Isolated antigen-binding proteins and uses thereof

Abstract

The present application relates to an antigen-binding protein comprising at least one CDR of a heavy chain variable region comprising the amino acid sequence shown in any one of SEQ ID NOs: 73 and 74, and at least one CDR of a light chain variable region comprising the amino acid sequence shown in any one of SEQ ID NOs: 75 and 76.

Description

[Technical Field]

[0001] This application relates to the biomedical field, specifically to antigen binding proteins and uses thereof. [Background technology]

[0002] Allergies (including atopy) and other allergic disorders are inappropriate or excessive immune responses to foreign antigens. Inappropriate immune responses include misdirected immune responses against the body's own components, resulting in autoimmune disorders. Allergic disorders (also known as allergic diseases) are the sixth most common chronic disease worldwide. They affect various tissues and organs in the body and, once established, cause a variety of unpleasant symptoms, significantly reducing or even endangering quality of life. For example, allergic reactions, allergic rhinitis, asthma, atopic dermatitis, food allergies, and rubella affect 20% of the total population in many countries, and the prevalence of these diseases is gradually increasing (Wuthrich B., Int. Arch. Allergy Appl. Tmmunol., 90, pp. 3-10, 1989). Allergic diseases are broadly divided into IgE-mediated and non-IgE-mediated types, with immunoglobulin E (IgE)-mediated hypersensitivity reactions prevalent. The high-affinity receptor for IgE (FcεRI) is key in mediating allergic symptoms. In addition to mast cells and basophils, FcεRI is also found on many other cell types (including eosinophils and platelets) and antigen-presenting cells (e.g., monocytes and dendritic cells). Because IgE plays a key role in mediating most allergic reactions, there is a great need to develop treatment strategies for allergic diseases that control IgE levels. Summary of the Invention

[0003] The present application provides an antigen-binding protein having one or more of the following advantages: 1) good stability, which makes the antigen-binding protein of the present application more suitable for drug discovery and more favorable for industrialized production; and 2) the antigen-binding protein of the present application has enhanced affinity for the neonatal receptor FcRn, resulting in a longer half-life, thereby achieving the goal of extending the administration cycle.

[0004] In some embodiments, the antigen binding protein of the present application comprises at least one complementarity determining region (CDR) of a heavy chain variable region (VH) comprising the amino acid sequence set forth in either SEQ ID NO: 73 or 74, and at least one complementarity determining region (CDR) of a light chain variable region (VL) comprising the amino acid sequence set forth in either SEQ ID NO: 75 or 76.

[0005] In some embodiments, the antigen binding protein of the present application is 1) Approx. 2.4×10 -9 Binds to IgE with a KD value of less than M 2) have one or more of the following properties: they are capable of inhibiting the binding of IgE to its receptors FcεRIa and / or FcεRII.

[0006] In some embodiments, the antigen binding proteins of the present application bind to human IgE.

[0007] In some embodiments, the antigen binding protein of the present application comprises a heavy chain HCDR3, wherein said HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:3.

[0008] In some embodiments, the antigen binding protein of the present application further comprises a heavy chain HCDR2, wherein said HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:77.

[0009] In some embodiments, in the antigen binding protein of the present application, the HCDR2 comprises the amino acid sequence set forth in either SEQ ID NO: 2 or 26.

[0010] In some embodiments, the antigen binding protein of the present application further comprises a heavy chain HCDR1, wherein said HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:1.

[0011] In some embodiments, the antigen binding protein of the present application comprises a heavy chain HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 77, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 3.

[0012] In some embodiments, the antigen binding protein of the present application comprises heavy chain HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence set forth in either SEQ ID NO: 2 or 26, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 3.

[0013] In some embodiments, the antigen-binding protein of the present application comprises a heavy chain framework region 1 (H-FR1), wherein the C-terminus of the H-FR1 is linked directly or indirectly to the N-terminus of the HCDR1, and the H-FR1 comprises the amino acid sequence set forth in either SEQ ID NO: 4 or 17.

[0014] In some embodiments, the antigen binding protein of the present application further comprises heavy chain frame region 2 (H-FR2), wherein said H-FR2 is located between said HCDR1 and said HCDR2, and said H-FR2 comprises the amino acid sequence set forth in any of SEQ ID NOs: 81, 5, or 27.

[0015] In some embodiments, in the antigen binding protein of the present application, the H-FR2 comprises the amino acid sequence set forth in any of SEQ ID NOs: 5, 18, 27, or 36.

[0016] In some embodiments, the antigen binding protein of the present application further comprises heavy chain frame region 3 (H-FR3), wherein the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises the amino acid sequence set forth in either SEQ ID NO: 6 or 19.

[0017] In some embodiments, the antigen binding protein of the present application further comprises heavy chain framework region 4 (H-FR4), wherein the N-terminus of said H-FR4 is linked to the C-terminus of said HCDR3, and said H-FR4 comprises the amino acid sequence set forth in either SEQ ID NO: 7 or 20.

[0018] In some embodiments, the antigen binding protein of the present application comprises a heavy chain variable region VH, wherein the VH comprises the amino acid sequence set forth in either SEQ ID NO: 73 or 74.

[0019] In some embodiments, the antigen binding protein of the present application comprises a heavy chain variable region, VH, wherein the VH comprises the amino acid sequence set forth in any of SEQ ID NOs: 8, 21, 28, 37, or 41.

[0020] In some embodiments, the antigen binding protein of the present application comprises a light chain LCDR3, wherein said LCDR3 comprises the amino acid sequence set forth in SEQ ID NO:79.

[0021] In some embodiments, in the antigen binding protein of the present application, the LCDR3 comprises the amino acid sequence set forth in either SEQ ID NO: 11 or 30.

[0022] In some embodiments, the antigen binding protein of the present application further comprises a light chain LCDR2, wherein said LCDR2 comprises the amino acid sequence set forth in SEQ ID NO:78.

[0023] In some embodiments, in the antigen binding protein of the present application, the LCDR2 comprises the amino acid sequence set forth in either SEQ ID NO: 10 or 29.

[0024] In some embodiments, the antigen binding protein of the present application further comprises a light chain LCDR1, wherein said LCDR1 comprises the amino acid sequence set forth in SEQ ID NO:9.

[0025] In some embodiments, the antigen binding protein of the present application comprises a light chain LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO:9, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO:78, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO:79.

[0026] In some embodiments, the antigen binding protein of the present application comprises a light chain LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 9, the LCDR2 comprises the amino acid sequence set forth in either SEQ ID NO: 10 or 29, and the LCDR3 comprises the amino acid sequence set forth in either SEQ ID NO: 11 or 30.

[0027] In some embodiments, the antigen binding protein of the present application comprises a light chain framework region 1 (L-FR1), wherein the C-terminus of said L-FR1 is linked directly or indirectly to the N-terminus of said LCDR1, and said L-FR1 comprises the amino acid sequence set forth in any of SEQ ID NOs: 31, 12, or 80.

[0028] In some embodiments, in the antigen binding protein of the present application, the L-FR1 comprises the amino acid sequence set forth in any of SEQ ID NOs: 12, 22, 31, or 38.

[0029] In some embodiments, the antigen binding protein of the present application further comprises a light chain frame region 2 (L-FR2), wherein the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 comprises the amino acid sequence set forth in any of SEQ ID NOs: 13, 23, or 32.

[0030] In some embodiments, the antigen binding proteins described herein further comprise a light chain frame region 3 (L-FR3), wherein said L-FR3 is located between said LCDR2 and said LCDR3, and said L-FR3 comprises the amino acid sequence set forth in any of SEQ ID NOs: 33, 14, or 82.

[0031] In some embodiments, in the antigen binding protein of the present application, the L-FR3 comprises the amino acid sequence set forth in any of SEQ ID NOs: 14, 24, 33, or 39.

[0032] In some embodiments, the antigen binding protein of the present application further comprises a light chain framework region 4 (L-FR4), wherein the N-terminus of the L-FR4 is linked to the C-terminus of the LCDR3, and the L-FR4 comprises the amino acid sequence set forth in either SEQ ID NO: 34 or 15.

[0033] In some embodiments, the antigen binding protein of the present application comprises a light chain variable region, VL, wherein the VL comprises the amino acid sequence set forth in either SEQ ID NO: 75 or 76.

[0034] In some embodiments, the antigen binding protein of the present application comprises a light chain variable region, VL, wherein the VL comprises the amino acid sequence set forth in any of SEQ ID NOs: 16, 35, 25, and 40.

[0035] In some embodiments, the antigen binding protein of the present application comprises the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 77, the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 3, the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 9, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 78, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 79.

[0036] In some embodiments, the antigen binding protein of the present application comprises the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence set forth in either SEQ ID NO: 2 or 26, the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 3, the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 9, the LCDR2 comprises the amino acid sequence set forth in either SEQ ID NO: 10 or 29, and the LCDR3 comprises the amino acid sequence set forth in either SEQ ID NO: 11 or 30.

[0037] In some embodiments, the antigen binding protein of the present application comprises a heavy chain variable region, VH, and a light chain variable region, VL, wherein the VH comprises the amino acid sequence set forth in either SEQ ID NO: 73 or 74, and the VL comprises the amino acid sequence set forth in either SEQ ID NO: 75 or 76.

[0038] In some embodiments, the antigen binding protein of the present application comprises a heavy chain variable region, VH, and a light chain variable region, VL, wherein the VH comprises the amino acid sequence set forth in any of SEQ ID NOs: 8, 21, 28, 37, or 41, and the VL comprises the amino acid sequence set forth in any of SEQ ID NOs: 16, 35, 25, or 40.

[0039] In some embodiments, the antigen binding protein of the present application comprises an antibody or an antigen binding fragment thereof.

[0040] In some embodiments of the antigen-binding proteins of the present application, the antigen-binding fragment comprises a Fab, a Fab', a F(ab)2, an Fv fragment, a F(ab')2, a scFv, a di-scFv, and / or a dAb.

[0041] In some embodiments, the antigen binding protein of the present application comprises a heavy chain constant region, and the heavy chain constant region is derived from human IgG.

[0042] In some embodiments, the antigen binding protein of the present application comprises a heavy chain constant region, and the heavy chain constant region is derived from human IgG1.

[0043] In some embodiments, the antigen binding proteins of the present application comprise a heavy chain constant region, wherein the heavy chain constant region comprises the following amino acid mutations compared to the amino acid sequence set forth in SEQ ID NO: 42: M135Y, S137T, and / or T139E.

[0044] In some embodiments, in the antigen binding protein of the present application, the heavy chain constant region comprises the amino acid sequence set forth in either SEQ ID NO: 42 or 44.

[0045] In some embodiments, the antigen binding protein of the present application comprises a heavy chain, and the heavy chain comprises the amino acid sequence set forth in any of SEQ ID NOs: 45, 47, or 49.

[0046] In some embodiments, the antigen binding protein of the present application comprises a light chain constant region, and said light chain constant region comprises the amino acid sequence set forth in SEQ ID NO:43.

[0047] In some embodiments, the antigen binding protein of the present application comprises a light chain, and the light chain comprises the amino acid sequence set forth in either SEQ ID NO: 46 or 48.

[0048] In some embodiments, the antigen binding protein of the present application comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in any of SEQ ID NOs: 45, 47, or 49, and the light chain comprises the amino acid sequence set forth in any of SEQ ID NOs: 46 or 48.

[0049] In another aspect, the present application further provides a polypeptide comprising the antigen binding protein of the present application.

[0050] In another aspect, the present application further provides an immunoconjugate comprising the antigen-binding protein of the present application.

[0051] In another aspect, the present application further provides an isolated nucleic acid molecule encoding an antigen binding protein of the present application.

[0052] In another aspect, the present application further provides a vector comprising the isolated nucleic acid molecule of the present application.

[0053] In another aspect, the present application further provides a cell comprising and / or expressing an antigen binding protein of the present application, a polypeptide of the present application, an immunoconjugate of the present application, an isolated nucleic acid molecule of the present application, or a vector of the present application.

[0054] In another aspect, the present application further provides a method of producing an antigen binding protein of the present application, comprising culturing a cell of the present application under conditions that allow expression of the antigen binding protein of the present application.

[0055] In another aspect, the present application further provides a pharmaceutical composition comprising an antigen-binding protein of the present application, a polypeptide of the present application, an immunoconjugate of the present application, an isolated nucleic acid molecule of the present application, a vector of the present application, a cell of the present application, and / or optionally a pharmaceutically acceptable carrier.

[0056] In another aspect, the present application further provides use of an antigen binding protein of the present application, a polypeptide of the present application, an immunoconjugate of the present application, an isolated nucleic acid molecule of the present application, a vector of the present application, a cell of the present application, and / or a pharmaceutical composition of the present application in the preparation of a medicament for preventing, alleviating, and / or treating an IgE-related disease or condition.

[0057] In another aspect, the present application further provides a method for preventing, alleviating, or treating an IgE-associated disease or condition, comprising administering to a subject in need thereof an antigen-binding protein of the present application, a polypeptide of the present application, an immunoconjugate of the present application, and / or a pharmaceutical composition of the present application.

[0058] In another aspect, the present application further provides an antigen binding protein of the present application, a polypeptide of the present application, an isolated nucleic acid molecule of the present application, a vector of the present application, a cell of the present application, an immunoconjugate of the present application, and / or a pharmaceutical composition of the present application for use in preventing, alleviating, or treating an IgE-related disease or condition.

[0059] In another aspect, the present application further provides a method for detecting or measuring IgE, comprising using an antigen binding protein of the present application or a polypeptide of the present application.

[0060] In another aspect, the present application further provides a kit for detecting or measuring IgE, the kit comprising an antigen-binding protein of the present application or a polypeptide of the present application.

[0061] Those skilled in the art will recognize other aspects and advantages of the present application from the following detailed description. The following detailed description merely illustrates and describes exemplary embodiments of the present application. Those skilled in the art will recognize that, given the teachings of the present application, they may modify the specific embodiments disclosed without departing from the spirit and scope of the invention(s) involved in the present application. Correspondingly, the drawings and description herein are illustrative only and not restrictive. [Brief explanation of the drawings]

[0062] Specific features of the invention herein are set forth in the appended claims. A better understanding of the features and advantages of the invention herein can be obtained by reference to the exemplary embodiments and drawings described in detail below, the brief description of which follows:

[0063] [Figure 1] 1 shows the results of the blocking activity of the antigen-binding protein (mouse monoclonal antibody) of the present application in blocking the binding of IgE to its receptor FcεRIa. [Figure 2] 1 shows the results of blocking activity of the antigen-binding proteins of the present application (chimeric antibodies and their corresponding mouse antibodies) in blocking the binding of IgE to its receptor FcεRIa. [Figure 3] 1 shows the results of blocking activity of antigen-binding proteins of the present application (humanized antibodies, their corresponding chimeric antibodies, and their corresponding murine antibodies) in blocking the binding of IgE to its receptor FcεRIa. [Figure 4]1 shows the results of detecting the blocking of IgE binding to its receptor FcεRIa by the antigen-binding protein of the present application (humanized SE2 antibody) and omalizumab. [Figure 5] 1 shows the results of detecting the blocking of IgE binding to its receptor FcεRIa by the antigen-binding protein of the present application (humanized SE5 antibody) and omalizumab. [Figures 6A-6B] 1 shows the binding affinity of the antigen-binding protein of the present application (humanized SE5ss antibody) to human IgE. [Figures 7A-7B] 1 shows the results of blocking activity of the antigen-binding protein of the present application (humanized SE5ss antibody) in blocking the binding of IgE to its receptors (FcεRIa and FcεRII). [Figure 8] 1 shows the results of detecting the inhibitory activity of the antigen-binding protein of the present application (humanized SE5ss antibody) in blocking IgE-mediated cell activation. [Figures 9A-9C] 1 shows the results of detecting the inhibitory activity of the antigen-binding protein of the present application (humanized SE5ss antibody) in blocking IgE-mediated cell activation by different allergens. [Figure 10] 1 shows the ability of the antigen-binding protein of the present invention (humanized SE5ss antibody) to suppress free human IgE in mice. [Figure 11] 1 shows the results of the antigen-binding protein of the present invention (humanized SE5ss antibody) alleviating changes in body temperature in a mouse immediate-type allergy model. [Figure 12] 1 shows the ability of the antigen-binding protein of the present application (humanized SE5ss antibody) to suppress free IgE in a cynomolgus monkey IgE suppression model. DETAILED DESCRIPTION OF THE INVENTION

[0064] Hereinafter, embodiments of the present invention will be described with reference to specific examples, and those skilled in the art will easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0065] Definition of Terms

[0066] In the present application, the term "antigen-binding protein" generally refers to a protein comprising an antigen-binding portion and, optionally, a scaffold or framework portion that enables the antigen-binding portion to adopt a conformation that promotes binding of the antigen-binding protein to an antigen. Antigen-binding proteins may include, but are not limited to, antibodies, antigen-binding fragments (Fab, Fab', F(ab)2, Fv fragments, F(ab')2, scFv, di-scFv, and / or dAb), immunoconjugates, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs, or fusion proteins, so long as they exhibit the required antigen-binding activity. In the present application, an "antigen-binding protein" may comprise an antigen-binding portion and, optionally, a scaffold or framework portion that enables the antigen-binding portion to adopt a conformation that promotes binding of the antigen-binding portion to an antigen.

[0067] As used herein, the terms "IgE" or "IgE immunoglobulin" or "immunoglobulin E" refer to a class of antibodies (or immunoglobulins (Ig)) typically produced by plasma cells. IgE molecules typically consist of two heavy chains (ε chains) and two light chains, where the ε chains may contain four Ig-like constant regions (Cε1-Cε4). As used herein, "IgE" may refer to intact IgE or fragments thereof, as well as functional variants, isoforms, species homologs, derivatives, analogs, and analogs that share at least one epitope with IgE. In humans, IgE is a polypeptide belonging to the antibody class primarily encoded by known immunoglobulin epsilon genes. IgE may include membrane-anchored forms (mIgE) or non-membrane-anchored forms, also known as circulating IgE. As used herein, "IgE" may be derived from a mammal, and may include human IgE. In the present application, the sequence number of said human IgE in the GenBank database is Gene ID:3497.

[0068] As used herein, the term "FcεRIα" generally refers to the α chain of the high-affinity receptor for the Fc region of immunoglobulin E (IgE) (FcεRI, also known as Fc epsilon RI). FcεRI is a tetrameric receptor complex capable of binding to the Fc region of the ε heavy chain of IgE and typically consists of one α chain (i.e., FcεRIα), one β chain (FcεRIβ), and two γ chains (FcεRIγ). Typically, the α chain functions as the binding site for antibodies (e.g., IgE), the γ chain functions as the initiation site for downstream signals, and the β chain can play a role in amplifying downstream signals. As used herein, "FcεRIα" may refer to the complete FcεRIα or a fragment thereof, or may refer to a functional variant, isoform, species homolog, derivative, or analog of FcεRIα, as well as an analog sharing at least one epitope with FcεRIα.

[0069] In this application, the term "KD" means "K D "," "K D ", "affinity constant" or "equilibrium dissociation constant" and is usually measured at equilibrium or dissociation rate constant (k d ) and the binding rate constant (k a ) is usually referred to as the binding rate constant (k a ), dissociation rate constant (k d ) and the equilibrium dissociation constant (K D) is used to represent the binding affinity of a binding protein (e.g., an antigen-binding protein described herein) to an antigen (e.g., human IgE). Methods for determining association and dissociation rate constants are known in the art. For example, the KD value may be measured using Octet, or other experimental routes and instruments, such as BIAcore (biomolecular interaction analysis). Alternatively, the KD value can be measured by electrochemiluminescence analysis-solution equilibrium titration (MSD-SET). Measurement methods are described in Estep P. et al., MAbs, 2013.5(2):pp.270-8. In the present application, the KD value can be detected using the KinExA method and software. Measurement methods are described in Jonathan K. Fleming et al., Methods Mol Biol. 2018, 1697:1-8.

[0070] In this application, the term "complementarity-determining region" or "CDR" generally refers to a complementarity-determining region within an antibody variable sequence. Three CDRs are present in each heavy and light chain variable region, designated CDR1, CDR2, and CDR3 for each variable region. As used herein, a combination of CDRs may refer to a set of three CDRs present in a single variable region capable of binding to an antigen. The exact boundaries of these CDRs are defined differently based on different systems. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., (1987) and (1991)) not only provides a clear residue numbering system for any variable region applicable to antibodies, but also provides precise residue boundaries defining the three CDRs. These CDRs may be referred to as Kabat CDRs. Chothia and colleagues (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987) and Chothia et al., Nature 342:877-883 (1989)) found that several subregions within the Kabat CDRs have nearly identical peptide backbone conformations despite great diversity at the amino acid sequence level. These subregions are designated L1, L2, and L3 or H1, H2, and H3, where "L" and "H" refer to the light and heavy chain regions, respectively. These regions may be referred to as Chothia CDRs, which have overlapping boundaries with the Kabat CDRs. Other boundaries defining CDRs that overlap with the Kabat CDRs are described in Padlan (FASEB J. 9:133-139 (1995)) and MacCallum (J Mol Biol 262(5):732-45 (1996)). Other CDR boundary definitions may not strictly adhere to either of the above systems but still overlap with the Kabat CDRs and do not significantly affect the prediction or experimental findings of antigen binding according to a particular residue or set of residues, or even the entire CDR, although they can be shortened or extended.The CDRs described herein may be defined using KABAT, but do not exclude the use of other methods to divide the CDR regions.

[0071] As used herein, the term "antibody" generally refers to an immunoglobulin, or a fragment, derivative, or variant thereof, including any polypeptide comprising an antigen-binding site, whether produced in vitro or in vivo. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, polyspecific, nonspecific, humanized, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, and grafted antibodies. As used herein, the term "antibody" may also include antibody fragments, such as Fab, F(ab'), Fv, scFv, Fd, dAb, and other antibody fragments that retain antigen-binding function (e.g., specific binding to human IgE). Antibodies described herein may include antibodies of human, murine, simian, and / or alpaca origin. As used herein, antibodies may also include functional variants, isoforms, species homologs, derivatives, or analogs thereof. For example, a variant herein may comprise an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.2%, at least 99.4%, at least 99.6%, at least 99.8%, or at least 99.9% identity to an antibody.

[0072] As used herein, the term "antigen-binding fragment" generally refers to a polypeptide fragment of an immunoglobulin or antibody that competes with the intact antibody (i.e., the intact antibody derived therefrom) for binding (i.e., specifically binding) to an antigen. Such antigen-binding fragments may include, but are not limited to, Fab, Fab', F(ab)2, F(ab')2, and Fv fragments, linear antibodies, single-chain antibodies, diabodies, and multispecific antibodies formed from antibody fragments.

[0073] As used herein, the term "variable region" or "variable domain" generally refers to portions of an antibody light chain and / or heavy chain that generally vary widely among different antibodies and may affect the binding specificity of the antibody to a particular antigen. Variable regions may include regions of relatively greater sequence variability called complementarity-determining regions (CDRs), and may also include regions that are more conserved, called frame regions (FRs).

[0074] As used herein, the term "directly or indirectly linked" generally refers to the relative terms "directly linked" and "indirectly linked." "Directly linked" generally refers to a direct link. For example, the direct link refers to a case where linked substances (e.g., amino acid sequence segments) are directly linked without any spacing component (e.g., an amino acid residue or a derivative thereof) between them. For example, amino acid sequence segment X and another amino acid sequence segment Y are directly linked via an amide bond formed by the C-terminal amino acid of amino acid sequence segment X and the N-terminal amino acid of amino acid sequence segment Y. "Indirectly linked" generally refers to a case where linked substances (e.g., amino acid sequence segments) are indirectly linked via a spacing component (e.g., an amino acid residue or a derivative thereof) between them. For example, in the antigen-binding protein described herein, the C-terminus of the L-FR1 and the N-terminus of the LCDR1 may be directly or indirectly linked.

[0075] As used herein, the term "immunoconjugate" generally refers to other moieties linked to an antigen-binding protein or fragment thereof. The immunoconjugate may include other polypeptides, therapeutic agents, probes, and / or other antibodies fused or coupled to the antigen-binding protein.

[0076] As used herein, the term "isolated nucleic acid molecule" generally refers to an isolated form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogues that have been isolated from their natural environment or artificially synthesized.

[0077] As used herein, the term "cell" generally refers to an individual cell, cell line, or cell culture that can contain or contain a plasmid or vector containing a nucleic acid molecule described herein, or that is capable of expressing an antigen-binding protein described herein. The cell may include the progeny of a single cell. Due to natural, accidental, or deliberate mutation, progeny cells may not necessarily be completely identical in morphology or genome to the original parent cell, but they may be capable of expressing an antigen-binding protein described herein. The cell may be obtained by transfecting cells in vitro with a vector described herein. The cell may be a prokaryotic cell (e.g., Escherichia coli) or a eukaryotic cell (e.g., a yeast cell, such as a COS cell, a Chinese hamster ovary (CHO) cell, a HeLa cell, a HEK293 cell, a COS-1 cell, a NS0 cell, or a myeloma cell).

[0078] As used herein, the term "pharmaceutical composition" generally refers to a composition suitable for administration to a patient, such as a human patient. For example, a pharmaceutical composition described herein may comprise an antigen-binding protein described herein, an immunoconjugate described herein, a nucleic acid molecule described herein, a vector described herein, and / or a cell described herein, and optionally a pharmaceutically acceptable carrier. The pharmaceutical composition may further comprise one or more suitable formulations of a (pharmaceutically effective) carrier, stabilizer, excipient, diluent, solubilizer, surfactant, emulsifier, and / or preservative. Acceptable components of the composition are non-toxic to recipients at the dosages and concentrations used. Pharmaceutical compositions of the present invention may include, but are not limited to, liquid, frozen, and lyophilized compositions.

[0079] As used herein, the term "vector" generally refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a protein is inserted to express the protein. A vector can transform, transduce, or transfect a host cell to express the genetic material carried within 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), phages such as lambda phage or M13 phage, and animal viruses. Animal viruses used as vectors include retroviruses (lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papilloma viruses, and papilloma vacuolar viruses (e.g., SV40). A vector may contain various elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. A vector may also contain an origin of replication. Vectors may also include components that facilitate cell entry, such as, but are not limited to, viral particles, liposomes, or protein coats.

[0080] In this application, the term "pharmaceutically acceptable carrier" generally includes pharmaceutically acceptable vectors, excipients, or stabilizers, which are non-toxic to cells or mammals exposed thereto at the dosages and concentrations used. Physiologically acceptable vectors may include suitable substances.

[0081] Details of the invention

[0082] antigen-binding proteins

[0083] According to one aspect, the present application provides an antigen-binding protein which may comprise at least one complementarity determining region (CDR) of a heavy chain variable region (VH) and at least one complementarity determining region (CDR) of a light chain variable region (VL), wherein the heavy chain variable region (VH) comprises the amino acid sequence set forth in either SEQ ID NO: 73 or 74, and the light chain variable region (VL) comprises the amino acid sequence set forth in either SEQ ID NO: 75 or 76.

[0084] For example, the antigen-binding protein of the present application may comprise one CDR of a heavy chain variable region, and the heavy chain variable region may comprise the amino acid sequence set forth in SEQ ID NO: 73 or a variant thereof.

[0085] For example, an antigen-binding protein of the present application may comprise two CDRs of a heavy chain variable region, wherein the heavy chain variable region may comprise the amino acid sequence set forth in SEQ ID NO: 73 or a variant thereof.

[0086] For example, the antigen binding protein of the present application may comprise three CDRs of a heavy chain variable region, and the heavy chain variable region may comprise the amino acid sequence set forth in SEQ ID NO: 73 or a variant thereof.

[0087] For example, the antigen binding protein of the present application may comprise one CDR of a heavy chain variable region, and the heavy chain variable region may comprise the amino acid sequence set forth in SEQ ID NO: 74 or a variant thereof.

[0088] For example, an antigen-binding protein of the present application may comprise two CDRs of a heavy chain variable region, wherein the heavy chain variable region may comprise the amino acid sequence set forth in SEQ ID NO: 74 or a variant thereof.

[0089] For example, the antigen binding protein of the present application may comprise three CDRs of a heavy chain variable region, and the heavy chain variable region may comprise the amino acid sequence set forth in SEQ ID NO: 74 or a variant thereof.

[0090] For example, the CDRs of the heavy chain variable region of the present application may be referred to as HCDRn, where n represents the CDR number, which may typically be 1, 2, or 3.

[0091] For example, the CDRs of a heavy chain variable region of the present application may comprise HCDR1, HCDR2, and HCDR3 as set forth herein.

[0092] For example, the antigen binding protein of the present application may comprise a heavy chain HCDR3, which may comprise the amino acid sequence shown in SEQ ID NO: 3 or a variant thereof.

[0093] For example, an antigen binding protein of the present application may comprise a heavy chain HCDR2, which may comprise the amino acid sequence set forth in SEQ ID NO: 77 or a variant thereof.

[0094] For example, the antigen-binding protein of the present application may comprise a heavy chain HCDR2, which may comprise the amino acid sequence set forth in either SEQ ID NO: 2 or 26, or a variant thereof.

[0095] For example, the antigen binding protein of the present application may comprise a heavy chain HCDR1, which may comprise the amino acid sequence shown in SEQ ID NO: 1 or a variant thereof.

[0096] For example, an antigen-binding protein of the present application may comprise a heavy chain HCDR3 and HCDR2, wherein the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 3 or a variant thereof, and the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 77 or a variant thereof.

[0097] For example, the antigen-binding protein of the present application may comprise a heavy chain HCDR3 and HCDR2, wherein the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 3 or a variant thereof, and the HCDR2 may comprise the amino acid sequence shown in either SEQ ID NO: 2 or 26 or a variant thereof.

[0098] For example, an antigen-binding protein of the present application may comprise a heavy chain HCDR2 and HCDR1, wherein said HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 77 or a variant thereof, and said HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO: 1 or a variant thereof.

[0099] For example, the antigen-binding protein of the present application may comprise a heavy chain HCDR2 and HCDR1, wherein the HCDR2 may comprise the amino acid sequence shown in either SEQ ID NO: 2 or 26 or a variant thereof, and the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 1 or a variant thereof.

[0100] For example, the antigen-binding protein of the present application may comprise heavy chain HCDR1, HCDR2 and HCDR3, wherein the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 1 or a variant thereof, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 77 or a variant thereof, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 3 or a variant thereof.

[0101] For example, the antigen-binding protein of the present application may comprise heavy chain HCDR1, HCDR2, and HCDR3, wherein the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 1 or a variant thereof, the HCDR2 may comprise the amino acid sequence shown in either SEQ ID NO: 2 or 26 or a variant thereof, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 3 or a variant thereof.

[0102] For example, the antigen binding protein of the present application may comprise one CDR of a light chain variable region, and the light chain variable region may comprise the amino acid sequence set forth in SEQ ID NO: 75 or a variant thereof.

[0103] For example, the antigen binding protein of the present application may comprise two CDRs of a light chain variable region, wherein the light chain variable region may comprise the amino acid sequence set forth in SEQ ID NO: 75 or a variant thereof.

[0104] For example, the antigen binding protein of the present application may comprise three CDRs of a light chain variable region, and said light chain variable region may comprise the amino acid sequence set forth in SEQ ID NO: 75 or a variant thereof.

[0105] For example, the antigen binding protein of the present application may comprise one CDR of a light chain variable region, and the light chain variable region may comprise the amino acid sequence set forth in SEQ ID NO: 76 or a variant thereof.

[0106] For example, an antigen binding protein of the present application may comprise two CDRs of a light chain variable region, wherein the light chain variable region may comprise the amino acid sequence set forth in SEQ ID NO: 76 or a variant thereof.

[0107] For example, the antigen binding protein of the present application may comprise three CDRs of a light chain variable region, and the light chain variable region may comprise the amino acid sequence set forth in SEQ ID NO: 76 or a variant thereof.

[0108] For example, the CDRs of the light chain variable region of the present application may be referred to as LCDRn, where n represents the CDR number and may typically be 1, 2, 3, or the like.

[0109] For example, the CDRs of the light chain variable region of the present application may comprise LCDR1, LCDR2, and LCDR3 as described herein.

[0110] For example, the antigen binding protein of the present application may comprise a light chain LCDR3, which may comprise the amino acid sequence set forth in SEQ ID NO: 79 or a variant thereof.

[0111] For example, the antigen-binding protein of the present application may comprise a light chain LCDR3, which may comprise the amino acid sequence set forth in either SEQ ID NO: 11 or 30, or a variant thereof.

[0112] For example, the antigen binding protein of the present application may comprise a light chain LCDR2, which may comprise the amino acid sequence set forth in SEQ ID NO: 78 or a variant thereof.

[0113] For example, the antigen-binding protein of the present application may comprise a light chain LCDR2, which may comprise the amino acid sequence set forth in either SEQ ID NO: 10 or 29, or a variant thereof.

[0114] For example, the antigen binding protein of the present application may comprise a light chain LCDR1, which may comprise the amino acid sequence shown in SEQ ID NO: 9 or a variant thereof.

[0115] For example, the antigen-binding protein of the present application comprises a light chain LCDR3 and LCDR2, wherein the LCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 79 or a variant thereof, and the LCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 78 or a variant thereof.

[0116] For example, the antigen-binding protein of the present application comprises a light chain LCDR3 and LCDR2, wherein the LCDR3 may comprise the amino acid sequence shown in either SEQ ID NO: 11 or 30 or a variant thereof, and the LCDR2 may comprise the amino acid sequence shown in either SEQ ID NO: 10 or 29 or a variant thereof.

[0117] For example, the antigen binding protein of the present application may comprise a light chain LCDR2 and LCDR1, wherein said LCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 78 or a variant thereof, and said LCDR1 may comprise the amino acid sequence set forth in SEQ ID NO: 9 or a variant thereof.

[0118] For example, the antigen-binding protein of the present application may comprise a light chain LCDR2 and LCDR1, wherein the LCDR2 may comprise the amino acid sequence shown in either SEQ ID NO: 10 or 29 or a variant thereof, and the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 9 or a variant thereof.

[0119] For example, the antigen-binding protein of the present application may comprise light chain LCDR1, LCDR2 and LCDR3, wherein the LCDR1 may comprise the amino acid sequence set forth in SEQ ID NO: 9 or a variant thereof, the LCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 78 or a variant thereof, and the LCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 79 or a variant thereof.

[0120] For example, the antigen-binding protein of the present application may comprise light chain LCDR1, LCDR2, and LCDR3, wherein the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 9 or a variant thereof, the LCDR2 may comprise the amino acid sequence shown in either SEQ ID NO: 10 or 29 or a variant thereof, and the LCDR3 may comprise the amino acid sequence shown in either SEQ ID NO: 11 or 30 or a variant thereof.

[0121] For example, the antigen-binding protein of the present application may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein said HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO: 1 or a variant thereof, said HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 77 or a variant thereof, said HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 3 or a variant thereof, said LCDR1 may comprise the amino acid sequence set forth in SEQ ID NO: 9 or a variant thereof, said LCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 78 or a variant thereof, and said LCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 79 or a variant thereof.

[0122] For example, the antigen-binding protein of the present application may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 1 or a variant thereof, the HCDR2 may comprise the amino acid sequence shown in any of SEQ ID NOs: 2 and 26 or a variant thereof, the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 3 or a variant thereof, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 9 or a variant thereof, the LCDR2 may comprise the amino acid sequence shown in any of SEQ ID NOs: 10 and 29 or a variant thereof, and the LCDR3 may comprise the amino acid sequence shown in any of SEQ ID NOs: 11 and 30 or a variant thereof.

[0123] Frame Region (FR)

[0124] For example, a light chain variable region of the present application may comprise the framework regions L-FR1, L-FR2, L-FR3, and / or L-FR4.

[0125] For example, a light chain variable region of the present application may comprise framework regions L-FR1, L-FR2, and L-FR3.

[0126] For example, a light chain variable region of the present application may comprise framework regions L-FR1, L-FR2, and L-FR4.

[0127] For example, a light chain variable region of the present application may comprise framework regions L-FR2, L-FR3, and L-FR4.

[0128] For example, a light chain variable region of the present application may comprise framework regions L-FR1, L-FR3, and L-FR4.

[0129] For example, a light chain variable region of the present application may comprise the framework regions L-FR1, L-FR2, L-FR3, and L-FR4.

[0130] For example, the C-terminus of L-FR1 of the present application may be directly or indirectly linked to the N-terminus of LCDR1 of the present application, for example, the C-terminus of L-FR1 of the present application may be directly linked to the N-terminus of LCDR1 of the present application, or the C-terminus of L-FR1 of the present application may be indirectly linked to the N-terminus of LCDR1 of the present application, for example, the indirect linking of the present application may include spacing of 1, 2, 3, 4, 5 or more amino acids between the C-terminal amino acid residue of L-FR1 of the present application and the N-terminal amino acid residue of LCDR1 of the present application, and these amino acids may be any naturally occurring or modified amino acid.

[0131] For example, L-FR1 of the present application may comprise the amino acid sequence shown in any one of SEQ ID NOs: 12, 31, or 80, or a variant thereof.

[0132] For example, L-FR1 of the present application may comprise the amino acid sequence shown in any one of SEQ ID NOs: 12, 31, 22, and 38, or a variant thereof.

[0133] For example, the L-FR2 of the present application may be located between the LCDR1 of the present application and the LCDR2 of the present application.

[0134] For example, the N-terminus of the L-FR2 of the present application may be directly or indirectly linked to the C-terminus of the LCDR1 of the present application, and the C-terminus of the L-FR2 of the present application may be directly or indirectly linked to the N-terminus of the LCDR2 of the present application.

[0135] For example, indirect linking may include spacing the two linked moieties (e.g., L-FR2 of the present application and LCDR1 of the present application, or L-FR2 of the present application and LCDR2 of the present application) apart by 1, 2, 3, 4, 5 or more amino acids, which may be any naturally occurring or modified amino acid.

[0136] For example, L-FR2 of the present application may comprise the amino acid sequence shown in any one of SEQ ID NOs: 13, 32, and 23, or a variant thereof.

[0137] For example, L -FR3 may be located between the LCDR2 of the present application and the LCDR3 of the present application.

[0138] For example, the N-terminus of the L-FR3 of the present application may be directly or indirectly linked to the C-terminus of the LCDR2 of the present application, and the C-terminus of the L-FR3 of the present application may be directly or indirectly linked to the N-terminus of the LCDR3 of the present application.

[0139] For example, indirect linking may include spacing the two linked moieties (e.g., L-FR3 of the present application and LCDR2 of the present application, or L-FR3 of the present application and LCDR3 of the present application) apart by 1, 2, 3, 4, 5 or more amino acids, which may be any naturally occurring or modified amino acid.

[0140] For example, L-FR3 of the present application may comprise the amino acid sequence shown in any one of SEQ ID NOs: 14, 33, and 82, or a variant thereof.

[0141] For example, L-FR3 of the present application may comprise the amino acid sequence shown in any one of SEQ ID NOs: 14, 33, 24, and 39, or a variant thereof.

[0142] For example, the N-terminus of the L-FR4 of the present application is directly or indirectly linked to the C-terminus of the LCDR3 of the present application.

[0143] For example, indirect linking may include spacing out 1, 2, 3, 4, 5 or more amino acids between the N-terminal amino acid residue of L-FR4 of the present application and the C-terminal amino acid residue of LCDR3 of the present application, and these amino acids may be any naturally occurring or modified amino acids.

[0144] For example, L-FR4 of the present application may comprise the amino acid sequence shown in either SEQ ID NO: 15 or 34, or a variant thereof.

[0145] For example, a heavy chain variable region of the present application may include framework regions H-FR1, H-FR2, H-FR3, and / or H-FR4.

[0146] For example, heavy The chain variable region is Work It may include regions H-FR1, H-FR2 and H-FR3.

[0147] For example, heavy The chain variable region is Work It may include regions H-FR1, H-FR2 and H-FR4.

[0148] For example, heavy The chain variable region is Work It may include regions H-FR2, H-FR3 and H-FR4.

[0149] For example, heavy The chain variable region is Work It may include regions H-FR1, H-FR3 and H-FR4.

[0150] For example, heavy The chain variable region is Work It may include regions H-FR1, H-FR2, H-FR3 and H-FR4.

[0151] For example, the C-terminus of H-FR1 of the present application may be directly or indirectly linked to the N-terminus of HCDR1 of the present application. For example, the C-terminus of H-FR1 of the present application may be directly linked to the N-terminus of HCDR1 of the present application, or the C-terminus of H-FR1 of the present application may be indirectly linked to the N-terminus of HCDR1 of the present application; for example, the indirect linking of the present application may include spacing out 1, 2, 3, 4, 5 or more amino acids between the C-terminal amino acid residue of H-FR1 of the present application and the N-terminal amino acid residue of HCDR1 of the present application, and these amino acids may be any naturally occurring or modified amino acid.

[0152] For example, H-FR1 of the present application may comprise the amino acid sequence shown in either SEQ ID NO: 4 or 17, or a variant thereof.

[0153] For example, H-FR2 of the present application may be located between HCDR1 of the present application and HCDR2 of the present application.

[0154] For example, the N-terminus of H-FR2 of the present application may be directly or indirectly linked to the C-terminus of HCDR1 of the present application, and the C-terminus of H-FR2 of the present application may be directly or indirectly linked to the N-terminus of HCDR2 of the present application.

[0155] For example, the indirect linking of the present application may include spacing the two linked moieties (e.g., H-FR2 of the present application and HCDR1 of the present application, or H-FR2 of the present application and HCDR2 of the present application) by 1, 2, 3, 4, 5 or more amino acids, and these amino acids may be any naturally occurring or modified amino acids.

[0156] For example, H-FR2 of the present application may comprise the amino acid sequence shown in any one of SEQ ID NOs: 5, 27, and 81, or a variant thereof.

[0157] For example, H-FR2 of the present application may comprise the amino acid sequence shown in any one of SEQ ID NOs: 5, 27, 18, and 36, or a variant thereof.

[0158] For example, the H-FR3 of the present application may be located between the HCDR2 of the present application and the HCDR3 of the present application.

[0159] For example, the N-terminus of H-FR3 of the present application may be directly or indirectly linked to the C-terminus of HCDR2 of the present application, and the C-terminus of H-FR3 of the present application may be directly or indirectly linked to the N-terminus of HCDR3 of the present application.

[0160] For example, indirect linking may include spacing the two portions to be linked (e.g., H-FR3 of the present application and HCDR2 of the present application, or H-FR3 of the present application and HCDR3 of the present application) by 1, 2, 3, 4, 5 or more amino acids, which may be any naturally occurring or modified amino acid.

[0161] For example, H-FR3 of the present application may comprise the amino acid sequence shown in either SEQ ID NO: 6 or 19, or a variant thereof.

[0162] For example, the N-terminus of the H-FR4 of the present application may be linked directly or indirectly to the C-terminus of the HCDR3 of the present application.

[0163] For example, indirect linking may include spacing out 1, 2, 3, 4, 5 or more amino acids between the N-terminal amino acid residue of L-FR4 of the present application and the C-terminal amino acid residue of LCDR3 of the present application, and these amino acids may be any naturally occurring or modified amino acids.

[0164] For example, H-FR4 of the present application may comprise the amino acid sequence shown in either SEQ ID NO: 7 or 20, or a variant thereof.

[0165] For example, when a specific amino acid sequence (e.g., a particular sequence) is referred to, the referenced sequence may encompass variants thereof. For example, variants herein may include amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.2%, at least 99.4%, at least 99.6%, at least 99.8%, or at least 99.9% identity to the amino acid sequence set forth in any of SEQ ID NOs: 4 to 7, 17 to 20, 27, or 36.

[0166] Variable Region, Constant Region, Heavy Chain and Light Chain

[0167] In the present application, the antigen-binding protein of the present application may comprise a heavy chain variable region (VH) and / or a light chain variable region (VL). For example, the heavy chain variable region of the present application may comprise the amino acid sequence set forth in either SEQ ID NO: 73 or 74, or a variant thereof. For example, the light chain variable region of the present application may comprise the amino acid sequence set forth in either SEQ ID NO: 75 or 76, or a variant thereof.

[0168] For example, when a specific amino acid sequence (e.g., a particular sequence) is referred to, the referenced sequence may encompass variants thereof. For example, variants herein may include amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.2%, at least 99.4%, at least 99.6%, at least 99.8%, or at least 99.9% identity to the amino acid sequence set forth in any of SEQ ID NOs: 73 to 76.

[0169] For example, the heavy chain variable region of the present application comprises the amino acid sequence set forth in either SEQ ID NO: 73 or 74, or a variant thereof. For example, the light chain variable region of the present application comprises the amino acid sequence set forth in either SEQ ID NO: 75 or 76, or a variant thereof.

[0170] For example, the heavy chain variable region of the present application comprises the amino acid sequence set forth in any one of SEQ ID NOs: 8, 21, 28, 37, or 41, or a variant thereof. For example, the light chain variable region of the present application comprises the amino acid sequence set forth in any one of SEQ ID NOs: 16, 35, 25, or 40, or a variant thereof.

[0171] For example, the heavy chain variable region of the present application comprises the nucleotide sequence set forth in any one of SEQ ID NOs: 57, 59, 61, 63, and 65, or a variant thereof. For example, the light chain variable region of the present application comprises the nucleotide sequence set forth in any one of SEQ ID NOs: 58, 60, 62, 64, and 66, or a variant thereof.

[0172] For example, when a specific amino acid and / or nucleotide sequence (e.g., a particular sequence) is referred to, the referenced sequence may encompass variants thereof. For example, variants herein may include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.2%, at least 99.4%, at least 99.6%, at least 99.8%, or at least 99.9% identity to the amino acid sequence set forth in any of SEQ ID NOs: 8, 16, 21, 25, 28, 35, 37, 40, 41, or 57-66.

[0173] For example, the antigen-binding protein of the present application may comprise an antibody heavy chain constant region, and the antibody heavy chain constant region of the present application may be a heavy chain constant region derived from human IgG. For example, the antigen-binding protein of the present application may comprise an antibody heavy chain constant region, and the antibody heavy chain constant region of the present application may be a heavy chain constant region derived from human IgG1. For example, the antibody heavy chain constant region of the present application may comprise the amino acid sequence set forth in SEQ ID NO: 42 or a variant thereof.

[0174] For example, when a specific amino acid sequence (e.g., a particular sequence) is referred to, the referenced sequence may encompass variants thereof. For example, variants herein may include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.2%, at least 99.4%, at least 99.6%, at least 99.8%, or at least 99.9% identity to the amino acid sequence set forth in SEQ ID NO:42.

[0175] For example, the heavy chain constant region variant of the present application may include a human IgG1 heavy chain constant region (amino acid sequence shown in SEQ ID NO: 42) as well as amino acid mutations M135Y, S137T, and / or T139E (based on the amino acid sequence of SEQ ID NO: 42).

[0176] For example, the antigen-binding protein of the present application may comprise an antibody light chain constant region, and the antibody light chain constant region of the present application may comprise a human Igκ (Kappa) constant region. For example, the antibody light chain constant region of the present application may comprise the amino acid sequence set forth in SEQ ID NO: 43 or a variant thereof.

[0177] For example, when a specific amino acid sequence (e.g., a particular sequence) is referred to, the referenced sequence may encompass variants thereof. For example, variants herein may include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.2%, at least 99.4%, at least 99.6%, at least 99.8%, or at least 99.9% identity to the amino acid sequence set forth in SEQ ID NO:43.

[0178] For example, the antigen binding protein of the present application may comprise an antibody heavy chain, and the antibody heavy chain of the present application may comprise the amino acid sequence set forth in any of SEQ ID NOs: 45, 47, or 49, or a variant thereof.

[0179] For example, the antigen binding protein of the present application may comprise an antibody heavy chain, and the antibody heavy chain of the present application may comprise the nucleotide sequence set forth in any one of SEQ ID NOs: 67, 69, or 71, or a variant thereof.

[0180] For example, when a specific amino acid and / or nucleotide sequence (e.g., a particular sequence) is referred to, the referenced sequence may encompass variants thereof. For example, variants herein may include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.2%, at least 99.4%, at least 99.6%, at least 99.8%, or at least 99.9% identity to the amino acid sequence set forth in any of SEQ ID NOs: 45, 47, 49, 67, 69, and 71.

[0181] For example, the antigen binding protein of the present application may comprise an antibody light chain, and the antibody light chain of the present application may comprise the amino acid sequence set forth in either SEQ ID NO: 46 or 48, or a variant thereof.

[0182] For example, the antigen binding protein of the present application may comprise an antibody light chain, and the antibody light chain of the present application may comprise the nucleotide sequence set forth in any one of SEQ ID NOs: 68, 70 or 72, or a variant thereof.

[0183] For example, when a specific amino acid and / or nucleotide sequence (e.g., a particular sequence) is referred to, the referenced sequence may encompass variants thereof. For example, variants herein may include amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.2%, at least 99.4%, at least 99.6%, at least 99.8%, or at least 99.9% identity to the amino acid sequence set forth in any of SEQ ID NOS: 45-49, 67-72.

[0184] For example, in the antigen-binding protein of the present application, the FR1, FR2, FR3, and FR4 may comprise an amino acid sequence selected from any one of the following groups:

[0185] 1) The H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 4, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 5, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 6, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 7, the L-FR1 comprises the amino acid sequence shown in SEQ ID NO: 12, the L-FR2 comprises the amino acid sequence shown in SEQ ID NO: 13, the L-FR3 comprises the amino acid sequence shown in SEQ ID NO: 14, and the L-FR4 comprises the amino acid sequence shown in SEQ ID NO: 15.

[0186] 2) The H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 4, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 27, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 6, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 7, the L-FR1 comprises the amino acid sequence shown in SEQ ID NO: 38, the L-FR2 comprises the amino acid sequence shown in SEQ ID NO: 32, the L-FR3 comprises the amino acid sequence shown in SEQ ID NO: 33, and the L-FR4 comprises the amino acid sequence shown in SEQ ID NO: 34.

[0187] 3) The H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 17, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 18, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 19, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 20, the L-FR1 comprises the amino acid sequence shown in SEQ ID NO: 22, the L-FR2 comprises the amino acid sequence shown in SEQ ID NO: 23, the L-FR3 comprises the amino acid sequence shown in SEQ ID NO: 24, and the L-FR4 comprises the amino acid sequence shown in SEQ ID NO: 15.

[0188] 4) The H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 17, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 36, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 19, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 20, the L-FR1 comprises the amino acid sequence shown in SEQ ID NO: 31, the L-FR2 comprises the amino acid sequence shown in SEQ ID NO: 23, the L-FR3 comprises the amino acid sequence shown in SEQ ID NO: 19, and the L-FR4 comprises the amino acid sequence shown in SEQ ID NO: 15.

[0189] 5) The H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 17, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 36, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 19, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 20, the L-FR1 comprises the amino acid sequence shown in SEQ ID NO: 38, the L-FR2 comprises the amino acid sequence shown in SEQ ID NO: 23, the L-FR3 comprises the amino acid sequence shown in SEQ ID NO: 39, and the L-FR4 comprises the amino acid sequence shown in SEQ ID NO: 15.

[0190] For example, the antigen binding protein of the present application may comprise the antibody heavy chain of the present application and the antibody light chain of the present application.

[0191] Properties of antigen-binding proteins

[0192] The antigen-binding protein of the present application is approximately 2.4 x 10 -9 It can bind to IgE (e.g., human IgE) with a KD value of M or less. The binding can be measured by the Octet method, the MSD-SET method, and / or the KinExA method, which are commonly used in the art. For example, the KD value can be measured by the MSD-SET method. The measurement method is described in Estep P. et al., MAbs, 2013.5(2):pp.270-8. The KD value can also be measured by the KinExA method. The measurement method is described in Jonathan K. Fleming et al., Methods Mol Biol. 2018, 1697:1-8.

[0193] For example, when measured by MSD-SET, the KD value of the antigen-binding protein that binds to the aforementioned human-derived IgE antigen is ≦2×10 -9 M, ≤1.5 × 10 -9 M, ≤ 1 × 10 -9 mm, ≤8×10 -10 M, ≤7 × 10 -10 M, ≤6 × 10 -10 M, ≤ 5 × 10 -10 M, ≤4.5 × 10 -10 M, ≤ 4 × 10 -10M, ≤3.5 × 10 -10 M, ≤ 3 × 10 -10 M, ≤2.5 × 10 -10 M, ≤ 2 × 10 -10 M, ≤1.8 × 10 -10 M, ≤1.5 × 10 -10 M, ≤ 1 × 10 -10 M, ≤9 × 10 -11 M, ≤8 × 10 -11 M, ≤7 × 10 -11 M, ≤6 × 10 -11 M, ≤ 5 × 10 -11 M, ≤ 4 × 10 -11 M, ≤ 3 × 10 -11 M, ≤ 2 × 10 -11 M, ≤ 1 × 10 -11 M, ≤9 × 10 -12 It may also be M.

[0194] For example, when measured by the KinExA method, the KD value of the antigen-binding protein of the present application that binds to the IgE is, for example, ≦3.5×10 -12 M, ≤3.4 × 10 -12 M, ≤3.3 × 10 -12 M, ≤3.2 × 10 -12 M, ≤3.1 × 10 -12 M, ≤ 3 × 10 -12 M, ≤2.9 × 10 -12 M, ≤2.8 × 10 -12 M, ≤2.7 × 10 -12 M, ≤2.6 × 10 -12 M, ≤2.5 × 10 -12 M, ≤2.4 × 10 -12 M, ≤2.3 × 10 -12 M, ≤2.2 × 10 -12 M, ≤2.1 × 10 -12 M, ≤ 2.0 × 10 -12 M, ≤1.9 × 10 -12 M, ≤1.8 × 10 -12 M, ≤1.7 × 10 -12 M, ≤1.6 × 10 -12 M, ≤1.5 × 10 -12 M, ≤1.4 × 10 -12 M, ≤1.3 × 10 -12 M, ≤ 1.2 × 10 -12 M or ≦1.1×10 -12It may also be M.

[0195] The antigen binding proteins of the present application are capable of inhibiting the binding of IgE to its receptors FcεRIa and / or FcεRII.

[0196] For example, IC of the antigen-binding protein of the present application that inhibits the binding of IgE to its receptor FcεRIa, as detected by ELISA. 50 Values ​​are ≦2000ng / mL, ≦1800ng / mL, ≦1500ng / mL, ≦1300ng / mL, ≦1000ng / mL, ≦950ng / mL, ≦900ng / mL, ≦850ng / mL, ≦800ng / mL. , ≦750ng / mL, ≦700ng / mL, ≦650ng / mL, ≦600ng / mL, ≦550ng / mL, ≦500ng / mL, ≦450ng / mL, ≦430ng / mL, ≦400ng / mL.

[0197] For example, IC of the antigen-binding protein of the present application that inhibits the binding of IgE to its receptor FcεRIa, as detected by ELISA. 50 The value is ≦4.9×10 -9 M, ≤4.5 × 10 -9 M, ≤4.0 × 10 -9 M, ≤3.5 × 10 -9 M, ≤3.0 × 10 -9 M, ≤2.5 × 10 -9 M, ≤ 2.0 × 10 -9 M, ≤1.5 × 10 -9 M, ≤ 1.0 × 10 -9 M, ≤9.0 × 10 -10 M, ≤8.0 × 10 -10 M, ≤7.0 × 10 -10 M, ≤6.0 × 10 -10 M, ≤5.0 × 10 -10 M, ≤4.0 × 10 -10 M, ≤3.0 × 10 -10 M, ≤2.5 × 10 -10 It may also be M.

[0198] For example, IC of the antigen-binding protein of the present application that inhibits the binding of IgE to its receptor FcεRIa, as detected by ELISA. 50 Values ​​are, for example, ≦35ng / mL, ≦34ng / mL, ≦33ng / mL, ≦32.5ng / mL, ≦32.5ng / mL, ≦32.4ng / mL, ≦32.3ng / mL, ≦ 32.2ng / mL, ≦32.1ng / mL, ≦32.0ng / mL, ≦31ng / mL, ≦30ng / mL, ≦29ng / mL, ≦28ng / mL, ≦27ng / mL, ≦2 6ng / mL, ≦25ng / mL, ≦24ng / mL, ≦23ng / mL, ≦22ng / mL, ≦21ng / mL, ≦20ng / mL, ≦19ng / mL, ≦18ng / mL, It may be ≦17ng / mL, ≦16ng / mL, ≦15ng / mL, ≦14ng / mL, ≦13ng / mL, ≦12ng / mL, ≦11ng / mL or ≦10ng / mL.

[0199] For example, IC of the antigen-binding protein of the present application that inhibits the binding of IgE to its receptor FcεRII, as detected by ELISA. 50 The value may be, for example, ≦4.0 μg / mL, ≦3.9 μg / mL, ≦3.8 μg / mL, ≦3.7 μg / mL, ≦3.6 μg / mL, ≦3.5 μg / mL, ≦3.4 μg / mL, ≦3.3 μg / mL, ≦3.2 μg / mL, ≦3.1 μg / mL or ≦3.0 μg / mL.

[0200] For example, the antigen-binding protein of the present application may comprise an antibody or an antigen-binding fragment thereof. For example, the antibody of the present application may include, but is not limited to, a recombinant antibody, a monoclonal antibody, a human antibody, a murine antibody, a humanized antibody, a chimeric antibody, a camelid single domain antibody, a bispecific antibody, a single chain antibody, a diantibody, a triabody, or a tetrabody.

[0201] For example, the antibody of the present application may be a humanized antibody. For example, the antigen-binding protein of the present application may immunospecifically bind to a relevant antigen (e.g., a human IgE antigen, and an antibody or variant, derivative, analog, or fragment thereof) having a frame region (FR) comprising an amino acid sequence essentially that of a human antibody and a complementarity-determining region (CDR) comprising an amino acid sequence essentially that of a non-human antibody. Here, "essentially," in the case of a CDR, may mean that the amino acid sequence of the CDR has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, or at least 99% identity to the amino acid sequence of the non-human antibody CDR. The humanized antibody of the present application may comprise essentially all of at least one, and usually two, variable domains (Fab, Fab', F(ab')2, Fv), in which all or essentially all of the CDR regions correspond to the CDR regions of a non-human immunoglobulin (i.e., antibody), and all or essentially all of the frame regions are frame regions having human immunoglobulin shared sequences. For example, a humanized antibody may further comprise at least a portion of an immunoglobulin constant region (e.g., Fc), typically a human immunoglobulin constant region. In some embodiments, a humanized antibody comprises at least a light chain or / and a heavy chain variable domain. The antibody may further comprise the CH1, hinge chain, CH2, CH3, and CH4 regions of the heavy chain. For example, a humanized antibody may comprise only a humanized light chain. For example, a humanized antibody may comprise only a humanized heavy chain. For example, a humanized antibody may comprise only a humanized variable domain of a light chain and / or a humanized heavy chain.

[0202] For example, antigen-binding fragments of the present application may include Fab, Fab', F(ab)2, Fv fragments, F(ab')2, scFv, di-scFv, and / or dAbs.

[0203] Polypeptides and immunoconjugates

[0204] In another aspect, the present application provides polypeptides that may comprise the antigen-binding proteins of the present application. The polypeptides of the present application may also comprise other peptide segments or protein fragments, for example, other framework or backbone moieties of a three-dimensional structure that facilitate binding of the antigen-binding protein to an antigen. In some cases, the polypeptides of the present application may also comprise other antigen-binding moieties and / or protein fragments with other functions. For example, the polypeptides of the present application may be fusion proteins, multispecific antibodies, and / or other recombinant proteins.

[0205] In another aspect, the present application provides immunoconjugates that may comprise the antigen-binding proteins or polypeptides of the present application. For example, the immunoconjugates of the present application may comprise other polypeptides, therapeutic agents (e.g., cytotoxic molecules), probes, and / or other antibodies fused or coupled to the antigen-binding protein. For example, the immunoconjugates described herein may comprise pharmaceutically acceptable markers, detection agents, and / or therapeutic agents.

[0206] Nucleic acids, vectors, host cells and production methods

[0207] In another aspect, the present application provides one or more isolated nucleic acid molecules capable of encoding the antigen-binding protein or polypeptide of the present application. For example, each nucleic acid molecule in the one or more nucleic acid molecules can completely encode the isolated antigen-binding protein or polypeptide, or can encode only a portion thereof. The nucleic acid molecules described in the present application may be isolated. In the present application, nucleic acids encoding the antigen-binding protein or polypeptide can be produced by various methods known in the art.

[0208] In another aspect, the present application provides one or more vectors containing one or more nucleic acid molecules described herein. Each vector may contain one or more of the nucleic acid molecules. The vector may also contain other genes, for example, a marker gene that allows selection of the vector in an appropriate host cell under appropriate conditions. The vector may also contain various elements for controlling expression, including a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selection element, and a reporter gene. The vector may also contain a replication origin. The vector may also include, for example, a plasmid, a cosmid, a virus, a phage, or other vectors commonly used in genetic engineering.

[0209] In another aspect, the present application provides cells that may contain one or more nucleic acid molecules and / or one or more vectors described herein. For example, each or each cell may contain one or more nucleic acid molecules or vectors described herein. For example, each or each cell may contain a plurality (e.g., two or more) or multiple species (e.g., two or more) of nucleic acid molecules or vectors described herein. For example, a vector described herein can be introduced into the cells, such as prokaryotic cells (e.g., bacterial cells), CHO cells, NS / 0 cells, HEK293 cells, or other eukaryotic cells, such as plant-derived cells, fungi, or yeast cells. A vector described herein can be introduced into the cells by methods known in the art, such as electroporation, lipofectine transfection, or lipofectamine transfection.

[0210] 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), phages such as λ phage or M13 phage, and animal viruses. Animal viruses used in vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex viruses), poxviruses, baculoviruses, papilloma viruses, and papilloma vacuolar viruses (such as SV40). The vectors may also include various expression control elements, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. The vectors may also include a replication origin. The vectors may also include components that aid in cell entry, such as, but not limited to, viral particles, liposomes, or protein coats.

[0211] For example, the cell may comprise a prokaryotic cell, a yeast cell, or a higher eukaryotic cell. Prokaryotes suitable for this purpose include gram-negative and gram-positive bacteria, such as enterobacteria, e.g., Escherichia coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, Serratia, and Shigella, as well as Bacillus, Pseudomonas, and Streptomyces.

[0212] For example, the cells may comprise a mammalian host cell line, such as monkey kidney cells, a human embryonic kidney cell line, baby hamster kidney cells, Chinese hamster ovary cells, mouse Sertoli cells, human cervical carcinoma cells (HELA), canine kidney cells, human lung cells, human liver cells, mouse breast carcinoma cells, or NSO cells.

[0213] For example, the cells may further include cells transfected in vitro with a vector described herein. For example, the cells may be bacterial cells (e.g., E. 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, or myeloma cells. In some embodiments, the cells may be mammalian cells. For example, the mammalian cells may be HEK293 cells.

[0214] Pharmaceutical compositions and applications

[0215] In another aspect, the present application further provides a method of producing an antigen binding protein of the present application, which may comprise culturing a cell described herein under conditions that allow expression of the antigen binding protein described herein.

[0216] In another aspect, the present application further provides use of an antigen binding protein of the present application, a nucleic acid molecule of the present application, a vector of the present application, a cell of the present application, and / or a pharmaceutical composition of the present application in the preparation of a medicament for preventing, alleviating, and / or treating an IgE-related disease or condition.

[0217] In another aspect, the present application further provides a method for preventing, alleviating, or treating an IgE-associated disease or condition, which may comprise administering to a subject in need thereof an antigen binding protein of the present application, a nucleic acid molecule of the present application, a vector of the present application, a cell of the present application, and / or a pharmaceutical composition of the present application.

[0218] In another aspect, the present application further provides an antigen binding protein of the present application, a nucleic acid molecule of the present application, a vector of the present application, a cell of the present application, and / or a pharmaceutical composition of the present application that can be used to prevent, alleviate, or treat an IgE-related disease or condition.

[0219] For example, the IgE-related disease or condition may include a disease or condition for which Omalizumab (trade name Xolair) is approved and suitable for treatment worldwide (e.g., the United States, China, Europe, etc.). For example, the disease or condition may include an allergic disease.

[0220] For example, the subject may include humans and non-human animals, for example, but not limited to, a cat, dog, horse, pig, cow, sheep, rabbit, mouse, rat, or monkey.

[0221] In another aspect, the present application further provides a pharmaceutical composition comprising an antigen binding protein of the present application, a nucleic acid molecule of the present application, a vector of the present application and / or a cell of the present application, and optionally a pharmaceutically acceptable carrier.

[0222] For example, the pharmaceutical composition may additionally comprise one or more other therapeutic agents suitable for the treatment or prevention of an IgE-related disease or condition.

[0223] For example, the pharmaceutically acceptable carrier may enhance or stabilize the composition or facilitate preparation of the composition. For example, the pharmaceutically acceptable carrier may include physiologically compatible solvents, dispersion media, coating materials, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc.

[0224] For example, the administration can be performed by different ways, such as intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.

[0225] For example, the pharmaceutical compositions of the present application can be administered by a variety of methods known in the art, which vary depending on the desired outcome, route, and / or mode of administration. For example, administration may be intravenous, intramuscular, intraperitoneal, or subcutaneous, or adjacent to the target site. For example, the pharmaceutical compositions may be formulated for administration into the vitreous of the eye. Depending on the route of administration, the antigen-binding proteins (e.g., antibodies, bispecific, and multispecific molecules) may be coated with a material to protect the compound from the effects of acids or other natural conditions that may inactivate the compound.

[0226] For example, the pharmaceutical compositions of the present application may be sterile and fluid. Proper fluidity can be maintained, for example, by the use of coating materials (such as lecithin), by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0227] For example, the composition may contain isotonic agents, such as sugars, polyols such as mannitol, sorbitol, or sodium chloride. For example, prolonged absorption of the injectable pharmaceutical composition can be achieved by including in the pharmaceutical composition an agent which delays absorption (for example, aluminum monostearate or gelatin).

[0228] For example, for storage, the present pharmaceutical compositions may be prepared by mixing the antigen-binding protein of the present application, the nucleic acid molecule of the present application, the vector of the present application, and / or the cell of the present application, and optionally a pharmaceutically acceptable vector, excipient, or stabilizer. For example, the pharmaceutical compositions of the present application may be in the form of a lyophilized formulation or an aqueous solution.

[0229] For example, a pharmaceutically acceptable carrier herein may include a pharmaceutically acceptable vector, excipient, or stabilizer.

[0230] For example, acceptable vectors, excipients, or stabilizers herein are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, acetate, and other organic acids.

[0231] For example, pharmaceutical compositions containing the antigen-binding proteins of the present application may be in a water-soluble form.

[0232] For example, pharmaceutical compositions for internal administration can be sterile. This can be readily achieved by filtration through sterile filtration membranes or other methods. For example, administration of pharmaceutical compositions comprising the antigen-binding proteins of the present application in the form of a sterile aqueous solution can be achieved by a variety of methods, including, but not limited to, oral, subcutaneous, intravenous, intranasal, intraaural, transdermal, topical (e.g., gels, ointments, detergents, creams, etc.), intraperitoneal, intramuscular, intrapulmonary, parenteral, rectal, or intraocular. In some cases, for example, when treating wounds, inflammation, etc., the antigen-binding proteins of the present application can be applied directly as a solution or spray.

[0233] For example, the pharmaceutical compositions of the present application can be prepared by methods known and conventionally practiced in the art. For example, the pharmaceutical compositions of the present application can be manufactured under GMP conditions. Typically, a therapeutically effective amount or effective amount of IgE-binding protein is used in the pharmaceutical compositions of the present application. For example, the IgE-binding protein can be prepared in a pharmaceutically acceptable dosage form by conventional methods known to those skilled in the art. The dosage regimen is adjusted to provide the optimum desired response (e.g., a therapeutic response). The actual dosage level of the active ingredient in the pharmaceutical compositions of the present application can be varied to obtain an amount of the active ingredient that is not toxic to the patient and is effective to achieve the desired therapeutic response, composition, and mode of administration for the particular patient. The selected dosage level will depend on various pharmacokinetic factors, such as the activity of the particular composition or its ester, salt, or amide used in the present application, the route of administration, the time of administration, the excretion rate of the particular compound used, the duration of treatment, other drugs, compounds, and / or substances used in combination with the particular composition used, the age, sex, weight, condition, general health, and previous medical history of the subject being treated, and other factors.

[0234] For example, subcutaneous administration may be used when the patient is able to self-administer the pharmaceutical composition. Many protein therapeutics are not effective enough to be prepared in a therapeutically effective amount in the maximum volume allowed for subcutaneous administration. The antigen-binding proteins disclosed herein are adaptable to subcutaneous administration. For example, they have increased potency, improved plasma half-life, and improved solubility.

[0235] As is known in the art, protein therapeutics can be delivered by IV infusion or bolus injection, and the antigen binding proteins disclosed herein can also be delivered using such methods.

[0236] In another aspect, the present application further provides a method for detecting IgE in a sample, the method comprising administering an antigen binding protein described herein.

[0237] For example, a sample obtained from a subject is contacted with an antigen-binding protein (e.g., an IgE-binding protein) of the present application. For example, the IgE-binding protein can be labeled with a detectable label or reporter molecule, or an anti-IgE-binding protein can be used as a capture ligand to selectively isolate IgE from a patient sample. Alternatively, an unlabeled anti-IgE-binding protein can be bound to a secondary antibody for detection applications, the secondary antibody itself bearing a detectable label. The detectable label or reporter molecule can be 3 H, 14 C. 32 P, 35 S or 125 The IgE may be a radioisotope such as I, a fluorescent or chemiluminescent moiety such as fluorescein isothiocyanate, rhodamine, or an enzyme such as alkaline phosphatase, β-galactosidase, horseradish peroxidase, or luciferase; certain exemplary assays that can be used to detect or measure IgE in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).

[0238] Samples that can be used for IgE detection herein may include any tissue or fluid sample from a subject under normal or pathological conditions that contains a detectable amount of IgE protein or its fragments. For example, the IgE protein level of a particular sample obtained from a healthy subject (e.g., a subject not suffering from an IgE-related disease) is measured to first establish a baseline or standard level of IgE. This baseline IgE level is then compared to the IgE level measured in a sample obtained from an individual suspected of suffering from an IgE-related disease or who has symptoms associated with that condition. The IgE-binding protein may contain no additional label or may contain an N- or C-terminal label. For example, the label may be biotin. In binding assays, the location of the label (if present) can determine the orientation of the peptide relative to the surface to which it binds. For example, if a surface is coated with anti-biotin protein, a peptide containing an N-terminal biotin will release the C-terminal portion of the peptide from the surface.

[0239] Without being limited by any theory, the following examples are merely intended to illustrate the technical solutions of the present invention, and are not intended to limit the scope of the present invention.

[0240] Example

[0241] Example 1: Production of the antigen-binding protein of the present application

[0242] 1-1 Production of anti-human IgE mouse monoclonal antibody

[0243] (1) Mouse immunization

[0244] Four-week-old BALB / c mice were subcutaneously injected with 20 μg of recombinantly expressed human IgE Fc region (CH2-4) protein (IgE-Fc, SEQ ID NO: 42) in Freund's complete adjuvant. Injections were given every 3-4 weeks for a total of five injections. Finally, a single intraperitoneal injection of 20 μg of recombinant human IgE-Fc protein was administered.

[0245] (2) Measurement of binding of mouse serum to human IgE-Fc antigen by ELISA

[0246] A microplate reader (Costar) was coated with recombinant human IgE-Fc protein and left overnight at room temperature. The coating solution was discarded, and each well was blocked for 0.5 hours with 2.5% nonfat milk dissolved in phosphate-buffered saline (PBS). The wells were then washed with PBS containing 0.05% Tween-20. Diluted serum and an irrelevant antibody (negative control) were then added and incubated for 1 hour at room temperature. The wells were then washed with PBS containing 0.05% Tween-20. Then, 50 μl of HRP-conjugated sheep anti-mouse IgG polyclonal antibody (Jackson Laboratory) was added to each well as a detection antibody. After serum testing, mice containing high levels of anti-recombinant human IgE-Fc protein antibody serum were identified.

[0247] (3) Cell fusion, screening, and production of mouse monoclonal antibodies

[0248] After identifying mice containing high levels of anti-human IgE-Fc antibody serum by serum testing, the spleens of these mice were removed and fused to the mouse myeloma Sp2 / 0 cell line. 8 Sp2 / 0 cells and 5 x 10 8 The spleen cells were mixed with 100 cells and fused in a 50% polyethylene glycol (PEG, molecular weight 1450) and 5% dimethyl sulfoxide (DMSO) solution. The number of spleen cells was increased to 7.5 × 10 in Iscove's medium (containing 10% fetal bovine serum, 100 units / mL penicillin, 100 μg / mL chromicin, 10 mM hypoxanthine, 0.4 μM aminopterin, and 1.6 mM thymidine). 5The solution was adjusted to 1 / mL and 0.2 mL was added to each well of a 96-well culture plate. The plate was placed in an incubator at 37°C with 5% CO2. After 10 days, the medium in each well was removed and the ability of the mouse antibody to bind to human IgE-Fc was tested, thereby screening positive wells for binding to human IgE-Fc. Furthermore, the fused cells in the wells containing antibodies capable of binding to human IgE-Fc were subcloned and similarly screened by ELISA to obtain hybridoma cell lines expressing high-affinity mouse monoclonal antibodies.

[0249] Purified mouse monoclonal antibodies were produced to obtain antibody samples for testing. Cell clones producing specific antibodies were cultured in RPMI 1640 medium supplemented with 10% FCS. At a cell density of approximately 5 x 10 5 When the cell density reached 100 cells / ml, the medium was replaced with serum-free medium. After 2-4 days, the culture medium was centrifuged and the culture supernatant was collected. Protein G columns were used to purify the antibodies. The monoclonal antibody eluate was dialyzed against 150 mM NaCl. The dialyzed solution was filtered through a 0.2 μm filter and sterilized to obtain the antibody samples to be tested. Monoclonal antibodies SE3 (11 / 5), SE5 (4 / 23), SE2, and SE5 were produced and obtained.

[0250] 1-2 Production of anti-human IgE chimeric antibodies

[0251] (1) Cloning of the heavy chain variable region of a mouse antibody

[0252] To obtain the coding DNA sequences for the heavy and light chain variable regions of the mouse antibody, mRNA was isolated from mouse hybridoma cells using an mRNA purification kit (NEB), and cDNA (SMARTer RACE kit, Clontech) was then prepared. The heavy chain variable region DNA fragment was cloned from the cDNA by polymerase chain reaction (PCR). The 5'-primer used in PCR was a mixture of 0.4 μM Primer 1 (5'-ctaatacgactcactatagggcAAGCAGTGGTATCAACGCAGAGT-3', shown in SEQ ID NO: 53) and 2 μM Primer 2 (5'-ctaatacgactcactatagggc-3', shown in SEQ ID NO: 54). The 3'-primer used in PCR was Primer 3 (5'-catcccagggtcaccatggagtta-3', shown in SEQ ID NO: 55). Primer 3 is homologous to the mouse IgG1 heavy chain constant region and is antisense. The heavy chain variable region DNA fragment obtained after gel purification was cloned into a TOPO-TA vector (Invitrogen) and sequenced to obtain the amino acid sequence (shown in SEQ ID NO:8) and nucleotide sequence (shown in SEQ ID NO:57) of the variable region encoding the SE2 mouse hybridoma heavy chain. The amino acid sequence of its complementarity-determining regions, HCDR1 (shown in SEQ ID NO:1), HCDR2 (shown in SEQ ID NO:2), and HCDR3 (shown in SEQ ID NO:3), was determined according to the Kabat definition. The amino acid sequence (shown in SEQ ID NO:28) and nucleotide sequence (shown in SEQ ID NO:61) of the variable region encoding the SE5 mouse hybridoma heavy chain, and the amino acid sequence of its complementarity-determining regions, HCDR1 (shown in SEQ ID NO:1), HCDR2 (shown in SEQ ID NO:26), and HCDR3 (shown in SEQ ID NO:3), were also determined. The definition of complementarity-determining region was based on Kabat E. et al., Sequences of Proteins of Immunological Interest, 5th Edition, US Department of Health and Human Services, NIH Publication No. 91-3242.

[0253] (2) Cloning of the light chain variable region of a mouse antibody

[0254] Using a method similar to step (1), the light chain variable region DNA fragment was cloned from the cDNA using the above 5'-primer mixture (primer 1 and primer 2) and a 3'-primer (primer 4, 5'-gactgaggcacctccagatgttaa-3', shown in SEQ ID NO: 56) that is homologous to the mouse immunoglobulin light chain constant region and is an antisense primer. These obtained DNA fragments were cloned into a TOPO-TA vector and sequenced to obtain the variable region amino acid sequence (shown in SEQ ID NO: 16) and nucleotide sequence (shown in SEQ ID NO: 58) encoding the SE2 mouse hybridoma light chain, and the amino acid sequences of its complementarity-determining regions LCDR1 (shown in SEQ ID NO: 9), LCDR2 (shown in SEQ ID NO: 10), and LCDR3 (shown in SEQ ID NO: 11), as well as the variable region amino acid sequence (shown in SEQ ID NO: 35) and nucleotide sequence (shown in SEQ ID NO: 62) encoding the SE5 mouse hybridoma light chain, and the amino acid sequences of its complementarity-determining regions LCDR1 (shown in SEQ ID NO: 9), LCDR2 (shown in SEQ ID NO: 29), and LCDR3 (shown in SEQ ID NO: 30).

[0255] (3) Production of chimeric antibodies

[0256] The heavy and light chain variable region sequences of the mouse monoclonal antibody obtained by cloning in step 2 were seamlessly cloned into pCDNA3.1 (purchased from Miaoling Biology) eukaryotic expression vectors containing a signal peptide (SEQ ID NO: 50) and a human IgG1 heavy chain constant region (SEQ ID NO: 42) or a signal peptide (SEQ ID NO: 50) and a human Kappa light chain constant region (SEQ ID NO: 43), respectively. After DNA sequencing confirmed the sequence was correct, the vectors were amplified in DH5α bacteria and the plasmids were prepared using the NucleoBond Xtra Midi Plus (Macherey-Nagel) plasmid extraction kit. The resulting chimeric monoclonal antibody heavy and light chain plasmids were co-transfected into Expi293 cells (purchased from Thermo Fisher) using PEI (PolyScience) to transiently express the monoclonal antibody. The transiently expressed antibody was purified using a protein A column, and the monoclonal antibody eluate was dialyzed against PBS. The dialyzed solution was filtered through a 0.2 μm filter and sterilized to obtain the antibody sample to be tested.

[0257] 1-3 Production of humanized anti-human IgE antibodies

[0258] (1) Humanization of anti-human IgE mouse monoclonal antibody

[0259] Based on the homology-modeled antibody variable region structure, important amino acid positions that may affect antigen-antibody interactions were determined, and restoration mutations were made to the important amino acid positions. Humanized amino acid sequences of the SE2 and SE5 antibody variable regions were obtained by CDR grafting. The amino acid sequence of the humanized SE2 heavy chain variable region is set forth in SEQ ID NO: 21, the amino acid sequence of the humanized SE2 light chain variable region is set forth in SEQ ID NO: 25, the amino acid sequence of the humanized SE5 heavy chain variable region is set forth in SEQ ID NO: 37, and the amino acid sequence of the humanized SE5 light chain variable region is set forth in SEQ ID NO: 40. The DNA sequences obtained after reverse translation of the amino acid sequences were synthesized, and the humanized nucleotide sequences of the SE2 heavy chain variable region are set forth in SEQ ID NO: 21, the SE2 light chain variable region is set forth in SEQ ID NO: 60, the SE5 heavy chain variable region is set forth in SEQ ID NO: 63, and the SE5 light chain variable region is set forth in SEQ ID NO: 64. The resulting DNA was used to construct a recombinant antibody expression vector.

[0260] (2) Production of humanized anti-human IgE antibodies

[0261] The heavy and light chain variable regions of the humanized monoclonal antibody obtained in step 1 were seamlessly cloned into pCDNA3.1 (purchased from Miaoling Biology) eukaryotic expression vectors containing a signal peptide (shown in SEQ ID NO: 50) and a human IgG1 heavy chain constant region (shown in SEQ ID NO: 42) or a signal peptide (shown in SEQ ID NO: 50) and a human Kappa light chain constant region (shown in SEQ ID NO: 43), respectively. After DNA sequencing confirmed the correctness, the plasmids were prepared using a NucleoBond Xtra Midi Plus (Macherey-Nagel) plasmid extraction kit. The prepared humanized monoclonal antibody heavy and light chain plasmids were co-transfected into Expi293 cells (purchased from Thermo Fisher) using PEI (PolyScience) to transiently express the monoclonal antibody. The transiently expressed antibody was purified using a protein A column, and the monoclonal antibody eluate was dialyzed against PBS. The dialyzed solution was filtered through a 0.2 μm filter and sterilized to obtain the antibody sample to be tested.

[0262] (3) Humanized antibody after modification

[0263] Sequence analysis of the antibody variable region revealed that N53 in the SE5 heavy chain CDR2 region (shown in SEQ ID NO: 26) is a potential N-glycosylation site. N53S / T57S mutations were made to humanized SE5, and M135Y, S137T, and / or T139E (YTE) modifications were simultaneously made to the Fc region of SE5 (SEQ ID NO: 42) to enhance the affinity of the antibody molecule for the neonatal receptor FcRn under acidic conditions, thereby extending the half-life of the antibody molecule in vivo and achieving the goal of extending the administration cycle. The modified humanized molecule is called SE5ss.

[0264] Example 2: Detection of blocking activity of the antigen-binding protein (mouse monoclonal antibody) of the present application

[0265] The blocking activity of purified mouse monoclonal antibodies against the binding of human IgE-Fc to its receptor, FcεRIa, was detected by ELISA. Specifically, recombinantly expressed human FcεRIa-Fc protein (amino acid sequence shown in SEQ ID NO: 51) was diluted with PBS to a final concentration of 2.5 μg / mL, and 100 μL / well of the diluted solution was used to coat a microplate reader (purchased from Corning, catalog number 42592) at 4°C overnight. The next day, the coating solution was discarded, and the wells were blocked with 2.5% nonfat milk in PBS for 1 hour. The wells were then washed with PBS containing 0.05% Tween 20. A 1 μg / mL premix solution containing biotin-labeled human IgE-Fc (amino acid sequence shown in SEQ ID NO: 52) and gradient diluted mouse or control antibodies, which had been mixed for 10 minutes, was added, and the mouse or control antibody was diluted two-fold from 5 μg / mL using mouse or control antibody bulk beads (purchased from Novartis, catalog number: Xolair) prepared according to Example 1 (i.e., to 5 μg / mL, 2.5 μg / mL, 1.25 μg / mL, 0.625 μg / mL, and 0.002441 μg / mL), followed by incubation at room temperature for 1 hour. After discarding the supernatant, the wells were washed with PBS containing 0.05% Tween 20 and detected using HRP-labeled streptavidin (purchased from Seiko Seibutsu, catalog number: D111054-0001) diluted 1:1000. The detection results are shown in Figure 1. Each of the purified mouse monoclonal antibodies had the ability to block the binding of human IgE-Fc to its receptor FcεRIa to different degrees, and both SE2 and SE5 had stronger blocking activity than the control antibody omalizumab.

[0266] Example 3: Detection of blocking activity of the antigen-binding protein (chimeric antibody) of the present application

[0267] To detect the activity of the recombinantly expressed chimeric monoclonal antibody obtained by cloning, the blocking activity of the purified chimeric antibody in blocking the binding of human IgE-Fc to its receptor, FcεRIa, was detected by an ELISA method similar to that described in Example 2. Specifically, recombinantly expressed human FcεRIa-Fc protein (amino acid sequence shown in SEQ ID NO: 51) was diluted with PBS to a final concentration of 1 μg / mL, and 100 μL / well of the diluted solution was used to coat a microplate reader (purchased from Corning, catalog number: 42592) and left overnight at 4°C. The next day, the coating solution was discarded, and the wells were blocked with 2.5% non-fat milk dissolved in PBS for 1 hour, and then washed with PBS containing 0.05% Tween 20. A 100 ng / mL premix solution containing biotin-labeled human IgE-Fc (amino acid sequence shown in SEQ ID NO: 52) and gradient-diluted chimeric or mouse antibodies, which had been mixed for 10 minutes in advance, was added. The chimeric or mouse antibodies were diluted 3-fold starting from 2 μg / mL (i.e., 2 μg / mL, 666.67 ng / mL, 222.22 ng / mL, 74.07 ng / mL, 24.69 ng / mL, 8.23 ​​ng / mL, 2.74 ng / mL, and finally 0 ng / mL), and incubated at room temperature for 1 hour. After discarding the supernatant, the wells were washed with PBS containing 0.05% Tween 20. Detection was performed using HRP-labeled streptavidin (purchased from Seiko Seibutsu, catalog number D111054-0001) diluted 1:1000. The inhibition rate (%) of antibody concentration x = (OD of antibody concentration 0). 450 Readout - OD at antibody concentration x 450 Reading) / OD at 0 antibody concentration 450 The inhibition rates for different antibodies at different concentrations were calculated by multiplying the readings by 100. The detection results are shown in Figure 2. The chimeric recombinant monoclonal antibodies constructed using the mouse monoclonal variable region sequences obtained by cloning had blocking activity similar to that of the mouse parent antibody purified by the hybridoma.

[0268] Example 4: Detection of blocking activity of the antigen-binding protein of the present invention (humanized anti-human IgE antibody)

[0269] 1) The blocking activity of purified mouse, chimeric, and humanized antibodies against the binding of human IgE-Fc to its receptor, FcεRIa, was detected using an ELISA method similar to that described in Example 2. Specifically, recombinantly expressed human FcεRIa-Fc protein (amino acid sequence shown in SEQ ID NO: 51) was diluted with PBS to a final concentration of 1 μg / mL, and 100 μL / well of this was used to coat a microplate reader (purchased from Corning, catalog number 42592) and left overnight at 4°C. The next day, the coating solution was discarded, and the wells were blocked with 2.5% nonfat milk dissolved in PBS for 1 hour, followed by washing with PBS containing 0.05% Tween 20. A 100 ng / mL premix containing biotin-labeled human IgE-Fc (amino acid sequence shown in SEQ ID NO: 52) and gradient diluted antibodies (each diluted 3-fold starting from 1 μg / mL (i.e., 1 μg / mL, 333.33 ng / mL, 111.11 ng / mL, 37.04 ng / mL, 12.35 ng / mL, 4.12 ng / mL, 1.37 ng / mL, and finally 0 ng / mL)) was added and incubated at room temperature for 1 hour. After discarding the supernatant, the wells were washed with PBS containing 0.05% Tween 20 and detected with HRP-labeled streptavidin (purchased from Seiko Seibutsu, catalog number: D111054-0001). The detection results are shown in Figures 3 to 5, and show that the recombinantly expressed humanized monoclonal antibodies SE2 and SE5 had blocking activity similar to that of their corresponding chimeric and mouse parent antibodies, respectively (Figure 3).

[0270] 2) By the method of step 1) of this example, the blocking activity of the SE2 humanized antibody, the SE5 humanized antibody, and omalizumab to block the binding of human IgE-Fc to its receptor FcεRIa was detected. In the comparison experiment between the SE2 humanized antibody and omalizumab, the antibody concentration was diluted three-fold starting from 3 μg / mL (i.e., 3 μg / mL, 1 μg / mL, 333.33 ng / mL, 111.11 ng / mL, 37.04 ng / mL, 12.35 ng / mL, 4.12 ng / mL, 1.37 ng / mL, and finally 0 ng / mL). In the comparison experiment between the SE5 humanized antibody and omalizumab, the antibody concentration was diluted three-fold starting from 10 nM (i.e., 10 nM, 3.33 nM, 1.11 nM, 370.37 pM, 123.46 pM, 41.15 pM, 13.72 pM, and finally 0 pM). The SE5 comparison experiment included a control antibody (i.e., the control in Figure 5, the isotype Her2 antibody pertuzumab, whose sequence was derived from the IMGT antibody library).

[0271] The following formula: Inhibition rate (%) of antibody concentration X = (OD at antibody concentration 0) 450 Readout - OD at antibody concentration x 450 The inhibition rates for different antibodies and concentrations were calculated by dividing the OD450 reading at antibody concentration 0 by 100. The results are shown in Figures 4 and 5. The SE2 and SE5 humanized antibodies both had stronger blocking activity than the control antibody omalizumab, and the blocking IC values ​​of SE2 and omalizumab were 50 The values ​​were approximately 416.7 ng / mL and 2110 ng / mL, respectively, and the blocking IC values ​​of SE5 and omalizumab were approximately 416.7 ng / mL and 2110 ng / mL, respectively. 50 The values ​​were approximately 266.2 pM and 4996 pM, respectively.

[0272] Example 5: Detection of affinity of the antigen-binding protein of the present invention (humanized anti-human IgE antibody)

[0273] The equilibrium affinity of humanized antibodies and human IgE was detected using ultrasensitive factor electrochemiluminescence assay-solution equilibrium titration (MSD-SET), as described in Estep P. et al., MAbs, 2013.5(2):pp.270-8. The solution equilibrium titration (SET) was performed in PBS + 0.1% BSA buffer without IgG (PBSF), in which the antigen human IgE (purchased from Abcam, catalog number: ab65866) was kept constant at 10-100 pM and incubated with 3-fold serial dilutions of the humanized antibodies starting from 0.1-100 nM. Specifically, when measuring SE2 and SE5, the antigen human IgE was kept constant at 13.33 pM, and the humanized antibodies SE2 and SE5 were serially diluted 3-fold from 166.67 pM to 0.0085 pM. When measuring omalizumab, the antigen human IgE was kept constant at 133.3 pM, and omalizumab was serially diluted 3-fold from 66.67 nM to 1.13 pM. 20 nM of antibody diluted in PBS was coated onto a standard-binding MSD-ECL plate (purchased from MSD, catalog number: L15XA-3) and left overnight at 4°C. The MSD-ECL plate was blocked with 1% casein for 30 minutes and simultaneously shaken at 700 rpm. The MSD-ECL plate was then washed three times with wash buffer (PBS + 0.05% Tween 20). Samples incubated with 2-fold serial dilutions of antigen human IgE and humanized antibody were added to the MSD-ECL plate and incubated for 150 s with shaking at 700 rpm, followed by one wash. 250 ng / mL of PBSF containing sulfo-tagged streptavidin was added to the MSD-ECL plate. After 5 minutes, the MSD-ECL plate was washed three times with wash buffer (PBS + 0.05% Tween 20). 1x Reader Buffer T (purchased from MSD, catalog number: R92TC-1) was read on an MSD Sector Imager instrument (model number: MESO QuickPlex SQ 120). The percentage of free antigen was plotted as a function of titrated antibody in Prism-GraphPad 6 software, and the KD was extracted by fitting to a quadratic equation. The measurement results are shown in Table 1 below. [Table 1]

[0274] As can be seen from the results shown in Table 1, the equilibrium affinities of the humanized recombinant monoclonal antibodies SE2 and SE5 for binding to human IgE were approximately 10.880 pM and 9.995 pM, respectively, while the control antibody omalizumab was approximately 2489 pM, showing significantly improved affinities.

[0275] Example 6: Detection of affinity of the antigen-binding protein of the present application (modified humanized anti-human IgE antibody)

[0276] KinExA was used to detect the equilibrium affinity of the humanized antibodies and the control antibody (Omalizumab) for human IgE (derived from U266 cells (ATCC), production method see Mol Immunol. 1986, 23(2):159-67). Solution equilibrium titration (SET) was performed in PBS + 0.1% BSA buffer without IgG (PBSF), among which the antigen human IgE-Biotin (biotin-labeled human IgE, production method Thermofisher EZ-Link TMThe Sulfo-NHS-LC-Biotinylation Kit (see the instruction manual) was maintained at a constant concentration of 20 pM to 5 nM and incubated with two-fold serial dilutions of the antibody from 2 nM to 1 μM. Specifically, when measuring the humanized antibody SE5ss, the antigen human IgE-Biotin was maintained at a constant concentration of 20 pM and serially diluted two-fold from 2 nM to 30.52 fM. The mixed sample was incubated at room temperature for 30 hours. When measuring the control antibody Omalizumab, the antigen human IgE-Biotin was maintained at a constant concentration of 5 nM and serially diluted two-fold from 1 μM to 15.26 pM. The mixed sample was incubated at room temperature for 20 hours. A sample incubated with the antigen human IgE-Biotin and two-fold serial dilutions of antibody was loaded onto antibody-coated beads in a KinExA instrument (model number: 4000). Any free antigen human IgE-Biotin in the sample that did not bind to the antibody bound to the beads. A fluorescent secondary antibody, Alexa Fluor 647 Streptavidin, was then sequentially loaded onto the beads. The instrument detected the fluorescent signal on the beads, which was then fitted using KinExA software to obtain KD values. The measurement results are shown in Figures 6A-B. The experimental results indicated that the equilibrium binding affinities of the humanized recombinant monoclonal antibodies SE5ss and Omalizumab prepared in Example 1 to human IgE were approximately 2.08 pM and 1.79 nM, respectively, indicating that SE5ss had a much higher affinity than Omalizumab.

[0277] Example 7: Detection of blocking activity of the antigen-binding protein of the present invention (modified humanized anti-human IgE antibody)

[0278] (1) Blocking activity of the antigen-binding protein of the present invention to block the binding of human IgE to its receptor FcεRIa

[0279] The blocking activity of the SE5ss monoclonal antibody against human IgE binding to its receptor, FcεRIa, was detected by ELISA. Specifically, recombinantly expressed human FcεRIa-Fc protein was diluted to 1 μg / mL in PBS, and 100 μL / well of the diluted solution was coated onto a microplate reader (Corning, Cat. No. 42592) and incubated overnight at 4°C. The next day, the coating solution was discarded, and the plate was blocked with PBS containing 2.5% nonfat milk at 25°C for 1 hour. After blocking, the plate was washed three times with PBST (PBS + 0.05% Tween 20). After premixing for 1 hour, a 100 ng / mL premix containing human IgE and gradient-diluted antibodies to be detected (SE5ss, omalizumab, and human IgG) was added and incubated for 1 hour at 25°C. The antibodies to be detected were serially diluted three-fold from 10 μg / mL to 0.17 ng / mL in 10-point increments, with a 0 μg / mL point added. After incubation, the plate was washed three times with PBST. Detection was performed with a goat anti-human IgE antibody (Invitrogen, catalog number: A18795) diluted 1:5000 and a rabbit anti-goat IgG antibody labeled with HRP (Seiko, catalog number: D110117-0100) diluted 1:2000. The OD was measured. 450 The values ​​were analyzed using a four-parameter regression model to determine the IC value of the antibody. 50 The results are shown in Figure 7A. Both SE5ss and Omalizumab prepared in Example 1 can effectively block the binding of human IgE to the FcεRIα receptor, and the IC 50 The agonists' potencies were 32.38 ng / mL and 1009 ng / mL, respectively, and the ability of SE5ss to block the binding of human IgE to the FcεRIα receptor was far greater than that of omalizumab.

[0280] (2) Blocking activity of the antigen-binding protein of the present invention to block human IgE and its receptor, FcεRII (CD23) receptor

[0281] The blocking activity of SE5ss against human IgE and its receptor FcεRII (CD23) was detected using an ELISA method similar to step (1). Specifically, recombinant human FcεRII (CD23, purchased from Yiqiao Shenzhou, catalog number: 10261-H07H) protein was diluted to 1.5 μg / mL in PBS and 100 μL / well was used to coat a microplate reader (Corning, catalog number: 42592). The next day, the coating solution was discarded and the plate was blocked with 1% casein for 2 hours at room temperature. Biotin-labeled recombinantly expressed β-lactoglobulin-specific human IgE (bIgEk, Structure 15, 1413-1421, 2007) and β-lactoglobulin (Sigma, catalog number: L3908) were homogeneously mixed at equal volumes (molar ratio of approximately 1:10) (both concentrations 60 μg / mL) and incubated at 37°C for 1 hour. The antibodies (SE5ss, omalizumab, and human IgG) were serially diluted 3-fold in seven steps from 300 μg / mL to 0.41 μg / mL, with a 0 μg / mL point added. The concentration of the bIgEk-Biotin and β-lactoglobulin complex was kept constant at 30 μg / mL. After incubation, the mixture was washed three times with PBST. Detection was performed with Streptavidin-HRP antibody (purchased from Seikoh, Cat. No. D110513-0100) diluted at 1:8000. OD 450 The values ​​were analyzed using a four-parameter regression model to determine the IC value for each antibody. 50 The results are shown in Figure 7B. The experimental results show that both SE5ss and Omalizumab prepared in Example 1 can effectively block the binding of human IgE to the FcεRII (CD23) receptor, and the IC 50 The values ​​were 3.756 μg / mL and 3.444 μg / mL, respectively, indicating that the abilities of both compounds to block the binding of human IgE to FcεRII protein were similar.

[0282] (3) Detection of the inhibitory activity of the antigen-binding protein of the present invention on human IgE-mediated reporter gene activation in RBL-2H3-FcεRIα-NFATLuc cells

[0283] Construction of the RBL-2H3-FcεRIα-NFATLuc cell line: To detect IgE-mediated cell activation and blockade of IgE cellular activity by the antigen-binding protein of this application, a luciferase reporter gene-expressing cell line, designated RBL-2H3-FcεRIα-NFATLuc, expressing the human FcεRIα receptor and NFAT-driven NFAT was constructed based on the rat basophil RBL-2H3 cell line (Chinese Academy of Sciences Cell Bank). The construction process was described in Analytical and Bioanalytical Chemistry, volume 412, pages 1901-1914 (2020); Allergy 2010, 65:1266-1273; J Immunol, July 1, 1996, 157(1)221-230.

[0284] Logarithmic growth phase RBL-2H3-FcεRIα-NFATLuc cells were seeded into a 96-well flat-bottom plate at approximately 5E4 cells / well in 50 μL of medium. A premixed solution of human IgE and gradient concentrations of the target antibodies (SE5ss, omalizumab, and human IgG) was then added and incubated at 37°C in a 5% CO2 incubator for approximately 20 hours. Human IgE was kept constant at a final concentration of 1000 ng / mL, and the target antibodies were serially diluted 3-fold in eight steps from 45 μg / mL to 0.021 μg / mL, with an additional 0 μg / mL point. The next day, the plate was washed twice with PBS, and complete medium containing 10 μg / mL of anti-human IgE antibody (purchased from Invitrogen, catalog number A18795) was added to each well and incubated in the incubator for approximately 3 hours. After completion, 100 μL of One-Lite™ (Nouweizan) detection reagent was added to each well, and after waiting for about 5 minutes, the wells were transferred to a 96-well white plate. The plate was then detected using a microplate reader luminescence detection module. The results were used to determine the IC value of the antibody using a four-parameter regression model fitting inhibition curve. 50The results are shown in Figure 8. Both SE5ss and Omalizumab were able to inhibit human IgE-mediated effector cell reporter gene activation, and the IC 50 The inhibitory effects of SE5ss were 0.2641 μg / mL and 0.6093 μg / mL, respectively, indicating that the inhibitory effect of SE5ss was significantly stronger than that of omalizumab.

[0285] (4) Detection of the inhibitory activity of the antigen-binding protein of the present invention on RBL-2H3-FcεRIα-NFATLuc cell activation induced by allergic human serum from different allergens.

[0286] RBL-2H3-FcεRIα-NFATLuc cells in logarithmic growth phase were seeded into 96-well flat-bottom plates at approximately 5E4 cells / well in 50 μL of medium. Gradient concentrations of the antibodies (SE5ss, omalizumab, and human IgG) and human sera for different allergens were added to the cells, and the cells were incubated in a 37°C, 5% CO2 incubator for approximately 20 hours. Because the IgE content of each serum for a specific allergen differed, different concentrations of serum and antibody were used. The concentration used for the mite-allergic human serum (PlasmaLab, catalog number: PL25740) was 1.5%, and the antibodies were serially diluted 3-fold in nine steps from a final concentration of 10,000 ng / mL to 5 ng / mL. The concentration used for the shrimp-allergic human serum (PlasmaLab, catalog number: PL24819) was 3%, and omalizumab and human IgG were serially diluted 3-fold in 12 steps from a final concentration of 1,200.00 ng / mL to 0.01 ng / mL. SE5ss was serially diluted 3-fold in 11 steps from a concentration of 133.33 ng / mL to 0.002 ng / mL. The concentration used for the walnut-allergic human serum (PlasmaLab, catalog number: PL27530) was 1.5%, and the antibodies were serially diluted 3-fold in nine steps from a final concentration of 10,000 ng / mL to 1.5 ng / mL. 0 μg / mL was added as a control for all three groups. The next day, the plates were washed twice with PBS, and 100 μL of complete medium containing 10 μg / mL of the corresponding allergen (mite allergen purchased from Greerlabs, catalog number XPB82D3A25; shrimp allergen purchased from Wolcavi, catalog number M110227; walnut allergen purchased from Wolcavi, catalog number M110343) was added to each well and incubated in an incubator for approximately 3 hours. After incubation, 100 μL of One-Lite TM After adding the detection reagent and waiting for about 5 minutes, the plate was transferred to a 96-well white plate and detected using a microplate reader luminescence detection module. The results were used to calculate the IC value of the antibody using a four-parameter regression model fitting inhibition curve. 50The experimental results showed that both SE5ss and Omalizumab prepared in Example 1 could inhibit the activation of RBL-2H3-FcεRIα-NFATLuc cell reporter mediated by each allergic serum, and the IC 50 were 19.38 ng / mL and 177.9 ng / mL, respectively (Fig. 9A), and the IC 50 The IC50 values ​​for walnut allergy serum suppression were 1.917 ng / mL and 229.8 ng / mL, respectively (Fig. 9B), and the IC50 values ​​for walnut allergy serum suppression were 51.57 ng / mL and 264.2 ng / mL, respectively (Fig. 9C), demonstrating that the inhibitory ability of SE5ss was superior to that of omalizumab.

[0287] Example 8: Detection of the activity of the antigen-binding protein of the present invention (modified humanized anti-human IgE antibody) in vivo

[0288] (1) Pharmacodynamic evaluation of the antigen-binding protein of the present application in a human IgE-reconstituted mouse model

[0289] A human IgE reconstitution model was established to evaluate the inhibitory effect of SE5ss on free IgE in mice. Balb / c mice (purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.) were used in the study. 44 mice were randomly divided into four groups based on sex and weight: negative control, vehicle control, SE5ss 0.5 μg, SE5ss 1 μg, SE5ss 2 μg, and omalizumab 2 μg. The negative control group consisted of four mice, and each of the other groups consisted of eight mice. Each mouse was intravenously injected with 2 μg of human IgE (derived from U266 cultures, in-house, lot number 5 / 20 / 29). The negative control group received an intravenous injection of the same volume of PBST (HyClone, lot number C520004-0001). Two hours later, different doses of SE5ss and omalizumab (Novartis, lot number SPX27) were intravenously injected. Blood samples were collected from the submandibular vein of all mice on the day before administration, 1 hour before administration, 1 hour after administration, and 4 hours after administration. The content of free IgE in the serum samples was measured using MSD. The method is briefly described below. An MSD plate was coated with FcεRIα-Fc (in-house, lot number: 069-109-11), the sample to be measured was added, and the plate was incubated at 37°C for 1 hour, followed by washing three times with PBST. A biotin-labeled mouse anti-human IgE antibody (in-house, Clone #AE10-10) was added, and the plate was incubated at 37°C for 1 hour, followed by washing three times with PBST. Detection was performed using MSD sulfo-tag labeled streptavidin (MSD, lot number: R32AD-1). SE5ss solvent (in-house, lot number 129-098-01) was used as a negative control in the test. The results are shown in Figure 10. Both SE5ss and Omalizumab prepared in Example 1 at different concentrations were able to significantly inhibit free IgE in the mice (P<0.05), and the inhibitory effect showed a dose-dependent effect, with the inhibitory effect of 0.5 μg / mouse SE5ss being greater than that of 2 μg / mouse Omalizumab (P<0.05).

[0290] (2) Pharmacodynamic evaluation of the antigen-binding protein of the present invention in an exogenous reconstituted immediate-type allergic reaction model

[0291] To establish an exogenous reconstituted immediate-type allergic reaction mouse model, human-FcεRIA transgenic mice (purchased from Shanghai Model Organisms Center, Inc.) were used in the study. Different doses of SE5ss were intraperitoneally injected, and an initial pharmacodynamic evaluation of SE5ss was performed by measuring changes in the mice's internal temperature. According to gender and baseline body temperature, 51 human-FcεRIA transgenic mice were randomly divided into a negative control group, a vehicle control group, an SE5ss 5 μg / mouse group, an SE5ss 10 μg / mouse group, an SE5ss 20 μg / mouse group, and an omalizumab 20 μg / mouse group. The negative control group consisted of six mice, and each of the other groups consisted of nine mice. Animals in each group were intraperitoneally injected with different doses of SE5ss and omalizumab (Novartis, lot number SPX27). One hour after administration, each group of animals received an intraperitoneal injection of 20 μg of human IgE per animal. 24 hours after administration, they received an intraperitoneal injection of 20 μg of anti-human IgE (ε-specific chain) goat anti-human IgE (Sigma, Lot No. I6284-1 mg) per animal to establish an immediate-type allergic reaction model. Mouse body temperatures were measured before and 20, 40, 60, and 90 minutes after administration of the anti-human IgE antibody. SE5ss vehicle was used as a negative control. The results are shown in Figure 11. After administration of the anti-human IgE antibody, the body temperatures of mice in the vehicle control group began to decrease, reaching a nadir at 40 minutes, and then gradually recovered. All doses of SE5ss inhibited the decrease in mouse body temperature, demonstrating a dose-dependent effect. Among them, SE5ss 10 μg / mouse has a pharmacological effect equivalent to Omalizumab 20 μg / mouse, and the SE5ss 20 μg / mouse group can maintain the mouse body temperature within the normal range.

[0292] (3) Pharmacodynamic evaluation of the antigen-binding protein of the present application in a cynomolgus monkey IgE suppression model

[0293] Different doses of SE5ss were intravenously injected into cynomolgus monkeys to evaluate their ability to inhibit free IgE in cynomolgus monkeys. Eighteen cynomolgus monkeys (Shanghai Institute of Materia Medica, Chinese Academy of Sciences) were randomly divided into three groups (3 per sex per group) based on sex and weight. SE5ss was administered subcutaneously at 1 mg / kg, 5 mg / kg, or 25 mg / kg, respectively. Serum samples were collected before administration, 1 hour after administration, and on days 14, 28, 42, and 56, and the free IgE content was measured. The results are shown in Figure 12. SE5ss at different concentrations was able to significantly suppress free IgE in the cynomolgus monkeys, and the suppressive ability showed a dose-dependent effect. In the 25 mg / kg group, free IgE fell to 2.4% of the pre-administration level one hour after administration. On days 14 and 28, free IgE was below the lower limit of detection and recovered to 19.1% and 34.8% of the baseline on days 42 and 56.

[0294] The foregoing detailed description is provided by way of interpretation and example only and is not intended to limit the scope of the appended claims. Presently, various variations of the embodiments recited herein will be apparent to those skilled in the art and are intended to fall within the scope of the appended claims and their equivalents. The present invention includes the following preferred embodiments. (1) 1. An antigen-binding protein comprising: at least one CDR of a heavy chain variable region and at least one CDR of a light chain variable region; the heavy chain variable region comprises the amino acid sequence shown in either SEQ ID NO: 73 or 74, and the light chain variable region comprises the amino acid sequence shown in either SEQ ID NO: 75 or 76; Antigen-binding proteins. (2) 1) Approx. 2.4×10 -9 It can bind to IgE with a KD value of less than M 2) have one or more of the following properties: they can inhibit the binding of IgE to its receptors FcεRIa and / or FcεRII; (1) The antigen-binding protein according to (1). (3) The IgE is human IgE. (2) The antigen-binding protein according to (1). (4) a heavy chain HCDR3, wherein the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 3; The antigen-binding protein according to any one of (1) to (3). (5) and further comprising a heavy chain HCDR2, wherein said HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 77. The antigen-binding protein according to any one of (1) to (4). (6) The HCDR2 comprises an amino acid sequence set forth in either SEQ ID NO: 2 or 26. (5) The antigen-binding protein according to (5). (7) Further comprising a heavy chain HCDR1, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 1; The antigen-binding protein according to any one of (1) to (6). (8) a heavy chain comprising HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 77, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 3; The antigen-binding protein according to any one of (1) to (7). (9) The heavy chain comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence shown in either SEQ ID NO: 2 or 26, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 3. The antigen-binding protein according to any one of (1) to (8). (10) a heavy chain H-FR1, wherein the C-terminus of the H-FR1 is directly or indirectly linked to the N-terminus of the HCDR1, and the H-FR1 comprises an amino acid sequence set forth in either SEQ ID NO: 4 or 17; The antigen-binding protein according to any one of (1) to (9). (11) The invention further comprises a heavy chain H-FR2, wherein the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 81, 5, or 27. The antigen-binding protein according to any one of (1) to (10). (12) The H-FR2 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 5, 18, 27, or 36. (11) The antigen-binding protein according to (11). (13) The heavy chain further comprises a heavy chain H-FR3, wherein the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises an amino acid sequence set forth in either SEQ ID NO: 6 or 19. The antigen-binding protein according to any one of (1) to (12). (14) The heavy chain further comprises H-FR4, wherein the N-terminus of the H-FR4 is linked to the C-terminus of the HCDR3, and the H-FR4 comprises an amino acid sequence set forth in either SEQ ID NO: 7 or 20. The antigen-binding protein according to any one of (1) to (13). (15) It comprises a heavy chain variable region VH, wherein the VH comprises an amino acid sequence set forth in either SEQ ID NO: 73 or 74. The antigen-binding protein according to any one of (1) to (14). (16) a heavy chain variable region VH, wherein the VH comprises an amino acid sequence set forth in any one of SEQ ID NOs: 8, 21, 28, 37, or 41; The antigen-binding protein according to any one of (1) to (15). (17) a light chain LCDR3, wherein the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 79; The antigen-binding protein according to any one of (1) to (16). (18) The LCDR3 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 11 and 30. (17) The antigen-binding protein according to (17). (19) Further comprising a light chain LCDR2, wherein the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 78. The antigen-binding protein according to any one of (1) to (18). (20) The LCDR2 comprises an amino acid sequence set forth in either SEQ ID NO: 10 or 29. (19) The antigen-binding protein according to (19). (21) Further comprising a light chain LCDR1, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 9; The antigen-binding protein according to any one of (1) to (20). (22) a light chain comprising LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 9, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 78, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 79; The antigen-binding protein according to any one of (1) to (21). (23) The light chain comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 9, the LCDR2 comprises the amino acid sequence shown in either SEQ ID NO: 10 or 29, and the LCDR3 comprises the amino acid sequence shown in either SEQ ID NO: 11 or 30. The antigen-binding protein according to any one of (1) to (22). (24) a light chain L-FR1, wherein the C-terminus of the L-FR1 is directly or indirectly linked to the N-terminus of the LCDR1, and the L-FR1 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 31, 12, or 80; The antigen-binding protein according to any one of (1) to (23). (25) The L-FR1 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 12, 22, 31, or 38. (24) The antigen-binding protein according to (24). (26) The antibody further comprises a light chain L-FR2, wherein the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 13, 23, or 32. The antigen-binding protein according to any one of (1) to (25). (27) The antibody further comprises a light chain L-FR3, wherein the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 33, 14, or 82. The antigen-binding protein according to any one of (1) to (26). (28) The L-FR3 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 14, 24, 33, or 39; (27) The antigen-binding protein according to (27). (29) Further comprising a light chain L-FR4, wherein the N-terminus of the L-FR4 is linked to the C-terminus of the LCDR3, and the L-FR4 comprises an amino acid sequence set forth in either SEQ ID NO: 34 or 15; The antigen-binding protein according to any one of (1) to (28). (30) It comprises a light chain variable region VL, wherein the VL comprises an amino acid sequence set forth in either SEQ ID NO: 75 or 76. The antigen-binding protein according to any one of (1) to (29). (31) The light chain variable region VL comprises an amino acid sequence set forth in any one of SEQ ID NOs: 16, 35, 25, and 40. The antigen-binding protein according to any one of (1) to (30). (32) The HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 77, the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 3, the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 9, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 78, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 79, The antigen-binding protein according to any one of (1) to (31). (33) The HCDR1 comprises the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence shown in any of SEQ ID NOs: 2 and 26, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 3, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 9, the LCDR2 comprises the amino acid sequence shown in any of SEQ ID NOs: 10 and 29, and the LCDR3 comprises the amino acid sequence shown in any of SEQ ID NOs: 11 and 30, The antigen-binding protein according to any one of (1) to (32). (34) It comprises a heavy chain variable region VH and a light chain variable region VL, wherein the VH comprises the amino acid sequence shown in either SEQ ID NO: 73 or 74, and the VL comprises the amino acid sequence shown in either SEQ ID NO: 75 or 76. The antigen-binding protein according to any one of (1) to (33). (35) The antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the amino acid sequence shown in any one of SEQ ID NOs: 8, 21, 28, 37, or 41, and the VL comprises the amino acid sequence shown in any one of SEQ ID NOs: 16, 35, 25, or 40. The antigen-binding protein according to any one of (1) to (34). (36) comprising an antibody or antigen-binding fragment thereof, The antigen-binding protein according to any one of (1) to (35). (37) the antigen-binding fragment comprises a Fab, a Fab', a F(ab)2, an Fv fragment, a F(ab')2, a scFv, a di-scFv, and / or a dAb; (36) The antigen-binding protein according to (36). (38) a heavy chain constant region, wherein the heavy chain constant region is derived from human IgG; The antigen-binding protein according to any one of (1) to (37). (39) a heavy chain constant region, wherein the heavy chain constant region is derived from human IgG1; The antigen-binding protein according to any one of (1) to (38). (40) a heavy chain constant region, wherein the heavy chain constant region comprises the following amino acid mutations compared to the amino acid sequence set forth in SEQ ID NO: 42: M135Y, S137T, and / or T139E; The antigen-binding protein according to any one of (1) to (39). (41) The heavy chain constant region comprises the amino acid sequence set forth in either SEQ ID NO: 42 or 44. The antigen-binding protein according to any one of (1) to (40). (42) a heavy chain, and the heavy chain comprises an amino acid sequence set forth in any of SEQ ID NOs: 45, 47, or 49; The antigen-binding protein according to any one of (1) to (41). (43) a light chain constant region, wherein the light chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 43; The antigen-binding protein according to any one of (1) to (42). (44) a light chain, and the light chain comprises the amino acid sequence set forth in either SEQ ID NO: 46 or 48; The antigen-binding protein according to any one of (1) to (43). (45) a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in any of SEQ ID NOs: 45, 47, or 49, and the light chain comprises the amino acid sequence set forth in any of SEQ ID NOs: 46 or 48; The antigen-binding protein according to any one of (1) to (44). (46) A polypeptide comprising the antigen-binding protein according to any one of (1) to (45). (47) An immune complex comprising the antigen-binding protein according to any one of (1) to (45). (48) An isolated nucleic acid molecule encoding the antigen-binding protein according to any one of (1) to (45). (49) A vector comprising the nucleic acid molecule according to (48). (50) A cell comprising and / or expressing the antigen-binding protein according to any one of (1) to (45), the polypeptide according to (46), the immune complex according to (47), the nucleic acid molecule according to (48), or the vector according to (49). (51) A method for producing the antigen-binding protein according to any one of (1) to (45), comprising: A method comprising culturing the cell according to (50) under conditions that allow the antigen-binding protein according to any one of (1) to (45) to be expressed. (52) 1. A pharmaceutical composition comprising: A pharmaceutical composition comprising the antigen-binding protein according to any one of (1) to (45), the polypeptide according to (46), the immune complex according to (47), the nucleic acid molecule according to (48), the vector according to (49), the cell according to (50), and / or optionally a pharmaceutically acceptable carrier. (53) Use of the antigen-binding protein according to any one of (1) to (45), the polypeptide according to (46), the immune complex according to (47), the nucleic acid molecule according to (48), the vector according to (49), the cell according to (50) and / or the pharmaceutical composition according to (52) in the preparation of a medicament for preventing, alleviating and / or treating an IgE-related disease or condition. (54) 1. A method for preventing, alleviating, or treating an IgE-related disease or condition, comprising: A method comprising administering to a subject in need thereof the antigen-binding protein according to any one of (1) to (45), the polypeptide according to (46), the immune complex according to (47), and / or the pharmaceutical composition according to (52). (55) The antigen-binding protein according to any one of (1) to (45), the polypeptide according to (46), the nucleic acid molecule according to (48), the vector according to (49), the cell according to (50), the immune complex according to (47), and / or the pharmaceutical composition according to (52), which are used for preventing, alleviating, or treating a disease or condition associated with IgE. (56) A method for detecting or measuring IgE, comprising: A method comprising using the antigen-binding protein according to any one of (1) to (45) or the polypeptide according to (46). (57) A kit for detecting or measuring IgE, comprising: A kit comprising the antigen-binding protein according to any one of (1) to (45) or the polypeptide according to (46).

Claims

1. An antigen-binding protein capable of binding to IgE, comprising: the antigen-binding protein is an antibody or an antigen-binding fragment thereof, the antigen-binding protein comprising a heavy chain variable region VH and a light chain variable region VL; the VH comprises HCDR1, HCDR2, and HCDR3, the VL comprises LCDR1, LCDR2, and LCDR3, and The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are each selected from the group consisting of the following: (1) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 containing the amino acid sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, respectively; (2) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 containing the amino acid sequences set forth in SEQ ID NO: 1, SEQ ID NO: 26, SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 29, and SEQ ID NO: 30, respectively; and (3) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 containing the amino acid sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 29, and SEQ ID NO: 30, respectively; comprising an amino acid sequence selected from the group consisting of: An antigen-binding protein capable of binding to IgE.

2. The VH comprises an amino acid sequence set forth in any one of SEQ ID NOs: 8, 21, 28, 37, or 41, and the VL comprises an amino acid sequence set forth in any one of SEQ ID NOs: 16, 35, 25, or 40.

2. The antigen-binding protein of claim 1.

3. the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, and di-scFv; 2. The antigen-binding protein of claim 1.

4. a heavy chain constant region, wherein the heavy chain constant region is derived from human IgG; 2. The antigen-binding protein of claim 1.

5. a heavy chain constant region, wherein the heavy chain constant region is derived from human IgG1; 2. The antigen-binding protein of claim 1.

6. a heavy chain constant region, wherein the heavy chain constant region comprises the following amino acid mutations compared to the amino acid sequence set forth in SEQ ID NO: 42: M135Y, S137T, and / or T139E; 2. The antigen-binding protein of claim 1.

7. a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in any of SEQ ID NOs: 45, 47, or 49, and the light chain comprises the amino acid sequence set forth in any of SEQ ID NOs: 46 or 48; 2. The antigen-binding protein of claim 1.

8. A polypeptide comprising the antigen-binding protein of claim 1.

9. An immune complex comprising the antigen-binding protein of claim 1.

10. 10. An isolated nucleic acid molecule encoding the antigen-binding protein of claim 1.

11. A vector comprising the nucleic acid molecule of claim 10.

12. A cell comprising and / or expressing the nucleic acid molecule of claim 10.

13. 1. A pharmaceutical composition comprising:

10. A pharmaceutical composition comprising the antigen-binding protein of claim 1 and a pharmaceutically acceptable carrier.

14. 14. A pharmaceutical composition according to claim 13 for use in the prevention, alleviation or treatment of an IgE-related disease or condition.

15. 1. A method for detecting or measuring IgE, comprising:

10. A method comprising using the antigen binding protein of claim 1.

16. A kit for detecting or measuring IgE, comprising: A kit comprising the antigen-binding protein of claim 1.

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