Antigen binding protein targeting TSLP and use

Abstract

Provided are an isolated antigen-binding protein targeting TSLP, a preparation method and a use thereof.

Description

Antigen binding proteins targeting TSLP and their applications Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to antigen-binding proteins targeting TSLP and their applications. Background Art

[0002] Thymic stromal lymphopoietin (TSLP) is an interleukin-7 (IL-7)-like cytokine. Mature hTSLP consists of 131 amino acid residues with a typical four-stranded α-helical bundle structure. It is primarily produced in epithelial cells, smooth muscle cells, keratinocytes, stromal cells, fibroblasts, mast cells (MC), monocytes / macrophages, granulocytes, and dendritic cells (DC). As an important allergic factor secreted by epithelial cells, TSLP induces Th2 responses through interactions with locally infiltrating DCs and T cells. TSLP can also shift the inflammatory response from Th1 to Th2 by inhibiting the Th1 pathway, playing a crucial role in coordinating and balancing the inflammatory response.

[0003] The TSLP receptor complex is a heterodimer composed of the TSLP receptor (TSLPR) and the IL-7 receptor α (IL-7Ra), both of which are highly expressed on myeloid dendritic cells. TSLP binds to TSLPR on the cell membrane and then to IL-7Rα, forming a stable TSLP-TSLPR-IL7Ra receptor complex. The intracellular domain of the TSLPR receptor in this complex recruits and activates JAK2, which, in conjunction with JAK1 recruited by IL7Rα, activates downstream signaling molecules. After TSLP binds to surface receptors on myeloid dendritic cells, dendritic cells secrete IL-8 and eotaxin-2 to recruit neutrophils and eosinophils, and TARC and MDC to recruit Th2 cells. Furthermore, TSLP-activated DCs induce CD4+ T cells to differentiate into Th2 cells. Th2 cells are capable of producing IL4, IL-5, IL-13, and TNF. These cytokines promote the production of IgE, eosinophils, and mucus, initiating allergic reactions and triggering diseases such as asthma and atopic dermatitis.

[0004] Currently, there are few studies on TSLP-related diseases, and there is an urgent need to obtain antibodies targeting TSLP. Summary of the Invention

[0005] The present application provides an isolated antigen-binding protein that targets TSLP and has the following advantages: (1) it can block the binding of TSLP to the cell surface TSLPR receptor, (2) it can bind to human and animal TSLP proteins, (3) it has good thermal stability, and (4) it can block the production of TARC.

[0006] The present application also provides nucleic acid molecules encoding the isolated antigen-binding proteins, expression vectors, host cells, and methods for preparing the isolated antigen-binding proteins. The isolated antigen-binding proteins described herein can be used to prevent, alleviate, and / or treat diseases and / or conditions.

[0007] In one aspect, the present application provides an isolated antigen-binding protein comprising HCDR3, wherein the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 3.

[0008] In certain embodiments, the isolated antigen-binding protein comprises HCDR2, the amino acid sequence of which is shown in SEQ ID NO: 2.

[0009] In certain embodiments, the isolated antigen-binding protein comprises HCDR1, and the amino acid sequence of the HCDR1 is shown in SEQ ID NO: 1.

[0010] In certain embodiments, the isolated antigen-binding protein comprises H-FR1, the C-terminus of H-FR1 is directly or indirectly linked to the N-terminus of HCDR1, and the amino acid sequence of H-FR1 is selected from SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9.

[0011] In certain embodiments, the isolated antigen-binding protein comprises H-FR2, wherein the H-FR2 is located between the HCDR1 and the HCDR2, and the amino acid sequence of the H-FR2 is selected from SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13.

[0012] In certain embodiments, the isolated antigen binding protein comprises H-FR3, wherein the H-FR3 is located between the HCDR2 and the HCDR3, and the amino acid sequence of the H-FR3 is selected from SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18.

[0013] In certain embodiments, the isolated antigen-binding protein comprises H-FR4, the N-terminus of the H-FR4 is connected to the C-terminus of the HCDR3, and the amino acid sequence of the H-FR4 is selected from SEQ ID NO: 19 and SEQ ID NO: 20.

[0014] In certain embodiments, the isolated antigen binding protein comprises a VH, the amino acid sequence of which is selected from SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, and SEQ ID NO:35.

[0015] In certain embodiments, the isolated antigen-binding protein comprises LCDR3, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:6.

[0016] In certain embodiments, the isolated antigen-binding protein comprises LCDR2, and the amino acid sequence of LCDR2 is shown in SEQ ID NO: 5 (GAR).

[0017] In certain embodiments, the isolated antigen-binding protein comprises LCDR1, and the amino acid sequence of LCDR1 is shown in SEQ ID NO:4.

[0018] In certain embodiments, the isolated antigen-binding protein comprises L-FR1, the C-terminus of the L-FR1 is directly or indirectly linked to the N-terminus of the LCDR1, and the amino acid sequence of the L-FR1 is selected from SEQ ID NO: 21 and SEQ ID NO: 22.

[0019] In certain embodiments, the isolated antigen-binding protein comprises L-FR2, wherein the L-FR2 is located between the LCDR1 and the LCDR2, and the amino acid sequence of the L-FR2 is selected from SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25.

[0020] In certain embodiments, the isolated antigen binding protein comprises L-FR3, wherein the L-FR3 is located between the LCDR2 and the LCDR3, and the amino acid sequence of the L-FR3 is selected from SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28 and SEQ ID NO:29.

[0021] In certain embodiments, the isolated antigen-binding protein comprises L-FR4, the N-terminus of the L-FR4 is linked to the C-terminus of the LCDR3, and the amino acid sequence of the L-FR4 may be SEQ ID NO: 30.

[0022] In certain embodiments, the isolated antigen binding protein comprises a VL, and the amino acid sequence of the VL is selected from SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:39.

[0023] In certain embodiments, the isolated antigen binding protein comprises any one group of VH and VL selected from the following:

[0024] 1) 1) VH: SEQ ID NO: 31, VL: SEQ ID NO: 36;

[0025] 2) VH: SEQ ID NO: 32, VL: SEQ ID NO: 37;

[0026] 3) VH: SEQ ID NO: 32, VL: SEQ ID NO: 38;

[0027] 4) VH: SEQ ID NO: 32, VL: SEQ ID NO: 39;

[0028] 5) VH: SEQ ID NO: 33, VL: SEQ ID NO: 37;

[0029] 6) VH: SEQ ID NO: 33, VL: SEQ ID NO: 38;

[0030] 7) VH: SEQ ID NO: 33, VL: SEQ ID NO: 39;

[0031] 8) VH: SEQ ID NO: 34, VL: SEQ ID NO: 37;

[0032] 9) VH: SEQ ID NO: 34, VL: SEQ ID NO: 38;

[0033] 10) VH: SEQ ID NO: 34, VL: SEQ ID NO: 39;

[0034] 11) VH: SEQ ID NO: 35, VL: SEQ ID NO: 37;

[0035] 12) VH: SEQ ID NO: 35, VL: SEQ ID NO: 38; and

[0036] 13) VH: SEQ ID NO: 35, VL: SEQ ID NO: 39.

[0037] In certain embodiments, the isolated antigen binding protein comprises an antibody heavy chain constant region.

[0038] In certain embodiments, the antibody heavy chain constant region is derived from a human IgG heavy chain constant region.

[0039] In certain embodiments, the antibody heavy chain constant region is derived from a human IgG1 heavy chain constant region.

[0040] In certain embodiments, the isolated antigen binding protein comprises an antibody light chain constant region.

[0041] In certain embodiments, the antibody light chain constant region is derived from a human Ig kappa constant region.

[0042] In certain embodiments, the isolated antigen binding protein comprises an antibody or an antigen binding fragment thereof.

[0043] In certain embodiments, the antigen-binding fragment comprises a Fab, a Fab', an Fv fragment, a F(ab')2, a F(ab)2, a scFv, a di-scFv and / or a dAb.

[0044] In certain embodiments, the antibody is selected from one or more of the following groups: a monoclonal antibody, a chimeric antibody, a humanized antibody, and a fully human antibody.

[0045] On the other hand, the present application also provides a chimeric antigen receptor, which comprises a targeting portion, wherein the targeting portion comprises the antigen binding protein described in the present application.

[0046] On the other hand, the present application also provides a polypeptide molecule comprising the isolated antigen-binding protein or the chimeric antigen receptor.

[0047] In certain embodiments, the polypeptide molecule comprises a fusion protein.

[0048] On the other hand, the present application also provides an immunoconjugate comprising the isolated antigen-binding protein.

[0049] On the other hand, the present application also provides one or more isolated nucleic acid molecules encoding the isolated antigen binding protein, the chimeric antigen receptor or the polypeptide molecule.

[0050] On the other hand, the present application also provides a vector comprising the nucleic acid molecule.

[0051] On the other hand, the present application also provides a cell comprising the isolated antigen-binding protein, the chimeric antigen receptor, the polypeptide molecule, the immunoconjugate, the nucleic acid molecule or the vector.

[0052] On the other hand, the present application also provides a pharmaceutical composition comprising the isolated antigen-binding protein, the chimeric antigen receptor, the polypeptide molecule, the immunoconjugate, the nucleic acid molecule, the vector, and / or the cell, and optionally a pharmaceutically acceptable carrier.

[0053] On the other hand, the present application also provides a method for preparing the isolated antigen-binding protein, which comprises culturing the cell under conditions allowing the antigen-binding protein to be expressed.

[0054] On the other hand, the present application also provides the use of the isolated antigen-binding protein, the chimeric antigen receptor, the polypeptide molecule, the immunoconjugate, the nucleic acid molecule, the vector, the cell and / or the pharmaceutical composition in the preparation of a medicament for preventing, alleviating and / or treating a disease and / or condition.

[0055] In certain embodiments, the disease and / or condition comprises a TSLP-associated disease.

[0056] In certain embodiments, the disease and / or condition comprises an inflammatory disease or a tumor.

[0057] In certain embodiments, the disease and / or condition comprises a TSLP-associated inflammatory disease or tumor.

[0058] On the other hand, the present application also provides a method for detecting TSLP in a sample, which comprises administering the isolated antigen-binding protein, the chimeric antigen receptor, the polypeptide molecule, the immunoconjugate, the nucleic acid molecule, the vector, the cell and / or the pharmaceutical composition.

[0059] On the other hand, the present application also provides a reagent or kit for detecting TSLP in a sample, which comprises the isolated antigen-binding protein, the chimeric antigen receptor, the polypeptide molecule, the immunoconjugate, the nucleic acid molecule, the vector, the cell and / or the pharmaceutical composition.

[0060] On the other hand, the present application also provides the use of the isolated antigen-binding protein, the chimeric antigen receptor, the polypeptide molecule, the immunoconjugate, the nucleic acid molecule, the vector, the cell and / or the pharmaceutical composition in preparing a kit for detecting the presence and / or content of TSLP in a sample.

[0061] Those skilled in the art can easily discern other aspects and advantages of the present application from the detailed description below. In the detailed description below, only exemplary embodiments of the present application are shown and described. As will be appreciated by those skilled in the art, the content of this application enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application relates. Accordingly, the descriptions in the drawings and specification of this application are merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] The specific features of the invention involved in this application are shown in the appended claims. The features and advantages of the invention involved in this application can be better understood by referring to the exemplary embodiments described in detail below and the accompanying drawings. A brief description of the drawings is as follows:

[0063] FIG1 shows the binding activity curve and EC50 value of 900792 and related humanized proteins described in this application with TSLP on the cell membrane surface.

[0064] FIG2 shows the curves and related IC50 values ​​of 900792 and related humanized proteins described in this application blocking the binding of TSLP to cell surface receptors.

[0065] FIG3 shows the functional activity curves and related IC50 values ​​of 900792 and related humanized proteins described in this application for inhibiting the binding of TSLP to cell surface receptors.

[0066] FIG4 shows the ELISA test results of the 900792 humanized protein described in this application for inhibiting TARC activity.

[0067] FIG5 shows the ELISA test results of the 900792 humanized protein described in this application for inhibiting TARC activity. DETAILED DESCRIPTION

[0068] The following describes the implementation of the present invention through specific embodiments. People familiar with this technology can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0069] Definition of terms

[0070] As used herein, the term "TSLP," also known as "thymic stromal lymphopoietin," is an interleukin-7 (IL-7)-like cytokine. Mature hTSLP consists of 131 amino acid residues and exhibits a typical four-stranded α-helical bundle structure. The term "TSLP" encompasses "full-length," unprocessed TSLP, as well as any form of TSLP produced by cellular processing. As used herein, the term "TSLP" encompasses full-length, wild-type TSLP, as well as its mutants, fragments, variants, isoforms, and homologs.

[0071] In this application, the term "isolated" generally refers to a substance obtained artificially from its natural state. If a substance or component is "isolated" in nature, it may be that its natural environment has been altered, or that the substance has been separated from its natural environment, or both. For example, a polynucleotide or polypeptide that is naturally present in a living animal and has not been separated is considered isolated. The term "isolated" does not exclude the presence of artificial or synthetic substances, nor does it exclude the presence of other impure substances that do not affect the activity of the substance.

[0072] In the present application, the term "isolated antigen-binding protein" generally refers to a protein with antigen-binding ability obtained by artificial means from a natural state. The "isolated antigen-binding protein" may comprise a portion that binds to an antigen and, optionally, a framework or framework portion that allows the antigen-binding portion to adopt a conformation that promotes the binding of the antigen-binding portion to the antigen. The antigen-binding protein may comprise, for example, a protein framework region (FR) derived from an antibody or an alternative protein framework region or an artificial framework region having a transplanted CDR or CDR derivative. Such frameworks include, but are not limited to, antibody-derived framework regions comprising, for example, mutations introduced to stabilize the three-dimensional structure of the antigen-binding protein and fully synthetic framework regions comprising, for example, biocompatible polymers. Examples of antigen binding proteins include, but are not limited to, human antibodies, humanized antibodies; chimeric antibodies; recombinant antibodies; single-chain antibodies; bifunctional antibodies; trifunctional antibodies; tetrafunctional antibodies; Fab, Fab', Fv fragment, F(ab')2, F(ab)2, scFv, di-scFv, dAb, IgD antibody; IgE antibody; IgM antibody; IgG1 antibody; IgG2 antibody; IgG3 antibody; or IgG4 antibody and fragments thereof.

[0073] In this application, the term "CDR", also known as "complementarity determining region", generally refers to a region in an antibody variable domain whose sequence is highly variable and / or forms a structure-defining loop. Typically, an antibody comprises six CDRs; three in VH (HCDR1, HCDR2, HCDR3), and three in VL (LCDR1, LCDR2, LCDR3). In certain embodiments, naturally occurring camel antibodies consisting only of heavy chains can function normally and stably in the absence of light chains. Antibody CDRs can be determined by a variety of coding systems, such as CCG, Kabat, Chothia, IMGT, Kabat / Chothia, etc. These coding systems are known in the art. For example, the amino acid sequence numbering of the antigen-binding protein can be divided according to the IMGT numbering system. For example, the CDRs of the antigen-binding protein can be divided according to the Kabat numbering system.

[0074] In this application, the term "FR" generally refers to the more highly conserved portion of an antibody variable domain, which is referred to as a framework region. Typically, the variable domains of natural heavy and light chains each comprise four FR regions, i.e., four in VH (H-FR1, H-FR2, H-FR3, and H-FR4), and four in VL (L-FR1, L-FR2, L-FR3, and L-FR4).

[0075] In this application, the terms "variable domain" and "variable region" are used interchangeably and generally refer to a portion of an antibody heavy chain and / or light chain. The variable domains of the heavy and light chains may be referred to as "V H ” and “V L ” (or “VH” and “VL”, respectively). These domains are generally the most variable parts of an antibody (relative to other antibodies of the same type) and contain the antigen-binding site.

[0076] In the present application, the term "chimeric antigen receptor" (CAR) generally refers to a recombinant polypeptide comprising at least an extracellular domain, a transmembrane region, and an intracellular domain that specifically binds to an antigen or target. For example, a hinge region is included between the extracellular domain and the transmembrane region. For example, the chimeric antigen receptor may include a signal peptide. The binding of the extracellular domain of CAR to the target antigen on the surface of the target cell causes CAR clustering and transmits the activation stimulus to the CAR-containing cell. CAR redirects the specificity of the immune effector cell and triggers proliferation, cytokine production, phagocytosis and / or production of molecules that can mediate cell death of cells expressing the target antigen in a manner independent of major histocompatibility (MHC). For example, the extracellular structure may include the above-mentioned antigen binding protein. For example, the extracellular structure may specifically bind to TSLP

[0077] Throughout this application, the terms "polypeptide molecule," "polypeptide," and "peptide" are used interchangeably and generally refer to a polymer of amino acid residues. The term "fusion protein" generally refers to a polypeptide having at least two covalently linked moieties. Each moiety can be a polypeptide with a distinct property. This property can be a biological property, such as in vitro or in vivo activity. It can also be a simple chemical or physical property, such as binding to a target molecule or catalysis of a reaction. The two moieties can be directly linked by a single peptide bond or through a peptide linker.

[0078] In this application, the term "nucleic acid molecule" generally refers to nucleotides of any length in isolated form, either deoxyribonucleotides or ribonucleotides, or analogs thereof, isolated from their natural environment or artificially synthesized.

[0079] In this application, the term "vector" generally refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a protein can be inserted and the protein can be expressed. A vector can be used to transform, transduce, or transfect a host cell, allowing the genetic material elements it carries to be expressed in the host cell. For example, vectors may include: plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages such as lambda phage or M13 phage, and animal viruses. Types of animal viruses used as vectors may include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, a vector may also contain a replication initiation site. Vectors may also include components that assist in their entry into cells, such as viral particles, liposomes, or protein coats, but are not limited to these substances.

[0080] In this application, the term "cell" generally refers to a single cell, cell line or cell culture that may be or has been a recipient of a subject's plasmid or vector, including nucleic acid molecules of the present invention or vectors of the present invention. Cells may include the offspring of a single cell. Due to natural, accidental or intentional mutations, offspring may not necessarily be identical to the original mother cell (in the form of total DNA complement or in the genome). Cells may include cells transfected in vitro with the vectors described herein. Cells may be bacterial cells (e.g., Escherichia coli), yeast cells or other eukaryotic cells, such as COS cells, Chinese hamster ovary (CHO) cells, CHO-K1 cells, LNCAP cells, HeLa cells, HEK293 cells, COS-1 cells, NS0 cells. In certain embodiments, cells are mammalian cells. In certain embodiments, mammalian cells are HEK293 cells.

[0081] In the present application, the term "immunoconjugate" generally refers to a conjugate formed by conjugating (e.g., covalently linked via a linker molecule) other agents (e.g., chemotherapeutic agents, radioactive elements, cytostatic agents, and cytotoxic agents) to the antibody or its antigen-binding fragment, which can deliver the other agents to target cells (e.g., tumor cells) through the specific binding of the antibody or its antigen-binding fragment to antigens on the target cells.

[0082] In this application, the term "pharmaceutical composition" generally refers to a composition for preventing / treating a disease or condition. The pharmaceutical composition may comprise the isolated antigen-binding protein described herein, the nucleic acid molecule described herein, the carrier described herein and / or the cell described herein, and optionally a pharmaceutically acceptable adjuvant. In addition, the pharmaceutical composition may also comprise a suitable formulation of one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and / or preservatives. The acceptable ingredients of the composition are preferably non-toxic to the recipient at the dosage and concentration used. The pharmaceutical composition of the present invention includes, but is not limited to, liquid, frozen and lyophilized compositions.

[0083] In this application, the term "pharmaceutically acceptable carrier" generally includes pharmaceutically acceptable carriers, excipients or stabilizers that are non-toxic to cells or mammals exposed thereto at the dosages and concentrations employed. Physiologically acceptable carriers may include, for example, buffers, antioxidants, low molecular weight (less than about 10 residues) polypeptides, proteins, hydrophilic polymers, amino acids, monosaccharides, disaccharides and other carbohydrates, chelating agents, sugar alcohols, salt-forming counterions, such as sodium; and / or nonionic surfactants.

[0084] In this application, the term "subject" generally refers to a human or non-human animal, including but not limited to a cat, dog, horse, pig, cow, sheep, rabbit, mouse, rat, or monkey.

[0085] In the present application, the proteins, polypeptides and / or amino acid sequences involved should also be understood to include at least the following scope: variants or homologs that have the same or similar functions as the proteins or polypeptides.

[0086] In the present application, the variant may be, for example, a protein or polypeptide in which one or more amino acids are substituted, deleted, or added in the amino acid sequence of the protein and / or polypeptide (e.g., an antibody or fragment thereof that specifically binds to a TSLP protein). For example, the functional variant may comprise a protein or polypeptide having an amino acid change by at least 1, for example, 1-30, 1-20, or 1-10, or for example, 1, 2, 3, 4, or 5 amino acid substitutions, deletions, and / or insertions. The functional variant may substantially retain the biological properties of the protein or polypeptide prior to the change (e.g., substitution, deletion, or addition). For example, the functional variant may retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (e.g., antigen binding ability) of the protein or polypeptide prior to the change. For example, the substitution may be a conservative substitution.

[0087] In the present application, the homolog can be a protein or polypeptide having at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology with the amino acid sequence of the protein and / or the polypeptide (e.g., an antibody or fragment thereof that specifically binds to a TSLP protein).

[0088] In the present application, described homology generally refers to the similarity, similarity or association between two or more sequences.Can calculate " sequence homology percentage ratio " in the following manner: two sequences to be compared are compared in a comparison window, determine that there is identical nucleic acid base (for example, A, T, C, G, I) or identical amino acid residue (for example, Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) number in the position to obtain the number of matching positions, the number of matching positions is divided by the total number of positions (that is, window size) in the comparison window, and the result is multiplied by 100, to produce the sequence homology percentage ratio.Comparison carried out in order to determine the sequence homology percentage ratio can be realized by various ways known in the art.

[0089] In this application, the term "include" generally means to include, encompass, contain or encompass. In some cases, it also means "to be", "to be composed of..."

[0090] In this application, the term "about" generally refers to a variation within a range of 0.5%-10% above or below the specified value, for example, a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified value.

[0091] Detailed Description of the Invention

[0092] Isolated antigen binding protein

[0093] The CDR of an antibody, also known as the complementarity determining region, is part of the variable region. The amino acid residues in this region can contact the antigen or antigenic epitope. Antibody CDRs can be determined using a variety of coding systems, such as CCG, Kabat, Chothia, IMGT, and a combination of Kabat / Chothia. These coding systems are known in the art, and for details, see, for example, http: / / www.bioinf.org.uk / abs / index.html#kabatnum. Those skilled in the art can use different coding systems to determine the CDR region based on the sequence and structure of the antibody. Using different coding systems, there may be differences in the CDR region. In this application, the CDR covers CDR sequences obtained by any CDR division method; it also covers variants thereof, wherein the variant includes the amino acid sequence of the CDR being substituted, deleted, and / or having one or more amino acids added. For example, 1-30, 1-20 or 1-10, and for example 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid substitutions, deletions and / or insertions; homologs thereof are also encompassed, and the homologs can be amino acid sequences having at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology to the amino acid sequence of the CDR. In certain embodiments, the CDRs are identified by the IMGT numbering scheme.

[0094] In one aspect, the present application provides an isolated antigen-binding protein comprising HCDR3, wherein the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 3.

[0095] In the present application, the isolated antigen-binding protein may further comprise HCDR2, the amino acid sequence of which is shown in SEQ ID NO: 2.

[0096] In the present application, the isolated antigen-binding protein may further comprise HCDR1, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0097] In the present application, the isolated antigen-binding protein may comprise HCDR3, HCDR2 and HCDR1. For example, the amino acid sequence of HCDR3 of the isolated antigen-binding protein is shown in SEQ ID NO: 3, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 2, and the amino acid sequence of HCDR1 is shown in SEQ ID NO: 1.

[0098] In the present application, the isolated antigen-binding protein may comprise LCDR3, the amino acid sequence of which is shown in SEQ ID NO: 6.

[0099] In the present application, the isolated antigen-binding protein may further comprise LCDR2, the amino acid sequence of which is shown in SEQ ID NO: 5 (GAR).

[0100] In the present application, the isolated antigen-binding protein may further comprise LCDR1, the amino acid sequence of which is shown in SEQ ID NO:4.

[0101] In the present application, the isolated antigen-binding protein may comprise LCDR3, LCDR2, and LCDR1. For example, the amino acid sequence of LCDR3 of the isolated antigen-binding protein is shown in SEQ ID NO:6, the amino acid sequence of LCDR2 is shown in SEQ ID NO:5, and the amino acid sequence of LCDR1 is shown in SEQ ID NO:4.

[0102] In the present application, the isolated antigen-binding protein may comprise HCDR3, HCDR2, HCDR1, LCDR3, LCDR2 and LCDR1. For example, the amino acid sequence of HCDR3 of the isolated antigen-binding protein described in the present application is shown in SEQ ID NO: 3, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 2, the amino acid sequence of HCDR1 is shown in SEQ ID NO: 1, the amino acid sequence of LCDR3 is shown in SEQ ID NO: 6, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 5 (GAR), and the amino acid sequence of LCDR1 is shown in SEQ ID NO: 4.

[0103] In the present application, the isolated antigen-binding protein may comprise H-FR1, the C-terminus of the H-FR1 may be directly or indirectly connected to the N-terminus of the HCDR1, and the amino acid sequence of the H-FR1 may be as shown in SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9.

[0104] In the present application, the isolated antigen-binding protein may comprise H-FR2, the H-FR2 may be located between the HCDR1 and the HCDR2, and the amino acid sequence of the H-FR2 may be as shown in SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13.

[0105] In the present application, the isolated antigen-binding protein may comprise H-FR3, the H-FR3 may be located between the HCDR2 and the HCDR3, and the amino acid sequence of the H-FR3 may be as shown in SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18.

[0106] In the present application, the isolated antigen-binding protein may comprise H-FR4, the N-terminus of the H-FR4 may be connected to the C-terminus of the HCDR3, and the amino acid sequence of the H-FR4 may be as shown in SEQ ID NO: 19 and SEQ ID NO: 20.

[0107] In the present application, the antigen binding protein may comprise H-FR1, H-FR2, H-FR3 and H-FR4.

[0108] For example, H-FR1, H-FR2, H-FR3 and H-FR4 of the isolated antigen-binding protein may respectively comprise the amino acid sequences shown in H-FR1: SEQ ID NO: 7, H-FR2: SEQ ID NO: 10, H-FR3: SEQ ID NO: 14, and H-FR4: SEQ ID NO: 19.

[0109] For example, H-FR1, H-FR2, H-FR3 and H-FR4 of the isolated antigen-binding protein may respectively comprise the amino acid sequences shown in H-FR1: SEQ ID NO: 8, H-FR2: SEQ ID NO: 11, H-FR3: SEQ ID NO: 15, and H-FR4: SEQ ID NO: 20.

[0110] For example, H-FR1, H-FR2, H-FR3 and H-FR4 of the isolated antigen-binding protein may respectively comprise the amino acid sequences shown in H-FR1: SEQ ID NO: 9, H-FR2: SEQ ID NO: 12, H-FR3: SEQ ID NO: 16, and H-FR4: SEQ ID NO: 20.

[0111] For example, H-FR1, H-FR2, H-FR3 and H-FR4 of the isolated antigen-binding protein may respectively comprise H-FR1: SEQ ID NO: 8, H-FR2: SEQ ID NO: 13, H-FR3: SEQ ID NO: 17, H-FR4: SEQ ID NO: 20.

[0112] For example, H-FR1, H-FR2, H-FR3 and H-FR4 of the isolated antigen-binding protein may respectively comprise H-FR1: SEQ ID NO: 9, H-FR2: SEQ ID NO: 12, H-FR3: SEQ ID NO: 18, H-FR4: SEQ ID NO: 20.

[0113] In the present application, the isolated antigen-binding protein may comprise L-FR1, the C-terminus of the L-FR1 may be directly or indirectly connected to the N-terminus of the LCDR1, and the amino acid sequence of the L-FR1 may be as shown in SEQ ID NO: 21 and SEQ ID NO: 22.

[0114] In the present application, the isolated antigen-binding protein may comprise L-FR2, which may be located between the LCDR1 and the LCDR2. The amino acid sequence of the L-FR2 may be as shown in SEQ ID NO: 23, SEQ ID NO: 24 and SEQ ID NO: 25.

[0115] In the present application, the isolated antigen-binding protein may comprise L-FR3, the L-FR3 may be located between the LCDR2 and the LCDR3, and the amino acid sequence of the L-FR3 may be as shown in SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28 and SEQ ID NO:29.

[0116] In the present application, the isolated antigen-binding protein may comprise L-FR4, the N-terminus of the L-FR4 may be connected to the C-terminus of the LCDR3, and the amino acid sequence of the L-FR4 may be as shown in SEQ ID NO: 30.

[0117] In the present application, the antigen binding protein may comprise L-FR1, L-FR2, L-FR3 and L-FR4.

[0118] For example, L-FR1, L-FR2, L-FR3 and L-FR4 of the isolated antigen-binding protein may respectively comprise the amino acid sequences shown in L-FR1: SEQ ID NO: 21, L-FR2: SEQ ID NO: 23, L-FR3: SEQ ID NO: 26, and L-FR4: SEQ ID NO: 30.

[0119] For example, L-FR1, L-FR2, L-FR3 and L-FR4 of the isolated antigen-binding protein may respectively comprise the amino acid sequences shown in L-FR1: SEQ ID NO: 22, L-FR2: SEQ ID NO: 24, L-FR3: SEQ ID NO: 27, and L-FR4: SEQ ID NO: 30.

[0120] For example, L-FR1, L-FR2, L-FR3 and L-FR4 of the isolated antigen-binding protein may respectively comprise the amino acid sequences shown in L-FR1: SEQ ID NO: 22, L-FR2: SEQ ID NO: 24, L-FR3: SEQ ID NO: 28, and L-FR4: SEQ ID NO: 30.

[0121] For example, L-FR1, L-FR2, L-FR3 and L-FR4 of the isolated antigen-binding protein may respectively comprise L-FR1: SEQ ID NO: 22, L-FR2: SEQ ID NO: 25, L-FR3: SEQ ID NO: 29, L-FR4: SEQ ID NO: 30.

[0122] In the present application, the isolated antigen-binding protein may comprise VH, and the VH may comprise the amino acid sequences shown in SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34 and SEQ ID NO:35.

[0123] In the present application, the isolated antigen-binding protein may comprise a VL, and the VL may comprise the amino acid sequence shown in SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38 and SEQ ID NO:39.

[0124] In the present application, the isolated antigen-binding protein may comprise the VH and VL.

[0125] For example, the VH may comprise the amino acid sequence shown in SEQ ID NO:31, and the VL may comprise the amino acid sequence shown in SEQ ID NO:36.

[0126] For example, the VH may comprise the amino acid sequence shown in SEQ ID NO:32, and the VL may comprise the amino acid sequence shown in SEQ ID NO:37.

[0127] For example, the VH may comprise the amino acid sequence shown in SEQ ID NO:32, and the VL may comprise the amino acid sequence shown in SEQ ID NO:38.

[0128] For example, the VH may comprise the amino acid sequence shown in SEQ ID NO:32, and the VL may comprise the amino acid sequence shown in SEQ ID NO:39.

[0129] For example, the VH may comprise the amino acid sequence shown in SEQ ID NO: 33, and the VL may comprise the amino acid sequence shown in SEQ ID NO: 37.

[0130] For example, the VH may comprise the amino acid sequence shown in SEQ ID NO:33, and the VL may comprise the amino acid sequence shown in SEQ ID NO:38.

[0131] For example, the VH may comprise the amino acid sequence shown in SEQ ID NO:33, and the VL may comprise the amino acid sequence shown in SEQ ID NO:39.

[0132] For example, the VH may comprise the amino acid sequence shown in SEQ ID NO: 34, and the VL may comprise the amino acid sequence shown in SEQ ID NO: 37.

[0133] For example, the VH may comprise the amino acid sequence shown in SEQ ID NO:34, and the VL may comprise the amino acid sequence shown in SEQ ID NO:38.

[0134] For example, the VH may comprise the amino acid sequence shown in SEQ ID NO:34, and the VL may comprise the amino acid sequence shown in SEQ ID NO:39.

[0135] For example, the VH may comprise the amino acid sequence shown in SEQ ID NO: 35, and the VL may comprise the amino acid sequence shown in SEQ ID NO: 37.

[0136] For example, the VH may comprise the amino acid sequence shown in SEQ ID NO: 35, and the VL may comprise the amino acid sequence shown in SEQ ID NO: 38.

[0137] For example, the VH may comprise the amino acid sequence shown in SEQ ID NO:35, and the VL may comprise the amino acid sequence shown in SEQ ID NO:39.

[0138] In the present application, the isolated antigen-binding protein may comprise at least one CDR of the VH described herein. In the present application, the isolated antigen-binding protein may comprise at least one CDR of the VL described herein. The CDRs may be obtained by dividing according to any division method.

[0139] In the present application, the isolated antigen-binding protein may comprise HCDR1, HCDR2 and HCDR3 of the VH described herein. The VH may comprise the amino acid sequence shown in any one of SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34 and SEQ ID NO:35.

[0140] In the present application, the isolated antigen-binding protein may comprise LCDR1, LCDR2 and LCDR3 in the VL described herein. The VL may comprise the amino acid sequence shown in any one of SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39.

[0141] In the present application, the isolated antigen-binding protein may include an antibody heavy chain constant region. The antibody heavy chain constant region may be derived from a human IgG heavy chain constant region. In certain embodiments, the isolated antigen-binding protein may include an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from a human IgG1 heavy chain constant region.

[0142] In the present application, the isolated antigen-binding protein may include an antibody light chain constant region. The antibody light chain constant region may be derived from a human Igκ constant region.

[0143] In the present application, the isolated antigen-binding protein may comprise an antibody or an antigen-binding fragment thereof.

[0144] In certain embodiments, the antigen-binding fragment may include a Fab, a Fab', an Fv fragment, a F(ab')2, a F(ab)2, a scFv, a di-scFv and / or a dAb.

[0145] In certain embodiments, the antibody may include a monoclonal antibody, a chimeric antibody, a humanized antibody, and / or a fully human antibody.

[0146] In addition, it should be noted that the isolated antigen-binding proteins described herein may include heavy chain and / or light chain sequences that have one or more conservative sequence modifications thereto. The so-called "conservative sequence modifications" refer to amino acid modifications that do not significantly affect or change the binding properties of the antibody. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the isolated antigen-binding proteins described herein by standard techniques known in the art, such as point mutations and PCR-mediated mutations. Conservative amino acid substitutions are the replacement of amino acid residues with amino acid residues having similar side chains. Groups of amino acid residues with similar side chains are known in the art. These amino acid residue groups include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar 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). In certain embodiments, one or more amino acid residues in the CDR region of the isolated antigen-binding protein described herein can be replaced with other amino acid residues from the same side chain group. Those skilled in the art will appreciate that some conservative sequence modifications will not eliminate antigen binding.

[0147] Chimeric antigen receptors, polypeptide molecules, nucleic acid molecules, vectors, cells, immunoconjugates and pharmaceutical compositions

[0148] On the other hand, the present application also provides a chimeric antigen receptor (CAR), which may comprise a targeting portion that binds to the TSLP protein.

[0149] In another aspect, the present application provides a polypeptide molecule, which may comprise the isolated antigen-binding protein described herein.

[0150] In certain embodiments, the polypeptide molecule may comprise a fusion protein. In certain embodiments, the polypeptide molecule may be a fusion protein.

[0151] In another aspect, the present application provides an isolated nucleic acid molecule that can encode an isolated antigen-binding protein described herein. For example, the nucleic acid molecule can be produced or synthesized by: (1) in vitro amplification, such as by polymerase chain reaction (PCR); (2) cloning and recombination; (3) purification, such as by enzymatic digestion and gel electrophoresis fractionation; or (4) synthesis, such as by chemical synthesis.

[0152] On the other hand, the present application provides a vector that can contain the nucleic acid molecules described herein. In addition, the vector can also contain other genes, such as marker genes that allow the vector to be selected in appropriate host cells and under appropriate conditions. In addition, the vector can also contain expression control elements that allow the coding region to be correctly expressed in an appropriate host. Such control elements are well known to those skilled in the art and can include, for example, promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation. The vector can be transformed, transduced, or transfected into host cells so that the genetic material elements it carries are expressed in the host cells. The vector can include, for example, a plasmid, a cosmid, a virus, a bacteriophage, or other vectors commonly used in, for example, genetic engineering. For example, the vector is an expression vector. In addition, the vector can also include components that assist its entry into cells, such as viral particles, liposomes, or protein coats, but is not limited to these substances.

[0153] On the other hand, the application provides a kind of cell, it can comprise the nucleic acid molecule described in the application or the vector described in the application.In certain embodiments, each kind or each host cell can comprise one or a kind of nucleic acid molecule or vector described in the application.In certain embodiments, each kind or each host cell can comprise multiple (for example, 2 or more) or multiple (for example, 2 or more) nucleic acid molecules or vector described in the application.For example, the vector described in the application can be introduced into the host cell, for example, eukaryotic cell, such as cell, fungus or yeast cell from plant etc.In certain embodiments, the cell can be bacterial cell (for example, Escherichia coli), yeast cell or other eukaryotic cell, for example COS cell, Chinese hamster ovary (CHO) cell, CHO-K1 cell, LNCAP cell, HeLa cell, 293T cell, COS-1 cell, SP2 / 0 cell, NS0 cell or myeloma cell.The vector described in the application can be introduced into the host cell by methods known in the art, for example, electroporation, lipofectine transfection, lipofectamin transfection etc.

[0154] On the other hand, the present application also provides an immunoconjugate, which may comprise the isolated antigen-binding protein described in the present application.

[0155] On the other hand, the present application also provides a pharmaceutical composition, which may comprise the isolated antigen-binding protein described herein, the polypeptide molecule described herein, the immunoconjugate described herein, the nucleic acid molecule described herein, the vector described herein and / or the cell described herein, and optionally a pharmaceutically acceptable carrier.

[0156] In certain embodiments, the pharmaceutical composition may also include one or more (pharmaceutically effective) adjuvants, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and / or suitable formulations of preservatives. The acceptable ingredients of the composition are preferably non-toxic to the recipient at the dosage and concentration used. The pharmaceutical composition of the present invention includes but is not limited to liquid, frozen and lyophilized compositions.

[0157] In certain embodiments, the pharmaceutical composition may be a bispecific or multispecific molecule comprising the isolated antigen-binding protein described herein. In certain embodiments, in addition to the molecule that binds to TSLP, the other molecules in the bispecific or multispecific molecule may bind to targets unrelated to TSLP.

[0158] In certain embodiments, the pharmaceutical composition may also contain more than one active compound, generally those having complementary activities that do not adversely affect each other. The type and effective amount of such drugs may depend, for example, on the amount and type of antagonist present in the formulation, and the clinical parameters of the subject.

[0159] In certain embodiments, the pharmaceutically acceptable carrier can include any and all solvents, dispersion media, coatings, isotonic agents, and absorption delaying agents that are compatible with pharmaceutical administration and are generally safe and non-toxic.

[0160] Preparation method

[0161] In another aspect, the present application provides a method for preparing the antigen-binding protein. The method may include culturing the host cell described herein under conditions that allow for expression of the antigen-binding protein. For example, the method may be performed using an appropriate culture medium, at an appropriate temperature and for an appropriate time, as is known to those skilled in the art.

[0162] Any method suitable for producing monoclonal antibodies can be used to produce the antigen-binding proteins of the present application. For example, animals can be immunized with linked or naturally occurring TSLP or fragments thereof. Suitable immunization methods can be used, including adjuvants, immunostimulants, and repeated booster immunizations, and one or more routes can be used. For example, hybridoma preparation methods can be used to obtain spleen cells from immunized mice and fuse them with SP2 / 0 myeloma cells, and hybridoma cell lines can be identified through HAT screening.

[0163] Any suitable form of TSLP can be used as an immunogen (antigen) to generate non-human antibodies specific for TSLP and screen for biological activity. For example, the stimulating immunogen can be full-length mature human TSLP, including its native homodimer, or a peptide containing a single or multiple epitopes. The immunogen can be used alone or in combination with one or more immunogenicity enhancers known in the art.

[0164] Chimeric human antibodies can be selected from any class of immunoglobulins, including IgM, IgD, IgG, IgA, and IgE. In the present application, the antibody can be an IgG antibody, and the IgG1 subtype can be used. The optimization of the necessary constant domain sequence can be achieved by screening the antibody using the biological assays described in the examples below to produce the desired biological activity. Similarly, any class of light chain can be used in the compounds and methods of the present application. For example, a kappa chain or a variant thereof can be used in the compounds and methods of the present application.

[0165] Methods and uses

[0166] On the other hand, the present application provides the use of the isolated antigen-binding protein, the chimeric antigen receptor, the polypeptide molecule, the nucleic acid molecule, the vector, the cell, the immunoconjugate and / or the pharmaceutical composition in the preparation of a medicament for preventing and / or treating a disease and / or condition.

[0167] On the other hand, the present application also provides a method for preventing and / or treating a disease and / or condition, which may comprise administering the isolated antigen-binding protein, the chimeric antigen receptor, the polypeptide molecule, the nucleic acid molecule, the vector, the cell, the immunoconjugate and / or the pharmaceutical composition described herein to a subject in need thereof.

[0168] On the other hand, the isolated antigen-binding protein, the polypeptide molecule, the nucleic acid molecule, the vector, the cell, the immunoconjugate and / or the pharmaceutical composition described herein can be used to prevent and / or treat diseases and / or disorders.

[0169] In the present application, the disease and / or disorder may be a TSLP-related disease and / or disorder.

[0170] In the present application, the disease and / or disorder may be an inflammatory disease or a tumor.

[0171] In the present application, the disease and / or may be a TSLP-related inflammatory disease or tumor.

[0172] On the other hand, the present application also provides a method for detecting TSLP in a sample, which comprises administering the isolated antigen-binding protein, the polypeptide molecule, the nucleic acid molecule, the vector, the cell, the immunoconjugate and / or the pharmaceutical composition.

[0173] In some cases, the method for detecting TSLP in a sample is an in vitro method. In some cases, the method for detecting TSLP in a sample is for non-therapeutic purposes. In some cases, the method for detecting TSLP in a sample is not a diagnostic method.

[0174] On the other hand, the present application also provides a reagent or kit for detecting TSLP in a sample, which comprises the isolated antigen-binding protein, the polypeptide molecule, the nucleic acid molecule, the vector, the cell, the immunoconjugate and / or the pharmaceutical composition.

[0175] On the other hand, the present application also provides the use of the isolated antigen-binding protein, the polypeptide molecule, the nucleic acid molecule, the vector, the cell, the immunoconjugate and / or the pharmaceutical composition in preparing a kit for detecting the presence and / or content of TSLP in a sample.

[0176] Without intending to be bound by any theory, the following examples are merely intended to illustrate the fusion protein, preparation method, and use of the present application, and are not intended to limit the scope of the present invention.

[0177] Example

[0178] Example 1 Preparation of Mouse Monoclonal Antibody Against Human TSLP - Primary Antibody

[0179] Preparation of hybridoma cells producing mouse monoclonal antibodies

[0180] The method for preparing mouse monoclonal antibodies utilizes the hybridoma preparation technique developed by Kohler and Milstein in 1975 (Nature, 1975, 256:495-497). Human TSLP His-tagged protein (ACRO, TSP-H52Ha) was first used as the immunizing antigen. Freund's Adjuvant, Complete (Sigma cat no. F5881, also known as FCA) and Freund's Adjuvant, Incomplete (Sigma cat no. F5506, also known as FICA) were used in Freund's adjuvant for immunization. Multiple BALB / c and CD1 mice were immunized subcutaneously at multiple sites. After four immunizations, serum was collected and assayed for titer by ELISA, and for binding and functional activity by FACS. The best mice were selected to obtain spleen cells for fusion with SP2 / 0 myeloma cells. Hybridoma cell lines were screened by HAT, and the cell culture supernatant was taken for FACS detection to screen out monoclonal hybridoma cell lines that specifically bind to human TSLP and monkey TSLP. Monoclonal cell lines that block TSLP-TSLPR signals and inhibit Hu-TSLP-stimulated proliferation of Baf3-overexpressing TSLPR / IL7Ra cells were screened again. The selected monoclonal cell lines were affinity screened (Biacore), and finally monoclonal hybridoma cell lines with anti-human TSLP antibodies were obtained for sequence analysis. The screening data are listed in Table 1.

[0181] Table 1: Hybridoma screening data results

[0182] Example 2 Cloning and humanization of anti-TSLP antibody variable region gene sequences

[0183] 2.1 Cloning of variable region genes of antibodies in hybridoma cells

[0184] Total RNA was extracted from mouse hybridoma cells using a QIAGEN RNA extraction kit (Cat. No. 74181) according to the manufacturer's instructions. Based on the principles of TAKARA's 5'RACE technology, cDNA sequences of the mouse antibody variable regions expressed by the hybridoma cell lines were cloned. Briefly, cDNAs specific for the heavy and light chain variable region genes were synthesized using the SMARTer 5'RACE Synthesis Kit (TAKARA, Cat. No. 634859) according to the manufacturer's instructions. The 5' and 3' ends of the cDNA sequences were modified with PCR primers designed to add appropriate leader sequences to the heavy and light chain variable region cDNAs, respectively, enabling seamless cloning of the resulting PCR products into the existing recombinant antibody expression vectors pHB-Fc and pHB-CK. The pHB-Fc expression vector contains the human IgG1 heavy chain constant region gene sequence, with the CH2 region mutations L234A and L235A (Eu numbering) that attenuate antibody ADCC activity. The pHB-CK vector contains the human kappa light chain constant region gene sequence. The PCR amplified heavy and light chain variable regions were cloned into an expression vector using an In-fusion cloning reagent (TAKARA, Catalog No. 639650) to generate a human-mouse chimeric antibody expression vector. This vector was then transformed into competent E. coli DH5α cells (Yisheng Biotechnology, Catalog No. FYE607-80VL). Single clones were selected for Sanger sequencing, and the variable region sequence of hybridoma clone 77E6 was determined. The variable region sequence of the resulting anti-TSLP chimeric antibody 900792 is as follows:

[0185] Table 2 CDR sequences of mouse anti-TSLP antibodies (using IMGT numbering rules)

[0186] 2.2 Expression of chimeric antibodies

[0187] The expression vector obtained in 2.1 was amplified in E. coli, and sufficient plasmid was prepared using an endotoxin-free plasmid extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd., Catalog No. DP117) for transient transfection and expression of the chimeric antibody. The host cells used for expression were CHO-S cells (Thermo Fisher Scientific, Catalog No. R80007). The two heavy chain vectors and the light chain vector were mixed with polyetherimide (PEI, Polysciences, Catalog No. 24765-1) to form liposome complexes. The complexes were then transfected into CHO-S cells and cultured in an incubator for 5-7 days. The cell culture supernatant was collected by centrifugation and purified using a Protein A affinity chromatography column to obtain the human-mouse chimeric antibody.

[0188] 2.3 Humanization of mouse anti-human TSLP antibody

[0189] The humanization of antigen-binding proteins uses a 3D modeling method: first, the three-dimensional structure of the mouse antigen-binding protein is modeled, the optimal structural model is selected, and 5-10 optimal structural solutions are selected using the homology modeling method. The loop region is generally modeled using the homology modeling method. If the CDR amino acid sequence alignment result shows less than 50% Identity, the CDR3 structural model is built using the de novo modeling method. Use PDB BLAST to retrieve the 10 antibody crystal structure models with the closest sequence (structural resolution higher than 2.5 angstroms), compare the automatic modeling model, and select the optimal structural model. The variable region sequence of the antigen-binding protein is then compared with the available sequences in the NCBI IgBlast database. Through identification and analysis, the human framework region (FR region) suitable for constructing the CDR transplanted heavy and light chains is finally determined.

[0190] During the modification, based on the conserved amino acid residues in the FR region of human antibodies and the important amino acid residues in the FR region of antibodies, the modification sites were designed, and the variable regions of the heavy and light chains of the antigen-binding protein 900792 of this application were respectively humanized. The designed humanized sequences should meet the requirements of not affecting the structural stability of the antibody, not affecting the binding of the antigen-binding protein to the antigen, not introducing protein modification sites such as glycosylation and phosphorylation, not introducing sites that are easily oxidized or aminated, and enhancing structural stability. After analysis, a total of 4 humanized heavy chain sequences and 3 humanized light chain sequences were designed for the mouse antigen-binding protein sequence of 900792. The sequences are shown in the following table:

[0191] 900792 heavy chain humanized sequence:

[0192] 900792 light chain humanized sequence:

[0193] Note: The italic underlined sequences are antibody CDR sequences, and the CDR sequence division method is based on the IMGT format.

[0194] The four humanized 900792 heavy chain sequences and three humanized 900792 light chain sequences were randomly combined to generate 12 900792 humanized antibodies, which were expressed and purified in CHO-S cells. Using flow cytometry, Biacore, and other assays, the 900792 humanized proteins were screened for their ability to bind to the TSLP antigen, their ability to block the binding of TSLP to its receptor, their nonspecific binding properties, and their thermal stability. Several high-performance humanized anti-TSLP antigen-binding proteins were obtained.

[0195] Example 3 Detection of anti-TSLP humanized antibodies

[0196] 3.1 Detection of the binding activity of humanized protein 900792 to cells expressing human TSLP

[0197] The binding activity of 900792 humanized protein to cells expressing human TSLP (Baf3-hu-TSLP-3G11, Huabo Biotechnology) was detected.

[0198] All antigen-binding proteins were diluted to 30 μg / ml with 1% BSA in PBS (1% BSA / PBS) and then diluted 3-fold in 10 gradients for a total of 11 concentrations. 20 μL was added to each well of a 96-well U-shaped plate. A negative control (1% BSA / PBS alone) was also set up simultaneously. A suspension of cells expressing human TSLP (Baf3-hu-TSLP-3G11, Huabo Bio) in the logarithmic growth phase was centrifuged (300 g for 5 min), the culture medium was discarded, and the cells were resuspended in 1% BSA / PBS to a viable cell density of 1×10 6 / mL, 20 μL (2×104 cells) per well was added to a 96-well U-shaped plate containing anti-TSLP antigen binding protein and incubated at room temperature for 30 min. After incubation, the 96-well U-shaped plate was centrifuged (300g × 3 min) and the supernatant was discarded. Then, 100 μl of 1% BSA / PBS was added to each well of the 96-well U-shaped plate to resuspend the cells, centrifuged (300g × 3 min), the supernatant was discarded, and the cells were washed twice. Then, 20 μl of 1:200 diluted PE-goat anti-human-Fc (Jackson Immuno Research, #109-115-098) was added to each well and incubated at room temperature in the dark for 15 min; the incubated 96-well U-shaped plate was centrifuged (300g × 3 min) and the supernatant was discarded. Then, 100 μl of 1% BSA / PBS was added to each well of the 96-well U-shaped plate to resuspend the cells, centrifuged (300g × 3 min), the supernatant was discarded, and the cells were washed three times. Finally, 100 μL of 1% BSA / PBS was added to each well and the fluorescence intensity of the PE channel was detected using a flow cytometer (BD, #CantoⅡ).

[0199] The experimental results are shown in Figure 1:

[0200] According to the binding activity curves and EC50 values ​​of 900792 and related humanized proteins with TSLP on the cell membrane surface, the binding activity of 900792 humanized protein and cells expressing human TSLP (Baf3-hu-TSLP-3G11, Huabo Biotechnology) is comparable.

[0201] 3.2 Experiments on blocking TSLP binding to cell surface TSLPR receptors using humanized protein 900792

[0202] The 900792 humanized protein was tested for its ability to block the binding of TSLP cytokine to cells expressing human TSLP receptor and IL7Ra (Baf3-huTSLPR-IL7Ra-3H9-2, Huabo Bio).

[0203] Biotinylated Human TSLP (Acro, #TSP-H82EB-200UG) was diluted to 1.6 μg / ml in PBS containing 1% BSA. All antigen-binding proteins were diluted to 30 μg / ml. The mixture was then diluted 2-fold in 10 steps, for a total of 11 concentrations. 10 μl of Biotinylated Human TSLP and 10 μl of antigen-binding protein were mixed and added to a 96-well U-shaped plate (1% BSA and Biotinylated Human TSLP were mixed as negative and positive control wells). Baf3-huTSLPR-IL7Ra-3H9-2 cells were diluted to 1×10 6 20 μl of the cell suspension at 500 μg / well was added to the 96-well U-shaped plate, mixed and incubated at room temperature for 30 min, centrifuged (300 g × 3 min), and the supernatant was discarded. Then, 100 μl of 1% BSA / PBS was added to each well of the 96-well U-shaped plate to resuspend the cells, centrifuged (300 g × 3 min), and the supernatant was discarded. After washing once, 20 μl of 1:200 diluted anti-biotin antibody APC-SA (BD, #554067) was added to each well and incubated at room temperature for 30 min (no addition was made to the negative control wells). After centrifugation (300 g × 3 min), the supernatant was discarded. Then, 100 μl of 1% BSA / PBS was added to each well of the 96-well U-shaped plate to resuspend the cells, centrifuged (300 g × 3 min), and the supernatant was discarded. After washing twice, the cells were finally resuspended in 100 μL of 1% BSA / PBS per well. The fluorescence intensity of the APC channel was detected by flow cytometry (BD, #CantoⅡ).

[0204] The experimental results are shown in Figure 2:

[0205] According to the curves of 900792 and related humanized proteins blocking the binding of TSLP to cell surface receptors and the relevant IC50 values, it is shown that 900792 and its related humanized proteins have comparable effects in blocking human TSLP and cells expressing human TSLP receptors (Baf3-huTSLPR-IL7Ra-3H9-2, Huabo Bio), among which 900792-huH2L2, 900792-huH3L2, and 900792-huH4L3 have better blocking effects.

[0206] 3.3 Detection of the inhibitory effect of 900792 humanized protein on human TSLP functional activity

[0207] The functional activity of 900792 humanized protein in inhibiting the binding of human TSLP cytokine to cells expressing human TSLP receptor and IL7Ra co-receptor (Baf3-huTSLPR-IL7Ra-3H9-2, Huabo Biotechnology) was detected.

[0208] Human TSLP (Acro, #TSP-H52Ha) was prepared at 3 ng / ml, and 50 μl was added to each well of a 96-well black plate (negative control wells were only added with cell culture medium: RPMI160 + 10% FBS + 10 mM HEPES and mixed with the following cells). At the same time, all antigen binding proteins were diluted to 50 μg / ml with culture medium and then diluted 8 times in 8 gradients, for a total of 9 concentrations. 50 μl was added to each well of a 96-well black plate containing human TSLP and mixed. Cells (Baf3-huTSLPR-IL7Ra-3H9-2, Huabo Biotechnology) were resuspended at 2×10 5 cells / ml, 50 μl was added to the black plate (a separate control well was set up in which only cells and human TSLP were mixed and incubated), mixed and placed in a 37°C 5% CO2 incubator for 40-48 hours, and after the incubation, centrifuged (300g×3min) and discarded 50 μl of the supernatant, and 100 μl of CTG ( Luminescent Cell Viability Assay, Promega, #G7573), wrapped in tin foil, and vibrated for 10 min before full wavelength detection.

[0209] The experimental results are shown in Figure 3:

[0210] According to the functional activity curves and related IC50 values ​​of 900792 and related humanized proteins in inhibiting the binding of TSLP to cell surface receptors, it is shown that 900792 and its related humanized proteins can inhibit the functional activity of human TSLP binding to cells expressing human TSLP receptor and human IL7Ra co-receptor (Baf3-huTSLPR-IL7Ra-3H9-2, Huabo Bio), among which 900792-huH1L3, 900792-huH2L3, 900792-huH3L2, and 900792-huH4L3 showed better inhibitory effects.

[0211] 3.4 Binding affinity testing of humanized protein 900792 to human and monkey TSLP proteins

[0212] Multi-cycle kinetic analysis was performed using a CM5 chip immobilized with an anti-His antibody to capture human TSLP antigen (ACRO, Cat#: TSP-H52Ha, Lot#: 2685a-82QF1-H2) and monkey TSLP antigen (R127A, R130S) protein. His tag (ACRO, Cat#: TSP-C52H4, Lot#: 3385a-9CHF1-QH) served as ligands, and serially diluted antibody samples served as analytes. Kinetic constants, including association rate constant (ka), dissociation rate constant (kd), and dissociation equilibrium rate constant (KD), were analyzed using a 1:1 Binding Model (Flow Rate: 30 μl / min), 120 s Association, 600 s Dissociation, and a 1:1 Binding Model-Fit local analysis.

[0213] The experimental results are shown in Tables 3 and 4:

[0214] The 900792 humanized proteins all bound to human and monkey TSLP proteins, and the binding affinity of most 900792 humanized proteins to human and monkey TSLP proteins was not significantly different from that of the 900792 mouse chimeric protein.

[0215] Among them, the heavy chain sequence of the 900792 chimera is SEQ ID NO:40, and the light chain sequence is SEQ ID NO:41; the sequence of 900792-huH1 is SEQ ID NO:42; the sequence of 900792-huH2 is SEQ ID NO:43; the sequence of 900792-huH3 is SEQ ID NO:44; the sequence of 900792-huH4 is SEQ ID NO:45; the sequence of 900792-huL1 is SEQ ID NO:46; the sequence of 900792-huL2 is SEQ ID NO:47; and the sequence of 900792-huL3 is SEQ ID NO:48.

[0216] Table 3: Binding affinity test of 900792 and humanized proteins to human TSLP protein

[0217] Table 4: Binding affinity test of 900792 and humanized protein to monkey TSLP protein

[0218] 3.5 Detection of nonspecific adsorption effects of 900792 humanized protein

[0219] The SPR method was used to determine the nonspecific adsorption effect of antigen-binding proteins on non-target molecules.

[0220] Quality control results for lysozyme solution from chicken egg and trypsin inhibitor type 1-S from Glycine max using a polyclonal rabbit anti-lysozyme and anti-trypsin inhibitor antibody, respectively, confirmed that the activity of egg white lysozyme and soybean trypsin inhibitor after amino-coupling immobilization on a CM5 chip was normal, indicating that the immobilized ligand activity was maintained throughout the experiment. The PI of trypsin inhibitor type 1-S from Glycine max was 4.5, and that of lysozyme was 11.3. In the HBS-EP buffer system at pH 7.4, trypsin inhibitor type 1-S from Glycine max has a strong negative charge, while lysozyme has a strong positive charge.

[0221] The experimental results are shown in Table 5:

[0222] The binding response values ​​of all tested samples with Lysozyme and Trypsin were less than 20RU, indicating that there was no obvious nonspecific electrostatic binding effect among the tested samples.

[0223] Table 5 Comparison of nonspecific binding amount of samples

[0224] 3.6 Thermal stability testing of humanized protein 900792

[0225] The experimental steps for the thermal stability of anti-TSLP antigen binding protein are as follows:

[0226] The melting temperature (Tm) and aggregation temperature (Tagg) of the anti-TSLP antigen binding protein were detected using a protein stability analyzer (Uncle, UNCHAINED LABS, US). The temperature increase range of Tm and Tagg was 25°C to 95°C, and the temperature increase rate was 0.3°C.

[0227] The experimental results are shown in Table 6:

[0228] The thermal stability test results of 900792 humanized protein showed that the thermal stability data Tm and Tagg of anti-TSLP humanized protein performed well.

[0229] Table 6 Thermal stability data of anti-TSLP antigen binding protein Tm, Tagg value detection

[0230] 3.7 Detection of TARC functional activity blocked by humanized protein 900792

[0231] TSLP (Thymic stromal lymphopoietin) can stimulate dendritic cells or monocytes to produce the chemokine TARC (Thymus and activation-regulated chemokine), thereby recruiting type II helper T cells and causing a type II inflammatory response. TARC is not produced in the resting state. In an in vitro culture system, TSLP is used to stimulate dendritic cells and monocytes in PBMCs, and the amount of TARC produced is measured. Neutralization of TSLP with anti-TSLP antibodies blocks TARC production, showing a dose-dependent relationship with a gradient of anti-TSLP antibody concentrations, thereby evaluating the neutralizing activity of the antibodies.

[0232] Detection method: First dilute the anti-TSLP antibody to 2 μg / mL, then dilute it 2-fold to a total of 7 concentrations, add 50 μl / well to a 96-well plate, then dilute TSLP to 10 ng / mL, add 50 μl / well to the above well plate, mix and incubate for 30 minutes, recover the cells, centrifuge at 500g for 5 minutes, remove the supernatant, resuspend in complete medium and count, adjust the density to 2×10 6 cells / mL, 100 μl / well was added to the cell plate. The incubated antigen-antibody mixture was added to the cells at 100 μl / well. The cells were cultured at 37°C in a 5% CO2 incubator for 48 hours, and the supernatant was collected by centrifugation. TARC content in the cell culture supernatant was determined by ELISA.

[0233] The experimental results are shown in Figures 4 and 5.

[0234] The functional activity of 900792 humanized protein in blocking TARC is nearly 10 times better than that of the positive control drug Tezepelumab (sequence derived from patent US8163284).

[0235] The 900792 humanized proteins were able to effectively block the functional activity of TARC, and their activity was similar to that of the chimeric antibody.

Claims

1. An isolated antigen binding protein targeting TSLP and comprising HCDR1, HCDR2 and HCDR3 of the heavy chain variable region VH; wherein, The amino acid sequence of HCDR1 is shown in SEQ ID NO: 1, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 2, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:

3.

2. The isolated antigen-binding protein according to claim 1, comprising H-FR1, wherein the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1, and the amino acid sequence of the H-FR1 is optionally selected from SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:

9.

3. The isolated antigen-binding protein according to any one of claims 1 to 2, comprising H-FR2, wherein the H-FR2 is located between the HCDR1 and the HCDR2, and the amino acid sequence of the H-FR2 is optionally selected from SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO:

13.

4. The isolated antigen-binding protein according to any one of claims 1 to 3, comprising H-FR3, wherein the H-FR3 is located between the HCDR2 and the HCDR3, and the amino acid sequence of the H-FR3 is optionally selected from SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO:

18.

5. The isolated antigen-binding protein according to any one of claims 1 to 4, comprising H-FR4, the N-terminus of which is connected to the C-terminus of the HCDR3, and the amino acid sequence of the H-FR4 is optionally selected from SEQ ID NO: 19 and SEQ ID NO:

20.

6. The isolated antigen-binding protein according to any one of claims 1 to 5, comprising H-FR1, H-FR2, H-FR3 and H-FR4, wherein the H-FR1, H-FR2, H-FR3 and H-FR4 are selected from any one of the following groups of amino acid sequences: 1) H-FR1: SEQ ID NO: 7, H-FR2: SEQ ID NO: 10, H-FR3: SEQ ID NO: 14, H-FR4: SEQ ID NO: 19; 2) H-FR1: SEQ ID NO: 8, H-FR2: SEQ ID NO: 11, H-FR3: SEQ ID NO: 15, H-FR4: SEQ ID NO: 20; 3) H-FR1: SEQ ID NO: 9, H-FR2: SEQ ID NO: 12, H-FR3: SEQ ID NO: 16, H-FR4: SEQ ID NO: 20; 4) H-FR1: SEQ ID NO: 8, H-FR2: SEQ ID NO: 13, H-FR3: SEQ ID NO: 17, H-FR4: SEQ ID NO: 20; and 5) H-FR1: SEQ ID NO: 9, H-FR2: SEQ ID NO: 12, H-FR3: SEQ ID NO: 18, H-FR4: SEQ ID NO:

20.

7. The isolated antigen-binding protein according to any one of claims 1 to 6, comprising an antibody heavy chain variable region VH, wherein the amino acid sequence of said VH is optionally selected from SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34 and SEQ ID NO:

35.

8. The isolated antigen-binding protein according to any one of claims 1 to 7, comprising LCDR1, LCDR2 and LCDR3 of the light chain variable region VL; wherein, The amino acid sequence of LCDR1 is shown in SEQ ID NO:4, the amino acid sequence of LCDR2 is shown in SEQ ID NO:5 (GAR), and the amino acid sequence of LCDR3 is shown in SEQ ID NO:

6.

9. The isolated antigen-binding protein according to any one of claims 1 to 8, comprising L-FR1, wherein the C-terminus of the L-FR1 is directly or indirectly connected to the N-terminus of the LCDR1, and the amino acid sequence of the L-FR1 is optionally selected from SEQ ID NO: 21 and SEQ ID NO:

22.

10. The isolated antigen-binding protein according to any one of claims 1 to 9, comprising L-FR2, wherein the L-FR2 is located between the LCDR1 and the LCDR2, and the amino acid sequence of the L-FR2 is selected from SEQ ID NO: 23, SEQ ID NO: 24 and SEQ ID NO:

25.

11. The isolated antigen-binding protein according to any one of claims 1 to 10, comprising L-FR3, wherein the L-FR3 is located between the LCDR2 and the LCDR3, and the amino acid sequence of the L-FR3 is selected from SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO:

29.

12. The isolated antigen-binding protein according to any one of claims 1 to 11, comprising L-FR4, the N-terminus of the L-FR4 being connected to the C-terminus of the LCDR3, and the amino acid sequence of the L-FR4 being SEQ ID NO:

30.

13. The isolated antigen-binding protein according to any one of claims 1 to 12, comprising L-FR1, L-FR2, L-FR3 and L-FR4, wherein the L-FR1, L-FR2, L-FR3 and L-FR4 are selected from any one of the following groups of amino acid sequences: 1) L-FR1: SEQ ID NO: 21, L-FR2: SEQ ID NO: 23, L-FR3: SEQ ID NO: 26, L-FR4: SEQ ID NO: 30; 2) L-FR1: SEQ ID NO: 22, L-FR2: SEQ ID NO: 24, L-FR3: SEQ ID NO: 27, L-FR4: SEQ ID NO: 30; 3) L-FR1: SEQ ID NO: 22, L-FR2: SEQ ID NO: 24, L-FR3: SEQ ID NO: 28, L-FR4: SEQ ID NO: 30; and 4) L-FR1: SEQ ID NO:22, L-FR2: SEQ ID NO:25, L-FR3: SEQ ID NO:29, L-FR4: SEQ ID NO:

30.

14. The isolated antigen-binding protein according to any one of claims 1 to 13, comprising an antibody heavy chain variable region VL, wherein the amino acid sequence of said VL is optionally selected from SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO:

39.

15. The isolated antigen-binding protein according to any one of claims 1 to 14, comprising any group of VH and VL selected from the following: 1) VH: SEQ ID NO: 31, VL: SEQ ID NO: 36; 2) VH: SEQ ID NO: 32, VL: SEQ ID NO: 37; 3) VH: SEQ ID NO: 32, VL: SEQ ID NO: 38; 4) VH: SEQ ID NO: 32, VL: SEQ ID NO: 39; 5) VH: SEQ ID NO: 33, VL: SEQ ID NO: 37; 6) VH: SEQ ID NO: 33, VL: SEQ ID NO: 38; 7) VH: SEQ ID NO: 33, VL: SEQ ID NO: 39; 8) VH: SEQ ID NO: 34, VL: SEQ ID NO: 37; 9) VH: SEQ ID NO: 34, VL: SEQ ID NO: 38; 10) VH: SEQ ID NO: 34, VL: SEQ ID NO: 39; 11) VH: SEQ ID NO: 35, VL: SEQ ID NO: 37; 12) VH: SEQ ID NO: 35, VL: SEQ ID NO: 38; and 13) VH: SEQ ID NO: 35, VL: SEQ ID NO:

39.

16. The isolated antigen binding protein of any one of claims 1-15, comprising an antibody heavy chain constant region.

17. The isolated antigen binding protein of claim 16, wherein the antibody heavy chain constant region is derived from a human IgG heavy chain constant region.

18. The isolated antigen binding protein according to any one of claims 16-17, wherein the antibody heavy chain constant region is derived from a human IgG1 heavy chain constant region.

19. The isolated antigen binding protein of any one of claims 1-18, comprising an antibody light chain constant region.

20. The isolated antigen binding protein of claim 19, wherein the antibody light chain constant region is derived from a human Ig kappa constant region.

21. The isolated antigen binding protein of any one of claims 1-20, comprising an antibody or an antigen binding fragment thereof.

22. The isolated antigen binding protein of claim 21, wherein the antigen binding fragment comprises a Fab, a Fab', a Fv fragment, a F(ab')2, a F(ab)2, a scFv, a di-scFv and / or a dAb.

23. The isolated antigen-binding protein of claim 21, wherein the antibody is selected from one or more of the following groups: a monoclonal antibody, a chimeric antibody, a humanized antibody, and a fully human antibody.

24. A chimeric antigen receptor comprising a targeting moiety comprising the isolated antigen binding protein of any one of claims 1-23.

25. A polypeptide molecule comprising the isolated antigen binding protein of any one of claims 1-23 or the chimeric antigen receptor of claim 24.

26. An immunoconjugate comprising the isolated antigen binding protein of any one of claims 1-23.

27. An isolated nucleic acid molecule or molecules encoding the isolated antigen binding protein of any one of claims 1-23, the chimeric antigen receptor of claim 24, or the polypeptide molecule of claim 25.

28. A vector comprising the nucleic acid molecule of claim 27.

29. A cell comprising the isolated antigen binding protein of any one of claims 1-23, the chimeric antigen receptor of claim 24, the polypeptide molecule of claim 25, the immunoconjugate of claim 26, the nucleic acid molecule of claim 27, or the vector of claim 28.

30. A pharmaceutical composition comprising the isolated antigen binding protein of any one of claims 1-23, the chimeric antigen receptor of claim 24, the polypeptide molecule of claim 25, the immunoconjugate of claim 26, the nucleic acid molecule of claim 27, the vector of claim 28 and / or the cell of claim 29, and optionally a pharmaceutically acceptable carrier.

31. A method of making the isolated antigen binding protein of any one of claims 1-23, the method comprising culturing the cell of claim 29 under conditions such that the antigen binding protein is expressed.

32. Use of the isolated antigen binding protein of any one of claims 1-23, the chimeric antigen receptor of claim 24, the polypeptide molecule of claim 25, the immunoconjugate of claim 26, the nucleic acid molecule of claim 27, the vector of claim 28, the cell of claim 29 and / or the pharmaceutical composition of claim 30 in the preparation of a medicament for preventing, alleviating and / or treating a disease and / or condition.

33. The use according to claim 32, wherein the disease and / or disorder comprises a TSLP-related disease.

34. Use according to claim 32 or 33, wherein the disease and / or disorder comprises an inflammatory disease or a tumor.

35. A method for detecting TSLP in a sample, the method comprising administering the isolated antigen binding protein of any one of claims 1-23, the chimeric antigen receptor of claim 24, the polypeptide molecule of claim 25, the immunoconjugate of claim 26, the nucleic acid molecule of claim 27, the vector of claim 28, the cell of claim 29 and / or the pharmaceutical composition of claim 30.

36. A reagent or kit for detecting TSLP in a sample, comprising the isolated antigen binding protein of any one of claims 1-23, the chimeric antigen receptor of claim 24, the polypeptide molecule of claim 25, the immunoconjugate of claim 26, the nucleic acid molecule of claim 27, the vector of claim 28, the cell of claim 29 and / or the pharmaceutical composition of claim 30.

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