Anti-TSLP antibodies and uses thereof

Abstract

The present disclosure provides an antibody or antigen binding fragment thereof, wherein the antibody or antigen binding fragment thereof specifically binds to TSLP. According to the present invention, the antibody can effectively inhibit the activation effect of the TSLP on the STATs and JAK2 signal channel of the HTSLP Reporter Cell Line cell, the antibody and the Tezepelumab can effectively inhibit the proliferation promotion effect of the TSLP on BaF3-TSLPR / IL7R alpha cell, the antibody does not intersect with the Tezepelumab binding epitope, does not block the MKK34 antibacterial peptide with antibacterial activity at the C terminal, and is expected to retain the external bacterial infection resistance of the TSLP.

Description

[0001] This application is a divisional application of the invention patent application for anti-TSLP antibodies and their uses, with the application date of December 26, 2023; application number: 202311832040.8; and the invention name:

[0002] Reference to a sequence listing

[0003] This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference. Technical Field

[0004] The present disclosure relates to the field of genetic engineering and antibodies, and more specifically to an antibody or an antigen-binding fragment thereof that specifically binds to TSLP and its use. The present application develops a new anti-TSLP antibody and provides the use of the antibody in the prevention, diagnosis, treatment, tracking and / or prognosis of TSLP-related diseases. Background Art

[0005] Thymic stromal lymphopoietin (TSLP) was first discovered in the culture supernatant of mouse thymic stromal cells. It is a B lymphocyte growth factor mainly expressed by epithelial cells. TSLP belongs to the IL-2 family of cytokines and is most similar to IL-7. IL-2 family cytokines include IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. They are all globular proteins containing four short-chain α helices and play an important role in promoting and maintaining T lymphocyte populations (Expert Rev Clin Immunol. 2014, 10(11): 1463–1474. Nature Communications, 2017, 8.). The latest research results show that TSLP is an "alarm protein" of the human immune barrier, regulating immune function in surface barriers such as skin keratinocytes, lung and intestinal epithelial cells. TSLP responds to pathogenic bacteria stimulation and activates immature dendritic cells, mast cells, basophils, eosinophils and lymphocytes, causing them to present a type 2 polarized phenotype. Abnormal TSLP signaling can cause a variety of serious health problems in the human body. It has been proven that TSLP is related to diseases such as asthma, atopic dermatitis, atopic rhinitis, psoriasis, chronic obstructive pulmonary disease (COPD), and eosinophilic esophagitis (Nat Immunol. 2010, 11(4): 289–293. Expert Rev Clin Immunol. 2014, 10(11): 1463–1474. Drugs (2020), 80, 449–458. Adv Immunol. 2009; 101: 1–25.).

[0006] IL-2 family cytokines bind to their own specific receptor subunits, and share the common receptor subunit γC. However, TSLP binds to two receptors, TSLPR (CRLF2) and IL-7Ralpha, and does not bind to the γC receptor (Expert Rev Clin Immunol. 2014, 10 (11): 1463-1474. Adv Immunol. 2009, 101: 1-25.). TSLP binds to TSLPR and IL-7Ralpha synergistically. TSLP binds to TSLPR with an affinity of about 32nM. However, TSLP does not bind directly to IL-7Ralpha (> 100nM). TSLP binds to IL-7Ralpha only after it binds to TSLPR to form a TSLP-TSLPR complex, with an affinity of about 29nM (Nature Communications, 2017, 8.).

[0007] There is a variable promoter in the TSLP gene region, which can express two proteins, long form TSLP (lfTSLP) and short form TSLP (sfTSLP) (including signal peptide). Compared with lfTSLP, the N-terminus of sfTSLP is truncated, retaining only "one and a half" α-helical structure. Studies have shown that the C-termini of lfTSLP and sfTSLP contain MKK34 antimicrobial peptides with antibacterial activity, and sfTSLP has stronger antibacterial activity. sfTSLP is the main form of TSLP expressed in normal human tissues. It is only constitutively expressed in normal oral mucosa, skin epidermis, salivary glands, and intestinal epithelial cells, exerting antibacterial activity; under inflammatory conditions, the expression of sfTSLP decreases (detected in pathological tissues of atopic dermatitis and segmental ileitis). However, lfTSLP is not expressed in normal tissues, but only in atopic dermatitis, asthma, ulcerative colitis, and oral mucosa of smoking (Pharmaceuticals 2016,9,41). When allergens stimulate epithelial cells, TSLP expression in the cells is upregulated, thereby creating a Th2-prone microenvironment, secreting Th2-type cytokines, generating inflammatory Th2 cells, and triggering allergic inflammatory responses. Studies have shown that when TSLP molecules bind to heterodimers composed of TSLPR and IL-7R-α, they activate JAK1 and JAK2, thereby activating signal transducers and activators of transcription 5 and 3 (STAT5, 3) and initiating the expression of downstream genes. TSLP is widely found in a variety of allergic diseases, such as allergic rhinitis, allergic asthma, eczema, etc. In addition, multiple TSLP SNP mutations have been detected in patients with atopic dermatitis, asthma, and eosinophilic esophagitis, which lead to high expression of lfTSLP. And these mutations are familial (Expert Rev Clin Immunol. 2014, 10(11): 1463–1474.). Therefore, it is of great significance to develop antibodies against lfTSLP to block diseases such as atopic dermatitis, asthma, eosinophilic esophagitis, and ulcerative colitis caused by abnormal expression of lfTSLP. At the same time, screening for MKK34 antimicrobial peptides with antibacterial activity without blocking the C-terminus is expected to retain the ability of TSLP to fight against external bacterial infections.

[0008] Tezepelumab, an antibody currently on the market, binds to the C-terminus of TSLP, which may affect the antimicrobial peptide activity of TSLP and lead to side effects. In addition, studies have shown that TSLP-trap molecules that simultaneously block TSLP, TSLPR, and IL-7Ralpha are 20-30 times more potent than Tezepelumab in inhibiting TSLP-induced STAT5 (Nature Communications, 2017, 8.). Therefore, the development of TSLP antibodies with different epitopes and stronger biological activity is expected to enhance drug efficacy and achieve better clinical treatment results.

[0009] Citation or identification of any document in this application is not an admission that such document is available as prior art to the present disclosure. Summary of the invention

[0010] In view of the above problems, the present disclosure provides antibodies, preparation methods, compositions, etc. The benefits provided by the present disclosure are widely applicable to the field of antibody therapy and diagnosis, and can be used in combination with antibodies that react with various targets. The present invention provides antibodies or antigen-binding fragments thereof that can specifically bind to TSLP, preferably chimeric monoclonal antibodies or humanized monoclonal antibodies.

[0011] In one aspect, the present disclosure provides an isolated antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof specifically binds to TSLP and comprises a heavy chain variable region and a light chain variable region,

[0012] The heavy chain variable region comprises:

[0013] CDRL1 comprising or consisting of a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to one of SEQ ID NOs: 16 and 22; and / or,

[0014] CDRL2 comprising or consisting of a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to one of SEQ ID NOs: 17 and 23; and / or,

[0015] The CDRL3 comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to one of SEQ ID NOs: 18 and 24, or consists of one of SEQ ID NOs: 18 and 24.

[0016] Further, in some embodiments of the present invention, the antibody or fragment, wherein,

[0017] The CDRH1 comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to one of SEQ ID NOs: 13 and 19; and / or,

[0018] The CDRH2 comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to one of SEQ ID NOs: 14 and 20; and / or,

[0019] The CDRH3 comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to one of SEQ ID NOs: 15 and 21, or consists of one of SEQ ID NOs: 15 and 21.

[0020] Further, in some embodiments of the present invention, the antibody or fragment, wherein the light chain variable region comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to one of SEQ ID NOs: 8 and 10, or consists of one of SEQ ID NOs: 8 and 10.

[0021] Further, in some embodiments of the present invention, the antibody or fragment, wherein the heavy chain variable region comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to one of SEQ ID NOs: 7, 9 or consists of one of SEQ ID NOs: 7, 9.

[0022] Further, in some embodiments of the present invention, the antibody or fragment also includes a heavy chain constant region, wherein the heavy chain constant region comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with one of SEQ ID NO: 11 or consists of one of SEQ ID NO: 11.

[0023] Further, in some embodiments of the present invention, the antibody or fragment further comprises a light chain constant region, which light chain constant region comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 12 or consists of SEQ ID NO: 12.

[0024] Further, in some embodiments of the present invention, the antibody or fragment comprises:

[0025] (a) a light chain comprising or consisting of a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to one of SEQ ID NOs: 4, 6;

[0026] (b) a heavy chain comprising a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to one of SEQ ID NOs: 3, 5 or consisting of one of SEQ ID NOs: 3, 5.

[0027] Further, in some embodiments of the present invention, the antibody or fragment comprises:

[0028] (a) a light chain having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% sequence identity to or consisting of SEQ ID NO:4; and a heavy chain having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to or consisting of one of SEQ ID NO:3 and 5;

[0029] (b) a light chain having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:6, or a light chain consisting of SEQ ID NO:6; and a heavy chain having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to one of SEQ ID NOs:3 and 5, or a heavy chain consisting of one of SEQ ID NOs:3 and 5.

[0030] Further, in some embodiments of the present invention, the antibody or fragment comprises:

[0031] (a) a light chain comprising or consisting of SEQ ID NO: 4; and a heavy chain comprising or consisting of SEQ ID NO: 3;

[0032] (b) a light chain comprising or consisting of SEQ ID NO: 6; and a heavy chain comprising or consisting of SEQ ID NO: 5.

[0033] Further, in some embodiments of the present invention, the antibody or fragment, wherein the antibody is a whole antibody, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fv fragment, or a single-chain Fv fragment (scFv).

[0034] Further, in some embodiments of the present invention, the antibody or fragment, wherein the antibody is a whole antibody or a monoclonal antibody.

[0035] Further, in some embodiments of the present invention, the antibody or fragment, wherein the antibody is a chimeric antibody or a humanized antibody or an improved antibody such as an improved chimeric antibody.

[0036] Further, in some embodiments of the present invention, the antibody or fragment, wherein the humanized antibody comprises:

[0037] a light chain variable region comprising or consisting of a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:26;

[0038] A heavy chain variable region comprising a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 25 or consisting of one of SEQ ID NO: 25.

[0039] Further, in some embodiments of the present invention, the antibody or fragment, wherein the humanized antibody comprises:

[0040] a light chain comprising or consisting of a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to one of SEQ ID NO:28;

[0041] A heavy chain comprising or consisting of a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to one of SEQ ID NO: 27.

[0042] In one aspect, the present disclosure provides an isolated nucleic acid molecule comprising a nucleic acid sequence encoding an antibody or fragment as described above.

[0043] In one aspect, the present disclosure provides a vector comprising the nucleic acid molecule.

[0044] In one aspect, the present disclosure provides a host cell comprising the nucleic acid molecule or the vector as described above.

[0045] In one aspect, the present disclosure provides a conjugate comprising the antibody or fragment described above coupled to at least one detectable label.

[0046] In one aspect, the present disclosure provides an antibody-drug conjugate (ADCs) comprising an antibody, which includes one or more drug moieties covalently linked to the antibody or fragment directly or via a linker.

[0047] On the one hand, the present disclosure provides a multispecific molecule, which comprises the antibody or antigen binding portion; preferably, the multispecific molecule specifically binds to TSLP; further preferably, the multispecific molecule also comprises at least one molecule having a second binding specificity for a second target.

[0048] In one aspect, the present disclosure provides a pharmaceutical composition or kit comprising the antibody or fragment, or the nucleic acid molecule, or the vector, or the host cell, or the conjugate, or the antibody-drug conjugate, or the multispecific molecule; and a pharmaceutically acceptable carrier.

[0049] In one aspect, the present disclosure provides a method for preparing the antibody or fragment, comprising the following steps:

[0050] (i) expressing said antibody or fragment in said host cell; and optionally

[0051] (ii) isolating the antibody or antigen-binding fragment thereof from the host cell.

[0052] On the other hand, the present disclosure provides use of the antibody or fragment, the nucleic acid molecule, the vector, the host cell, the conjugate, the antibody-drug conjugate, the multispecific molecule, the pharmaceutical composition or the kit in the preparation of a kit for diagnosing, detecting or monitoring diseases related to TSLP expression.

[0053] On the other hand, the present disclosure provides use of the antibody or fragment, the nucleic acid molecule, the vector, the host cell, the conjugate, the antibody-drug conjugate, the multispecific molecule, the pharmaceutical composition or the kit in the preparation of a drug for treating a disease associated with TSLP expression or determining the prognosis thereof.

[0054] In another aspect, the use described in the present disclosure, wherein the disease associated with TSLP expression is cancer, and the cancer is selected from: Hodgkin's lymphoma, breast cancer, pancreatic cancer, melanoma, cervical cancer, skin T-cell lymphoma, gastric cancer, lung cancer, B-cell lymphoma, including NHL, pre-B-cell lymphocytic leukemia / lymphoma and mature B-cell tumors, such as B-cell chronic lymphocytic leukemia (CLL) / small lymphocytic lymphoma (SLL), B-cell prolymphoma, The following are the major types of leukemia: lymphoplasmacytic lymphoma, mantle cell lymphoma (MCL), follicular lymphoma (FL), including low-grade, intermediate-grade, and high-grade FL, cutaneous follicle center lymphoma, marginal zone B-cell lymphoma (MALT-type, nodal, and splenic), hairy cell leukemia, diffuse large B-cell lymphoma, Burkitt's lymphoma, plasmacytoma, plasma cell myeloma, post-transplant lymphoproliferative disorder, Waldenstrom's macroglobulinemia, and anaplastic large cell lymphoma (ALCL).

[0055] In another aspect, the use described in the present disclosure, wherein the disease associated with TSLP expression is an autoimmune disease, and the autoimmune disease is selected from the following group: systemic lupus erythematosus, rheumatoid arthritis, psoriatic arthritis, axial spondyloarthritis, myasthenia gravis, polymyositis, psoriasis, pemphigus, vitiligo, multiple sclerosis, narcolepsy, neuromyelitis optica, type 1 diabetes, hyperthyroidism, hypothyroidism, Crohn's disease, ulcerative colitis, celiac disease, autoimmune gastritis, primary biliary cholangitis, autoimmune hepatitis and lupus nephritis.

[0056] In another aspect, the use described in the present disclosure, wherein the disease associated with TSLP expression is an inflammatory disease, and the inflammatory disease is selected from the following group: asthma, atopic dermatitis, chronic obstructive pulmonary disease (COPD), eosinophilic esophagitis (EoE), nasal polyps, chronic spontaneous urticaria, Ig-driven diseases, IgA nephropathy, lupus nephritis, eosinophilic gastritis, chronic sinusitis without nasal polyps and idiopathic pulmonary fibrosis (IFF).

[0057] The antibody of the present invention can effectively inhibit the activation effect of TSLP on the STATs and JAK2 signaling pathways of H_TSLP Reporter Cell Line cells. The antibody of the present invention and Tezepelumab can both effectively inhibit the proliferation-promoting effect of TSLP on BaF3-TSLPR / IL7Rα cells. The antibody of the present invention does not cross the binding epitope of Tezepelumab and will not block the MKK34 antimicrobial peptide with antibacterial activity at the C-terminus, and is expected to retain the ability of TSLP to resist external bacterial infection.

[0058] The 4G7 and 4C4 antibodies of the present invention can effectively inhibit the activation of TSLP on the STATs and JAK2 signaling pathways of H_TSLP Reporter Cell Line cells, wherein the inhibitory signaling pathway activation activity of 4C4 is 59 times that of Tezepelumab, and the inhibitory signaling pathway activation activity of 4G7 is 26 times that of Tezepelumab.

[0059] The 4G7, 4C4 antibodies and Tezepelumab of the present invention can effectively inhibit the proliferation-promoting effect of TSLP on BaF3-TSLPR / IL7Rα cells, and the inhibitory activity of 4C4 is 11.3 times that of Tezepelumab, and the inhibitory activity of 4G7 is 6.5 times that of Tezepelumab.

[0060] The 4G7 V60 of the present invention is a humanized antibody. Both 4G7 V60 and Tezepelumab can effectively inhibit the activation of TSLP on the STATs and JAK2 signaling pathways of H_TSLP Reporter Cell Line cells. The inhibitory signaling pathway activity of 4G7 V60 is increased by 4743 times compared with Tezepelumab.

[0061] The antibody 4G7 of the present invention does not cross the binding epitope of Tezepelumab and will not block the MKK34 antimicrobial peptide with antibacterial activity at the C-terminus, and is expected to retain the ability of TSLP to resist external bacterial infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] The accompanying drawings are used to provide further understanding of the present disclosure and constitute a part of the specification. They are used to explain the present disclosure together with the embodiments of the present disclosure and do not constitute a limitation of the present disclosure.

[0063] Figure 1A-1B The results of the detection of the binding activity between the antibody of the present invention and TSLP are shown;

[0064] Figure 2A-2B The results of the blocking activity test of the antibody of the present invention on TSLP and TSLPR at the protein level are shown;

[0065] Figure 3A-3B The results of the detection of the activity of the antibody of the present invention in blocking TSLP binding to BaF3-TSLPR / IL7Rα cells are shown;

[0066] Figure 4A-4B The results of the detection of the activation activity of the antibody of the present invention in inhibiting TSLP on the STATs and JAK2 signaling pathways of H_TSLP Reporter Cell Line cells are shown;

[0067] Figure 5A-Figure 5B The results of the activity test of the antibody of the present invention inhibiting the proliferation-promoting effect of TSLP on BaF3-TSLPR / IL7Rα cells are shown;

[0068] Figure 6 The results of the humanized antibody of the present invention inhibiting the activation activity of TSLP on the STATs and JAK2 signaling pathways of H_TSLP Reporter Cell Line cells are shown. DETAILED DESCRIPTION

[0069] In order to make those skilled in the art better understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only illustrative embodiments of a part of the present invention, rather than all embodiments. Therefore, the present invention is not limited to the specific embodiments illustrated. In addition, any section titles used herein are not to be construed as limiting the subject matter described.

[0070] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention will have the meanings commonly understood by those of ordinary skill in the art. In addition, unless the context requires otherwise, terms in the singular shall include plural forms, and terms in the plural shall include singular forms. More specifically, as used in this specification and the appended claims, unless the context clearly indicates otherwise, the singular forms "a", "an" and "the" include plural indicators. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "comprising" and other forms (such as "including" and "containing") is not restrictive. In addition, the ranges provided in the specification and the appended claims include endpoints and all values ​​between the endpoints.

[0071] definition

[0072] In order to better understand the present invention, definitions and explanations of relevant terms are provided below.

[0073] The term "antibody" or "Ab" generally refers to a Y-shaped tetrameric protein comprising two heavy (H) and two light (L) polypeptide chains held together by covalent disulfide bonds and non-covalent interactions. The light chains of antibodies can be divided into κ or λ light chains. The heavy chains can be divided into μ, δ, γ, α or ε, which define the isotype of the antibody as IgM, IgD, IgG, IgA or IgE, respectively. In the light and heavy chains, the variable region is connected to the constant region by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of 3 domains (CH1, CH2 and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The VH and VL regions can be further divided into hypervariable regions (called complementary determining regions, CDRs) separated by relatively conservative regions (called framework regions, FRs for short). Each VH and VL consists of 3 CDRs and 4 FRs in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 from N-terminus to C-terminus. The variable regions (VH and VL) of each heavy chain / light chain pair form antigen binding sites / parts, respectively. The distribution of amino acids in various regions or domains follows the numbering definitions in common systems such as Kabat, IMGT or Chothia. In the specific embodiments of the present disclosure, the determination of CDR sequences uses the numbering definitions in the Kabat system. The CDRs on VH are CDRH1, CDRH2 and CDRH3, and the CDRs on VL are CDRL1, CDRL2 and CDRL3.

[0074] Antibodies in the present disclosure also include antigen binding portions (interchangeably used with the term "antigen binding fragment"). Antigen binding portions refer to polypeptides comprising fragments of intact antibodies that retain the ability to specifically bind to an antigen that specifically binds to a full-length or intact antibody, and / or that compete with a full-length antibody for binding to the same antigen. Under some conditions, antigen binding portions include Fab, Fab', F(ab')2, Fd, Fv, dAb and complementary determining region (CDR) fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, diabodies, and antibodies that contain at least a portion of an antibody sufficient to confer specific antigen binding ability to a polypeptide. The antigen binding portion of an antibody can be obtained from a given antibody by conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods), and can be screened for specificity in the same manner as intact antibodies.

[0075] The term "isotype" refers to the antibody class (eg, IgM or IgG1) encoded by the heavy chain constant region genes.

[0076] The term "monoclonal antibody" or "mAb" refers to an antibody molecule / preparation of single molecular composition. A monoclonal antibody displays a single binding specificity and affinity for a particular epitope. The antibodies of the present invention may be derived from different species, including but not limited to mouse, rat, rabbit, guinea pig and human.

[0077] The term "epitope" refers to an antigenic determinant in a molecule, which refers to a portion of a molecule that is recognized by the immune system (eg, by an antibody), such as a discrete three-dimensional site on an antigen recognized by the immune system. In the present invention, the epitope is, for example, TSLP protein.

[0078] The term "chimeric antibody" as used herein refers to an antibody whose variable region sequence is from one species and the constant region sequence is from another species, for example, an antibody in which the variable region sequence is derived from a mouse antibody and the constant region sequence is derived from a human antibody.

[0079] The term "humanized antibody" is intended to refer to antibodies in which CDR sequences / antigen binding portions or sites derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. In addition, additional framework region modifications may also be made within the human framework sequences.

[0080] The term "KD value" is the equilibrium dissociation constant between an antibody and its antigen, i.e., the ratio of koff / kon or kd / ka (determined by SPR technology). Therefore, the lower the KD value (the lower the concentration), the higher the affinity of the antibody. Therefore, the "KD value" can be used to measure the binding affinity between an antibody and its antigen.

[0081] The terms "TSLP" and "TSLP antigen" are used interchangeably herein and include any variants, isoforms, and species homologs of human TSLP that are naturally expressed by cells or expressed on cells transfected with the TSLP gene. In some embodiments, binding of the antibodies of the present disclosure to the TSLP antigen mediates killing of cells expressing TSLP (e.g., tumor cells) by inactivating TSLP. Killing of cells expressing TSLP may occur by one or more of the following mechanisms: cell death / apoptosis induction, ADCC, and CDC.

[0082] The term "anti-TSLP antibody" or "TSLP antibody" refers to an antibody capable of binding to a TSLP antigen or to a cell expressing TSLP as defined herein.

[0083] The term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and an antigen to which it is directed. In certain embodiments, an antibody that specifically binds to an antigen (or has specificity for an antigen) means that the antibody binds to the antigen with a specificity of less than about 10 -5 M, for example, less than about 10 -6 M, 10-7 M, 10 -8 M, 10 -9 M or 10 -10 In some embodiments of the present invention, the term "targeting" refers to specific binding.

[0084] The term "isolated" refers to a state obtained from a natural state by artificial means. If a certain "isolated" substance or component exists in nature, it may be because its natural environment has changed, or the substance has been separated from the natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide exists naturally in a living organism, and the same polynucleotide or polypeptide with high purity separated from this natural state is called an isolated polynucleotide or polypeptide. The term "isolated" neither excludes mixed artificial or synthetic substances, nor excludes other impure substances that do not affect the activity of the isolated substance. For example, an isolated antibody may be substantially free of other cellular materials and / or chemicals.

[0085] The term "vector" refers to a nucleic acid vector into which a polynucleotide can be inserted. When a vector allows the expression of a protein encoded by a polynucleotide inserted therein, the vector is called an expression vector. The vector can be transformed, transduced or transfected into a host cell to express the genetic material elements carried in the host cell. Vectors are well known to those skilled in the art, and include but are not limited to plasmids, bacteriophages, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage and animal viruses. Animal viruses that can be used as vectors include but are not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papovaviruses (such as SV40). The vector may contain multiple elements for controlling expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may contain an origin of replication. As for the vector for expressing the antibody, a vector type in which the antibody heavy chain and light chain exist in different vectors or a vector type in which the heavy chain and light chain exist in the same vector can be used.

[0086] The term "host cell" refers to a cell system that can be engineered to produce a target protein, protein fragment or peptide. Host cells include, but are not limited to, cultured cells, such as cultured mammalian cells derived from rodents (rat, mouse, guinea pig or hamster), such as CHO, BHK, NSO, SP2 / 0, YB2 / 0; or human tissue or hybridoma cells, yeast cells and insect cells, as well as cells contained in transgenic animals or cultured tissues. The term covers not only specific test cells, but also the offspring of such cells. Because certain modifications may occur in subsequent generations due to mutations or environmental influences, such offspring may be different from the parental cells, but are still included in the scope of the term "host cell".

[0087] The term "identity" refers to the relationship between the sequences of two or more polypeptide molecules (or protein molecules) or two or more nucleic acid molecules determined by alignment and comparison of the sequences. "Percent identity" refers to the percentage of identical residues between amino acids or nucleotides in the compared molecules and is calculated based on the size of the smallest molecules being compared. For these calculations, gaps in the alignment, if any, are preferably addressed by a specific mathematical model or computer program (i.e., an "algorithm"). Methods that can be used to calculate the identity of aligned nucleic acids or polypeptides include those described in Computational Molecular Biology, (Lesk, AM, ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, DW, ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, AM, and Griffin, HG, eds.), 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M. Stockton Press; and Carillo et al, 1988, SIAM J. Applied Math. 48:1073.

[0088] The term "immunogenicity" refers to the ability to stimulate the formation of specific antibodies or sensitized lymphocytes in an organism. It not only refers to the property of antigens to stimulate the activation, proliferation and differentiation of specific immune cells to ultimately produce immune effector substances such as antibodies and sensitized lymphocytes, but also refers to the specific immune response of antibodies or sensitized T lymphocytes that can be formed in the immune system of an organism after stimulating the organism with an antigen. Immunogenicity is the most important property of an antigen. Whether an antigen can successfully induce the generation of an immune response in a host depends on three factors: the nature of the antigen, the reactivity of the host and the means of immunity.

[0089] The term "transfection" refers to the process of introducing nucleic acids into eukaryotic cells, particularly mammalian cells. Protocols and techniques for transfection include, but are not limited to, lipid transfection and chemical and physical methods such as electroporation. Many transfection techniques are well known in the art and disclosed herein. See, for example, Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, supra; Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al, 1981, Gene 13:197.

[0090] The term "hybridoma" and the term "hybridoma cell line" are used interchangeably. When referring to the term "hybridoma" and the term "hybridoma cell line", they also include subclones and progeny cells of the hybridoma.

[0091] The term "immune effector function" includes any function mediated by components of the immune system that results in inhibition of tumor growth and / or inhibition of tumorigenesis, including inhibition of tumor dissemination and metastasis. Preferably, the immune effector function results in killing tumor cells. Preferably, the immune effector function in the present invention is an antibody-mediated effector function. Such functions include complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), inducing apoptosis in cells carrying tumor-associated antigens (e.g., by binding of antibodies to surface antigens) and / or inhibiting the proliferation of cells carrying tumor-associated antigens, preferably ADCC and / or CDC. Antibodies can also simply act by binding to tumor-associated antigens on the surface of tumor cells. For example, antibodies can block the function of tumor-associated antigens or induce apoptosis simply by binding to tumor-associated antigens on the surface of tumor cells.

[0092] The term "antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig bound to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) enables these cytotoxic effector cells to specifically bind to target cells bearing antigens and subsequently kill the target cells with cytotoxins. Antibodies "arm" cytotoxic cells and are absolutely required for this killing. NK cells, the main cells that mediate ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. In order to assess the ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Pat. Nos. 5,500,362 or 5,821,337, can be performed. Effector cells that can be used for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, ADCC activity of the molecule of interest can be assessed in vivo, for example, in an animal model such as that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

[0093] The term "complement dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to an antibody (of the appropriate subclass) that binds to its cognate antigen. To assess complement activation, a CDC assay, such as that described in Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996), can be performed.

[0094] The term "subject" includes any mammal, such as a human or non-human animal, preferably a human.

[0095] The terms "treatment" and "treatment" as used herein in the context of treating a condition generally refer to treatment and therapy of a human or animal in which some desired therapeutic effect is achieved, for example, inhibition of disease progression, including decreased rate of progression, stagnation of rate of progression, regression of disease, improvement of disease, and cure of disease. Treatment as a preventive measure (i.e., prevention) is also included. For cancer, "treatment" may refer to inhibiting or slowing tumor or malignant cell growth, proliferation, or metastasis, or some combination thereof. For tumors, "treatment" includes removing all or part of a tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying the development of a tumor, or some combination thereof.

[0096] The term "therapeutically effective amount" relates to an amount of an active compound or a material, composition or dosage form comprising an active compound, which, when administered according to the desired treatment regimen, is effective for producing some desired therapeutic effect commensurate with a reasonable benefit / risk ratio. For example, when used in conjunction with the treatment or detection of a disease or condition of interest, an "effective amount" or "effective dose" refers to an amount or concentration of an antibody or antigen-binding fragment thereof that is effective for treating or detecting the disease or condition.

[0097] The term "pharmaceutically acceptable" means that the carrier, diluent, excipient and / or salt thereof is chemically and / or physically compatible with the other ingredients of the formulation and physiologically compatible with the recipient.

[0098] The term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active agent and is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19 th Ed. Pennsylvania: Mack Publishing Company, 1995), and includes but is not limited to pH adjusters, surfactants, adjuvants and ionic strength enhancers. For example, pH adjusters include but are not limited to phosphate buffer; surfactants include but are not limited to cationic, anionic or nonionic surfactants, such as Tween-80; ionic strength enhancers include but are not limited to sodium chloride.

[0099] The term "adjuvant" refers to a non-specific immunopotentiator, which can enhance the immune response to the antigen in the organism or change the type of immune response when it is delivered to the organism together with the antigen or delivered to the organism in advance. There are multiple adjuvants, including but not limited to aluminum adjuvants (such as aluminum hydroxide), Freund's adjuvants (such as Freund's complete adjuvant and Freund's incomplete adjuvant), Corynebacterium brevis, lipopolysaccharide, cytokines, etc. Freund's adjuvant is the most commonly used adjuvant in current animal experiments. Aluminum hydroxide adjuvant is more commonly used in clinical trials.

[0100] The term "autoimmune disease" refers to a disease caused by the body's immune response to its own antigens, resulting in damage to its own tissues, including but not limited to systemic lupus erythematosus, rheumatoid arthritis, psoriatic arthritis, axial spondyloarthritis, myasthenia gravis, polymyositis, psoriasis, pemphigus, vitiligo, multiple sclerosis, narcolepsy, neuromyelitis optica, type 1 diabetes, hyperthyroidism, hypothyroidism, Crohn's disease, ulcerative colitis, celiac disease, autoimmune gastritis, primary biliary cholangitis, autoimmune hepatitis and lupus nephritis.

[0101] The term "inflammatory disease" refers to a general term for diseases in which inflammation is the main destructive factor. Inflammation is a biological response of tissue to harmful stimuli, which is a pathological change and is accompanied by three events of tissue degeneration, circulatory disturbances and fluid exudation, as well as hypertrophy. Examples of inflammatory diseases include acute and chronic diseases, including but not limited to asthma, scleroderma, systemic lupus erythematosus, rheumatoid arthritis, Churg-Strauss syndrome, Wegener's granulomatosis, Goodpasture's hemorrhage-nephritis syndrome, hypersensitivity pneumonitis, atopic dermatitis, rhinitis, ankylosing spondylitis, rheumatic fever, fibromyalgia, psoriatic arthritis, chronic nephritis, Sjögren's syndrome, and multiple sclerosis, allergic dermatitis, allergic conjunctivitis, atopic dermatitis fibrosis, and inflammatory bowel disease.

[0102] The term "cancer" refers to any tumor or malignant cell growth, proliferation or metastasis-mediated medical condition, whether solid or non-solid, such as leukemia, benign, pre-malignant or malignant, including but not limited to Hodgkin lymphoma, breast cancer, pancreatic cancer, melanoma, cervical cancer, cutaneous T-cell lymphoma, gastric cancer, lung cancer, B-cell lymphoma, including NHL, pre-B-cell lymphocytic leukemia / lymphoma and mature B-cell neoplasms, such as B-cell chronic lymphocytic leukemia (CLL) / small lymphocytic leukemia Lymphoma (SLL), B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, mantle cell lymphoma (MCL), follicular lymphoma (FL), including low-grade, intermediate-grade and high-grade FL, cutaneous follicle center lymphoma, marginal zone B-cell lymphoma (MALT-type, nodal and splenic), hairy cell leukemia, diffuse large B-cell lymphoma, Burkitt's lymphoma, plasmacytoma, plasma cell myeloma, post-transplant lymphoproliferative disorder, Waldenstrom's macroglobulinemia and anaplastic large cell lymphoma (ALCL).

[0103] In the present disclosure, the amino acid at a certain position may be allowed to vary, for example, [K / R] indicates that the amino acid at the position may be either K or R; another example is [S / *], where the * indicates a deletion, indicating that the amino acid at the position may be either S or deleted.

[0104] Anti-TSLP Antibodies

[0105] In some aspects, the invention includes isolated antibodies or antigen-binding fragments thereof.

[0106] In the context of the present application, "antibodies" may include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized and primatized antibodies, CDR-grafted antibodies, human antibodies, recombinantly produced antibodies, intracellular antibodies, bifunctional antibodies, multispecific antibodies, bispecific antibodies, monovalent antibodies, multivalent antibodies, anti-idiotypic antibodies, synthetic antibodies, including mutant proteins and variants thereof, improved antibodies; and derivatives thereof (including Fc fusion proteins and other modifications), as well as any other immunoreactive molecules, as long as they show preferential association or binding with TSLP proteins. In addition, unless the context otherwise dictates, the term also includes antibodies of all classes (i.e., IgA, IgD, IgE, IgG, and IgM) and all subclasses (i.e., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). In a preferred embodiment, the antibody is a monoclonal antibody. In a more preferred embodiment, the antibody is a chimeric monoclonal antibody or a humanized monoclonal antibody or an improved chimeric monoclonal antibody.

[0107] The variable regions and CDRs in an antibody sequence can be identified according to general rules developed in the art (eg, as described above, the Kabat numbering system or by aligning the sequence with a database of known variable regions).

[0108] In some embodiments, the isolated antibody or antigen-binding fragment thereof may comprise conservative substitutions or modifications or replacements (e.g., conservative replacements), deletions or additions (e.g., replacements, deletions or additions of 1, 2, 3, 4 or 5 amino acids; or conservative replacements of up to 20, up to 15, up to 10, or up to 5 amino acids) of amino acids in the variable region of the heavy chain and / or light chain, or have at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100% sequence identity with the antibody or antigenic fragment thereof from which it is derived. Amino acid deletion variants comprising deletions at the N-terminus and / or C-terminus of the protein are also referred to as N-terminal and / or C-terminal truncation variants. It is understood in the art that certain modifications of conservative sequences that do not eliminate antigen binding may be performed. See, e.g., Brummell et al. (1993) Biochem 32:1180-8; de Wildt et al. (1997) Prot. Eng. 10:835-41; Komissarov et al. (1997) J. Biol. Chem. 272:26864-26870; Hall et al. (1992) J. Immunol. 149:1605-12; Kelley and O'Connell (1993) Biochem. 32:6862-35; Adib-Conquy et al. (1998) Int. Immunol. 10:341-6 and Beers et al. (2000) Clin. Can. Res. 6:2835-43.

[0109] As described above, the term "conservative substitution / conservative replacement" as used herein refers to an amino acid substitution / replacement that does not adversely affect or change the basic properties of the protein / polypeptide comprising the amino acid sequence. For example, conservative substitution / conservative replacement can be introduced by standard techniques known in the art (e.g., site-directed mutagenesis and PCR-mediated mutagenesis). Conservative amino acid substitutions include substitutions in which an amino acid residue is replaced by another amino acid residue with a similar side chain, such as a physical or functionally similar residue (e.g., having a similar size, shape, charge, chemical properties including the ability to form covalent bonds or hydrogen bonds, etc.) to a corresponding amino acid residue. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine and histidine), amino acids with acidic side chains (e.g., aspartic acid and glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with beta-branched side chains (e.g., threonine, valine, isoleucine) and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, the corresponding amino acid residue is preferably substituted by another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10): 879-884 (1999); and Burks et al., Proc. Natl. Acad. Sci. USA 94: 412-417 (1997), which are incorporated herein by reference).

[0110] According to the present invention, if reference is made to comprising a specific antibody heavy chain and / or a specific antibody light chain (e.g., chains comprising specific CDR sequences), the two heavy chains and / or the two light chains of the antibody are preferably constituted by the specific antibody heavy chain and / or the specific antibody light chain, respectively.

[0111] Regardless of how the antibody is produced, methods for testing the ability of the antibody to bind to an antigen (e.g., TSLP) are known in the art and include any antibody-antigen binding assay, such as radioimmunoassay (RIA), ELISA, Western blot, immunoprecipitation, SPR, and competitive inhibition assays (see, e.g., Janeway et al., infra, and U.S. Patent Application Publication No. 2002 / 0197266 and the above section on competition assays).

[0112] According to the present invention, an antibody is capable of binding to a predetermined target (e.g., a TSLP protein or a cell expressing TSLP) if the antibody has significant affinity for the predetermined target in a standard assay (e.g., the assay described herein). To test the binding of a monoclonal antibody to a living cell expressing TSLP, flow cytometry can be used. Preferably, the antibody is capable of binding to a target expressed on the cell surface in a flow cytometry analysis (FACS) analysis if the antibody detectably binds to the target (TSLP protein or a cell expressing TSLP). Preferably, an antibody in the present invention detectably binds to the target if it is present at a concentration of 10 μg / mL or less, 5 μg / mL or less, 3 μg / mL or less, 2 μg / mL or less, 1 μg / mL or less, 0.5 μg / mL or less.

[0113] In some embodiments, the EC50 binding properties of the antibody or fragment may be: binding to human TSLP or monkey TSLP with an EC50 of 5 μg / mL or less; binding to human TSLP or monkey TSLP with an EC50 of 3 μg / mL or less; or binding to human TSLP or monkey TSLP with an EC50 of 2 μg / mL or less; or binding to human TSLP or monkey TSLP with an EC50 of 1 μg / mL or less; or binding to human TSLP or monkey TSLP with an EC50 of 0.5 μg / mL or less; binding to human TSLP or monkey TSLP with an EC50 of 0.3 μg / mL or less; or binding to human TSLP or monkey TSLP with an EC50 of 0.1 μg / mL or less.

[0114] In some embodiments, the antibody is a monoclonal antibody.

[0115] In some embodiments, the antibody is a chimeric antibody or a humanized antibody or an improved antibody such as an improved chimeric antibody.

[0116] In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises a constant region of IgG. The constant region of IgG is preferably selected from IgG1, IgG2, IgG3, or IgG4 constant region. The constant region of IgG is more preferably selected from the constant region of IgG1.

[0117] In the art, various methods are used to modify the desired properties of antibodies without changing them, such as the method used in the present disclosure of recombining the light chain and heavy chain of the antibody, replacing amino acids, etc. For example, the sequences of the present invention, including chimeric antibody sequences or humanized antibody sequences, can be subjected to conservative amino acid substitutions.

[0118] Antibodies interact with target antigens primarily through amino acid residues located in the complementary determining regions (CDRs) of the heavy and light chains. For this reason, the amino acid sequences of CDRs are more diverse than other sequences between antibodies. Since CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of specific naturally occurring antibodies by constructing expression vectors that contain CDR sequences from the specific naturally occurring antibodies, which are transplanted onto framework sequences from different antibodies with different properties (see, e.g., Riechmann, L. et al. (1998) Nature 332: 323-327; Jones, P. et al. (1986) Nature 321: 522-525; and Queen, C. et al. (1989) Proc. Natl. Acad. Sci. USA 86: 10029-10033). Such framework sequences can be obtained from public DNA databases that include germline antibody gene sequences. These germline sequences are different from mature antibody gene sequences because they do not contain fully assembled variable genes. The germline gene sequence will also have sequences that differ from the high affinity secondary repertoire antibody at individual locations evenly throughout the variable region.

[0119] Mouse antibodies are highly immunogenic in humans, resulting in reduced therapeutic efficacy when used repeatedly, with the main immunogenicity mediated by the heavy chain constant region. If the individual antibodies are chimerized or humanized, the immunogenicity of mouse antibodies in humans can be reduced or completely avoided.

[0120] Chimeric antibodies refer to antibodies in which different parts are from different animal species, for example, antibodies with variable regions from mouse antibodies and human immunoglobulin constant regions. The variable regions of mouse antibody heavy and light chains are linked together with the constant regions of human heavy and light chains to obtain the chimeric nature of the antibody (for example, as described in Kraus et al., in Methods in Molecular Biology series, Recombinant antibodies for cancer therapy ISBN-0-89603-918-8). In a preferred embodiment, chimeric antibodies are produced by connecting human kappa light chain constant regions to mouse light chain variable regions. In another preferred embodiment, chimeric antibodies can be produced by connecting human lambda light chain constant regions to mouse light chain variable regions.

[0121] Humanized antibodies are antibodies in which CDR sequences / antigen-binding portions or sites derived from the germline of another mammalian species, such as a mouse, are grafted onto human framework sequences.

[0122] In order to reduce the immunogenicity of the antibody to humans, the sequence of the TSLP antibody of the present disclosure is used to produce a humanized anti-TSLP antibody, and the CDR region of an anti-TSLP antibody from other sources is combined with a human framework region (e.g., human immunoglobulin) to form the humanized anti-TSLP antibody of the present disclosure. The humanized antibody is expected to retain the function of binding to human TSLP as well as the function of binding to monkey TSLP.

[0123] Preparation or generation of antibodies

[0124] The antibodies of the present invention can be produced by a variety of techniques, including conventional monoclonal antibody methods, such as the standard somatic cell hybridization technique of Kohler and Milstein, Nature 256: 495 (1975). Although hybridoma technology is preferred, in principle, other techniques for producing monoclonal antibodies can be used, such as virus or oncogene transformation of B lymphocytes or phage display technology using antibody gene libraries, somatic cell hybridization methods, and for example, obtaining through genetic engineering recombinant technology. For example, DNA molecules encoding the heavy chain and light chain genes of the antibodies of the present invention are obtained by chemical synthesis or PCR amplification, the resulting DNA molecules are inserted into expression vectors, and then transfected into host cells, and then, the transfected host cells are cultured under specific conditions, and the antibodies of the present invention are expressed.

[0125] Other preferred animal systems for preparing hybridomas that secrete monoclonal antibodies are rat and rabbit systems (described, for example, in Spieker-Polet et al., Proc. Natl. Acad. Sci. USA 92: 9348 (1995), see also Rossie et al.. Am. J. Clin. Pathol. 124: 295 (2005)). Hybridoma production in mice is a very well established method. Immunization protocols and techniques for isolating immunized spleen cells for fusion are known in the art. Fusion partners (e.g., mouse myeloma cells) and fusion methods are also known.

[0126] Monoclonal antibodies can be prepared using a variety of techniques known in the art, including hybridoma technology, recombinant technology, phage display technology, transgenic animals or some combination thereof. For example, hybridomas and biochemical and genetic engineering techniques recognized in the art can be used to produce monoclonal antibodies, as described in detail in An, Zhiqiang (ed.) Therapeutic Monoclonal Antibodies: From Bench to Clinic, John Wiley and Sons, 1st ed.2009; Shire et al. (eds.) Current Trends in Monoclonal Antibody Development and Manufacturing, Springer Science + Business Media LLC, 1st ed.2010; Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed.1988; Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY, 1981), each of which is incorporated herein by reference in its entirety.

[0127] It should be understood that the selected binding sequence can be further altered, for example, to increase affinity for the target, humanize the target binding sequence, improve its production in cell culture, reduce its immunogenicity in vivo, generate multispecific antibodies, etc., and antibodies comprising altered target binding sequences are also antibodies of the present invention.

[0128] In some embodiments, the method of producing the antibodies or fragments described herein comprises the following steps:

[0129] (i) expressing the antibody or fragment in a host cell; and optionally

[0130] (ii) isolating the antibody or antigen-binding fragment thereof from the host cell.

[0131] In a preferred embodiment, anti-TSLP monoclonal antibodies are prepared by using hybridomas.

[0132] In order to obtain hybridomas that produce antibodies of the invention, such as human monoclonal antibodies of the invention, splenocytes and / or lymph node cells from immunized mice can be isolated and fused to a suitable immortalized cell line, such as a mouse myeloma cell line. The resulting hybridomas are screened for the production of antigen-specific antibodies. The production of hybridomas is well known in the art. See, for example, Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York.

[0133] The antibodies of the present invention can also be produced in host cell transfectomas using, for example, a combination of recombinant DNA technology and gene transfection methods well known in the art (e.g., Morrison, S. (1985) Science 229: 1202). In some embodiments, DNA encoding partial or full-length light and heavy chains obtained by standard molecular biology techniques is inserted into one or more expression vectors so that the genes are operably linked to transcriptional and translational regulatory sequences. In this context, the term "operably linked" is intended to mean that the antibody gene is linked to the vector so that the transcriptional and translational control sequences within the vector perform their intended functions of regulating transcription and translation of the antibody gene.

[0134] The antibody light chain gene and the antibody heavy chain gene can be inserted into the same or different expression vectors.In some embodiments, the variable region is used to produce the full-length antibody gene of any antibody isotype by inserting it into the expression vector of the heavy chain constant region and the light chain constant region encoding the desired isotype, so that the VH segment is operably connected to the CH segment and the VL segment in the carrier are operably connected to the CL segment in the carrier.In addition or alternatively, the recombinant expression vector can encode the signal peptide that promotes the secretion of the antibody chain from the host cell.The antibody chain gene can be cloned into a vector so that the signal peptide is connected to the amino terminal of the antibody chain gene.The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

[0135] To express the light and heavy chains, expression vectors encoding the heavy and light chains are transfected into host cells by standard techniques. The various forms of the term "transfection" are intended to encompass various techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, etc. The antibodies of the present invention can be expressed in prokaryotic or eukaryotic host cells, such as mammalian host cells, which can assemble and secrete appropriately folded and immunologically active antibodies.

[0136] Mammalian host cells for expressing the recombinant antibodies of the invention include Chinese hamster ovary cells (CHO cells) (including dhfr CHO cells described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77: 4216-4220) used with a DHFR selection marker (e.g., as described in RJ Kaufman and PA Sharp (1982) J. MoI. Biol. 159: 601-621), NSO myeloma cells, COS cells and SP2 cells. In particular, for use with NSO myeloma, another expression system is the GS gene expression system disclosed in WO 87 / 04462, WO 89 / 01036 and EP 338,841. When a recombinant expression vector encoding an antibody gene is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a period of time sufficient to allow the antibody to be expressed in the host cell or secreted into the culture medium in which the host cell is grown. The antibody can be recovered from the culture medium using standard protein purification methods.

[0137] In another preferred embodiment, transgenic or transchromosomal mice carrying parts of the human immune system (rather than the mouse system) can be used to generate human monoclonal antibodies against TSLP.

[0138] Another strategy for generating monoclonal antibodies is to directly isolate the gene encoding the antibody from the lymphocytes producing antibodies of a defined strategy, see for example Babcock et al., 1996; A novel strategy for generating monoclonal antibodies from single, isolated lymphocytes producing antibodies of defined strategy. For details of recombinant antibody engineering, see also Welschof and Krau, Recombinant antibodies for cancer therapy ISBN-0-89603-918-8 and Benny KC Lo Antibody Engineering ISBN 1-58829-092-1.

[0139] To prepare chimeric antibodies, murine immunoglobulin variable regions can be linked to human immunoglobulin constant regions using methods known in the art (see, e.g., U.S. Pat. No. 4,816,567 to Cabilly et al.). The isolated nucleic acid encoding the VH region can be converted into a full-length heavy chain gene by operably linking the nucleic acid encoding the VH to another DNA molecule encoding the heavy chain constant region (CH1, CH2, and CH3). The sequences of human heavy chain constant region genes are known in the art (see, e.g., Kabat et al. (1991), Sequences Of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242). The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, but more preferably an IgG1 or IgG4 constant region. The isolated nucleic acid encoding the VL region can be converted into a full-length light chain gene (as well as a Fab light chain gene) by operably linking the DNA encoding the VL to another DNA molecule encoding the light chain constant region CL. The sequences of human light chain constant region genes are known in the art (see, e.g., Kabat et al., supra), and DNA fragments comprising these regions can be obtained by standard PCR amplification. In a preferred embodiment, the light chain constant region can be a kappa or lambda constant region, but is generally preferably a kappa constant region. Once DNA fragments encoding VH and VL segments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, such as converting variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In these operations, the DNA fragment encoding VL or VH is operably connected to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. The term "operably connected" as used herein is intended to indicate that two DNA fragments are connected so that the amino acid sequences encoded by the two DNA fragments remain in frame.

[0140] To prepare humanized antibodies, mouse CDR regions can be inserted into human framework sequences using methods known in the art (see Winter, U.S. Pat. No. 5,225,539; Queen et al., U.S. Pat. No. 5,530,101; U.S. Pat. No. 5,585,089; U.S. Pat. No. 5,693,762; and Lo, Benny, KC, editor, in Antibody Engineering: Methods and Protocols, volume 248, Humana Press, New Jersey, 2004). Alternatively, transgenic animals can be used that are capable of not producing endogenous immunoglobulins after immunization and are capable of producing a complete human antibody repertoire. For example, it has been reported that the homozygous deletion of the antibody heavy-chain joining region (JH) gene in chimeric and germ-line mutant mice can completely inhibit endogenous antibody production, and then transfer of the human germ-line immunoglobulin gene array into the germ-line mutant mice will result in the mice producing human antibodies when challenged with antigen (see, e.g., Jakobovits et al., 1993, Proc. Natl. Acad. Sci. USA 90:2551; Jakobovits et al., 1993, Nature 362:255-258; Bruggermann et al., 1993, Year in Immunology 7:33; and Duchosal et al., 1992, Nature 355:258). Non-limiting examples of the above transgenic animals include, HuMAb mice (Medarex, Inc.), which contain human immunoglobulin gene miniloci encoding unrearranged human heavy chain (μ and γ) and κ light chain immunoglobulin sequences, plus targeted mutations that inactivate endogenous μ and κ chain loci (see, e.g., Lonberg et al. (1994) Nature 368 (6474): 856-859); or "KM mice TM" carrying human heavy chain transgenes and human light chain transchromosomes (see patent application WO02 / 43478). Other methods for antibody humanization include phage display technology (Hoogenboom et al., 1991, J. Mol. Biol. 227: 381; Marks et al., J. Mol. Biol. 1991, 222: 581-597; Vaughan et al., 1996, Nature Biotech 14: 309).

[0141] Nucleic acid molecules encoding the antibodies of the present invention

[0142] In some aspects, the present invention relates to an isolated nucleic acid molecule comprising a nucleic acid sequence encoding the above-mentioned isolated antibody or fragment thereof as disclosed herein.

[0143] Nucleic acids of the present invention can be obtained using standard molecular biology techniques. For hybridoma-expressed antibodies (e.g., hybridomas prepared by transgenic mice carrying human immunoglobulin genes as further described below), cDNA encoding the light and heavy chains of antibodies prepared by hybridomas can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from immunoglobulin gene libraries (e.g., using phage display technology), nucleic acids encoding such antibodies can be recovered from gene libraries.

[0144] In order to prepare chimeric antibodies, methods known in the art can be used to connect the mouse immunoglobulin variable region to the human immunoglobulin constant region (see, for example, U.S. Pat. No. 4,816,567 to Cabilly et al.). The isolated nucleic acid encoding the VH region can be converted into a full-length heavy chain gene by operably connecting the nucleic acid encoding the VH to another DNA molecule encoding the heavy chain constant region (CH1, CH2 and CH3), and DNA fragments containing these regions can be obtained by standard PCR amplification. The isolated nucleic acid encoding the VL region can be converted into a full-length light chain gene (and a Fab light chain gene) by operably connecting the DNA encoding the VL to another DNA molecule encoding the light chain constant region CL. Once the DNA fragments encoding the VH and VL segments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, such as converting the variable region gene into a full-length antibody chain gene, a Fab fragment gene or a scFv gene. In these operations, the DNA fragment encoding VL or VH is operably connected to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker.

[0145] In some embodiments, the present invention relates to an isolated nucleic acid molecule comprising a nucleic acid sequence encoding the heavy chain variable region of an isolated antibody as disclosed herein.

[0146] In some embodiments, the present invention relates to an isolated nucleic acid molecule comprising a nucleic acid sequence encoding the light chain variable region of an isolated antibody as disclosed herein.

[0147] In one aspect, the present disclosure provides a vector comprising the nucleic acid molecule as described above.

[0148] In one aspect, the present disclosure provides a host cell comprising the nucleic acid molecule or the vector as described above.

[0149] Conjugate

[0150] In one aspect, the present disclosure provides a conjugate comprising an antibody or fragment thereof as described above coupled to at least one detectable marker. Detectable markers include, but are not limited to: (i) providing a detectable signal; (ii) interacting with a second marker to modify the detectable signal provided by the first or second marker, such as FRET (Fluorescence Resonance Energy Transfer); (iii) affecting mobility (e.g., electrophoretic mobility) by charge, hydrophobicity, shape or other physical parameters, or (iv) providing a capture moiety, such as affinity, antibody / antigen or ion complexation.

[0151] Suitable structures as labels include fluorescent labels, luminescent labels, chromophore labels, radioisotope labels, isotope labels, preferably stable isotope labels, isobaric labels, enzyme labels (such as horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), particle labels (especially metal particle labels, magnetic particle labels, polymer particle labels), organic small molecules (such as biotin, receptor ligands or binding molecules (such as cell adhesion proteins or phosphatidylcholine), which can be used to bind The marker sequence containing nucleic acid and / or amino acid residues can be detected by the marker. The marker includes, but is not limited to, barium sulfate, iodine, iopanoic acid, amiopromide calcium, diatrizoate sodium, diatrizoate meglumine, meglumine, sodium tyropanate, and radiodiagnostic agents (including positron emitters, (e.g., fluorine-18 and carbon-11), gamma emitters (e.g., iodine-123, iodine-125, technetium-99m, iodine-131 and indium-111), nuclear magnetic resonance nuclides (e.g., fluorine and gadolinium)), luminescent substances (e.g., isoluminol and acridinium esters), fluorescent substances (e.g., fluorescein and rhodamine), and colored substances (e.g., latex particles and colloidal gold).

[0152] Detectable labels as described above can be detected by methods known in the art. For example, fluorescent markers can be detected using a photodetector to detect emitted light. Enzyme labels are generally detected by providing a substrate to the enzyme and detecting the reaction product produced by the action of the enzyme on the substrate. In certain embodiments, such labels can be suitable for immunological detection (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescent immunoassay, chemiluminescent immunoassay, etc.). In certain embodiments, detectable labels as described above can be connected to antibodies or antigen-binding fragments thereof of the present invention by linkers of varying lengths to reduce potential steric hindrance.

[0153] Antibody Drug Conjugates / Immunoconjugates

[0154] In one aspect, the present disclosure provides an antibody-drug conjugate comprising an antibody, which includes one or more drug moieties / therapeutic agents, wherein the drug moieties are directly or via a linker (e.g., covalently linked) to an antibody or fragment thereof as described above. In the antibody-drug conjugate of the present application, the linker structure for conjugating the anti-TSLP antibody to the drug is not particularly limited, as long as the resulting antibody-drug conjugate can be used.

[0155] Because antibody-drug conjugates have the ability to selectively deliver one or more drugs to target tissues (e.g., tumor-associated antigens, such as tumors affected by TSLP), antibody-drug conjugates can improve the therapeutic efficacy of the antibodies or antigen-binding fragments thereof of the present invention in treating diseases (e.g., cancer).

[0156] Multispecific molecules

[0157] The antibodies or antigen-binding fragments thereof of the present invention can be used to form multispecific molecules (e.g., bispecific molecules). The antibodies or antigen-binding fragments thereof of the present invention can be part of a multispecific molecule (e.g., a bispecific molecule), wherein the bispecific or multispecific molecule comprises a second functional module (e.g., a second antibody) or a third functional module (e.g., a third antibody) having a binding specificity different from that of the antibodies or antigen-binding fragments thereof of the present invention, thereby being able to bind to at least two different binding sites and / or target molecules. For example, the antibodies or antigen-binding fragments thereof of the present invention can be connected to a second antibody or antigen-binding fragment thereof that can specifically bind to any protein that can be used as a potential target for combined therapy. To produce the bispecific or multispecific molecules, the antibodies or antigen-binding fragments thereof of the present invention can be connected (e.g., by chemical coupling, genetic fusion, non-covalent association or other means) to one or more other binding molecules (e.g., additional antibodies, antibody fragments, peptides, or binding mimetics).

[0158] Thus, in some aspects, the invention provides a multispecific molecule comprising an antibody or antigen-binding fragment thereof of the invention.

[0159] In certain preferred embodiments, the multispecific molecule specifically binds to TSLP (eg, human TSLP or monkey TSLP) and specifically binds to one or more additional targets.

[0160] In certain preferred embodiments, the multispecific molecule further comprises at least one molecule having a second binding specificity for a second target (eg, a second antibody).

[0161] In certain preferred embodiments, the multispecific molecule is a bispecific antibody.

[0162] Pharmaceutical composition

[0163] In some aspects, the present invention relates to a pharmaceutical composition. The present disclosure provides a pharmaceutical composition or kit comprising the antibody or fragment as described above, the nucleic acid molecule as described above, the vector as described above, the host cell as described above, the conjugate as described above, the antibody-drug conjugate as described above, the multispecific molecule as described above; and a pharmaceutically acceptable carrier.

[0164] The pharmaceutical composition may optionally contain one or more additional pharmaceutically active ingredients, such as another antibody or drug. The pharmaceutical composition of the present invention may also be administered in combination with, for example, another immunostimulant, anticancer agent, antiviral agent or vaccine, such that the anti-TSLP antibody enhances the immune response to the vaccine. Pharmaceutically acceptable carriers may include, for example, pharmaceutically acceptable liquid, gel or solid carriers, aqueous media, non-aqueous media, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending / dispersing agents, chelating agents, diluents, adjuvants, excipients or non-toxic auxiliary substances, combinations of various components known in the art or more.

[0165] Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavoring agents, thickeners, colorants, emulsifiers or stabilizers such as sugars and cyclodextrins. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, mercaptoglycerol, thioglycolic acid, mercaptosorbitol, butylmethylanisole, butylated hydroxytoluene and / or propyl arsenate. As disclosed in the present invention, in a solvent containing an antibody or its antigen-binding fragment of the composition disclosed in the present invention containing one or more antioxidants such as methionine that reduce the antibody or its antigen-binding fragment, it may be oxidized. Redox can prevent or reduce the reduction of binding affinity, thereby enhancing antibody stability and extending shelf life. Therefore, in some embodiments, the present invention provides a composition comprising one or more antibodies or their antigen-binding fragments and one or more antioxidants such as methionine. The present invention further provides methods wherein the antibodies or antigen-binding fragments thereof are mixed with one or more antioxidants such as methionine, whereby the antibodies or antigen-binding fragments thereof can be protected from oxidation to extend their shelf life and / or increase their activity.

[0166] To further illustrate, pharmaceutically acceptable carriers can include, for example, aqueous vehicles such as sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, or dextrose and lactated Ringer's injection, non-aqueous vehicles such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil, antimicrobial agents at bacteriostatic or fungistatic concentrations, isotonic agents such as sodium chloride or dextrose, buffers such as phosphate or citrate buffers, antioxidants such as sodium bisulfate, local anesthetics such as procaine hydrochloride, suspending and dispersing agents such as sodium carboxymethylcellulose, hydroxypropyl methylcellulose or polyvinylpyrrolidone, emulsifiers such as polysorbate 80 (TWEEN-80), partitioning agents or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid), ethanol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid or lactic acid. Antimicrobial agents used as carriers can be added to pharmaceutical compositions in multiple dose containers comprising phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl parabens, thimerosal, benzalkonium chloride and benzethonium chloride. Suitable excipients can include, for example, water, saline, dextrose, glycerol or ethanol. Suitable nontoxic auxiliary substances can include, for example, wetting agents or emulsifiers, pH buffers, stabilizers, solubility enhancers or reagents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate or cyclodextrins.

[0167] Administration, formulation and dosage

[0168] The pharmaceutical compositions of the present invention can be administered to a subject in need thereof in vivo by various routes, including but not limited to oral, intravenous, intraarterial, subcutaneous, parenteral, intranasal, intramuscular, intracranial, intracardiac, intraventricular, intratracheal, oral, rectal, intraperitoneal, intradermal, topical, transdermal and intrathecal, or by implantation or inhalation. The compositions of the present invention can be formulated into solid, semisolid, liquid or gaseous preparations; including but not limited to tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalants and aerosols. Suitable preparations and routes of administration can be selected according to the intended application and treatment regimen.

[0169] Suitable formulations for enteral administration include hard or soft gelatin capsules, pills, tablets (including coated tablets), elixirs, suspensions, syrups or inhalants and controlled release forms thereof.

[0170] Preparations suitable for parenteral administration (e.g., by injection) include aqueous or non-aqueous, isotonic, pyrogen-free, sterile liquids (e.g., solutions, suspensions) in which the active ingredient is dissolved, suspended, or otherwise provided (e.g., in liposomes or other microparticles). These liquids may additionally contain other pharmaceutically acceptable ingredients, such as antioxidants, buffers, preservatives, stabilizers, bacteriostats, suspending agents, thickeners, and solutes that make the preparation isotonic with the blood (or other relevant body fluids) of the intended recipient. Examples of excipients include, for example, water, alcohol, polyols, glycerol, vegetable oils, etc. Examples of isotonic carriers suitable for such preparations include sodium chloride injection, Ringer's solution, or lactated Ringer's injection. Similarly, specific dosage regimens (including dosage, time, and repetition) will depend on specific individuals and individual medical history as well as empirical considerations such as pharmacokinetics (e.g., half-life, clearance, etc.).

[0171] The requirements for effective pharmaceutical carriers for injectable formulations / compositions are well known to those of ordinary skill in the art (see, e.g., Pharmaceutics and Pharmacy Practice, JB Lippincott Company, Philadelphia, PA, eds. Banker and Chalmers, pp. 238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pp. 622-630 (1986)).

[0172] The frequency of administration can be determined and adjusted during the course of treatment and is based on reducing the number of proliferating or tumorigenic cells, maintaining the reduction of such tumor cells, reducing the proliferation of tumor cells or delaying the development of metastasis. In some embodiments, the dose administered can be adjusted or reduced to control potential side effects and / or toxicity. Alternatively, a sustained continuous release formulation of the therapeutic composition of the present invention may be suitable.

[0173] Those skilled in the art will appreciate that the appropriate dosage may vary from patient to patient. Determining the optimal dosage generally involves a balance between the level of therapeutic benefit and any risk or adverse side effects. The selected dosage level will depend on a variety of factors, including but not limited to the activity of the specific compound, administration, administration time, compound clearance rate, duration of treatment, other drugs, compounds and / or materials used in combination, severity of the condition, and species, sex, age, weight, condition, general health and previous medical history of the patient. However, the dosage is generally selected to achieve a local concentration at the site of action that achieves the desired effect without causing substantial harmful or adverse side effects.

[0174] Generally, the antibodies or antigen-binding fragments thereof of the present invention can be administered in various ranges. These include about 4 μg / kg body weight to about 100 mg / kg body weight per dose; about 20 μg / kg body weight to about 50 mg / kg body weight per dose; about 50 μg / kg body weight to about 10 mg / kg body weight per dose. Other ranges include about 100 μg / kg body weight to about 20 mg / kg body weight per dose and about 0.5 mg / kg body weight to about 15 mg / kg body weight per dose. In certain embodiments, the dosage is at least about 100 μg / kg body weight, at least about 250 μg / kg body weight, at least about 750 μg / kg body weight, at least about 3 mg / kg body weight, at least about 5 mg / kg body weight, at least about 10 mg / kg body weight.

[0175] In certain preferred embodiments, the course of treatment involving the antibodies or antigen-binding fragments thereof of the present invention will include multiple doses of the selected pharmaceutical product administered over a period of weeks or months. More specifically, the antibodies or antigen-binding fragments thereof of the present invention may be administered every day, every two days, every four days, every week, every ten days, every two weeks, every three weeks, every month, every six weeks, every two months, every ten weeks or every three months. In this regard, it is understood that the dosage may be changed or the interval may be adjusted based on patient response and clinical practice.

[0176] Compatible formulations for parenteral administration (e.g., intravenous injection) will include an antibody or antigen-binding fragment thereof as disclosed herein at a concentration of about 5 μg / mL to about 100 mg / mL. In certain selected embodiments, the concentration of the antibody or antigen-binding fragment thereof will include 10 μg / mL, 20 μg / mL, 50 μg / mL, 60 μg / mL, 80 μg / mL, 100 μg / mL, 200 μg / μg / mL, 300 μg / mL, 400 μg / mL, 500 μg / mL, 600 μg / mL, 800 μg / mL, 900 μg / mL or 1 mg / mL. In other preferred embodiments, the antibody-drug conjugate will include 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 8 mg / mL, 10 mg / mL, 12 mg / mL, 14 mg ml, 16 mg / mL, 18 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL or 100 mg / mL.

[0177] The antibodies of the invention can be co-administered with one or more other therapeutic agents (e.g., cytotoxic agents, radiotoxic agents, anti-tumor agents, anti-angiogenic agents or immunosuppressants) to reduce the induction of an immune response against the antibodies of the invention. The antibody can be linked to the therapeutic agent (as an immune complex) or can be administered separately from the therapeutic agent.

[0178] In the context of administering a treatment, the term "combination" or "co-administration" as used herein refers to the use of more than one treatment or therapeutic agent. The use of the term "combination" does not limit the order in which the treatment or therapeutic agent is administered to a subject. The treatment or therapeutic agent may be administered before, simultaneously, or after the second treatment or therapeutic agent is administered to the patient. Preferably, the treatment or therapeutic agent is administered to the subject in a certain order, amount, and / or within a certain time interval so that the treatment or therapeutic agent can work together. In a specific embodiment, the treatment or therapeutic agent is administered to the subject in a certain order, amount, and / or within a certain time interval so that they provide an increased benefit than if they were administered in other ways (particularly independently of each other). Preferably, the increased benefit is a synergistic effect.

[0179] Medical Uses

[0180] The antibodies, antibody compositions and methods of the invention have many in vitro and in vivo uses, including, for example, detection of TSLP or enhancement of immune responses. For example, these molecules can be administered to cultured cells in vitro or ex vivo, or, for example, administered to human subjects in vivo.

[0181] Preferred subjects include mammals, such as humans / patients. Mammals in the context of the present invention are humans, non-human primates, domesticated animals such as dogs, cats, sheep, cows, goats, pigs, horses, etc., laboratory animals such as mice, rats, rabbits, guinea pigs, etc., and captive animals, such as zoo animals.

[0182] Treating conditions associated with TSLP expression

[0183] In some aspects, the invention provides methods of treating a disorder in a mammal comprising administering to a subject (eg, a human) in need of treatment a therapeutically effective amount of an antibody or antigen-binding fragment thereof disclosed herein.

[0184] According to the present invention, "disease associated with TSLP expression" means that the expression of TSLP in cells of diseased tissues or organs is increased or decreased compared to the state in healthy tissues or organs. Increase or decrease refers to an increase or decrease or at least 10%, in particular at least 20%, at least 50%, at least 100%, at least 200%, at least 500%, at least 1000%, at least 10000% or even higher. For example, the disease is cancer or an autoimmune disease or an inflammatory disease.

[0185] In some aspects, the present disclosure provides a method for treating a disease associated with TSLP expression or determining the prognosis thereof in a subject, comprising administering an effective dose of the antibody or antigen-binding fragment thereof, the nucleic acid molecule, the vector, the host cell, the conjugate, the antibody-drug conjugate, the multispecific molecule, or the pharmaceutical composition or kit to a subject in need thereof.

[0186] In some aspects, the present disclosure provides the antibody or antigen-binding fragment thereof, the nucleic acid molecule, the vector, the host cell, the conjugate, the antibody-drug conjugate, the multispecific molecule, or the pharmaceutical composition or kit for use in a method for treating a disease associated with TSLP expression or determining the prognosis thereof in a subject.

[0187] In some aspects, the present disclosure provides use of the antibody or antigen-binding fragment thereof, the nucleic acid molecule, the vector, the host cell, the conjugate, the antibody-drug conjugate, the multispecific molecule, or the pharmaceutical composition or kit in the preparation of an agent (or drug) for treating a disease associated with TSLP expression or determining the prognosis thereof.

[0188] In one embodiment, the disease associated with TSLP expression includes a neoplastic disease, such as cancer.

[0189] In some embodiments, the disease associated with TSLP expression is cancer, and the cancer is preferably selected from the group consisting of Hodgkin's lymphoma, breast cancer, pancreatic cancer, melanoma, cervical cancer, skin T-cell lymphoma, gastric cancer, lung cancer, B-cell lymphoma, including NHL, pre-B-cell lymphocytic leukemia / lymphoma and mature B-cell tumors, such as B-cell chronic lymphocytic leukemia (CLL) / small lymphocytic lymphoma (SLL), B-cell prolymphocytic lymphoma (P ...CL), B-cell prolymphocytic lymphoma (PCL), B-cell prolymphocytic lymphoma (PCL), B-cell prolymphocytic lymphoma (PCL), B-cell prolymphocytic lymphoma (PCL), B-cell pro Leukemia, lymphoplasmacytic lymphoma, mantle cell lymphoma (MCL), follicular lymphoma (FL), including low-, intermediate-, and high-grade FL, cutaneous follicle center lymphoma, marginal zone B-cell lymphoma (MALT-type, nodal, and splenic), hairy cell leukemia, diffuse large B-cell lymphoma, Burkitt's lymphoma, plasmacytoma, plasma cell myeloma, post-transplant lymphoproliferative disorder, Waldenstrom's macroglobulinemia, and anaplastic large cell lymphoma (ALCL).

[0190] The antibodies or antigen-binding fragments thereof can be used alone as a monotherapy or can be used in combination with chemotherapy or radiation therapy.

[0191] The antibodies or antigen-binding fragments thereof can be used in combination with anticancer agents, cytotoxic agents, or chemotherapeutic agents.

[0192] The term "anticancer agent" or "antiproliferative agent" means any agent that can be used to treat a cell proliferative disorder such as cancer, and includes, but is not limited to, cytotoxic agents, cytostatic agents, anti-angiogenic agents, radiotherapy and radiotherapeutic agents, targeted anticancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormone therapy, radiotherapy, anti-metastatic agents, and immunotherapeutic agents. It should be understood that in selected embodiments as described above, such anticancer agents may comprise a conjugate and may be combined with the disclosed site-specific antibodies prior to administration. More specifically, in certain embodiments, the selected anticancer agent is linked to an unpaired cysteine ​​of an engineered antibody to provide an engineered conjugate as described herein. Therefore, such an engineered conjugate is explicitly contemplated within the scope of the present invention. In other embodiments, the disclosed anticancer agent will be administered in combination with a site-specific conjugate comprising a different therapeutic agent as described above.

[0193] In some embodiments, the disease associated with TSLP expression is an autoimmune disease, preferably an autoimmune disease selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, psoriatic arthritis, axial spondyloarthritis, myasthenia gravis, polymyositis, psoriasis, pemphigus, vitiligo, multiple sclerosis, narcolepsy, neuromyelitis optica, type 1 diabetes, hyperthyroidism, hypothyroidism, Crohn's disease, ulcerative colitis, celiac disease, autoimmune gastritis, primary biliary cholangitis, autoimmune hepatitis and lupus nephritis.

[0194] In some embodiments, the disease associated with TSLP expression is an inflammatory disease selected from the group consisting of asthma, atopic dermatitis, chronic obstructive pulmonary disease (COPD), eosinophilic esophagitis (EoE), nasal polyps, chronic spontaneous urticaria, Ig-driven diseases, IgA nephropathy, lupus nephritis, eosinophilic gastritis, chronic sinusitis without nasal polyps, and idiopathic pulmonary fibrosis (IFF).

[0195] diagnosis

[0196] The present invention provides in vitro and in vivo methods for detecting, diagnosing or monitoring proliferative disorders and methods for screening cells from patients to identify tumor cells including tumorigenic cells. Such methods include identifying individuals with cancer for treatment or monitoring the progression of cancer, including contacting the patient or a sample obtained from the patient (in vivo or in vitro) with an antibody as described herein, and detecting the presence or absence or binding level of the antibody bound to a bound or free target molecule in the sample. In some embodiments, the antibody will comprise a detectable label or reporter molecule as described herein.

[0197] In some aspects, the present disclosure provides a method for diagnosing, detecting or monitoring a disease associated with TSLP expression, comprising administering an effective dose of the antibody or antigen-binding fragment thereof, the nucleic acid molecule, the vector, the host cell, the conjugate, the antibody-drug conjugate, the multispecific molecule, or the pharmaceutical composition or kit to a subject in need thereof.

[0198] In some aspects, the present disclosure provides the antibody or antigen-binding fragment thereof, the nucleic acid molecule, the vector, the host cell, the conjugate, the antibody-drug conjugate, the multispecific molecule, or the pharmaceutical composition or kit for use in a method for diagnosing, detecting or monitoring a disease associated with TSLP expression in a subject.

[0199] In yet another aspect, the present disclosure provides use of the antibody or antigen-binding fragment thereof, the nucleic acid molecule, the vector, the host cell, the conjugate, the antibody-drug conjugate, the multispecific molecule, or the pharmaceutical composition or kit in the preparation of an agent (or drug) for diagnosing, detecting or monitoring a disease associated with TSLP expression.

[0200] Samples can be analyzed by a variety of assays, such as radioimmunoassays, enzyme immunoassays (e.g., ELISA), competitive binding assays, fluorescent immunoassays, immunoblotting assays, Western blot analysis, and flow cytometry assays. Compatible in vivo diagnostics or diagnostic assays can include imaging or monitoring techniques known in the art, such as magnetic resonance imaging, computerized tomography (e.g., CAT scans), positron emission tomography (e.g., PET scans), radiography, ultrasound, etc., known to those skilled in the art.

[0201] The methods described in the present invention for detecting or monitoring TSLP expression or the level of TSLP-expressing cells in vitro can also be used for non-diagnostic purposes.

[0202] Preferred subjects include mammals, such as humans / patients in need thereof.

[0203] The sample from the subject is blood, excreta (urine or feces), oral or nasal secretions, or alveolar lavage fluid, tissue fluid, sweat, or extracts thereof from the subject.

[0204] Pharmaceutical packaging and kits

[0205] Also provided are pharmaceutical packaging and kits containing one or more containers of one or more doses of an antibody or an antigen-binding fragment thereof. In certain embodiments, a unit dose is provided, wherein the unit dose contains a predetermined amount of a composition comprising, for example, an antibody or an antigen-binding fragment thereof, with or without one or more other agents. For other embodiments, such a unit dose is supplied in a disposable prefilled syringe for injection. In other embodiments, the composition contained in the unit dose may contain saline, sucrose or the like; a buffer such as phosphate, etc.; and / or be formulated in a stable and effective pH range. Alternatively, in certain embodiments, the conjugate composition may be provided as a lyophilized powder, which may be reconstituted after adding a suitable liquid (e.g., sterile water or saline). In certain preferred embodiments, the composition comprises one or more substances that inhibit protein aggregation, including but not limited to sucrose and arginine. Any label on or associated with the container indicates that the encapsulated conjugate composition is used to treat a selected tumor disease condition.

[0206] Such kits typically include a pharmaceutically acceptable formulation of the engineered conjugate in a suitable container, and optionally include one or more anticancer agents or other agents in the same or different containers. The kit may also contain other pharmaceutically acceptable formulations for diagnosis or combined therapy.

[0207] More specifically, the kit can have a single container containing an antibody or antigen-binding fragment thereof of the present disclosure, with or without additional components, or they can have different containers for each required reagent. In the case of providing a combination therapeutic agent for conjugation, a single solution can be premixed in a molar equivalent combination or one component more than another. Alternatively, the conjugate of the kit and any optional anticancer agent can be stored separately in different containers before being administered to a patient. The kit can also include a second / third container device for containing a sterile pharmaceutically acceptable buffer or other diluent such as bacteriostatic water for injection (BWFI), phosphate buffered saline (PBS), Ringer's solution, and glucose solution.

[0208] When the components of the kit are provided in one or more liquid solutions, the liquid solution is preferably an aqueous solution, particularly preferably a sterile aqueous solution or a saline solution. However, the components of the kit may be provided as dry powders. When the reagents or components are provided in dry powder form, the powder may be reconstituted by adding a suitable solvent. It is contemplated that the solvent may also be provided in another container.

[0209] Example

[0210] Incorporated by Reference

[0211] Each patent document and scientific document mentioned herein is incorporated by reference in its entirety for all purposes.

[0212] Equivalence

[0213] The present invention may be embodied in other specific ways without departing from its spirit or essential characteristics. Therefore, the above embodiments should be regarded as illustrative in all cases, rather than limiting the invention described herein. Therefore, the scope of the present invention is indicated by the appended claims rather than by the above description, and it is intended that all changes within the equivalent meaning and scope of the claims are included therein.

[0214] Example 1 Animal immunization and TSLP antibody screening

[0215] 10-12 week old New Zealand rabbits were selected, and the human TSLP full-length protein (Acro Biosystems, TSP-H52Ha) was emulsified with Freund's adjuvant and injected subcutaneously at multiple points in the animals, once every two weeks, for a total of 4-5 immunizations. Depending on the serum titer, the selected high titer animals were intraperitoneally injected with antigen solution or subjected to shock immunization 3 days before peripheral blood collection. PBMCs were separated from the collected rabbit peripheral blood, and the antibody light chain and heavy chain variable region sequences were obtained through the single B cell screening technology platform. The expression vector constructed by the PCR method was co-transfected into 293T cells, and the cell culture supernatant was collected. Multiple positive clones that recognized human TSLP were screened by ELISA and FACS methods.

[0216] By selecting some positive clones, the light and heavy chain genes were cloned into expression vectors containing human light chain constant regions or human IgG1 heavy chain constant regions, respectively, eukaryotic expression vectors were constructed and a series of antibody sequences were sequenced.

[0217] Example 2 Expression and purification of candidate chimeric antibodies

[0218] The light chain and heavy chain variable region sequences of the candidate antibody sequences were connected to appropriate signal peptides at the N-termini, and connected to the light and heavy chain constant regions at the C-termini, respectively. After codon optimization, they were cloned into the expression vector pCDNA3.4, and co-transfected into ExpiCHO-S cells (Thermo Fisher) for expression according to the operating procedures provided by the manufacturer. The supernatant was collected and purified by Protein A to obtain candidate antibody proteins 4C4 and 4G7.

[0219] 4C4 full length heavy chain (SEQ ID NO: 3)

[0220] QTLKESGGRLVTPGTPLTLTCTVSGFSLS SNAMS WVRQAPGKGLEWIG IIGSSDITYYATWAKG RFTISKT

[0221] STTVDLKITSPTTEDTATYFCAR EAYADGFDP WGPGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL

[0222] VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV

[0223] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH

[0224] NAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS

[0225] RDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS

[0226] CSVMHEALHNHYTQKSLSLSPGK

[0227] 4C4 full-length light chain (SEQ ID NO: 4)

[0228] DVVMTQTPSSVSAAVGGTVTINC RASEDIESYLA WYQQKPGQPPKLLIY GASDLAS GVPSRFKGSGSGT

[0229] DYTLTISGVQCDDAATYYC QTSYYSTSGDTFFA FGGGTEVVVKRTVAAPSVFIFPPSDEQLKSGTASVVC

[0230] LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS

[0231] SPVTKSFNRGEC

[0232] 4G7 full-length heavy chain (SEQ ID NO: 5)

[0233] QSVEESGGRLVTPGTPLTLTCTVSGIDLS SNSMS WVRQAPGKGLAWIG SISTVGNTFYASWAKG RFTISKT

[0234] STTVDLKITSPTTEDTATYFCAR IRDDYGDFLVFYAFDP WGPGTLVTISSASTKGPSVFPLAPSSKSTSGGT

[0235] AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT

[0236] KVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY

[0237] VDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP

[0238] QVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR

[0239] WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0240] 4G7 full length light chain (SEQ ID NO: 6)

[0241] AAVMTQTPASVSVAVGGTVTINC QASEDIDSYLA WYQQKPGQPPKLLIY YASNLAS GVPSRFKGSGSGK

[0242] QFTLTISGVQCADAATYYC QGGFYTTIGDSA FGGGTEVVVKRTVAAPSVFIFPPSDEQLKSGTASVVCLL

[0243] NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP

[0244] VTKSFNRGEC

[0245] The area marked with a horizontal line is the CDR area.

[0246] Example 3 ELISA analysis of chimeric antibody protein binding activity

[0247] The purified candidate antibodies were subjected to ELISA to detect protein binding activity. Human TSLP antigen protein (AcroBiosystems, TSP-H5255) was dispensed into a 96-well ELISA plate at a concentration of 1 μg / mL, 100 μl / well, and incubated at 4°C overnight; after washing 3 times with PBST, 100 μl of 3% BSA blocking solution was added, and incubated at 37°C for 1 hour; after washing 3 times with PBST, 50 μl of gradient diluted antibody sample, reference product (Tezepelumab, heavy chain as shown in SEQ ID NO: 1, light chain as shown in SEQ ID NO: 2) and negative control IgG (2 μg / ml starting concentration, 3-fold gradient dilution, a total of 10 concentration points) were added, and incubated at 37°C for 1 hour; after washing 3 times with PBST, Anti-Human IgG was added HRP (1:10000 dilution) 100 μl, incubate at 37°C for 0.5 h; after washing 4 times with PBST, add 50 μl of TMB colorimetric solution, develop at 37°C for 8 min, add 50 μl / well of ELISA stop solution to terminate the reaction, and measure the absorbance value at a wavelength of 450 nm with an enzyme reader.

[0248] The results are as follows Figure 1A and Figure 1BIt was shown that the anti-TSLP antibodies of the present invention can specifically bind to human TSLP with high binding force, and the binding activity of 4C4 and 4G7 to human TSLP protein is better than that of the reference product Tezepelumab.

[0249] Example 4 ELISA analysis of blocking activity of candidate antibodies at protein level

[0250] The ability of the candidate antibodies of the present invention to block the binding of the reference human TSLP to the receptor TSLP-R was determined by competitive ELISA. The human TSLP-R antigen protein (Acro Biosystems, TSP-H52Ha) was dispensed into a 96-well ELISA plate at a concentration of 1 μg / mL, 100 μl / well, and incubated overnight at 4°C; after washing 3 times with PBST, 100 μl of 3% BSA blocking solution was added and incubated at 37°C for 1 hour; after washing 3 times with PBST, the anti-TSLP antibody of the present invention, the reference product or the negative control IgG was diluted (6 μg / ml starting concentration, 3-fold gradient dilution, a total of 8 concentration points). After washing the plate, 100 μL / well of the antibody / IL4Rαhis mixture was added to the plate coated with the reference product. Incubate at 37°C for 40 minutes, add 50 μl of serially diluted candidate antibody samples, reference products (Tezepelumab) and negative control IgG (6 μg / ml starting concentration, 4-fold serial dilution, a total of 7 concentration points), and incubate at 37°C for 1 hour; after washing with PBST for 3 times, add 100 μl of Anti-Human IgG HRP (1:10000 dilution) and incubate at 37°C for 0.5 hour; after washing with PBST for 4 times, add 50 μl of TMB colorimetric solution, develop at 37°C for 8 minutes, add 50 μl / well of ELISA stop solution to terminate the reaction, and measure the absorbance value at a wavelength of 450 nm with an enzyme reader.

[0251] The results are as follows Figure 2A and Figure 2B The results show that the 4C4 antibody of the present invention can well block the binding of TSLP and TSLPR, and its blocking ability is better than that of the reference product Tezepelumab. The 4G7 antibody of the present invention has a weaker ability to block the binding of TSLP and TSLPR.

[0252] Example 5: Candidate antibodies block the binding of TSLP to BaF3-TSLPR / IL7Rα cells

[0253] BaF3-TSLPR / IL7Rα cells are a stable cell line with high expression of TSLP receptor. Flow cytometry was used to detect whether rabbit anti-human TSLP antibody blocked the binding of TSLP to BaF3-TSLPR / IL7Rα cells. Specific operation: Add candidate antibody (4*1250ng / ml, 2-fold gradient dilution, 8 concentration points) to 96-well plate, 50μl / well; add TSLP (Acro Biosystems, catalog number: TSP-H52Ha) solution (4*40ng / ml), 50μl / well; incubate at 37℃ for 15 minutes; continue to add BaF3-TSLPR / IL7Rα cells, 1E6 / ml, 100μl / well; incubate at 2-8℃ for 1 hour; wash 3 times; add goat anti-mouse IgG Fc specific-FITC antibody (sigma-Aldrich, catalog number: F4143) diluted at a ratio of 1:300, incubate at 2-8℃ in the dark for 0.5 hour, wash 3 times, add staining buffer to resuspend cells, and use flow cytometry (BD FACS Lyric) to detect cell fluorescence intensity (MFI). Use the formula: blocking percentage (%) = (MFI TSLP对照 -MFI 样品 ) / (MFI TSLP对照 -MFI cell control )*100% to calculate the sample blocking efficiency. Where: MFI 样品 Indicates the wells with TSLP, anti-TSLP antibody, and cells; MFI cell control represents the wells with cells added only; MFI TSLP对照 The final concentration of the antibody was used as the horizontal axis, and the blocking percentage (%) of the corresponding concentration of the antibody was used as the vertical axis for nonlinear fitting to calculate the IC50 value. Figure 3A and Figure 3B The results showed that the IC50 of 4G7 and 4C4 antibodies in BaF3-TSLPR / IL7Rα cell-level blocking activity was better than that of Tezepelumab. The cell-level blocking activity of 4C4 was 1.5 times that of Tezepelumab, and the cell-level blocking activity of 4G7 was about 2.4 times that of Tezepelumab, but the maximum blocking percentage did not reach 100%.

[0254] Example 6: Effect of candidate antibodies on inhibiting TSLP activation of H_TSLP Reporter Cell Line cell STATs and JAK2 signaling pathways

[0255] H_TSLP Reporter Cell Line reporter gene cell line is a Luciferase reporter gene cell line constructed based on the Jak2-Stat signaling pathway. After TSLP binds to TSLPR, the dimer enhances the recruitment of IL-7Rα, forms an extracellular ternary complex, and Jak2 is activated. Jak2 further mediates the phosphorylation of Stats, causing Stats to be transported into the nucleus, thereby activating the expression of luciferase (Luciferase). This method can effectively detect the inhibition of TSLP activation by anti-TSLP antibodies on H_TSLP Reporter Cell Line cells. Specific operation: Wash the H_TSLPReporter Cell Line cells (Jiman Bio, Catalog No.: GM-C15572) in the logarithmic growth phase once with 1640+1% FBS+1% PS and adjust the cell density to 2E6 / ml, inoculate into a white 96-well cell culture plate (Corning, Catalog No.: 3917), 50μl / well; incubate overnight at 37°C, 5% CO2. The next day, an appropriate amount of TSLP (Acro Biosystems, catalog number: TSP-H52Ha) and different concentrations of candidate anti-TSLP antibodies (starting concentration 4*25nM, 10-fold gradient dilution, 8 concentration points) were mixed in equal volumes and incubated at 37°C in the dark for 15 minutes. The TSLP and antibody mixture was added to a 96-well plate inoculated with H_TSLP Reporter Cell Line cells and incubated at 37°C, 5% CO2 for 6 hours. Bright-Lumi TM II Firefly Luciferase Reporter Gene Assay Kit (Biyuntian, Cat. No.: RG052M), 100 μl / well; incubate at room temperature in the dark for 5-10 minutes. Use a multifunctional microplate reader (MD, SpectraMax i3X) to detect the LUM value. Use the formula: Inhibition rate (%) = (RLU TSLP对照 -RLU 样品 ) / (RLU TSLP对照 -RLU cell control )*100% to calculate the sample inhibition efficiency. Where: RLU 样品 Indicates the wells with TSLP, anti-TSLP antibody, and cells; RLU cell control Represents wells with cells only; RLU TSLP对照 The y-axis represents the wells with TSLP and cells. The final antibody concentration was used as the abscissa and the corresponding antibody inhibition rate (%) was used as the ordinate for nonlinear fitting to calculate the IC50 value. Figure 4A and Figure 4BThe results showed that 4G7 and 4C4 antibodies can effectively inhibit the activation of TSLP on the STATs and JAK2 signaling pathways of H_TSLP Reporter Cell Line cells. The inhibitory activity of 4C4 on the activation of the signaling pathway is 59 times that of Tezepelumab, and the inhibitory activity of 4G7 on the activation of the signaling pathway is 26 times that of Tezepelumab.

[0256] Example 7: Candidate antibodies inhibit the proliferation-promoting effect of TSLP on BaF3-TSLPR / IL7Rα cells

[0257] BaF3-TSLPR / IL7Rα cells are a stable cell line with high expression of TSLP receptors and rely on mIL3 for proliferation. Without the addition of mIL3, TSLP can effectively promote the proliferation of BaF3-TSLPR / IL7Rα cells. This method can be used to effectively evaluate the effect of anti-TSLP antibodies on inhibiting the proliferation of BaF3-TSLPR / IL7Rα cells. Specific operation: 1640+10% FBS+1% PS analysis buffer was used to prepare 4*0.625ng / ml TSLP (Acro Biosystems, catalog number: TSP-H52Ha) solution, added to 96-well U-bottom plate, 50μl / well; diluted the candidate antibody to different concentrations with 1640+10% FBS+1% PS analysis buffer (starting concentration 4*166.67ng / ml, 6-fold gradient dilution, 8 concentration points) and added to 96-well U-bottom plate with TSLP, 50μl / well, incubated at 37°C for 15 minutes; resuspended BaF3-TSLPR / IL7Rα with 1640+10% FBS+1% PS analysis buffer and adjusted the density to 5E4 / ml, added to the above 96-well U-bottom plate, 100μl / well; incubated at 37°C for 72 hours; transferred the cells at the end of incubation to a new white 96-well plate, 100μl / well; used CellCounting-Lite2.0 Luminescent Cell ViabilityAssay (Novozyme, Catalog No.: DD1101-02), 100 μl / well, incubate at room temperature in the dark for 5-10 minutes, and read the LUM value on a multifunctional microplate reader. The formula is: Inhibition rate (%) = (RLU TSLP对照 -RLU 样品 ) / (RLU TSLP对照 -RLU cell control )*100% to calculate the sample inhibition efficiency. Where: RLU 样品 Indicates the wells with TSLP, anti-TSLP antibody, and cells; RLU cell control Represents wells with cells only; RLU TSLP对照The y-axis represents the wells with TSLP and cells. The final antibody concentration was used as the abscissa and the corresponding antibody inhibition rate (%) was used as the ordinate for nonlinear fitting to calculate the IC50 value. Figure 5A and Figure 5B The results showed that 4G7, 4C4 antibodies and Tezepelumab can effectively inhibit the proliferation-promoting effect of TSLP on BaF3-TSLPR / IL7Rα cells, and the inhibitory activity of 4C4 is 11.3 times that of Tezepelumab, and the inhibitory activity of 4G7 is 6.5 times that of Tezepelumab.

[0258] Example 8: Epitope competition experiment between candidate antibodies and Tezepelumab

[0259] Experimental principle: Through BLI technology, the SA probe is used to capture the biotinylated primary antibody, bind to the antigen, and then bind to the secondary antibody for epitope grouping.

[0260] Experimental steps: (1) SA probe binds to the biotinylated primary antibody Tezepelumab; (2) binds to the antigen human TSLP; (3) then binds to the solvent (blank control) or the non-biotinylated secondary antibody 4C4 or 4G7.

[0261] Experimental results: 4C4 and Tezepelumab bind to human TSLP epitopes that overlap, and 4G7 and Tezepelumab bind to different epitopes of human TSLP (as shown in Table 1). According to the public literature (Nat Commun. 2017; 8: 14937.), Tezepelumab binds to the C-terminal region. The antibody 4G7 screened by the present invention does not overlap with the Tezepelumab binding epitope, and will not block the MKK34 antimicrobial peptide with antibacterial activity at the C-terminal end, and is expected to retain the ability of TSLP to resist external bacterial infection.

[0262] Table 1 Second antibody binding signal values

[0263]

[0264] Example 9: Humanization of candidate antibodies

[0265] Humanized antibody transformation: The light chain and heavy chain variable region genes of 4G7 were homologously compared with the human IgG germline sequence, IGHV3-66*01 / IGHJ5*01 was selected as the heavy chain CDR transplantation template, and the CDR region of the 4G7 heavy chain (i.e., HCDR1, HCDR2, and HCDR3) was transplanted into the framework region of IGHV3-66*01 / IGHJ5*01; IGKV1-6*01 / IGKJ4*01 was selected as the light chain CDR transplantation template, and the CDR region of the 4G7 light chain (i.e., LCDR1, LCDR2, and LCDR3) was transplanted into the framework region of IGKV1-6*01 / IGKJ4*01; specific sites in the framework region were reversely mutated to obtain the 4G7 V60 humanized antibody variable region. The sequence of the humanized heavy chain variable region is shown in SEQ ID NO: 25; the amino acid sequence of the humanized light chain variable region is shown in SEQ ID NO: 26. The heavy chain and light chain genes of 4G7 V60 humanized antibody were synthesized and cloned into the expression vector of pCDNA3.4. The heavy chain expression plasmid and light chain expression plasmid of humanized antibody were co-transfected into CHO-S cells. After 4-8 days after transfection, the culture supernatant was harvested and purified with Protein A to obtain humanized antibody.

[0266] Example 9: Anti-human TSLP humanized antibody 4G7 V60 inhibits TSLP activation of H_TSLP Reporter CellLine cell STATs and JAK2 signaling pathways

[0267] H_TSLP Reporter Cell Line reporter gene cell line is a Luciferase reporter gene cell line constructed based on the Jak2-Stat signaling pathway. After TSLP binds to TSLPR, the dimer enhances the recruitment of IL-7Rα, forms an extracellular ternary complex, and Jak2 is activated. Jak2 further mediates the phosphorylation of Stats, causing Stats to be transported into the nucleus, thereby activating the expression of luciferase (Luciferase). This method can effectively detect the inhibition of TSLP activation by anti-TSLP antibodies on H_TSLP Reporter Cell Line cells. Specific operation: Wash the H_TSLPReporter Cell Line cells (Jiman Bio, Catalog No.: GM-C15572) in the logarithmic growth phase once with 1640+1% FBS+1% PS and adjust the cell density to 2E6 / ml, inoculate into a white 96-well cell culture plate (Corning, Catalog No.: 3917), 50μl / well; incubate overnight at 37°C, 5% CO2. The next day, an appropriate amount of TSLP (Acro Biosystems, catalog number: TSP-H52Ha) and different concentrations of anti-human TSLP humanized antibody 4G7 V60 (starting concentration 4*25nM, 10-fold gradient dilution, 8 concentration points) were mixed in equal volumes and incubated at 37°C in the dark for 15 minutes. The TSLP and antibody mixture was added to a 96-well plate inoculated with H_TSLP Reporter Cell Line cells and incubated at 37°C, 5% CO2 for 6 hours. Bright-Lumi TM II Firefly Luciferase Reporter Gene Assay Kit (Biyuntian, Catalog No.: RG052M), 100μl / well; incubate at room temperature in the dark for 5-10 minutes. Use a multifunctional microplate reader (MD, SpectraMax i3X) to detect the LUM value. The sample inhibition efficiency is calculated using the formula: Inhibition rate (%) = (RLUTSLP control-RLU sample) / (RLUTSLP control-RLUcell control)*100%. Among them: RLU sample represents the wells with TSLP, anti-human TSLP humanized antibody 4G7 V60, and cells; RLUcell control represents the wells with cells only; RLUTSLP control represents the wells with TSLP and cells. Use the final antibody concentration as the horizontal axis and the corresponding concentration antibody inhibition rate (%) as the vertical axis for nonlinear fitting to calculate the IC50 value. The results are shown in Figure 6The results showed that the 4G7 V60 antibody can effectively inhibit the activation of TSLP on the STATs and JAK2 signaling pathways of H_TSLP Reporter Cell Line cells. The activity of 4G7 V60 in inhibiting signaling pathways is significantly better than that of Tezepelumab. Compared with Tezepelumab, the activity of 4G7 V60 is 4743 times higher.

[0268] Incorporated by Reference

[0269] Each patent document and scientific document mentioned herein is incorporated by reference in its entirety for all purposes.

[0270] Equivalence

[0271] The present invention may be embodied in other specific ways without departing from its spirit or essential characteristics. Therefore, the above embodiments should be regarded as illustrative in all cases, rather than limiting the invention described herein. Therefore, the scope of the present invention is indicated by the appended claims rather than by the above description, and it is intended that all changes within the equivalent meaning and scope of the claims are included therein.

Claims

1. An isolated antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof specifically binds to TSLP and comprises a heavy chain variable region and a light chain variable region, The heavy chain variable region and the light chain variable region comprise one of the following combinations: (1) CDRH1 as set forth in SEQ ID NO: 13, CDRH2 as set forth in SEQ ID NO: 14, CDRH3 as set forth in SEQ ID NO: 15; and CDRL1 as set forth in SEQ ID NO: 16, CDRL2 as set forth in SEQ ID NO: 17, and CDRL3 as set forth in SEQ ID NO: 18; (2) CDRH1 as shown in SEQ ID NO: 19, CDRH2 as shown in SEQ ID NO: 20, CDRH3 as shown in SEQ ID NO: 21; and CDRL1 as shown in SEQ ID NO: 22, CDRL2 as shown in SEQ ID NO: 23, and CDRL3 as shown in SEQ ID NO:

24.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody comprises one of the following combinations: (1) a light chain variable region as set forth in SEQ ID NO: 8 and a heavy chain variable region as set forth in SEQ ID NO: 7; (2) the light chain variable region shown in SEQ ID NO: 10 and the heavy chain variable region shown in SEQ ID NO:

9.

3. The antibody or antigen-binding fragment thereof according to claim 2, further comprising a heavy chain constant region as shown in SEQ ID NO: 11 and a light chain constant region as shown in SEQ ID NO:

12.

4. The antibody or antigen-binding fragment thereof according to claim 3, wherein the antibody comprises one of the following combinations: (1) a light chain as shown in SEQ ID NO: 4 and a heavy chain as shown in SEQ ID NO: 3; (2) The light chain shown in SEQ ID NO:6 and the heavy chain shown in SEQ ID NO:

5.

5. The antibody or antigen-binding fragment thereof according to any of the preceding claims, wherein the fragment is a Fab fragment, a Fab' fragment, a F(ab')2 fragment, or a Fv fragment; The antibody is a monoclonal antibody or a humanized antibody.

6. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding the antibody or antigen-binding fragment thereof as claimed in any one of claims 1 to 5. A vector comprising the nucleic acid molecule according to claim 6 .

8. A host cell comprising the nucleic acid molecule according to claim 6 or the vector according to claim 7.

9. A conjugate comprising the antibody or fragment of any one of claims 1 to 5 coupled to at least one detectable label.

10. A multispecific molecule comprising the antibody or antigen binding portion of any one of claims 1-5; preferably, the multispecific molecule specifically binds to TSLP; further preferably, the multispecific molecule further comprises at least one molecule having a second binding specificity for a second target.

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