WO2023070502A1 - 分离的抗原结合蛋白及其用途 - Google Patents

分离的抗原结合蛋白及其用途 Download PDF

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WO2023070502A1
WO2023070502A1 PCT/CN2021/127316 CN2021127316W WO2023070502A1 WO 2023070502 A1 WO2023070502 A1 WO 2023070502A1 CN 2021127316 W CN2021127316 W CN 2021127316W WO 2023070502 A1 WO2023070502 A1 WO 2023070502A1
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amino acid
seq
acid sequence
sequence shown
binding protein
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PCT/CN2021/127316
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English (en)
French (fr)
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孙乃超
周若芸
马海立
刘运华
杨红舟
郭若闻
高琪
刘恒
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天辰生物医药(苏州)有限公司
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Priority to PCT/CN2021/127316 priority Critical patent/WO2023070502A1/zh
Priority to KR1020247017592A priority patent/KR20240091042A/ko
Priority to TW110140767A priority patent/TW202317616A/zh
Publication of WO2023070502A1 publication Critical patent/WO2023070502A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression

Definitions

  • This application relates to the field of biomedicine, in particular to an antigen-binding protein and its use.
  • Allergies including atopy and other hypersensitivity disorders are inappropriate or excessive immune responses to foreign antigens. Inappropriate immune responses include those that are misdirected against the body's own components, leading to autoimmune diseases.
  • Allergic disease also known as allergic disease
  • Allergic disease is the sixth largest chronic disease in the world, affecting various tissues and organs of the body. Once it occurs, it will cause various symptoms, seriously reduce the quality of life and even endanger life. For example, anaphylaxis, allergic rhinitis, asthma, atopic dermatitis, food allergies, and rubella afflict up to 20% of the population in many countries and are increasing in prevalence (Wuthrich B., Int. Arch. Allergy AppI.
  • Allergic diseases are broadly divided into IgE-mediated (immunoglobulin-mediated) and IgE-non-mediated (immunoglobulin-nonmediated), and hypersensitivity reactions mediated by immunoglobulin E (IgE) account for the majority.
  • IgE immunoglobulin E
  • the high-affinity receptor for IgE (Fc ⁇ RI) is key in mediating allergic manifestations.
  • Fc ⁇ RI is also found in many other cell types (including eosinophils, platelets) and antigen-presenting cells (eg, monocytes and dendritic cells).
  • IgE plays an important role in mediating most allergic reactions, it is imperative to develop therapeutic strategies for allergic diseases that control IgE levels.
  • the application provides an antigen-binding protein, which has one or more of the following advantages: 1) good stability, the antigen-binding protein of the application has high druggability, and is more conducive to industrial production; 2) the antigen-binding protein of the application The affinity of the protein to the neonatal receptor FcRn is enhanced, and the half-life is longer, which can achieve the purpose of prolonging the administration cycle.
  • the antigen binding protein of the present application comprises at least one complementarity determining region (CDR) of the variable region of the heavy chain, and the variable region of the heavy chain (VH) comprises any of SEQ ID NO:73,74.
  • CDR complementarity determining region
  • VH variable region of the heavy chain
  • the antigen binding proteins of the present application have one or more of the following properties:
  • the antigen binding proteins of the present application bind to human IgE.
  • the antigen binding protein of the present application comprises heavy chain HCDR3, and described HCDR3 comprises the aminoacid sequence shown in SEQ ID NO:3.
  • the antigen binding protein of the present application further comprises a heavy chain HCDR2, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:77.
  • the HCDR2 in the antigen binding protein of the present application comprises the amino acid sequence shown in any one of SEQ ID NO:2,26.
  • the antigen binding protein of the present application further comprises a heavy chain HCDR1, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1.
  • the antigen binding protein of the present application comprises heavy chain HCDR1, HCDR2 and HCDR3; wherein said HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1, and said HCDR2 comprises the amino acid sequence shown in SEQ ID NO:77 Amino acid sequence, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:3.
  • the antigen binding protein of the present application comprises heavy chain HCDR1, HCDR2 and HCDR3; wherein said HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1, and said HCDR2 comprises SEQ ID NO:2, 26 The amino acid sequence shown in any one, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:3.
  • the antigen binding protein of the present application comprises heavy chain framework region 1 (H-FR1), the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1, and the H- FR1 comprises the amino acid sequence shown in any one of SEQ ID NO:4,17.
  • H-FR1 heavy chain framework region 1
  • the antigen binding protein of the present application further comprises a heavy chain framework region 2 (H-FR2), the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises SEQ The amino acid sequence shown in any one of ID NO:81, 5 or 27.
  • H-FR2 heavy chain framework region 2
  • the H-FR2 in the antigen binding protein of the present application comprises the amino acid sequence shown in any one of SEQ ID NO:5, 18, 27 or 36.
  • the antigen binding protein of the present application further comprises a heavy chain framework region 3 (H-FR3), the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises SEQ The amino acid sequence shown in any one of ID NO:6,19.
  • H-FR3 heavy chain framework region 3
  • the antigen binding protein of the present application further comprises a heavy chain framework region 4 (H-FR4), the N-terminus of the H-FR4 is connected to the C-terminus of the HCDR3, and the H-FR4 comprises The amino acid sequence shown in any one of SEQ ID NO:7,20.
  • H-FR4 heavy chain framework region 4
  • the antigen binding protein of the present application comprises a heavy chain variable region VH, said VH comprising the amino acid sequence shown in any one of SEQ ID NO:73,74.
  • the antigen binding protein of the present application comprises a heavy chain variable region VH comprising the amino acid sequence shown in any one of SEQ ID NO: 8, 21, 28, 37 or 41.
  • the antigen binding protein of the present application comprises a light chain LCDR3 comprising the amino acid sequence shown in SEQ ID NO:79.
  • the LCDR3 in the antigen binding protein of the present application comprises the amino acid sequence shown in any one of SEQ ID NO:11,30.
  • the antigen binding protein of the present application also comprises a light chain LCDR2, and said LCDR2 comprises the amino acid sequence shown in SEQ ID NO:78.
  • the LCDR2 in the antigen binding protein of the present application comprises the amino acid sequence shown in any one of SEQ ID NO:10,29.
  • the antigen binding protein of the present application further comprises a light chain LCDR1, and said LCDR1 comprises the amino acid sequence shown in SEQ ID NO:9.
  • the antigen binding protein of the present application comprises light chain LCDR1, LCDR2 and LCDR3; Wherein said LCDR1 comprises the amino acid sequence shown in SEQ ID NO:9, and said LCDR2 comprises the aminoacid sequence shown in SEQ ID NO:78 Amino acid sequence, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:79.
  • the antigen binding protein of the present application comprises light chain LCDR1, LCDR2 and LCDR3; Wherein said LCDR1 comprises the amino acid sequence shown in SEQ ID NO:9, and said LCDR2 comprises SEQ ID NO:10,29 The amino acid sequence shown in any one, and the LCDR3 comprises the amino acid sequence shown in any one of SEQ ID NO:11,30.
  • the antigen binding protein of the present application comprises light chain framework region 1 (L-FR1), the C-terminus of said L-FR1 is directly or indirectly linked to the N-terminus of said LCDR1, and said L- FR1 comprises the amino acid sequence shown in any one of SEQ ID NO: 31, 12 or 80.
  • L-FR1 light chain framework region 1
  • the L-FR1 described in the antigen binding protein of the present application comprises the amino acid sequence shown in any one of SEQ ID NO: 12, 22, 31 or 38.
  • the antigen binding protein of the present application further comprises a light chain framework region 2 (L-FR2), the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 comprises SEQ The amino acid sequence shown in any one of ID NO:13, 23 or 32.
  • L-FR2 light chain framework region 2
  • the antigen binding protein of the present application further comprises a light chain framework region 3 (L-FR3), the L-FR3 is located between the LCDR2 and the LCDR3, and the L- FR3 comprises the amino acid sequence shown in any one of SEQ ID NO: 33, 14 or 82.
  • L-FR3 light chain framework region 3
  • the L-FR3 in the antigen binding protein of the present application comprises the amino acid sequence shown in any one of SEQ ID NO: 14, 24, 33 or 39.
  • the antigen binding protein of the present application further comprises a light chain framework region 4 (L-FR4), the N-terminus of the L-FR4 is connected to the C-terminus of the LCDR3, and the L-FR4 comprises The amino acid sequence shown in any one of SEQ ID NO:34,15.
  • L-FR4 light chain framework region 4
  • the antigen-binding protein of the present application comprises a light chain variable region VL, said VL comprising the amino acid sequence shown in any one of SEQ ID NO:75,76.
  • the antigen binding protein of the present application comprises a light chain variable region VL, said VL comprising the amino acid sequence shown in any one of SEQ ID NO:16,35,25,40.
  • 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 shown in SEQ ID NO: 1, and the HCDR2 comprises SEQ ID
  • the HCDR3 includes the amino acid sequence shown in SEQ ID NO:3
  • the LCDR1 includes the amino acid sequence shown in SEQ ID NO:9
  • the LCDR2 includes the amino acid sequence shown in SEQ ID NO:78
  • the amino acid sequence and the LCDR3 comprise the amino acid sequence shown in SEQ ID NO:79.
  • 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 shown in SEQ ID NO: 1, and the HCDR2 comprises SEQ ID
  • the amino acid sequence shown in any one of NO:2,26, described HCDR3 comprises the aminoacid sequence shown in SEQ ID NO:3, and described LCDR1 comprises the aminoacid sequence shown in SEQ ID NO:9, and described LCDR2 comprises SEQ
  • the amino acid sequence shown in any one of ID NO:10,29 and described LCDR3 comprise the amino acid sequence shown in any one of SEQ ID NO:11,30.
  • the antigen-binding protein of the present application comprises a heavy chain variable region VH and a light chain variable region VL, said VH comprising the amino acid sequence shown in any one of SEQ ID NO:73,74, and The VL comprises the amino acid sequence shown in any one of SEQ ID NO:75,76.
  • the antigen binding protein of the present application comprises heavy chain variable region VH and light chain variable region VL, and described VH comprises any one of SEQ ID NO:8,21,28,37 or 41
  • the amino acid sequence shown and the VL comprises the amino acid sequence shown in any one of SEQ ID NO:16,35,25,40.
  • the antigen binding proteins of the present application include antibodies or antigen binding fragments thereof.
  • the antigen-binding fragments of the antigen-binding proteins of the present application include Fab, Fab', F(ab)2, Fv fragments, F(ab')2, scFv, di-scFv and/or dAb .
  • 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.
  • 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.
  • the antigen binding protein of the present application comprises a heavy chain constant region, wherein compared with the amino acid sequence shown in SEQ ID NO: 42, the heavy chain constant region comprises the following amino acid mutations: M135Y, S137T and /or T139E.
  • the heavy chain constant region in the antigen binding protein of the present application comprises the amino acid sequence shown in any one of SEQ ID NO:42,44.
  • the antigen binding protein of the present application comprises a heavy chain, and the heavy chain comprises the amino acid sequence shown in any one of SEQ ID NO:45, 47 or 49.
  • the antigen binding protein of the present application comprises a light chain constant region, and the light chain constant region comprises the amino acid sequence shown in SEQ ID NO:43.
  • the antigen binding protein of the present application comprises a light chain, and the light chain comprises the amino acid sequence shown in any one of SEQ ID NO:46 or 48.
  • 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 shown in any one of SEQ ID NO: 45, 47 or 49, and the light chain Comprising the amino acid sequence shown in any one of SEQ ID NO:46 or 48.
  • the present application also provides a polypeptide comprising the antigen-binding protein of the present application.
  • the present application also provides an immunoconjugate comprising the antigen-binding protein of the present application.
  • the present application also provides an isolated nucleic acid molecule encoding the antigen-binding protein of the present application.
  • the present application also provides a vector comprising the isolated nucleic acid molecule of the present application.
  • the present application also provides cells comprising and/or expressing the antigen-binding protein of the present application, the polypeptide of the present application, the immunoconjugate of the present application, the isolated nucleic acid molecule of the present application or the vector of the present application.
  • the present application also provides a method for preparing the antigen-binding protein of the present application, the method comprising culturing the cells of the present application under the conditions for expressing the antigen-binding protein of the present application.
  • the present application also provides a pharmaceutical composition, which comprises the antigen-binding protein of the present application, the polypeptide of the present application, the immunoconjugate of the present application, the isolated nucleic acid molecule of the present application, the carrier of the present application, the cells and/or optionally a pharmaceutically acceptable carrier.
  • the present application also provides the antigen-binding protein of the present application, the polypeptide of the present application, the immunoconjugate of the present application, the isolated nucleic acid molecule of the present application, the carrier of the present application, the cell of the present application and/or the present application.
  • the present application also provides a method for preventing, alleviating or treating an IgE-related disease or disorder, the method comprising administering the antigen-binding protein of the present application, the polypeptide of the present application, the polypeptide of the present application to a subject in need.
  • Immunoconjugates and/or pharmaceutical compositions of the present application are also provided.
  • the present application also provides the antigen binding protein of the present application, the polypeptide of the present application, the isolated nucleic acid molecule of the present application, the carrier of the present application, the cell of the present application, the immunoconjugate of the present application and/or the present application.
  • the pharmaceutical composition of the application is used for preventing, alleviating or treating IgE-related diseases or conditions.
  • the present application also provides a method for detecting or measuring IgE, the method comprising using the antigen-binding protein of the present application or the polypeptide of the present application.
  • the present application also provides a kit for detecting or measuring IgE, the kit comprising the antigen-binding protein of the present application or the polypeptide of the present application.
  • Figure 1 shows the result of the blocking activity of the antigen binding protein (mouse monoclonal antibody) of the present application to block the binding of IgE to its receptor Fc ⁇ RIa.
  • Figure 2 shows the results of the blocking activity of the antigen-binding proteins of the present application (chimeric antibodies and their corresponding murine antibodies) blocking the binding of IgE to its receptor Fc ⁇ RIa.
  • Figure 3 shows the results of the blocking activity of the antigen-binding proteins of the present application (humanized antibody, its corresponding chimeric antibody and corresponding murine antibody) blocking the binding of IgE to its receptor Fc ⁇ RIa.
  • Figure 4 shows the detection results of the antigen binding protein (humanized SE2 antibody) of the present application and omalizumab blocking the binding of IgE to its receptor Fc ⁇ RIa.
  • Figure 5 shows the detection results of the antigen-binding protein (humanized SE5 antibody) of the present application and omalizumab blocking the binding of IgE to its receptor Fc ⁇ RIa.
  • Figures 6A-6B show the binding affinity of the antigen-binding protein of the present application (humanized SE5ss antibody) to human IgE.
  • FIGS 7A-7B show the results of the blocking activity of the antigen binding protein (humanized SE5ss antibody) of the present application to block the binding of IgE to its receptors (Fc ⁇ RIa and Fc ⁇ RII).
  • Fig. 8 shows the detection results of the inhibitory activity of the antigen-binding protein of the present application (humanized SE5ss antibody) blocking IgE-mediated cell activation.
  • Figures 9A-9C show the test results of the inhibitory activity of the antigen-binding protein (humanized SE5ss antibody) of the present application in blocking cell activation mediated by different allergens IgE.
  • Figure 10 shows the ability of the antigen-binding protein of the present application (humanized SE5ss antibody) to inhibit free human IgE in mice.
  • Figure 11 shows the results of the antigen-binding protein of the present application (humanized SE5ss antibody) alleviating body temperature changes in a mouse model of instant hypersensitivity.
  • Figure 12 shows the ability of the antigen-binding protein of the present application (humanized SE5ss antibody) to inhibit free IgE in a cynomolgus monkey IgE inhibition model.
  • antigen binding protein generally refers to a protein comprising an antigen-binding moiety, and optionally a scaffold or backbone moiety that allows the antigen-binding moiety to adopt a conformation that facilitates binding of the antigen-binding protein to the 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 (such as bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs, or fusion proteins, etc., as long as they exhibit the desired antigen-binding activity.
  • an "antigen binding protein” of the present application may comprise a moiety that binds an antigen and, optionally, a scaffold or framework portion that allows the antigen binding moiety to adopt a conformation that facilitates binding of the antigen binding moiety to an antigen.
  • IgE or "IgE immunoglobulin” or “immunoglobulin E” generally refers to a class of antibodies (or immunoglobulins (Ig)) produced by plasma cells.
  • IgE monomers usually consist of two heavy chains ( ⁇ chains) and two light chains, wherein the ⁇ chains may contain four Ig-like constant regions (C ⁇ 1-C ⁇ 4).
  • IgE may be intact IgE or fragments thereof, may be functional variants of IgE, isoforms, species homologues, derivatives, analogs, and have at least one common epitope with IgE analogues of .
  • IgE belongs to the polypeptide class of antibodies essentially encoded by the well-known immunoglobulin epsilon genes. IgE can include membrane-anchored (mIgE), or non-membrane-anchored, also known as circulating IgE. In the present application, the term "IgE" may be derived from mammals, and IgE may include human IgE. In the present application, the sequence number of the human IgE in the GenBank database is Gene ID: 3497.
  • Fc ⁇ RI ⁇ generally refers to the alpha chain of the high affinity receptor (Fc ⁇ RI, also known as Fc epsilon RI, ) which is the Fc region of immunoglobulin E (IgE).
  • Fc ⁇ RI is a tetrameric receptor complex capable of binding the Fc region of the ⁇ heavy chain of IgE, which usually consists of one ⁇ chain (ie Fc ⁇ RI ⁇ ), one ⁇ chain (Fc ⁇ RI ⁇ ) and two ⁇ chains (Fc ⁇ RI ⁇ ).
  • the ⁇ chain can serve as the binding site of an antibody (for example, IgE), the ⁇ chain can serve as the initiation site of the downstream signal, and the ⁇ chain can play the role of amplifying the downstream signal.
  • Fc ⁇ RI ⁇ may be complete Fc ⁇ RI ⁇ or its fragments, may be functional variants, isoforms, species homologues, derivatives, analogs of Fc ⁇ RI ⁇ , and have at least one common epitope with Fc ⁇ RI ⁇ analogues of .
  • KD also known as “ KD ", " KD “, "affinity constant” or “equilibrium dissociation constant”
  • association rate constant ( ka ) the association rate constant ( ka )
  • dissociation rate constant (k d ) the association rate constant (k d )
  • equilibrium dissociation constant (K D ) can be used to express the binding protein (such as the antigen binding protein described herein) to the antigen (such as human IgE) binding affinity.
  • the KD value can be determined by Octet, and can also be determined using other experimental approaches and instruments such as BIAcore (Biomolecular Interaction Analysis).
  • electrochemiluminescence analysis-solution equilibrium titration MSD-SET
  • the measurement method is as described in Estep P. et al., MAbs, 2013.5(2):p.270-8.
  • the KD value can be detected by the KinExA method and software, and the measurement method is as described in Jonathan K. Fleming et al., Methods Mol Biol. 2018; 1697:1-8.
  • CDR complementarity determining region
  • CDR1 complementarity determining region
  • CDR2 complementarity determining region
  • CDR3 complementarity determining region
  • a CDR combination may refer to a set of 3 CDRs that occur in a single variable domain capable of binding antigen. The exact boundaries of these CDRs have been defined differently from system to system. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest National Institutes of Health, Bethesda, Md.
  • Chothia CDRs regions may be referred to as Chothia CDRs, which have overlapping borders with Kabat CDRs.
  • Other boundaries defining the CDRs that overlap the Kabat CDRs have been described by Padlan (FASEB J.9:133-139 (1995)) and MacCallum (J Mol Biol 262(5):732-45 (1996)). In other CDR boundary definitions that may not strictly follow one of the above systems, but will still overlap with Kabat CDRs, they Can be shortened or lengthened.
  • the CDR described in this article can be defined using KABAT, but it does not exclude the use of other methods to divide the CDR region.
  • the term “antibody” generally refers to an immunoglobulin or fragment thereof or derivative or variant thereof, encompassing any polypeptide comprising an antigen combining site, whether produced in vitro or in vivo.
  • the term includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, nonspecific, humanized, single-stranded, chimeric, synthetic, recombinant, hybrid , mutated and transplanted antibodies.
  • the term “antibody” may also include antibody fragments such as Fab, F(ab')2, Fv, scFv, Fd, dAb and other antibody fragments that retain antigen binding function (e.g., specifically bind human IgE) .
  • the antibodies described herein may include antibodies derived from humans, mice, monkeys and/or alpacas.
  • the antibody may include functional variants, isoforms, species homologues, derivatives or analogs thereof.
  • variants of the present application may include 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%, Amino acid sequences that are at least 99.2%, at least 99.4%, at least 99.6%, at least 99.8%, or at least 99.9% identical.
  • antigen-binding fragment generally refers to polypeptide fragments of immunoglobulins or antibodies that compete with intact antibodies (ie, with intact antibodies from which they are derived) for antigen binding (ie, specific binding).
  • the 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 multiple antibodies formed by antibody fragments. specific antibody.
  • variable region or “variable domain” generally refers to a part of an antibody's light chain and/or heavy chain, which usually varies greatly among different antibodies and may affect the antibody's specificity. Antigen binding specificity.
  • the variable region can contain regions with large sequence changes called complementarity determining regions (CDR), and the variable region can also contain relatively conserved regions, usually called framework regions (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • directly or indirectly connected generally refers to the relative “directly connected” or “indirectly connected”.
  • Directly connected generally means a direct connection.
  • the direct connection may be the case where there is no spacer (such as amino acid residues or derivatives thereof) between the connected substances (such as amino acid sequence segments); for example, the amino acid sequence segment X and another amino acid Sequence segment Y is directly linked by an amide bond formed between the C-terminal amino acid of amino acid sequence segment X and the N-terminal amino acid of amino acid sequence segment Y.
  • “Indirect connection” usually refers to the situation that there is a spacer (such as amino acid residue or its derivative) between the connected substances (such as amino acid sequence segments) and they are indirectly connected.
  • the C-terminal of the L-FR1 may be directly or indirectly connected to the N-terminal of the LCDR1.
  • immunoconjugate generally refers to other components linked to an antigen-binding protein or fragment thereof.
  • the immunoconjugates may include other polypeptides, therapeutic agents, probes and/or other antibodies fused or conjugated to the antigen binding protein.
  • isolated nucleic acid molecule generally refers to nucleotides of any length in isolated form, deoxyribonucleotides or ribonucleotides, or analogs isolated from their natural environment or artificially synthesized.
  • the term "cell” generally refers to an individual cell, cell line or cell culture that can or has contained a plasmid or vector comprising a nucleic acid molecule described herein, or is capable of expressing an antigen binding protein described herein .
  • the cells may include progeny of a single cell. Due to natural, accidental or deliberate mutations, the progeny cells may not necessarily be completely identical in morphology or genome to the original parent cells, but it is sufficient to be able to express the antigen-binding protein described in this application.
  • the cells can be obtained by transfecting cells in vitro with the vectors described in this application.
  • the cells can be prokaryotic cells (such as Escherichia coli) or eukaryotic cells (such as yeast cells, such as COS cells, Chinese hamster ovary (CHO) cells, HeLa cells, HEK293 cells, COS-1 cells, NSO cells or myeloma cells).
  • prokaryotic cells such as Escherichia coli
  • eukaryotic cells such as yeast cells, such as COS cells, Chinese hamster ovary (CHO) cells, HeLa cells, HEK293 cells, COS-1 cells, NSO cells or myeloma cells.
  • the term "pharmaceutical composition” generally refers to a composition suitable for administration to a patient, eg a human patient.
  • the pharmaceutical composition described herein may comprise the antigen binding protein described herein, the immunoconjugate described herein, the nucleic acid molecule described herein, the carrier described herein and/or The cells described herein, and optionally a pharmaceutically acceptable carrier.
  • the pharmaceutical composition may also comprise one or more suitable (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and/or preservatives. preparations.
  • the acceptable ingredients of the composition are nontoxic to recipients at the dosages and concentrations employed.
  • Pharmaceutical compositions of the present invention may include, but are not limited to, liquid, frozen and lyophilized compositions.
  • vector generally refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a protein is inserted and the protein is expressed.
  • the vector can be expressed by transforming, transducing or transfecting the host cell, so that the genetic material elements carried by it can be expressed in the host cell.
  • vectors may include: plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or P1-derived artificial chromosome (PAC); phage such as lambda phage or M13 phage and Animal viruses, etc.
  • Types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillary polyoma vacuoles Viruses (such as SV40).
  • retroviruses including lentiviruses
  • adenoviruses such as herpes simplex virus
  • poxviruses such as herpes simplex virus
  • baculoviruses such as herpes simplex virus
  • baculoviruses such as baculoviruses
  • papillomaviruses such as SV40
  • a vector may contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes.
  • the vector may also contain an origin of replication.
  • Vectors may also
  • pharmaceutically acceptable carrier generally includes a pharmaceutically acceptable carrier, excipient or stabilizer that is inert to the cells or mammals to which it is exposed at the dosage and concentration employed. poisonous.
  • a physiologically acceptable carrier may include suitable substances.
  • the application provides an antigen binding protein, which may comprise at least one complementarity determining region (CDR) of a heavy chain variable region, said heavy chain variable region (VH) comprising such as SEQ ID NO: 73, 74 The amino acid sequence shown in any one; And it comprises at least one complementarity determining region (CDR) of light chain variable region (VL), and described light chain variable region comprises as any in SEQ ID NO:75,76 The amino acid sequence shown in item.
  • CDR complementarity determining region
  • VH heavy chain variable region
  • VL light chain variable region
  • described light chain variable region comprises as any in SEQ ID NO:75,76 The amino acid sequence shown in item.
  • the antigen binding protein of the present application may comprise a CDR of the heavy chain variable region, which may comprise the amino acid sequence shown in SEQ ID NO: 73 or a variant thereof.
  • the antigen binding protein of the present application may comprise two CDRs in the heavy chain variable region, which may comprise the amino acid sequence shown in SEQ ID NO: 73 or a variant thereof.
  • the antigen binding protein of the present application may comprise three CDRs in the heavy chain variable region, which may comprise the amino acid sequence shown in SEQ ID NO: 73 or a variant thereof.
  • an antigen binding protein of the present application may comprise a CDR of a heavy chain variable region, which may comprise an amino acid sequence as shown in SEQ ID NO: 74 or a variant thereof.
  • the antigen binding protein of the present application may comprise two CDRs in the heavy chain variable region, which may comprise the amino acid sequence shown in SEQ ID NO: 74 or a variant thereof.
  • the antigen binding protein of the present application may comprise three CDRs in the heavy chain variable region, which may comprise the amino acid sequence shown in SEQ ID NO: 74 or a variant thereof.
  • the CDRs in the heavy chain variable region of the present application may be called HCDRn, where n represents the number of the CDRs, usually 1, 2 or 3.
  • the CDRs in the heavy chain variable region of the present application may include HCDR1, HCDR2 and HCDR3 described in the present application.
  • the antigen binding protein of the present application may comprise heavy chain HCDR3, and said HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 3 or a variant thereof.
  • the antigen binding protein of the present application can comprise heavy chain HCDR2, and described HCDR2 can comprise the aminoacid sequence shown in SEQ ID NO:77 or its variant.
  • the antigen binding protein of the present application may comprise heavy chain HCDR2, and said HCDR2 may comprise the amino acid sequence shown in any one of SEQ ID NO: 2, 26 or a variant thereof.
  • the antigen binding protein of the present application may comprise heavy chain HCDR1, and said HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 1 or a variant thereof.
  • the antigen binding protein of the present application can comprise heavy chain HCDR3 and HCDR2, described HCDR3 can comprise the aminoacid sequence shown in SEQ ID NO:3 or its variant, and described HCDR2 can comprise the aminoacid sequence shown in SEQ ID NO:77 Amino acid sequence or its variants.
  • the antigen binding protein of the present application may comprise heavy chain HCDR3 and HCDR2, said HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 3 or a variant thereof, and said HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 2, 26 The amino acid sequence shown in any one or its variant.
  • the antigen binding protein of the present application may comprise heavy chain HCDR2 and HCDR1, said HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:77 or a variant thereof, and said HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:1 Amino acid sequence or its variants.
  • the antigen binding protein of the present application may comprise heavy chain HCDR2 and HCDR1, said HCDR2 may comprise the amino acid sequence shown in any one of SEQ ID NO:2, 26 or its variant, and said HCDR1 may comprise SEQ ID The amino acid sequence shown in NO: 1 or its variant.
  • the antigen binding protein of the present application may comprise heavy chain HCDR1, HCDR2 and HCDR3; wherein said HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 1 or a variant thereof, and said HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 77
  • the amino acid sequence shown in SEQ ID NO: 3 or a variant thereof, the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 3 or a variant thereof.
  • the antigen binding protein of the present application may comprise heavy chain HCDR1, HCDR2 and HCDR3; wherein said HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 1 or a variant thereof, and said HCDR2 may comprise SEQ ID NO: 2,
  • the amino acid sequence shown in any one of 26 or its variant, described HCDR3 can comprise the amino acid sequence shown in SEQ ID NO:3 or its variant.
  • the antigen binding protein of the present application may comprise a CDR of the light chain variable region, which may comprise the amino acid sequence shown in SEQ ID NO: 75 or a variant thereof.
  • the antigen binding protein of the present application may comprise two CDRs in the light chain variable region, which may comprise the amino acid sequence shown in SEQ ID NO: 75 or a variant thereof.
  • the antigen binding protein of the present application may comprise three CDRs in the light chain variable region, which may comprise the amino acid sequence shown in SEQ ID NO: 75 or a variant thereof.
  • the antigen binding protein of the present application may comprise a CDR of the light chain variable region, which may comprise the amino acid sequence shown in SEQ ID NO: 76 or a variant thereof.
  • the antigen binding protein of the present application may comprise two CDRs in the variable region of the light chain, which may comprise the amino acid sequence shown in SEQ ID NO: 76 or a variant thereof.
  • the antigen binding protein of the present application may comprise three CDRs in the light chain variable region, which may comprise the amino acid sequence shown in SEQ ID NO: 76 or a variant thereof.
  • the CDRs in the light chain variable region of the present application may be called LCDRn, where n represents the number of the CDRs, usually 1, 2 or 3, etc.
  • the CDRs in the light chain variable region of the present application may include LCDR1, LCDR2 and LCDR3 described herein.
  • the antigen binding protein of the present application may comprise a light chain LCDR3, and said LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 79 or a variant thereof.
  • the antigen binding protein of the present application may comprise a light chain LCDR3, and said LCDR3 may comprise the amino acid sequence shown in any one of SEQ ID NO: 11, 30 or a variant thereof.
  • the antigen-binding protein of the present application may comprise a light chain LCDR2, which may comprise the amino acid sequence shown in SEQ ID NO: 78 or a variant thereof.
  • the antigen binding protein of the present application may comprise a light chain LCDR2, and said LCDR2 may comprise the amino acid sequence shown in any one of SEQ ID NO: 10, 29 or a variant thereof.
  • the antigen-binding protein of the present application may comprise a light chain LCDR1, and said LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 9 or a variant thereof.
  • the antigen-binding protein of the present application comprises light chain LCDR3 and LCDR2, said LCDR3 can comprise the amino acid sequence shown in SEQ ID NO:79 or its variant, and said LCDR2 can comprise the amino acid shown in SEQ ID NO:78 sequence or its variants.
  • the antigen binding protein of the present application comprises light chain LCDR3 and LCDR2, and described LCDR3 can comprise the aminoacid sequence shown in any one of SEQ ID NO:11,30 or its variant, and described LCDR2 can comprise SEQ ID NO : the amino acid sequence shown in any one of 10 and 29 or a variant thereof.
  • the antigen binding protein of the present application can comprise light chain LCDR2 and LCDR1, described LCDR2 can comprise the aminoacid sequence shown in SEQ ID NO:78 or its variant, and described LCDR1 can comprise the aminoacid sequence shown in SEQ ID NO:9 Amino acid sequence or its variants.
  • the antigen binding protein of the present application can comprise light chain LCDR2 and LCDR1
  • described LCDR2 can comprise the amino acid sequence shown in any one of SEQ ID NO:10,29 or its variant
  • described LCDR1 can comprise SEQ ID The amino acid sequence shown in NO:9 or its variant.
  • the antigen binding protein of the present application can comprise light chain LCDR1, LCDR2 and LCDR3, wherein said LCDR1 can comprise the amino acid sequence shown in SEQ ID NO:9 or its variant, and described LCDR2 can comprise SEQ ID NO:78
  • the amino acid sequence shown in SEQ ID NO:79 or a variant thereof, the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:79 or a variant thereof.
  • the antigen binding protein of the present application can comprise light chain LCDR1, LCDR2 and LCDR3, wherein said LCDR1 can comprise the amino acid sequence shown in SEQ ID NO:9 or its variant, and described LCDR2 can comprise SEQ ID NO:10, The amino acid sequence shown in any one of 29 or its variant, the LCDR3 can comprise the amino acid sequence shown in any one of SEQ ID NO:11,30 or its variant.
  • the antigen binding protein of the present application can comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein said HCDR1 can comprise the amino acid sequence shown in SEQ ID NO:1 or its variant, and said HCDR2 can comprise SEQ ID
  • the amino acid sequence shown in NO:77 or its variant, described HCDR3 can comprise the amino acid sequence shown in SEQ ID NO:3 or its variant, and described LCDR1 can comprise the amino acid sequence shown in SEQ ID NO:9 or its variant Variants
  • the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 78 or a variant thereof and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 79 or a variant thereof.
  • the antigen binding protein of the present application can comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein said HCDR1 can comprise the amino acid sequence shown in SEQ ID NO:1 or its variant, and said HCDR2 can comprise SEQ ID
  • said HCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 3 or its variant
  • described LCDR1 can comprise SEQ ID NO: 9
  • the amino acid sequence shown in or its variant, the LCDR2 can comprise the amino acid sequence shown in any one of SEQ ID NO:10,29 or its variant and the LCDR3 can comprise any one of SEQ ID NO:11,30
  • the amino acid sequence shown in item or its variant can comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein said HCDR1 can comprise the amino acid sequence shown in SEQ ID NO:1 or its variant, and said HCDR2 can comprise SEQ ID
  • the light chain variable regions of the present application may include framework regions L-FR1, L-FR2, L-FR3, and/or L-FR4.
  • the light chain variable region of the present application may include the framework regions L-FR1, L-FR2 and L-FR3.
  • the light chain variable region of the present application may include the framework regions L-FR1, L-FR2 and L-FR4.
  • the light chain variable region of the present application may include the framework regions L-FR2, L-FR3 and L-FR4.
  • the light chain variable region of the present application may include the framework regions L-FR1, L-FR3 and L-FR4.
  • the light chain variable region of the present application may include the framework regions L-FR1, L-FR2, L-FR3 and L-FR4.
  • the C-terminal of the L-FR1 of the present application can be directly or indirectly connected to the N-terminal of the LCDR1 of the present application, for example, the C-terminal of the L-FR1 of the present application can be directly connected to the N-terminal of the LCDR1 of the present application, for example,
  • the C-terminal of the L-FR1 of the present application can be indirectly connected with the N-terminal of the LCDR1 of the present application, for example, the indirect connection of the present application can include the C-terminal amino acid residue of the L-FR1 of the present application and the N-terminal of the LCDR1 of the present application
  • the amino acid residues are separated by 1, 2, 3, 4, 5 or more amino acids, which may be any amino acid naturally occurring or modified.
  • the L-FR1 of the present application may comprise the amino acid sequence shown in any one of SEQ ID NO: 12, 31 or 80 or a variant thereof.
  • the L-FR1 of the present application may comprise the amino acid sequence shown in any one of SEQ ID NO: 12, 31, 22, 38 or a variant thereof.
  • L-FR2 of the present application may be located between LCDR1 of the present application and LCDR2 of the present application.
  • the N-terminal of the L-FR2 of the present application can be directly or indirectly connected to the C-terminal of the LCDR1 of the present application
  • the C-terminal of the L-FR2 of the present application can be directly or indirectly connected to the N-terminal of the LCDR2 of the present application.
  • indirect connection may include 1, 2, or 3 parts between the two connected parts (for example, 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). , 4, 5 or more amino acids, which may be any naturally occurring or modified amino acid.
  • the L-FR2 of the present application may comprise the amino acid sequence shown in any one of SEQ ID NO: 13, 32, 23 or a variant thereof.
  • -FR3 of the present application may be located between LCDR2 of the present application and LCDR3 of the present application.
  • the N-terminal of the L-FR3 of the present application can be directly or indirectly connected to the C-terminal of the LCDR2 of the present application
  • the C-terminal of the L-FR3 of the present application can be directly or indirectly connected to the N-terminal of the LCDR3 of the present application.
  • the indirect connection may include 1, 2, or 3 parts between the two connected parts (for example, the L-FR3 of the present application and the LCDR2 of the present application, or the L-FR3 of the present application and the LCDR3 of the present application). , 4, 5 or more amino acids, which may be any naturally occurring or modified amino acid.
  • the L-FR3 of the present application may comprise the amino acid sequence shown in any one of SEQ ID NO: 14, 33, 82 or a variant thereof.
  • the L-FR3 of the present application may comprise the amino acid sequence shown in any one of SEQ ID NO: 14, 33, 24, 39 or a variant thereof.
  • the N-terminal of L-FR4 of the present application is directly or indirectly connected to the C-terminal of LCDR3 of the present application.
  • the indirect connection may include 1, 2, 3, 4, 5 or more between the N-terminal amino acid residues of the L-FR4 of the present application and the C-terminal amino acid residues of the LCDR3 of the present application.
  • Amino acids which may be any amino acid naturally occurring or modified.
  • the L-FR4 of the present application may comprise the amino acid sequence shown in any one of SEQ ID NO: 15, 34 or a variant thereof.
  • the heavy chain variable regions of the present application may include framework regions H-FR1, H-FR2, H-FR3, and/or H-FR4.
  • the light chain variable region of the present application may include framework regions H-FR1, H-FR2 and H-FR3.
  • the light chain variable region of the present application may include framework regions H-FR1, H-FR2 and H-FR4.
  • the light chain variable region of the present application may include framework regions H-FR2, H-FR3 and H-FR4.
  • the light chain variable region of the present application may include framework regions H-FR1, H-FR3 and H-FR4.
  • chain variable regions of the present application may include framework regions H-FR1, H-FR2, H-FR3 and H-FR4.
  • the C-terminal of the H-FR1 of the present application may be directly or indirectly connected to the N-terminal of the HCDR1 of the present application.
  • the C-terminal of the H-FR1 of the present application can be directly connected with the N-terminal of the HCDR1 of the present application, for example, the C-terminal of the H-FR1 of the present application can be indirectly connected with the N-terminal of the HCDR1 of the present application, for example, the The indirect connection may include 1, 2, 3, 4, 5 or more amino acids between the C-terminal amino acid residues of the H-FR1 of the present application and the N-terminal amino acid residues of the HCDR1 of the present application , these amino acids may be any amino acids that occur naturally or are modified.
  • the H-FR1 of the present application may comprise the amino acid sequence shown in any one of SEQ ID NO: 4, 17 or a variant thereof.
  • the H-FR2 of the present application may be located between the HCDR1 of the present application and the HCDR2 of the present application.
  • the N-terminal of the H-FR2 of the present application can be directly or indirectly connected to the C-terminal of the HCDR1 of the present application
  • the C-terminal of the H-FR2 of the present application can be directly or indirectly connected to the N-terminal of the HCDR2 of the present application.
  • the indirect connection of the present application may include that the two parts connected (for example, the H-FR2 of the present application and the HCDR1 of the present application, or the H-FR2 of the present application and the HCDR2 of the present application) are separated by 1 or 2 , 3, 4, 5 or more amino acids, which may be any naturally occurring or modified amino acids.
  • the H-FR2 of the present application may comprise the amino acid sequence shown in any one of SEQ ID NO:5, 27, 81 or a variant thereof.
  • the H-FR2 of the present application may comprise the amino acid sequence shown in any one of SEQ ID NO:5, 27, 18, 36 or a variant thereof.
  • the H-FR3 of the present application may be located between the HCDR2 of the present application and the HCDR3 of the present application.
  • the N-terminal of the H-FR3 of the present application can be directly or indirectly connected to the C-terminal of the HCDR2 of the present application
  • the C-terminal of the H-FR3 of the present application can be directly or indirectly connected to the N-terminal of the HCDR3 of the present application.
  • the indirect connection may include 1, 2, or 3 parts between the two parts connected (for example, the H-FR3 of the present application and the HCDR2 of the present application, or the H-FR3 of the present application and the HCDR3 of the present application). , 4, 5 or more amino acids, which may be any naturally occurring or modified amino acid.
  • the H-FR3 of the present application may comprise the amino acid sequence shown in any one of SEQ ID NO: 6, 19 or a variant thereof.
  • the N-terminal of the H-FR4 of the present application may be directly or indirectly connected to the C-terminal of the HCDR3 of the present application.
  • the indirect connection may include 1, 2, 3, 4, 5 or more between the N-terminal amino acid residues of the L-FR4 of the present application and the C-terminal amino acid residues of the LCDR3 of the present application.
  • Amino acids which may be any amino acid naturally occurring or modified.
  • the H-FR4 of the present application may comprise the amino acid sequence shown in any one of SEQ ID NO: 7, 20 or a variant thereof.
  • a variant of the present application may comprise at least 80%, at least 85%, at least 90%, at least 95% of the amino acid sequence shown in any one of SEQ ID NO: 4-7, 17-20, 27 or 36 Amino acid sequences that are 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% identical.
  • variable region constant region, heavy chain and light chain
  • the antigen binding protein of the present application may comprise a heavy chain variable region (VH) and/or a light chain variable region (VL).
  • VH heavy chain variable region
  • VL light chain variable region
  • the heavy chain variable region of the present application may comprise the amino acid sequence shown in any one of SEQ ID NO: 73, 74 or a variant thereof.
  • the light chain variable region of the present application may comprise the amino acid sequence shown in any one of SEQ ID NO:75, 76 or a variant thereof.
  • a variant of the present application may comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97% of the amino acid sequence shown in any one of SEQ ID NO: 73-76 , an amino acid sequence that is 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% identical.
  • the heavy chain variable region of the present application comprises the amino acid sequence shown in any one of SEQ ID NO: 73, 74 or a variant thereof.
  • the light chain variable region of the present application comprises the amino acid sequence shown in any one of SEQ ID NO: 75, 76 or a variant thereof.
  • the heavy chain variable region of the present application comprises the amino acid sequence shown in any one of SEQ ID NO: 8, 21, 28, 37 or 41 or a variant thereof.
  • the light chain variable region of the present application comprises the amino acid sequence shown in any one of SEQ ID NO: 16, 35, 25, 40 or a variant thereof.
  • the heavy chain variable region of the present application comprises the nucleotide sequence shown in any one of SEQ ID NO: 57, 59, 61, 63, 65 or a variant thereof.
  • the light chain variable region of the present application comprises the nucleotide sequence shown in any one of SEQ ID NO:58, 60, 62, 64, 66 or variants thereof.
  • a variant of the present application may comprise 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% identical amino acid sequences.
  • 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 derived from a human IgG heavy chain constant region.
  • 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 derived from a human IgG1 heavy chain constant region.
  • the heavy chain constant region of the antibody of the present application may comprise the amino acid sequence shown in SEQ ID NO: 42 or a variant thereof.
  • variants of the present application may comprise at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least Amino acid sequences that are 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% identical.
  • the heavy chain constant region variant of the present application can be included on the basis of the human IgG1 heavy chain constant region (amino acid sequence such as SEQ ID NO: 42), comprising the following amino acid mutations: M135Y, S137T and/or T139E (amino acid sequence numbering Based on the sequence SEQ ID NO: 42).
  • amino acid sequence such as SEQ ID NO: 42
  • amino acid mutations M135Y, S137T and/or T139E (amino acid sequence numbering Based on the sequence SEQ ID NO: 42).
  • the antigen binding protein of the present application may comprise an antibody light chain constant region
  • the antibody light chain constant region of the present application may comprise a human Ig ⁇ (Kappa) constant region.
  • the light chain constant region of the antibody of the present application may comprise the amino acid sequence shown in SEQ ID NO: 43 or a variant thereof.
  • variants of the present application may comprise at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least Amino acid sequences that are 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% identical.
  • the antigen binding protein of the present application may comprise an antibody heavy chain
  • the antibody heavy chain of the present application may comprise the amino acid sequence shown in any one of SEQ ID NO: 45, 47 or 49 or a variant thereof.
  • the antigen-binding protein of the present application may comprise an antibody heavy chain
  • the antibody heavy chain of the present application may comprise the nucleotide sequence shown in any one of SEQ ID NO:67, 69, or 71 or a variant thereof.
  • a variant of the present application may comprise at least 80%, at least 85%, at least 90%, at least 95% of the amino acid sequence shown in any one of SEQ ID NO:45, 47, 49, 67, 69, 71 Amino acid sequences that are 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% identical.
  • the antigen binding protein of the present application may comprise an antibody light chain
  • the antibody light chain of the present application may comprise the amino acid sequence shown in any one of SEQ ID NO: 46 or 48 or a variant thereof.
  • the antigen-binding protein of the present application may comprise an antibody light chain
  • the antibody light chain of the present application may comprise the nucleotide sequence shown in any one of SEQ ID NO: 68, 70 or 72 or a variant thereof.
  • a variant of the present application may comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96% of the amino acid sequence shown in any one of SEQ ID NO: 45-49, 67-72 Amino acid sequences that are 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% identical.
  • the antigen-binding protein of the present application wherein said FR1, FR2, FR3 and FR4 may comprise an amino acid sequence selected from any one of the following groups:
  • 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, and the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:6 Amino acid sequence, 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, and the L-FR2 comprises the amino acid sequence shown in SEQ ID NO:13
  • the amino acid sequence shown, the L-FR3 includes the amino acid sequence shown in SEQ ID NO: 14, and the L-FR4 includes the amino acid sequence shown in SEQ ID NO: 15.
  • 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, and the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:6 Amino acid sequence, 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, and the L-FR2 comprises the amino acid sequence shown in SEQ ID NO:32
  • the amino acid sequence shown, the L-FR3 includes the amino acid sequence shown in SEQ ID NO:33, and the L-FR4 includes the amino acid sequence shown in SEQ ID NO:34.
  • 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
  • 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 amino acid sequence shown, the L-FR3 includes the amino acid sequence shown in SEQ ID NO:24
  • the L-FR4 includes the amino acid sequence shown in SEQ ID NO:15.
  • 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
  • Amino acid sequence 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 amino acid sequence shown, the L-FR3 includes the amino acid sequence shown in SEQ ID NO: 19, and the L-FR4 includes the amino acid sequence shown in SEQ ID NO: 15.
  • the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:17, 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, and 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.
  • an antigen binding protein of the present application may comprise an antibody heavy chain of the present application and an antibody light chain of the present application.
  • the antigen-binding protein of the present application can bind to IgE (such as human IgE) with a KD value of about 2.4 ⁇ 10 -9 M or less.
  • the binding can be determined by Octet method, MSD-SET method and/or KinExA method commonly used in the art.
  • the KD value can be determined by MSD-SET, and the measurement method is as described in Estep P. et al., MAbs, 2013.5 (2): p.270-8.
  • the KD value can also be determined by the KinExA method, and the measurement method As described by Jonathan K. Fleming et al., Methods Mol Biol. 2018;1697:1-8.
  • the KD value of the antigen-binding protein binding to human-derived IgE antigen can be ⁇ 2 ⁇ 10 -9 M, ⁇ 1.5 ⁇ 10 -9 M, ⁇ 1 ⁇ 10 -9 M, ⁇ 8 ⁇ 10 -10 M, ⁇ 7 ⁇ 10 -10 M, ⁇ 6 ⁇ 10 -10 M, ⁇ 5 ⁇ 10 -10 M, ⁇ 4.5 ⁇ 10 -10 M , ⁇ 4 ⁇ 10 -10 M, ⁇ 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,
  • the KD value of the antigen-binding protein of the present application for binding to the IgE can be, 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
  • the antigen binding protein of the present application can inhibit the binding of IgE to its receptors Fc ⁇ RIa and/or Fc ⁇ RII.
  • the antigen binding protein of the present application can inhibit the binding of IgE to its receptor Fc ⁇ RIa with an IC value of ⁇ 2000ng/mL, ⁇ 1800ng/mL, ⁇ 1500ng/mL, ⁇ 1300ng/mL, ⁇ 1000ng/mL mL, ⁇ 950ng/mL, ⁇ 900ng/mL, ⁇ 850ng/mL, ⁇ 800ng/mL, ⁇ 750ng/mL, ⁇ 700ng/mL, ⁇ 650ng/mL, ⁇ 600ng/mL, ⁇ 550ng/mL, ⁇ 500ng/mL mL, ⁇ 450ng/mL, ⁇ 430ng/mL, ⁇ 400ng/mL.
  • the antigen binding protein of the present application can inhibit the binding of IgE to its receptor Fc ⁇ RIa with an IC 50 value of ⁇ 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 M.
  • the antigen binding protein of the present application can inhibit the binding of IgE to its receptor Fc ⁇ RIa IC50 value can be, 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 mL, ⁇ 28ng/mL, ⁇ 27ng/mL, ⁇ 26ng/mL, ⁇ 25ng/mL, ⁇ 24ng/mL, ⁇ 23ng/mL, ⁇ 22ng/mL, ⁇ 21ng/mL, ⁇ 20ng/mL, ⁇ 19ng/mL mL, ⁇ 18ng/mL, ⁇ 17
  • the antigen binding protein of the present application can inhibit the binding of IgE to its receptor Fc ⁇ RII with an IC50 value of, for example, ⁇ 4.0 ⁇ g/mL, ⁇ 3.9 ⁇ g/mL, ⁇ 3.8 ⁇ g/mL, ⁇ 3.7 ⁇ g/mL, ⁇ 3.6 ⁇ g/mL, ⁇ 3.5 ⁇ g/mL, ⁇ 3.4 ⁇ g/mL, ⁇ 3.3 ⁇ g/mL, ⁇ 3.2 ⁇ g/mL, ⁇ 3.1 ⁇ g/mL, or ⁇ 3.0 ⁇ g/mL.
  • an IC50 value of, for example, ⁇ 4.0 ⁇ g/mL, ⁇ 3.9 ⁇ g/mL, ⁇ 3.8 ⁇ g/mL, ⁇ 3.7 ⁇ g/mL, ⁇ 3.6 ⁇ g/mL, ⁇ 3.5 ⁇ g/mL, ⁇ 3.4 ⁇ g/mL, ⁇ 3.3 ⁇ g/mL, ⁇ 3.2 ⁇ g/mL, ⁇ 3.1
  • an antigen binding protein of the present application may comprise an antibody or an antigen binding fragment thereof.
  • antibodies of the present application may include, but are not limited to, recombinant antibodies, monoclonal antibodies, human antibodies, murine antibodies, humanized antibodies, chimeric antibodies, camelized single domain antibodies, bispecific antibodies, single chain antibodies, diabody, triabody or tetrabody.
  • the antibodies of the present application may be humanized antibodies.
  • the antigen-binding protein of the present application can immunospecifically bind to a related antigen (such as a human IgE antigen and comprise a framework region (FR) that essentially has the amino acid sequence of a human antibody and a complementary FR that essentially has the amino acid sequence of a non-human antibody. Determining regions (CDRs) of antibodies or variants, derivatives, analogs or fragments thereof).
  • Essentially here in the context of CDRs may mean that the amino acid sequence of the CDR is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97% identical to the amino acid sequence of the CDR of a non-human antibody , at least 98%, at least 98.5%, or at least 99% identical.
  • the humanized antibodies of the present application may comprise substantially all of at least one and usually two variable domains (Fab, Fab', F(ab')2, Fv), wherein all or substantially all of the CDR regions correspond to non-human immune domains.
  • the CDR regions and all or substantially all of the framework regions of a globulin are framework regions having human immunoglobulin consensus sequences.
  • the humanized antibody also will comprise at least a portion of an immunoglobulin constant region (eg, Fc), typically that of a human immunoglobulin, for example.
  • a humanized antibody contains at least the variable domain of a light chain or a heavy chain.
  • Antibodies may also include the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain.
  • a humanized antibody may contain only humanized light chains.
  • a humanized antibody can contain only a humanized heavy chain.
  • a humanized antibody may contain only the light chain and/or the humanized variable domain of a humanized heavy chain.
  • antigen-binding fragments of the present application may include Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv and/or dAb.
  • the present application provides a polypeptide, which may comprise the antigen-binding protein of the present application.
  • the polypeptides of the present application may also include other peptides or protein fragments, for example, they may also include other frameworks or framework parts that facilitate the conformation of the antigen-binding protein to bind to the antigen.
  • the polypeptide of the present application may also include a part that binds to other antigens, and/or a protein fragment with other functions.
  • the polypeptides of the present application can be fusion proteins, multispecific antibodies, and/or other recombinant proteins.
  • the present application provides an immunoconjugate, which may comprise the antigen-binding protein of the present application or the polypeptide of the present application.
  • immunoconjugates of the present application may include other polypeptides, therapeutic agents (eg, cytotoxic molecules), probes, and/or other antibodies fused or conjugated to the antigen binding protein.
  • the immunoconjugates described herein can include pharmaceutically acceptable labels, detection agents and/or therapeutics.
  • the application provides isolated one or more nucleic acid molecules that encode an antigen binding protein or polypeptide of the application.
  • each of the one or more nucleic acid molecules may encode the entirety of the isolated antigen binding protein or the polypeptide, or may encode a portion thereof.
  • the nucleic acid molecules described herein can be isolated.
  • the nucleic acid encoding the antigen-binding protein or the polypeptide can be prepared by various methods known in the art.
  • the present application provides one or more vectors comprising one or more nucleic acid molecules described herein.
  • Each vector may contain one or more such nucleic acid molecules.
  • other genes may be included in the vector, such as marker genes that allow selection of the vector in appropriate host cells and under appropriate conditions.
  • the vector may contain a variety of expression-controlling elements, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes.
  • the vector may also contain an origin of replication.
  • the vectors may include, for example, plasmids, cosmids, viruses, phages, or other vectors commonly used in, for example, genetic engineering.
  • the present application provides a cell, which may comprise one or more nucleic acid molecules described herein and/or one or more vectors described herein.
  • each or each cell may comprise one or more of the nucleic acid molecules or vectors described herein.
  • each or each cell may contain multiple (eg, 2 or more) or multiple (eg, 2 or more) nucleic acid molecules or vectors described herein.
  • the vectors described herein can be introduced into said cells, such as prokaryotic cells (e.g., bacterial cells), CHO cells, NS/0 cells, HEK293 cells, or other eukaryotic cells, such as cells from plants, fungi or yeast cells etc.
  • the vectors described in this application can be introduced into the cells by methods known in the art, such as electroporation, lipofectine transfection, lipofectamin transfection and the like.
  • vectors can 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, etc.
  • 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, etc.
  • Types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillary polyoma vacuoles Viruses (such as SV40).
  • the vector may contain various expression-controlling elements, including a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selection element, and a reporter gene.
  • the vector may also contain an origin of replication.
  • the vector may also include components that facilitate its entry into cells, such as, but not exclusively, viral particles, liposomes or protein coats.
  • the cells may include prokaryotic cells, yeast cells, or higher eukaryotic cells.
  • Prokaryotes suitable for this purpose include Gram-negative and Gram-positive bacteria, for example, enterobacteria such as Escherichia coli, Enterobacter, Erwinia, Klebsiella, Proteus (Proteus), Salmonella, Serratia, and Shigella, as well as Bacillus, Pseudomonas, and Streptomyces.
  • the cells can include mammalian host cell lines.
  • mammalian host cell lines for example, monkey kidney cells, human embryonic kidney cell lines, baby hamster kidney cells, Chinese hamster ovary cells, mouse Sertoli cells, human cervical cancer cells (HELA), canine kidney cells, human lung cells, human Liver cells, mouse breast cancer cells or NSO cells.
  • HELA human cervical cancer cells
  • the cells may also include cells transfected with the vector of the present invention in vitro.
  • 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.
  • the cells may be mammalian cells.
  • the mammalian cells can be HEK293 cells.
  • the present application also provides a method for preparing the antigen-binding protein of the present application, and the method may include culturing the cell described in the present application under the condition that the antigen-binding protein described in the present application is expressed.
  • the present application also provides the application of the antigen-binding protein of the present application, the nucleic acid molecule of the present application, the carrier of the present application, the cells of the present application and/or the pharmaceutical composition of the present application in the preparation of medicines. Medications are used to prevent, alleviate and/or treat IgE-related diseases or conditions.
  • the present application also provides a method for preventing, alleviating or treating IgE-related diseases or disorders.
  • the method of the present application may include administering the antigen-binding protein of the present application, the nucleic acid molecule of the present application, the nucleic acid molecule of the present application to a subject in need.
  • the present application also provides the antigen binding protein of the present application, the nucleic acid molecule of the present application, the carrier of the present application, the cell of the present application and/or the pharmaceutical composition of the present application, which can be used for prevention, alleviation or treatment IgE-associated disease or condition.
  • IgE-related diseases or conditions may include diseases or conditions for which Omalizumab (trade name Xolair) has been approved worldwide (eg, the United States, China, Europe, etc.) and is applicable for treatment.
  • the disease or condition can include allergic diseases.
  • the subject can include humans and non-human animals.
  • the subject may include, but is not limited to, cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats, or monkeys.
  • the application also provides a pharmaceutical composition, which comprises the antigen binding protein of the application, the nucleic acid molecule of the application, the carrier of the application and/or the cell of the application, and optionally a pharmaceutically acceptable carrier agent.
  • a pharmaceutical composition may additionally contain one or more other therapeutic agents suitable for the treatment or prevention of an IgE-associated disease or condition.
  • the pharmaceutically acceptable carrier enhances or stabilizes the composition, or facilitates preparation of the composition.
  • the pharmaceutically acceptable carrier may include physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like.
  • the administration can be carried out in different ways, such as intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.
  • the pharmaceutical compositions of the present application can be administered by various methods known in the art, which vary depending on the desired result, the route of administration, and/or the mode of administration.
  • administration can be intravenous, intramuscular, intraperitoneal, or subcutaneous, or administration adjacent to the target site.
  • the pharmaceutical compositions are formulated so that they can be administered intravitreally into the eye.
  • the antigen binding proteins e.g., antibodies, bispecific and multispecific molecules
  • the antigen binding proteins can be coated with materials to protect the compound from acid or other natural conditions that can inactivate the compound.
  • compositions of the present application can be sterile and fluid.
  • Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, or in the case of dispersions, by maintaining the required particle size, and by the use of surfactants.
  • isotonic agents are included in the composition, for example, sugars, polyalcohols such as mannitol, sorbitol or sodium chloride.
  • prolonged absorption of the injectable pharmaceutical compositions can be brought about by including in the pharmaceutical compositions an agent delaying absorption (eg, aluminum monostearate or gelatin).
  • the antigen binding protein of the present application, the nucleic acid molecule of the present application, the carrier of the present application and/or the cell of the present application are mixed with optional pharmaceutically acceptable carriers, excipients or stabilizers and prepared for storage.
  • the form of the pharmaceutical composition of the present application may be a lyophilized preparation or an aqueous solution.
  • a pharmaceutically acceptable carrier of the present application may include a pharmaceutically acceptable carrier, excipient or stabilizer.
  • acceptable carriers, excipients or stabilizers herein are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate citrates, acetates and other organic acids.
  • a pharmaceutical composition comprising an antigen binding protein of the present application may be in water-soluble form.
  • compositions for in vivo administration can be sterile. This is readily accomplished by filtration through sterile filtration membranes or other methods.
  • administration of a pharmaceutical composition comprising an antigen binding protein of the present application in the form of a sterile aqueous solution can be performed in a variety of ways including, but not limited to, orally, subcutaneously, intravenously, intranasally, intraaurally, transdermally, topically (e.g., coagulated gels, ointments, lotions, creams, etc.), intraperitoneally, intramuscularly, intrapulmonarily, parenterally, rectally, or intraocularly.
  • the antigen binding proteins of the present application can be directly applied as a solution or spray.
  • the pharmaceutical compositions of the present application can be prepared according to methods well known and routinely practiced in the art.
  • the pharmaceutical composition of the present application can be prepared under GMP conditions.
  • a therapeutically effective or effective dose of an IgE binding protein is used in the pharmaceutical compositions of the present application.
  • IgE binding proteins can be formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art. Dosage regimens are adjusted to provide the optimum desired response (eg, a therapeutic response). Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present application may be varied in order to obtain an amount of the active ingredient effective to achieve the desired therapeutic response, composition, and mode of administration for a particular patient without being toxic to the patient.
  • the selected dosage level will depend on various pharmacokinetic factors, for example, the activity of the particular composition used herein or its ester, salt or amide, the route of administration, the time of administration, the rate of excretion of the particular compound being used, the therapeutic Duration, other drugs, compounds and/or substances used in combination with the particular composition used, age, sex, weight, condition, general health and prior medical history of the subject being treated, and other factors.
  • subcutaneous administration can be used in situations where the patient can self-administer the pharmaceutical composition.
  • Many protein therapeutics are not potent enough to allow formulation of a therapeutically effective dose for subcutaneous administration of the maximum acceptable volume.
  • the antigen binding proteins disclosed herein may be suitable for subcutaneous administration, eg, may have increased potency, increased plasma half-life, and increased solubility.
  • protein therapeutics can be delivered by IV infusion or bolus injection.
  • Antigen binding proteins disclosed herein can also be delivered using such methods.
  • the present application also provides a method for detecting IgE in a sample, the method comprising administering the antigen-binding protein described in the present application.
  • a sample obtained from a subject is contacted with an antigen binding protein (eg, an IgE binding protein) of the present application.
  • an antigen binding protein eg, an IgE binding protein
  • the IgE binding protein is labeled with a detectable label or reporter molecule or an anti-IgE binding protein is used as a capture ligand to selectively isolate IgE from a patient sample.
  • an unlabeled anti-IgE binding protein can be used in detection applications in conjunction with a secondary antibody, which is itself detectably labeled.
  • the detectable label or reporter molecule may be a radioactive isotope such as 3 H, 14 C, 32 P, 35 S or 125 I; or a fluorescent or chemiluminescent moiety such as fluorescein isothiocyanate, rhodamine; or Enzymes such as alkaline phosphatase, beta-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).
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • FACS fluorescence-activated cell sorting
  • Samples that may be used in the IgE detection of the present application may include any tissue or fluid sample obtained from a subject under normal or pathological conditions, which contains detectable amounts of IgE protein or fragments thereof.
  • the level of IgE protein in a particular sample obtained from a healthy subject eg, a subject not suffering from an IgE-related disease
  • This baseline level of IgE is then compared to the IgE level measured in a sample obtained from an individual suspected of having an IgE-related disease or disorder or symptoms associated with the disorder.
  • the IgE binding protein may be free of additional tags, or may contain N-terminal or C-terminal tags.
  • the label is biotin.
  • the position of the label can determine the orientation of the peptide relative to the surface on which the peptide is bound. For example, if the surface is coated with avidin, a peptide containing N-terminal biotin will move the C-terminal portion of the peptide away from the surface.
  • Embodiment 1 Antigen-binding protein preparation of the present application
  • mice Four-week-old BALB/c mice were injected subcutaneously with 20 ⁇ g of recombinantly expressed Fc region (CH2-4) protein of human IgE (IgE-Fc, SEQ ID NO:42) in complete Freund's adjuvant. Injections are given every three to four weeks for a total of five injections. Finally, a single injection of 20 ⁇ g of recombinant human IgE-Fc protein was given intraperitoneally.
  • CH2-4 recombinantly expressed Fc region protein of human IgE protein of human IgE
  • the enzyme plate (Costar) was coated with recombinant human IgE-Fc protein and left overnight at room temperature. The coating solution was discarded, the wells were blocked with 2.5% skim milk dissolved in phosphate buffered saline (PBS) for 0.5 hours, and the wells were washed with PBS containing 0.05% Tween-20. Then add diluted serum and irrelevant antibody (Irrevalent mAb) as a negative control, incubate at room temperature for 1 hour, wash the wells with PBS containing 0.05% Tween (Tween)-20, and then add 50 ⁇ l of HRP-labeled goat antibody to each well. Mouse IgG polyclonal antibody (Jackson Laboratory) was used as detection antibody. After sera testing, mice containing high levels of anti-recombinant human IgE-Fc protein antibody serum were identified.
  • PBS phosphate buffered saline
  • mice containing high levels of anti-human IgE-Fc antibody serum the mouse spleen was removed and fused with the mouse myeloma Sp2/0 cell line. 5 ⁇ 10 8 Sp2/0 cells and 5 ⁇ 10 8 splenocytes were mixed for fusion in 50% polyethylene glycol (PEG, molecular weight 1450) and 5% dimethylsulfoxide (DMSO) solution.
  • PEG polyethylene glycol
  • DMSO dimethylsulfoxide
  • Iscove medium containing 10% fetal bovine serum, 100 units/mL penicillin, 100 ⁇ g/mL streptomycin, 10 mM hypoxanthine, 0.4 ⁇ M aminopterin and 1.6 mM thymidine
  • Iscove medium containing 10% fetal bovine serum, 100 units/mL penicillin, 100 ⁇ g/mL streptomycin, 10 mM hypoxanthine, 0.4 ⁇ M aminopterin and 1.6 mM thymidine
  • mRNA was isolated from mouse hybridoma cells using the mRNA purification kit (NEB) to prepare cDNA (SMARTer RACE kit, Clontech). Heavy chain variable region DNA fragments were cloned from cDNA by polymerase chain reaction (PCR).
  • the 5'-primer mixture of PCR 0.4 ⁇ M of primer 1 (5'-ctaatacgactcactatagggcAAGCAGTGGTATCAACGCAGAGT-3', as shown in SEQ ID NO:53) and 2 ⁇ M of primer 2 (5'-ctaatacgactcactatagggc-3', as shown in SEQ ID NO:54).
  • the 3'-primer of PCR is primer 3 (5'-catcccagggtcaccatggagtta-3', shown in SEQ ID NO:55). Primer 3 is homologous and antisense to the mouse IgG1 heavy chain constant region.
  • variable region DNA fragment obtained after gel purification was cloned into the TOPO-TA vector (Invitrogen) and sequenced to obtain the variable region amino acid sequence (as shown in SEQ ID NO: 8) encoding the SE2 mouse hybridoma heavy chain And nucleotide sequence (as shown in SEQ ID NO:57), determine the aminoacid sequence HCDR1 (as shown in SEQ ID NO:1) of its complementarity determining region by Kabat definition mode, HCDR2 (as shown in SEQ ID NO:2 ) and HCDR3 (as shown in SEQ ID NO:3), and the variable region amino acid sequence (as shown in SEQ ID NO:28) and nucleotide sequence (as shown in SEQ ID NO: 61), and the amino acid sequences HCDR1 (as shown in SEQ ID NO: 1), HCDR2 (as shown in SEQ ID NO: 26) and HCDR3 (as shown in SEQ ID NO: 3) of the complementarity
  • step (1) use the above 5'-primer mixture (primer 1 and primer 2) and another 3'-primer (primer 4 , 5'-gactgaggcacctccagatgttaa-3', as shown in SEQ ID NO:56), clone the light chain variable region DNA fragment from cDNA.
  • DNA fragments are cloned into the TOPO-TA vector and sequenced to obtain the amino acid sequence (as shown in SEQ ID NO: 16) and nucleotide sequence (as shown in SEQ ID NO: 16) of the variable region encoding SE2 mouse hybridoma light chain :58), and the amino acid sequence LCDR1 (as shown in SEQ ID NO: 9), LCDR2 (as shown in SEQ ID NO: 10) and LCDR3 (as shown in SEQ ID NO: 11) of its complementary determining region, And the variable region amino acid sequence (as shown in SEQ ID NO: 35) and the nucleotide sequence (as shown in SEQ ID NO: 62) of coding SE5 mouse hybridoma light chain, and the amino acid sequence LCDR1 of its complementarity determining region (as shown in SEQ ID NO:9), LCDR2 (as shown in SEQ ID NO:29) and LCDR3 (as shown in SEQ ID NO:30).
  • the heavy chain and light chain variable region sequences of the murine monoclonal antibody cloned in step 2) were inserted into the sequence containing the signal peptide (as shown in SEQ ID NO: 50) and the human IgG1 heavy chain constant using a seamless cloning method, respectively.
  • region as shown in SEQ ID NO:42
  • pCDNA3.1 purchased from Miaoling Bio
  • the plasmid was prepared using the NucleoBond Xtra Midi Plus (MACHEREY-NAGEL) plasmid extraction kit.
  • the prepared chimeric monoclonal antibody heavy chain and light chain plasmids were co-transfected into Expi293 cells (purchased from Thermo fisher) using PEI (PolyScience) to transiently express the monoclonal antibody.
  • Transiently expressed antibodies were purified using protein A columns and the monoclonal antibody eluate was dialyzed against PBS. The dialyzed solution was filter sterilized through a 0.2 ⁇ m filter to obtain the antibody sample to be tested.
  • the humanized nucleotide sequence of the SE2 heavy chain variable region is shown in SEQ ID NO: 21, and the humanized nucleotide sequence of the SE2 light chain variable region As shown in SEQ ID NO:60, the nucleotide sequence of the humanized variable region of the heavy chain of SE5 is shown in SEQ ID NO:63, and the nucleotide sequence of the humanized nucleotide sequence of the variable region of the light chain of SE5 is shown in SEQ ID NO:64 As shown, the resulting DNA was used for recombinant antibody expression vector construction.
  • the heavy chain and light chain variable regions of the humanized monoclonal antibody obtained in step 1) were inserted into the human IgG1 heavy chain constant region containing the signal peptide (as shown in SEQ ID NO: 50) using a seamless cloning method, respectively. (as shown in SEQ ID NO:42), or pCDNA3.1 (purchased from Miao Spirit organisms) eukaryotic expression vector, after DNA sequencing confirmed the correctness, use the NucleoBond Xtra Midi Plus (MACHEREY-NAGEL) plasmid extraction kit to prepare the plasmid.
  • the prepared humanized monoclonal antibody heavy chain and light chain plasmids were co-transfected into Expi293 cells (purchased from Thermo fisher) using PEI (PolyScience) to transiently express the monoclonal antibody.
  • Transiently expressed antibodies were purified using protein A columns and the monoclonal antibody eluate was dialyzed against PBS. The dialyzed solution was filter sterilized through a 0.2 ⁇ m filter to obtain the antibody sample to be tested.
  • N53 in the CDR2 region of the heavy chain of SE5 (as shown in SEQ ID NO:26) is a potential N-glycosylation site, and N53S/T57S mutations were carried out on humanized SE5
  • M135Y, S137T and/or T139E (YTE) were modified to the Fc region of SE5 (SEQ ID NO: 42) to enhance the affinity of the antibody molecule to the neonatal receptor FcRn under acidic conditions, thereby extending the antibody molecule in The half-life in the body can achieve the purpose of prolonging the administration cycle.
  • the modified humanized molecule is called SE5ss.
  • the blocking activity of the purified mouse monoclonal antibody in blocking the binding of human IgE-Fc to its receptor Fc ⁇ RIa was detected by ELISA method.
  • the specific method is that the recombinantly expressed human Fc ⁇ RIa-Fc protein (amino acid sequence shown in SEQ ID NO: 51) is diluted to a final concentration of 2.5 ⁇ g/mL with PBS, and 100 ⁇ L/well is coated with a microtiter plate (purchased from Corning, Cat. No. : 42592), warm overnight at 4°C. The next day, the coating solution was discarded, blocked with 2.5% skimmed milk dissolved in PBS for 1 hour, and washed with PBS containing 0.05% Tween 20.
  • the ELISA method similar to that of Example 2 was used to test the blocking activity of the purified chimeric antibody to block the binding of human IgE-Fc to its receptor Fc ⁇ RIa.
  • the specific method is that the recombinantly expressed human Fc ⁇ RIa-Fc protein (amino acid sequence shown in SEQ ID NO: 51) is diluted to a final concentration of 1 ⁇ g/mL with PBS, and 100 ⁇ L/well is coated with a microtiter plate (purchased from Corning, Cat. No.: 42592), warm overnight at 4°C.
  • the coating solution was discarded, blocked with 2.5% skimmed milk dissolved in PBS for 1 hour, and washed with PBS containing 0.05% Tween 20.
  • Inhibition rate of antibody concentration X (%) (OD 450 reading value of antibody concentration 0 - OD 450 reading value of antibody concentration X) / OD 450 reading value of antibody concentration 0 ⁇ 100, the calculation is different Antibody, inhibition rate at different concentrations.
  • the test results are shown in Figure 2.
  • the chimeric recombinant monoclonal antibody constructed from the cloned mouse monoclonal variable region sequence has similar blocking activity to the mouse parent antibody purified from its hybridoma.
  • Example 2 1) Using an ELISA method similar to Example 2 to detect the blocking activity of the purified murine antibody, chimeric antibody and humanized antibody in blocking the binding of human IgE-Fc to its receptor Fc ⁇ RIa.
  • the specific method is that the recombinantly expressed human Fc ⁇ RIa-Fc protein (amino acid sequence as shown in SEQ ID NO: 51) is diluted to a final concentration of 1 ⁇ g/mL with PBS, and 100 ⁇ L/well is coated with a microtiter plate (purchased from Corning, article number: 42592), warm overnight at 4°C.
  • step 1) of this example the blocking activity of SE2 humanized antibody, SE5 humanized antibody and omalizumab in blocking the binding of human IgE-Fc to its receptor Fc ⁇ RIa was detected.
  • the antibody concentration was diluted 3 times from 3 ⁇ g/mL (ie 3 ⁇ g/mL, 1 ⁇ g/mL, 333.33 ng/mL, 111.11 ng/mL, 37.04 ng/mL mL, 12.35ng/mL, 4.12ng/mL, 1.37ng/mL, and finally 0ng/mL); in the comparative experiment of SE5 humanized antibody and omalizumab, the antibody concentration was diluted 3 times from 10nM ( ie 10nM, 3.33nM, 1.11nM, 370.37pM, 123.46pM, 41.15pM, 13.72pM, and finally OpM).
  • Inhibition rate (%) of antibody concentration X (OD 450 reading value of antibody concentration 0 - OD 450 reading value of antibody concentration X) / OD450 reading value of antibody concentration 0 ⁇ 100, calculate different antibodies , Inhibition rate at different concentrations.
  • the results are shown in Figure 4-5, SE2 humanized antibody and SE5 humanized antibody have stronger blocking activity than the control antibody omalizumab, and the blocking IC 50 values of SE2 and omalizumab were respectively
  • the blocking IC 50 values of SE5 and omalizumab were about 416.7ng/mL and 2110ng/mL, about 266.2pM and 4996pM, respectively.
  • MSD-SET hypersensitive factor electrochemiluminescence analysis-solution equilibrium titration method
  • the antigen human IgE remained constant at 13.33pM, and the humanized antibodies SE2 and SE5 were serially diluted threefold from 166.67pM to 0.0085pM; when measured against omalizumab, the antigen human IgE remained constant At 133.3 pM, omalizumab was serially diluted 3-fold starting at 66.67 nM to 1.13 pM.
  • Antibody diluted at 20 nM in PBS was coated onto a standard binding MSD-ECL plate (purchased from MSD, Cat. No.: L15XA-3) overnight at 4°C. The MSD-ECL plate was blocked with 1% Casein for 30 minutes while shaking at 700 rpm.
  • the MSD-ECL plate was then washed 3 times with wash buffer (PBS+0.05% Tween 20).
  • the samples incubated with 2-fold serial dilutions of the antigen human IgE and the humanized antibody were added to the MSD-ECL plate, incubated for 150s under shaking at 700rpm, and then washed once.
  • PBSF containing 250 ng/mL sulfo-tag-labeled streptavidin was added to the MSD-ECL plate, and after 5 minutes, the MSD-ECL plate was washed 3 times with washing buffer (PBS+0.05% Tween 20).
  • Table 1 The equilibrium affinity of humanized antibody and control antibody to human IgE detected by MSD-SET
  • KinExA was used to detect the equilibrium affinity of humanized antibody and control antibody (Omalizumab) to human IgE (derived from U266 cells (ATCC), see Mol Immunol. 1986, 23(2):159-67 for the preparation method).
  • Solution equilibrium titration (SET) was carried out in PBS+0.1% IgG-free BSA buffer solution (PBSF), wherein the antigen human IgE-Biotin (biotin-labeled human IgE, the preparation method refers to Thermofisher EZ-Link TM Sulfo-NHS-LC -Biotinylation Kit Instructions) kept constant at 20pM-5nM, and incubated with two-fold serial dilution of antibody from 2nM-1 ⁇ M.
  • PBSF PBS+0.1% IgG-free BSA buffer solution
  • the antigen human IgE-Biotin was kept constant at 20pM, and the humanized antibody SE5ss was serially diluted two-fold from 2nM to 30.52fM, and the mixed samples were incubated at room temperature for 30 hours; for the control
  • the antigen human IgE-Biotin was kept constant at 5nM, and Omalizumab was serially diluted twice from 1 ⁇ M to 15.26pM, and the mixed samples were incubated at room temperature for 20 hours.
  • the sample incubated with the antigen human IgE-Biotin and the two-fold serial dilution of the antibody flows through the beads coated with the antibody in the KinExA instrument (model: 4000), and the free antigen human IgE-Biotin in the sample that is not bound to the antibody will be combined with the beads After binding the coated antibody, continue to flow the fluorescent secondary antibody Alexa Fluor 647 Streptavidin through the beads.
  • the instrument detects the fluorescent signal on the beads and obtains the KD value through KinExA software fitting. The measurement results are shown in Figures 6A-B.
  • Example 7 Antigen-binding protein of the present application (modified anti-human IgE humanized antibody) blocking activity detection
  • the antigen-binding protein of the present application blocks the binding activity of human IgE to its receptor Fc ⁇ RIa
  • the blocking activity of SE5ss monoclonal antibody in blocking the binding of human IgE to its receptor Fc ⁇ RIa was detected by ELISA method.
  • the specific method is as follows: the recombinantly expressed human Fc ⁇ RIa-Fc protein was diluted to 1 ⁇ g/mL with PBS, 100 ⁇ L/well was coated with a microtiter plate (Corning, catalog number: 42592), and incubated overnight at 4°C.
  • the antigen binding protein of the present application blocks the blocking activity of human IgE and its receptor Fc ⁇ RII (CD23) receptor
  • step (1) the blocking activity of SE5ss blocking human IgE and its receptor Fc ⁇ RII (CD23) was detected.
  • the specific method is as follows: Dilute recombinant human Fc ⁇ RII (CD23, purchased from Yiqiao Shenzhou, Cat. No.: 10261-H07H) protein with PBS to 1.5 ⁇ g/mL, and coat 100 ⁇ L/well on a microtiter plate (Corning, Cat. No.: 42592). Discard the coating solution the next day, and block with 1% casein for 2 hours at room temperature.
  • RBL-2H3-Fc ⁇ RI ⁇ -NFATLuc cell line construction In order to detect IgE-mediated cell activation and the antigen-binding protein of the present application to block the cellular level activity of IgE, rat basophil RBL-2H3 cell line (Cell Bank of Chinese Academy of Sciences) ), a cell line expressing human Fc ⁇ RI ⁇ receptor and NFAT-driven luciferase (Luciferase) reporter gene was constructed, named: RBL-2H3-Fc ⁇ RI ⁇ -NFATLuc.
  • the RBL-2H3-Fc ⁇ RI ⁇ -NFATLuc cells in the logarithmic growth phase were spread on a 96-well flat bottom plate, with about 5E4 cells per well and 50uL of medium. Then add premixed human IgE and gradient concentrations of the antibodies to be tested (SE5ss, Omalizumab and human IgG), and incubate for about 20 hours at 37° C. in a 5% CO 2 incubator.
  • the constant final concentration of human IgE is 1000 ng/mL, the final concentration of the antibody to be tested starts from 45ug/mL, 3-fold serial dilution of 8 points to 0.021ug/mL, plus 0ug/mL point.
  • the RBL-2H3-Fc ⁇ RI ⁇ -NFATLuc cells in the logarithmic growth phase were spread on a 96-well flat bottom plate, with about 5E4 cells per well and 50uL of medium.
  • the antibodies to be tested (SE5ss, Omalizumab, and human IgG) at gradient concentrations and human serum from different allergens were sequentially added to the cells, and incubated in a 37°C, 5% CO2 incubator for about 20 hours. Since the IgE content of specific allergens in each serum is different, different concentrations of serum and antibodies were used.
  • Dust mite allergic human serum (PlasmaLab, product number: PL25740) was used at a concentration of 1.5%, and the final antibody concentration was 3-fold serially diluted from 10,000 ng/mL to 5 ng/mL; shrimp allergic human serum (PlasmaLab, product number: PL24819) was used The concentration is 3%, the final concentration of Omalizumab and human IgG is diluted 12 points from 1200.00ng/mL to 0.01ng/mL, the concentration of SE5ss is from 133.33ng/mL, serially diluted 11 points to 0.002ng /mL; walnut-allergic human serum (PlasmaLab, product number: PL27530) is used at a concentration of 1.5%, and the final antibody concentration starts from 10,000 ng/mL, 3-fold serial dilution of 9 points to 1.5 ng/mL, and 0 ug/mL is added to all three groups point as a comparison.
  • the plate was washed twice with PBS, and 100 ⁇ L of the corresponding allergen containing 10 ⁇ g/mL was added to each well (dust mite allergen, purchased from Greerlabs, product number: XPB82D3A25; shrimp allergen, purchased from Wokawi, product number: M110227; walnut Allergens, purchased from Wokawi, product number: M110343) complete medium, put in the incubator and incubate for about 3 hours.
  • dust mite allergen purchased from Greerlabs, product number: XPB82D3A25
  • shrimp allergen purchased from Wokawi, product number: M110227
  • walnut Allergens purchased from Wokawi, product number: M110343
  • Example 8 Antigen-binding protein of the present application (modified anti-human IgE humanized antibody) activity detection in animals
  • a human IgE reconstruction model was established to evaluate the inhibitory effect of SE5ss on free IgE in mice.
  • the experiment used Balb/c mice (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.), and 44 mice were randomly divided into negative control group, vehicle control group, SE5ss 0.5 ⁇ g group, and SE5ss 1 ⁇ g group according to sex and body weight. , SE5ss 2 ⁇ g group and Omalizumab 2 ⁇ g group, in which the negative control group consisted of 4 mice, and the rest of the groups had 8 mice in each group.
  • mice Each mouse was intravenously injected with 2 ⁇ g of human IgE (derived from U266 culture and purification, In house, batch number 5/20/29), and the negative control group was intravenously injected with the same volume of PBST (HyClone, batch number C520004-0001); 2 hours later Different doses of SE5ss and positive drug Omalizumab (Novartis, lot number SPX27) were injected intravenously. On the day before administration, 1 hour before administration, 1 hour after administration and 4 hours after administration, blood was collected from the jaw vein of all mice, and MSD was used to detect the content of free IgE in serum samples of mice.
  • PBST HyClone, batch number C520004-0001
  • the method is briefly described as follows : The MSD plate was coated with Fc ⁇ RI ⁇ -Fc (In house, batch number: 069-109-11), added the sample to be tested, incubated at 37 degrees for 1 hour, washed 3 times with PBST, added biotin-labeled mouse anti-human IgE antibody (In house , Clone#AE10-10), incubated at 37 degrees for 1 hour, washed 3 times with PBST, and detected with MSD sulfo-tag labeled streptavidin (MSD, batch number: R32AD-1).
  • MSD MSD sulfo-tag labeled streptavidin
  • SE5ss and Omalizumab of different concentrations prepared according to Example 1 can significantly inhibit free IgE in mice (P ⁇ 0.05), and the inhibitory ability is dose-dependent.
  • the inhibitory ability of IgE was greater than that of Omalizumab at 2 ⁇ g/mouse (P ⁇ 0.05).
  • human-Fc ⁇ RIA transgenic mice purchased from Shanghai N forcing Model Biotechnology Co., Ltd.
  • 51 human-Fc ⁇ RIA transgenic mice were randomly divided into negative control group, vehicle control group, SE5ss 5 ⁇ g/mouse group, SE5ss 10 ⁇ g/mouse group, SE5ss 20 ⁇ g/mouse group and Omalizumab 20 ⁇ g/mouse group according to sex and baseline body temperature. There were 6 mice in the control group, and 9 mice in the other groups.
  • SE5ss 10 ⁇ g/mouse has the same pharmacological effect as Omalizumab 20 ⁇ g/mouse, and SE5ss 20 ⁇ g/mouse group can maintain the body temperature of mice in the normal range.

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Abstract

本申请涉及一种抗原结合蛋白，其包含重链可变区的至少一个CDR，所述重链可变区包含如SEQ ID NO:73、74中任一项所示的氨基酸序列；且其包含轻链可变区的至少一个CDR，所述轻链可变区包含如SEQ ID NO:75、76中任一项所示的氨基酸序列。

Description

分离的抗原结合蛋白及其用途 技术领域
本申请涉及生物医药领域，具体的涉及一种抗原结合蛋白及其用途。
背景技术
过敏(包括特应性)和其他过敏反应性疾病是对外来抗原不适当的或过度的免疫反应。不适当的免疫反应包括那些被误导的针对人体自身成分的免疫反应，导致自身免疫性疾病。过敏性疾病(也称作变应性疾病)是全球第六大慢性疾病，影响机体多种组织器官，一旦发作会引起各种不适症状，严重降低生活质量甚至危及生命。例如过敏性反应、过敏性鼻炎、哮喘、遗传性过敏皮炎、食物过敏和风疹，在许多国家中给占人口总数20％的人群带来痛苦，并且疾病流行的程度在逐渐增加(Wuthrich B.，Int.Arch.Allergy AppI.Tmmunol.,90,pp3_10,1989)。过敏性疾病大体上分为IgE-介导的(immunoglobulin-mediated)与IgE-非介导的(immunoglobulin-nonmediated)，由免疫球蛋白E(IgE)介导的超敏反应占多数。IgE的高亲和力受体(FcεRI)是介导变应性表现的关键。除了肥大细胞和嗜碱性粒细胞以外，FcεRI还见于许多其他细胞类型(包括嗜酸性粒细胞、血小板)和抗原呈递细胞(例如单核细胞和树突细胞)。由于IgE在介导大部分变应性反应中起重要作用，开发针对控制IgE水平的过敏性疾病的治疗策略是十分必要的。
发明内容
本申请提供了一种抗原结合蛋白，其具备以下的一种或多种优势：1)稳定性好，本申请的抗原结合蛋白成药性高，更有利于工业化生产；2)本申请的抗原结合蛋白对新生儿受体FcRn的亲和力增强，半衰期更长，可以达到延长给药周期的目的。
在某些实施方式中，本申请的抗原结合蛋白包含重链可变区的至少一个互补决定区(CDR)，所述重链可变区(VH)包含如SEQ ID NO:73、74中任一项所示的氨基酸序列；且其包含轻链可变区(VL)的至少一个互补决定区(CDR)，所述轻链可变区包含如SEQ ID NO:75、76中任一项所示的氨基酸序列。
在某些实施方式中，本申请的抗原结合蛋白具有下述性质中的一种或多种：
1)能够以约2.4×10 -9M或更小的KD值与IgE相结合；
2)能够抑制IgE与其受体FcεRIa和/或FcεRII的结合。
在某些实施方式中，本申请的抗原结合蛋白与人IgE结合。
在某些实施方式中，本申请的抗原结合蛋白包含重链HCDR3，所述HCDR3包含SEQ ID NO:3所示的氨基酸序列。
在某些实施方式中，本申请的抗原结合蛋白还包含重链HCDR2，所述HCDR2包含SEQ ID NO:77所示的氨基酸序列。
在某些实施方式中，本申请的抗原结合蛋白中所述HCDR2包含SEQ ID NO:2、26中任一项所示的氨基酸序列。
在某些实施方式中，本申请的抗原结合蛋白还包含重链HCDR1，所述HCDR1包含SEQ ID NO:1所示的氨基酸序列。
在某些实施方式中，本申请的抗原结合蛋白包含重链HCDR1、HCDR2和HCDR3；其中所述HCDR1包含SEQ ID NO:1所示的氨基酸序列，所述HCDR2包含SEQ ID NO:77所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:3所示的氨基酸序列。
在某些实施方式中，本申请的抗原结合蛋白包含重链HCDR1、HCDR2和HCDR3；其中所述HCDR1包含SEQ ID NO:1所示的氨基酸序列，所述HCDR2包含SEQ ID NO:2、26中任一项所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:3所示的氨基酸序列。
在某些实施方式中，本申请的抗原结合蛋白包含重链框架区1(H-FR1)，所述H-FR1的C末端与所述HCDR1的N末端直接或间接相连，且所述H-FR1包含SEQ ID NO:4、17中任一项所示的氨基酸序列。
在某些实施方式中，本申请的抗原结合蛋白还包含重链框架区2(H-FR2)，所述H-FR2位于所述HCDR1与所述HCDR2之间，且所述H-FR2包含SEQ ID NO:81、5或27中任一项所示的氨基酸序列。
在某些实施方式中，本申请的抗原结合蛋白中所述H-FR2包含SEQ ID NO:5、18、27或36中任一项所示的氨基酸序列。
在某些实施方式中，本申请的抗原结合蛋白还包含重链框架区3(H-FR3)，所述H-FR3位于所述HCDR2与所述HCDR3之间，且所述H-FR3包含SEQ ID NO:6、19中任一项所示的氨基酸序列。
在某些实施方式中，本申请的抗原结合蛋白还包含重链框架区4(H-FR4)，所述H-FR4的N末端与所述HCDR3的C末端相连，且所述H-FR4包含SEQ ID NO:7、20中任一项所示的氨基酸序列。
在某些实施方式中，本申请的抗原结合蛋白包含重链可变区VH，所述VH包含SEQ ID NO:73、74中任一项所示的氨基酸序列。
在某些实施方式中，本申请的抗原结合蛋白包含重链可变区VH，所述VH包含SEQ ID NO:8、21、28、37或41中任一项所示的氨基酸序列。
在某些实施方式中，本申请的抗原结合蛋白包含轻链LCDR3，所述LCDR3包含SEQ ID NO:79所示的氨基酸序列。
在某些实施方式中，本申请的抗原结合蛋白中所述LCDR3包含SEQ ID NO:11、30中任一项所示的氨基酸序列。
在某些实施方式中，本申请的抗原结合蛋白还包含轻链LCDR2，所述LCDR2包含SEQ ID NO:78所示的氨基酸序列。
在某些实施方式中，本申请的抗原结合蛋白中所述LCDR2包含SEQ ID NO:10、29中任一项所示的氨基酸序列。
在某些实施方式中，本申请的抗原结合蛋白还包含轻链LCDR1，所述LCDR1包含SEQ ID NO:9所示的氨基酸序列。
在某些实施方式中，本申请的抗原结合蛋白包含轻链LCDR1、LCDR2和LCDR3；其中所述LCDR1包含SEQ ID NO:9所示的氨基酸序列，所述LCDR2包含SEQ ID NO:78所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:79所示的氨基酸序列。
在某些实施方式中，本申请的抗原结合蛋白包含轻链LCDR1、LCDR2和LCDR3；其中所述LCDR1包含SEQ ID NO:9所示的氨基酸序列，所述LCDR2包含SEQ ID NO:10、29中任一项所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:11、30中任一项所示的氨基酸序列。
在某些实施方式中，本申请的抗原结合蛋白包含轻链框架区1(L-FR1)，所述L-FR1的C末端与所述LCDR1的N末端直接或间接相连，且所述L-FR1包含SEQ ID NO:31、12或80中任一项所示的氨基酸序列。
在某些实施方式中，本申请的抗原结合蛋白中所述L-FR1包含SEQ ID NO:12、22、31或38中任一项所示的氨基酸序列。
在某些实施方式中，本申请的抗原结合蛋白还包含轻链框架区2(L-FR2)，所述L-FR2位于所述LCDR1与所述LCDR2之间，且所述L-FR2包含SEQ ID NO:13、23或32中任一项所示的氨基酸序列。
在某些实施方式中，本申请的所述的抗原结合蛋白还包含轻链框架区3(L-FR3)，所述 L-FR3位于所述LCDR2与所述LCDR3之间，且所述L-FR3包含SEQ ID NO:33、14或82中任一项所示的氨基酸序列。
在某些实施方式中，本申请的抗原结合蛋白中所述L-FR3包含SEQ ID NO:14、24、33或39中任一项所示的氨基酸序列。
在某些实施方式中，本申请的抗原结合蛋白还包含轻链框架区4(L-FR4)，所述L-FR4的N末端与所述LCDR3的C末端相连，且所述L-FR4包含SEQ ID NO:34、15中任一项所示的氨基酸序列。
在某些实施方式中，本申请的抗原结合蛋白包含轻链可变区VL，所述VL包含SEQ ID NO:75、76中任一项所示的氨基酸序列。
在某些实施方式中，本申请的抗原结合蛋白包含轻链可变区VL，所述VL包含SEQ ID NO:16、35、25、40中任一项所示的氨基酸序列。
在某些实施方式中，本申请的抗原结合蛋白包含所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，其中所述HCDR1包含SEQ ID NO:1所示的氨基酸序列，所述HCDR2包含SEQ ID NO:77所示的氨基酸序列，所述HCDR3包含SEQ ID NO:3所示的氨基酸序列，所述LCDR1包含SEQ ID NO:9所示的氨基酸序列，所述LCDR2包含SEQ ID NO:78所示的氨基酸序列和所述LCDR3包含SEQ ID NO:79所示的氨基酸序列。
在某些实施方式中，本申请的抗原结合蛋白包含所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，其中所述HCDR1包含SEQ ID NO:1所示的氨基酸序列，所述HCDR2包含SEQ ID NO:2、26中任一项所示的氨基酸序列，所述HCDR3包含SEQ ID NO:3所示的氨基酸序列，所述LCDR1包含SEQ ID NO:9所示的氨基酸序列，所述LCDR2包含SEQ ID NO:10、29中任一项所示的氨基酸序列和所述LCDR3包含SEQ ID NO:11、30中任一项所示的氨基酸序列。
在某些实施方式中，本申请的抗原结合蛋白包含重链可变区VH和轻链可变区VL，所述VH包含SEQ ID NO:73、74中任一项所示的氨基酸序列，且所述VL包含SEQ ID NO:75、76中任一项所示的氨基酸序列。
在某些实施方式中，本申请的抗原结合蛋白包含重链可变区VH和轻链可变区VL，所述VH包含SEQ ID NO:8、21、28、37或41中任一项所示的氨基酸序列，且所述VL包含SEQ ID NO:16、35、25、40中任一项所示的氨基酸序列。
在某些实施方式中，本申请的抗原结合蛋白包括抗体或其抗原结合片段。
在某些实施方式中，本申请的抗原结合蛋白中所述抗原结合片段包括Fab，Fab’，F(ab)2、 Fv片段、F(ab’)2，scFv，di-scFv和/或dAb。
在某些实施方式中，本申请的抗原结合蛋白包括重链恒定区，且所述重链恒定区源自人IgG。
在某些实施方式中，本申请的抗原结合蛋白包括重链恒定区，且所述重链恒定区源自人IgG1。
在某些实施方式中，本申请的抗原结合蛋白包括重链恒定区，其中与SEQ ID NO:42所示的氨基酸序列相比，所述重链恒定区包含下述氨基酸突变：M135Y、S137T和/或T139E。
在某些实施方式中，本申请的抗原结合蛋白中所述重链恒定区包含SEQ ID NO:42、44中任一项所示的氨基酸序列。
在某些实施方式中，本申请的抗原结合蛋白包含重链，且所述重链包含SEQ ID NO:45、47或49中任一项所示的氨基酸序列。
在某些实施方式中，本申请的抗原结合蛋白包括轻链恒定区，且所述轻链恒定区包含SEQ ID NO:43所示的氨基酸序列。
在某些实施方式中，本申请的抗原结合蛋白包含轻链，且所述轻链包含SEQ ID NO:46或48中任一项所示的氨基酸序列。
在某些实施方式中，本申请的抗原结合蛋白包含重链和轻链，其中所述重链包含SEQ ID NO:45、47或49中任一项所示的氨基酸序列，且所述轻链包含SEQ ID NO:46或48中任一项所示的氨基酸序列。
另一方面，本申请还提供了多肽，其包含本申请的抗原结合蛋白。
另一方面，本申请还提供了免疫缀合物，其包含本申请的抗原结合蛋白。
另一方面，本申请还提供了分离的核酸分子，其编码本申请的的抗原结合蛋白。
另一方面，本申请还提供了载体，其包含本申请的的分离的核酸分子。
另一方面，本申请还提供了细胞，其包含和/或表达本申请的抗原结合蛋白、本申请的多肽、本申请的免疫缀合物、本申请的分离的核酸分子或本申请的载体。
另一方面，本申请还提供了制备本申请的抗原结合蛋白的方法，所述方法包括在使得本申请的抗原结合蛋白表达的条件下，培养本申请的细胞。
另一方面，本申请还提供了药物组合物，其包含本申请的抗原结合蛋白、本申请的多肽、本申请的免疫缀合物、本申请的分离的核酸分子、本申请的载体、本申请的细胞和/或任选地药学上可接受的载剂。
另一方面，本申请还提供了本申请的抗原结合蛋白、本申请的多肽、本申请的免疫缀合 物、本申请的分离的核酸分子、本申请的载体、本申请的细胞和/或本申请的药物组合物在制备药物中的用途，所述药物用于预防、缓解和/或治疗IgE相关的疾病或病症。
另一方面，本申请还提供了预防、缓解或治疗IgE相关的疾病或病症的方法，所述方法包括向有需要的受试者施用本申请的抗原结合蛋白、本申请的多肽、本申请的免疫缀合物和/或本申请的药物组合物。
另一方面，本申请还提供了本申请的抗原结合蛋白、本申请的多肽、本申请的分离的核酸分子、本申请的载体、本申请的细胞、本申请的免疫缀合物和/或本申请的药物组合物，其用于预防、缓解或治疗IgE相关的疾病或病症。
另一方面，本申请还提供了一种用于检测或测定IgE的方法，所述方法包括使用本申请的抗原结合蛋白或本申请的多肽。
另一方面，本申请还提供了一种用于检测或测定IgE的试剂盒，所述试剂盒包括本申请的抗原结合蛋白或本申请的多肽。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明书如下：
图1显示的是本申请的抗原结合蛋白(鼠源单克隆抗体)阻断IgE与其受体FcεRIa结合的阻断活性结果。
图2显示的是本申请的抗原结合蛋白(嵌合抗体及其相应鼠源抗体)阻断IgE与其受体FcεRIa结合的阻断活性结果。
图3显示的是本申请的抗原结合蛋白(人源化抗体、其相应嵌合抗体及相应鼠源抗体)阻断IgE与其受体FcεRIa结合的阻断活性结果。
图4显示的是本申请的抗原结合蛋白(人源化SE2抗体)与奥马珠单抗阻断IgE与其受体FcεRIa结合的检测结果。
图5显示的是本申请的抗原结合蛋白(人源化SE5抗体)与奥马珠单抗阻断IgE与其受体FcεRIa结合的检测结果。
图6A-6B显示的是本申请的抗原结合蛋白(人源化SE5ss抗体)与人IgE结合的亲和力。
图7A-7B显示的是本申请的抗原结合蛋白(人源化SE5ss抗体)阻断IgE与其受体(FcεRIa和FcεRII)结合的阻断活性结果。
图8显示的是本申请的抗原结合蛋白(人源化SE5ss抗体)阻断IgE介导的细胞激活的抑制活性检测结果。
图9A-9C显示的是本申请的抗原结合蛋白(人源化SE5ss抗体)阻断不同过敏原IgE介导的细胞激活的抑制活性检测结果。
图10显示的是本申请的抗原结合蛋白(人源化SE5ss抗体)在小鼠体内对游离人IgE的抑制能力。
图11显示的是本申请的抗原结合蛋白(人源化SE5ss抗体)在小鼠速发过敏模型中缓解体温变化的结果。
图12显示的是本申请的抗原结合蛋白(人源化SE5ss抗体)在食蟹猴IgE抑制模型中对游离IgE的抑制能力。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，本领域技术人员可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
术语定义
在本申请中，术语“抗原结合蛋白”通常指包含结合抗原部分的蛋白质，以及任选地允许结合抗原的部分采用促进抗原结合蛋白与抗原结合的构象的支架或骨架部分。抗原结合蛋白可包括但不限于抗体、抗原结合片段(Fab、Fab’、F(ab)2、Fv片段、F(ab’)2、scFv、di-scFv和/或dAb)、免疫缀合物、多特异性抗体(例如双特异性抗体)、抗体片段、抗体衍生物、抗体类似物或融合蛋白等，只要它们显示出所需的抗原结合活性即可。本申请的“抗原结合蛋白”可以包含结合抗原的部分和任选地，允许抗原结合部分采用促进所述抗原结合部分结合抗原的构象的支架或构架部分。
在本申请中，术语“IgE”或“IgE免疫球蛋白”或“免疫球蛋白E”通常是指一类由浆细胞产生的抗体(或免疫球蛋白(Ig))。IgE的单体通常由两条重链(ε链)和两条轻链组成， 其中ε链可包含4个Ig样恒定区(Cε1-Cε4)。在本申请中，“IgE”可以是完整的IgE或其片段，可以是IgE的功能性变体、同工型、物种同源物、衍生物、类似物，以及具有至少一个与IgE共同表位的类似物。在人类中，IgE属于基本上由公知免疫球蛋白epsilon基因编码的抗体类别的多肽。IgE可以包括膜锚定的(mIgE)，或非膜锚定的，也称为循环IgE。在本申请中，术语“IgE”可以来源于哺乳动物，IgE可以包括人IgE。在本申请中，所述人IgE在GenBank数据库中的序列号为Gene ID:3497。
在本申请中，术语“FcεRIα”通常是指是免疫球蛋白E(IgE)的Fc区的高亲和受体(FcεRI，也称作Fc epsilon RI，)的α链。FcεRI是能够结合IgE的ε重链的Fc区的四聚体受体复合物，其通常由一个α链(即FcεRIα)，一个β链(FcεRIβ)和两个γ链(FcεRIγ)组成。通常情况下，α链可以作为抗体(例如，IgE)的结合位点，γ链可以作为下游信号起始的位点，β链可以起到放大下游信号的作用。在本申请中，“FcεRIα”可以是完整的FcεRIα或其片段，可以是FcεRIα的功能性变体、同工型、物种同源物、衍生物、类似物，以及具有至少一个与FcεRIα共同表位的类似物。
在本申请中，术语“KD”，也称作“K D”、“K D”、“亲和常数”或“平衡解离常数”，通常是指在滴定测量中在平衡时、或者通过将解离速率常数(k d)除以结合速率常数(k a)所获得的值。通常，可使用结合速率常数(k a)、解离速率常数(k d)和平衡解离常数(K D)表示结合蛋白(例如本申请所述的抗原结合蛋白)对抗原(例如人IgE)的结合亲和力。确定结合和解离速率常数的方法为本领域熟知。例如，可以通过Octet测定所述KD值，也可以使用其他实验途径和仪器例如BIAcore(生物分子相互作用分析)测定。另外，电化学发光分析-溶液平衡滴定法(MSD-SET)可以测定所述KD值。测量方法如Estep P.等人,MAbs,2013.5(2):p.270-8所述。在本申请中，可以采用KinExA方法和软件检测KD值，测量方法如Jonathan K.Fleming等人，Methods Mol Biol.2018；1697:1-8所述。
在本申请中，术语“互补决定区”或术语“CDR”通常是指在抗体可变序列内的互补性决定区。在重链和轻链的每个可变区中存在3个CDR，所述CDR对于每个可变区命名为CDR1、CDR2和CDR3。如本文使用的，CDR组合可指在能够结合抗原的单个可变区中出现的3个CDR的组。这些CDR的确切边界已根据不同系统不同地限定。由Kabat(Kabat等人，Sequences of Proteins of Immunological Interest National Institutes of Health，Bethesda，Md.(1987)和(1991))描述的系统，不仅提供了可应用于抗体的任何可变区的明确残基编号系统，还提供了限定3个CDR的精确残基边界。这些CDR可以被称为Kabat CDR。Chothia和同事(Chothia和Lesk，J.Mol.Biol.196：901-917(1987)以及Chothia等人， Nature 342：877-883(1989))发现Kabat CDR内的某些亚部分采取几乎相同的肽主链构象，尽管在氨基酸序列水平上具有大的多样性。这些亚部分命名为L1、L2和L3或H1、H2和H3，其中“L”和“H”分别指轻链和重链区域。这些区域可以被称为Chothia CDR，所述Chothia CDR具有与Kabat CDR重叠的边界。与Kabat CDR重叠的限定CDR的其他边界已由Padlan(FASEB J.9：133-139(1995))和MacCallum(J Mol Biol 262(5)：732-45(1996))描述。在其他的CDR边界定义可能不严格地遵循上述系统之一，但仍将与Kabat CDR重叠，尽管按照特定残基或残基组或甚至整个CDR并不显著影响抗原结合的预测或实验发现，它们可以缩短或加长。本文所述CDR，可使用KABAT进行定义，但不排除采用其他方法进行CDR区域的划分。
在本申请中，术语“抗体”通常指免疫球蛋白或其片段或其衍生物或其变体，涵盖包括抗原结合位点的任何多肽，无论其是在体外还是体内产生的。该术语包括但不限于多克隆的、单克隆的、单特异性的、多特异性的、非特异性的、人源化的、单链的、嵌合的、合成的、重组的、杂化的、突变的和移植的抗体。在本申请中，术语“抗体”也可以包括抗体片段，比如Fab、F(ab')2、Fv、scFv、Fd、dAb和保持抗原结合功能(例如，特异性结合人IgE)的其它抗体片段。本申请所述抗体可以包括来源于人、来源于鼠、来源于猴和/或来源于羊驼的抗体。在本申请中，所述抗体可以包括其功能性变体、同工型、物种同源物、衍生物或类似物。例如，本申请的中的变体可以包括与抗体具有至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少98.5％、至少99％、至少99.2％、至少99.4％、至少99.6％、至少99.8％或至少99.9％同一性的氨基酸序列。
在本申请中，术语“抗原结合片段”通常是指与抗原结合(即特异性结合)的完整抗体(即与它们所衍生的完整抗体)竞争的免疫球蛋白或抗体的多肽片段。所述抗原结合片段可以包括但不限于：Fab、Fab’、F(ab)2、F(ab’)2和Fv片段，线性抗体，单链抗体，双体抗体，以及由抗体片段形成的多特异性抗体。
在本申请中，术语“可变区”或“可变结构域”通常是指抗体轻链和/或重链的一部分，其通常在不同的抗体中有较大差异，且可能影响抗体与特定抗原的结合特异性。可变区可以包含序列变化较大的区域称作互补决定区(CDR)，可变区也可以包含比较保守的区域通常称作框架区(FR)。
在本申请中，术语“直接或间接相连”通常是指相对的“直接相连”或“间接相连”。“直接相连”通常是指直接连接。例如，所述直接相连可以为相连的物质(例如氨基酸序列区段)之间没有间隔成分(例如氨基酸残基或其衍生物)而直接相连接的情况；例如氨基酸序列区段 X与另一氨基酸序列区段Y通过氨基酸序列区段X的C端氨基酸与氨基酸序列区段Y的N端氨基酸形成的酰胺键直接连接。“间接相连”通常是指相连的物质(例如氨基酸序列区段)之间有间隔成分(例如氨基酸残基或其衍生物)而间接相连接的情况例如，在本申请所述的抗原结合蛋白中，所述L-FR1的C末端与所述LCDR1的N末端可以直接或间接相连。
在本申请中，术语“免疫缀合物”通常指与抗原结合蛋白或其片段相连接的其他组分。所述免疫缀合物可以包括与抗原结合蛋白融合或偶联的其他的多肽、治疗剂、探针和/或其他抗体。
在本申请中，术语“分离的核酸分子”通常是指任何长度的分离形式的核苷酸，脱氧核糖核苷酸或核糖核苷酸，或从其天然环境分离的或人工合成的类似物。
在本申请中，术语“细胞”通常是指可以或已经含有包括本申请所述的核酸分子的质粒或载体，或者能够表达本申请所述的抗原结合蛋白的个体细胞，细胞系或细胞培养物。所述细胞可以包括单个细胞的子代。由于天然的，意外的或故意的突变，子代细胞与原始亲本细胞在形态上或在基因组上可能不一定完全相同，但能够表达本申请所述的抗原结合蛋白即可。所述细胞可以通过使用本申请所述的载体体外转染细胞而得到。所述细胞可以是原核细胞(例如大肠杆菌)，也可以是真核细胞(例如酵母细胞，例如COS细胞，中国仓鼠卵巢(CHO)细胞，HeLa细胞，HEK293细胞，COS-1细胞，NS0细胞或骨髓瘤细胞)。
在本申请中，术语“药物组合物”通常是指涉及适合施用于患者、例如人患者的组合物。例如，本申请所述的药物组合物，其可以包含本申请所述的抗原结合蛋白、本申请所述的免疫缀合物、本申请所述的核酸分子、本申请所述的载体和/或本申请所述的细胞，以及任选地药学上可接受的载剂。此外，所述药物组合物还可以包含一种或多种(药学上有效的)载剂、稳定剂、赋形剂、稀释剂、增溶剂、表面活性剂、乳化剂和/或防腐剂的合适的制剂。组合物的可接受成分在所用剂量和浓度下对接受者无毒。本发明的药物组合物可以包括但不限于液体、冷冻和冻干组合物。
在本申请中，术语“载体”通常指将编码某蛋白的多聚核苷酸插入其中并使蛋白获得表达的一种核酸运载工具。载体可通过转化、转导或转染宿主细胞，使其携带的遗传物质元件在宿主细胞内表达得以表达。例如，载体可包括：质粒、噬菌粒、柯斯质粒、人工染色体如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。用作载体的动物病毒种类有逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可能含有多种控制表达的元件，包括 启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。载体还有可能包括有协助其进入细胞的成分，如病毒颗粒、脂质体或蛋白外壳，但不仅仅只有这些物质。
在本申请中，术语“药学上可接受的载剂”通常包括药剂学可接受的载体、赋形剂或稳定剂，它们在所采用的剂量和浓度对暴露于其的细胞或哺乳动物是无毒的。生理学可接受的载体可包括合适的物质。
发明详述
抗原结合蛋白
一方面，本申请提供了一种抗原结合蛋白，其可包含重链可变区的至少一个互补决定区(CDR)，所述重链可变区(VH)包含如SEQ ID NO:73、74中任一项所示的氨基酸序列；且其包含轻链可变区(VL)的至少一个互补决定区(CDR)，所述轻链可变区包含如SEQ ID NO:75、76中任一项所示的氨基酸序列。
例如，本申请的抗原结合蛋白可以包含重链可变区的一个CDR，所述重链可变区可以包含如SEQ ID NO:73所示的氨基酸序列或其变体。
例如，本申请的抗原结合蛋白可以包含重链可变区中的两个CDR，所述重链可变区可以包含如SEQ ID NO:73所示的氨基酸序列或其变体。
例如，本申请的抗原结合蛋白可以包含重链可变区中的三个CDR，所述重链可变区可以包含如SEQ ID NO:73所示的氨基酸序列或其变体。
例如，本申请的抗原结合蛋白可以包含重链可变区的一个CDR，所述重链可变区可以包含如SEQ ID NO:74所示的氨基酸序列或其变体。
例如，本申请的抗原结合蛋白可以包含重链可变区中的两个CDR，所述重链可变区可以包含如SEQ ID NO:74所示的氨基酸序列或其变体。
例如，本申请的抗原结合蛋白可以包含重链可变区中的三个CDR，所述重链可变区可以包含如SEQ ID NO:74所示的氨基酸序列或其变体。
例如，本申请的重链可变区中的CDR可以称为HCDRn，n表示CDR的编号，通常可以为1，2或3。
例如，本申请的重链可变区中的CDR可以包括本申请所述的HCDR1、HCDR2和HCDR3。
例如，本申请的抗原结合蛋白可包含重链HCDR3，所述HCDR3可包含SEQ ID NO:3所示的氨基酸序列或者其变体。
例如，本申请的抗原结合蛋白可包含重链HCDR2，所述HCDR2可包含SEQ ID NO:77 所示的氨基酸序列或者其变体。
例如，本申请的抗原结合蛋白可包含重链HCDR2，所述HCDR2可包含SEQ ID NO:2、26中任一项所示的氨基酸序列或者其变体。
例如，本申请的抗原结合蛋白可包含重链HCDR1，所述HCDR1可包含SEQ ID NO:1所示的氨基酸序列或者其变体。
例如，本申请的抗原结合蛋白可包含重链HCDR3和HCDR2，所述HCDR3可包含SEQ ID NO:3所示的氨基酸序列或者其变体，且所述HCDR2可包含SEQ ID NO:77所示的氨基酸序列或者其变体。
例如，本申请的抗原结合蛋白可包含重链HCDR3和HCDR2，所述HCDR3可包含SEQ ID NO:3所示的氨基酸序列或者其变体，且所述HCDR2可包含SEQ ID NO:2、26中任一项所示的氨基酸序列或者其变体。
例如，本申请的抗原结合蛋白可包含重链HCDR2和HCDR1，所述HCDR2可包含SEQ ID NO:77所示的氨基酸序列或者其变体，且所述HCDR1可包含SEQ ID NO:1所示的氨基酸序列或者其变体。
例如，本申请的抗原结合蛋白可包含重链HCDR2和HCDR1，所述HCDR2可包含SEQ ID NO:2、26中任一项所示的氨基酸序列或者其变体，且所述HCDR1可包含SEQ ID NO:1所示的氨基酸序列或者其变体。
例如，本申请的抗原结合蛋白可包含重链HCDR1、HCDR2和HCDR3；其中所述HCDR1可包含SEQ ID NO:1所示的氨基酸序列或者其变体，所述HCDR2可包含SEQ ID NO:77所示的氨基酸序列或者其变体，所述HCDR3可包含SEQ ID NO:3所示的氨基酸序列或者其变体。
例如，本申请的抗原结合蛋白可包含重链HCDR1、HCDR2和HCDR3；其中所述HCDR1可包含SEQ ID NO:1所示的氨基酸序列或者其变体，所述HCDR2可包含SEQ ID NO:2、26中任一项所示的氨基酸序列或者其变体，所述HCDR3可包含SEQ ID NO:3所示的氨基酸序列或者其变体。
例如，本申请的抗原结合蛋白可以包含轻链可变区的一个CDR，所述轻链可变区可以包含如SEQ ID NO:75所示的氨基酸序列或其变体。
例如，本申请的抗原结合蛋白可以包含轻链可变区中的两个CDR，所述轻链可变区可以包含如SEQ ID NO:75所示的氨基酸序列或其变体。
例如，本申请的抗原结合蛋白可以包含轻链可变区中的三个CDR，所述轻链可变区可以 包含如SEQ ID NO:75所示的氨基酸序列或其变体。
例如，本申请的抗原结合蛋白可以包含轻链可变区的一个CDR，所述轻链可变区可以包含如SEQ ID NO:76所示的氨基酸序列或其变体。
例如，本申请的抗原结合蛋白可以包含轻链可变区中的两个CDR，所述轻链可变区可以包含如SEQ ID NO:76所示的氨基酸序列或其变体。
例如，本申请的抗原结合蛋白可以包含轻链可变区中的三个CDR，所述轻链可变区可以包含如SEQ ID NO:76所示的氨基酸序列或其变体。
例如，本申请的轻链可变区中的CDR可以称为LCDRn，n表示CDR的编号，通常可以为1，2或3等。
例如，本申请的轻链可变区中的CDR可以包括本申请所述的LCDR1、LCDR2和LCDR3。
例如，本申请的抗原结合蛋白可包含轻链LCDR3，所述LCDR3可包含SEQ ID NO:79所示的氨基酸序列或者其变体。
例如，本申请的抗原结合蛋白可包含轻链LCDR3，所述LCDR3可包含SEQ ID NO:11、30中任一项所示的氨基酸序列或者其变体。
例如，本申请的抗原结合蛋白可包含轻链LCDR2，所述LCDR2可包含SEQ ID NO:78所示的氨基酸序列或者其变体。
例如，本申请的抗原结合蛋白可包含轻链LCDR2，所述LCDR2可包含SEQ ID NO:10、29中任一项所示的氨基酸序列或者其变体。
例如，本申请的抗原结合蛋白可包含轻链LCDR1，所述LCDR1可包含SEQ ID NO:9所示的氨基酸序列或者其变体。
例如，本申请的抗原结合蛋白包含轻链LCDR3和LCDR2，所述LCDR3可包含SEQ ID NO:79所示的氨基酸序列或者其变体，且所述LCDR2可包含SEQ ID NO:78所示的氨基酸序列或者其变体。
例如，本申请的抗原结合蛋白包含轻链LCDR3和LCDR2，所述LCDR3可包含SEQ ID NO:11、30中任一项所示的氨基酸序列或者其变体，且所述LCDR2可包含SEQ ID NO:10、29中任一项所示的氨基酸序列或者其变体。
例如，本申请的抗原结合蛋白可包含轻链LCDR2和LCDR1，所述LCDR2可包含SEQ ID NO:78所示的氨基酸序列或者其变体，且所述LCDR1可包含SEQ ID NO:9所示的氨基酸序列或者其变体。
例如，本申请的抗原结合蛋白可包含轻链LCDR2和LCDR1，所述LCDR2可包含SEQ  ID NO:10、29中任一项所示的氨基酸序列或者其变体，且所述LCDR1可包含SEQ ID NO:9所示的氨基酸序列或者其变体。
例如，本申请的抗原结合蛋白可包含轻链LCDR1、LCDR2和LCDR3，其中所述LCDR1可包含SEQ ID NO:9所示的氨基酸序列或者其变体，所述LCDR2可包含SEQ ID NO:78所示的氨基酸序列或者其变体，所述LCDR3可包含SEQ ID NO:79所示的氨基酸序列或者其变体。
例如，本申请的抗原结合蛋白可包含轻链LCDR1、LCDR2和LCDR3，其中所述LCDR1可包含SEQ ID NO:9所示的氨基酸序列或者其变体，所述LCDR2可包含SEQ ID NO:10、29中任一项所示的氨基酸序列或者其变体，所述LCDR3可包含SEQ ID NO:11、30中任一项所示的氨基酸序列或者其变体。
例如，本申请的抗原结合蛋白可以包含HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，其中所述HCDR1可包含SEQ ID NO:1所示的氨基酸序列或其变体，所述HCDR2可包含SEQ ID NO:77所示的氨基酸序列或其变体，所述HCDR3可包含SEQ ID NO:3所示的氨基酸序列或其变体，所述LCDR1可包含SEQ ID NO:9所示的氨基酸序列或其变体，所述LCDR2可包含SEQ ID NO:78所示的氨基酸序列或其变体和所述LCDR3可包含SEQ ID NO:79所示的氨基酸序列或其变体。
例如，本申请的抗原结合蛋白可以包含HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，其中所述HCDR1可包含SEQ ID NO:1所示的氨基酸序列或其变体，所述HCDR2可包含SEQ ID NO:2、26中任一项所示的氨基酸序列或其变体，所述HCDR3可包含SEQ ID NO:3所示的氨基酸序列或其变体，所述LCDR1可包含SEQ ID NO:9所示的氨基酸序列或其变体，所述LCDR2可包含SEQ ID NO:10、29中任一项所示的氨基酸序列或其变体和所述LCDR3可包含SEQ ID NO:11、30中任一项所示的氨基酸序列或其变体。
框架区(FR)
例如，本申请的轻链可变区可以包括框架区L-FR1，L-FR2，L-FR3，和/或L-FR4。
例如，本申请的轻链可变区可以包括框架区L-FR1，L-FR2和L-FR3。
例如，本申请的轻链可变区可以包括框架区L-FR1，L-FR2和L-FR4。
例如，本申请的轻链可变区可以包括框架区L-FR2，L-FR3和L-FR4。
例如，本申请的轻链可变区可以包括框架区L-FR1，L-FR3和L-FR4。
例如，本申请的轻链可变区可以包括框架区L-FR1，L-FR2，L-FR3和L-FR4。
例如，本申请的L-FR1的C末端可以与本申请的LCDR1的N末端直接或间接相连，例 如，本申请的L-FR1的C末端可以与本申请的LCDR1的N末端直接相连，例如，本申请的L-FR1的C末端可以与本申请的LCDR1的N末端间接相连，例如，本申请的间接相连可以包括本申请的L-FR1的C末端氨基酸残基与本申请的LCDR1的N末端氨基酸残基之间相隔1个、2个、3个、4个、5个或更多个氨基酸，这些氨基酸可以是天然存在的或经修饰的任意氨基酸。
例如，本申请的L-FR1可以包含SEQ ID NO:12、31或80中任一项所示的氨基酸序列或其变体。
例如，本申请的L-FR1可以包含SEQ ID NO:12、31、22、38中任一项所示的氨基酸序列或其变体。
例如，本申请的L-FR2可以位于本申请的LCDR1与本申请的LCDR2之间。
例如，本申请的L-FR2的N末端可以与本申请的LCDR1的C末端直接或间接相连，并且本申请的L-FR2的C末端可以与本申请的LCDR2的N末端直接或间接相连。
例如，间接相连可以包括相连的两个部分(例如，本申请的L-FR2与本申请的LCDR1，或者本申请的L-FR2与本申请的LCDR2)之间相隔1个、2个、3个、4个、5个或更多个氨基酸，这些氨基酸可以是天然存在的或经修饰的任意氨基酸。
例如，本申请的L-FR2可以包含SEQ ID NO:13、32、23中任一项所示的氨基酸序列或其变体。
例如，本申请的-FR3可以位于本申请的LCDR2与本申请的LCDR3之间。
例如，本申请的L-FR3的N末端可以与本申请的LCDR2的C末端直接或间接相连，并且本申请的L-FR3的C末端可以与本申请的LCDR3的N末端直接或间接相连。
例如，间接相连可以包括相连的两个部分(例如，本申请的L-FR3与本申请的LCDR2，或者本申请的L-FR3与本申请的LCDR3)之间相隔1个、2个、3个、4个、5个或更多个氨基酸，这些氨基酸可以是天然存在的或经修饰的任意氨基酸。
例如，本申请的L-FR3可以包含SEQ ID NO:14、33、82中任一项所示的氨基酸序列或其变体。
例如，本申请的L-FR3可以包含SEQ ID NO:14、33、24、39中任一项所示的氨基酸序列或其变体。
例如，本申请的L-FR4的N末端与本申请的LCDR3的C末端直接或间接相连。
例如，间接相连可以包括本申请的L-FR4的N末端氨基酸残基与本申请的LCDR3的C末端氨基酸残基之间相隔1个、2个、3个、4个、5个或更多个氨基酸，这些氨基酸可以是 天然存在的或经修饰的任意氨基酸。
例如，本申请的L-FR4可以包含SEQ ID NO:15、34中任一项所示的氨基酸序列或其变体。
例如，本申请的重链可变区可以包括框架区H-FR1，H-FR2，H-FR3，和/或H-FR4。
例如，本申请的轻链可变区可以包括框架区H-FR1，H-FR2和H-FR3。
例如，本申请的轻链可变区可以包括框架区H-FR1，H-FR2和H-FR4。
例如，本申请的轻链可变区可以包括框架区H-FR2，H-FR3和H-FR4。
例如，本申请的轻链可变区可以包括框架区H-FR1，H-FR3和H-FR4。
例如，本申请的链可变区可以包括框架区H-FR1，H-FR2，H-FR3和H-FR4。
例如，本申请的H-FR1的C末端可以与本申请的HCDR1的N末端直接或间接相连。例如，本申请的H-FR1的C末端可以与本申请的HCDR1的N末端直接相连，例如，本申请的H-FR1的C末端可以与本申请的HCDR1的N末端间接相连，例如，本申请的间接相连可以包括本申请的H-FR1的C末端氨基酸残基与本申请的HCDR1的N末端氨基酸残基之间相隔1个、2个、3个、4个、5个或更多个氨基酸，这些氨基酸可以是天然存在的或经修饰的任意氨基酸。
例如，本申请的H-FR1可以包含SEQ ID NO:4、17中任一项所示的氨基酸序列或其变体。
例如，本申请的H-FR2可以位于本申请的HCDR1与本申请的HCDR2之间。
例如，本申请的H-FR2的N末端可以与本申请的HCDR1的C末端直接或间接相连，并且本申请的H-FR2的C末端可以与本申请的HCDR2的N末端直接或间接相连。
例如，本申请的间接相连可以包括相连的两个部分(例如，本申请的H-FR2与本申请的HCDR1，或者本申请的H-FR2与本申请的HCDR2)之间相隔1个、2个、3个、4个、5个或更多个氨基酸，这些氨基酸可以是天然存在的或经修饰的任意氨基酸。
例如，本申请的H-FR2可以包含SEQ ID NO:5、27、81中任一项所示的氨基酸序列或其变体。
例如，本申请的H-FR2可以包含SEQ ID NO:5、27、18、36中任一项所示的氨基酸序列或其变体。
例如，本申请的H-FR3可以位于本申请的HCDR2与本申请的HCDR3之间。
例如，本申请的H-FR3的N末端可以与本申请的HCDR2的C末端直接或间接相连，并且本申请的H-FR3的C末端可以与本申请的HCDR3的N末端直接或间接相连。
例如，间接相连可以包括相连的两个部分(例如，本申请的H-FR3与本申请的HCDR2，或者本申请的H-FR3与本申请的HCDR3)之间相隔1个、2个、3个、4个、5个或更多个氨基酸，这些氨基酸可以是天然存在的或经修饰的任意氨基酸。
例如，本申请的H-FR3可以包含SEQ ID NO:6、19中任一项所示的氨基酸序列或其变体。
例如，本申请的H-FR4的N末端可以与本申请的HCDR3的C末端直接或间接相连。
例如，间接相连可以包括本申请的L-FR4的N末端氨基酸残基与本申请的LCDR3的C末端氨基酸残基之间相隔1个、2个、3个、4个、5个或更多个氨基酸，这些氨基酸可以是天然存在的或经修饰的任意氨基酸。
例如，本申请的H-FR4可以包含SEQ ID NO:7、20中任一项所示的氨基酸序列或其变体。
例如，在提及具体的氨基酸序列时(例如特定的序列)，所指代的序列还可以涵盖其变体。例如，本申请的变体可以包含与SEQ ID NO:4-7、17-20、27或36中任一项所示的氨基酸序列具有至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少98.5％、至少99％、至少99.2％、至少99.4％、至少99.6％、至少99.8％或至少99.9％同一性的氨基酸序列。
可变区，恒定区，重链和轻链
在本申请中，本申请的抗原结合蛋白可包含重链可变区(VH)和/或轻链可变区(VL)。例如，本申请的重链可变区可包含SEQ ID NO:73、74中任一项所示的氨基酸序列或其变体。例如，本申请的轻链可变区可包含SEQ ID NO:75、76中任一项所示的氨基酸序列或其变体。
例如，在提及具体的氨基酸序列时(例如特定的序列)，所指代的序列还可以涵盖其变体。例如，本申请的变体可以包含与SEQ ID NO:73-76中任一项所示的氨基酸序列具有至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少98.5％、至少99％、至少99.2％、至少99.4％、至少99.6％、至少99.8％或至少99.9％同一性的氨基酸序列。
例如，本申请的重链可变区包含SEQ ID NO:73、74任一项所示的氨基酸序列或其变体。例如，本申请的轻链可变区包含SEQ ID NO:75、76任一项所示的氨基酸序列或其变体。
例如，本申请的重链可变区包含SEQ ID NO:8、21、28、37或41任一项所示的氨基酸序列或其变体。例如，本申请的轻链可变区包含SEQ ID NO:16、35、25、40任一项所示的氨基酸序列或其变体。
例如，本申请的重链可变区包含SEQ ID NO:57、59、61、63、65中任一项所示的核苷 酸序列或其变体。例如，本申请的轻链可变区包含SEQ ID NO:58、60、62、64、66中任一项所示的核苷酸序列或其变体。
例如，在提及具体的氨基酸和/或核苷酸序列时(例如特定的序列)，所指代的序列还可以涵盖其变体。例如，本申请的变体可以包含与SEQ ID NO:8、16、21、25、28、35、37、40、41或57-66中任一项所示的氨基酸序列具有至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少98.5％、至少99％、至少99.2％、至少99.4％、至少99.6％、至少99.8％或至少99.9％同一性的氨基酸序列。
例如，本申请的抗原结合蛋白可以包括抗体重链恒定区，且本申请的抗体重链恒定区可以源自人IgG重链恒定区。例如，本申请的抗原结合蛋白可以包括抗体重链恒定区，且本申请的抗体重链恒定区可以源自人IgG1重链恒定区。例如，本申请的抗体重链恒定区可以包含SEQ ID NO:42所示的氨基酸序列或其变体。
例如，在提及具体的氨基酸序列时(例如特定的序列)，所指代的序列还可以涵盖其变体。例如，本申请的变体可以包含与SEQ ID NO:42所示的氨基酸序列具有至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少98.5％、至少99％、至少99.2％、至少99.4％、至少99.6％、至少99.8％或至少99.9％同一性的氨基酸序列。
例如，本申请的重链恒定区变体可以包括在人IgG1重链恒定区(氨基酸序列如SEQ ID NO:42)基础上，包含下述氨基酸突变：M135Y、S137T和/或T139E(氨基酸序列编号以序列SEQ ID NO:42为基础)。
例如，本申请的抗原结合蛋白可以包括抗体轻链恒定区，且本申请的抗体轻链恒定区可以包括人Igκ(Kappa)恒定区。例如，本申请的抗体轻链恒定区可以包含SEQ ID NO:43所示的氨基酸序列或其变体。
例如，在提及具体的氨基酸序列时(例如特定的序列)，所指代的序列还可以涵盖其变体。例如，本申请的变体可以包含与SEQ ID NO:43所示的氨基酸序列具有至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少98.5％、至少99％、至少99.2％、至少99.4％、至少99.6％、至少99.8％或至少99.9％同一性的氨基酸序列。
例如，本申请的抗原结合蛋白可以包含抗体重链，且本申请的抗体重链可以包含SEQ ID NO:45、47或49中任一项所示的氨基酸序列或其变体。
例如，本申请的抗原结合蛋白可以包含抗体重链，且本申请的抗体重链可以包含SEQ ID NO:67、69、或71中任一项所示的核苷酸序列或其变体。
例如，在提及具体的氨基酸和/或核苷酸序列时(例如特定的序列)，所指代的序列还可以 涵盖其变体。例如，本申请的变体可以包含与SEQ ID NO:45、47、49、67、69、71中任一项所示的氨基酸序列具有至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少98.5％、至少99％、至少99.2％、至少99.4％、至少99.6％、至少99.8％或至少99.9％同一性的氨基酸序列。
例如，本申请的抗原结合蛋白可以包含抗体轻链，且本申请的抗体轻链可以包含SEQ ID NO:46或48中任一项所示的氨基酸序列或其变体。
例如，本申请的抗原结合蛋白可以包含抗体轻链，且本申请的抗体轻链可以包含SEQ ID NO:68、70或72中任一项所示的核苷酸序列或其变体。
例如，在提及具体的氨基酸和/或核苷酸序列时(例如特定的序列)，所指代的序列还可以涵盖其变体。例如，本申请的变体可以包含与SEQ ID NO:45-49、67-72中任一项所示的氨基酸序列具有至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少98.5％、至少99％、至少99.2％、至少99.4％、至少99.6％、至少99.8％或至少99.9％同一性的氨基酸序列。
例如，本申请的抗原结合蛋白，其中所述FR1，FR2，FR3和FR4可以包含选自下述任意一组的氨基酸序列：
1)所述H-FR1包含SEQ ID NO:4所示的氨基酸序列，所述H-FR2包含SEQ ID NO:5所示的氨基酸序列，所述H-FR3包含SEQ ID NO:6所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:7所示的氨基酸序列；所述L-FR1包含SEQ ID NO:12所示的氨基酸序列，所述L-FR2包含SEQ ID NO:13所示的氨基酸序列，所述L-FR3包含SEQ ID NO:14所示的氨基酸序列，且所述L-FR4包含SEQ ID NO:15所示的氨基酸序列。
2)所述H-FR1包含SEQ ID NO:4所示的氨基酸序列，所述H-FR2包含SEQ ID NO:27所示的氨基酸序列，所述H-FR3包含SEQ ID NO:6所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:7所示的氨基酸序列；所述L-FR1包含SEQ ID NO:38所示的氨基酸序列，所述L-FR2包含SEQ ID NO:32所示的氨基酸序列，所述L-FR3包含SEQ ID NO:33所示的氨基酸序列，且所述L-FR4包含SEQ ID NO:34所示的氨基酸序列。
3)所述H-FR1包含SEQ ID NO:17所示的氨基酸序列，所述H-FR2包含SEQ ID NO:18所示的氨基酸序列，所述H-FR3包含SEQ ID NO:19所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:20所示的氨基酸序列；所述L-FR1包含SEQ ID NO:22所示的氨基酸序列，所述L-FR2包含SEQ ID NO:23所示的氨基酸序列，所述L-FR3包含SEQ ID NO:24所示的氨基酸序列，且所述L-FR4包含SEQ ID NO:15所示的氨基酸序列。
4)所述H-FR1包含SEQ ID NO:17所示的氨基酸序列，所述H-FR2包含SEQ ID NO:36所示的氨基酸序列，所述H-FR3包含SEQ ID NO:19所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:20所示的氨基酸序列；所述L-FR1包含SEQ ID NO:31所示的氨基酸序列，所述L-FR2包含SEQ ID NO:23所示的氨基酸序列，所述L-FR3包含SEQ ID NO:19所示的氨基酸序列，且所述L-FR4包含SEQ ID NO:15所示的氨基酸序列。
5)所述H-FR1包含SEQ ID NO:17所示的氨基酸序列，所述SEQ ID NO:36所示的氨基酸序列，所述H-FR3包含SEQ ID NO:19所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:20所示的氨基酸序列；所述L-FR1包含SEQ ID NO:38所示的氨基酸序列，所述L-FR2包含SEQ ID NO:23所示的氨基酸序列，所述L-FR3包含SEQ ID NO:39所示的氨基酸序列，且所述L-FR4包含SEQ ID NO:15所示的氨基酸序列。
例如，本申请的抗原结合蛋白可以包含本申请的抗体重链和本申请的抗体轻链。
抗原结合蛋白的性质
本申请的抗原结合蛋白能够以约2.4×10 -9M或更小的KD值与IgE(例如人IgE)相结合。可以通过本领域常用的Octet方法、MSD-SET法和/或KinExA方法对所述结合进行测定。例如，所述KD值可以通过MSD-SET测定，测量方法如Estep P.等人,MAbs,2013.5(2):p.270-8所述，所述KD值也可以通过KinExA方法测定，测量方法如Jonathan K.Fleming等人，Methods Mol Biol.2018；1697:1-8所述。
例如，通过MSD-SET测定，所述的抗原结合蛋白结合源自人的IgE抗原的KD值可以为≤2×10 -9M、≤1.5×10 -9M、≤1×10 -9M M、≤8×10 -10M、≤7×10 -10M、≤6×10 -10M、≤5×10 -10M、≤4.5×10 - 10M、≤4×10 -10M、≤3.5×10 -10M、≤3×10 -10M、≤2.5×10 -10M、≤2×10 -10M、≤1.8×10 -10M、≤1.5×10 - 10M、≤1×10 -10M、≤9×10 -11M、≤8×10 -11M、≤7×10 -11M、≤6×10 -11M、≤5×10 -11M、≤4×10 -11M、≤3×10 -11M、≤2×10 -11M、≤1×10 -11M、≤9×10 -12M。
例如，通过KinExA法进行测定时，本申请的抗原结合蛋白结合所述IgE的KD值可以为例如，≤3.5×10 -12M、≤3.4×10 -12M、≤3.3×10 -12M、≤3.2×10 -12M、≤3.1×10 -12M、≤3×10 -12M、≤2.9×10 -12M、≤2.8×10 -12M、≤2.7×10 -12M、≤2.6×10 -12M、≤2.5×10 -12M、≤2.4×10 -12M、≤2.3×10 - 12M、≤2.2×10 -12M、≤2.1×10 -12M、≤2.0×10 -12M、≤1.9×10 -12M、≤1.8×10 -12M、≤1.7×10 -12M、≤1.6×10 - 12M、≤1.5×10 -12M、≤1.4×10 -12M、≤1.3×10 -12M、≤1.2×10 -12M或≤1.1×10 -12M。
本申请的抗原结合蛋白能够抑制IgE与其受体FcεRIa和/或FcεRII的结合。
例如，通过ELISA方法检测，本申请的抗原结合蛋白抑制IgE与其受体FcεRIa的结合的IC 50值可以为≤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。
例如，通过ELISA方法检测，本申请的抗原结合蛋白抑制IgE与其受体FcεRIa的结合的IC 50值可以为≤4.9×10 -9M、≤4.5×10 -9M、≤4.0×10 -9M、≤3.5×10 -9M、≤3.0×10 -9M、≤2.5×10 -9M、≤2.0×10 -9M、≤1.5×10 -9M、≤1.0×10 -9M、≤9.0×10 -10M、≤8.0×10 -10M、≤7.0×10 -10M、≤6.0×10 -10M、≤5.0×10 -10M、≤4.0×10 -10M、≤3.0×10 -10M、≤2.5×10 -10M。
例如，通过ELISA方法检测时，本申请的抗原结合蛋白抑制IgE与其受体FcεRIa的结合的IC 50值可以为例如，≤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、≤26ng/mL、≤25ng/mL、≤24ng/mL、≤23ng/mL、≤22ng/mL、≤21ng/mL、≤20ng/mL、≤19ng/mL、≤18ng/mL、≤17ng/mL、≤16ng/mL、≤15ng/mL、≤14ng/mL、≤13ng/mL、≤12ng/mL、≤11ng/mL或≤10ng/mL。
例如，通过ELISA方法检测时，本申请的抗原结合蛋白抑制IgE与其受体FcεRII的结合的IC 50值可以为例如，≤4.0μg/mL、≤3.9μg/mL、≤3.8μg/mL、≤3.7μg/mL、≤3.6μg/mL、≤3.5μg/mL、≤3.4μg/mL、≤3.3μg/mL、≤3.2μg/mL、≤3.1μg/mL或≤3.0μg/mL。
例如，本申请的抗原结合蛋白可以包括抗体或其抗原结合片段。例如，本申请的抗体可以包括但不限于重组抗体、单克隆抗体、人抗体、鼠源抗体、人源化抗体、嵌合抗体、骆驼化单结构域抗体、双特异性抗体、单链抗体、双抗体、三抗体或四抗体。
例如，本申请的抗体可以为人源化抗体。例如，本申请的抗原结合蛋白可以为免疫特异性结合至相关抗原(例如人IgE抗原且包含基本上具有人类抗体的氨基酸序列的框架区(FR)及基本上具有非人类抗体的氨基酸序列的互补决定区(CDR)的抗体或其变异体、衍生物、类似物或片段)。此处的“基本上”在CDR的情况下可以是指CDR的氨基酸序列与非人类抗体CDR的氨基酸序列至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少98.5％或至少99％同一。本申请的人源化抗体基本上可以包含所有至少一个且通常两个可变域(Fab、Fab′、F(ab′)2、Fv)，其中所有或基本上所有CDR区对应于非人类免疫球蛋白(即抗体)的CDR区且所有或基本上所有框架区为具有人类免疫球蛋白共有序列的框架区。例如，人源化抗体还可包含至少一部分免疫球蛋白恒定区(例如，Fc)，通常为人类免疫球蛋白的恒定区。在一些实施例中，人源化抗体至少含有轻链或及重链的可变域。抗体还可包括重链的CH1、铰链、CH2、CH3及CH4区。例如，人源化抗体可以仅含人源化轻链。例如，人源化抗体可以仅含人源化重链。例如，人源化抗体可以仅含轻链和/或人源化重链的 人源化可变域。
例如，本申请的抗原结合片段可以包括Fab，Fab’，F(ab)2、Fv片段、F(ab’)2，scFv，di-scFv和/或dAb。
多肽和免疫缀合物
另一方面，本申请提供了一种多肽，其可以包含本申请的抗原结合蛋白。本申请的多肽还可以包括其他肽段或蛋白片段，例如，其还可以包含促进抗原结合蛋白与抗原结合的构象的其他架或骨架部分。在某些情况下，本申请的多肽还可以包含与其他抗原结合的部分，和/或具有其他功能的蛋白片段。例如，本申请的多肽可以是融合蛋白，多特异性抗体，和/或其他重组蛋白。
另一方面，本申请提供了一种免疫缀合物，其可以包含本申请的抗原结合蛋白或本申请的多肽。例如，本申请的免疫缀合物可以包括与所述抗原结合蛋白融合或偶联的其他的多肽、治疗剂(例如，细胞毒性分子)、探针和/或其他抗体。例如，本申请所述免疫缀合物可以包括药学上可接受的标记物、检测剂和/或治疗物。
核酸、载体、宿主细胞和制备方法
另一方面，本申请提供了分离的一种或多种核酸分子，所述一种或多种核酸分子可编码本申请的抗原结合蛋白或多肽。例如，所述一种或多种核酸分子中的每一个核酸分子可以编码完整的所述分离的抗原结合蛋白或所述的多肽，也可以编码其中的一部分。本申请所述的核酸分子可以为分离的。在本申请中，可以通过本领域已知的多种方法来制备编码所述的抗原结合蛋白或所述的多肽的核酸。
另一方面，本申请提供了一种或多种载体，其包含本申请所述的一种或多种核酸分子。每种载体中可包含一种或多种所述核酸分子。此外，所述载体中还可包含其他基因，例如允许在适当的宿主细胞中和在适当的条件下选择该载体的标记基因。此外，所述载体可以含有多种控制表达的元件，包括启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，所述载体还可以含有复制起始位点。此外，所述载体可以包括，例如质粒、粘粒、病毒、噬菌体或者在例如遗传工程中通常使用的其他载体。
另一方面，本申请提供了一种细胞，所述细胞可包含本申请所述的一种或多种核酸分子和/或本申请所述的一种或多种载体。例如，每种或每个细胞可包含一个或一种本申请所述的核酸分子或载体。例如，每种或每个细胞可包含多个(例如，2个或以上)或多种(例如，2种或以上)本申请所述的核酸分子或载体。例如，可将本申请所述的载体引入所述细胞中，例如原核细胞(例如，细菌细胞)、CHO细胞、NS/0细胞、HEK293细胞，或者其他真核细 胞，如来自植物的细胞、真菌或酵母细胞等。可通过本领域已知的方法将本申请所述的载体引入所述细胞中，例如电穿孔、lipofectine转染、lipofectamin转染等。
例如，载体可以包括：质粒；噬菌粒；柯斯质粒；人工染色体如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。用作载体的动物病毒种类有逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。又例如，所述载体可以含有多种控制表达的元件，包括启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，所述载体还可以含有复制起始位点。此外，所述载体还可以包括有协助其进入细胞的成分，如病毒颗粒、脂质体或蛋白外壳，但不仅仅只有这些物质。
例如，所述细胞可以包括原核细胞、酵母菌细胞或高等真核细胞。适用于此目的的原核生物包括格兰氏阴性菌和格兰氏阳性菌，例如，肠细菌，例如大肠杆菌、肠杆菌、欧文氏菌(Erwinia)、克雷臼氏杆菌(Klebsiella)、变形菌(Proteus)、沙门氏菌(Salmonella)、粘质沙雷氏菌(Serratia)、和志贺氏杆菌(Shigella),以及杆菌、假单胞菌和链霉菌。
例如，所述细胞可以包括哺乳动物宿主细胞系。例如，猴肾脏细胞，人类胚胎肾脏细胞系，幼小仓鼠肾脏细胞，中国仓鼠卵巢细胞小鼠塞尔托利(sertoli)细胞，人类子宫颈癌细胞(HELA)，犬肾脏细胞，人类肺细胞，人类肝脏细胞，小鼠乳腺癌细胞或NSO细胞。
例如，所述细胞还可以包括用本发明所述的载体在体外转染的细胞。例如，所述细胞可以是细菌细胞(例如，大肠杆菌)、酵母细胞或其它真核细胞，例如COS细胞、中国仓鼠卵巢(CHO)细胞、CHO-K1细胞、LNCAP细胞、HeLa细胞、HEK293细胞、COS-1细胞、NS0细胞或骨髓瘤细胞。在某些实施方案中，所述细胞可以为哺乳动物细胞。例如，所述哺乳动物细胞可以为HEK293细胞。
药物组合物、应用
另一方面，本申请还提供了制备本申请的抗原结合蛋白的方法，所述方法可以包括在使得本申请所述的抗原结合蛋白表达的条件下，培养本申请所述的细胞。
另一方面，本申请还提供了本申请的抗原结合蛋白、本申请的核酸分子、本申请的载体、本申请的细胞和/或本申请的药物组合物在制备药物中的用途，本申请的药物用于预防、缓解和/或治疗IgE相关的疾病或病症。
另一方面，本申请还提供了预防、缓解或治疗IgE相关的疾病或病症的方法，本申请方法可以包括向有需要的受试者施用本申请的抗原结合蛋白、本申请的核酸分子、本申请的载 体、本申请的细胞和/或本申请的药物组合物。
另一方面，本申请还提供了本申请的抗原结合蛋白、本申请的核酸分子、本申请的载体、本申请的细胞和/或本申请的药物组合物，其可以用于预防、缓解或治疗IgE相关的疾病或病症。
例如，IgE相关的疾病或病症可以包括Omalizumab(商品名Xolair)在世界范围(例如，美国、中国、欧洲等)内获得批准的可适用于治疗的疾病或病症。例如，所述疾病或病症可以包括过敏性疾病。
例如，所述受试者可以包括人类和非人类动物。例如，所述受试者可以包括但不限于猫、狗、马、猪、奶牛、羊、兔、小鼠、大鼠或猴。
另一方面，本申请还提供了药物组合物，其包含本申请的抗原结合蛋白、本申请的核酸分子、本申请的载体和/或本申请的细胞，以及任选地药学上可接受的载剂。
例如，药物组合物可以另外地含有适合治疗或预防IgE相关的疾病或病症的一种或多种其它治疗剂。
例如，所述药学上可接受的载剂增强或稳定组合物，或促进组合物的制备。例如，所述药学上可接受的载剂可以包括生理上相容的溶剂、分散介质、包衣材料、抗细菌剂和抗真菌剂、等渗剂和吸收延迟剂等。
例如，所述施用可以通过不同的方式进行，例如静脉内、腹膜内、皮下、肌肉内、局部或真皮内施用。
例如，本申请的药物组合物可以通过本领域已知的多种方法施用，取决于期望结果、施用途径和/或施用模式改变。例如，施用可以为静脉内、肌内、腹膜内或皮下、或邻近靶位点施用。例如，配制所述的药物组合物使得可将它们玻璃体内施用入眼。例如，取决于施用途径，可以将所述的抗原结合蛋白(例如，抗体、双特异性和多特异性分子)用材料包覆以保护化合物免于酸或其它天然条件的作用，所述作用可以使化合物失活。
例如，本申请的药物组合物可以为无菌的且为流体。例如，通过使用包衣材料(诸如卵磷脂)、或在分散液的情况下通过保持所要求的颗粒大小、以及通过使用表面活性剂可以保持适当的流动性。
例如，在组合物中包括等渗剂，例如，糖、多元醇，诸如甘露糖醇、山梨糖醇或氯化钠。例如，可注射的所述药物组合物的长期吸收可以通过在所述药物组合物中包括延迟吸收的试剂(例如，单硬脂酸铝或明胶)来实现。
例如，本申请的抗原结合蛋白、本申请的核酸分子、本申请的载体和/或本申请的细胞与 任选的药学可接受载体、赋形剂或稳定剂混合而制备以贮存。例如，本申请的药物组合物的形式可以为冻干制剂或水溶液。
例如，本申请的药学上可接受的载剂可以包括药学可接受载体、赋形剂或稳定剂。
例如，本申请的可接受的载体、赋形剂或稳定剂以采用的剂量和浓度对接受者是无毒的，并且包括缓冲剂例如磷酸盐柠檬酸盐、乙酸盐和其他有机酸。
例如，包含本申请的抗原结合蛋白的药物组合物可以是水溶性形式。
例如，用于体内施用的药物组合物可以是无菌的。这可以通过无菌过滤膜过滤或其他方法而容易地实现。例如，以无菌水溶液形式施用包含本申请的抗原结合蛋白的药物组合物可以多种方式进行，包括但不限于口服、皮下、静脉内、鼻内、耳内、透皮、局部(例如，凝胶、油膏、洗剂、乳霜等)、腹膜内、肌肉内、肺内、胃肠外、直肠或眼内。在一些情况下，例如治疗伤口、炎症等，本申请的抗原结合蛋白可以作为溶液或喷雾剂直接应用。
例如，本申请的药物组合物可以根据本领域熟知且常规实践的方法制备。例如，本申请的药物组合物可以在GMP条件下制备。通常，在本申请的药物组合物中使用IgE结合蛋白的治疗有效剂量或有效剂量。例如，通过本领域技术人员已知的常规方法可将IgE结合蛋白配制成药学上可接受的剂量形式。调节剂量方案以提供最佳的期望响应(例如，治疗响应)。本申请的药物组合物中活性成分的实际剂量水平可发生改变以便获得有效实现特定患者所期望的治疗响应、组合物和施用模式而对患者不具有毒性的活性成分的量。选择的剂量水平取决于多种药代动力学因素，例如，本申请使用的特定组合物或其酯、盐或酰胺的活性，施用途径，施用时间，正使用的特定化合物的排泄速率，治疗的持续时间，与使用的特定组合物组合使用的其它药物、化合物和/或物质，治疗的受试者的年龄、性别、体重、状态、一般健康和先前病史以及其它因素。
例如，皮下施用可以用于其中患者可以自己施用药物组合物的情况。许多蛋白治疗剂不足够有效以允许配制皮下施用最大可接受体积的治疗有效剂量。本申请公开的抗原结合蛋白可适用于皮下施用，例如，可以增加的效价、提高的血浆半衰期和提高的溶解度。
本领域已知，蛋白治疗剂可以通过IV输注或快速浓注来递送。本申请公开的抗原结合蛋白也可以使用此类方法递送。
另一方面本申请还提供了检测样品中IgE的方法，所述方法包括施用本申请中所述的抗原结合蛋白。
例如，将获取自受试者的样品接触本申请的抗原结合蛋白(例如，IgE结合蛋白)。例如，其中用可检测标记或报告分子标记IgE结合蛋白或将抗IgE结合蛋白用作捕获配体以选择性 地从患者样品分离IgE。或者，未标记的抗IgE结合蛋白可以结合第二抗体用于检测应用中，所述第二抗体自身是具有可检测标记的。可检测标记或报告分子可以是放射性同位素，例如如 3H、 14C、 32P、 35S或 125I；还可以是荧光或化学发光部分，例如异硫氰酸荧光素、若丹明；或酶，例如碱性磷酸酶、β-半乳糖苷酶、辣根过氧化物酶或荧光素酶；可以用于检测或测量样品中的IgE的特定示例性分析包括酶联免疫吸附分析(ELISA)、放射性免疫分析(RIA)和荧光激活的细胞分选(FACS)。
可以用于本申请的IgE检测中的样品可以包括获取自正常或病理状况下的受试者的任何组织或流体样品，其含有可检测含量的IgE蛋白或其片段。例如，可以将测量获自健康受试者(例如，未患IgE相关疾病的受试者)的特定样品的IgE蛋白的水平，以最初建立IgE的基线或标准水平。随后将这个IgE的基线水平相对于获取自疑似患有IgE相关疾病或病症或与该病症相关症状的个体的样品中测量的IgE水平进行比较。IgE结合蛋白可以不含另外的标记，也可以含有N-末端或C-末端标记。例如，所述标记是生物素。在结合分析中，标记(如果存在的话)的位置可以确定肽相对于其上结合肽的表面的方向。例如，如果表面包被了抗生物素蛋白，则含有N-末端生物素的肽将使得肽的C-末端部分远离表面。
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请发明的各个技术方案，而不用于限制本申请发明的范围。
实施例
实施例1本申请的抗原结合蛋白制备
1-1抗人IgE鼠源单克隆抗体制备
(1)小鼠免疫
向四周龄的BALB/c小鼠皮下注射20μg重组表达的人IgE的Fc区域(CH2-4)蛋白(IgE-Fc，SEQ ID NO:42)(在弗氏完全佐剂中)。每三至四星期注射一次，一共注射五次。最后在腹膜内单次注射20μg重组人IgE-Fc蛋白。
(2)ELISA法测定小鼠血清与人IgE-Fc抗原的结合
以重组人IgE-Fc蛋白包被酶标板(Costar)，室温过夜。弃去包被溶液，用溶解在磷酸盐缓冲盐水(PBS)的2.5％的脱脂奶封闭各孔0.5小时，用含有0.05％吐温(Tween)-20的PBS洗孔。然后分别加入稀释血清和作为阴性对照的非相关抗体(Irrevalent mAb)，室温孵育1小时，用含有0.05％吐温(Tween)-20的PBS洗孔，然后每孔加入50μl的HRP标记的羊抗鼠IgG多克隆抗体(Jackson Laboratory)作为检测抗体。经血清测试后，鉴定出含有高水平抗重 组人IgE-Fc蛋白抗体血清的小鼠。
(3)细胞融合、筛选和鼠源单克隆抗体制备
经血清测试鉴定出含有高水平抗人IgE-Fc抗体血清的小鼠后，取出该小鼠脾脏与小鼠骨髓瘤Sp2/0细胞株相融合。混合5×10 8的Sp2/0细胞与5×10 8的脾细胞在50％聚乙二醇(PEG，分子量为1450)和5％二甲基亚枫(DMSO)溶液中融合。用Iscove培养基(含有10％胎牛血清，100单位/mL青霉素，100μg/mL链霉素，10mM次黄嘌呤，0.4μM氨基蝶呤和1.6mM胸苷)来调整脾脏细胞数至7.5x 105/mL，以0.2ml加入96孔培养板的孔内。置于37℃，5％CO2的培养箱内。10天后，取出各孔内的培养基测试其中鼠源抗体对人IgE-Fc的结合能力，以此筛选出与人IgE-Fc结合的阳性孔。再将上述含有能与人IgE-Fc结合抗体的孔内融合细胞进行亚克隆，同样以ELISA方法筛选得到表达高亲和力鼠单克隆抗体的杂交瘤细胞株。
制备纯化的鼠单克隆抗体，以获得待测试的抗体样品。在补充10％FCS的RPMI1640培养基中培养产生特异性抗体的细胞克隆。当细胞密度达到大约5×10 5个细胞/ml时，用无血清培养基替换该培养基。2至4天后，将培养过的培养基离心，以收集培养物上清液。使用蛋白G柱用于纯化抗体。用150mM的NaCl透析单克隆抗体洗脱液。通过0.2μm滤器将透析的溶液过滤除菌，以获得待测试的抗体样品。制备得到单克隆抗体SE3(11/5)、SE5(4/23)、SE2、SE5。
1-2抗人IgE嵌合抗体制备
(1)鼠源抗体的重链可变区的克隆
为获得鼠源抗体的重链和轻链可变区编码DNA序列，用mRNA纯化试剂盒(NEB)从小鼠杂交瘤细胞分离出mRNA，以此制备cDNA(SMARTer RACE试剂盒，Clontech)。通过聚合酶链式反应(PCR)从cDNA中克隆重链可变区DNA片段。PCR的5'-引物使用混合液:0.4μM的引物1(5'-ctaatacgactcactatagggcAAGCAGTGGTATCAACGCAGAGT-3'，如SEQ ID NO:53所示)和2μM的引物2(5'-ctaatacgactcactatagggc-3'，如SEQ ID NO:54所示)。PCR的3'-引物为引物3(5'-catcccagggtcaccatggagtta-3'，如SEQ ID NO:55所示)。引物3与小鼠IgG1重链恒定区同源反义。将胶纯化后得到的重链可变区DNA片段克隆入TOPO-TA载体(Invitrogen)并测序，得到编码SE2小鼠杂交瘤重链的可变区氨基酸序列(如SEQ ID NO:8所示)和核苷酸序列(如SEQ ID NO:57所示)，通过Kabat定义方式确定其互补决定区的氨基酸序列HCDR1(如SEQ ID NO:1所示),HCDR2(如SEQ ID NO:2所示)和HCDR3(如SEQ ID NO:3所示)，和编码SE5小鼠杂交瘤重链的可变区氨基酸序列(如SEQ ID NO:28所示)和核苷酸序列(如SEQ ID NO:61所示)，及其互补决定区的氨基酸序列HCDR1(如SEQ  ID NO:1所示),HCDR2(如SEQ ID NO:26所示)和HCDR3(如SEQ ID NO:3所示)。互补决定区的定义参见Kabat E.等人的Sequences of Proteins of Immunological Interest第5版U.S.Department of Health and Human Services,NIH Publication No.91-3242。
(2)鼠源抗体的轻链可变区的克隆
以与步骤(1)相似的方法，使用上述5'-引物混合液(引物1和引物2)和另一个与小鼠免疫球蛋白轻链恒定区同源反义的3'-引物(引物4，5’-gactgaggcacctccagatgttaa-3’，如SEQ ID NO:56所示)，从cDNA中克隆轻链可变区DNA片段。将这些得到的DNA片段克隆入TOPO-TA载体并测序，得到编码SE2小鼠杂交瘤轻链的可变区氨基酸序列(如SEQ ID NO:16所示)和核苷酸序列(如SEQ ID NO:58所示)，及其互补决定区的氨基酸序列LCDR1(如SEQ ID NO:9所示),LCDR2(如SEQ ID NO:10所示)和LCDR3(如SEQ ID NO:11所示)，和编码SE5小鼠杂交瘤轻链的可变区氨基酸序列(如SEQ ID NO:35所示)和核苷酸序列(如SEQ ID NO:62所示)，及其互补决定区的氨基酸序列LCDR1(如SEQ ID NO:9所示),LCDR2(如SEQ ID NO:29所示)和LCDR3(如SEQ ID NO:30所示)。
(3)嵌合型抗体制备
将步骤2)克隆得到的鼠源单克隆抗体的重链和轻链可变区序列分别使用无缝克隆的方法插入到含有信号肽(如SEQ ID NO:50所示)和人IgG1重链恒定区(如SEQ ID NO:42所示)，或者含有信号肽(如SEQ ID NO:50所示)和人Kappa轻链恒定区(如SEQ ID NO:43所示)的pCDNA3.1(购自淼灵生物)真核表达载体中，经DNA测序确认正确后，在DH5α菌株中扩增后，使用NucleoBond Xtra Midi Plus(MACHEREY-NAGEL)质粒中抽试剂盒制备质粒。将制备好的嵌合型单克隆抗体重链和轻链质粒使用PEI(PolyScience)共转Expi293细胞(购自Thermo fisher)，瞬时表达单克隆抗体。使用蛋白A柱纯化瞬时表达的抗体，用PBS透析单克隆抗体洗脱液。通过0.2μm滤器将透析的溶液过滤除菌，以获得待测试的抗体样品。
1-3抗人IgE人源化抗体制备
(1)抗人IgE鼠源单克隆抗体人源化
以同源建模的抗体可变区结构为基础，确定可能影响抗原-抗体相互作用的关键氨基酸位点，并对关键氨基酸位点进行回复突变，使用CDR移植的方法获得SE2和SE5抗体可变区的人源化氨基酸序列，人源化SE2重链可变区氨基酸序列如SEQ ID NO:21所示，人源化SE2轻链可变区氨基酸序列如SEQ ID NO:25所示，人源化SE5重链可变区氨基酸序列如SEQ ID NO:37所示，人源化SE5轻链可变区氨基酸序列如SEQ ID NO:40所示。对氨基酸序列进行逆翻译后得到的DNA序列并进行合成，SE2重链可变区人源化核苷酸序列如SEQ ID NO:21 所示，SE2轻链可变区人源化核苷酸序列如SEQ ID NO:60所示，SE5重链可变区人源化核苷酸序列如SEQ ID NO:63所示，SE5轻链可变区人源化核苷酸序列如SEQ ID NO:64所示，所得到的DNA用于重组抗体表达载体构建。
(2)抗人IgE人源化抗体制备
将步骤1)得到的人源化单克隆抗体的重链和轻链可变区分别使用无缝克隆的方法插入到含有信号肽(如SEQ ID NO:50所示)和人IgG1重链恒定区(如SEQ ID NO:42所示)，或者含有信号肽(如SEQ ID NO:50所示)和人Kappa轻链恒定区(如SEQ ID NO:43所示)的pCDNA3.1(购自淼灵生物)真核表达载体中，经DNA测序确认正确后，使用NucleoBond Xtra Midi Plus(MACHEREY-NAGEL)质粒中抽试剂盒制备质粒。将制备好的人源化单克隆抗体重链和轻链质粒使用PEI(PolyScience)共转Expi293细胞(购自Thermo fisher)，瞬时表达单克隆抗体。使用蛋白A柱纯化瞬时表达的抗体，用PBS透析单克隆抗体洗脱液。通过0.2μm滤器将透析的溶液过滤除菌，以获得待测试的抗体样品。
(3)改造后的人源化抗体
对抗体可变区的序列分析发现，SE5重链CDR2区(如SEQ ID NO:26所示)的N53为潜在的N-糖基化位点，对人源化SE5进行了N53S/T57S突变改造，同时对SE5的Fc区域(SEQ ID NO:42)进行了M135Y、S137T和/或T139E(YTE)改造，以增强抗体分子在酸性条件下与新生儿受体FcRn的亲和力，从而延长抗体分子在体内的半衰期，达到延长给药周期的目的，改造后的人源化分子称为SE5ss。
实施例2本申请抗原结合蛋白(鼠源单克隆抗体)阻断活性检测
使用ELISA方法检测纯化的鼠源单克隆抗体阻断人IgE-Fc与其受体FcεRIa结合的阻断活性。具体方法为，将重组表达的人FcεRIa-Fc蛋白(如SEQ ID NO:51所示氨基酸序列)使用PBS稀释到终浓度2.5μg/mL，100μL/孔包被酶标板(购自Corning，货号：42592)，4℃温过夜。第二天弃去包被液，用溶解在PBS中的2.5％的脱脂奶封闭1小时，用含有0.05％吐温(Tween)20的PBS洗孔。加入预先混合10分钟、含有1μg/mL的生物素标记的人IgE-Fc(如SEQ ID NO:52所示氨基酸序列)和梯度稀释的鼠源或对照抗体的预混液，由实施例1制备所得的鼠源抗体或对照抗体奥马珠(购自Novartis，货号：Xolair)从5μg/mL做2倍梯度稀释(即5μg/mL，2.5μg/mL，1.25μg/mL，0.625μg/mL直到0.002441μg/mL，)，室温孵育1小时。弃去上清后用含有0.05％吐温(Tween)20的PBS洗孔，使用HRP标记的链霉亲和素(购自生工生物，货号：D111054-0001)1:1000稀释后检测。检测结果如图1所示，纯化得到的各鼠源单克隆抗体均能够不同程度的阻断人IgE-Fc与其受体FcεRIa结合的能力，SE2、 SE5均较对照抗体奥马珠单抗有着更强的阻断活性。
实施例3本申请抗原结合蛋白(嵌合抗体)阻断活性检测
为进一步验证克隆得到并重组表达的嵌合单克隆抗体的活性，使用与实施例2相似的ELISA方法检测纯化的嵌合抗体阻断人IgE-Fc与其受体FcεRIa结合的阻断活性。具体方法为，将重组表达的人FcεRIa-Fc蛋白(如SEQ ID NO:51所示氨基酸序列)使用PBS稀释到终浓度1μg/mL，100μL/孔包被酶标板(购自Corning，货号：42592)，4℃温过夜。第二天弃去包被液，用溶解在PBS的2.5％脱脂奶封闭1小时，用含有0.05％吐温(Tween)20的PBS洗孔。加入预先混合10分钟、含有100ng/mL的生物素标记的人IgE-Fc(如SEQ ID NO:52所示氨基酸序列)和梯度稀释的嵌合抗体或鼠源抗体的预混液，嵌合抗体或鼠源抗体从2μg/mL开始做3倍梯度稀释(即2μg/mL、666.67ng/mL、222.22ng/mL、74.07ng/mL、24.69ng/mL、8.23ng/mL、2.74ng/mL，最后为0ng/mL)，室温孵育1小时。弃去上清后用含有0.05％吐温(Tween)20的PBS洗孔，使用HRP标记的链霉亲和素(购自生工生物，货号：D111054-0001)1:1000稀释后检测。根据如下公式：抗体浓度X的抑制率(％)＝(抗体浓度为0的OD 450读值-抗体浓度为X的OD 450读值)/抗体浓度为0的OD 450读值×100，计算不同抗体、不同浓度下的抑制率。检测结果如图2所示，由克隆得到鼠源单克隆可变区序列构建的嵌合重组单克隆抗体，与其杂交瘤纯化的鼠源母抗体有相近的阻断活性。
实施例4本申请的抗原结合蛋白(抗人IgE人源化抗体)阻断活性检测
1)使用与实施例2近似的ELISA方法检测纯化的鼠源抗体、嵌合抗体以及人源化抗体阻断人IgE-Fc与其受体FcεRIa结合的阻断活性。具体方法为，将重组表达的人FcεRIa-Fc蛋白(如SEQ ID NO:51所示氨基酸序列)使用PBS稀释到终浓度1μg/mL，100μL/孔包被酶标板(购自Corning，货号：42592)，4℃温过夜。第二天弃去包被液，用溶解在PBS的2.5％的脱脂奶封闭1小时，用含有0.05％吐温(Tween)20的PBS洗孔。加入预先混合10分钟、含有100ng/mL的生物素标记的人IgE-Fc(如SEQ ID NO:52所示氨基酸序列)和梯度稀释的各个抗体的预混液，各抗体均从1μg/mL做3倍梯度稀释(即1μg/mL、333.33ng/mL、111.11ng/mL、37.04ng/mL、12.35ng/mL、4.12ng/mL、1.37ng/mL，最后点为0ng/mL)，室温孵育1小时。弃去上清后用含有0.05％吐温(Tween)20的PBS洗孔，使用HRP标记的链霉亲和素(购自生工生物，货号：D111054-0001)检测。检测结果如图3至图5所示，重组表达的人源化单克隆SE2和SE5，分别与其相应的嵌合抗体及鼠源母抗体有相近的阻断活性(图3)。
2)按照本实施例步骤1)的方法检测SE2人源化抗体、SE5人源化抗体和奥马珠单抗阻断人IgE-Fc与其受体FcεRIa结合的阻断活性。在SE2人源化抗体和奥马珠单抗的比较实验 中，抗体浓度从3μg/mL做3倍梯度稀释(即3μg/mL，1μg/mL，333.33ng/mL，111.11ng/mL，37.04ng/mL、12.35ng/mL、4.12ng/mL、1.37ng/mL，最后为0ng/mL)；在SE5人源化抗体和奥马珠单抗的比较实验中，抗体浓度从10nM做3倍梯度稀释(即10nM，3.33nM，1.11nM，370.37pM、123.46pM、41.15pM、13.72pM，最后为0pM)。SE5比较实验中包括对照抗体(即图5中对照，其为同型her2抗体pertuzumab，序列来源于IMGT抗体库)。
根据如下公式：抗体浓度X的抑制率(％)＝(抗体浓度为0的OD 450读值-抗体浓度为X的OD 450读值)/抗体浓度为0的OD450读值×100，计算不同抗体、不同浓度下的抑制率。结果如图4-5所示，SE2人源化抗体和SE5人源化抗体较对照抗体奥马珠单抗均有着更强的阻断活性，其中SE2和奥马珠单抗的阻断IC 50值分别约为416.7ng/mL和2110ng/mL，SE5和奥马珠单抗的阻断IC 50值分别约为266.2pM和4996pM。
实施例5本申请的抗原结合蛋白(抗人IgE人源化抗体)亲和力检测
使用超敏因子电化学发光分析-溶液平衡滴定法(MSD-SET)检测人源化抗体和人IgE的平衡亲和力，测量方法如Estep P.等人,MAbs,2013.5(2):p.270-8所述。溶液平衡滴定(SET)在PBS+0.1％不含IgG的BSA缓冲液(PBSF)中进行，其中抗原人IgE(购自：abcam，货号：ab65866)保持恒定在10-100pM，且与从0.1-100nM起始的人源化抗体3倍连续稀释液一起孵育。具体地，针对SE2和SE5测定时，抗原人IgE保持恒定在13.33pM，人源化抗体SE2和SE5从166.67pM连续三倍稀释至0.0085pM；针对奥马珠单抗测定时，抗原人IgE保持恒定在133.3pM，奥马珠单抗从66.67nM开始3倍连续稀释至1.13pM。将稀释于PBS中的20nM的抗体包被至标准结合MSD-ECL板(购自MSD，货号：L15XA-3)上在4℃下过夜。用1％的Casein将MSD-ECL板封闭30分钟，同时以700rpm振荡。然后将MSD-ECL板用洗涤缓冲液(PBS+0.05％Tween 20)洗涤3次。将抗原人IgE与人源化抗体2倍连续稀释液孵育的样品加入MSD-ECL板，700rpm振荡下孵育150s，之后洗涤一次。向MSD-ECL板加入含有250ng/mL磺基标签标记的链霉亲和素的PBSF，5分钟之后，将MSD-ECL板用洗涤缓冲液洗涤(PBS+0.05％Tween 20)洗涤3次。加入1x阅读缓冲液T(购自MSD，货号：R92TC-1)在MSD Sector Imager仪器(型号：MESO QuickPlex SQ 120)上读数。在Prism-GraphPad6软件中，将游离抗原百分比绘制为滴定的抗体的函数，并拟合至二次方程式以提取KD。测量结果见下表1。
表1 MSD-SET法检测人源化抗体和对照抗体与人IgE的平衡亲和力
Figure PCTCN2021127316-appb-000001
Figure PCTCN2021127316-appb-000002
由表1中所示结果可知，人源化重组单克隆抗体SE2和SE5与人IgE结合的平衡亲和力分别约为10.880pM和9.995pM，对照抗体奥马珠单抗约为2489pM，亲和力有显著提高。
实施例6本申请的抗原结合蛋白(改造后抗人IgE人源化抗体)亲和力检测
使用KinExA检测人源化抗体和对照抗体(Omalizumab)与人IgE(来源于U266细胞(ATCC)，制备方法参见Mol Immunol.1986,23(2):159-67)的平衡亲和力。溶液平衡滴定(SET)在PBS+0.1％不含IgG的BSA缓冲液(PBSF)中进行，其中抗原人IgE-Biotin(生物素标记人IgE，制备方法参考Thermofisher EZ-Link TM Sulfo-NHS-LC-Biotinylation Kit使用说明)保持恒定在20pM-5nM，且与从2nM-1μM抗体两倍连续稀释液一起孵育。具体地，针对人源化抗体SE5ss测定时，抗原人IgE-Biotin保持恒定在20pM，人源化抗体SE5ss从2nM开始连续两倍稀释至30.52fM，将混合样品在室温下孵育30小时；针对对照抗体Omalizumab测定时，抗原人IgE-Biotin保持恒定在5nM，Omalizumab从1μM开始连续两倍稀释至15.26pM，将混合样品在室温下孵育20小时。将抗原人IgE-Biotin与抗体两倍连续稀释液孵育的样品在KinExA仪器(型号：4000)中流过用抗体包被的珠子，样品中未与抗体结合的游离抗原人IgE-Biotin就会与珠子上包被的抗体结合，继续将连有荧光的二抗Alexa Fluor 647 Streptavidin流过珠子，仪器通过检测珠子上的荧光信号并通过KinExA软件拟合得到KD值。测量结果见图6A-B。实验结果表明，根据实施例1制备的人源化重组单克隆抗体SE5ss和Omalizumab与人IgE结合的平衡亲和力分别约为2.08pM和1.79nM，SE5ss亲和力远高于Omalizumab。
实施例7本申请的抗原结合蛋白(改造后抗人IgE人源化抗体)阻断活性检测
(1)本申请的抗原结合蛋白阻断人源IgE与其受体FcεRIa结合的阻断活性
使用ELISA方法检测SE5ss单克隆抗体阻断人源IgE与其受体FcεRIa结合的阻断活性。具体方法为，将重组表达的人FcεRIa-Fc蛋白用PBS稀释到1μg/mL，100μL/孔包被酶标板(Corning，货号：42592)，4℃孵育过夜。第二天弃去包被液，使用含2.5％脱脂牛奶的PBS 25℃封闭1小时，结束后用PBST(PBS+0.05％Tween 20)洗涤3次，之后加入预先混合1小时、含有100ng/mL的人源IgE和梯度稀释的待检抗体(SE5ss、Omalizumab和人源IgG)的预混液，25℃孵育1小时，待检抗体终浓度从10ug/mL起，3倍连续稀释10个点至0.17ng/mL，再加0ug/mL的点，结束后用PBST洗3次。使用1:5000稀释的山羊抗人IgE抗体 (Invitrogen，货号：A18795)和1:2000稀释的HRP标记的兔抗山羊IgG抗体(生工，货号：D110117-0100)进行检测。测得OD 450值用四参数回归模型分析，计算抗体的IC 50。结果如图7A所示，根据实施例1制备的SE5ss和Omalizumab均能有效阻断人源IgE与FcεRIα受体的结合，IC 50分别是32.38ng/mL和1009ng/mL，SE5ss阻断人源IgE与FcεRIα受体结合的能力远远好于Omalizumab。
(2)本申请的抗原结合蛋白阻断人源IgE与其受体FcεRII(CD23)受体的阻断活性
使用与步骤(1)相似的ELISA方法，检测SE5ss阻断人源IgE与其受体FcεRⅡ(CD23)的阻断活性。具体方法为，将重组人FcεRⅡ(CD23，购于翊翘神州，货号：10261-H07H)蛋白用PBS稀释至1.5μg/mL，100μL/孔包被酶标板(Corning，货号：42592)。第二天弃去包被液，使用1％casein室温封闭2小时。将生物素标记的重组表达β乳球蛋白特异性人IgE(bIgEk，Structure 15,1413–1421,2007)和β-乳球蛋白(sigma，货号：L3908)等体积(摩尔比大约1：10)混匀(浓度均为60μg/mL)，37℃孵育1小时后和梯度浓度的抗体再混合，37℃孵育1小时，待测抗体(SE5ss、Omalizumab和人源IgG)终浓度从300ug/mL起，3倍连续稀释7个点到0.41μg/mL，再加0ug/mL的点，bIgEk-Biotin和β-乳球蛋白复合物的浓度恒定为30μg/mL，结束后用PBST洗3次。使用1:8000稀释的Streptavidin-HRP抗体(购自生工，货号：D110513-0100)进行检测。测得OD 450值用四参数回归模型分析，计算各抗体的IC 50。结果如图7B所示，实验结果表明根据实施例1制备的SE5ss和Omalizumab均能有效阻断人源IgE与FcεRⅡ(CD23)受体的结合，IC 50分别是3.756μg/mL和3.444μg/mL，二者阻断人源IgE与FcεRⅡ蛋白结合的能力与相近。
(3)本申请的抗原结合蛋白对人源IgE介导的RBL-2H3-FcεRIα-NFATLuc细胞报告基因激活的抑制活性检测
RBL-2H3-FcεRIα-NFATLuc细胞株构建：为检测IgE介导的细胞激活及本申请的抗原结合蛋白阻断IgE的细胞水平活性，在大鼠嗜碱性细胞RBL-2H3细胞系(中科院细胞库)基础上，构建了表达人FcεRIα受体和NFAT驱动的荧光素酶(Luciferase)报告基因细胞株，命名为：RBL-2H3-FcεRIα-NFATLuc。构建过程参见：Analytical and Bioanalytical Chemistry volume 412,pages1901-1914(2020)；Allergy 2010；65:1266–1273；J Immunol July 1,1996,157(1)221-230。
取对数生长期的RBL-2H3-FcεRIα-NFATLuc细胞铺于96孔平底板，每孔约5E4个细胞，50uL培养基。之后加入预先混合的人源IgE和梯度浓度的待检抗体(SE5ss、Omalizumab和人源IgG)的预混液，37℃、5％CO 2培养箱孵育20小时左右。人源IgE恒定终浓度为1000 ng/mL，待检抗体终浓度从45ug/mL起，3倍连续稀释8个点到0.021ug/mL，再加0ug/mL的点。第二天板子用PBS清洗两遍，每孔加入含10μg/mL抗人IgE抗体的(购自Invitrogen，货号：A18795)完全培养基，放于培养箱孵育3小时左右。结束后每孔加入100μL One-Lite TM(诺唯赞)检测试剂，等待约5分钟后转移到96孔白板中，使用酶标仪发光检测模块进行检测，结果用四参数回归模型拟合抑制曲线，计算抗体的IC 50。结果如图8所示，结果说明SE5ss和Omalizumab均可抑制人源IgE介导的效应细胞报告基因激活，IC 50分别为0.2641μg/mL和0.6093μg/mL，SE5ss的抑制能力明显强于Omalizumab。
(4)本申请的抗原结合蛋白抑制不同过敏原过敏的人血清诱发的RBL-2H3-FcεRIα-NFATLuc细胞激活的活性检测
取对数生长期的RBL-2H3-FcεRIα-NFATLuc细胞铺于96孔平底板，每孔约5E4个细胞，50uL培养基。将梯度浓度的待测抗体(SE5ss、Omalizumab和人源IgG)和不同过敏源人血清依次加到细胞中，37℃、5％CO2培养箱孵育20小时左右。由于各血清中特异性过敏原的IgE含量不同，所以使用了不同浓度的血清及抗体。尘螨过敏人血清(PlasmaLab，货号：PL25740)使用浓度是1.5％，抗体终浓度从10000ng/mL起3倍连续稀释9个点至5ng/mL；虾过敏人血清(PlasmaLab，货号：PL24819)使用浓度是3％，Omalizumab和人源IgG终浓度从1200.00ng/mL起3倍稀释12个点到0.01ng/mL，SE5ss的浓度从133.33ng/mL起，3倍连续稀释11个点至0.002ng/mL；胡桃过敏人血清(PlasmaLab，货号：PL27530)使用浓度是1.5％，抗体终浓度从10000ng/mL起，3倍连续稀释9个点至1.5ng/mL，三组均加上0ug/mL的点作为对照。第二天板子用PBS清洗两遍，每孔加入100uL含10μg/mL对应过敏原(尘螨过敏原，购于Greerlabs，货号：XPB82D3A25；虾过敏原，购于沃卡威，货号：M110227；胡桃过敏原，购于沃卡威，货号：M110343)的完全培养基，放于培养箱孵育3小时左右。结束后每孔加入100μL One-Lite TM检测试剂，等待约5分钟后转移到96孔白板中，使用酶标仪发光检测模块进行检测，结果用四参数回归模型拟合抑制曲线，计算抗体的IC 50。实验结果表明，根据实施例1制备的SE5ss和Omalizumab都能抑制各过敏血清介导的RBL-2H3-FcεRIα-NFATLuc细胞报告激活，其中对尘螨过敏血清抑制的IC 50分别为19.38ng/mL和177.9ng/mL(图9A)；对虾过敏血清抑制的IC 50分别为1.917ng/mL和229.8ng/mL(图9B)；对胡桃过敏血清抑制的IC50分别为51.57ng/mL和264.2ng/mL(图9C)，SE5ss的抑制能力均明显优于Omalizumab。
实施例8本申请的抗原结合蛋白(改造后抗人IgE人源化抗体)在动物体内活性检测
(1)本申请的抗原结合蛋白在人源IgE重建小鼠模型中的药效学评估
建立人源IgE重建模型评估SE5ss对小鼠体内游离IgE的抑制作用。试验采用Balb/c小鼠(购自北京维通利华实验动物技术有限公司)，将44只小鼠根据性别和体重随机分为阴性对照组、溶媒对照组、SE5ss 0.5μg组、SE5ss 1μg组、SE5ss 2μg组和Omalizumab 2μg组，其中阴性对照组为4只小鼠，其余各组均为每组8只。每只小鼠静脉注射2μg人源IgE(来源于U266培养纯化，In house，批号5/20/29)，其中阴性对照组静脉注射同等体积的PBST(HyClone，批号C520004-0001)；2小时后静脉注射不同剂量的SE5ss和阳性药Omalizumab(Novartis，批号SPX27)。分别于给药前一天，给药前1小时，给药后1小时和给药后4小时对所有小鼠进行下颌静脉采血，使用MSD检测小鼠血清样本中游离IgE的含量，方法简述如下：MSD板包被FcεRIα-Fc(In house，批号：069-109-11)，加入待测样本，37度孵育1小时后PBST洗涤3次，加入生物素标记的鼠抗人IgE抗体(In house，Clone#AE10-10)，37度孵育1小时后PBST洗涤3次，采用MSD sulfo-tag labeled streptavidin进行检测(MSD，批号：R32AD-1)。试验采用SE5ss溶媒(In house，批号129-098-01)作为阴性对照。结果如图10所示，根据实施例1制备的不同浓度的SE5ss和Omalizumab均能明显的抑制小鼠体内游离IgE(P<0.05)，且抑制能力呈剂量依赖效应，0.5μg/只SE5ss对游离IgE的抑制能力大于2μg/只的Omalizumab(P<0.05)。
(2)本申请的抗原结合蛋白在外源重建速发型过敏反应模型中药效学评估
试验使用human-FcεRIA转基因小鼠(购自上海南方模式生物科技股份有限公司)建立外源重建速发型过敏反应小鼠模型，腹腔注射不同剂量的SE5ss，通过小鼠体内温度的变化，对SE5ss进行初步药效学评估。51只human-FcεRIA转基因小鼠根据性别及基线体温随机分为阴性对照组、溶媒对照组、SE5ss 5μg/只组、SE5ss 10μg/只组、SE5ss 20μg/只组和Omalizumab 20μg/只组，其中阴性对照组为6只，其余各组为9只小鼠。各组动物腹腔注射给予不同剂量的SE5ss和Omalizumab(Novartis，批号SPX27)。给药1小时后腹腔注射给予各组动物人源IgE 20μg/只，给药24小时后腹腔注射给予抗-人IgE(ε-特定链)山羊抗20μg/只(Sigma，批号I6284-1mg)，建立速发型过敏反应模型。分别于给抗人源IgE抗体前和后20分钟、40分钟、60分钟和90分钟对小鼠的体温进行测定。试验采用SE5ss溶媒作为阴性对照。结果如图11所示，给予抗人源IgE抗体后，溶媒对照组小鼠体温开始下降，在40分钟时达到体温最低点，后续缓慢回升。SE5ss各剂量组均能抑制小鼠体温下降，且呈剂量依赖效应。其中SE5ss 10μg/只与Omalizumab 20μg/只药理学效应相当，SE5ss 20μg/只组可以将小鼠体温维持在正常范围。
(3)本申请的抗原结合蛋白在食蟹猴IgE抑制模型中的药效学评估
在食蟹猴体内静脉注射不同剂量的SE5ss，对其抑制食蟹猴游离IgE的能力进行评价。将18只食蟹猴(中科院上海药物研究所)，根据性别和体重随机分为3组，3/性别/组，单次皮下注射分别给予1mg/kg、5mg/kg、25mg/kg SE5ss，并于给药前、给药后1小时、第14天、第28天、第42天和第56天的采集血清样本并测定游离IgE的含量。结果如图12所示，不同浓度的SE5ss均能明显抑制食蟹猴体内游离IgE，且抑制能力呈剂量依赖效应25mg/kg组在给药后1小时，游离IgE下降至给药前的2.4％，在第14和28天游离IgE均低于检测下限，在第42和56天回升至基线的19.1％和34.8％。
前述详细说明是以解释和举例的方式提供的，并非要限制所附权利要求的范围。目前本申请所列举的实施方式的多种变化对本领域普通技术人员来说是显而易见的，且保留在所附的权利要求和其等同方式的范围内。

Claims (57)

  1. 抗原结合蛋白，其包含重链可变区的至少一个CDR，所述重链可变区包含如SEQ ID NO:73、74中任一项所示的氨基酸序列；且其包含轻链可变区的至少一个CDR，所述轻链可变区包含如SEQ ID NO:75、76中任一项所示的氨基酸序列。
  2. 根据权利要求1所述的抗原结合蛋白，其具有下述性质中的一种或多种：
    1)能够以约2.4×10 -9M或更小的KD值与IgE相结合；
    2)能够抑制IgE与其受体FcεRIa和/或FcεRII的结合。
  3. 根据权利要求2中所述的抗原结合蛋白，其中所述IgE为人IgE。
  4. 根据权利要求1-3中任一项所述的抗原结合蛋白，其包含重链HCDR3，所述HCDR3包含SEQ ID NO:3所示的氨基酸序列。
  5. 根据权利要求1-4中任一项所述的抗原结合蛋白，其还包含重链HCDR2，所述HCDR2包含SEQ ID NO:77所示的氨基酸序列。
  6. 根据权利要求5所述的抗原结合蛋白，其中所述HCDR2包含SEQ ID NO:2、26中任一项所示的氨基酸序列。
  7. 根据权利要求1-6中任一项所述的抗原结合蛋白，其还包含重链HCDR1，所述HCDR1包含SEQ ID NO:1所示的氨基酸序列。
  8. 根据权利要求1-7中任一项所述的抗原结合蛋白，其包含重链HCDR1、HCDR2和HCDR3；其中所述HCDR1包含SEQ ID NO:1所示的氨基酸序列，所述HCDR2包含SEQ ID NO:77所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:3所示的氨基酸序列。
  9. 根据权利要求1-8中任一项所述的抗原结合蛋白，其包含重链HCDR1、HCDR2和HCDR3；其中所述HCDR1包含SEQ ID NO:1所示的氨基酸序列，所述HCDR2包含SEQ ID NO:2、26中任一项所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:3所示的氨基酸序列。
  10. 根据权利要求1-9中任一项所述的抗原结合蛋白，其包含重链H-FR1，所述H-FR1的C末端与所述HCDR1的N末端直接或间接相连，且所述H-FR1包含SEQ ID NO:4、17中任一项所示的氨基酸序列。
  11. 根据权利要求1-10中任一项所述的抗原结合蛋白，其还包含重链H-FR2，所述H-FR2位于所述HCDR1与所述HCDR2之间，且所述H-FR2包含SEQ ID NO:81、5或27中任一项所示的氨基酸序列。
  12. 根据权利要求11所述的抗原结合蛋白，其中所述H-FR2包含SEQ ID NO:5、18、27或36中任一项所示的氨基酸序列。
  13. 根据权利要求1-12中任一项所述的抗原结合蛋白，其还包含重链H-FR3，所述H-FR3位于所述HCDR2与所述HCDR3之间，且所述H-FR3包含SEQ ID NO:6、19中任一项所示的氨基酸序列。
  14. 根据权利要求1-13中任一项所述的抗原结合蛋白，其还包含重链H-FR4，所述H-FR4的N末端与所述HCDR3的C末端相连，且所述H-FR4包含SEQ ID NO:7、20中任一项所示的氨基酸序列。
  15. 根据权利要求1-14中任一项所述的抗原结合蛋白，其包含重链可变区VH，所述VH包含SEQ ID NO:73、74中任一项所示的氨基酸序列。
  16. 根据权利要求1-15中任一项所述的抗原结合蛋白，其包含重链可变区VH，所述VH包含SEQ ID NO:8、21、28、37或41中任一项所示的氨基酸序列。
  17. 根据权利要求1-16中任一项所述的抗原结合蛋白，其包含轻链LCDR3，所述LCDR3包含SEQ ID NO:79所示的氨基酸序列。
  18. 根据权利要求17所述的抗原结合蛋白，其中所述LCDR3包含SEQ ID NO:11、30中任一项所示的氨基酸序列。
  19. 根据权利要求1-18中任一项所述的抗原结合蛋白，其还包含轻链LCDR2，所述LCDR2包含SEQ ID NO:78所示的氨基酸序列。
  20. 根据权利要求19所述的抗原结合蛋白，其中所述LCDR2包含SEQ ID NO:10、29中任一项所示的氨基酸序列。
  21. 根据权利要求1-20中任一项所述的抗原结合蛋白，其还包含轻链LCDR1，所述LCDR1包含SEQ ID NO:9所示的氨基酸序列。
  22. 根据权利要求1-21中任一项所述的抗原结合蛋白，其包含轻链LCDR1、LCDR2和LCDR3；其中所述LCDR1包含SEQ ID NO:9所示的氨基酸序列，所述LCDR2包含SEQ ID NO:78所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:79所示的氨基酸序列。
  23. 根据权利要求1-22中任一项所述的抗原结合蛋白，其包含轻链LCDR1、LCDR2和LCDR3；其中所述LCDR1包含SEQ ID NO:9所示的氨基酸序列，所述LCDR2包含SEQ ID NO:10、29中任一项所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:11、30中任一项所示的氨基酸序列。
  24. 根据权利要求1-23中任一项所述的抗原结合蛋白，其包含轻链L-FR1，所述L-FR1的C末端与所述LCDR1的N末端直接或间接相连，且所述L-FR1包含SEQ ID NO:31、12或80中任一项所示的氨基酸序列。
  25. 根据权利要求24所述的抗原结合蛋白，其中所述L-FR1包含SEQ ID NO:12、22、31或38中任一项所示的氨基酸序列。
  26. 根据权利要求1-25中任一项所述的抗原结合蛋白，其还包含轻链L-FR2，所述L-FR2位于所述LCDR1与所述LCDR2之间，且所述L-FR2包含SEQ ID NO:13、23或32中任一项所示的氨基酸序列。
  27. 根据权利要求1-26中任一项所述的抗原结合蛋白，其还包含轻链L-FR3，所述L-FR3位于所述LCDR2与所述LCDR3之间，且所述L-FR3包含SEQ ID NO:33、14或82中任一项所示的氨基酸序列。
  28. 根据权利要求27所述的抗原结合蛋白，其中所述L-FR3包含SEQ ID NO:14、24、33或39中任一项所示的氨基酸序列。
  29. 根据权利要求1-28中任一项所述的抗原结合蛋白，其还包含轻链L-FR4，所述L-FR4的N末端与所述LCDR3的C末端相连，且所述L-FR4包含SEQ ID NO:34、15中任一项所示的氨基酸序列。
  30. 根据权利要求1-29中任一项所述的抗原结合蛋白，其包含轻链可变区VL，所述VL包含SEQ ID NO:75、76中任一项所示的氨基酸序列。
  31. 根据权利要求1-30中任一项所述的抗原结合蛋白，其包含轻链可变区VL，所述VL包含SEQ ID NO:16、35、25、40中任一项所示的氨基酸序列。
  32. 根据权利要求1-31中任一项所述的抗原结合蛋白，其包含所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，其中所述HCDR1包含SEQ ID NO:1所示的氨基酸序列，所述HCDR2包含SEQ ID NO:77所示的氨基酸序列，所述HCDR3包含SEQ ID NO:3所示的氨基酸序列，所述LCDR1包含SEQ ID NO:9所示的氨基酸序列，所述LCDR2包含SEQ ID NO:78所示的氨基酸序列和所述LCDR3包含SEQ ID NO:79所示的氨基酸序列。
  33. 根据权利要求1-32中任一项所述的抗原结合蛋白，其包含所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，其中所述HCDR1包含SEQ ID NO:1所示的氨基酸序列，所述HCDR2包含SEQ ID NO:2、26中任一项所示的氨基酸序列，所述HCDR3包含SEQ ID NO:3所示的氨基酸序列，所述LCDR1包含SEQ ID NO:9所示的氨基酸序列，所述LCDR2包含SEQ ID NO:10、29中任一项所示的氨基酸序列和所述LCDR3包含SEQ ID NO:11、30中任一项所示的氨基酸序列。
  34. 根据权利要求1-33中任一项所述的抗原结合蛋白，其包含重链可变区VH和轻链可变区VL，所述VH包含SEQ ID NO:73、74中任一项所示的氨基酸序列，且所述VL包含 SEQ ID NO:75、76中任一项所示的氨基酸序列。
  35. 根据权利要求1-34中任一项所述的抗原结合蛋白，其包含重链可变区VH和轻链可变区VL，所述VH包含SEQ ID NO:8、21、28、37或41中任一项所示的氨基酸序列，且所述VL包含SEQ ID NO:16、35、25、40中任一项所示的氨基酸序列。
  36. 根据权利要求1-35中任一项所述的抗原结合蛋白，其包括抗体或其抗原结合片段。
  37. 根据权利要求36所述的抗原结合蛋白，其中所述抗原结合片段包括Fab，Fab’，F(ab)2、Fv片段、F(ab’)2，scFv，di-scFv和/或dAb。
  38. 根据权利要求1-37中任一项所述的抗原结合蛋白，其包括重链恒定区，且所述重链恒定区源自人IgG。
  39. 根据权利要求1-38中任一项所述的抗原结合蛋白，其包括重链恒定区，且所述重链恒定区源自人IgG1。
  40. 根据权利要求1-39中任一项所述的抗原结合蛋白，其包括重链恒定区，其中与SEQ ID NO:42所示的氨基酸序列相比，所述重链恒定区包含下述氨基酸突变：M135Y、S137T和/或T139E。
  41. 根据权利要求1-40中任一项所述的抗原结合蛋白，其重链恒定区包含SEQ ID NO:42、44中任一项所示的氨基酸序列。
  42. 根据权利要求1-41中任一项所述的抗原结合蛋白，其包含重链，且所述重链包含SEQ ID NO:45、47或49中任一项所示的氨基酸序列。
  43. 根据权利要求1-42中任一项所述的抗原结合蛋白，其包括轻链恒定区，且所述轻链恒定区包含SEQ ID NO:43所示的氨基酸序列。
  44. 根据权利要求1-43中任一项所述的抗原结合蛋白，其包含轻链，且所述轻链包含SEQ ID NO:46或48中任一项所示的氨基酸序列。
  45. 根据权利要求1-44中任一项所述的抗原结合蛋白，其包含重链和轻链，其中所述重链包含SEQ ID NO:45、47或49中任一项所示的氨基酸序列，且所述轻链包含SEQ ID NO:46或48中任一项所示的氨基酸序列。
  46. 多肽，其包含权利要求1-45中任一项所述的抗原结合蛋白。
  47. 免疫缀合物，其包含权利要求1-45中任一项所述的抗原结合蛋白。
  48. 分离的核酸分子，其编码根据权利要求1-45中任一项所述的抗原结合蛋白。
  49. 载体，其包含权利要求48所述的核酸分子。
  50. 细胞，其包含和/或表达权利要求1-45中任一项所述的抗原结合蛋白、权利要求46所述的多肽、权利要求47所述的免疫缀合物、权利要求48所述的核酸分子或权利要求49所 述的载体。
  51. 制备权利要求1-45中任一项所述的抗原结合蛋白的方法，所述方法包括在使得权利要求1-45中任一项所述的抗原结合蛋白表达的条件下，培养权利要求50所述的细胞。
  52. 药物组合物，其包含权利要求1-45中任一项所述抗原结合蛋白、权利要求46所述的多肽、权利要求47所述的免疫缀合物、权利要求48所述的核酸分子、权利要求49所述的载体、权利要求50所述的细胞和/或任选地药学上可接受的载剂。
  53. 权利要求1-45中任一项所述的抗原结合蛋白、权利要求46所述的多肽、权利要求47所述的免疫缀合物、权利要求48所述的核酸分子、权利要求49所述的载体、权利要求50所述的细胞和/或权利要求52所述的药物组合物在制备药物中的用途，所述药物用于预防、缓解和/或治疗IgE相关的疾病或病症。
  54. 预防、缓解或治疗IgE相关的疾病或病症的方法，所述方法包括向有需要的受试者施用权利要求1-45中任一项所述的抗原结合蛋白、权利要求46所述的多肽、权利要求47所述的免疫缀合物和/或权利要求52所述的药物组合物。
  55. 权利要求1-45中任一项所述的抗原结合蛋白、权利要求46所述的多肽、权利要求48所述的核酸分子、权利要求49所述的载体、权利要求50所述的细胞、权利要求47所述的免疫缀合物和/或权利要求52所述的药物组合物，其用于预防、缓解或治疗IgE相关的疾病或病症。
  56. 一种用于检测或测定IgE的方法，所述方法包括使用权利要求1-45中任一项所述的抗原结合蛋白或权利要求46所述的多肽。
  57. 一种用于检测或测定IgE的试剂盒，所述试剂盒包括权利要求1-45中任一项所述的抗原结合蛋白或权利要求46所述的多肽。
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