Anti-tslp antibodies and uses thereof

Abstract

The present disclosure provides an antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof specifically binds to TSLP. The antibody of the present application can effectively inhibit the activation of TSLP on the STATs and JAK2 signaling pathway of H_TSLP Reporter Cell Line cells, and both the antibody of the present application and Tezepelumab can effectively inhibit the proliferation promoting effect of TSLP on BaF3-TSLPR / IL7Rα cells. The antibody of the present application does not cross the binding epitope of Tezepelumab, does not block the antibacterial peptide MKK34 with antibacterial activity at the C-terminus, and is expected to retain the ability of TSLP to resist bacterial infection from the outside.

Description

[0001] This application is a divisional application of the patent application for the invention with the application date of December 26, 2023, the application number of 202311832040.8, and the invention name of Anti-TSLP antibody and its use.

[0002] Reference to Sequence Listing

[0003] The instant 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 antigen-binding fragment thereof that specifically binds to TSLP and uses thereof. 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 test of TSLP-related diseases. BACKGROUND

[0005] Thymic stromal lymphopoietin (TSLP) was first discovered in the supernatant of mouse thymic stromal cell culture, and 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. The IL-2 family of cytokines includes IL-2, IL-4, IL-7, IL-9, IL-15, IL-21, which are all globular proteins containing four short alpha helices, and all 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 a "warning protein" of the human immune barrier, which regulates immune function in surface barriers such as skin keratinocytes, lung and intestinal epithelial cells. TSLP responds to pathogenic bacterial stimulation, activates immature dendritic cells, mast cells, basophils, eosinophils and lymphocytes, and makes them exhibit a type 2 polarized phenotype. Abnormal TSLP signaling can cause a variety of serious health problems in humans, and it has been proven that TSLP is related to diseases such as asthma, atopic dermatitis, atopic rhinitis, psoriasis, chronic obstructive pulmonary disease (COPD), eosinophilic esophagitis, etc. (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 respective specific receptor subunits, and share the common receptor subunit yc. But TSLP binds to TSLPR (CRLF2), IL-7Ralpha two receptors, and does not bind to yc receptor (Expert Rev Clin Immunol. 2014, 10(11): 1463-1474. Adv Immunol. 2009, 101: 1-25.). The binding of TSLP to TSLPR and IL-7Ralpha is synergistic. TSLP binds to TSLPR with an affinity of about 32 nM. While TSLP does not directly bind to IL-7Ralpha (> 100 nM). TSLP binds to IL-7Ralpha with an affinity of about 29 nM only after TSLP binds to TSLPR to form a TSLP-TSLPR complex (Nature Communications, 2017, 8.).

[0007] The TSLP gene region has a variable promoter, which can express two proteins (including signal peptides) of long form TSLP (lfTSLP) and short form TSLP (sfTSLP). The sfTSLP is truncated at the N-terminal compared with the lfTSLP, and only retains "one and a half" alpha helix structure. Studies have shown that the C-terminal of both lfTSLP and sfTSLP have MKK34 antibacterial peptides with antibacterial activity, and sfTSLP has more powerful antibacterial activity. sfTSLP is the main form of TSLP expressed in normal tissues of the human body, which is constitutively expressed in normal oral mucosa, skin epidermis, salivary glands, intestinal epithelial cells, and plays an antibacterial activity; under inflammatory conditions, the expression amount of sfTSLP decreases (detected in the pathological tissues of atopic dermatitis and Crohn's disease). While lfTSLP is not expressed in normal tissues, it is only expressed in atopic dermatitis, asthma, ulcerative colitis, and smoking oral mucosa (Pharmaceuticals 2016, 9, 41). When allergens stimulate epithelial cells, TSLP expression in the cells is up-regulated, thereby generating a microenvironment with Th2 generation tendency, secreting Th2 type cytokines, generating inflammatory reactive Th2 cells, and then triggering allergic inflammatory response. Studies have shown that when the TSLP molecule binds to the heterodimer composed of TSLPR and IL-7R-alpha, JAK1 and JAK2 are activated, thereby activating signal transducers and transcriptional activators 5 and 3 (STAT5, 3), and initiating the expression of downstream genes. TSLP is widely present in various allergic diseases, such as allergic rhinitis, allergic asthma, eczema, etc. In addition, multiple SNP mutations of TSLP were detected in patients with atopic dermatitis, asthma, and eosinophilic esophagitis, which led to high expression of lfTSLP. And these mutations have family heredity (Expert Rev Clin Immunol. 2014, 10(11): 1463-1474.). Therefore, it is of great significance to develop antibodies against lfTSLP to block the atopic dermatitis, asthma, eosinophilic esophagitis, ulcerative colitis and other diseases caused by abnormal expression of lfTSLP. At the same time, it is expected to retain the ability of TSLP to resist bacterial infection by screening for antibodies that do not block the C-terminal MKK34 antibacterial peptide with antibacterial activity.

[0008] The antibody Tezepelumab that has been marketed so far binds to the C-terminal end of TSLP, which can affect the antibacterial peptide activity of TSLP and thus cause side effects. In addition, studies have shown that TSLP-trap molecules that block TSLP with TSLPR and IL-7Ralpha at the same time are 20-30 times stronger 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 the efficacy and achieve better clinical treatment effect.

[0009] The 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

[0010] To solve the above problems, the present disclosure provides antibodies, methods for preparing the same, compositions, and the like. 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 disclosure 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 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: 18 and 24.

[0016] Further, in some embodiments of the application, the antibody or fragment, wherein the heavy chain variable region 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: 1-7.

[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 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: 15 and 21.

[0020] Further, in some embodiments of the application, the antibody or fragment, wherein the heavy chain variable region 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: 1-7.

[0021] Further, in some embodiments of the application, the antibody or fragment, wherein the heavy chain variable region 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: 7, 9.

[0022] Further, in some embodiments of the application, the antibody or fragment, further comprises a heavy chain constant region, wherein the heavy chain constant region 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: 11.

[0023] Further, in some embodiments of the application, the antibody or fragment, further comprises a light chain constant region, the light chain constant region 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 SEQ ID NO: 12.

[0024] Further, in some embodiments of the application, the antibody or fragment, wherein 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 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: 3, 5.

[0027] Further, in some embodiments of the application, the antibody or fragment, wherein 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 SEQ ID NO: 4 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 one of SEQ ID NOs: 3 and 5 or consisting of one of SEQ ID NOs: 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 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 consisting of one of SEQ ID NOs: 3 and 5.

[0030] Further, in some embodiments of the application, the antibody or fragment, wherein comprising:

[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 application, 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 application, the antibody or fragment, wherein the antibody is a whole antibody, a monoclonal antibody.

[0035] Further, in some embodiments of the application, 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 application, the antibody or fragment, wherein the humanized antibody comprises:

[0037] a heavy chain variable region comprising a sequence with 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: 25 or consisting of one of SEQ ID NOs: 25.

[0038] a heavy chain variable region comprising a sequence with 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: 25 or consisting of one of SEQ ID NOs: 25.

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

[0040] a heavy chain comprising a sequence with 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: 27 or consisting of one of SEQ ID NOs: 27.

[0041] a heavy chain comprising a sequence with 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: 27 or consisting of one of SEQ ID NOs: 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.

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

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

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

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

[0047] In one aspect, the present disclosure provides a multispecific molecule comprising the antibody or antigen binding portion; 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.

[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 of making the antibody or fragment, comprising the steps of:

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

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

[0052] In another aspect, the present disclosure provides use of the antibody or fragment, the nucleic acid molecule, or the vector, or the host cell, or the conjugate, or the antibody drug conjugate, or the multispecific molecule, or the pharmaceutical composition or kit in the manufacture of a medicament for treating or determining the prognosis of a disease associated with TSLP expression.

[0053] In another aspect, the present disclosure provides use of the antibody or fragment, the nucleic acid molecule, or the vector, or the host cell, or the conjugate, or the antibody drug conjugate, or the multispecific molecule, or the pharmaceutical composition or kit in the manufacture of a medicament for treating or determining the prognosis of a disease associated with TSLP expression.

[0054] In yet another aspect, the use described by the present disclosure, wherein the disease associated with TSLP expression is a cancer selected from the group consisting of Hodgkin's lymphoma, breast cancer, pancreatic cancer, melanoma, cervical cancer, cutaneous T-cell lymphoma, gastric cancer, lung cancer, B-cell lymphoma, including NHL, pre-B-cell lymphoblastic leukemia / lymphoma, and mature B-cell neoplasms, such as B-cell chronic lymphocytic leukemia (CLL) / small lymphocytic 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 type), 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 yet another aspect, the use described by the present disclosure, wherein the disease associated with TSLP expression is 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.

[0056] In yet another aspect, the use described by the present disclosure, wherein 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 rhinosinusitis without nasal polyps, and idiopathic pulmonary fibrosis (IFF).

[0057] The antibody of the present application can effectively inhibit the activation of TSLP on H_TSLP Reporter Cell Line cells STATs and JAK2 signaling pathway, and the antibody of the present application and Tezepelumab can effectively inhibit the proliferation promoting effect of TSLP on BaF3-TSLPR / IL7Ra cells, the antibody of the present application does not cross with Tezepelumab binding epitope, and does not block the C-terminal MKK34 antibacterial peptide with antibacterial activity, and is expected to retain the ability of TSLP to resist external bacterial infection.

[0058] The 4G7 and 4C4 antibodies of the present application can effectively inhibit the activation of the TSLP on the H_TSLP Reporter Cell Line cell STATs and JAK2 signal pathway, wherein the 4C4 inhibits the signal pathway activation activity 59 times that of Tezepelumab, and the 4G7 inhibits the signal pathway activation activity 26 times that of Tezepelumab.

[0059] The 4G7 and 4C4 antibodies of the present application and Tezepelumab can effectively inhibit the proliferation promoting effect of TSLP on BaF3-TSLPR / IL7R alpha cells, and the 4C4 inhibits the activity 11.3 times that of Tezepelumab, and the 4G7 inhibits the activity 6.5 times that of Tezepelumab.

[0060] The 4G7 V60 of the present application is a humanized antibody, and the 4G7 V60 and Tezepelumab can effectively inhibit the activation of the TSLP on the H_TSLP Reporter Cell Line cell STATs and JAK2 signal pathway, and the 4G7 V60 inhibits the signal pathway activity 4743 times that of Tezepelumab.

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

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

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

[0064] Figures 2A-2B The blocking activity detection results of the antibody of the present application on the protein level of TSLP and TSLPR are shown;

[0065] Figures 3A-3B The blocking activity detection results of the antibody of the present application on the binding activity of TSLP and BaF3-TSLPR / IL7R alpha cells are shown;

[0066] Figures 4A-4B The activation activity detection results of the antibody of the present application on the H_TSLP Reporter Cell Line cell STATs and JAK2 signal pathway are shown;

[0067] Figures 5A-5B Results of detection of the activity of the antibody of the present application to inhibit the proliferation-promoting effect of TSLP on BaF3-TSLPR / IL7Rα cells are shown;

[0068] Figure 6 Results of detection of the activity of the humanized antibody of the present application to inhibit the activation of STATs and JAK2 signaling pathway of H_TSLP Reporter Cell Line cells by TSLP are shown. DETAILED DESCRIPTION

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

[0070] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings commonly understood by a person of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. More specifically, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. In this application, the use of "or" means "and / or" unless stated otherwise. Furthermore, the use of the term "including" as well as other forms such as "include", "includes" and "included" is not limiting. Also, ranges provided in the specification are inclusive of the endpoints and all values between the endpoints.

[0071] Definitions

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

[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 noncovalent interactions. The light chains of an antibody can be classified as kappa or lambda light chains. The heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, which define the isotype of the antibody as IgM, IgD, IgG, IgA, or IgE, respectively. Within 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 further comprises a "D" region of about 3 or more amino acids. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region is composed of three domains (CH1, CH2, and CH3). Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The VH and VL regions can be further subdivided into variable regions (called complementarity determining regions, CDRs) spaced apart by relatively conserved regions (called framework regions, FRs). Each VH and VL is composed of three CDRs and four FRs, in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4, from N- to C-terminus. The variable regions of each heavy / light chain pair form an antigen binding site / portion, respectively. The distribution of amino acids in the various regions or domains follows the numbering definitions in common systems such as Kabat, IMGT, or Chothia, and in specific embodiments of the present disclosure, the determination of CDR sequences uses the numbering definition 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 (used interchangeably with the term "antigen-binding fragment"). An antigen-binding portion refers to a polypeptide that comprises a fragment of an intact antibody that retains the ability to specifically bind with the antigen to which the full-length or intact antibody specifically binds, and / or that competes with the full-length antibody for binding to the same antigen. In some conditions, antigen-binding portions include Fab, Fab', F(ab')2, Fd, Fv, dAb, and complementarity determining region (CDR) fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, diabodies, and comprises at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide. An antigen-binding portion of an antibody can be obtained from a given antibody by conventional techniques, such as recombinant DNA technology or enzymatic or chemical cleavage, and can be screened for specificity in the same manner as an intact antibody.

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

[0076] The term "monoclonal antibody" or "mAb" refers to antibody molecules / preparations of single molecular composition. Monoclonal antibodies display single binding specificity and affinity for a particular epitope. Antibodies of the present application can 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 on a molecule, i.e., a portion of a molecule that is recognized, e.g., by an antibody, as foreign, e.g., a three-dimensional site on an antigen that makes contact with a combining site of an immunoglobulin molecule. In the present application, the epitope is, for example, a TSLP protein.

[0078] The term "chimeric antibody" as used herein refers to an antibody whose variable region sequences are from one species and the constant region sequences are from another species, e.g., an antibody in which the variable region sequences are derived from a mouse antibody and the constant region sequences are 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, like a mouse, have been grafted onto human framework sequences. Additionally, additional framework region modifications can 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 (as determined by SPR technology). Thus, the lower the KD value (the lower the concentration), the higher the affinity of the antibody. Thus, "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 variant, isoform, and species homolog of human TSLP that is expressed by a cell natively or on a cell transfected with a TSLP gene. In some embodiments, binding of an antibody of the present disclosure to a TSLP antigen mediates killing of a TSLP-expressing cell (e.g., a tumor cell) by inactivating TSLP. Killing of a TSLP-expressing cell can occur through one or more of the following mechanisms: induction of cell death / apoptosis, ADCC, and CDC.

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

[0083] The term "specifically binds" refers to a nonrandom binding reaction between two molecules, such as the reaction between an antibody and its antigen. In certain embodiments, an antibody that specifically binds to (or has specificity for) an antigen refers to an antibody that binds to the antigen with a dissociation constant (KD) 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 M or smaller KD values bind the antigen. In some embodiments of the application, the term "targeting" refers to specific binding.

[0084] The term "isolated" refers to a state of being obtained by artificial means from a natural state. If a certain "isolated" material or component exists naturally, it can be because its natural environment has changed, or the material has been separated from the natural environment, or both. For example, a certain polynucleotide or polypeptide that is not isolated naturally exists in a certain living organism, and a high-purity version of the same polynucleotide or polypeptide separated from the natural state is referred to as an isolated polynucleotide or polypeptide. The term "isolated" does not exclude mixed artificial or synthetic substances, nor does it exclude other impurities that do not affect the activity of the isolated substance. For example, an isolated antibody can be substantially free of other cellular material and / or chemical substances.

[0085] The term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. When the vector allows the expression of a protein encoded by the inserted polynucleotide, the vector is referred to as an expression vector. The vector can be expressed in a host cell by transformation, transduction or transfection of the 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), pox viruses, baculoviruses, papillomaviruses, papovaviruses (such as SV40). The vector can comprise a plurality of 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 can comprise an origin of replication. For vectors expressing antibodies, vector types in which the heavy and light chains of the antibody exist in different vectors or vector types in which the heavy and light chains exist in the same vector can be used.

[0086] The term "host cell" refers to a cellular system that can be engineered to produce a protein of interest, protein fragment, or peptide. Host cells include, but are not limited to, cultured cells, for example, mammalian cultured cells derived from rodents (rats, mice, guinea pigs, or hamsters), 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 within a transgenic animal or cultured tissue. The term encompasses not only the particular subject cell but also its progeny. Because certain modifications can occur in succeeding generations due to mutations or environmental influences, such progeny can not be identical to the parent cell, but are still included within the scope of the term "host cell."

[0087] The term "identity" refers to a relationship between two or more polypeptide molecules (or protein molecules) or between two or more nucleic acid molecules, as determined by comparing and aligning sequences. "Percent identity" refers to the percentage of identical residues between the compared molecules, and is calculated based on the size of the smallest of the molecules being compared. For these calculations, gaps in the alignment, if any, are addressed by a particular mathematical model or computer program (i.e., "algorithm"). Methods that can be used to calculate the identity of aligned nucleic acid or polypeptide sequences include those described in Computational Molecular Biology, (Lesk, A.M., ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, D.W., ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, A.M., and Griffin, H.G., 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 refers not only to the property of an antigen to stimulate specific immune cells to activate, proliferate and differentiate to ultimately produce immunologic effectors such as antibodies and sensitized lymphocytes, but also to the specific immune response of antibodies or sensitized T lymphocytes that can be formed in the immune system of an organism after the organism has been stimulated with an antigen. Immunogenicity is the most important property of an antigen. Whether an antigen is able to 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 immunization.

[0089] The term "transfection" refers to the process of introducing nucleic acids into eukaryotic cells, in particular mammalian cells. Protocols and techniques for transfection include, but are not limited to, lipofection and chemical and physical methods such as electroporation. Many transfection techniques are well known in the art and are disclosed herein. See, e.g., 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 terms "hybridoma" and "hybridoma cell line" are used interchangeably. When referring to the terms "hybridoma" and "hybridoma cell line", they also include subclones and progeny cells of the hybridomas.

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

[0092] The term "antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which a secreted Ig molecule mediates the lysis of a target cell by a cytotoxic cell, such as a natural killer (NK) cell, neutrophil, or macrophage after binding of the Ig molecule to Fc receptors (FcRs) present on these cells. The antibody "arms" the cytotoxic cell, and is absolutely required for such killing. The primary cells for mediating ADCC, NK cells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII, and FcγRIII. To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in US Patent No. 5,500,362 or US Patent No. 5,821,337 can be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest can be assessed in vivo, e.g., 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 lysis of a target cell in the presence of complement. Activation of the classical complement pathway begins by the binding of the first component of the complement system (Clq) to antibody (of the appropriate subclass) which is bound to its homologous 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 "treat" as used herein in the context of treating a condition, generally refer to the treatment and therapy of a human or an animal subject, in which some desired therapeutic effect is achieved, such as, for example, the inhibition of the progress of the condition, including a decrease in the rate of progress, a halt in the rate of progress, a regression of the condition, an amelioration of the condition, and a cure of the condition. Treatment as a prophylactic measure (i.e., prophylaxis) is also included. For cancer, "treatment" can refer to the inhibition or slowing of growth, proliferation, or metastasis of a tumor or malignant cells, or some combination thereof. For a tumor, "treatment" includes removal of all or part of a tumor, inhibition or slowing of tumor growth and metastasis, prevention or delay of tumor development, or some combination thereof.

[0096] The term "therapeutically effective amount" relates to the amount of an active compound, or a material, composition or dosage form containing an active compound, which is effective for producing some desired therapeutic effect in accordance with the treatment regimen desired. For example, an "effective amount" or "effective dose" when used in connection with treating or detecting a disease or disorder refers to the amount or concentration of an antibody or antigen-binding fragment thereof effective to treat or detect the disease or disorder.

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

[0098] The term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is compatible, in pharmacological and / or physiological terms, with the subject and active agent, as is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusting agents, surfactants, adjuvants, and ionic strength enhancers. For example, pH adjusting agents include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic, or non-ionic surfactants, such as Tween-80; ionic strength enhancers include, but are not limited to, sodium chloride. th ed.Pennsylvania:Mack Publishing Company,1995), and includes, but is not limited to, pH adjusting agents, surfactants, adjuvants, and ionic strength enhancers. For example, pH adjusting agents include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic or non-ionic 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 immune enhancer that can enhance or alter the type of immune response to an antigen in an organism when delivered with the antigen to the organism or when delivered to the organism in advance. There are a variety of adjuvants, including but not limited to aluminum adjuvants (e.g., aluminum hydroxide), Freund's adjuvants (e.g., Freund's complete adjuvant and Freund's incomplete adjuvant), Corynebacterium parvum, lipopolysaccharide, cytokines, etc. Freund's adjuvant is the most commonly used adjuvant in animal experiments at present. Aluminum hydroxide adjuvant is more commonly used in clinical trials.

[0100] The term "autoimmune disease" refers to a disease caused by the immune reaction of the body to its own antigens, leading to damage to the body's 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, etc.

[0101] The term "inflammatory disease" refers to a collective term for diseases in which inflammation is the major destructive factor. Inflammation is a biological response of tissues to harmful stimuli, is a pathological process, and is accompanied by three events of tissue degeneration, circulatory disturbance, 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 syndrome, hypersensitivity pneumonitis, atopic dermatitis, rhinitis, ankylosing spondylitis, rheumatic fever, fibromyalgia, psoriatic arthritis, chronic nephritis, Sjogren's syndrome, and multiple sclerosis, atopic dermatitis, allergic conjunctivitis, atopic dermatitis fibrosis, and inflammatory bowel disease, etc.

[0102] The term "cancer" refers to any neoplastic or malignant cell growth, proliferation, or metastasis-mediated solid and non-solid tumors such as leukemias, which can be benign, pre-malignant, or malignant, including but not limited to Hodgkin's lymphoma, breast cancer, pancreatic cancer, melanoma, cervical cancer, cutaneous T-cell lymphoma, gastric cancer, lung cancer, B-cell lymphoma, including NHL, pre-B-cell lymphoblastic leukemia / lymphoma, and mature B-cell neoplasms, such as B-cell chronic lymphocytic leukemia (CLL) / small lymphocytic 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 type), 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, an amino acid at a certain position can be allowed to vary, for example, [K / R] indicates that the amino acid at the position can be K or R; for another example, [S / *] indicates that the amino acid at the position can be S or absent.

[0104] Anti-TSLP antibodies

[0105] In some aspects, the present application includes an isolated antibody or antigen binding fragment thereof.

[0106] In the context of the present application, "antibody" can include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized and primatized antibodies, CDR- grafted antibodies, human antibodies, recombinantly produced antibodies, intrabodies, bifunctional antibodies, multispecific antibodies, bispecific antibodies, monovalent antibodies, multivalent antibodies, anti-idiotypic antibodies, synthetic antibodies, including muteins and variants thereof, improved antibodies; and derivatives thereof (including Fc fusion proteins and other modifications), as well as any other immunoreactive molecule that exhibits preferential association or binding to TSLP protein. Furthermore, unless otherwise specified by context, the term also includes antibodies of all classes (i.e., IgA, IgD, IgE, IgG and IgM) and all subclasses (i.e., IgGl, IgG2, IgG3, IgG4, IgAl 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 the antibody sequences can be identified according to general rules that have been developed in the art (as described above, e.g., the Kabat numbering system or by aligning the sequences with databases of known variable regions).

[0108] In some embodiments, the isolated antibody or antigen-binding fragment thereof can comprise conservative substitutions or modifications or substitutions of amino acids (e.g., conservative substitutions), deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids; or conservative substitutions of up to 20, up to 15, up to 10, or up to 5 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 antigen fragment thereof from which it is derived. Amino acid deletion variants comprising deletions at the N- and / or C-terminus of the protein are also referred to as N- and / or C-terminal truncation variants. It is understood in the art that certain conservative sequence modifications can be made which do not abrogate antigen binding. 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 amino acid substitutions / replacements that do not adversely affect or alter the essential properties of a protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions / replacements can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions in which an amino acid residue is replaced with another amino acid residue having a similar side chain, e.g., a substitution of a physically or functionally similar residue (e.g., a residue having similar size, shape, charge, chemical properties including ability to form covalent or hydrogen bonds, etc.) for the corresponding amino acid residue. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), uncharged hydrophobic side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a corresponding amino acid residue is preferably replaced with 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 application, if reference is made to an antibody comprising a specific antibody heavy chain and / or a specific antibody light chain (e.g., a chain comprising a specific CDR sequence), the two heavy chains and / or two light chains of the antibody are preferably each composed of the specific antibody heavy chain and / or the specific antibody light chain, respectively.

[0111] Regardless of how the antibody is produced, methods of testing the ability of an 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 assay (see, e.g., Janeway et al., infra, and U.S. Patent Application Publication No. 2002 / 0197266 and the above section on competitive assays).

[0112] According to the present application, an antibody is capable of binding to a predetermined target (e.g., a TSLP protein or a TSLP-expressing cell) if the antibody has significant affinity for the predetermined target in a standard assay (e.g., an assay described in the present application), and to test the binding of a monoclonal antibody to a living cell expressing TSLP, flow cytometry can be used. Preferably, in a flow cytometry analysis (FACS) assay, the binding of the antibody to a target expressed on the surface of a cell is determined, and the antibody is capable of binding to the target if the antibody binds detectably to the target (a TSLP protein or a TSLP-expressing cell) with "affinity." Preferably, the antibody of the present application binds detectably to the target if the antibody 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 antibody or fragment has an EC50binding property of binding human TSLP or monkey TSLP with an EC50of 5 μg / mL or less; binding human TSLP or monkey TSLP with an EC50of 3 μg / mL or less; or binding human TSLP or monkey TSLP with an EC50of 2 μg / mL or less; or binding human TSLP or monkey TSLP with an EC50of 1 μg / mL or less; or binding human TSLP or monkey TSLP with an EC50of 0.5 μg / mL or less; binding human TSLP or monkey TSLP with an EC50of 0.3 μg / mL or less; or binding human TSLP or monkey TSLP with an EC50of 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 an IgG. The constant region of the IgG is preferably selected from the group consisting of a constant region of IgGl, IgG2, IgG3, or IgG4. The constant region of the IgG is more preferably selected from the group consisting of a constant region of IgGl.

[0117] In the art, various means are employed to modify without changing the desired properties of an antibody, such as the means of recombining the light and heavy chains of an antibody employed in the present disclosure, making substitutions of amino acids, and the like. For example, the sequences of the present application, 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 complementarity determining regions (CDRs) of the heavy and light chains. For this reason, the amino acid sequences of the CDRs are more diverse between antibodies than other sequences. Since the CDR sequences are responsible for most antibody-antigen interactions, it is possible to express a recombinant antibody that mimics the properties of a particular naturally occurring antibody by constructing an expression vector that contains the CDR sequences from the particular naturally occurring antibody grafted onto framework sequences from a different antibody 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. U.S.A. 86:10029-10033). Such framework sequences can be obtained from public DNA databases that include germline antibody gene sequences. These germlines are sequences different from mature antibody gene sequences because they do not contain a complete set of assembled variable genes. Germline gene sequences will also have sequences at individual positions across the variable region that are different from a high affinity secondary repertoire antibody.

[0119] Mouse antibodies are highly immunogenic in humans, leading to a decrease in therapeutic efficacy upon repeated applications, mediated primarily through the heavy chain constant region. If each antibody is chimerized or humanized, the immunogenicity of mouse antibodies in humans can be reduced or completely avoided.

[0120] Chimeric antibodies are antibodies that have different portions from different animal species, e.g., an antibody having a variable region from a mouse antibody and a human immunoglobulin constant region. Chimeric antibodies are obtained by joining together the variable regions of the heavy and light chains of a mouse antibody with the constant regions of a human heavy and light chain (e.g., 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, a chimeric antibody is produced by joining a human kappa light chain constant region to a mouse light chain variable region. In another preferred embodiment, a chimeric antibody can be produced by joining a human lambda light chain constant region to a mouse light chain variable region.

[0121] Humanized antibodies, on the other hand, 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 a human framework sequence.

[0122] To reduce the immunogenicity of the antibodies to humans, humanized anti-TSLP antibodies are produced using the sequences of the TSLP antibodies of the present disclosure, utilizing the CDR regions of other sources of anti-TSLP antibodies in combination with human-derived framework regions (e.g., human immunoglobulins) to form the humanized anti-TSLP antibodies of the present disclosure, which are expected to retain the function of binding to human TSLP as well as the function of binding to monkey TSLP.

[0123] Preparation or production of antibodies

[0124] Antibodies of the present application can be produced by a variety of techniques, including conventional monoclonal antibody methodology, such as the standard somatic cell hybridization technique of Kohler and Milstein, Nature 256:495 (1975). Although the hybridoma technique is preferred, in principle, other techniques for producing monoclonal antibodies also can be employed, such as viral or oncogenic transformation of B lymphocytes or phage display techniques using antibody gene libraries, somatic cell hybridization methods, and recombinant techniques, such as those involving the engineering of genes by gene recombination techniques. For example, DNA molecules encoding the heavy and light chain genes of the antibodies of the present application are obtained, either by chemical synthesis or by PCR amplification, inserted into expression vectors, and then transfected into host cells, which are then cultured under conditions suitable for the expression of the antibodies of the present application.

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

[0126] Monoclonal antibodies can be produced 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, monoclonal antibodies can be produced using hybridoma and art-recognized biochemical and genetic engineering techniques, 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, N.Y., 1981), each of which is hereby incorporated by reference in its entirety.

[0127] It will be appreciated that selected binding sequences can be further altered, for example to improve affinity for the target, to humanize the target binding sequence, to improve its production in cell culture, to reduce its immunogenicity in vivo, to create a multispecific antibody, etc., and antibodies comprising altered target binding sequences are also antibodies of the application.

[0128] In some embodiments, a method of producing an antibody or fragment of the present disclosure comprises the steps of:

[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, the anti-TSLP monoclonal antibody is produced by using a hybridoma.

[0132] To obtain hybridomas producing antibodies of the application, e.g., human monoclonal antibodies of the application, spleen and / or lymph node cells from immunized mice can be isolated and fused to suitable immortalized cell lines, e.g., mouse myeloma cell lines. The resulting hybridomas are screened for production of antigen-specific antibodies. The production of hybridomas is well known in the art. See, e.g., Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York.

[0133] Antibodies of the application can also be produced in host cell transfectomas using, e.g., 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 the partial or full light and heavy chains will be inserted into one or more expression vectors such that the genes are operably linked to transcriptional and translational regulatory sequences. In this context, the term "operably linked" is intended to mean that an antibody gene is ligated into a vector in such a way that transcriptional and translational control sequences within the vector

[0134] Antibody light chain genes and antibody heavy chain genes can be inserted into the same or different expression vectors. In some embodiments, the variable region is used to produce a full-length antibody gene of any antibody isotype by inserting it into an expression vector that already encodes a heavy chain constant region and a light chain constant region of the desired isotype, such that the V segment is operably linked to the CH segment within the vector and the VL segment is operably linked to the CL segment within the vector. Additionally or alternatively, the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain(s) from a host cell. The antibody chain genes can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus 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, the expression vectors encoding the heavy and light chains are transfected into host cells by standard techniques. The various forms of the term "transfect" are intended to encompass a variety of techniques for getting exogenous DNA into a prokaryotic or eukaryotic host cell such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like. Antibodies of the application can be expressed in prokaryotic or eukaryotic host cells, e.g., mammalian host cells, which can assemble and secrete properly folded and immunologically active antibodies.

[0136] Mammalian host cells for expressing the recombinant antibodies of the application include Chinese hamster ovary cells (CHO cells) used with a DHFR selectable marker (e.g., as described in R.J. Kaufman and P.A. Sharp (1982) J. MoI. Biol. 159:601-621) including dhfr CHO cells described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, 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 recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, the antibody is produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or to permit the secretion of the antibody into the culture medium in which the host cells are grown. Antibody can be recovered from the culture medium using standard protein purification methods.

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

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

[0139] To create a chimeric antibody, the murine immunoglobulin variable regions can be linked to human immunoglobulin constant regions using methods known in the art (see, e.g., U.S. Patent No. 4,816,567 to Cabilly et al.). An isolated nucleic acid encoding a VHand can be converted to a full-length heavy chain gene by operably linking the VHand to another DNA molecule encoding heavy chain constant regions (CHI, CH2, and CH3). 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, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). The heavy chain constant region can be an IgGl, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but more preferably is an IgGl or IgG4 constant region. An isolated nucleic acid encoding a VLregion can be converted to a full-length light chain gene (as well as a Fab light chain gene) by operably linking the VLto another DNA molecule encoding a light chain constant region, CL. Sequences of human light chain constant region genes are known in the art (see, e.g., Kabat et al., supra), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. In preferred embodiments, the light chain constant region can be a kappa or lambda constant region, but a kappa constant region is generally preferred. Once DNA fragments encoding VHand VLsegments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example, to convert the variable region genes to full-length antibody chain genes, Fab fragment genes or scFv genes. In these manipulations, DNA fragments encoding VLor VHand another protein-encoding DNA fragment, such as an antibody constant region or a flexible linker, are operably linked. The term "operably linked," as used in this document, is intended to mean the joining together of two DNA fragments in such a way that the amino acid sequence encoded by the two DNA fragments remains in frame.

[0140] To make humanized antibodies, the murine CDR regions can be inserted into human framework sequences using methods known in the art (see U.S. Patent No. 5,225,539 to Winter; U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,762 to Queen et al.; and Lo, Benny, K.C., editor, in Antibody Engineering: Methods and Protocols, volume 248, Humana Press, New Jersey, 2004). Alternatively, transgenic animals can also be utilized that are capable of producing a complete repertoire of human antibodies upon immunization and that are incapable of producing endogenous immunoglobulins. 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 results in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array VDJ and C mu to the mutant mice results in the production of entirely human antibodies in response to antigen challenge (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 such transgenic animals include the HuMAb mouse (Medarex, Inc.) which contains human immunoglobulin genes miniloci that encode unrearranged human heavy (mu and gamma) and kappa light chain immunoglobulin sequences, together with targeted mutations that inactivate the endogenous mu and kappa chain loci (see, e.g., Lonberg et al. (1994) Nature 368(6474):856-859); or the "KM mouse™" (see patent application WO 02 / 43478) which carries human heavy chain transgene and human light chain transchromosome. Other methods of humanizing antibodies 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 antibodies of the invention

[0142] In some aspects, the present application relates to an isolated nucleic acid molecule comprising a nucleic acid sequence encoding an isolated antibody or fragment thereof as described above in the present disclosure.

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

[0144] To make chimeric antibodies, the murine immunoglobulin variable regions can be linked to human immunoglobulin constant regions using methods known in the art (see, e.g., U.S. Patent No. 4,816,567 to Cabilly et al.). An isolated nucleic acid encoding a VHand can be converted to a full-length heavy chain gene by operably linking the nucleic acid encoding the VHand another DNA molecule encoding heavy chain constant regions (CHI, CH2, and CH3), and DNA fragments comprising these regions can be obtained by standard PCR amplification. An isolated nucleic acid encoding a VLcan be converted to a full-length light chain gene (as well as a Fab light chain gene) by operably linking the DNA encoding the VLto another DNA molecule encoding a light chain constant region, CL. Once DNA fragments

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

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

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

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

[0149] Conjugates

[0150] In one aspect, the present disclosure provides a conjugate comprising an antibody or fragment thereof as previously described coupled to at least one detectable label. Detectable labels include, but are not limited to: (i) provide a detectable signal; (ii) interact with a second label to modify the detectable signal provided by the first or second label, e.g., FRET (Fluorescence Resonance Energy Transfer); (iii) affect mobility (e.g., electrophoretic mobility) by charge, hydrophobicity, shape, or other physical parameter, or (iv) provide a capture moiety, e.g., an affinity, antibody / antigen, or ionic complex.

[0151] Suitable structures for labels are, for example, fluorescent labels, luminescent labels, chromophoric labels, radioisotope labels, isotopic labels, preferably stable isotopic labels, isobaric labels, enzymatic labels (e.g., horseradish peroxidase, alkaline phosphatase, beta-galactosidase, urease, glucose oxidase, etc.), particulate labels (especially metal particulate labels, magnetic particulate labels, polymeric particulate labels), small organic molecules (e.g., biotin, a ligand or binding molecule for a receptor (e.g., a cell adhesion protein or a phospholipid), a label sequence comprising nucleic acid and / or amino acid residues that can be detected using a binding agent, etc. Labels include, without limitation, barium sulfate, ioxitalamic acid, iopanoic acid, calcium iodipamide, sodium amidotrizoate, meglumine amidotrizoate, meglumine metrizamide, sodium tyropanoate, 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 ester), fluorescent substances (e.g., fluorescein and rhodamine), 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 labels can be detected using a light detector to detect emitted light. Enzymatic labels are generally detected by providing a substrate for the enzyme and detecting the reaction product produced by action of the enzyme on the substrate. In certain embodiments, such labels can be adapted for use in immunoassays (e.g., enzyme-linked immunoassays, radioimmunoassays, fluorescent immunoassays, chemiluminescent immunoassays, etc.). In certain embodiments, detectable labels as described above can be linked to antibodies or antigen-binding fragments thereof of the present disclosure via 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, the drug moieties being linked (e.g., covalently linked) to the antibody or fragment thereof as previously described either directly or via a linker. The linker structure for conjugating the anti-TSLP antibody to the drug in the antibody-drug conjugates of the present application is not particularly limited as long as the resulting antibody-drug conjugate can be used.

[0155] Because of the ability of the antibody-drug conjugates to selectively deliver one or more drugs to a target tissue (e.g., an antigen associated with a tumor, such as a tumor affected by TSLP), the antibody-drug conjugates can improve the therapeutic efficacy of the antibodies or antigen-binding fragments thereof of the present application in treating a disease (e.g., cancer).

[0156] Multispecific molecules

[0157] The antibodies or antigen-binding fragments thereof of the present application can be used to form multispecific molecules (e.g., bispecific molecules). The antibodies or antigen-binding fragments thereof of the present application can be part of a multispecific molecule (e.g., a bispecific molecule) that comprises a second functional module (e.g., a second antibody) or a third functional module (e.g., a third antibody) having a different binding specificity than the antibodies or antigen-binding fragments thereof of the present application, thereby being able to bind at least two different binding sites and / or target molecules. For example, the antibodies or antigen-binding fragments thereof of the present application can be linked to a second antibody or antigen-binding fragment thereof that is capable of specifically binding to any protein that can serve as a potential target for a combination therapy. To generate such bispecific or multispecific molecules, the antibodies or antigen-binding fragments thereof of the present application can be linked (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 present application provides a multispecific molecule comprising an antibody or antigen-binding fragment thereof of the present application.

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

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

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

[0162] Pharmaceutical compositions

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

[0164] The pharmaceutical composition can optionally contain one or more additional pharmaceutically active ingredients, such as another antibody or drug. The pharmaceutical composition of the present application can 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. The pharmaceutically acceptable carrier can include, for example, a pharmaceutically acceptable liquid, gel or solid carrier, aqueous medium, non-aqueous medium, antimicrobial agent, isotonic agent, buffer, antioxidant, anesthetic, suspending / dispersing agent, chelating agent, diluent, adjuvant, excipient or non-toxic auxiliary substance, combinations of various components known in the art or more.

[0165] Suitable components can include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavorings, thickening agents, colorants, emulsifiers or stabilizers such as sugars and cyclodextrins. Suitable antioxidants can include, for example, methionine, ascorbic acid, EDTA, sodium thiothio sulfate, platinum, catalase, citric acid, cysteine, mercaptoglycerol, mercaptoacetic acid, mercaptosorbitol, butylated hydroxyanisole, butylated hydroxytoluene and / or propyl arsenate. As disclosed herein, an antibody or antigen-binding fragment thereof comprising one or more antioxidants such as methionine can be oxidized in a solvent containing the antibody or antigen-binding fragment thereof of the present disclosure. Oxidation can prevent or reduce the reduction of binding affinity, thereby enhancing antibody stability and extending shelf life. Accordingly, in some embodiments, the present application provides a composition comprising one or more antibodies or antigen-binding fragments thereof and one or more antioxidants such as methionine. The present application further provides a variety of methods in which an antibody or antigen-binding fragment thereof is mixed with one or more antioxidants such as methionine, such that the antibody or antigen-binding fragment thereof can be prevented from being oxidized to extend its shelf life and / or increase activity.

[0166] For further explanation, the pharmaceutically acceptable carrier 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 injection, non-aqueous vehicles such as fixed oils, cottonseed oil, corn oil, sesame oil, or peanut oil, antimicrobial or antifungal agents, isotonic agents such as sodium chloride or dextrose, buffers such as phosphate or citrate buffers, antioxidants such as sodium bisulfate, local anesthetics such as procine hydrochloride, suspending and dispersing agents such as sodium carboxymethylcellulose, hydropropylmethylcellulose, or polyvinylpyrrolidone, emulsifying agents such as poly sorbate 80 (TWEEN-80), sequestering 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 a pharmaceutical composition in a multi-dose container comprising phenolics or cresylics, 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 non-toxic auxiliary substances can include, for example, wetting or emulsifying agents, pH buffering agents, stabilizing agents, solubility enhancers, or agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrins.

[0167] Administration, formulation, and dosage

[0168] The pharmaceutical compositions of the present application can be administered to a subject in need thereof in vivo by a variety of routes including, but not limited to, oral, intravenous, intra-arterial, subcutaneous, parenteral, intranasal, intramuscular, intracranial, intracardiac, intraventricular, intratracheal, buccal, rectal, intraperitoneal, intradermal, topical, transdermal, and intrathecal, or by implantation or inhalation. The compositions of the present application can be formulated into preparations in solid, semi-solid, liquid, or gaseous forms; including, but not limited to, tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalers, and aerosols. The appropriate preparation and administration route can be selected depending on the intended application and therapeutic regimen.

[0169] Suitable preparations for enteral administration include hard or soft gelatin capsules, pills, tablets, including coated tablets, elixirs, suspensions, syrups, or inhalers, and controlled release dosage forms thereof.

[0170] Formulations 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 a liposome or other microparticulate). These liquids can additionally contain other pharmaceutically acceptable ingredients such as antioxidants, buffers, preservatives, stabilizers, bacteriostats, suspending agents, thickening agents, and solutes that render the formulations isotonic with the blood (or other relevant bodily fluid) of the intended recipient. Examples of excipients include, e.g., water, alcohols, polyols, glycerol, vegetable oils, and the like. Examples of isotonic carriers suitable for such formulations include sodium chloride injection, Ringer's solution, or lactated Ringer's injection. Similarly, the specific dosage regimen (including dosing, timing, and repetition) will depend on the particular individual and the individual's medical history, and empirical considerations such as pharmacokinetics (e.g., half-life, clearance rate, 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, J.B. Lippincott Company, Philadelphia, PA, Banker and Chalmers eds., 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 therapy and is based on reducing the number of proliferating or tumorigenic cells, maintaining such reduction in tumor cells, reducing proliferation of tumor cells, or delaying development of metastases. 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 application can be appropriate.

[0173] Those of skill will appreciate that an appropriate dosage can vary from patient to patient. Determining the optimal dosage will generally involve balancing the level of therapeutic benefit against any risk or deleterious side effects. The selected dosage level will depend on a variety of factors including, but not limited to, the activity of the particular compound, the route, time and

[0174] Generally, the antibodies or antigen binding fragments thereof of the present application can be administered in a variety of ranges. These include about 4 pg / kg body weight to about 100 mg / kg body weight per dose; about 20 pg / kg body weight to about 50 mg / kg body weight per dose; about 50 pg / kg body weight to about 10 mg / kg body weight per dose. Other ranges include about 100 pg / 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 pg / kg body weight, at least about 250 pg / kg body weight, at least about 750 pg / 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 application will comprise 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 application can be administered daily, every two days, every four days, weekly, every ten days, every two weeks, every three weeks, monthly, every six weeks, every two months, every ten weeks, or every three months. In this regard, it will be appreciated that the dosage or interval can be altered based on patient response and clinical practice.

[0176] Compatible formulations for parenteral administration (e.g., intravenous injection) will comprise the antibodies or antigen binding fragments thereof as disclosed herein at a concentration of about 5 pg / mL to about 100 mg / mL. In certain selected embodiments, the concentration of the antibodies or antigen binding fragments thereof will include 10 pg / mL, 20 pg / mL, 50 pg / mL, 60 pg / mL, 80 pg / mL, 100 pg / mL, 200 pg / pg / mL, 300 pg / mL, 400 pg / mL, 500 pg / mL, 600 pg / mL, 800 pg / mL, 900 pg / mL, or 1 mg / mL. In other preferred embodiments, the antibody conjugate drug 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 present application can be co-administered with one or more other therapeutic agents (e.g., cytotoxic agents, radiotoxic agents, antitumor agents, anti-angiogenic agents, or immunosuppressive agents) to reduce the induction of an immune response against the anti-TSLP antibodies of the present application. The antibodies can be linked to the therapeutic agents (as an immunoconjugate) or can be administered separately from the therapeutic agents.

[0178] In the context of administering a therapy, the term "combination" or "co-administration" as used herein refers to the use of more than one therapy or therapeutic agent. The use of the term "combination" does not restrict the order in which therapies or therapeutic agents are administered to a subject. A therapy or therapeutic agent can be administered prior to, simultaneously with, or after the administration of a second therapy or therapeutic agent to a patient. Preferably, the therapies or therapeutic agents are administered to a subject in an order, amount, and / or at an interval such that they can act together. In a particular embodiment, the therapies or therapeutic agents are administered to a subject in an order, amount, and / or at an interval such that they provide an increased benefit than if administered otherwise, 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 present application have a number of in vitro and in vivo uses, including, for example, detection of TSLP or enhancement of an immune response. For example, these molecules can be administered in vitro or ex vivo to cultured cells, or, for example, in vivo to a human subject.

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

[0182] Treatment of disorders associated with TSLP expression

[0183] In some aspects, the present application provides a method of treating a disorder in a mammal, comprising administering to a subject (e.g., 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 application, a "disease associated with TSLP expression" means an elevated or reduced expression of TSLP in cells of a diseased tissue or organ as compared to the state in a healthy tissue or organ. Elevated or reduced means an elevation or reduction of 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 more. For example, the disease is a cancer or an autoimmune disease or an inflammatory disease.

[0185] In some aspects, the present disclosure provides a method for treating or determining a prognosis of a disease associated with TSLP expression in a subject, comprising administering to a subject in need thereof an effective amount 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.

[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 or determining a prognosis of a disease associated with TSLP expression in a subject.

[0187] In some aspects, the present disclosure provides the 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 for the manufacture of a medicament (or drug) for treating or determining a prognosis of a disease associated with TSLP expression.

[0188] In one embodiment, the disease associated with TSLP expression comprises a tumor disease, e.g. a cancer.

[0189] In some embodiments, the disease associated with TSLP expression is a cancer, preferably selected from the group consisting of Hodgkin's lymphoma, breast cancer, pancreatic cancer, melanoma, cervical cancer, cutaneous T-cell lymphoma, gastric cancer, lung cancer, B-cell lymphoma, including NHL, pre-B-cell lymphoblastic leukemia / lymphoma and mature B-cell neoplasms, such as B-cell chronic lymphocytic leukemia (CLL) / small lymphocytic 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 type), hairy cell leukemia, diffuse large B-cell lymphoma, Burkitt's lymphoma, plasmacytoma, plasmacytoma of the bone marrow, post-transplant lymphoproliferative disorder, Waldenstrom's macroglobulinemia, and anaplastic large cell lymphoma (ALCL).

[0190] The antibody or antigen-binding fragment thereof can be used alone as a monotherapy or can be used in combination with chemotherapy or radiotherapy.

[0191] The antibody or antigen-binding fragment thereof can be used in combination with an anti-cancer agent, cytotoxic agent or chemotherapeutic agent.

[0192] The term "anti-cancer agent" or "anti-proliferative agent" means any agent useful in the treatment of 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 anti-cancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormonal therapies, radiation therapies, anti-metastatic agents, and immunotherapeutic agents. It will be appreciated that in selected embodiments as described above, such anti-cancer agents can comprise conjugates and can be combined with the disclosed site-specific antibodies prior to administration. More particularly, in certain embodiments, a selected anti-cancer agent will be linked to an unpaired cysteine of the engineered antibody to provide an engineered conjugate as described herein. Accordingly, such engineered conjugates are expressly contemplated within the scope of the present application. In other embodiments, the disclosed anti-cancer agents will be administered in combination with site-specific conjugates comprising different therapeutic agents 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, wherein the disease associated with TSLP expression is an inflammatory disease, the inflammatory disease is 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 rhinosinusitis without nasal polyps, and idiopathic pulmonary fibrosis (IFF).

[0195] Diagnosis

[0196] The present disclosure provides in vitro and in vivo methods for detecting, diagnosing or monitoring proliferative disorders and methods of screening cells from a patient to identify tumor cells, including tumorigenic cells. Such methods include identifying an individual having cancer for treatment or monitoring the progression of cancer, including contacting a patient or a sample obtained from a patient (in vivo or in vitro) with an antibody described herein and detecting the presence or absence or level of binding of the antibody to bound or free target molecules 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 of diagnosing, detecting or monitoring a disease associated with TSLP expression, comprising administering to a subject in need thereof an effective amount 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.

[0198] In some aspects, the present disclosure provides a method of diagnosing, detecting or monitoring a disease associated with TSLP expression in a subject, comprising administering to the subject an effective amount 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.

[0199] In 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 manufacture of a medicament (or drug) for diagnosing, detecting or monitoring a disease associated with TSLP expression.

[0200] The sample can be analyzed by a variety of assays, such as radioimmunoassay, enzyme immunoassay (e.g., ELISA), competitive binding assay, fluoroimmunoassay, immunoblot assay, Western blot analysis, and flow cytometry assay. Compatible in vivo diagnostic or diagnostic assays can include imaging or monitoring techniques known in the art, such as magnetic resonance imaging, computerized tomography (e.g., CAT scan), positron emission tomography (e.g., PET scan), radiography, ultrasound, and the like, as known to those skilled in the art.

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

[0202] Preferred subjects include mammals, such as a human / patient in need.

[0203] The sample from the subject is blood, excretion (urine or feces), oral or nasal secretions, or alveolar lavage fluid, interstitial fluid, sweat, or an extract thereof from the subject.

[0204] Pharmaceutical packaging and kits

[0205] Also provided are pharmaceutical packs and kits comprising one or more containers containing one or more dosages of an antibody or 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 antigen-binding fragment thereof, with or without one or more other agents. For other embodiments, such unit doses are supplied as single-unit dosage preparations in a ready-to-use form in a vial. In other embodiments, the composition contained in the unit dose can include saline, sucrose or the like; buffers such as phosphate, and / or the like; and / or be formulated in a pH range that is stable and effective. Alternatively, in certain embodiments, the conjugate composition can be supplied as a lyophilized powder that can be reconstituted upon addition of a suitable liquid (e.g., sterile water or a saline solution). In certain preferred embodiments, the composition comprises one or more substances that inhibit aggregation of the protein, including but not limited to sucrose and arginine. Any label on or associated with the container indicates that the conjugate composition contained therein is used for treating a selected neoplastic disease condition.

[0206] Such kits will typically comprise a pharmaceutically acceptable formulation of the engineered conjugate in a suitable container, and optionally one or more anti-cancer agents or other agents in the same or different container(s). The kits can also contain other pharmaceutically acceptable formulations for diagnostic or combination therapy.

[0207] More specifically, the kits 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 separate containers for each of the desired reagents. Where a combination therapeutic is provided for conjugation, the single solution can be premixed in molar equivalents or one component in excess of the other. Alternatively, the conjugate of the kit and any optional anticancer agent can be stored separately in different containers until administration to the patient. The kits can also include second / third container means for containing sterile pharmaceutical acceptable buffers or other diluents, e.g., bacteriostatic water for injection (BWFI), phosphate-buffered saline (PBS), Ringer's solution, and dextrose 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 can be provided as dry powders. When the reagents or components are provided in dry powder form, the powder can be reconstituted by the addition of a suitable solvent. It is envisaged that the solvent can also be provided in a further container.

[0209] Examples

[0210] Incorporation by Reference

[0211] The entire contents of each of the patent and scientific literature references herein are incorporated by reference herein for all purposes.

[0212] Equivalents

[0213] The present application can be embodied in other specific ways without departing from the spirit or essential characteristics thereof. Therefore, the illustrative embodiments described herein are not to be considered in all respects to be limiting of the application, rather, they are provided for illustrative purposes only. The scope of the application is to be indicated by the appended claims, rather than by the foregoing description, and all changes that come within the meaning and range of equivalents are intended to be embraced therein.

[0214] Example 1 Animal Immunization and TSLP Antibody Screening

[0215] Select 10-12 weeks New Zealand rabbits, use human TSLP full-length protein (Acro Biosystems, TSP-H52Ha) emulsified with Freund's adjuvant to subcutaneously inject the animals at multiple points, once every two weeks, a total of 4-5 times of immunization. According to the serum titer, 3 days before the collection of peripheral blood, the selected high titer animals are injected with antigen solution or subjected to impact immunization. The collected rabbit peripheral blood is separated to obtain PBMC, and then the antibody light chain and heavy chain variable region sequences are obtained by single B cell screening technology platform. The expression vector constructed by PCR method is co-transfected into 293T cells, and the cell culture supernatant is collected, and a plurality of positive clones recognizing human TSLP are screened by ELISA and FACS methods.

[0216] By preferred partial positive clones, the light and heavy chain genes were cloned into expression vectors containing human light chain constant region or human IgG1 heavy chain constant region, respectively, to construct eukaryotic expression vectors and sequence a series of antibody sequences.

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

[0218] The N-terminal of the light chain and heavy chain variable region sequences of the candidate antibody sequence were respectively connected with appropriate signal peptides, and the C-terminal was respectively connected with the light and heavy chain constant region. After codon optimization, they were respectively cloned into the expression vector pCDNA3.4, and ExpiCHO-S cells (Thermo) were co-transfected according to the manufacturer's operation method for expression. The supernatant was collected, and the candidate antibody proteins 4C4 and 4G7 were obtained after Protein A purification.

[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 heavy chain (SEQ ID NO: 1)

[0241] AAVMTQTPASVSVAVGGTVTINC QASEDIDSYLA WYQQKPGQPPKLLIY YASNLAS GVPSRFKGSGSGK

[0242] QFTLTISGVQCADAATYYC QGGFYTTIGDSA FGGGTEVVVKRTVAAPSVFIFPPSDEQLKSGTASVVCLL

[0243] NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP

[0244] VTKSFNRGEC

[0245] The CDR regions are underlined.

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

[0247] The above purified candidate antibodies were subjected to ELISA to detect protein level binding activity. Human TSLP antigen protein (AcroBiosystems, TSP-H5255) was aliquoted into a 96-well enzyme-labeled plate at a concentration of 1 μg / mL, 100 μl / well, 4°C overnight incubation; 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 samples, reference (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, 100 μl of Anti-Human IgG HRP (1:10000 dilution) was added, and incubated at 37°C for 0.5 hours; after washing 4 times with PBST, 50 μl of TMB color developing solution was added, and color developed at 37°C for 8 min, and the reaction was terminated by adding 50 μl / well of ELISA termination solution, and the absorbance value was determined at 450 nm wavelength on an enzyme-labeled instrument.

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

[0249] Example 4: ELISA analysis of candidate antibody protein level blocking activity

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

[0251] The results are shown in Figure 2A and Figure 2B It was shown that the 4C4 antibody of the present application can well block the binding of TSLP to TSLPR, and the blocking ability is better than that of the reference Tezepelumab. The 4G7 antibody of the present application has weaker blocking ability of TSLP to TSLPR.

[0252] Example 5: Candidate antibody blocking TSLP binding to BaF3-TSLPR / IL7Rα cells

[0253] BaF3-TSLPR / IL7Ra cells are a stable cell line with high expression of TSLP receptor. The blocking effect of rabbit anti-human TSLP antibody on the binding of TSLP to BaF3-TSLPR / IL7Ra cells is detected by flow cytometry. Specific operation: add candidate antibody (4*1250 ng / ml, 2-fold gradient dilution, 8 concentration points) to 96-well plate, 50 μl / well; add TSLP (Acro Biosystems, item number: TSP-H52Ha) solution (4*40 ng / ml), 50 μl / well; 37°C for 15 minutes; continue to add BaF3-TSLPR / IL7Ra cells, 1E6 / ml, 100 μl / well; 2-8°C for 1 hour; wash 3 times; add goat anti-mouse IgG Fc specific-FITC antibody (sigma-Aldrich, item number: F4143) diluted at a ratio of 1:300, incubate at 2-8°C for 0.5 hours in the dark, wash 3 times, then resuspend the cells with staining buffer, and detect the fluorescence intensity (MFI) of the cells using flow cytometry (BD FACS Lyric). The blocking efficiency of the sample is calculated by the formula: blocking percentage (%) = (MFI TSLP对照 -MFI 样品 ) / (MFI TSLP对照 -MFI cell control )*100%. Wherein: MFI 样品 represents the well with TSLP, anti-TSLP antibody and cells; MFI cell control represents the well with only cells; MFI TSLP对照 represents the well with TSLP and cells. The IC50 value is calculated by nonlinear fitting with the final concentration of antibody as the abscissa and the blocking percentage (%) of the corresponding concentration of antibody as the ordinate. The results are shown in Figure 3A and Figure 3B show that the IC50 of 4G7 and 4C4 antibodies in blocking activity at the level of BaF3-TSLPR / IL7Ra cells is better than that of Tezepelumab. The cell level blocking activity of 4C4 is 1.5 times that of Tezepelumab, and the cell level blocking activity of 4G7 is about 2.4 times that of Tezepelumab, but the maximum blocking percentage does not reach 100%.

[0254] Example 6: Effect of candidate antibody on inhibition of TSLP activation of H_TSLP Reporter Cell Line cells STATs and JAK2 signaling pathway

[0255] H_TSLP Reporter Cell Line is a Luciferase reporter cell line based on Jak2-Stat signaling pathway. After TSLP binds to TSLPR, the dimer enhances the recruitment of IL-7Rα, forming an extracellular ternary complex, and Jak2 is activated. Jak2 further mediates the phosphorylation of Stats, which is transported into the nucleus, thereby activating the expression of Luciferase. This method can effectively detect the inhibition of anti-TSLP antibodies on the activation of H_TSLP Reporter Cell Line cells by TSLP. Specific operation: the logarithmic growth period of H_TSLP Reporter Cell Line cells (GimBio, item number: GM-C15572) is washed once with 1640+1%FBS+1%PS and the cell density is adjusted to 2E6 / ml, inoculated into white 96-well cell culture plates (Corning, item number: 3917) at 50ul / well; incubate at 37°C, 5%CO2 for overnight. The next day, mix an appropriate amount of TSLP (Acro Biosystems, item number: TSP-H52Ha) with different concentrations of candidate anti-TSLP antibodies (starting concentration 4*25nM, 10-fold gradient dilution, 8 concentration points) in equal volume, and incubate at 37°C in the dark for 15 minutes. Add the TSLP and antibody mixture to the 96-well plate inoculated with H_TSLP Reporter Cell Line cells, and incubate at 37°C, 5%CO2 for 6 hours. Add Bright-Lumi TM II Firefly Luciferase Reporter Gene Detection Kit (Bi Yun Tian, item number: RG052M), 100ul / well; incubate at room temperature in the dark for 5-10 minutes. Use a multifunctional enzyme marker (MD, SpectraMax i3X) to detect LUM value. Calculate the sample inhibition efficiency with the formula: inhibition rate (%) = (RLU TSLP对照 -RLU 样品 ) / (RLU TSLP对照 -RLU cell control )*100%. Wherein: RLU 样品 represents the well added with TSLP, anti-TSLP antibody and cells; RLU cell control represents the well added with only cells; RLU TSLP对照 represents the well added with TSLP and cells. Take the final concentration of antibody as the abscissa and the inhibition rate (%) of the corresponding concentration of antibody as the ordinate to perform nonlinear fitting, and calculate the IC50 value. The results are as follows Figure 4A and Figure 4BResults: 4G7, 4C4 antibodies can effectively inhibit the activation of TSLP on H_TSLP Reporter Cell Line cells STATs and JAK2 signaling pathway, and the inhibition of 4C4 on the activation of the signaling pathway is 59 times that of Tezepelumab, and the inhibition 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 / IL7Ra cells

[0257] BaF3-TSLPR / IL7Ra cells are a stable cell line with high expression of TSLP receptor and depend on mIL3 for proliferation. When mIL3 is not added, TSLP can effectively promote the proliferation of BaF3-TSLPR / IL7Ra cells. This method can effectively evaluate the inhibition of anti-TSLP antibodies on the proliferation-promoting effect of TSLP on BaF3-TSLPR / IL7Ra cells. Specific operation: 4*0.625ng / ml TSLP (Acro Biosystems, Cat No: TSP-H52Ha) solution was prepared with 1640+10%FBS+1%PS analysis buffer, added to 96-well U-bottom plate, 50ul / well; the candidate antibodies were diluted to different concentrations (starting concentration 4*166.67ng / ml, 6-fold gradient dilution, 8 concentration points) with 1640+10%FBS+1%PS analysis buffer and added to the 96-well U-bottom plate with TSLP, 50ul / well, 37℃ incubated for 15 minutes; resuspend BaF3-TSLPR / IL7Ra with 1640+10%FBS+1%PS analysis buffer and adjust the density to 5E4 / ml, add to the above 96-well U-bottom plate, 100ul / well; 37℃ incubate for 72 hours; transfer the incubated cells to a new white 96-well plate, 100ul / well; use CellCounting-Lite2.0 Luminescent Cell Viability Assay (Novozyme, Cat No: DD1101-02) to detect, 100ul / well, room temperature, avoid light, incubate for 5-10 minutes, read LUM value on multifunctional enzyme label instrument. Calculate the inhibition efficiency of the sample with the formula: inhibition rate (%) = (RLU TSLP对照 -RLU 样品 ) / (RLU TSLP对照 -RLU cell control )*100%. Wherein: RLU 样品 represents the well with TSLP, anti-TSLP antibody, and cells; RLU cell control represents the well with only cells; RLU TSLP对照The wells added with TSLP and cells. The IC50 value was calculated by taking the final concentration of antibody as the abscissa and the inhibition rate (%) of the corresponding concentration of antibody as the ordinate for non-linear fitting. The results are as follows Figure 5A and Figure 5B It is shown that 4G7, 4C4 antibody and Tezepelumab can effectively inhibit the proliferation promoting effect of TSLP on BaF3-TSLPR / IL7Ra cells, and the inhibition activity of 4C4 is 11.3 times that of Tezepelumab, and the inhibition activity of 4G7 is 6.5 times that of Tezepelumab.

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

[0259] Experimental principle: Through BLI technology, the biotinylated first antibody is captured by SA probe, combined with antigen, and then combined with the second antibody for epitope grouping.

[0260] Experimental steps: (1) SA probe combined with biotinylated first antibody Tezepelumab; (2) combined with antigen human TSLP; (3) combined with solvent (blank control) or non-biotinylated second antibody 4C4 or 4G7, respectively.

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

[0262] Table 1: Second antibody binding signal value

[0263]

[0264] Example 9: Humanization of candidate antibodies

[0265] Antibody humanization: the light chain and heavy chain variable region genes of 4G7 were homology aligned with human IgG germline sequences, IGHV3-66*01 / IGHJ5*01 was selected as the heavy chain CDR grafting template, and the CDR regions (i.e. HCDR1, HCDR2 and HCDR3) of the heavy chain of 4G7 were grafted into the framework region of IGHV3-66*01 / IGHJ5*01; IGKV1-6*01 / IGKJ4*01 was selected as the light chain CDR grafting template, and the CDR regions (i.e. LCDR1, LCDR2 and LCDR3) of the light chain of 4G7 were grafted into the framework region of IGKV1-6*01 / IGKJ4*01; back mutations were made at specific sites of the framework region to obtain the 4G7 V60 humanized antibody variable region. The sequence of the humanized heavy chain variable region is shown as SEQ ID NO: 25; the amino acid sequence of the humanized light chain variable region is shown as SEQ ID NO: 26. The heavy chain and light chain genes of 4G7 V60 humanized antibody were synthesized and cloned into the expression vector pCDNA3.4. The humanized antibody heavy chain expression plasmid and light chain expression plasmid were co-transfected into CHO-S cells. After 4-8 days after transfection, the culture supernatant was harvested and subjected to one-step purification with Protein A to obtain the humanized antibody.

[0266] Example 9: Anti-human TSLP humanized antibody 4G7 V60 inhibits the effect of TSLP on activating H_TSLP Reporter CellLine cell STATs and JAK2 signaling pathway

[0267] H_TSLP Reporter Cell Line is a Luciferase reporter cell line based on Jak2-Stat signaling pathway. After TSLP binds to TSLPR, the dimer enhances the recruitment of IL-7Rα, forming an extracellular ternary complex, and Jak2 is activated. Jak2 further mediates the phosphorylation of Stats, which is transported into the nucleus, thereby activating the expression of Luciferase. This method can effectively detect the inhibition of anti-TSLP antibodies on the activation of H_TSLP Reporter Cell Line cells by TSLP. Specific operation: the logarithmic growth period of H_TSLP Reporter Cell Line cells (GimBio, item number: GM-C15572) is washed once with 1640+1%FBS+1%PS and the cell density is adjusted to 2E6 / ml, inoculated into white 96-well cell culture plates (Corning, item number: 3917) at 50ul / well; incubate at 37°C, 5%CO2 for overnight. The next day, mix an appropriate amount of TSLP (Acro Biosystems, item number: TSP-H52Ha) with different concentrations of anti-human TSLP humanized antibody 4G7 V60 (starting concentration 4*25nM, 10-fold gradient dilution, 8 concentration points) in equal volume, and incubate at 37°C for 15 minutes in the dark. Add the TSLP and antibody mixture to the 96-well plate inoculated with H_TSLP Reporter Cell Line cells, and incubate at 37°C, 5%CO2 for 6 hours. Add Bright-Lumi TM II Firefly Luciferase Reporter Gene Detection Kit (Bi Yun Tian, item number: RG052M), 100ul / well; incubate at room temperature for 5-10 minutes in the dark. Use a multifunctional enzyme marker (MD, SpectraMax i3X) to detect LUM value. Calculate the sample inhibition efficiency with the formula: inhibition rate (%)=(RLUTSLP control-RLU sample) / (RLUTSLP control-RLUcell control)*100%. Wherein: RLU sample represents the well added with TSLP, anti-human TSLP humanized antibody 4G7 V60 and cells; RLUcell control represents the well added with only cells; RLUTSLP control represents the well added with TSLP and cells. Take the final concentration of antibody as the abscissa and the inhibition rate (%) of the corresponding concentration of antibody as the ordinate to perform nonlinear fitting, and calculate the IC50 value. The results are as follows Figure 6Display: 4G7 V60 antibody can effectively inhibit the activation of TSLP on H_TSLP Reporter Cell Line cell STATs and JAK2 signal pathway, 4G7 V60 inhibits signal pathway activity significantly better than Tezepelumab, compared with the two, the activity of 4G7 V60 is 4743 times higher than Tezepelumab.

[0268] incorporated by reference

[0269] The entire contents of each patent and scientific document referred to herein is incorporated by reference herein for all purposes.

[0270] equivalents

[0271] The application can be embodied in other specific ways without departing from the spirit or essential characteristics thereof. Therefore, the above embodiments are merely illustrative, and not restrictive. The scope of the application should be determined by the appended claims and their equivalents, rather than by the description above, and all changes that come within the meaning and range of equivalents are intended to be embraced 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 shown in SEQ ID NO: 13, CDRH2 as shown in SEQ ID NO: 14, CDRH3 as shown in SEQ ID NO: 15; and CDRL1 as shown in SEQ ID NO: 16, CDRL2 as shown in SEQ ID NO: 17, and CDRL3 as shown 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 of claim 1, wherein the antibody comprises one of the following combinations: (1) a light chain variable region as shown in SEQ ID NO: 8 and a heavy chain variable region as shown in SEQ ID NO: 7; (2) a light chain variable region as shown in SEQ ID NO: 10 and a heavy chain variable region as shown in SEQ ID NO:

9.

3. The antibody or antigen-binding fragment thereof of the immediately preceding claim, 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 of 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) a light chain as shown in SEQ ID NO: 6 and a heavy chain as shown in SEQ ID NO:

5.

5. The antibody or antigen-binding fragment thereof of any of the preceding claims, wherein the fragment is a Fab fragment, a Fab' fragment, a F(ab')2 fragment, 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 of any one of claims 1-5.

7. A vector comprising the nucleic acid molecule of claim 6.

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

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

10. A multispecific molecule comprising the antibody or antigen-binding fragment thereof of any one of claims 1-5, the multispecific molecule further comprising at least one molecule having a second binding specificity for a second target.

11. A pharmaceutical composition or kit comprising the antibody or antigen-binding fragment thereof of any one of claims 1-5, or the nucleic acid molecule of claim 6, or the vector of claim 7, or the host cell of claim 8, or the conjugate of claim 9, or the multispecific molecule of claim 10; and a pharmaceutically acceptable carrier.

12. A method of making the antibody or antigen-binding fragment thereof of any one of claims 1-5, comprising the steps of: (i) expressing the antibody or antigen-binding fragment thereof of any one of claims 1-5 in the host cell of claim 8; (ii) isolating the antibody or antigen-binding fragment thereof from the host cell.

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