed., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987 and Sequence Analysis Primer, Gribskov, M. and Devereux, J., ed., Stockton Press, New York, 1991 and Carillo, H. and Lipman, D., SIAM J. Applied Math., 48:1073 (1988). The preferred method for determining identity is to obtain the largest match between the sequences tested. Methods for determining identity are compiled in publicly available computer programs. Preferred computer program methods for determining the identity between two sequences include, but are not limited to, the GCG program package (Devereux, J. et al., 1984), BLASTP, BLASTN and FASTA (Altschul, S, F. et al., 1990). The BLASTX program is publicly available from NCBI and other sources (BLAST Manual, 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.

[0197] Preferably, the antibody described herein is one or more of a full-length antibody protein, an antigen-antibody binding domain protein fragment, a bispecific antibody, a multispecific antibody, a single-chain antibody (scFv), a single-domain antibody (sdAb), and a single-domain antibody (Signle-domain antibody), as well as a monoclonal antibody or a polyclonal antibody prepared from the above antibodies. The monoclonal antibody can be prepared by a variety of approaches and techniques, including hybridoma technology, phage display technology, single lymphocyte gene cloning technology, etc. The mainstream is to prepare monoclonal antibodies from wild-type or transgenic mice by hybridoma technology.

[0198] The full-length antibody protein is a conventional full-length antibody protein in the art, which includes 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 light chain variable region of the protein and the human heavy chain constant region and the human light chain constant region constitute a full-length human antibody protein. Preferably, the full-length antibody protein is IgG1, IgG2, IgG3 or IgG4 type.

[0199] The antibody of the present invention may be a double-chain or single-chain antibody, and may be selected from animal-derived antibodies, chimeric antibodies, and humanized antibodies, more preferably humanized antibodies, human-animal chimeric antibodies, and more preferably fully humanized antibodies.

[0200] The antibody derivatives of the present invention can be single-chain antibodies and / or antibody fragments, such as Fab, Fab', (Fab')2 or other known antibody derivatives in the field, as well as any one or more of IgA, IgD, IgE, IgG and IgM antibodies or other subtypes of antibodies.

[0201] The single-chain antibody is a conventional single-chain antibody in the art, which includes a heavy chain variable region, a light chain variable region and a short peptide of 15 to 20 amino acids.

[0202] Wherein, the animal is preferably a mammal, such as a mouse.

[0203] The antibody of the present invention may be a chimeric antibody, a humanized antibody, a CDR-grafted and / or modified antibody targeting TSLP (eg, human TSLP, cynomolgus monkey TSLP, or mouse TSLP).

[0204] In the above content of the present invention, the number of added, deleted, modified and / or substituted amino acids is preferably not more than 40% of the total number of amino acids in the initial amino acid sequence, more preferably not more than 35%, more preferably 1-33%, more preferably 5-30%, more preferably 10-25%, more preferably 15-20%.

[0205] In the above content of the present invention, more preferably, the number of the added, deleted, modified and / or substituted amino acids may be 1-7, more preferably 1-5, more preferably 1-3, more preferably 1-2.

[0206] Recombinant protein

[0207] The present invention also provides a recombinant protein, which includes one or more of the heavy chain CDR1 (VH-CDR1), heavy chain CDR2 (VH-CDR2) and heavy chain CDR3 (VH-CDR3) of the antibody of the present invention, and / or one or more of the light chain CDR1 (VL-CDR1), light chain CDR2 (VL-CDR2) and light chain CDR3 (VL-CDR3) of the antibody of the present invention.

[0208] 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 antibody heavy chain constant region or a 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.

[0209] In another preferred embodiment, the recombinant protein includes the antibody of the present invention.

[0210] The recombinant protein is a conventional protein in the art, preferably, it is one or more of a full-length antibody protein, an antigen-antibody binding domain protein fragment, a bispecific antibody, a multispecific antibody, a single chain antibody (scFv), a single domain antibody (sdAb) and a single-domain antibody (Signle-domainantibody), as well as a monoclonal antibody or a polyclonal antibody prepared from the above antibodies.

[0211] The single-chain antibody is a conventional single-chain antibody in the art, which includes a heavy chain variable region, a light chain variable region and a short peptide of 15 to 20 amino acids.

[0212] The antigen-antibody binding domain protein fragment is a conventional antigen-antibody binding domain protein fragment in the art, which includes a light chain variable region, a light chain constant region and an Fd segment of a heavy chain constant region. Preferably, the antigen-antibody binding domain protein fragment is Fab and F(ab')2.

[0213] The single-domain antibody is a conventional single-domain antibody in the art, which includes a heavy chain variable region and a heavy chain constant region.

[0214] The single-domain antibody is a conventional single-domain antibody in the art, which only includes the heavy chain variable region.

[0215] Wherein, the preparation method of the recombinant protein is a conventional preparation method in the art. The preparation method is preferably: obtaining by separation from an expression transformant that recombinantly expresses the protein or obtaining by artificially synthesizing a protein sequence. The separation from the expression transformant that recombinantly expresses the protein is preferably the following method: cloning a polynucleotide molecule encoding the protein and having a point mutation into a recombinant vector, transforming the obtained recombinant vector into a transformant, obtaining a recombinant expression transformant, and culturing the obtained recombinant expression transformant to obtain the recombinant protein.

[0216] Polynucleotide

[0217] The present invention also provides a polynucleotide encoding the above-mentioned antibody or recombinant protein of the present invention or a chimeric antigen receptor (CAR) construct of the antibody of the present invention.

[0218] The method for preparing the polynucleotide is a conventional method in the art, and preferably comprises the following steps: obtaining a nucleic acid molecule encoding the above protein by gene cloning technology, or obtaining a nucleic acid molecule encoding the above protein by artificial full sequence synthesis.

[0219] Those skilled in the art know that the base sequence encoding the amino acid sequence of the above-mentioned protein can be appropriately introduced with substitution, deletion, change, insertion or addition to provide a polynucleotide homologue. The polynucleotide homologue of the present invention can be prepared by replacing, deleting or adding one or more bases of the gene encoding the protein sequence within the range of maintaining the antibody activity.

[0220] Carrier

[0221] The present invention also provides a recombinant expression vector comprising the polynucleotide.

[0222] The recombinant expression vector can be obtained by conventional methods in the art, that is, the polynucleotide molecule of the present invention is connected to various expression vectors. The expression vector is any conventional vector in the art, as long as it can carry the aforementioned polynucleotide molecule. The vector preferably includes various plasmids, cosmids, phages or virus vectors, etc.

[0223] The present invention also provides a recombinant expression transformant comprising the recombinant expression vector.

[0224] Wherein, the preparation method of the recombinant expression transformant is a conventional preparation method in the art, preferably: the above-mentioned recombinant expression vector is transformed into a host cell to obtain it. The host cell is various host cells conventional in the art, as long as it can satisfy the above-mentioned recombinant expression vector to stably replicate itself, and the polynucleotide carried can be effectively expressed. Preferably, the host cell is E.coli TG1 or E.coli BL21 cell (expressing single-chain antibody or Fab antibody), or HEK293 or CHO cell (expressing full-length IgG antibody). The aforementioned recombinant expression plasmid is transformed into a host cell to obtain a preferred recombinant expression transformant of the present invention. Wherein the transformation method is a conventional transformation method in the art, preferably a chemical transformation method, a heat shock method or an electroporation method.

[0225] Antibody preparation

[0226] The sequence of the DNA molecule of the antibody of the present invention or its fragment can be obtained by conventional techniques, such as PCR amplification or genomic library screening. In addition, the coding sequences of the light chain and the heavy chain can be fused together to form a single-chain antibody.

[0227] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.

[0228] In addition, artificial synthesis methods can also be used to synthesize related sequences, especially when the fragment length is shorter. Usually, a long fragment of sequence can be obtained by synthesizing multiple small fragments first and then connecting them.

[0229] At present, the DNA sequence encoding the antibody (or its fragment, or its derivative) of the present invention can be obtained completely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention by chemical synthesis.

[0230] The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.

[0231] The host cell can be a prokaryotic cell; or a lower eukaryotic cell; or a higher eukaryotic cell, such as a mammalian cell.

[0232] Typically, the transformed host cells are cultured under conditions suitable for the expression of the antibodies of the present invention, and then purified using conventional separation and purification methods well known to those skilled in the art to obtain the antibodies of the present invention.

[0233] The resulting monoclonal antibodies can be identified by conventional means. For example, the binding specificity of the monoclonal antibodies can be determined by immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). The binding affinity of the monoclonal antibodies can be determined, for example, by the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).

[0234] The antibodies of the present invention can be expressed in cells, on cell membranes, or secreted outside the cells. If necessary, the recombinant protein can be separated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art.

[0235] application

[0236] The present invention also provides uses of the antibodies, recombinant proteins, chimeric antigen receptor (CAR) constructs and / or immune cells of the present invention, for example, for preparing diagnostic preparations or preparing drugs.

[0237] Preferably, the drug is a drug for preventing and / or treating diseases associated with abnormal TSLP expression or function.

[0238] In the present invention, the disease associated with TSLP expression or abnormal function is a conventional disease associated with TSLP expression or abnormal function in the art. Preferably, the disease associated with TSLP expression or abnormal function is an allergic disease, a tumor, an immune disease, or a combination thereof. Preferably, the disease associated with TSLP expression or abnormal function is selected from the group consisting of asthma (such as moderate to severe asthma), atopic dermatitis, urticaria, chronic obstructive pulmonary disease (COPD), chronic rhinitis with nasal polyps, eosinophilic esophagitis, or a combination thereof.

[0239] Test Uses and Kits

[0240] The antibodies of the present invention can be used in detection applications, for example, for detecting a sample to provide diagnostic information.

[0241] In the present invention, the sample (specimen) used includes cells, tissue samples and biopsy specimens. The term "biopsy" used in the present invention should include all types of biopsies known to those skilled in the art. Therefore, the biopsy used in the present invention may include, for example, a resection sample of a tumor, a tissue sample prepared by an endoscopic method or a puncture or needle biopsy of an organ.

[0242] Samples used in the present invention include fixed or preserved cell or tissue samples.

[0243] The present invention also provides a kit containing the antibody (or fragment thereof) of the present invention. In a preferred embodiment of the present invention, the kit further comprises a container, instructions for use, a buffer, etc. In a preferred embodiment, the antibody of the present invention can be fixed to a detection plate.

[0244] Pharmaceutical composition

[0245] The present invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition, which contains the above-mentioned antibody or its active fragment or its fusion protein or corresponding immune cells, and a pharmaceutically acceptable carrier.

[0246] The antibody of the present invention can also be expressed in cells by nucleotide sequences for cell therapy.

[0247] The pharmaceutical composition of the present invention is a pharmaceutical composition for preventing and / or treating diseases associated with abnormal TSLP expression or function.

[0248] The pharmaceutical composition of the present invention contains a safe and effective amount of the monoclonal antibody of the present invention and a pharmaceutically acceptable carrier or excipient. The pharmaceutical preparation should match the administration method. The dosage of the active ingredient is a therapeutically effective amount. In addition, the polypeptide of the present invention can also be used together with other therapeutic agents.

[0249] In one embodiment of the present invention, the polypeptide of the present invention can be used in combination with other therapeutic agents for treating and / or preventing diseases, such as therapeutic agents for treating and / or preventing allergic diseases, tumors, or immune diseases.

[0250] In the present invention, preferably, the pharmaceutical composition of the present invention further comprises one or more pharmaceutical carriers. The pharmaceutical carrier is a conventional pharmaceutical carrier in the art, and the pharmaceutical carrier can be any suitable physiologically or pharmaceutically acceptable pharmaceutical excipient. The pharmaceutical excipient is a conventional pharmaceutical excipient in the art, preferably including a pharmaceutically acceptable excipient, filler or diluent, etc.

[0251] In the present invention, preferably, the amount of the pharmaceutical composition administered is an effective amount, which is an amount that can alleviate or delay the progression of a disease, degenerative or damaging condition. The effective amount can be determined on an individual basis and will be based in part on considerations of the symptoms to be treated and the results sought. Those skilled in the art can determine the effective amount by using the above factors on an individual basis and using experiments that do not exceed routine.

[0252] The present invention provides the use of the above-mentioned pharmaceutical composition in the preparation of a drug for preventing and / or treating a disease associated with TSLP expression or dysfunction. Preferably, the disease associated with TSLP expression or dysfunction is an allergic disease, a tumor, an immune disease, or a combination thereof.

[0253] Method and composition for detecting TSLP in a sample

[0254] The present invention also provides a method for detecting TSLP in a sample (for example, detecting cells overexpressing TSLP), comprising the following steps: contacting the above-mentioned antibody with the sample to be tested in vitro, and detecting whether the above-mentioned antibody and the sample to be tested combine to form an antigen-antibody complex.

[0255] The meaning of overexpression is conventional in the art, referring to the overexpression of TSLP RNA or protein in the sample to be tested (due to increased transcription, post-transcriptional processing, translation, post-translational processing and changes in protein degradation), as well as local overexpression and increased functional activity (such as in the case of increased enzymatic hydrolysis of the substrate) due to changes in protein transport patterns (increased cell membrane localization).

[0256] In the present invention, the above-mentioned detection method of whether the antigen-antibody complex is formed by binding is a conventional detection method in the art, preferably a flow cytometry (FACS) detection.

[0257] The present invention provides a composition for detecting TSLP in a sample, which comprises the above-mentioned antibody, recombinant protein, immune cell, or a combination thereof as an active ingredient. Preferably, it also comprises a compound composed of the functional fragments of the above-mentioned antibody as an active ingredient.

[0258] The main advantages of the present invention include:

[0259] 1) The anti-TSLP antibody of the present invention can bind to TSLP proteins of different species and has high affinity for both human and cynomolgus monkey TSLP proteins.

[0260] 2) The anti-TSLP antibody of the present invention can effectively block the binding of TSLP to its receptor TSLPR molecule, as well as the binding to the TSLPR / IL7Rα receptor on the cell surface.

[0261] 3) The anti-TSLP antibody of the present invention can inhibit the proliferation of target cells that highly express TSLPR / IL7Rα, and inhibit the expression of the STAT5 gene in the TSLP downstream signaling pathway.

[0262] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0263] Example 1 Preparation of TSLP and TSLPR

[0264] For animal immunization and antibody sequence screening, human and cynomolgus macaque sequences encoding His- and Fc-tags were synthesized into pTT5 vectors to construct expression plasmids for subsequent transfection of Expi293F (Thermo).

[0265] The specific transfection steps are as follows: Expi293F cells with a viability of >95% were transfected at 0.8×10 6 / ml was inoculated into the expression medium and placed in 6% CO2, 130rpm, 37℃ for further culture for 24 hours. After 24 hours, the sterile plasmid to be transfected was sterilized with a 0.22μm filter and the concentration was measured. The 1mg / ml transfection reagent polyethyleneimine (PEI) was sterilized with a 0.22μm filter for standby use. The mass ratio of PEI to plasmid was 2:1. Taking the transfection of 200ml Expi293F cells as an example, 10ml of Opti-MEM and 200μg of plasmid were mixed and allowed to stand for 5min; another 10ml of Opti-MEM and 400μl PEI were mixed and allowed to stand for 5min. The plasmid mixture and PEI mixture were mixed and allowed to stand at room temperature for 15min. The plasmid and PEI mixture was slowly added to the 200ml Expi293F cells and placed in a shaker at 6% CO2, 130rpm, 37℃ for culture. 250 mM sodium valproate solution was added at 0.8% of the transfection volume 18-24 hours after transfection, and 5% of the volume of feed medium was added on the 1st and 3rd days of transfection. On the 5th day of transfection, the cell supernatant was collected by centrifugation at 4000 rpm for 15 minutes, filtered, and purified.

[0266] The relevant amino acid sequence is shown below:

[0267] People TSLP:

[0268] MFPFALLYVLSVSFRKIFILQLVGLVLTYDFTNCDFEKIKAAYLSTISKDLITYMSGTKSTEFNNTVSCSNRPHCLTEIQSLTFNPTAGCASLAKEMFAMKTKAALAIWCPGYSETQINATQAMKK A R S KVTTNKCLEQVSQLQGLWRRFNRPLLKQQ(SEQ ID NO.1)

[0269] Cynomolgus monkey TSLP:

[0270] MKSLGQSKKEEVSFRKFFIFQFVGLVLTYDFTNCDFQKIEADYLRTISKDLITYMSGTKSTDFNNTVSCSNRPHCLTEIQSLTFNPTPRCASLAKEMFARKTKATLALWCPGYSETQINATQAMKK A R S KVTTNKCLEQVSQLLGLWRRFIRTLLKKQ(SEQ ID NO.2)

[0271] Note: The underlined amino acids are mutated to avoid Furin protease cleavage

[0272] Human TSLPR (extracellular region):

[0273] MGRLVLLWGAAVFLLGGWMALGQGGAAEGVQIQIIYFNLETVQVTWNASKYSRTNLTFHYRFNGDEAYDQCTNYLLQEGHTSGCLLDAEQRDDILYFSIRNGTHPVFTASRWMVYYLKPSSPKHVRFSWHQDAVTVTCSDLSYGDLLYEVQYRSPFDTEWQSKQENTCNVTIEGLDAEKCYSFWVRV KAMEDVYGPDTYPSDWSEVTCWQRGEIRDACAETPTPPKPKLSKFILISSLAILLMVSLLLLSLWKLWRVKKFLIPSVPDPKSIFPGLFEIHQGNFQEWITDTQNVAHLHKMAGAEQESGPEEPLVVQLAKTEAESPRMLDPQTEEKEASGGSLQLPHQPLQGGDVVTIGGFTFVMNDRSYVAL(SEQ ID NO.3)

[0274] Cynomolgus monkey TSLPR (extracellular region):

[0275] MGRLVLLWGAAVFLLGSWMALGQVATGEGLQIQIIYFNLETVQVTWNASHYPRSNLSFHYKFSRDEAYDQCTVYILQEGHTSGCLLDAEQQDDILYFSIRNGTHPVFTASRWIFYYLKPSSPKQVSFSWH QDAVTVTCSDLSYRGLLYEVQYRSPFDTEWQSKQENTCNVTIEDLDAEKCYAFRARVKAMEDAYGPDTYPSDWSEVTCWQRGKTRDSCPEPRTPPPKPKLSKFMLVSSLAILLMVCLLLLSLRKLWR(SEQ ID NO.4)

[0276] Example 2 Purification of TSLP and TSLPR recombinant proteins

[0277] Purification of His-tagged fusion protein: centrifuge the cell expression supernatant sample at high speed to remove impurities, filter, balance the nickel column with 20mM PB+0.5M NaCl solution, and wash 10 times the column volume. Load the filtered supernatant sample onto the column. Wash the column with 20mM PB+0.5M NaCl solution containing 20mM imidazole until the A280 reading drops to the baseline. Elute the target protein with 20mM PB+0.5M NaCl solution containing 250mM imidazole, and collect the elution peak. Replace the buffer with PBS, and divide it for use after SDS-PAGE identification.

[0278] Purification of Fc-tagged recombinant protein: The cell expression supernatant sample was centrifuged at high speed to remove impurities, and the recombinant protein expression supernatant was purified using a Protein A column. The column was rinsed with PBS until the A280 reading dropped to the baseline. The target protein was eluted with 100mM glycine, pH 3.0, and neutralized with 1M Tris-HCl, pH 8.0. The buffer was replaced with PBS, and the obtained protein was identified as correct by SDS-PAGE and then aliquoted for use.

[0279] Example 3 Construction of human TSLPR / IL7Rα receptor co-expressing cells

[0280] In order to screen antibodies that can block TSLP binding to TSLP receptor, CHO-K1 and BaF3 cell lines expressing both human TSLP receptor and human IL7Rα (TSLPR / IL7Rα) were constructed. The target gene TSLPR / IL7Rα was packaged by lentivirus and cloned into the target cell line to form a stable high-expression cell line. First, the human TSLPR and human IL7Rα genes were cloned into pCDH-CMV-MCS-EF1-puro and pCDH-CMV-MCS-EF1-Neo (SBI, CD500B-1) plasmids, respectively, and then human TSLPR was cloned into CHO-K1 and BaF3 cell lines by lentiviral infection, and selected and cultured for three weeks under 10μg / ml puromycin (puromycin, Gibco, US) selection pressure. On this basis, a second round of infection was performed, and the human IL7Rα gene was cloned into the cells, and screened with 1mg / ml G418 (Gibco, US) and 10μg / ml puromycin for two to three weeks. Finally, CHO-K1 and BaF3 monoclonal cell lines that highly expressed both TSLPR and IL7RR were screened by flow cytometry.

[0281] The sequence information of the two receptors is as follows:

[0282] Human TSLPR: as shown in SEQ ID NO.3.

[0283] Human IL7Rα:

[0284] MTILGTTFGMVFSLLQVVSGESGYAQNGDLEDAELDDYSFSCYSQLEVNGSQHSLTCAFEDPDVNITNLEFEICGALVEVKCLNFRKLQEIYFIETKKFLLIGKSNICVKVGEKS LTCKKIDLTTIVKPEAPFDLSVVYREGANDFVVTFNTSHLQKKYVKVLMHDVAYRQEKDENKWTHVNLSSTKLTLLQRKLQPAAMYEIKVRSIPDHYFKGFWSEWSPSYYFRTPEI NNSSGEMDPILLTISILSFFSVALLVILACVLWKKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGFLQDTFPQQLEESEKQRLGGDVQSP NCPSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMSSFYQNQ(SEQ ID NO.5)

[0285] Example 4 Discovery and Screening of Anti-human TSLP Antibodies

[0286] Anti-human TSLP monoclonal antibodies were produced by immunizing mice. The experiments used SJL white mice, female, 6-8 weeks old (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., Animal Production License No.: SCXK (Beijing) 2012-0001). Feeding environment: SPF grade. After the mice were purchased, they were raised in the laboratory environment for 1 week, with a 12 / 12 hour light / dark cycle, a temperature of 20-25°C, and a humidity of 40-60%. The acclimated mice were immunized with recombinant proteins huTSLP-Fc (25 μg), huTSLP-his (12.5 μg) and cynoTSLP-his (12.5 μg) with TiterMax, Alum or CpG adjuvants. After the 4th-5th immunization, mice with high antibody titers in the serum and titers tending to a plateau were selected, and after being killed, spleen cells were taken and fused with myeloma cells. The spleen lymphocytes were fused with myeloma cells Sp2 / 0 cells (CRL-8287TM) using an optimized PEG-mediated fusion procedure to obtain hybridoma cells.

[0287] The initial screening was conducted using ELISA binding experiments for human and monkey TSLP, experiments to block human TSLP binding to its receptor TSLPR, etc. After the hybridoma cells were transferred to a 24-well plate, the supernatant was rescreened. In addition to the above experiments, experiments to inhibit the upregulation of reporter gene signals induced by TSLP and the proliferation of BaF3 cells induced by TSLP were added to screen positive clones. After two rounds of subcloning, hybridoma clones were obtained for antibody production and purified by affinity methods.

[0288] The monoclonal hybridoma cell lines 64B8F8 and 217H11C5 with good activity were screened, and the hybridoma cells in the logarithmic growth phase were collected, and RNA was extracted with NucleoZol (MN) and reverse transcribed. The cDNA obtained by reverse transcription was amplified by PCR and sent to a sequencing company for sequencing. The mouse anti-TSLP antibody: 64B8F8, 217H11C5 sequence was obtained by sequencing, and its amino acid sequence is as follows:

[0289] 64B8F8 mouse antibody sequence information:

[0290] 64B8F8-mouse-VH:

[0291] EVQLQQSGAELVRSGASVKLSCTASGFNIK DNYMH WVKQRPEQGLEWI G WIDPENGNTEYVPKFQA KATMTADTSSNTVYLHLSSLTSEDTAVYYCNA R FYGSGYRNYFDY WGQGTTLTVSS (SEQ ID NO. 6)

[0292] 64B8F8-mouse-VL:

[0293] QIVLTQSPAIMSTSPGEKVTMTC SATSSVTYMF WYQQKPGSSPRLLIY DT SNLAS GVPFRFSGSGSGTTSSSLTISRMEAEDAATYYC QQWNTYPPT FGAGTK LELK (SEQ ID NO.7)

[0294] 217H11C5 mouse antibody sequence information:

[0295] 217H11C5-mouse-VH:

[0296] QIQLVQSGPELKKPGETVKISCKASGYNFI TSGMS WVKQAPGKGLKWM G WINTYSGVATYADDLKGRFAFSLETSASTASLQINNLKIEDTATYFCAR GEP PAY WGQGTLVTVSA (SEQ ID NO. 8)

[0297] 217H11C5-mouse-VL:

[0298] DILLTQSPAILSVSPGERVSFSC RASQSIGTSIH WYQQRTNGSPRLLIK YA SESIS GIPSRFSGSGSGTDFTLSINSVESEDIADYFC QESNSWPTWT FGGGTKLE IK (SEQ ID NO.9)

[0299] Therein, a single underline indicates a CDR region.

[0300] Example 5 Humanized Design of Anti-human TSLP Antibody

[0301] In this embodiment, the antibody is engineered to transform the non-human antibody into a humanized antibody, thereby improving its safety and efficacy in clinical applications. The structure of the antibody is adjusted by genetic engineering technology and protein engineering technology to make it have better immune tolerance in the human body and enhance its affinity and specificity. The antibodies involved are 64B8F8 and 217H11C5 antibodies. The humanized antibody engineering method is as follows:

[0302] Selection of human antibody framework: Screening and selecting appropriate human antibody framework by comparing protein primary sequence and antibody structure;

[0303] CDR replacement: Use the CDR region of 64B8F8 and 217H11C5 mouse antibodies to replace the CDR region of the selected human antibody framework to obtain the variable region sequence of the framework region transplanted antibody;

[0304] Referring to the simulated three-dimensional structures of 64B8F8 and 217H11C5 mouse antibodies, some key residues of the replaced VH and VL were reverse mutated, and humanized antibody molecules were obtained by cross-screening the VH and VL reverse mutant sequences;

[0305] The constant region of the non-human antibody was replaced with a human constant region, the heavy chain was constructed as an IgG4 chimeric antibody, and the light chain was constructed as an IgG4 kappa chain constant region.

[0306] The heavy and light chain constant region sequences of the control antibody AMG157 are as follows:

[0307] AMG157-VH:

[0308] QMQLVESGGGVVQPGRSLRLSCAASGFTFRTYGMHWVRQAPGKGLEW VAVIWYDGSNKHYADSVKGRFTITRDNSKNTLNLQMNSLRAEDTAVYYCA RAPQWELVHEAFDIWGQGTMVTVSS**(SEQ ID NO.61)

[0309] AMG157-VL:

[0310] SYVLTQPPSVSVAPGQTARITCGGNNLGSKSVHWYQQKPGQAPVLVVY DDSDRPSWIPERFSGSNSGNTATLTISRGEAGDEADYYCQVWDSSSDHVVFG GGTKLTVL**(SEQ ID NO.62)

[0311] Unless otherwise specified, the subtype of humanized antibodies and control antibodies is IgG4.

[0312] The IgG4 heavy chain constant region sequence is as follows:

[0313] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG

[0314] VHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESK

[0315] YGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQF

[0316] NWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVS

[0317] NKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDI

[0318] AVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVM

[0319] HEALHNHYTQKSLSLSLGK**(SEQ ID NO.10)

[0320] The IgG4 light chain constant region sequence is as follows:

[0321] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC**(SEQ ID NO.11)

[0322] Among them, ** represents the stop codon.

[0323] 5.1 Selection and back mutation of the human FR region of the 64B8F8 mouse antibody

[0324] (1) Selection and back mutation of human FR regions

[0325] The FR of the humanized VH of 64B8F8 selected IGHV1-46*01+IGHJ4*01, and the FR of the humanized VL selected IGKV3-11*01+IGKJ2*01. The CDR region of the 64B8F8 mouse antibody was transplanted onto the above human template, and the obtained sequence is as follows:

[0326] >64B8F8-VH0

[0327] QVQLVQSGAEVKKPGASVKVSCKASGFNIK DNYMH WVRQAPGQGLE WMG IIDPENGNTSYAQKFQG RVTMTRDTTSTVYMELSSLRSEDTAVYYCN A RFYGSGYRNYFDY WGQGTLVTVSS (SEQ ID NO. 12)

[0328] >64B8F8-VL0

[0329] EIVLTQSPATLSLSPGERATLSC SATSSVTYMF WYQQKPGQAPRLLIY DT SNLAS GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC QQWNTYPPT FGQGTKL EIK (SEQ ID NO. 22)

[0330] The structure of the parent antibody was modeled by the MOE homology modeling program. Humanized antibodies were designed using CDR transplantation, and the CDRs of the parent antibody were transplanted into the humanized framework to obtain humanized light chains and humanized heavy chains for each parent antibody. Eight heavy chains (VH1, VH2, VH3, VH4, VH5, VH6, VH7, VH8) and four light chains (VL1, VL2, VL3 and VL4) were paired for affinity ranking experiments. The combinations are detailed in Tables 1 and 2

[0331] The heavy chain constant region was constructed as a chimeric antibody of IgG4, and the light chain was constructed as an IgG4 kappa chain constant region.

[0332] The IgG4 heavy chain constant region sequence is (SEQ ID NO.10)

[0333] The IgG4 light chain constant region sequence is (SEQ ID NO.11)

[0334] The amino acid sequences of the humanized light chain and heavy chain variable regions are as follows:

[0335] >64B8F8-VH1

[0336]

[0337] >64B8F8-VH2

[0338]

[0339] >64B8F8-VH3

[0340]

[0341] >64B8F8-VH4

[0342]

[0343] >64B8F8-VH5

[0344]

[0345] >64B8F8-VH6

[0346] QVQLVQSGAEVKKPGASVKVSCKASGFNIK DNYMH WVKQRPGQGLEWMG IIDPENGNTSYAQKFQG RVTMTRDTTSTVYMELSSLRSEDTAVYYCNA RFYGSGYRNYFDY WGQGTLVTVSS (SEQ ID NO. 18)

[0347] >64B8F8-VH7

[0348]

[0349] >64B8F8-VH8

[0350]

[0351] The N-terminus of the above heavy chain variable region sequences can be connected to the signal peptide METDTLLLWVLLLWVPGSTG (SEQ ID NO. 21).

[0352] Light chain

[0353] >64B8F8-VL1

[0354] EIVLTQSPATLSLSPGERATLSC SATSSVTYMF WYQQKPGQAPRLLIY DT SNLAS GIPARFSGSGSGTDSTLTISSLEPEDFAVYYC QQWNTYPPT FGQGTKL EIK (SEQ ID NO. 23)

[0355] >64B8F8-VL2

[0356]

[0357] >64B8F8-VL3

[0358]

[0359] >64B8F8-VL4

[0360]

[0361] The N-terminus of the above light chain variable region sequences can be connected to the signal peptide METDTLLLWVLLLWVPGS TG (SEQ ID NO. 21).

[0362] Note: Single underline indicates CDR region, bold+italic indicates back mutation.

[0363] The above VL and VH were combined with the light chain and heavy chain constant regions of human germline to obtain the full-length sequence antibody.

[0364] Different combinations of antibody molecules were further obtained through genetic engineering technology, protein engineering technology, cell culture technology and protein purification technology. The above two groups of sequences were divided into two batches for antibody preparation according to Table 1 and Table 2 below.

[0365] Table 1 First Batch Composition

[0366] 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

[0367] Table 2 The second batch of combinations

[0368] 64B8F8-VH5 64B8F8-VH6 64B8F8-VH7 64B8F8-VH8 64B8F8-VL1 SZ247 SZ248 SZ249 SZ250

[0369] The above-mentioned different antibody molecules were all tested by ELISA and cell function experiments to verify the binding activity and functional activity of different antibody molecules.

[0370] (2) Affinity maturation of 64B8F8 humanized antibody

[0371] As an important drug and diagnostic reagent in the field of biomedicine, the affinity of antibody drugs is a key factor affecting biological function and efficacy. Therefore, improving the affinity of antibodies to target antigens is an important goal to improve antibody quality and efficacy. In this embodiment, antibody molecules that can block TSLP / TSLPR binding are screened and optimized. The affinity maturation method includes the following steps:

[0372] Antigen-antibody complex structure analysis: Determine the antigen-antibody complex structure through cyno-EM, computer molecular docking and other means;

[0373] Point mutation design: Based on the structural information of the antibody-antigen complex, a point mutation scheme is designed to replace the amino acid residues at the binding site of the antibody in a targeted manner;

[0374] Implementation of point mutation: Using genetic engineering technology, the designed point mutation is introduced into the gene sequence of the antibody to synthesize the mutated antibody;

[0375] Affinity assessment: Use SPR or BLI technology to assess the binding affinity between the mutated antibody and the target antigen, and screen for antibody candidates with higher affinity;

[0376] Functional validation: The biological functions of affinity matured antibodies are validated, including antigen neutralization activity, intracellular signal transduction, etc., to verify their effectiveness in biological processes.

[0377] Table 3 Affinity maturation mutations

[0378]

[0379]

[0380] The constant region heavy chain was constructed as a chimeric antibody of IgG4, and the light chain was constructed as an IgG4 kappa chain constant region. The sequence of the IgG4 heavy chain constant region is (SEQ ID NO.10)

[0381] The IgG4 light chain constant region sequence is (SEQ ID NO.11)

[0382] The affinity matured variable region sequences are as follows:

[0383] >64B8F8-VH7-M1

[0384] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0385] <h2 style=";text-align:left;direction:ltr"> >64B8F8-VH7-M2<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0386] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0387] <h2 style=";text-align:left;direction:ltr"> >64B8F8-VH7-M3<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0388] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0389] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0390] <h2 style=";text-align:left;direction:ltr"> >64B8F8-VH7-M4<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0391] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0392] <h2 style=";text-align:left;direction:ltr"> >64B8F8-VH7-M5<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0393] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0394] <h2 style=";text-align:left;direction:ltr"> >64B8F8-VH7-M6<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0395] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0396] <h2 style=";text-align:left;direction:ltr"> >64B8F8-VH7-M7<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0397] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0398] <h2 style=";text-align:left;direction:ltr"> >64B8F8-VH7-M8<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0399] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0400] <h2 style=";text-align:left;direction:ltr"> >64B8F8-VH7-M9<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0401] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0402] <h2 style=";text-align:left;direction:ltr"> >64B8F8-VH7-M10<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0403] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0404] <h2 style=";text-align:left;direction:ltr"> >64B8F8-VH7-M11<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0405] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0406] <h2 style=";text-align:left;direction:ltr"> >64B8F8-VH7-M12<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0407] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0408] <h2 style=";text-align:left;direction:ltr"> >64B8F8-VH7-M13<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0409] <h2 style=";text-align:left;direction:ltr">

[0410] >64B8F8-VH7-M14

[0411]

[0412] >64B8F8-VH7-M15

[0413]

[0414] >64B8F8-VH7-M16

[0415]

[0416] >64B8F8-VH7-M17

[0417]

[0418] >64B8F8-VH7-M18

[0419]

[0420] >64B8F8-VH7-M19

[0421] QVQLVQSGAEVKKPGASVKVSCKASGFNIK DNYMH WVKQRPGQGLEWMG WIDPENGNTEYVPKFQG KVTMTADNSTNTVYMELSSLRSEDTAVYYCNA RFYGSGYRNYFDY WGQGTLVTVSS(SEQ ID NO.45)

[0422] >64B8F8-VH7-M20

[0423]

[0424] >64B8F8-VH7-M21

[0425]

[0426] >64B8F8-VH7-M22

[0427]

[0428] >64B8F8-VH7-M23

[0429]

[0430] >64B8F8-VH7-M24

[0431]

[0432] >64B8F8-VH7-M25

[0433]

[0434] >64B8F8-VH7-M26

[0435]

[0436] >64B8F8-VH7-M27

[0437]

[0438] >64B8F8-VH7-M28

[0439]

[0440] >64B8F8-VH7-M29

[0441]

[0442]

[0443] >64B8F8-VH7-M30

[0444]

[0445] >64B8F8-VH7-M31

[0446]

[0447] >64B8F8-VH7-M32

[0448]

[0449] >64B8F8-VH7-M33

[0450]

[0451] >64B8F8-VH7-M34

[0452]

[0453] Note: Single underline indicates CDR regions, bold+italics indicate back mutations.

[0454] The N-terminus of the above affinity matured variable region sequences can be connected to the signal peptide METDTLLLWVLLLW VPGSTG (SEQ ID NO. 21).

[0455] The variable region CDRs of 64B8F8 and its humanized and affinity matured antibodies are shown in Table 4.

[0456] Table 4 Variable region CDRs of 64B8F8 and its humanized and affinity matured antibodies

[0457]

[0458]

[0459]

[0460] (3) Production of chimeric and humanized antibodies

[0461] DNA sequences encoding the heavy and light chains of the antibody were synthesized to construct expression plasmids for full-length IgG antibodies. Antibody expression was performed in 4 mL CHO cell culture, and the supernatant was purified using a protein A affinity column. The purified antibody buffer was exchanged into PBS using a PD-10 desalting column.

[0462] (4) Multi-concentration affinity determination of chimeric antibodies and selected humanized antibodies

[0463] The affinity for Human TSLP-his was determined by selecting chimeric antibodies and the top 3 antibodies (antibodies with smaller KD values ​​were preferred, and antibodies with smaller kd values ​​were considered comprehensively when KD values ​​were equivalent) using the surface plasmon resonance (SPR) biosensor Biacore T200 (GE Healthcare). The antibodies were captured on the sensor chip by the Fc capture method. Human TSLP-his was used as the analyte. The data of dissociation (kd) and association (ka) rate constants were obtained using the Biacore T200 evaluation software. The equilibrium dissociation constant (KD) was calculated from the ratio of kd to ka.

[0464] Table 5 Antibody affinity

[0465]

[0466]

[0467] The affinity test results showed that after affinity maturation, the affinity of 64B8F8 antibody was improved compared with the chimeric antibody in binding to Cyno TSLP.

[0468] 5.2 Selection and back mutation of the human FR region of the 217H11C5 mouse antibody

[0469] (1) Selection and back mutation of human FR regions

[0470] The FR of the humanized VH of 217H11C5 was selected as IGHV9-3*01+IGH-J3*01, and the FR of the humanized VL was selected as IGKV5-48*01+IGKJ1*01. The CDR region of the 217H11C5 mouse antibody was transplanted onto the above human template.

[0471] The structure of the parent antibody was modeled by the MOE homology modeling program. Humanized antibodies were designed using CDR transplantation, and the CDRs of the parent antibody were transplanted into the humanized framework to obtain humanized light chains and humanized heavy chains for each parent antibody. Four heavy chains (VH1, VH2, VH3, VH4) and four light chains (VL1, VL2, VL3 and VL4) were paired for affinity ranking experiments.

[0472] The heavy chain constant region was constructed as a chimeric antibody of IgG4, and the light chain was constructed as an IgG4 kappa chain constant region.

[0473] The IgG4 heavy chain constant region sequence is (SEQ ID NO.10)

[0474] The IgG4 light chain constant region sequence is (SEQ ID NO.11)

[0475] The amino acid sequences of the humanized light chain and heavy chain variable regions are as follows:

[0476] >217H11C5-VH1

[0477] QVQLVQSGSELKKPGASVKVSCKASGYNFI TSGMS WVRQAPGQGLEW MG WINTYSGVATYADDLKG RFVFSLDTSVSTAYLQISSLKAEDTAVYYCAR GEPPAY WGQGTLVTVSS (SEQ ID NO. 64)

[0478] >217H11C5-VH2

[0479] QVQLVQSGSELKKPGASVKVSCKASGYNFI TSGMS WVRQAPGQGLKW MG WINTYSGVATYADDLKG RFVFSLDTSVSTAYLQISSLKAEDTAVYFCAR GEPPAY WGQGTLVTVSS (SEQ ID NO. 65)

[0480] >217H11C5-VH3

[0481] QVQLVQSGSELKKPGASVKVSCKASGYNFI TSGMSWVRQAPGQGLKW MG WINTYSGVATYADDLKG RFVFSLDTSVSTASLQISSLKAEDTAVYFCAR G EPPAY WGQGTLVTVSS(SEQ ID NO.66)

[0482] >217H11C5-VH4

[0483] QVQLVQSGSELKKPGASVKVSCKASGYNFI TSGMS WVKQAPGQGLKW MG WINTYSGVATYADDLKG RFVFSLDTSVSTASLQISSLKAEDTAVYFCAR G EPPAY WGQGTLVTVSS(SEQ ID NO.67)

[0484] >217H11C5-VL1

[0485] EIVLTQSPDFQSVTPKEKVTITC RASQSIGTSIH WYQQKPDQSPKLLIK YA SESIS GVPSRFSGSGSGTDFTLTINSLEAEDAATYYC QESNSWPTWT FGGGTK GAME(SEQ ID NO.68)

[0486] >217H11C5-VL2

[0487] EIVLTQSPDFQSVTPKEKVTITC RASQSIGTSIH WYQQKPDQSPKLLIK YA SESIS GIPSRFSGSGSGTDFTLTINSLEAEDAATYFC QESNSWPTWT FGGGTKL EIK(SEQ ID NO.69)

[0488] >217H11C5-VL3

[0489] EIVLTQSPDFQSVTPKEKVTITC RASQSIGTSIH WYQQKPDQSPRLLIK YA SESIS GVPSRFSGSGSGTDFTLTINSLEAEDAADYFC QESNSWPTWT FGGGTK GAME(SEQ ID NO.70)

[0490] >217H11C5-VL4

[0491] EIVLTQSPDFQSVTPKEKVTFTC RASQSIGTSIHWYQQKPDQSPRLLIK Y ASESIS GIPSRFSGSGSGTDFTLTINSLEAEDAADYFC QESNSWPTWT FGGGTK LEIK (SEQ ID NO. 71)

[0492] The N-terminus of the above humanized variable region sequences can be connected to the signal peptide MGWSCIILFLVATATGVHS (SEQ ID NO.63).

[0493] The variable region CDRs of 217H11C5 and its humanized and affinity matured antibodies are shown in Table 6.

[0494] Table 6 Variable region CDRs of 217H11C5 and its humanized and affinity matured antibodies

[0495]

[0496]

[0497] (2) Production of chimeric and humanized antibodies

[0498] DNA sequences encoding antibody heavy and light chains were synthesized to construct expression plasmids containing full-length IgG. Antibody expression was performed in 4 mL CHO cell (ATCC) cultures and the supernatant was purified using a protein A affinity column. The purified antibody buffer was exchanged into PBS using a PD-10 desalting column.

[0499] (3) Affinity determination of chimeric and humanized antibodies

[0500] The affinity of purified antibodies binding to Human TSLP-his was determined separately using the surface plasmon resonance (SPR) biosensor Biacore T200 (GE Healthcare). The antibodies were immobilized on a sensor chip by Fc capture. Human TSLP-his was used as the analyte. The data for dissociation (kd) and association (ka) rate constants were obtained using the Biacore T200 evaluation software. The equilibrium dissociation constant (KD) was calculated from the ratio of kd to ka. Antibodies were sorted by their dissociation rate constant (dissociation rate, kd).

[0501] Table 7 Affinity of 217H11C5 humanized antibody

[0502]

[0503]

[0504] The above humanized antibodies were sorted by single-point affinity, and the antibodies with stronger affinity in the above table were selected for further study.

[0505] (4) Multi-concentration affinity determination of chimeric antibodies and selected humanized antibodies

[0506] The affinity for Human TSLP-his was determined by selecting chimeric antibodies and the top 3 antibodies (antibodies with smaller KD values ​​were preferred, and antibodies with smaller kd values ​​were considered comprehensively when KD values ​​were equivalent) using the surface plasmon resonance (SPR) biosensor Biacore T200 (GE Healthcare). The antibodies were captured on the sensor chip by the Fc capture method. Human TSLP-his was used as the analyte. The data of dissociation (kd) and association (ka) rate constants were obtained using the Biacore T200 evaluation software. The equilibrium dissociation constant (KD) was calculated from the ratio of kd to ka.

[0507] Table 8 Antibody affinity

[0508]

[0509] The above preferred sequences were subjected to affinity tests at multiple concentrations, and the antibodies with stronger affinity in the above table were selected for further study.

[0510] Example 6 ELISA detection of binding of anti-human TSLP antibodies to human TSLP and cynomolgus monkey TSLP

[0511] Coat the reaction wells with hTSLP-his and cynoTSLP-His diluted with PBS at a concentration of 2μg / ml, 100μL per well. Seal with a sealing film and let stand overnight at 2-8°C. Remove the sealing film, add 200μL of PBST to each well to wash the plate, and repeat 3 times. Add 200μl of 3% BSA to each well, seal with a sealing film, and let stand at 37°C for 2h. Remove the sealing film, add 200μL of PBST to each well to wash the plate, and repeat 3 times. The starting concentration of the sample is 20000ng / mL, and 8 dilutions are prepared by 4-fold dilution, 100μL / well, and the diluent is PBS+1% BSA.

[0512] Detect antigen-antibody binding: incubate the antibody to be detected with the antigen at 37°C for 1h, and use AMG157 as a positive control. Remove the sealing film, add 200μL of PBST to each well to wash the plate, and repeat 3 times. Add HRP-IgG antibody (diluted 1:5000 with PBS+1% BSA), 100μL to each well, and let stand at 37°C for 1h. Remove the sealing film, add 200μL of PBST to each well to wash the plate, and repeat 4 times. Add 100μL of single-component TMB colorimetric solution to each well, seal with the sealing film, and let stand at room temperature in the dark for 10min. Remove the sealing film and add 100μL of ELISA stop solution to each well. Use an enzyme reader to detect OD450. Use Graphpad Prism to process the data: the average OD450 absorbance value of each concentration sample is used as the Y-axis, the sample concentration value is the X-axis, and a four-parameter fit is performed to obtain the EC50 and fitting constant R 2 .

[0513] The results are as follows Figures 1 - 4 As shown, both humanized antibodies and affinity matured antibodies can specifically bind to human / cynomolgus monkey TSLP protein with high affinity.

[0514] Example 7 BIACORE detection of binding of anti-TSLP humanized antibodies to human / cynomolgus monkey TSLP

[0515] The affinity of purified antibodies binding to Human TSLP-his was determined separately using a surface plasmon resonance (SPR) biosensor Biacore T200 (GE Healthcare). The antibodies were immobilized on a sensor chip by Fc capture. Human TSLP-his was used as an analyte. The data for dissociation (kd) and association (ka) rate constants were obtained using Biacore T200 evaluation software. The equilibrium dissociation constant (KD) was calculated from the ratio of kd to ka. The test results are shown in Tables 5, 7, and 8.

[0516] Example 8 ELSA-based anti-TSLP antibody blocking TSLP binding to TSLP receptor experiment

[0517] Coat the reaction wells with hTSLPR-Fc and cynoTSLPR-Fc diluted with PBS at a concentration of 2μg / mL, 100μL per well. Seal with a sealing film and let stand overnight at 2-8°C. Remove the sealing film, add 200μL of PBST to each well to wash the plate, and repeat 3 times. Add 200μL of 1% BSA to each well, seal with a sealing film, and let stand at 37°C for 2h. Remove the sealing film, add 200μL of PBST to each well to wash the plate, and repeat 3 times. The starting concentration of the sample is 10000ng / mL (configuration concentration 20000ng / mL), and 8 dilutions are prepared with 4-fold dilutions and 50μL / well. The diluent is 1% BSA-PBS.

[0518] Add TSLP-His at a final concentration of 100 ng / ml (configuration concentration 200 ng / ml), 50 μL / well; add cynoTSLP-His at a final concentration of 20 ng / mL (configuration concentration 40 ng / ml), 50 μl / well to detect the blocking effect of the antibody on TSLP binding to TSLPR. AMG157 antibody was used as a positive control. The reaction solution was incubated at 37°C for 1 hour. Remove the plate seal, add 200 μL of PBST to each well to wash the plate, and repeat 3 times. Add HRP-his antibody (diluted 1:5000 with PBS+1% BSA), 100 μL to each well, and let stand at 37°C for 1 hour. Remove the plate seal, add 200 μL of PBST to each well to wash the plate, and repeat 4 times. Add 100 μL of single-component TMB colorimetric solution to each well, seal the plate seal, and let stand at room temperature in the dark for 10 minutes. Remove the plate seal, and add 100 μL of ELISA stop solution to each well. OD450 was detected by microplate reader. Graphpad Prism was used to process the data: the average OD450 absorbance value of each concentration sample was used as the Y axis, the sample concentration value was used as the X axis, and four-parameter fitting was performed to obtain IC50 and fitting constant R 2 .

[0519] The results are as follows Figures 5 - 8 As shown in Figure 2, both humanized antibodies and affinity matured antibodies can effectively block the ligand-receptor binding between human / cynomolgus monkey TSLP protein and its receptor (TSLPR).

[0520] Example 9: Experiment of TSLP antibody blocking TSLP binding to TSLP receptor based on FACS

[0521] The CHOK1-TSLPR+IL7R cells prepared in Example 3 were collected, the culture medium was replaced with PBS+1% FBS, and the cell density was adjusted to 4*10 6 / mL, inoculated into 96-well V-shaped plates at 50μl / well, centrifuged and discarded the supernatant. Dilute TSLP-His ligand to a concentration of 0.4μg / ml (final concentration of 0.1μg / ml), 25μl / well; gradient dilution of antibody, initial concentration of 200μg / ml (final concentration of 50μg / ml), 5-fold dilution, 25μl / well to prepare 8 dilutions. Add ligand and antibody in a 1:1 ratio, i.e., 25μl:25μl, to 96-well V-shaped plates; incubate at 37 degrees for 1h. Centrifuge at 1500rpm for 5min, discard the supernatant, wash the cells twice; dilute APC-His secondary antibody (1:100), add to 96-well V-shaped plates, 100μl / well, incubate at 37 degrees for 30min, wash the cells twice. Flow cytometry detection, Graphdpad prism data processing.

[0522] The results are as follows Figures 9 - 10As shown, both humanized antibodies and affinity matured antibodies can effectively block the ligand-receptor binding between human TSLP protein and TSLP receptor expressed on the cell surface.

[0523] Example 10 Anti-TSLP antibody inhibits TSLP-induced proliferation of BaF3-TLSPR / IL7R cells

[0524] Collect the BaF3-TSLPR / IL7R cells prepared in Example 3: centrifuge at 1000 r / min for 5 min, collect the cells in the logarithmic growth phase, wash the cells twice with 1640+10% FBS medium, resuspend the cells with 1640+10% FBS and adjust the cell concentration to 5×10 4 / mL. According to 2.5*10 3 / well, 50 μL / well, inoculate cells into 96-well plates, diluent is 1640 + 10% FBS. Cell viability must be greater than 80%. TSLP antibody was diluted in a gradient manner and added to the 96-well culture plate inoculated with cells, 25 μL / well; TSLP antibody starting concentration was 40000 ng / ml (final concentration was 10000 ng / mL), and 8 dilutions were prepared by 4-fold dilution; TSLP-His was diluted to 0.4 ng / mL (final concentration was 0.1 ng / mL), and added to the 96-well culture plate inoculated with cells, 25 μL / well; two replicates for each concentration. The culture plate was incubated at 37°C for 72 hours.

[0525] Use of detection reagents 2.0 Luminescent Cell Viability Assay (Novozyme). Take out the cell culture plate to be tested from the incubator and leave it at room temperature for 30 minutes to allow the temperature of the culture plate to equilibrate to room temperature. Pipette 100μL CellCounting-Lite 2.0 and add it to 100μL of the cell culture to be tested. Oscillate and mix for 2-5 minutes to fully lyse the cells; leave it at room temperature for 10 minutes to stabilize the luminescent signal, and then perform the test. Detect the full-wavelength Luminesecnce (RLU) in the chemiluminescence mode of the microplate reader. Use Graphpad software to analyze the experimental data, with TSLP antibody concentration as the X-axis and the corresponding Luminesecnce (RLU) as the Y-axis. Use a four-parameter regression model to fit the effect curve.

[0526] The results are as follows Figures 11 - 12 As shown in Figure 2, both humanized antibodies and affinity matured antibodies can inhibit the proliferation of BaF3 cells induced by human TSLP protein.

[0527] Example 11 Anti-TSLP antibodies inhibit TSLP-induced BaF3-TLSPR / IL7R / STAT5-Luc reporter gene signal

[0528] Based on the TSLPR / IL-7R overexpressing BaF3 cells prepared by the method described in Example 3, the plasmid pGL4.52[luc2P / STAT5 RE / Hygro] was transfected into the receptor overexpressing cells of Example 3. The cells were inoculated one day before transfection so that the cell confluence reached 50%-80% on the day of transfection. On the day of transfection, the serum-free medium opti-MEM and the transfection reagent FUGENE6 were balanced to room temperature, and the transfection reagent and DNA complex were configured. During the configuration process, the FUGENE6 transfection reagent was first added to the MEM medium, incubated at room temperature for 5 minutes, and then the plasmid DNA was added, mixed evenly, and incubated at room temperature for 15 minutes. After 48 hours of transfection, antibiotics were added for pressure screening, and after the cells grew up, FACS and reporter gene detection were performed. When performing reporter gene detection, the cells were centrifuged one day in advance, the cells were washed once with 1640+10% FBS medium, the medium was replaced with 1640+10% FBS (without mIL3), and the cells were starved overnight. Harvest BaF3TSLPR+IL7R cells, centrifuge at 1000r / min for 5min, collect cells in the logarithmic growth phase, resuspend the cells in 1640+10% FBS and adjust the cell concentration to 4×10 5 / mL. Press 2x10 4 / well, 50 μl / well, inoculate cells into 96-well plates, diluent 1640 + 10% FBS. TSLP antibody was graded and added to the 96-well culture plate inoculated with cells, 25 μL / well; the starting concentration of TSLP antibody was 40000 ng / mL (the final concentration was 10000 ng / ml), and 8 dilutions were prepared by 10-fold dilution; TSLP-His was diluted to 0.4 ng / ml (the final concentration was 0.1 ng / ml), and added to the 96-well culture plate inoculated with cells, 25 μL / well; two replicates for each concentration. The culture plate was incubated at 37°C for 5 hours.

[0529] Use of detection reagents Luciferase Assay System (Cat.#E6120). Take out the cell culture plate to be tested from the incubator and leave it at room temperature for 10 minutes to allow the temperature of the culture plate to equilibrate to room temperature. Pipette 100μl of detection reagent and add it to 100μL of the cell culture to be tested. Oscillate and mix for 2-5 minutes to fully lyse the cells; leave it at room temperature for 10 minutes to stabilize the luminescent signal, and then perform the test. Detect the full wavelength Luminesecnce (RLU) in the chemiluminescence mode of the microplate reader. Data analysis: Use Graphpad software to analyze the experimental data, with TSLP antibody concentration as the X-axis and the corresponding Luminesecnce (RLU) as the Y-axis. Use a four-parameter regression model to fit the effect curve.

[0530] The results are as followsFigures 13 - 14 As shown in Figure 2, both humanized antibodies and affinity matured antibodies can inhibit the downstream STAT5 signaling pathway induced by TSLP.

[0531] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. An anti-TSLP antibody, comprising a heavy chain and a light chain, characterized in that: 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 shown in SEQ ID NO:74, 75 or 76, and VH-CDR3 shown in any one of SEQ ID NOs: 77-87; 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; Furthermore, 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 according to claim 1, characterized in that 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; Furthermore, the light chain variable region has the following complementarity determining regions (CDRs): 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 according to claim 1, characterized in that The heavy chain variable region has the following complementarity determining regions (CDRs): 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; Furthermore, the light chain variable region has the following complementarity determining regions (CDRs): 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 according to claim 1, characterized in that The heavy chain variable region has an amino acid sequence shown 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, 68-71.

5. The antibody according to claim 1, characterized in that 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 shown in SEQ ID NO:46 and a light chain variable region as shown in SEQ ID NO:25; (4) a heavy chain variable region as shown in SEQ ID NO:64 and a light chain variable region as shown in SEQ ID NO:68; (5) a heavy chain variable region as shown in SEQ ID NO:64 and a light chain variable region as shown in SEQ ID NO:69; (6) a heavy chain variable region as shown in SEQ ID NO:64 and a light chain variable region as shown 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 shown in SEQ ID NO:65 and a light chain variable region as shown 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) the heavy chain variable region shown in SEQ ID NO:65 and the light chain variable region shown in SEQ ID NO:70; (11) the heavy chain variable region shown in SEQ ID NO:65 and the light chain variable region shown in SEQ ID NO:71; (12) a heavy chain variable region as shown in SEQ ID NO:66 and a light chain variable region as shown in SEQ ID NO:68; (13) the heavy chain variable region shown in SEQ ID NO:66 and the light chain variable region shown in SEQ ID NO:69; (14) the heavy chain variable region shown in SEQ ID NO:66 and the light chain variable region shown in SEQ ID NO:70; (15) the heavy chain variable region shown in SEQ ID NO:66 and the light chain variable region shown in SEQ ID NO:71; (16) the heavy chain variable region shown in SEQ ID NO:67 and the light chain variable region shown in SEQ ID NO:68; (17) the heavy chain variable region shown in SEQ ID NO:67 and the light chain variable region shown in SEQ ID NO:69; (18) the heavy chain variable region shown in SEQ ID NO:67 and the light chain variable region shown in SEQ ID NO:70; (19) the heavy chain variable region shown in SEQ ID NO:67 and the light chain variable region shown in SEQ ID NO:71; (20) the heavy chain variable region shown in SEQ ID NO: 8 and the light chain variable region shown in SEQ ID NO: 9; or (21) The heavy chain variable region shown in SEQ ID NO:6 and the light chain variable region shown in SEQ ID NO:

7.

6. A recombinant protein, characterized in that The recombinant protein comprises: (i) the antibody according to claim 1; and (ii) optionally a tag sequence to facilitate expression and / or purification.

7. A chimeric antigen receptor (CAR) construct, wherein the antigen binding region of the chimeric antigen receptor construct includes a single-chain variable fragment (scFv) that specifically binds to TSLP, and the scFv has the heavy chain variable region and light chain variable region of the antibody as described in claim 1.

8. A recombinant immune cell, characterized in that: The immune cells express the exogenous CAR construct as described in claim 7.

9. A pharmaceutical composition comprising: (i) an active ingredient, wherein the active ingredient is selected from the group consisting of the antibody according to claim 1, the recombinant protein according to claim 6, the immune cell according to claim 8, or a combination thereof; and (ii) a pharmaceutically acceptable carrier.

10. Use of the antibody according to claim 1, or the recombinant protein according to claim 6, or the immune cell according to claim 8 and / or the pharmaceutical composition according to claim 9 in the preparation of a medicament for treating diseases associated with abnormal TSLP expression or function.

11. A polynucleotide, characterized in that The polynucleotide encodes a polypeptide selected from the group consisting of: (1) The antibody according to 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 contains the polynucleotide according to claim 11.

13. An engineered host cell, comprising the vector of claim 12 or the polynucleotide of claim 11 integrated into its genome.

14. A method for detecting TSLP in a sample, the method comprising the steps of: (1) contacting the sample with the antibody according to claim 1; (2) Detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of TSLP in the sample.

15. A detection board, comprising: A substrate and a test strip, wherein the test strip contains the antibody as claimed in claim 1, the recombinant protein as claimed in claim 6, the immune cell as claimed in claim 8, or a combination thereof.

16. A kit, comprising: (1) a first container, wherein the first container contains the antibody according to claim 1; and / or (2) a second container, wherein the second container contains a secondary antibody against the antibody of claim 1; or, The kit contains the detection plate as described in claim 15.

17. A method for preparing a recombinant polypeptide, the method comprising: (a) culturing the host cell according to claim 13 under conditions suitable for expression; (b) isolating a recombinant polypeptide from the culture, wherein the recombinant polypeptide is the antibody according to claim 1 or the recombinant protein according to claim 6.

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