WO2019096219A1 - Anticorps anti-il13 humanisé, son procédé de préparation et son utilisation - Google Patents

Anticorps anti-il13 humanisé, son procédé de préparation et son utilisation Download PDF

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WO2019096219A1
WO2019096219A1 PCT/CN2018/115694 CN2018115694W WO2019096219A1 WO 2019096219 A1 WO2019096219 A1 WO 2019096219A1 CN 2018115694 W CN2018115694 W CN 2018115694W WO 2019096219 A1 WO2019096219 A1 WO 2019096219A1
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seq
variable region
sequence
chain variable
light chain
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PCT/CN2018/115694
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Chinese (zh)
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杨沂蓁
宫世勇
吴建
康立山
梁绍勤
段清
刘礼乐
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上海开拓者生物医药有限公司
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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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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
    • 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
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • 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
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the invention relates to the field of antibodies, in particular to a humanized anti-IL-13 antibody, a preparation method and application thereof.
  • asthma Bronchial asthma
  • glucocorticoids such as steroids
  • beta-adrenergic receptor agonists assisting long-acting beta-adrenergic receptor agonists.
  • about 10% of patients cannot be relieved by routine airway inhalation.
  • These patients often need oral steroids with high side effects to control the disease, and are still associated with higher mortality.
  • the quality of life of patients greatly affected, but the direct medical costs and indirect costs that result are enormous burdens on the individual and the socio-economic.
  • IL-13 interleukin 13
  • IL-4 interleukin 4
  • IL-4 and IL-13 are produced by Th2 cells, which share the same receptor chain and have many similarities in function.
  • Th2 cells which share the same receptor chain and have many similarities in function.
  • Elevated levels of plasma IgE which are common in asthmatic patients, have also been shown to be the result of IL-13-stimulated B cell proliferation and differentiation.
  • IgE which are common in asthmatic patients, have also been shown to be the result of IL-13-stimulated B cell proliferation and differentiation.
  • R130Q of IL-13 it was also found that there is a natural variant R130Q of IL-13.
  • IL-13 drugs that inhibit IL-13, such as specific monoclonal antibodies that bind to IL-13.
  • These antibodies bind to human IL-13 protein (wild-type or R130Q variant) and are capable of inhibiting IL-13 and its receptor IL-13Ra1/IL-4Ra heterodimer or receptor IL-13Ra2 at the protein and cellular levels. Binding; can significantly inhibit IL-13-induced thymic activation, regulate chemokine secretion and periostin secretion, and expression of vascular cell adhesion molecule-1; and can significantly inhibit IL-13-induced airway hyperresponsiveness in mice. . Therefore, IL-13 antibodies can be used in the preparation of drugs for the prevention, diagnosis and treatment of diseases such as bronchial asthma.
  • Monoclonal antibodies are developing into new diagnostic and therapeutic drugs due to their advantages of targeting, specificity, specificity and high affinity.
  • early clinical trials revealed that the use of non-human monoclonal antibodies in humans often results in severe immune responses due to human anti-mouse antibody (HAMA) and human anti-rat antibody (HARA) responses, and antibodies are rapidly cleared.
  • HAMA human anti-mouse antibody
  • HAA human anti-rat antibody
  • antibodies with less immunogenicity were developed, including chimeric antibodies, humanized antibodies, and fully human antibodies.
  • therapeutic monoclonal antibody drugs can be divided into four types: murine antibody (unmanned amino acid sequence), chimeric antibody (60% to 70% humanized amino acid sequence), CDR graft antibody (90% to 95% humanized amino acid sequence) and fully human antibody (100% human amino acid sequence).
  • non-rat monoclonal antibodies can alleviate human anti-mouse antibody responses (HAMA and HARA responses) during human therapy, gradually eliminating the immunogenicity of heterologous antibodies, and maintaining high affinity for antigens.
  • HAMA and HARA responses human anti-mouse antibody responses
  • the pharmacokinetics of the antibodies have been improved, and these antibody drugs have been used in a large amount in the clinic for targeted therapy.
  • monoclonal antibodies can be developed by various methods and techniques, including hybridoma technology, phage display technology, single lymphocyte gene cloning technology, etc.
  • the mainstream is to prepare monoclonal antibodies from wild-type or transgenic mice by hybridoma technology.
  • the monoclonal antibody prepared by a conventional animal such as a mouse can clone the antibody heavy chain variable region and the light chain variable region gene by conventional molecular biological methods, and the variable region gene can be grafted to the human antibody constant region gene to form a human mouse.
  • Chimeric antibodies see U.S. Patent No. 4,816,567) to greatly reduce immunogenicity in human use.
  • the CDR domain of the murine antibody variable region can be grafted onto the human antibody framework, thereby reducing the murine antibody component to less than 5%, greatly increasing the safety of the antibody for use in humans.
  • the antibody obtained by this route is called a humanized antibody, and is a major product of the current antibody drug market (see U.S. Pat. No. 5,225,539, etc.).
  • the technical problem to be solved by the present invention is to overcome the deficiency of the current lack of IL-13 antibody in the prior art, and to provide a humanized anti-IL-13 antibody with high affinity and high specificity, a preparation method and application thereof.
  • the humanized anti-IL-13 antibody has high affinity to human IL-13 protein (wild type or R130Q variant) and is capable of inhibiting IL-13 and its receptor IL-13Ra1/IL- at the protein and cellular levels. 4Ra heterodimer or receptor IL-13Ra2 binds. These antibodies exhibited good biological activity in chemokine release assays and extracellular matrix protein release assays, cell adhesion molecule expression assays.
  • HAMA reaction human anti-mouse antibody response
  • a humanized antibody comprising a heavy chain variable region and/or a light chain variable region is provided;
  • the heavy chain variable region of the humanized antibody comprises the following three complementarity determining region CDRs:
  • the light chain variable region of the humanized antibody comprises the following three complementarity determining region CDRs:
  • any one of the above amino acid sequences further comprises a derivative sequence which optionally adds, deletes, modifies and/or substitutes at least one amino acid and is capable of retaining IL-13 binding affinity.
  • the amino acid sequence of any of the above CDRs comprises a derivative CDR sequence that has been added, deleted, modified and/or substituted 1, 2 or 3 amino acids, and which results in VH and VL containing the derived CDR sequences.
  • the constructed derivative antibody is capable of retaining the affinity for binding to IL-13.
  • the ratio of the affinity F1 of the derivative antibody to IL-13 binding to the affinity F0 of the corresponding non-derivatized antibody to IL-13 is 0.5-2, preferably 0.7-1.5, and more preferably 0.8-1.2.
  • the number of amino acids added, deleted, modified and/or substituted is from 1 to 5 (e.g., from 1 to 3, preferably from 1 to 2, more preferably 1).
  • the ratio of the immunogenic Z1 of the humanized antibody in human to the immunogenicity Z0 of a non-humanized antibody (such as a murine antibody) in humans (Z1/Z0) It is 0-0.5, preferably 0-0.2, more preferably 0-0.05 (e.g., 0.001-0.05).
  • the amino acid sequence of the heavy chain variable region CDR1 is at least 80% identical to the amino acid sequence set forth in SEQ ID No. 18, SEQ ID No. 2, and SEQ ID No. 10 of the Sequence Listing. Sequence homology of 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%,
  • the amino acid sequence of the heavy chain variable region CDR2 is at least 80%, 85%, 90%, 91 as the amino acid sequence shown in SEQ ID No. 19, SEQ ID No. 3, and SEQ ID No. 11 in the Sequence Listing. %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology, and
  • the amino acid sequence of the heavy chain variable region CDR3 is at least 80%, 85%, 90%, 91 as the amino acid sequence shown in SEQ ID No. 20, SEQ ID No. 4, and SEQ ID No. 12 in the Sequence Listing. %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology.
  • the amino acid sequence of the light chain variable region CDR1 is at least 80% identical to the amino acid sequence represented by SEQ ID No. 22, SEQ ID No. 6, and SEQ ID No. 14 in the sequence listing. Sequence homology of 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%,
  • the amino acid sequence of the light chain variable region CDR2 is at least 80%, 85%, 90%, 91 as the amino acid sequence shown in SEQ ID No. 23, SEQ ID No. 7, and SEQ ID No. 15 in the Sequence Listing. %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology,
  • the amino acid sequence of the light chain variable region CDR3 is at least 80%, 85%, 90%, 91 as the amino acid sequence shown in SEQ ID No. 24, SEQ ID No. 8, and SEQ ID No. 16 in the Sequence Listing. %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology.
  • the heavy chain variable region of the humanized antibody comprises the following three complementarity determining region CDRs:
  • the light chain variable region of the humanized antibody comprises the following three complementarity determining region CDRs:
  • the heavy chain variable region sequence of the humanized antibody is selected from the group consisting of SEQ ID NO: 17, 1 or 9; and/or
  • the light chain variable region sequence of the humanized antibody is selected from the group consisting of SEQ ID NO: 21, 5 or 13.
  • the heavy chain variable region sequence of the humanized antibody is SEQ ID NO: 17, and the light chain variable region sequence is SEQ ID NO:21.
  • the heavy chain variable region sequence of the humanized antibody is SEQ ID NO: 1
  • the light chain variable region sequence is SEQ ID NO: 5.
  • the heavy chain variable region sequence of the humanized antibody is SEQ ID NO:9 and the light chain variable region sequence is SEQ ID NO:13.
  • the heavy chain variable region of the humanized antibody comprises the following three complementarity determining region CDRs:
  • the light chain variable region of the humanized antibody comprises the following three complementarity determining region CDRs:
  • the heavy chain variable region sequence of the humanized antibody is set forth in SEQ ID NO: 57, 51, 52, 53, 54, 55 or 56; and/or the humanized antibody
  • the light chain variable region sequence is set forth in SEQ ID NO: 58, 59 or 60.
  • the heavy and light chain variable region sequences of the humanized antibody are shown in Table A:
  • Antibody number Heavy chain variable region SEQ ID NO.
  • Light chain variable region SEQ ID NO.
  • Hu-29D9H8-1 26 33 Hu-29D9H8-2 26 34 Hu-29D9H8-3 26 35 Hu-29D9H8-4 27 32 Hu-29D9H8-5 27 33 Hu-29D9H8-6 27 34 Hu-29D9H8-7 27 35 Hu-29D9H8-8 28 32 Hu-29D9H8-9 28 33 Hu-29D9H8-10 28 34 Hu-29D9H8-11 28 35 Hu-29D9H8-12 29 32
  • Hu-29D9H8-13 29
  • Hu-29D9H8-14 29
  • Hu-29D9H8-15 29
  • Hu-29D9H8-16 30
  • Hu-29D9H8-17 30
  • Hu-29D9H8-18 30
  • Hu-29D9H8-19 30
  • Hu-29D9H8-20 31
  • Hu-29D9H8-21 31
  • Hu-29D9H8-22 31
  • Hu-28A2E11-2 37
  • Hu-28A2E11-3 37
  • Hu-28A2E11-4 37
  • Hu-28A2E11-5 47
  • Hu-28A2E11-6 37
  • Hu-28A2E11-7 37
  • Hu-28A2E11-8 37
  • Hu-28A2E11-9 38
  • Hu-28A2E11-10 45 Hu-28A2E11-11
  • 46 Hu-28A2E11-12 38
  • Hu-28A2E11-19 39
  • Hu-28A2E11-20 39
  • Hu-28A2E11-21 40
  • Hu-28A2E11-22 40
  • Hu-28A2E11-23 40
  • Hu-28A2E11-24 40
  • Hu-28A2E11-25 40
  • Hu-28A2E11-26 41
  • Hu-28A2E11-27 41
  • Hu-28A2E11-28 41
  • Hu-28A2E11-29 41
  • Hu-28A2E11-31 42
  • Hu-28A2E11-32 42
  • Hu-28A2E11-33 42
  • Hu-28A2E11-34 42
  • Hu-35E2C3-1 51
  • Hu-35E2C3-2 51
  • Hu-35E2C3-3 51
  • Hu-35E2C3-4 52
  • Hu-35E2C3-5 52
  • Hu-35E2C3-6 52
  • Hu-35E2C3-7 53
  • Hu-35E2C3-15 55
  • 35E2C3-16 56
  • Hu-35E2C3-17 56
  • Hu-35E2C3-18 56
  • the heavy chain variable region sequence of the humanized antibody is as set forth in SEQ ID NO: 57, 51, 52, 53, 54, 55, or 56; and the humanized antibody The light chain variable region sequence is set forth in SEQ ID NO: 58, 59, or 60.
  • the humanized antibody further comprises a heavy chain constant region and/or a light chain constant region.
  • the heavy chain constant region is of human origin, and/or the light chain constant region is of human origin.
  • the humanized antibody is a monoclonal antibody.
  • the humanized antibody is a diabody, a single chain antibody, a single domain antibody, or a single region antibody.
  • the humanized antibody is an antibody full length protein, or an antigen binding fragment.
  • the humanized antibody is a bispecific antibody, or a multispecific antibody.
  • the heavy chain variable region of the humanized antibody further comprises a human framework region, and/or the light chain variable region of the humanized antibody further comprises a human framework region.
  • the heavy chain FR sequence on the heavy chain variable region of the humanized antibody is derived from the human germline heavy chain sequence comprising 1) VH1-f or VH4-59 FR1, FR2, FR3 The region, and 2) the combination of the human antibody heavy chain framework regions of the FR4 region of JH1 or JH6.
  • the light chain FR sequence on the light chain variable region of the humanized antibody is derived from the FR1, FR2, FR3 region of the human germline light chain comprising 1) L1, 7B or O12, and 2) Combination of human antibody light chain framework regions of the FR4 region of JK4 or JL1.
  • the heavy chain framework region of the humanized antibody is selected from the group consisting of germline: DP3 (VH1-f), DP4, DP7, DP8, DP9, DP10, DP31, DP33, DP35 (VH3-11) , DP45, DP46, DP47, DP48, DP49 (VH3-30), DP50, DP51 (VH3-48), DP53, DP54, DP65, DP66, DP67, DP68, DP69 or DP71 (VH4-59); and/or
  • the light chain framework region of the humanized antibody is selected from the group consisting of: L1, O2, O12, DPK1 (O18), DPK2, DPK3, DPK4, DPK5, DPK6, DPK7, DPK8, DPK9, DPK10, DPK12 (A2), DPK13, DPK15, DPK16, DPKI8, DPK19, DPK20, DPK21, DPK22, DPK23, DPK24 (B3), DPK25, DPK26, DPK28, DPL1, DPL7, DPL6, DPL2, DPL3, DPL5, DPL12, DPL11, DPL13, DPL10, D9 , DPL16, DPL24, DPL18, DPL19 (7b), DPL21, DPL22 or DPL20.
  • the heavy chain framework region of the humanized antibody is selected from the group consisting of germline: DP3 (VH1-f), DP4, DP7, DP8, DP9, DP10, DP31, DP33, DP35 (VH3-11) FR1, FR2 of DP45, DP46, DP47, DP48, DP49 (VH3-30), DP50, DP51 (VH3-48), DP53, DP54, DP65, DP66, DP67, DP68, DP69 or DP71 (VH4-59) FR3; and JH fragments JH1, JH2, JH3, JH4, JH4b, JH5 and JH6, in particular the FR4-encoded sequences of these lines, or the consensus sequence of the heavy-chain framework regions.
  • the light chain framework region of the humanized antibody is selected from the group consisting of: L1, O2, O12, DPK1 (O18), DPK2, DPK3, DPK4, DPK5, DPK6, DPK7, DPK8, DPK9, DPK10, DPK12 (A2), DPK13, DPK15, DPK16, DPKI8, DPK19, DPK20, DPK21, DPK22, DPK23, DPK24 (B3), DPK25, DPK26, DPK28, DPL1, DPL7, DPL6, DPL2, DPL3, DPL5, DPL12, DPL11, DPL13, DPL10, D9, DPL16, DPL24, DPL18, DPL19(7b), DPL21, DPL22 or DPL20, preferably FR1, FR2, FR3 of these germplasms; and Jk segments Jk1, Jk2, Jk3, Jk4 and JK5 In particular, the FR4 encoded sequences of these lines.
  • the heavy and light chain variable region sequences of the humanized antibody are shown in Table B:
  • Antibody number Heavy chain variable region SEQ ID NO.
  • Light chain variable region SEQ ID NO. Hu-35E2C3-19 57 58 Hu-35E2C3-9 53 60 Hu-35E2C3-10 54 58 Hu-35E2C3-11 54 59 .
  • the heavy and light chain variable region sequences of the humanized antibody are shown in Table C:
  • Antibody number Heavy chain variable region SEQ ID NO.
  • Light chain variable region SEQ ID NO. Hu-29D9H8-10 28 34 Hu-29D9H8-19 30 35 Hu-28A2E11-6 37 48 Hu-28A2E11-7 37 49 Hu-28A2E11-20 39 48 .
  • the antibody is in the form of a drug conjugate.
  • a recombinant protein comprising:
  • Antibody number Heavy chain variable region SEQ ID NO.
  • Light chain variable region SEQ ID NO. c29D9H8 1 5 Hu-29D9H8-1 26 33 Hu-29D9H8-2 26 34 Hu-29D9H8-3 26 35 Hu-29D9H8-4 27 32 Hu-29D9H8-5 27 33 Hu-29D9H8-6 27 34 Hu-29D9H8-7 27 35 Hu-29D9H8-8 28 32 Hu-29D9H8-9 28 33 Hu-29D9H8-10 28 34 Hu-29D9H8-11 28 35 Hu-29D9H8-12 29 32 Hu-29D9H8-13 29 33 Hu-29D9H8-14 29 34 Hu-29D9H8-15 29 35
  • Hu-29D9H8-16 30 32 Hu-29D9H8-17 30 33 Hu-29D9H8-18 30 34 Hu-29D9H8-19 30 35 Hu-29D9H8-20 31 32 Hu-29D9H8-21 31 33 Hu-29D9H8-22 31 34 Hu-29D9H8-23 31 35 c28A2E11 9 13 Hu-28A2E11-1 37 43 Hu-28A2E11-2 37 44 Hu-28A2E11-3 37 45 Hu-28A2E11-4 37 46 Hu-28A2E11-5 37 47 Hu-28A2E11-6 37 48 Hu-28A2E11-7 37 49 Hu-28A2E11-8 38 43 Hu-28A2E11-9 38 44 Hu-28A2E11-10 38 45 Hu-28A2E11-11 38 46 Hu-28A2E11-12 38 47 Hu-28A2E11-13 38 48 Hu-28A2E11-14 38 49 Hu-28A2E11-15 39 43 Hu-28A2E11-16 39 44 Hu-28A
  • Hu-28A2E11-21 40 Hu-28A2E11-22 40 44 Hu-28A2E11-23 40 45 Hu-28A2E11-24 40 46 Hu-28A2E11-25 40 47 Hu-28A2E11-26 41 43 Hu-28A2E11-27 41 44 Hu-28A2E11-28 41 45 Hu-28A2E11-29 41 46 Hu-28A2E11-31 42 43 Hu-28A2E11-32 42 44 Hu-28A2E11-33 42 45 Hu-28A2E11-34 42 46 c35E2C3 17 twenty one Hu-35E2C3-1 51 58 Hu-35E2C3-2 51 59 Hu-35E2C3-3 51 60 Hu-35E2C3-4 52 58 Hu-35E2C3-5 52 59 Hu-35E2C3-6 52 60 Hu-35E2C3-7 53 58 Hu-35E2C3-8 53 59 Hu-35E2C3-9 53 60 Hu-35E2C3-10 54 58 Hu-
  • Hu-35E2C3-16 56 58 Hu-35E2C3-17 56 59 Hu-35E2C3-18 56 60 Hu-35E2C3-19 57 58 Hu-35E2C3-20 57 59 Hu-35E2C3-21 57 60 .
  • the recombinant protein comprises:
  • a polynucleotide is provided, the polynucleotide encoding a polypeptide selected from the group consisting of:
  • polynucleotide is as shown in Table 4.
  • polypeptide selected from the group consisting of:
  • polynucleotide encoding the heavy chain variable region is set forth in SEQ ID NO: 117, 88, 97, 77, 61 or 69; and/or
  • the polynucleotide encoding the light chain variable region is set forth in SEQ ID NO: 118, 94, 108, 81, 65 or 73.
  • polynucleotide encoding the heavy chain variable region is set forth in SEQ ID NO: 117, and the polynucleotide encoding the light chain variable region is set forth in SEQ ID NO: 118; or
  • polynucleotide encoding the heavy chain variable region is set forth in SEQ ID NO: 88, and the polynucleotide encoding the light chain variable region is set forth in SEQ ID NO: 94;
  • polynucleotide encoding the heavy chain variable region is set forth in SEQ ID NO: 97, and the polynucleotide encoding the light chain variable region is set forth in SEQ ID NO: 108;
  • polynucleotide encoding the heavy chain variable region is set forth in SEQ ID NO: 77, and the polynucleotide encoding the light chain variable region is set forth in SEQ ID NO: 81;
  • polynucleotide encoding the heavy chain variable region is set forth in SEQ ID NO: 61, and the polynucleotide encoding the light chain variable region is set forth in SEQ ID NO: 65;
  • the polynucleotide encoding the heavy chain variable region is set forth in SEQ ID NO: 69, and the polynucleotide encoding the light chain variable region is set forth in SEQ ID NO:73.
  • a vector comprising the polynucleotide of any one of the third aspect of the invention.
  • a genetically engineered host cell comprising the vector or genome of the fourth aspect of the invention, wherein any one of the third aspects of the invention is integrated Polynucleotides as described.
  • an antibody conjugate comprising:
  • a coupling moiety coupled to the antibody moiety being selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.
  • an immune cell which is expressed or exposed to the humanized antibody of any one of the first aspects of the invention.
  • a pharmaceutical composition comprising:
  • an active ingredient which is selected from the group consisting of the humanized antibody of any one of the first aspect of the invention, the recombinant protein of the second aspect of the invention, and the sixth aspect of the invention.
  • an active ingredient which is selected from the group consisting of the humanized antibody of any one of the first aspect of the invention, the recombinant protein of the second aspect of the invention, and the sixth aspect of the invention.
  • the pharmaceutical composition comprises 0.01 to 99.99% of the humanized antibody of any one of the first aspects of the invention, the recombinant protein of the second aspect of the invention, and the present invention
  • an active ingredient selected from the group consisting of the humanized antibody of any one of the first aspect of the invention, the second aspect of the invention
  • the diagnostic reagent comprises a medicament, a reagent, a test plate or a kit.
  • the IL-13 associated disease is selected from the group consisting of bronchial asthma.
  • a method of detecting IL-13 protein in a sample in vitro comprising the steps of:
  • composition for detecting IL-13 protein in a sample in vitro comprising the humanized antibody of any one of the first aspects of the invention, the second of the invention.
  • a test board comprising: a substrate (support plate) and a test strip, the test strip comprising any one of the first aspects of the invention.
  • a method for preparing a recombinant polypeptide comprising:
  • the recombinant polypeptide is isolated from the culture, the recombinant polypeptide of the humanized antibody of any one of the first aspect of the invention or the recombinant protein of the second aspect of the invention.
  • a method of treating bronchial asthma comprising: using the humanized antibody of any one of the first aspects of the invention, the recombination of the second aspect of the invention.
  • the antibody, the antibody conjugate according to the sixth aspect of the invention, and the immune cell of the seventh aspect of the invention comprising: using the humanized antibody of any one of the first aspects of the invention, the recombination of the second aspect of the invention.
  • Figure 1 shows the results of examining the biological activity of recombinant IL-13 protein by the TARC secretion assay.
  • Figure 2 shows the results of ELISA for detecting serum antibody titers in mice after immunization with immunogen A.
  • Figure 3 shows the results of the reactivity of IL-13 leader antibody with human IL-13 in an ELISA (enzyme-linked immunosorbent assay).
  • Figure 4 shows the results of the reactivity of the IL-13 leader antibody with the human IL-13 variant (IL-13R130Q variant) in an ELISA (enzyme-linked immunosorbent assay).
  • Figure 5 is a graph showing the results of reactivity of IL-13 leader antibody with monkey IL-13 variant in ELISA (enzyme-linked immunosorbent assay).
  • Figure 6 shows the results of the reactivity of IL-13 leader antibody with murine IL-13 variant in ELISA (enzyme-linked immunosorbent assay).
  • Figure 7 is a graph showing the results of flow cytometric analysis of the expression level of hIL-13Ra1 protein in a HEK293 cell line overexpressing full-length human IL-13Ra1.
  • the antibody refers to a goat anti-human IL-13Ra1 antibody (purchased from RnD systems); the negative control refers to a goat IgG control.
  • Figure 8 is a graph showing the results of flow cytometric analysis of the expression level of hIL-4Ra protein in a HEK293 cell line overexpressing full-length human IL-4Ra.
  • the antibody refers to a murine anti-human IL-4Ra antibody (purchased from RnD systems); the negative control refers to a murine IgG control.
  • Figure 9 is a graph showing the results of flow cytometric analysis of the expression level of hIL-13Ra2 protein in a HEK293 cell line overexpressing full-length human IL-13Ra2.
  • the antibody refers to a goat anti-human IL-13Ra2 antibody (purchased from RnD systems); the negative control refers to a goat IgG control.
  • Figure 10 is a graph showing the results of FACS detection of IL-13 leader antibody blocking IL-13 binding to the cell surface receptor IL-13Ra1/IL-4Ra heterodimer.
  • Figure 11 shows the results of FACS detection of IL-13 leader antibody blocking IL-13 binding to the cell surface receptor IL-13Ra2.
  • Figure 12 is a graph showing the results of IL-13 leader antibody neutralizing IL-13-induced thymic activation-regulated chemokine (TARC) secretion.
  • Figure 13 is a result of ELISA detecting the reactivity of an IL-13 chimeric antibody with immunogen A.
  • Figure 14 is a graph showing the results of ELISA assay for the reactivity of IL-13 chimeric antibodies with IL-13R130Q variants.
  • Figure 15 is the result of ELISA detecting the reactivity of IL-13 chimeric antibodies with monkey IL-13 variants.
  • Figure 16 shows the results of FACS detection of IL-13 chimeric antibodies blocking IL-13 and the cell surface receptor IL-13Ra1/IL-4Ra heterodimer.
  • Figure 17 is a graph showing the results of FACS detection of IL-13 chimeric antibody blocking IL-13 binding to the cell surface receptor IL-13Ra2.
  • Figure 18 is a graph showing the results of IL-13 chimeric antibodies neutralizing IL-13-induced thymic activation-regulated chemokine (TARC) secretion.
  • Figure 19 is a graph showing the results of IL-13 chimeric antibody neutralizing IL-13-induced secretion of periostin.
  • Figure 20 is a graph showing the results of IL-13 chimeric antibody neutralizing IL-13-induced expression of vascular cell adhesion molecule-1.
  • Figure 21 is a graph showing the results of an IL-13 chimeric antibody inhibiting human IL-13-induced respiratory inflammation in mice.
  • Figure 22 is a sequence comparison of the humanized anti-IL-13 antibody Hu-29D9H8 heavy chain variable region Hu-29D9H8.VH1 and its variants with the c29D9H8 chimeric antibody VH and the human germline VH exon hVH1-F/JH1.
  • Figure 23 is a sequence of humanized anti-IL-13 antibody Hu-29D9H8 light chain variable region Hu-29D9H8.V K 1 and its variant and c29D9H8 chimeric antibody V K and human germline V K exon L1/JK4 Comparison.
  • Figure 24 is a sequence comparison of the humanized anti-IL-13 antibody Hu-28A2E11 heavy chain variable region Hu-28A2E11.VH1 and its variants with the c28A2E11 chimeric antibody VH and the human germline VH exon hVH1-F/JH1.
  • FIG 25 is a humanized anti-IL-13 antibody Hu-28A2E11 light chain variable region Hu-28A2E11.V L 1 and variants thereof and chimeric antibodies c28A2E11 human germline V L and V L exon 7B / JL1 sequence Comparison.
  • Figure 26 is a sequence comparison of the humanized anti-IL-13 antibody Hu-35E2C3 heavy chain variable region Hu-35E2C3.VH1 and its variants with the c35E2C3 chimeric antibody VH and the human germline VH exon hVH4-59/JH6.
  • Figure 27 is a sequence of humanized anti-IL-13 antibody Hu-35E2C3 light chain variable region Hu-35E2C3.V K 1 and its variant and c35E2C3 chimeric antibody V K and human germline V K exon O12/JK4 Comparison.
  • Figure 28 is a graph showing the results of the reactivity of the humanized anti-IL-13 antibody with immunogen A in an ELISA (enzyme-linked immunosorbent assay).
  • Figure 29 shows the results of the reactivity of the humanized anti-IL-13 antibody with immunogen A in an ELISA (enzyme-linked immunosorbent assay).
  • Figure 30 is the result of the reactivity of the humanized anti-IL-13 antibody with immunogen A in an ELISA (enzyme-linked immunosorbent assay).
  • Figure 31-1 Flow cytometry assay detects the binding of humanized anti-IL-13 antibody to block the binding of IL-13 to the cell surface receptor IL-13Ra1/IL-4Ra heterodimer.
  • Figure 31-2 shows the results of flow cytometric assay for detection of binding of humanized anti-IL-13 antibody to IL-13 binding to the cell surface receptor IL-13Ra2.
  • Figure 32-1 shows the results of a flow cytometric assay for detecting the binding of humanized anti-IL-13 antibody to block IL-13 binding to the cell surface receptor IL-13Ra1/IL-4Ra heterodimer.
  • Figure 32-2 shows the results of flow cytometric assay for detection of binding of humanized anti-IL-13 antibody to IL-13 binding to the cell surface receptor IL-13Ra2.
  • Figure 33-1 shows the results of a flow cytometric assay for detecting the binding of humanized anti-IL-13 antibody to block IL-13 binding to the cell surface receptor IL-13Ra1/IL-4Ra heterodimer.
  • Figure 33-2 shows the results of flow cytometric assay for detection of binding of humanized anti-IL-13 antibody to IL-13 binding to the cell surface receptor IL-13Ra2.
  • Figure 34-1 shows the results of a flow cytometric assay for detecting the binding of humanized anti-IL-13 antibody to block IL-13 binding to the cell surface receptor IL-13Ra1/IL-4Ra heterodimer.
  • Figure 34-2 shows the results of flow cytometric assay for detection of binding of humanized anti-IL-13 antibody to IL-13 binding to the cell surface receptor IL-13Ra2.
  • Figure 35-1 shows the results of a flow cytometric assay for detecting the binding of humanized anti-IL-13 antibody to block IL-13 binding to the cell surface receptor IL-13Ra1/IL-4Ra heterodimer.
  • Figure 35-2 shows the results of flow cytometry assay for detection of binding of humanized anti-IL-13 antibody to IL-13 binding to the cell surface receptor IL-13Ra2.
  • Figure 36-1 Flow cytometric analysis method detects the binding of humanized anti-IL-13 antibody to block the binding of IL-13 to the cell surface receptor IL-13Ra1/IL-4Ra heterodimer.
  • Figure 36-2 shows the results of flow cytometry assay for detection of binding of humanized anti-IL-13 antibody to IL-13 binding to the cell surface receptor IL-13Ra2.
  • Figure 37-1 shows the results of flow cytometric assay for detection of binding of humanized anti-IL-13 antibody to IL-13 binding to the cell surface receptor IL-13Ra1/IL-4Ra heterodimer.
  • Figure 37-2 shows the results of flow cytometry assay for detection of binding of humanized anti-IL-13 antibody to IL-13 binding to the cell surface receptor IL-13Ra2.
  • Figure 38-1 shows the results of a flow cytometric assay for detecting the binding of humanized anti-IL-13 antibody to block IL-13 binding to the cell surface receptor IL-13Ra1/IL-4Ra heterodimer.
  • Figure 38-2 shows the results of flow cytometric assay for detection of binding of humanized anti-IL-13 antibody to IL-13 binding to the cell surface receptor IL-13Ra2.
  • Figure 39-1 shows the results of a flow cytometric assay for detecting the binding of humanized anti-IL-13 antibody to block IL-13 binding to the cell surface receptor IL-13Ra1/IL-4Ra heterodimer.
  • Figure 39-2 shows the results of flow cytometric assay for detection of binding of humanized anti-IL-13 antibody to IL-13 binding to the cell surface receptor IL-13Ra2.
  • Figure 40-1 shows the results of a flow cytometric assay for detecting the binding of humanized anti-IL-13 antibody to block IL-13 binding to the cell surface receptor IL-13Ra1/IL-4Ra heterodimer.
  • Figure 40-2 shows the results of flow cytometric assay for detection of binding of humanized anti-IL-13 antibody to IL-13 binding to the cell surface receptor IL-13Ra2.
  • Figure 41-1 shows the results of flow cytometry assay for detection of binding of humanized anti-IL-13 antibody to IL-13 binding to the cell surface receptor IL-13Ra1/IL-4Ra heterodimer.
  • Figure 41-2 shows the results of flow cytometric assay for detection of binding of humanized anti-IL-13 antibody to IL-13 binding to the cell surface receptor IL-13Ra2.
  • Figure 41-1 shows the results of neutralizing IL-13-induced thymic activation-regulated chemokine (TARC) secretion by a humanized anti-IL-13 antibody.
  • Figure 42-2 shows the results of thymic activation-regulated chemokine (TARC) secretion induced by humanized anti-IL-13 antibody neutralizing human IL-13R130Q variant.
  • TARC thymic activation-regulated chemokine
  • Figure 43-1 shows the results of neutralizing IL-13-induced thymic activation-regulated chemokine (TARC) secretion by a humanized anti-IL-13 antibody.
  • Figure 43-2 shows the results of thymic activation-regulated chemokine (TARC) secretion induced by humanized anti-IL-13 antibody neutralizing human IL-13R130Q variant.
  • TARC thymic activation-regulated chemokine
  • Figure 44-1 shows the results of neutralizing IL-13-induced thymic activation-regulated chemokine (TARC) secretion by a humanized anti-IL-13 antibody.
  • Figure 44-2 shows the results of thymic activation-regulated chemokine (TARC) secretion induced by humanized anti-IL-13 antibody neutralizing human IL-13R130Q variant.
  • TARC thymic activation-regulated chemokine
  • Figure 45-1 shows the results of neutralizing IL-13-induced thymic activation-regulated chemokine (TARC) secretion by a humanized anti-IL-13 antibody.
  • Figure 45-2 shows the results of thymic activation-regulated chemokine (TARC) secretion induced by humanized anti-IL-13 antibody neutralizing human IL-13R130Q variant.
  • TARC thymic activation-regulated chemokine
  • Figure 46-1 shows the results of neutralizing IL-13-induced thymic activation-regulated chemokine (TARC) secretion by a humanized anti-IL-13 antibody.
  • Figure 46-2 shows the results of thymic activation-regulated chemokine (TARC) secretion induced by humanized anti-IL-13 antibody neutralizing human IL-13R130Q variant.
  • TARC thymic activation-regulated chemokine
  • Figure 47-1 shows the results of neutralizing IL-13-induced thymic activation-regulated chemokine (TARC) secretion by a humanized anti-IL-13 antibody.
  • Figure 47-2 shows the results of thymic activation-regulated chemokine (TARC) secretion induced by humanized anti-IL-13 antibody neutralizing human IL-13R130Q variant.
  • TARC thymic activation-regulated chemokine
  • Figure 48-1 shows the results of neutralizing IL-13-induced thymic activation-regulated chemokine (TARC) secretion by a humanized anti-IL-13 antibody.
  • Figure 48-2 shows the results of thymic activation-regulated chemokine (TARC) secretion induced by humanized anti-IL-13 antibody neutralizing human IL-13R130Q variant.
  • TARC thymic activation-regulated chemokine
  • Figure 49-1 shows the results of IL-13 humanized antibody inhibition of human IL-13-induced lung goblet cell metaplasia and mucus hypersecretion in mice.
  • Figure 49-2 shows the results of IL-13 humanized antibody inhibition of human IL-13-induced lung goblet cell metaplasia and mucus hypersecretion in mice.
  • the present inventors conducted a series of adjustments and improvements through extensive and intensive research using recombinant human IL-13 protein as an immunogen using conventional hybridoma preparation techniques (see Kohler and Milstein, Nature, 1975, 256:495).
  • a lead antibody to the IL-13 antibody is obtained.
  • an IL-13 antibody having a high affinity to a protein such as human IL-13 protein is obtained by preliminary production, purification and identification of the leader antibody.
  • the amino acid sequence of the heavy chain variable region of the obtained IL-13 antibody and the light chain variable region of the IL-13 antibody was obtained by sequencing by molecular biological methods.
  • the present invention utilizes a mouse antibody which has been analyzed in detail, and the CDR region bound to the antigen is grafted with a human antibody framework, and is reshaped by affinity to form a CDR-grafted antibody, ie, a humanized antibody, to maintain its specificity and Most of the affinity, while almost completely removing immunogenicity and side effects.
  • the present invention has been extensively screened and unexpectedly finally obtained an anti-IL-13 humanized monoclonal antibody Hu-35E2C3-19, and the experimental results show that the humanized monoclonal antibody can bind with high specificity.
  • the IL-13 antigen which has a high affinity (KD ⁇ 1 ⁇ 10 -8 M), and the antibody has a remarkable activity of inhibiting respiratory inflammation in mice, and has no visible side effects to mammals themselves.
  • the present invention has been completed on this basis.
  • the present invention can well alleviate a series of asthma symptoms caused by IL-13, thereby playing a role in treating asthma. Especially for severe asthma, it can help to treat conventional treatments or even replace conventional treatments.
  • the present invention provides a humanized anti-IL-13 antibody comprising:
  • the humanized anti-IL-13 antibody comprises a framework region of a human antibody framework region (framework region) residue.
  • the human antibody framework region comprises a heavy chain framework region and a light chain framework region
  • the human antibody light chain framework region residues may comprise germline L1, O2, O12, DPK1 (O18), DPK2, DPK3, DPK4, DPK5, DPK6 , DPK7, DPK8, DPK9, DPK10, DPK12 (A2), DPK13, DPK15, DPK16, DPKI8, DPK19, DPK20, DPK21, DPK22, DPK23, DPK24 (B3), DPK25, DPK26, DPK28, DPL1, DPL7, DPL6, DPL2 , DPL3, DPL5, DPL12, DPL11, DPL13, DPL10, D9, DPL16, DPL24, DPL18, DPL19 (7b), DPL21, DPL22 or DPL20, in particular FR1, FR2, FR3 of these species; and Jk segment Jk1 Jk2, Jk3, Jk4 and JK5, in particular the FR4-
  • Human antibody heavy chain framework region residues may comprise germline DP3 (VH1-f), DP4, DP7, DP8, DP9, DP10, DP31, DP33, DP35 (VH3-11), DP45, DP46, DP47, DP48, DP49 ( VH3-30), DP50, DP51 (VH3-48), DP53, DP54, DP65, DP66, DP67, DP68, DP69 or DP71 (VH4-59), especially FR1, FR2, FR3 of these lines; and JH fragment JH1, JH2, JH3, JH4, JH4b, JH5 and JH6, in particular the FR4-encoded sequences of these lines, or the consensus sequence of the heavy-chain framework regions.
  • Such framework region sequences can be obtained from public DNA databases including germline antibody gene sequences or published references.
  • the germline DNA sequences of human heavy and light chain variable region genes can be obtained from the "VBase” human germline sequence database (www.mrcco8.com.ac.uk/vbase), and in Kabat, EA et al., 1991. Sequences of Proteins of Immunological Interest, found in the 5th edition.
  • the light chain framework region of the humanized antibody is selected from the group consisting of: a human antibody variable region framework selected, wherein the light chain FR sequence on the antibody light chain variable region is derived from a human germline light chain comprising 1) a combination of the FR1, FR2, FR3 regions of L1, 7B or O12, and 2) the human antibody light chain framework region of the FR4 region of JK4 or JL1; the heavy chain FR sequence on the heavy chain variable region of the antibody, A combination of human antibody heavy chain framework regions derived from the human germline heavy chain sequence comprising 1) FR1, FR2, FR3 regions of VH1-f or VH4-59, and 2) FR4 regions of JH1 or JH6.
  • the selection of the human acceptor framework region should be similar to the framework region of the donor antibody, or the consensus sequence most similar to the variable region subfamily.
  • sequence mutations can be made in the donor and/or acceptor sequences to optimize antigen binding, functionality, codon usage, expression levels, and the like, including introduction of non-human residues into the framework regions.
  • the CDR is a CDR of a mouse selected from one or more CDRs of the light chain variable region set forth in SEQ ID NO. 5, 13 or 21, or SEQ One or more CDRs of the heavy chain variable region of ID NO. 1, 9 or 17.
  • the amino acid sequence of the heavy chain CDR1 is as shown in SEQ ID No. 2, SEQ ID No. 10 or SEQ ID No. 18 of the Sequence Listing, and more preferably, the amino acid sequence encoding the heavy chain CDR1.
  • the nucleotide sequence is shown as SEQ ID No. 62, SEQ ID No. 70 or SEQ ID No. 78 in the sequence listing;
  • amino acid sequence of the heavy chain CDR2 is shown in SEQ ID No. 3, SEQ ID No. 11 or SEQ ID No. 19 of the Sequence Listing, and more preferably, the nucleotide sequence encoding the amino acid sequence of the heavy chain CDR2 is as SEQ SEQ ID No. 63, SEQ ID No. 71 or SEQ ID No. 79 in the sequence listing;
  • amino acid sequence of the heavy chain CDR3 is as shown in SEQ ID No. 4, SEQ ID No. 12 or SEQ ID No. 20 of the Sequence Listing, and more preferably, the nucleotide sequence encoding the amino acid sequence of the heavy chain CDR3. As shown in SEQ ID No. 64, SEQ ID No. 72 or SEQ ID No. 80 in the Sequence Listing;
  • the amino acid sequence of the light chain CDR1 is as shown in SEQ ID No. 6, SEQ ID No. 14 or SEQ ID No. 22 of the Sequence Listing, and more preferably, the nucleotide sequence encoding the amino acid sequence of the light chain CDR1. As shown in SEQ ID No. 66, SEQ ID No. 74 or SEQ ID No. 82 in the sequence listing;
  • the amino acid sequence of the light chain CDR2 is as shown in SEQ ID No. 7, SEQ ID No. 15 or SEQ ID No. 23 of the Sequence Listing, and more preferably, the nucleotide sequence encoding the amino acid sequence of the light chain CDR2. As shown in SEQ ID No. 67, SEQ ID No. 75 or SEQ ID No. 83 in the Sequence Listing;
  • amino acid sequence of the light chain CDR3 is set forth in SEQ ID No. 8, SEQ ID No. 16 or SEQ ID No. 24 of the Sequence Listing; more preferably, the nucleotide sequence encoding the amino acid sequence of the light chain CDR3 As shown in SEQ ID No. 68, SEQ ID No. 76 or SEQ ID No. 84 in the sequence listing;
  • the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID No. 2 of the Sequence Listing
  • the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID No. 3 of the Sequence Listing and the amino acid sequence of the heavy chain CDR3.
  • SEQ ID No. 4 the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID No. 10 of the Sequence Listing
  • the amino acid sequence of the heavy chain CDR2 is as shown in SEQ ID No. 11 of the Sequence Listing.
  • the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID No. 12 of the Sequence Listing
  • the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID No.
  • amino acid sequence of the heavy chain CDR2 is SEQ ID:
  • amino acid sequence of the heavy chain CDR3 is shown in SEQ ID No. 20 of the Sequence Listing; the amino acid sequence of the light chain CDR1 is shown in SEQ ID No. 6 of the Sequence Listing, and the light chain CDR2
  • the amino acid sequence is shown in SEQ ID No. 7 of the Sequence Listing and the amino acid sequence of the light chain CDR3 is shown in SEQ ID No. 8 of the Sequence Listing; the amino acid sequence of the light chain CDR1 is shown in SEQ ID No. 14 of the Sequence Listing.
  • the amino acid sequence of the light chain CDR2 is as shown in SEQ ID No.
  • the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID No. 2 of the Sequence Listing
  • the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID No. 3 of the Sequence Listing and the amino acid sequence of the heavy chain CDR3.
  • SEQ ID No. 4 As shown in the Sequence Listing SEQ ID No. 4; and, the amino acid sequence of the light chain CDR1 is shown in SEQ ID No. 6 of the Sequence Listing, and the amino acid sequence of the light chain CDR2 is shown in SEQ ID No. 7 of the Sequence Listing.
  • the amino acid sequence of the light chain CDR3 is shown in SEQ ID No. 8 of the sequence listing; the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID No.
  • amino acid sequence of the heavy chain CDR2 is as in the sequence listing.
  • SEQ ID No. 11 and the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID No. 12 of the Sequence Listing; and the amino acid sequence of the light chain CDR1 is as shown in SEQ ID No. 14 of the Sequence Listing.
  • the amino acid sequence of the light chain CDR2 is shown in SEQ ID No. 15 of the Sequence Listing and the amino acid sequence of the light chain CDR3 is shown in SEQ ID No. 16 of the Sequence Listing;
  • amino acid sequence of the heavy chain CDR1 is SEQ ID No.
  • the amino acid sequence of the heavy chain CDR2 is as shown in SEQ ID No.
  • the humanized anti-IL-13 antibody comprises at least one heavy chain variable region and/or at least one light chain variable region.
  • the amino acid sequence of the heavy chain variable region is SEQ ID No. 1, SEQ ID No. 9, SEQ ID No. 17, SEQ ID No. 25, SEQ ID No. 26, SEQ ID No. 27, SEQ ID No. 28, SEQ ID No. 29, SEQ ID No. 30, SEQ ID No. 31, SEQ ID No. 36, SEQ ID No. 37, SEQ ID No. 38, SEQ ID No. 39, SEQ ID No. 40, SEQ ID No. 41, SEQ ID No. 42, SEQ ID No. 50, SEQ ID No. 51, SEQ ID No. 52, SEQ ID No. 53, SEQ ID No. 54, SEQ ID No. 55, SEQ ID No. 56 or SEQ ID No.
  • amino acid sequence of the light chain variable region is SEQ ID No. 5, SEQ ID No. 13, SEQ ID No. 21, SEQ ID in the sequence listing. No. 32, SEQ ID No. 33, SEQ ID No. 34, SEQ ID No. 35, SEQ ID No. 43, SEQ ID No. 44, SEQ ID No. 45, SEQ ID No. 46, SEQ ID No. 47. SEQ ID No. 48, SEQ ID No. 49, SEQ ID No. 58, SEQ ID No. 59 or SEQ ID No. 60; more preferably, an amino acid sequence encoding the heavy chain variable region
  • the nucleotide sequences are respectively SEQ ID No. 61, SEQ ID No. 69, SEQ ID No. 77, SEQ ID No.
  • SEQ ID No. 86 SEQ ID No. 87, SEQ ID No. 88, SEQ ID No. 89, SEQ ID No. 90, SEQ ID No. 91, SEQ ID No. 96, SEQ ID No. 97, SEQ ID No. .98, SEQ ID No. 99, SEQ ID No. 100, SEQ ID No. 101, SEQ ID No. 102, SEQ ID No. 110, SEQ ID No. 111, SEQ ID No. 112, SEQ ID No. 113 SEQ ID No. 114, SEQ ID No. 115, SEQ ID No. 116 or SEQ ID No.
  • nucleotide sequence encoding the amino acid sequence of the light chain variable region is as shown in the sequence listing ID No. 65, SEQ ID No. 73, SEQ ID No. 81, SEQ ID No. 92, SEQ ID No. 93, SEQ ID No. 94, SEQ ID No. 95, SEQ ID No. 103, SEQ ID No. .104, SEQ ID No. 105, SEQ ID No. 106, SEQ ID No. 107, SEQ ID No. 108, SEQ ID No. 109, SEQ ID No. 118, SEQ ID No. 119 or SEQ ID No. 120 Shown.
  • amino acid sequence of the heavy chain variable region is, as in the sequence listing, SEQ ID No. 1, SEQ ID No. 9, SEQ ID No. 17, SEQ ID No. 25, SEQ ID No. 26, SEQ ID No. 27, SEQ ID No. 28, SEQ ID No. 29, SEQ ID No. 30, SEQ ID No. 31, SEQ ID No. 36, SEQ ID No. 37, SEQ ID No. 38, SEQ ID No. 39, SEQ ID No. 40, SEQ ID No. 41, SEQ ID No. 42, SEQ ID No. 50, SEQ ID No. 51, SEQ ID No. 52, SEQ ID No. 53, SEQ ID No. 54.
  • amino acid sequence of the light chain variable region is, and SEQ ID No. 5, SEQ ID No. 13, SEQ ID No. 21, SEQ ID No. 32, SEQ ID No. 33, SEQ ID No. 34, SEQ ID No. 35, SEQ ID No. 43, SEQ ID No. 44, SEQ ID No. 45, SEQ ID No. 46, SEQ ID No. 47, SEQ ID No. 48, SEQ ID No. 49, SEQ ID No. 58, SEQ ID No. 59 or SEQ ID An amino acid sequence represented by No. 60 having at least 80% sequence homology; preferably, The amino acid sequence of the heavy chain variable region is, as in the sequence listing, SEQ ID No. 61, SEQ ID No.
  • SEQ ID No. 69 SEQ ID No. 77, SEQ ID No. 85, SEQ ID No. 86, SEQ ID No. .87, SEQ ID No. 88, SEQ ID No. 89, SEQ ID No. 90, SEQ ID No. 91, SEQ ID No. 96, SEQ ID No. 97, SEQ ID No. 98, SEQ ID No. 99 SEQ ID No. 100, SEQ ID No. 101, SEQ ID No. 102, SEQ ID No. 110, SEQ ID No. 111, SEQ ID No. 112, SEQ ID No. 113, SEQ ID No. 114, SEQ The amino acid sequence encoded by the nucleotide sequence represented by ID No. 115, SEQ ID No.
  • SEQ ID No. 116 or SEQ ID No. 117 has an amino acid sequence of 80% sequence homology; the amino acid sequence of the light chain variable region is And SEQ ID No. 65, SEQ ID No. 73, SEQ ID No. 81, SEQ ID No. 92, SEQ ID No. 93, SEQ ID No. 94, SEQ ID No. 95, SEQ ID No in the Sequence Listing .103, SEQ ID No. 104, SEQ ID No. 105, SEQ ID No. 106, SEQ ID No. 107, SEQ ID No. 108, SEQ ID No. 109, SEQ ID No. 118, SEQ ID No. 119
  • the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID No. 120 has 8 0% sequence homologous amino acid sequence.
  • the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 26 of the Sequence Listing, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No. 33 of the Sequence Listing; more preferably The nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 86 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 93 of the Sequence Listing. sequence;
  • the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 26 of the Sequence Listing, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 34 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 86 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 94 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 26 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 35 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 86 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 95 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 27 of the Sequence Listing, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 32 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 87 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 92 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 27 of the Sequence Listing, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 33 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 87 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 93 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 27 of the Sequence Listing, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 34 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 87 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 94 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 27 of the Sequence Listing, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 35 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 87 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 95 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 28 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is the sequence shown in SEQ ID No. 32 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 88 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 92 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 28 of the Sequence Listing, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 33 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 88 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 93 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 28 of the Sequence Listing, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 34 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 88 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is set forth in SEQ ID No. 94 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 28 of the Sequence Listing, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 35 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 88 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 95 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 29 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is the sequence shown in SEQ ID No. 32 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 89 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 92 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 29 of the Sequence Listing, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 33 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 89 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 93 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 29 of the Sequence Listing, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No. 34 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 89 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 94 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 29 of the Sequence Listing, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 35 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 89 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 95 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 30 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is the sequence shown in SEQ ID No. 32 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 90 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 92 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 30 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is the sequence shown in SEQ ID No. 33 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 90 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 93 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 30 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is the sequence shown in SEQ ID No. 34 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 90 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 94 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 30 of the Sequence Listing, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 35 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 90 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 95 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 31 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 32 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 91 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 92 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 31 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 33 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 91 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 93 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 31 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 34 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 91 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 94 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 31 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 35 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 91 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 95 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 37 of the Sequence Listing, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 43 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 97 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 103 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 37 of the Sequence Listing, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 44 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 97 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 104 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 37 of the Sequence Listing, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 45 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 97 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 105 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 37 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is the sequence shown in SEQ ID No. 46 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 97 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 106 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 37 of the Sequence Listing, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 47 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 97 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 107 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 37 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 48 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 97 of the Sequence Listing, and the nucleotide sequence of the light chain variable region is as shown in SEQ ID No. 108 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 37 of the Sequence Listing, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 49 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 97 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 109 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 38 of the Sequence Listing, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 43 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 98 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 103 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 38 of the Sequence Listing, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 44 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 98 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 104 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 38 of the Sequence Listing, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 45 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 98 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 105 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 38 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 46 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 98 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is set forth in SEQ ID No. 106 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 38 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 47 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 98 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 107 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 38 of the Sequence Listing, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 48 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 98 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 108 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 38 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 49 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 98 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 109 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 39 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 43 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 99 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is set forth in SEQ ID No. 103 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 39 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is the sequence shown in SEQ ID No. 44 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 99 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 104 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 39 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 45 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 99 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 105 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 39 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is the sequence shown in SEQ ID No. 46 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 99 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 106 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 39 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is the sequence shown in SEQ ID No. 47 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 99 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 107 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 39 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is the sequence shown in SEQ ID No. 48 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 99 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 108 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 40 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 43 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 100 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 103 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 40 of the Sequence Listing, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 44 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 100 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 104 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 40 of the Sequence Listing, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 45 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 100 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is set forth in SEQ ID No. 105 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 40 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is the sequence shown in SEQ ID No. 46 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 100 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 106 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 40 of the Sequence Listing, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 47 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 100 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 107 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 41 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is the sequence shown in SEQ ID No. 43 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 101 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 103 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 41 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 44 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 101 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 104 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 41 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 45 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 101 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 105 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 41 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 46 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 101 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is set forth in SEQ ID No. 106 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 42 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is the sequence shown in SEQ ID No. 43 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 102 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is set forth in SEQ ID No. 103 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 42 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 44 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 102 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 104 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 42 of the Sequence Listing, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 45 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 102 of the Sequence Listing, and the nucleotide sequence of the light chain variable region is set forth in SEQ ID No. 105 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 42 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is the sequence shown in SEQ ID No. 46 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 102 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is set forth in SEQ ID No. 106 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 51 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is the sequence shown in SEQ ID No. 58 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 111 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 118 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 51 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 59 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 111 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 119 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 51 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is the sequence shown in SEQ ID No. 60 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 111 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 120 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 52 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 58 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 112 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 118 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 52 of the Sequence Listing, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 59 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 112 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 119 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 52 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 60 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 112 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 120 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 53 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 58 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 113 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 118 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 53 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 59 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 113 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 119 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 53 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 60 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 113 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 120 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 54 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is the sequence shown in SEQ ID No. 58 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 114 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 118 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 54 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is the sequence shown in SEQ ID No. 59 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 114 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 119 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 54 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is the sequence shown in SEQ ID No. 60 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 114 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 120 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 55 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is the sequence shown in SEQ ID No. 58 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 115 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 118 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 55 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is the sequence shown in SEQ ID No. 59 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 115 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 119 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 55 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is the sequence shown in SEQ ID No. 60 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 115 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 120 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 56 of the Sequence Listing, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 58 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 116 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 118 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 56 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is the sequence shown in SEQ ID No. 59 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 116 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 119 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 56 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is the sequence shown in SEQ ID No. 60 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 116 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 120 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 57 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is the sequence shown in SEQ ID No. 58 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 117 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is set forth in SEQ ID No. 118 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 57 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 59 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 117 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is as shown in SEQ ID No. 119 of the Sequence Listing. Sequence shown;
  • the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 57 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is the sequence shown in SEQ ID No. 60 of the Sequence Listing;
  • the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID No. 117 of the Sequence Listing, and the nucleotide sequence of the variable region of the light chain is set forth in SEQ ID No. 120 of the Sequence Listing. Sequence shown;
  • Antibody number Heavy chain variable region SEQ ID NO.
  • Light chain variable region SEQ ID NO. c29D9H8 1 5 Hu-29D9H8-1 26 33 Hu-29D9H8-2 26 34 Hu-29D9H8-3 26 35 Hu-29D9H8-4 27 32 Hu-29D9H8-5 27 33 Hu-29D9H8-6 27 34
  • Hu-29D9H8-7 27
  • Hu-29D9H8-8 28
  • Hu-29D9H8-9 28
  • Hu-29D9H8-10 28
  • Hu-29D9H8-11 28
  • Hu-29D9H8-12 29
  • Hu-29D9H8-13 29
  • Hu-29D9H8-14 29
  • Hu-29D9H8-15 29
  • Hu-29D9H8-16 30
  • Hu-29D9H8-17 30
  • Hu-29D9H8-18 30
  • Hu-29D9H8-19 30
  • Hu-29D9H8-20 31
  • Hu-29D9H8-21 31
  • Hu-29D9H8-22 31
  • Hu-29D9H8-23 31
  • c28A2E11 9 13
  • Hu-28A2E11-1 37
  • Hu-28A2E11-2 37
  • Hu-28A2E11-3 37
  • Hu-28A2E11-4 46
  • Hu-28A2E11-5 37
  • Hu-28A2E11-12 38
  • Hu-28A2E11-13 38
  • Hu-28A2E11-14 38
  • Hu-28A2E11-15 39
  • Hu-28A2E11-16 39
  • Hu-28A2E11-17 39
  • Hu-28A2E11-18 39
  • Hu-28A2E11-19 39
  • Hu-28A2E11-20 39
  • Hu-28A2E11-21 43
  • Hu-28A2E11-22 40
  • Hu-28A2E11-23 40
  • Hu-28A2E11-24 40
  • Hu-35E2C3-7 53 58 Hu-35E2C3-8 53 59 Hu-35E2C3-9 53 60 Hu-35E2C3-10 54 58 Hu-35E2C3-11 54 59 Hu-35E2C3-12 54 60 Hu-35E2C3-13 55 58 Hu-35E2C3-14 55 59 Hu-35E2C3-15 55 60 Hu-35E2C3-16 56 58 Hu-35E2C3-17 56 59 Hu-35E2C3-18 56 60 Hu-35E2C3-19 57 58 Hu-35E2C3-20 57 59 Hu-35E2C3-21 57 60
  • the number in Table 1 ⁇ 2 is the sequence number "SEQ ID No.”
  • the amino acid sequence of the heavy chain protein variable region of c29D9H8 is SEQ ID No. 1 of the sequence listing, and the light chain protein of c29D9H8 is variable.
  • the amino acid sequence of the region is SEQ ID No. 5 of the Sequence Listing.
  • the humanized anti-IL-13 antibody preferably an antibody full-length protein, an antigen-antibody binding domain protein fragment, a bispecific antibody, a multi-specific antibody, a single chain antibody (Single chain antibody fragment, scFv) One or more of a single domain antibody (sdAb) and a single-region antibody (Signle-domain antibody), and a monoclonal antibody or a polyclonal antibody produced by the above antibody.
  • the monoclonal antibodies can be developed by a variety of pathways and techniques, including hybridoma technology, phage display technology, single lymphocyte gene cloning technology, etc.
  • the mainstream is the preparation of monoclonal antibodies from wild-type or transgenic mice by hybridoma technology.
  • the invention also includes superhumanized antibodies, diabody, and the like.
  • the antibody full-length protein is a conventional full-length antibody protein of the art, which includes a heavy chain variable region, a light chain variable region, a heavy chain constant region, and a light chain constant region.
  • the humanized anti-IL-13 antibody further comprises a human antibody heavy chain constant region and/or a human antibody light chain constant region.
  • the heavy chain variable region and the light chain variable region and the human heavy chain constant region and the human light chain constant region constitute a humanized antibody full length protein.
  • the humanized antibody heavy chain constant region is routine in the art and may comprise a constant region derived from a human constant region, further comprising a heavy chain constant region of human IgGl, IgG2, IgG3, IgG4 or variants thereof;
  • the humanized antibody light chain constant region is routine in the art and may comprise a constant region derived from a human constant region, further comprising a light chain constant region of a human kappa, lambda chain or variant thereof.
  • the single-chain antibody is a conventional single-chain antibody in the art, which includes the heavy chain variable region, the light chain variable region, and a short peptide of 15-20 amino acids.
  • the antigen-antibody binding domain protein fragment is a conventional antigen-antibody binding domain protein fragment of the art comprising a light chain variable region, a light chain constant region, and an Fd segment of a heavy chain constant region.
  • the antigen-antibody binding domain protein fragments are Fab and F(ab')2.
  • the single domain antibodies are conventional single domain antibodies in the art, including heavy chain variable regions and heavy chain constant regions.
  • the single region antibodies are conventional single region antibodies of the art which include only heavy chain variable regions.
  • the preparation method of the humanized anti-IL-13 antibody is a preparation method conventional in the art.
  • the preparation method is preferably obtained by isolating the expression transformant recombinantly expressing the humanized anti-IL-13 antibody or by artificially synthesizing the protein sequence.
  • the method for isolating the expression transformant recombinantly expressing the humanized anti-IL-13 antibody preferably obtains a method of cloning the nucleic acid molecule encoding the humanized anti-IL-13 antibody into a recombinant vector, and the resulting recombinant The vector is transformed into a transformant to obtain a recombinant expression transformant, and the recombinant expression transformant obtained by culturing can be isolated and purified to obtain the humanized anti-IL-13 antibody.
  • the preparation of representative humanized anti-IL-13 antibodies of the invention is described in Example 10.
  • Humanized antibodies are also broadly a class of chimeric antibodies in which the variable region residues responsible for antigen binding, including complementarity determining regions derived from non-human species, shortened complementarity determining regions, or any other residue involved in antigen binding. Whereas the remaining variable region residues, eg, the residues and constant regions of the framework regions, are at least partially derived from human antibody sequences. A subset of the framework region residues and constant region residues of the humanized antibody can be derived from a non-human source.
  • the variable region of a humanized antibody is also described as a humanized light chain variable region and/or heavy chain variable region.
  • Non-human species are generally species used for immunization with antigens, such as mice, rats, rabbits, non-human primates, or other non-human mammal species.
  • Humanized antibodies are generally less immunogenic than traditional chimeric antibodies and exhibit improved stability upon administration to humans.
  • Complementarity determining regions are residues of antibody variable regions involved in antigen binding.
  • CDRs Complementarity determining regions
  • Several numbering systems for identifying CDRs are commonly used, including, for example, Kabat definitions, Chothia definitions, and AbM definitions.
  • the Kabat definition is based on sequence variability
  • the Chothia definition is based on the position of the structural loop region
  • the AbM definition is a compromise between the Kabat and Chothia methods.
  • the light chain variable region has three CDR regions, the CDR1 is located at amino acids 24-34 (CDR1-L), the CDR2 is at amino acids 50-56 (CDR2-L), and the CDR3 is located.
  • Amino acids 89-97 (CDR3-L). Due to the change in the length of the variable region, in the different central or different subgroups, the 27th position may have 1-6 amino acids, and the 95th position may have 1-6 amino acids, which are added to the original number. English letters are numbered, such as: 27A, 27B, 95A, 95B, etc. According to the Kabat definition, the CDRs of the heavy chain variable region are defined by residues at positions 31 and 35B (CDR1-H), positions 50 and 65 (CDR2-H), and positions 95 and 102 (CDR3-H). (according to Kabat numbering).
  • the CDRs of the heavy chain variable region are defined by residues at positions 26 and 32 (CDR1-H), positions 52 and 56 (CDR2-H), and positions 95 and 102 (CDR3-H) (according to the Chothia number).
  • the CDRs of the heavy chain variable region are defined by the residues at positions 26 and 35B (CDR1-H), at positions 50 and 58 (CDR2-H), and at positions 95 and 102 (CDR3-H) (according to Kabat number).
  • a plurality of amino acids may be present at positions 35, 52, 82, and 100, and are numbered A, B, C, and the like. See Martin et al.
  • each CDR region is defined by Kabat.
  • SDRs Specificity determining regions
  • a framework (framework) residue is part of a light chain variable region or a heavy chain variable region and is an antibody variable region other than a hypervariable residue (a hypervariable residue multi-finger complementarity determining region or CDR) or a CDR residue A residue that serves as a scaffold for the antigen binding loop (CDR) of the variable domain.
  • the framework residues can be derived from naturally occurring human antibodies, and artificial framework region sequences representing consensus sequences between individual sequences can also be used. When a framework region for humanization is selected, sequences that are widely present in humans may be superior to less common sequences.
  • Additional mutations in the human framework receptor sequence can be made to restore murine residues believed to be involved in antigen contact and/or residues involved in the structural integrity of the antigen binding site, or to improve antibody expression.
  • Peptide structure prediction can be used to analyze humanized heavy chain variable region and light chain variable region sequences to identify and avoid post-translational protein modification sites introduced by humanized design.
  • the humanized antibodies can be prepared using any of a variety of methods, including inlays of complementarity determining regions (CDRs), transplantation, transplantation of shortened CDRs, transplantation of specificity determining regions (SDRs), and Frankenstein assembly.
  • CDRs complementarity determining regions
  • SDRs specificity determining regions
  • Frankenstein assembly a variety of methods, including inlays of complementarity determining regions (CDRs), transplantation, transplantation of shortened CDRs, transplantation of specificity determining regions (SDRs), and Frankenstein assembly.
  • the humanized antibody also includes a superhumanized antibody, which is a method for preparing a humanized antibody, which does not rely on the human framework sequence as an analysis point, but relies on comparing the normative CDRs of the non-human antibody.
  • the structural type and the CDR structure type of the human antibody particularly the human antibody encoded by the human germline sequence, from which a candidate human antibody sequence from which a suitable human framework sequence can be obtained is identified.
  • a human residue can replace a non-human residue in a CDR, wherein one or more changes have been introduced into the CDRs.
  • the inlay is based on the concept of reducing the amino acid sequence of potential immunogenicity in rodents or other non-human antibodies by reconstituting the solvent-accessible surface of the antibody with a human amino acid sequence.
  • a human amino acid sequence See Padlan (1991) Mol. Immunol. 28: 489-980.
  • Identification of the outer framework region residues of solvent accessible residues exposed to the surface in non-human antibodies (the residues are different from the framework regions of human antibodies) Those residues at the same position) and replacing the identified residues with amino acids occupying the same position in the human antibody for inlaying, ie, the embedded antibody, the surface residues of which are predominantly human sequences, and
  • the internal residues are primarily the original murine sequences.
  • Transplantation of CDRs is performed by replacing one or more CDRs of a receptor antibody (eg, a human antibody or other antibody comprising the desired framework residues) with CDRs of a donor antibody (eg, a non-human antibody).
  • the acceptor antibody can be selected based on the similarity of the framework residues between the candidate acceptor antibody and the donor antibody. For example, according to the Frankenstein method, a human framework region having substantial sequence homology to each framework region of a related non-human antibody is identified, and CDRs of non-human antibodies are grafted onto the complex of these different human framework regions.
  • the above methods can be combined to produce an anti-IL-13 antibody of any desired sequence.
  • the invention also provides a nucleic acid encoding the above-described humanized anti-IL-13 antibody, comprising a nucleic acid encoding the heavy chain variable region, and/or a nucleic acid encoding the light chain variable region.
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is SEQ ID No. 1, SEQ ID No. 9, SEQ ID No. 17, SEQ ID No. 25, SEQ ID No. 26 in the sequence listing. SEQ ID No. 27, SEQ ID No. 28, SEQ ID No. 29, SEQ ID No. 30, SEQ ID No. 31, SEQ ID No. 36, SEQ ID No. 37, SEQ ID No. 38, SEQ ID No. 39, SEQ ID No. 40, SEQ ID No. 41, SEQ ID No. 42, SEQ ID No. 50, SEQ ID No. 51, SEQ ID No. 52, SEQ ID No. 53, SEQ ID No. .54, SEQ ID No. 55, SEQ ID No.
  • the amino acid sequence encoded by the nucleic acid of the light chain variable region is SEQ ID No. 5, SEQ ID No. in the sequence listing. 13.
  • the nucleotide sequences of the nucleic acids of the variable region of the heavy chain are respectively SEQ ID No.
  • SEQ ID No. 69 SEQ I in the sequence listing.
  • D No. 77 SEQ ID No. 85, SEQ ID No. 86, SEQ ID No. 87, SEQ ID No. 88, SEQ ID No. 89, SEQ ID No. 90, SEQ ID No. 91, SEQ ID No. .96, SEQ ID No. 97, SEQ ID No. 98, SEQ ID No. 99, SEQ ID No. 100, SEQ ID No. 101, SEQ ID No. 102, SEQ ID No. 110, SEQ ID No. 111 , SEQ ID No. 112, SEQ ID No. 113, SEQ ID No. 114, SEQ ID No. 115, SEQ ID No.
  • nucleic acid encoding the light chain variable region
  • the nucleotide sequences are respectively SEQ ID No. 65, SEQ ID No. 73, SEQ ID No. 81, SEQ ID No. 92, SEQ ID No. 93, SEQ ID No. 94, SEQ ID No. in the Sequence Listing. 95, SEQ ID No. 103, SEQ ID No. 104, SEQ ID No. 105, SEQ ID No. 106, SEQ ID No. 107, SEQ ID No. 108, SEQ ID No. 109, SEQ ID No. 118, SEQ ID No. 119 or SEQ ID No. 120.
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 26 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the variable region of the light chain is SEQ ID No.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 86 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region As shown in the sequence listing SEQ ID No. 93;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 26 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the variable region of the light chain is shown in SEQ ID No. 34 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 86 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 94;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 26 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the variable region of the light chain is as shown in SEQ ID No. 35 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 86 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 95;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 27 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 32 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 87 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 92;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 27 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 33 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 87 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 93;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 27 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the variable region of the light chain is shown in SEQ ID No. 34 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 87 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 94;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 27 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 35 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 87 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 95;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 28 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the variable region of the light chain is shown in SEQ ID No. 32 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 88 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 92;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 28 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the variable region of the light chain is shown in SEQ ID No. 33 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 88 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 93;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 28 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the variable region of the light chain is shown in SEQ ID No. 34 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 88 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 94;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 28 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 35 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 88 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 95;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 29 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the variable region of the light chain is shown in SEQ ID No. 32 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 89 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, the sequence listing. SEQ ID No. 92;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 29 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 33 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 89 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, the sequence listing. SEQ ID No. 93;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 29 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the variable region of the light chain is shown in SEQ ID No. 34 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 89 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, the sequence listing. SEQ ID No. 94;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 29 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 35 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 89 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, the sequence listing. SEQ ID No. 95;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 30 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the variable region of the light chain is as shown in SEQ ID No. 32 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 90 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, the sequence listing. SEQ ID No. 92;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 30 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 33 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 90 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, the sequence listing. SEQ ID No. 93;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 30 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the variable region of the light chain is shown in SEQ ID No. 34 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 90 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, the sequence listing. SEQ ID No. 94;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 30 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the variable region of the light chain is as shown in SEQ ID No. 35 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 90 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, the sequence listing. SEQ ID No. 95;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 31 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the variable region of the light chain is as shown in SEQ ID No. 32 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 91 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, the sequence listing. SEQ ID No. 92;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 31 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 33 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 91 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, the sequence listing. SEQ ID No. 93;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 31 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 34 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 91 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, the sequence listing. SEQ ID No. 94;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 31 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 35 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 91 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, the sequence listing. SEQ ID No. 95;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 37 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the variable region of the light chain is as shown in SEQ ID No. 43 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 97 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, the sequence listing. SEQ ID No. 103;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 37 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 44 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 97 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, the sequence listing. SEQ ID No. 104;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 37 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 45 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 97 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, the sequence listing. SEQ ID No. 105;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 37 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the variable region of the light chain is shown in SEQ ID No. 46 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 97 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, the sequence listing. SEQ ID No. 106;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 37 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 47 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 97 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, the sequence listing. SEQ ID No. 107;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 37 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 48 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 97 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, the sequence listing. SEQ ID No. 108;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 37 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 49 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 97 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, the sequence listing. SEQ ID No. 109;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 38 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 43 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 98 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, the sequence listing. SEQ ID No. 103;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 38 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 44 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 98 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, the sequence listing. SEQ ID No. 104;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 38 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 45 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 98 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, the sequence listing. SEQ ID No. 105;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 38 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 46 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 98 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, the sequence listing. SEQ ID No. 106;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 38 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 47 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 98 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, the sequence listing. SEQ ID No. 107;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 38 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is shown in SEQ ID No. 48 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 98 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, the sequence listing. SEQ ID No. 108;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 38 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 49 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 98 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 109;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 39 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the variable region of the light chain is shown in SEQ ID No. 43 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 99 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 103;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 39 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the variable region of the light chain is as shown in SEQ ID No. 44 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 99 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 104;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 39 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 45 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 99 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 105;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 39 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the variable region of the light chain is shown in SEQ ID No. 46 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 99 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 106;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 39 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the variable region of the light chain is as shown in SEQ ID No. 47 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 99 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 107;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 39 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the variable region of the light chain is shown in SEQ ID No. 48 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 99 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 108;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 40 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 43 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 100 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as in the sequence listing. SEQ ID No. 103;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 40 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the variable region of the light chain is as shown in SEQ ID No. 44 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 100 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as in the sequence listing. SEQ ID No. 104;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 40 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 45 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 100 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as in the sequence listing. SEQ ID No. 105;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 40 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 46 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 100 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as in the sequence listing. SEQ ID No. 106;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 40 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 47 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 100 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as in the sequence listing. SEQ ID No. 107;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 41 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 43 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 101 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 103;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 41 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 44 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 101 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 104;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 41 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 45 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 101 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 105;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 41 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the variable region of the light chain is shown in SEQ ID No. 46 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 101 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 106;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 42 of the Sequence Listing
  • the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 43 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 102 of the sequence listing
  • the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 103;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 42 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 44 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 102 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 104;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 42 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 45 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 102 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 105;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 42 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the variable region of the light chain is as shown in SEQ ID No. 46 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 102 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 106;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 51 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the variable region of the light chain is as shown in SEQ ID No. 58 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 111 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 118;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 51 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the variable region of the light chain is shown in SEQ ID No. 59 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 111 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 119;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 51 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 60 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 111 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 120;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 52 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the variable region of the light chain is shown in SEQ ID No. 58 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 112 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 118;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 52 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the variable region of the light chain is as shown in SEQ ID No. 59 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 112 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 119;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 52 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the variable region of the light chain is as shown in SEQ ID No. 60 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 112 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 120;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 53 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 58 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 113 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 118;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 53 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 59 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 113 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 119;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 53 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 60 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 113 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 120;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 54 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is shown in SEQ ID No. 58 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 114 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, the sequence listing. SEQ ID No. 118;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 54 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the variable region of the light chain is as shown in SEQ ID No. 59 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 114 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, the sequence listing. SEQ ID No. 119;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 54 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 60 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 114 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, the sequence listing. SEQ ID No. 120;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 55 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the variable region of the light chain is as shown in SEQ ID No. 58 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 115 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, the sequence listing. SEQ ID No. 118;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 55 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the variable region of the light chain is as shown in SEQ ID No. 59 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 115 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, the sequence listing. SEQ ID No. 119;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 55 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the variable region of the light chain is as shown in SEQ ID No. 60 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 115 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, the sequence listing. SEQ ID No. 120;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is set forth in SEQ ID No. 56 of the Sequence Listing
  • the amino acid sequence encoded by the nucleic acid of the light chain variable region is set forth in SEQ ID No. 58 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 116 of the sequence listing
  • the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 118;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 56 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the variable region of the light chain is as shown in SEQ ID No. 59 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 116 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 119;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 56 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 60 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 116 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, a sequence listing. SEQ ID No. 120;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 57 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the variable region of the light chain is as shown in SEQ ID No. 58 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 117 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, the sequence listing. SEQ ID No. 118;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 57 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the variable region of the light chain is as shown in SEQ ID No. 59 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 117 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, the sequence listing. SEQ ID No. 119;
  • the amino acid sequence encoded by the nucleic acid of the heavy chain variable region is as shown in SEQ ID No. 57 of the Sequence Listing, and the amino acid sequence encoded by the nucleic acid of the light chain variable region is as shown in SEQ ID No. 60 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 117 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, the sequence listing. SEQ ID No. 120;
  • nucleotide sequences of the above antibodies are numbered as shown in Table 3-4:
  • Antibody number Heavy chain variable region SEQ ID NO.
  • Light chain variable region SEQ ID NO. c29D9H8 61 65 Hu-29D9H8-1 86 93 Hu-29D9H8-2 86 94 Hu-29D9H8-3 86 95 Hu-29D9H8-4 87 92 Hu-29D9H8-5 87 93 Hu-29D9H8-6 87 94 Hu-29D9H8-7 87 95
  • Hu-29D9H8-8 88 92 Hu-29D9H8-9 88 93 Hu-29D9H8-10 88 94 Hu-29D9H8-11 88 95 Hu-29D9H8-12 89 92 Hu-29D9H8-13 89 93 Hu-29D9H8-14 89 94 Hu-29D9H8-15 89 95 Hu-29D9H8-16 90 92 Hu-29D9H8-17 90 93 Hu-29D9H8-18 90 94 Hu-29D9H8-19 90 95 Hu-29D9H8-20 91 92 Hu-29D9H8-21 91 93 Hu-29D9H8-22 91 94 Hu-29D9H8-23 91 95 c28A2E11 69 73 Hu-28A2E11-1 97 103 Hu-28A2E11-2 97 104 Hu-28A2E11-3 97 105 Hu-28A2E11-4 97 106 Hu-28A2E11-5 97
  • Hu-28A2E11-13 98 108 Hu-28A2E11-14 98 109 Hu-28A2E11-15 99 103 Hu-28A2E11-16 99 104 Hu-28A2E11-17 99 105 Hu-28A2E11-18 99 106 Hu-28A2E11-19 99 107 Hu-28A2E11-20 99 108 Hu-28A2E11-21 100 103 Hu-28A2E11-22 100 104 Hu-28A2E11-23 100 105 Hu-28A2E11-24 100 106 Hu-28A2E11-25 100 107 Hu-28A2E11-26 101 103 Hu-28A2E11-27 101 104 Hu-28A2E11-28 101 105 Hu-28A2E11-29 101 106 Hu-28A2E11-31 102 103 Hu-28A2E11-32 102 104 Hu-28A2E11-33 102 105 Hu-28A2E11-34 102 106 c35E2C3
  • Hu-35E2C3-8 113 119 Hu-35E2C3-9 113 120 Hu-35E2C3-10 114 118 Hu-35E2C3-11 114 119 Hu-35E2C3-12 114 120 Hu-35E2C3-13 115 118 Hu-35E2C3-14 115 119 Hu-35E2C3-15 115 120 Hu-35E2C3-16 116 118 Hu-35E2C3-17 116 119 Hu-35E2C3-18 116 120 Hu-35E2C3-19 117 118 Hu-35E2C3-20 117 119 Hu-35E2C3-21 117 120
  • the numbers in Table 3-4 are the sequence number "SEQ ID No.”
  • the nucleotide sequence of the heavy chain protein variable region encoding c29D9H8 is SEQ ID No. 61 of the sequence listing
  • the light encoding c29D9H8 is light.
  • the nucleotide sequence of the variable region of the streptavidin is SEQ ID No. 65 of the Sequence Listing.
  • the preparation method of the nucleic acid is a preparation method conventional in the art, and preferably includes the steps of obtaining a nucleic acid molecule encoding the above humanized anti-IL-13 antibody by gene cloning technology, or by artificial full sequence synthesis method. A nucleic acid molecule encoding the above humanized anti-IL-13 antibody is obtained.
  • nucleic acid encoding a humanized anti-IL-13 antibody as described above may be suitably introduced with a substitution, deletion, alteration, insertion or addition to provide a homolog of a polynucleotide.
  • a homologue of a polynucleotide of the invention can be made by replacing, deleting or increasing one or more nucleotides of a nucleic acid encoding the humanized anti-IL-13 antibody while maintaining antibody activity.
  • the invention also provides a recombinant expression vector comprising the nucleic acid.
  • the recombinant expression vector can be obtained by a conventional method in the art, that is, the nucleic acid molecule of the present invention is ligated to various expression vectors.
  • the expression vector is a variety of vectors conventional in the art as long as it can accommodate the aforementioned nucleic acid molecule.
  • the vector preferably includes: various plasmids, cosmids, phage or viral vectors, and the like.
  • the present invention also provides a recombinant expression transformant comprising the above recombinant expression vector.
  • the preparation method of the recombinant expression transformant is a preparation method conventional in the art, and preferably, the recombinant expression vector is transformed into a host cell.
  • the host cell is a variety of host cells conventional in the art, as long as it satisfies the stable self-replication of the above recombinant expression vector, and the nucleic acid carried can be efficiently expressed.
  • the host cell is an E. coli TG1 or BL21 cell (expressing a single-chain antibody or a Fab antibody), or a CHO-K1 cell (expressing a full-length IgG antibody).
  • the recombinant expression plasmid of the present invention can be obtained by transforming the aforementioned recombinant expression plasmid into a host cell.
  • the conversion method is a conventional transformation method in the art, preferably a chemical conversion method, a heat shock method or an electrotransformation method.
  • the present invention provides a method for producing a humanized anti-IL-13 antibody, which comprises the steps of culturing the above recombinant expression transformant, or a cell, or a cell line, and obtaining a humanized anti-IL-13 antibody from the culture. .
  • the present invention also provides a method for detecting cells overexpressing IL-13 protein, comprising the steps of: contacting the above-mentioned protein with a sample to be tested in vitro, and detecting the binding of the above protein to the sample to be tested.
  • overexpression is conventional in the art, and refers to overexpression of RNA or protein of IL-13 protein in a sample to be tested (due to increased transcription, post-transcriptional processing, translation, post-translational processing, and protein degradation), and Local overexpression and increased functional activity due to changes in protein transport patterns (increased nuclear localization) (as in the case of increased enzymatic hydrolysis of the substrate).
  • the detection mode of the combination is a conventional detection method in the art, preferably FACS detection.
  • the present invention provides a composition for detecting cells overexpressing IL-13 protein, which comprises the above-described protein as an active ingredient.
  • a composition for detecting cells overexpressing IL-13 protein which comprises the above-described protein as an active ingredient.
  • it further comprises a compound consisting of functional fragments of the above proteins as an active ingredient.
  • the present invention provides the use of the above proteins for the preparation of a medicament.
  • the medicament is a medicament for preventing or treating bronchial asthma.
  • the present invention also provides a pharmaceutical composition comprising the above-mentioned protein as an active ingredient.
  • the pharmaceutical composition is a pharmaceutical composition for preventing or treating bronchial asthma.
  • the administration route of the pharmaceutical composition of the present invention is preferably administered by injection or orally.
  • the administration by injection preferably includes intravenous, intramuscular, intraperitoneal, intradermal or subcutaneous injection.
  • the pharmaceutical composition is in various forms conventional in the art, preferably in the form of a solid, semi-solid or liquid, and may be an aqueous solution, a non-aqueous solution or a suspension, more preferably a tablet, a capsule or a granule. , injection or infusion, etc.
  • the pharmaceutical compositions of the present invention further comprise one or more pharmaceutically acceptable carriers.
  • the pharmaceutical carrier is a conventional pharmaceutical carrier in the art, and the pharmaceutically acceptable carrier can be any suitable physiologically or pharmaceutically acceptable pharmaceutical adjuvant.
  • the pharmaceutical excipients are conventional pharmaceutical excipients in the art, and preferably include pharmaceutically acceptable excipients, fillers or diluents and the like. More preferably, the pharmaceutical composition comprises from 0.01 to 99.99% of the above protein and from 0.01 to 99.99% of a pharmaceutically acceptable carrier, the percentage being a percentage by mass of the pharmaceutical composition.
  • the pharmaceutical composition is administered in an amount effective to reduce or delay the progression of a disease, degenerative or damaging condition.
  • the effective amount can be determined on an individual basis and will be based in part on the consideration of the condition to be treated and the results sought.
  • One skilled in the art can determine the effective amount by using the above factors such as the individual basis and using no more than conventional experiments.
  • the present invention provides the use of the above protein for the preparation of a medicament for preventing or treating a disease associated with IL-13 expression or dysfunction.
  • the IL-13 expression or dysfunction-associated disease is a conventional IL-13 expression or dysfunction-related disease in the art.
  • it is bronchial asthma.
  • the present invention provides the use of the above pharmaceutical composition for the preparation of a medicament for preventing or treating a disease associated with IL-13 expression or dysfunction.
  • the room temperature described in the examples is room temperature conventional in the art, generally 20-25 °C.
  • the PBS buffer was PBS phosphate buffer, pH 7.4.
  • the protein of the present invention that is, the IL-13 antibody provided has high affinity, and the affinity with human IL-13 reaches KD ⁇ 1 ⁇ 10 -8 M;
  • TARC thymic activation-regulated chemokine
  • VCAM-1 vascular cell adhesion molecule-1
  • mice In the animal model of IL-13-induced airway inflammation in mice, it can significantly inhibit airway respiratory inflammation in mice and inhibit IL-13-induced airway hyperresponsiveness in mice;
  • the protein of the present invention which is expected to be provided, can be applied to a medicament for preventing or treating bronchial asthma.
  • Example 1 Expression and purification of human IL-13 and human IL-13R130Q variants
  • nucleotide sequence encoding Met1-Asn132 in the amino acid sequence encoding human IL-13 protein was added to the nucleotide encoding six histidine to obtain the his tag.
  • the nucleotide sequence of the recombinant human IL-13 protein (as shown in SEQ ID No. 121 of the Sequence Listing).
  • nucleotide sequence encoding Met1-Asn132 in the amino acid sequence of human IL-13 protein variant IL-13R130Q is added to a nucleotide encoding six histidines to obtain a recombinant human IL-encoded with his tag.
  • the nucleotide sequence of the 13R130Q variant (as shown in SEQ ID No. 122 of the Sequence Listing).
  • nucleotide sequence encoding the his-tag recombinant human IL-13 protein and the nucleotide sequence of the recombinant human IL-13R130Q variant encoding the his tag were cloned into the PCP vector (purchased from Invitrogen) and established according to the established standard molecular organisms. Methods For the preparation of plasmids, see Sambrook, J., Fritsch, EF, and Maniatis T. (1989). Molecular Cloning: A Laboratory Manual, Second Edition (Plainview, New York: Cold Spring Harbor Laboratory Press).
  • FreeStyle 293F cells purchased from Invitrogen for transient transfection (PEI, Polysciences)
  • FreeStyle TM 293 Invitrogen
  • the cell culture medium was collected, and the cell components were removed by centrifugation to obtain a culture supernatant of human-containing IL-13 containing his-tag or a culture supernatant of a human-containing IL-13R130Q variant containing his-tag.
  • the above culture supernatants were separately loaded onto a Ni-NTA affinity chromatography column (purchased from GE), and the his-tagged human IL-13 (i.e., immunogen A) and his-tagged human IL- in the supernatant were purified.
  • the 13R130Q variant was further purified using a molecular sieve column (purchased from GE) to remove impurities such as macromolecular aggregates.
  • Purified immunogen A and his-tagged human IL-13R130Q variant were stored in PBS phosphate buffer (pH 7.4), filtered through a 0.22 ⁇ m sterile filter, and stored at -80 °C.
  • the purified immunogen A was subjected to a TARC secretion test. Specific methods for TARC secretion assays are described in Miller et al. 2008, J Immunol Methods 334(1-2): 134-41.
  • A549 cells (purchased from ATCC) were cultured in 10% (w/w) fetal bovine serum F-12k medium (purchased from Gibco), and expanded to 75-90% in T-175 cell culture flasks. At the confluency, the medium was discarded, washed 1-2 times with PBS buffer, and then digested with trypsin-EDTA (purchased from Life Technology) and the cells were collected. The collected cells were resuspended in the medium, and after counting, the cells were diluted to 2 ⁇ 10 6 cells/mL, and 100 ⁇ L of each well was added to a 96-well cell culture plate (2 ⁇ 10 5 cells per well).
  • the plates were incubated overnight at 37 ° C in a 5% (v/v) CO 2 incubator.
  • the gradient diluted immunogen A was mixed with recombinant human TNF ⁇ (purchased from Peprotech) to obtain a mixture I (wherein TNF ⁇ accounted for 200 ng/mL of the final concentration of the mixture I).
  • the culture supernatant of the overnight cultured cell culture plate was discarded, and the above mixture I was added, and the cell culture plate was cultured overnight at 37 ° C in a 5% (v/v) CO 2 incubator.
  • the culture supernatant in the plate was aspirated, the cells were removed by centrifugation, and the concentration of TARC in the culture supernatant was determined using a TARC ELISA kit (purchased from RnD systems).
  • the experimental procedure was carried out in accordance with the instructions of the kit.
  • the murine anti-human TARC antibody was diluted to 2 ⁇ g/mL with PBS, and then added to a 96-well microtiter plate according to 100 ⁇ L per well. Incubate overnight at 4 ° C; wash the plate 2 times with the washing solution [PBS buffer containing 0.05% (w/w) Tween 20], and add the sample dilution [PBS buffer containing 1% (w/w) BSA] Liquid] 300 ⁇ L per well at room temperature for 1 hour; discard the blocking solution, dilute the standard dilution to 500 pg/mL with the sample dilution, dilute the dilution by six points, use the sample dilution as a blank control, and simultaneously cultivate the supernatant Dilute 8 times with sample dilution.
  • TMB substrate purchased from Huzhou Yingchuang
  • stop solution 1.0 N HCl
  • the OD 450 nm value was read by an ELISA plate reader (SpectraMax M5e, available from Molecular Device), and the light absorption value was calculated using the OD 540 nm value as a background, and the TARC concentration in the culture supernatant was calculated.
  • Table 5 shows that immunogen A can stimulate the secretion of thymocyte activation chemokine (TARC) by A549 cells, and the biological activity of immunogen A is basically consistent with the biological activity of commercial proteins.
  • IL-13 (Sino Bio) refers to the commercial IL-13 protein, which is purchased from Sino biological as a positive control; IL-13 (Batch 1) is the above-mentioned immunogen A.
  • Biotin-XX-NHS purchased from SIGMA-ALDRICH
  • the purified immunogen A prepared in Example 1 were mixed in a ratio of 3:1, and the reaction was shaken at room temperature for 30 minutes, and then a final concentration of 50 mM of NH 4 was added. The reaction was stopped by Cl to obtain biotin-labeled immunogen A.
  • the biotin-labeled immunogen A was then dialyzed into PBS buffer (pH 7.4), and the concentration was determined using Broadford reagent (purchased from Pierce) using BSA as a standard (for details, see Bradford, 1976, Anal Biochem 72: 248-54).
  • the experimental results are shown in Table 6.
  • the OD 595 nm value determined by biotin-labeled immunogen A was 0.49 using a standard fit curve.
  • the concentration of biotin-labeled immunogen A was calculated to be 0.319 mg/mL.
  • the biotin-labeled immunogen A was filtered through a 0.22 ⁇ m sterile filter, aseptically dispensed, and stored at -80 °C.
  • Example 2 Obtaining a lead antibody using hybridoma technology
  • Immunization with immunogen A Balb/c, SJL/J mice (supplied by Shanghai Slack), 6-8 weeks old, were used. Mice were housed under Specific pathogen Free (SPF) conditions after receipt. The initial immunization dose was 50 ⁇ g of immunogen A per mouse. The protein was emulsified with Freund's complete adjuvant and 0.25 mL was injected subcutaneously into the tail. After 2 weeks of initial immunization, boost the immunization. Immunogen A (25 ⁇ g per mouse) was emulsified with Freund's incomplete adjuvant and intraperitoneally injected with 0.25 mL. Each time the booster interval is 3 weeks.
  • SPPF Specific pathogen Free
  • mice Serum samples were collected one week after each boost, and antibody titers in serum were measured by ELISA and antibody activity in serum was measured by receptor ligand binding block assay. Mice with higher serum titers and better blocking of binding of immunogen A to the receptor will be preferred for cell fusion and hybridoma cell preparation, and the remaining mice will continue to boost the immunization reserve.
  • Table 7 shows that the sera of mice immunized with immunogen A have different degrees of binding to immunogen A, exhibiting an antigen-antibody reaction. Among them, the highest dilution of serum (ie, dilution factor) is about 100,000.
  • the blank control in Table 7 refers to 1% (w/w) BSA, and the batch TB2 refers to the serum of the mice on the seventh day after the second boost, and the data in the table is the OD 450 nm value.
  • the PCP expression vector expressing the recombinant human IL-13 protein constructed in Example 1 was coated onto a 1.0 ⁇ m gold colloidal bullet and immunized with a Helios gene gun (Bio-rad). Gold colloidal bullet preparation and immunization procedures were developed according to the Helios gene gun instructions.
  • Female SJL/J mice (supplied by Shanghai Slack), 6-8 weeks old, were housed under SPF conditions after receiving. All mice were immunized 3-4 times with a gene gun through the abdomen, 3-4 shots each time, 1.0 ⁇ g of plasmid per shot. The interval between the initial immunization and the first booster immunization was 2 weeks, and each booster interval was 3 weeks later.
  • mice immunized with immunogen A can achieve a titer of 1:1 or more after 2-3 immunizations, and can be used to collect lymphocytes for cell fusion and hybridoma preparation.
  • mice Prior to cell fusion, the mice were immunized with 50-100 ⁇ g of immunogen A per patient for the last immunization. Mice were sacrificed 3-5 days later and spleen cells were collected. Add NH 4 OH to a final concentration of 1% (w/w) to lyse red blood cells in spleen cell suspension, wash the cells 2-3 times with DMEM basal medium, and then mix the mouse bone marrow at a ratio of 5:1.
  • Tumor cells SP2/0 mixed purchased from ATCC
  • cell fusion using traditional PEG cell fusion method or high-efficiency electrofusion method see METHODS IN ENZYMOLOGY, VOL.220
  • the obtained cells are hybridoma cells.
  • the fused cells were diluted into DMEM selective medium containing 20% (w/w) fetal bovine serum and 1 ⁇ HAT, and added to a 96-well cell culture plate at 1 ⁇ 10 5 /20 ⁇ L per well, and placed. 5% (v/v) CO 2 in a 37 ° C incubator. After 10-14 days, the cell fusion plate was screened by ELISA, and the positive clone with OD 450nm >1.0 in the ELISA was amplified into 24-well plate to expand the culture. After 2-3 days, the 24-well plate was retested, including detection by ELISA.
  • the binding activity of the antibodies in Qingqing to immunogen A the detection of antibody blocking IL-13 and receptor binding activity by supernatant using flow cytometry, and the use of A549 thymic activation-regulating chemokine (TARC) secretion test
  • TARC A549 thymic activation-regulating chemokine
  • hybridoma cell culture supernatants in ELISA experiments with OD 450nm >1.0 receptor ligand binding block assay for human IL-13 and hIL-13Ra/hIL-4R heterodimer and/or human IL-13
  • the blockade of hIL-13Ra2 receptor binding reached 60%, and/or the A549 thymic activation-regulating chemokine (TARC) secretion test in which the hybridoma supernatant and human IL-13 induced TARC secretion, the inhibition rate reached 60%.
  • Hybridoma cells are eligible positive clones.
  • the optimal subclones were selected for expansion culture, liquid nitrogen cryopreservation, antibody production and purification as lead antibodies.
  • the clone numbers of these lead antibodies were 29D9H8, 28A2E11 and 35E2C3, respectively.
  • Hybridoma cells produce a lower concentration of antibodies, which are only about 1-10 ⁇ g/mL, and the concentration varies greatly. Moreover, the various proteins produced by the cell culture in the medium and the fetal bovine serum components contained in the medium have different degrees of interference with many biological activity analysis methods, so that small-scale (1-5 mg) antibody production purification is required.
  • the hybridoma cells prepared in Example 2 were expanded into T-75 cell culture flasks and domesticated for 2-3 passages using a production medium (Hybridoma serum free medium, purchased from Invitrogen).
  • the hybridoma cells are in good growth state, and the cell culture spinner is inoculated. 200-500 mL of production medium was added to each 2 liter culture spinner flask, and the seed density was 0.5-1.0 ⁇ 10 5 /mL. Cap the cap tightly and place the spinner on a roller shaker in a 37 ° C incubator and adjust to a speed of 3 rpm.
  • the cell culture medium was collected, centrifuged or filtered to remove the cells, and filtered through a 0.22-0.45 ⁇ m filter.
  • the treated cell culture supernatant can be immediately purified or frozen at -30 °C.
  • the monoclonal antibody in the hybridoma cell culture supernatant can be purified by Protein G affinity chromatography (Protein G, Protein G column) column. According to the size of the sample, prepare a corresponding volume of the column. For small volume purification of 200-300 mL, 1-2 mL of Protein G column is required.
  • the Protein G column was first equilibrated with equilibration buffer (PBS buffer, pH 7.4) and the culture supernatant was applied to a Protein G column with a flow rate of 3-4 mL/min. After loading, wash the column with 3-5 column volumes with equilibration buffer.
  • equilibration buffer PBS buffer, pH 7.4
  • the antibody bound to the column was eluted with an eluent (0.1 M glycine hydrochloride buffer, pH 2.5), and the elution was monitored with a UV detector. Collect the eluted antibody (according to the A280 UV absorption peak), add 10% (v/v) 1.0 M Tris-HCl buffer and neutralize the pH, then immediately dialyze overnight with PBS buffer, and change the solution once a day, and Continue dialysis for 2-3 hours. The dialyzed antibody was collected and sterile-filtered using a 0.22 ⁇ m filter to obtain an IL-13 monoclonal antibody, which was aseptically stored. Packing samples for protein concentration, purity, and endotoxin detection and analysis. The endotoxin concentration of the IL-13 monoclonal antibody was found to be less than 3.0 EU/mg. See Table 8 for the results of their partial detection analysis.
  • the lead antibodies obtained in Example 3 were each subjected to the following assay.
  • Enzyme-linked immunosorbent assay was used to detect the binding of lead antibody to immunogen A, IL-13R130Q variant, monkey IL-13 and murine IL-13.
  • Streptavidin was diluted with PBS to a final concentration of 1.0 ⁇ g/mL, and then added to a 96-well microtiter plate according to 100 ⁇ l per well. Incubate overnight at 4 ° C; wash the plate 2 times with a washing solution [PBS containing 0.05% (w/w) Tween 20], add blocking solution [containing 0.05% (w/w) Tween 20 and 2% (w /w) BSA in PBS buffer] blocked at 37 ° C for 1-2 hours.
  • the blocking solution was discarded, and the biotin-labeled immunogen A, IL-13R130Q variant (prepared in Example 1), monkey IL-13 (purchased from sino biological) and murine IL-13 (purchased from sino) prepared in Example 1 were discarded.
  • Biological Dilute to 0.5 ⁇ g/mL with sample diluent [PBS buffer containing 0.05% (w/w) Tween20 and 0.2% (w/w) BSA, respectively, add 50-100 ⁇ L per well to the plate, 37 Incubate for 1 hour at °C. The plate was washed 2-3 times with a washing solution [PBS buffer containing 0.01% (w/w) Tween20].
  • the gradient-diluted leader antibody prepared in Example 3 was added to 50-100 ⁇ L per well, and after incubation at 37 ° C for 1 hour, the plate was washed 2-3 times with a washing solution.
  • Horseradish peroxidase-labeled (HRP) human or mouse IgG secondary antibody (purchased from Sigma) was added, and after incubation at 37 ° C for 1 hour, the plate was washed with PBS buffer containing 0.05% (w/w) Tween20. Liquid] Wash the plate 2-3 times. 100 ⁇ L/well of TMB substrate was added and incubated for 15 minutes at room temperature, and 50 ⁇ L of 1.0 N HCl was added to each well to terminate.
  • the OD 450 nm value was read using an ELISA plate reader (SpectraMax M5e, available from Molecular Device). Some experimental results are shown in Figures 3-6 and Tables 9-11. Tables 9-11 illustrate that the leader antibody binds to recombinant human IL-13 protein, recombinant human IL-13R130Q variant, and recombinant monkey IL-13 at the ELISA level. However, it did not bind to mouse IL-13.
  • the IgG control was murine IgG and the data in the table is the OD 450 nm value.
  • the nucleotide sequences of human IL-13Ra1, human IL-13Ra2 and human IL-4Ra full-length genes were cloned into pIRES expression vector and packaged into lentiviruses.
  • the pIRES expression vector and lentivirus were purchased from Shanghai Jima Pharmaceutical Technology Co., Ltd. and operated according to the instructions), and the HEK293 cells were each subjected to lentivirus infection.
  • the human IL-13Ra1 and human IL-4Ra genes were co-transfected into the same HEK293 cell line to form IL-13Ra1/IL-4Ra heterodimers on the surface, and the cells were infected with 100 ⁇ g/mL Hygromycin B ( 37 mg, 5% (v/v) CO in DMEM medium purchased from MILLIPORE) and 0.25 ⁇ g/mL Puromycin (purchased from Invitrogen) in one or two and 10% (w/w) fetal bovine serum. 2 cultures were selected for 2 weeks. After 2 weeks, the infected cells were subcloned into 96-well culture plates by limiting dilution.
  • the monoclonal well cells were expanded into 6-well plates or culture flasks to obtain HEK293 cell lines expressing full-length human IL-13Ra1 and human IL-4Ra.
  • the amplified clones were assayed for receptor expression levels by flow cytometry using specific antibodies against each receptor (hIL-13Ra1 antibody, hIL-4Ra antibody, hIL-13Ra2 antibody purchased from RnD systems). And the ability to bind to the ligand IL-13 protein. Cell lines with better growth, higher expression levels, and stronger binding were selected to continue to expand culture and cryopreservation of liquid nitrogen.
  • HEK293 cells were infected with lentiviral particles containing human IL-13Ra2 gene, and the cell lines with good growth, high expression level and strong binding were selected and expanded in the same manner and stored in liquid nitrogen to obtain overexpression.
  • HEK293 cell line clone 4C1 overexpressing full-length human IL-13Ra1 and human IL-4Ra obtained in step 1 of Example 4 in Experiment B, and HEK293 cell line clone 1A1 overexpressing full-length human IL-13Ra2 in T-175 Incubate the culture in a cell culture flask to 75-90% confluence and discard the medium.
  • the culture medium to be expanded is DMEM containing 100 ⁇ g/mL Hygromycin B (purchased from MILLIPORE) and 0.25 ⁇ g/mL Puromycin (purchased from Invitrogen), one or two, and 10% (w/w) fetal bovine serum. Medium.
  • the conditions for the expanded culture were cultured at 37 ° C and 5% (v/v) CO 2 . Wash 1-2 times with PBS buffer, then digest and collect cells with recombinant enzyme cell dissociation solution (TrypLE, purchased from Life technology); wash cells with PBS buffer 1-2 times, perform cell counting and block cells Dilute [PBS containing 2% (w/w) fetal bovine serum] to 1-2 ⁇ 10 6 cells/mL, incubate on ice for 20-30 minutes, then use blocking solution [containing 2% (w/) w) fetal bovine serum in PBS buffer] washed twice. The collected cells were suspended in a blocking solution to 1 ⁇ 10 6 cells/mL, and added to a 96-well FACS reaction plate (i.e., 1 ⁇ 10 5 cells per well) at 100 ⁇ L per well.
  • the gradient-diluted leader antibody prepared in Example 3 (hereinafter collectively referred to as "lead antibody”) was mixed with the biotin-labeled immunogen A prepared in Example 1, and then added to the cells in 100 ⁇ L per well, and incubated on ice for 1-2 hours. .
  • the HEK293 cell line overexpressing the full-length human IL-13Ra1 and human IL-4Ra obtained in the step B of Example 4, Test B was added with a gradient dilution of the lead antibody and a biotin-labeled immunization with a final concentration of 30 ng/mL.
  • Tables 15-16 show that IL-13 antibody binds to human IL-13 and blocks human IL-13 and cell surface receptor IL-13Ra1/ Binding of hIL-4Ra heterodimer, or blocking the binding of IL-13 to the cell surface human IL-13Ra2 receptor.
  • the IgG control was murine IgG and the data in the table is the mean fluorescence intensity.
  • TARC thymic activation regulating chemokine
  • A549 cells (purchased from ATCC) were cultured in 10% (w/w) fetal bovine serum F-12k medium (purchased from Gibco) at 37 ° C, 5% (v/v) CO 2 , and cultured at When the culture was expanded to 75-90% confluence in T-175 cell culture flask, the medium was discarded, washed 1-2 times with PBS buffer, and then digested with trypsin solution (Trypsin-EDTA, purchased from Life technology). Collect cells. The collected cells were resuspended in the medium, and after counting, the cells were diluted to 2 ⁇ 10 6 cells/mL, and 100 ⁇ L of each well was added to a 96-well cell culture plate (2 ⁇ 10 5 cells per well).
  • the plates were incubated overnight at 37 ° C in a 5% (v/v) CO 2 incubator.
  • the gradient-diluted lead antibody prepared in Example 3 was mixed with the immunogen A prepared in Example 1 to obtain a mixture A; recombinant human TNF ⁇ (purchased from Peprotech) was used to contain 10% (w/w) fetal bovine serum.
  • the F-12k medium was mixed in an equal volume to obtain a mixture B in the medium.
  • the final concentration of the mixed recombinant human TNF ⁇ in the mixture B was 200 ng/mL.
  • the final concentration of the immunogen A in the total volume of the mixture A and the mixture B was 5 ng/mL
  • the final concentration of the recombinant human TNF ⁇ in the total volume of the mixture A and the mixture B was 200 ng/mL.
  • the cell culture plates were incubated overnight at 37 ° C in a 5% (v/v) CO 2 incubator. After 20 hours, the culture supernatant in the plate was aspirated, the cells were removed by centrifugation, and the concentration of TARC in the culture supernatant was determined using a TARC ELISA kit (purchased from RnD systems). The experimental procedure was carried out in accordance with the instructions of the kit (see Example 1 for details). Some experimental results are shown in Figure 12 and Table 17.
  • Table 17 shows that the leader antibody can neutralize the secretion of TARC by A549 cells induced by IL-13 and TNF ⁇ co-stimulation by binding to human IL-13.
  • the data in Table 17 is the concentration (pg/ml) of TARC in the culture supernatant, wherein the IgG control is murine IgG.
  • RNA isolation The hybridoma cells corresponding to the lead antibody prepared in Example 3 were resuscitated, cultured, and 1-5 ⁇ 10 7 cells were collected by centrifugation, mixed with 1 mL of Trizol and transferred to a 1.5 mL centrifuge tube, and allowed to stand at room temperature. Set for 5 minutes. 0.2 mL of chloroform was added, shaken for 15 seconds, allowed to stand for 2 minutes, centrifuged at 12000 g for 5 minutes at 4 ° C, and the supernatant was transferred to a new 1.5 mL centrifuge tube. 0.5 mL of isopropanol was added, and the liquid in the tube was gently mixed.
  • Reverse transcription and PCR 1 ⁇ g of total RNA was taken, 20 ⁇ L of the system was placed, reverse transcriptase was added, and the reaction was carried out at 42 ° C for 60 minutes, and the reaction was terminated by reaction at 7 ° C for 10 minutes.
  • a 50 ⁇ L PCR system was configured, including 1 ⁇ L of cDNA, 25 pmol of each primer, 1 ⁇ L of DNA polymerase, and a matching buffer system, 250 ⁇ mol dNTPs.
  • the PCR program was set up, pre-denatured at 95 ° C for 3 minutes, denatured at 95 ° C for 30 seconds, annealed at 55 ° C for 30 seconds, extended at 72 ° C for 35 seconds, and 35 cycles and then extended at 72 ° C for 5 minutes to obtain a PCR product (for reverse transcription).
  • the kit was PrimeScript RT Master Mix, available from Takara, item number RR036; the kit used for PCR was Q5 super-fidelity enzyme, available from NEB, item number M0492).
  • PCR product 5 ⁇ L was detected by agarose gel electrophoresis, and the positive sample was purified by column recovery kit (the recovery kit was Gel & PCR Clean-up, available from MACHEREY-NAGEL, Cat. No. 740609).
  • the ligation reaction was carried out: 50 ng of sample, 50 ng of T vector, 0.5 ⁇ L of ligase, 1 ⁇ L of buffer, 10 ⁇ L of reaction system, and reacted at 16 ° C for half an hour to obtain a ligation product (the linked kit was T4 DNA ligase, purchased from NEB, article number M0402).
  • amino acid sequence of the 29D9H8 heavy chain variable region is set forth in SEQ ID NO. 1 of the Sequence Listing, and the light chain variable region sequence is set forth in SEQ ID NO. 5 of the Sequence Listing.
  • the amino acid sequence of CDR1 of the heavy chain variable region is shown in positions 31 to 35 of SEQ ID No. 1, and the amino acid sequence of CDR2 is from position 50 to SEQ ID No. 1 in the sequence listing. As indicated at position 66, the amino acid sequence of CDR3 is shown in positions 99 to 109 of SEQ ID No. 1 of the Sequence Listing;
  • the amino acid sequence of CDR1 of the murine antibody light chain variable region is shown in positions 24 to 34 of SEQ ID No. 5 of the Sequence Listing, and the amino acid sequence of CDR2 is as shown in SEQ ID No. 5 of the Sequence Listing. As shown in positions 50 to 56, the amino acid sequence of CDR3 is shown in positions 89 to 97 of SEQ ID No. 5 of the Sequence Listing.
  • amino acid sequence of the 28A2E11 heavy chain variable region is set forth in SEQ ID NO. 9 of the Sequence Listing, and the light chain variable region sequence is set forth in SEQ ID NO. 13 of the Sequence Listing.
  • the amino acid sequence of CDR1 of the heavy chain variable region is shown in positions 31 to 35 of SEQ ID No. 9 of the Sequence Listing, and the amino acid sequence of CDR2 is the 50th to the SEQ ID No. 9 of the Sequence Listing. As indicated at position 66, the amino acid sequence of CDR3 is shown in positions 99 to 107 of SEQ ID No. 9 of the Sequence Listing;
  • the amino acid sequence of CDR1 of the murine antibody light chain variable region is shown in positions 23 to 36 of SEQ ID No. 13 of the Sequence Listing, and the amino acid sequence of CDR2 is as shown in SEQ ID No. 13 of the Sequence Listing. As shown in positions 52 to 58, the amino acid sequence of CDR3 is shown in positions 91 to 99 of SEQ ID No. 13 of the Sequence Listing.
  • amino acid sequence of the 35E2C3 heavy chain variable region is set forth in SEQ ID NO. 17 of the Sequence Listing, and the light chain variable region sequence is set forth in SEQ ID NO. 21 of the Sequence Listing.
  • the amino acid sequence of CDR1 of the heavy chain variable region is shown in positions 31 to 35 of SEQ ID No. 17 of the Sequence Listing, and the amino acid sequence of CDR2 is from position 50 to SEQ ID No. 17 of the Sequence Listing. As indicated at position 65, the amino acid sequence of CDR3 is shown in positions 98 to 107 of SEQ ID No. 17 of the Sequence Listing;
  • the amino acid sequence of CDR1 of the murine antibody light chain variable region is shown in positions 24 to 34 of SEQ ID No. 21 of the Sequence Listing, and the amino acid sequence of CDR2 is as shown in SEQ ID No. 21 of the Sequence Listing. As shown in positions 50 to 56, the amino acid sequence of CDR3 is shown in positions 89 to 97 of SEQ ID No. 21 of the Sequence Listing.
  • nucleotide sequence of the heavy chain variable region of 29D9H8 is set forth in SEQ ID NO. 61, and the nucleotide sequence of the light chain variable region is set forth in SEQ ID NO.
  • nucleotide sequence of the heavy chain variable region of 28A2E11 is set forth in SEQ ID NO. 69, and the nucleotide sequence of the light chain variable region is set forth in SEQ ID NO.
  • nucleotide sequence of the heavy chain variable region of 35E2C3 is set forth in SEQ ID NO. 77, and the nucleotide sequence of the light chain variable region is set forth in SEQ ID NO.
  • nucleotide sequence of the CDR1 domain in the heavy chain protein variable region encoding 29D9H8 is the 91st to 105th positions in SEQ ID No. 61 of the Sequence Listing;
  • the nucleotide sequence of the CDR2 domain in the heavy chain protein variable region encoding 29D9H8 is from positions 148 to 198 of the sequence listing SEQ ID No. 61;
  • nucleotide sequence of the CDR3 domain in the heavy chain protein variable region encoding 29D9H8 is from positions 295 to 327 in SEQ ID No. 61 of the Sequence Listing;
  • the nucleotide sequence of the CDR1 domain in the light chain protein variable region encoding 29D9H8 is from positions 70 to 102 of SEQ ID No. 65 of the Sequence Listing;
  • nucleotide sequence of the CDR2 domain of the light chain protein variable region encoding 29D9H8 is from positions 148 to 168 of SEQ ID No. 65 of the Sequence Listing;
  • nucleotide sequence of the CDR3 domain of the light chain protein variable region encoding 29D9H8 is position 265 to 291 of SEQ ID No. 65 of the Sequence Listing;
  • nucleotide sequence of the CDR1 domain in the heavy chain protein variable region encoding 28A2E11 is position 91 to 105 in SEQ ID No. 69 of the Sequence Listing;
  • the nucleotide sequence of the CDR2 domain of the heavy chain protein variable region encoding 28A2E11 is from positions 148 to 198 of SEQ ID No. 69 of the Sequence Listing;
  • nucleotide sequence of the CDR3 domain in the heavy chain protein variable region encoding 28A2E11 is from positions 295 to 321 of SEQ ID No. 69 of the Sequence Listing;
  • the nucleotide sequence of the CDR1 domain of the light chain protein variable region encoding 28A2E11 is from position 67 to position 108 of SEQ ID No. 73 of the Sequence Listing;
  • the nucleotide sequence of the CDR2 domain in the light chain protein variable region encoding 28A2E11 is from positions 154 to 174 of SEQ ID No. 73 of the Sequence Listing;
  • nucleotide sequence of the CDR3 domain of the light chain protein variable region encoding 28A2E11 is from positions 271 to 297 of SEQ ID No. 73 of the Sequence Listing;
  • nucleotide sequence of the CDR1 domain of the heavy chain protein variable region encoding 35E2C3 is position 91 to 105 of SEQ ID No. 77 of the Sequence Listing;
  • the nucleotide sequence of the CDR2 domain of the heavy chain protein variable region encoding 35E2C3 is 148th to 195th in SEQ ID No. 77 of the Sequence Listing;
  • nucleotide sequence of the CDR3 domain in the heavy chain protein variable region encoding 35E2C3 is position 292 to 321 in SEQ ID No. 77 of the Sequence Listing;
  • the nucleotide sequence of the CDR1 domain of the light chain protein variable region encoding 35E2C3 is from positions 70 to 102 of SEQ ID No. 81 of the Sequence Listing;
  • the nucleotide sequence of the CDR2 domain of the light chain protein variable region encoding 35E2C3 is from positions 148 to 168 of SEQ ID No. 81 of the Sequence Listing;
  • the nucleotide sequence of the CDR3 domain in the light chain protein variable region encoding 35E2C3 is from position 265 to position 291 in SEQ ID No. 81 of the Sequence Listing;
  • Example 3 A purified IL-13 antibody (lead antibody) was obtained from the culture supernatant of hybridoma cells. Among them, the mouse-human chimeric IL-13 antibody can be prepared from the lead antibody obtained in Example 3 by following the procedure described in the present example.
  • the IL-13 antibody heavy chain variable region and light chain variable region sequences were clarified according to the sequencing results of Example 5.
  • the heavy chain variable region sequence of the leader antibody obtained in Example 3 was recombined into an expression vector comprising a signal peptide and a human heavy chain antibody IgG1 constant region (in which the expression vector was purchased from Invitrogen), and the light chain of the IL-13 antibody was The variable region sequence was recombined into an expression vector comprising the signal peptide and the human antibody light chain kappa constant region or Lambda constant region (wherein the expression vector was purchased from Invitrogen) to obtain a recombinant plasmid (see above for the experimental principle and procedure of plasmid recombination) "Molecular Cloning Experiment Guide (Third Edition)", (US) J.
  • the recombinant plasmid having a purity increased by mass spectrometry using an alkali lysis kit purchased from MACHEREY-NAGEL was used at a mass of 500 ⁇ g or more, and filtered through a 0.22 ⁇ m filter (purchased from Millopore) for transfection.
  • 293E cells (purchased from Invitrogen) were cultured in medium Freestyle 293 expression medium (purchased from Invitrogen). The shaker was set to 37 ° C, 130 RPM and 8% CO 2 (v/v). Freestyle 293 expression medium was added with 10% (v/v) F68 (purchased from Invitrogen) to a final concentration of 0.1% (v/v) of F68 during transfection to obtain Freestyle 293 expression culture containing 0.1% (v/v) F68.
  • Base medium A. 5 mL of Medium A and 200 ⁇ g/mL of PEI (purchased from Sigma) were mixed to obtain Medium B.
  • IL-13 antibody After harvesting the IL-13 antibody, it was dialyzed against 1 PBS for 4 hours to avoid endotoxin contamination. After the end of dialysis, the concentration was measured using a spectrophotometer or a kit, the purity of the antibody was determined using HPLC-SEC, and the endotoxin content of the antibody was detected using an endotoxin test kit (purchased from Lonza). The obtained IL-13 antibody was characterized.
  • the chimeric antibodies c29D9H8, c28A2E11 and c35E2C3 were prepared using the 29D9H8, 28A2E11 and 35E2C3 leader antibodies, respectively.
  • the cloned front-end letter c represents a mouse-human chimeric antibody (hereinafter referred to as chimeric antibody).
  • Example 7 Assay of mouse-human chimeric IL-13 antibody
  • the mouse-human chimeric IL-13 antibody obtained in Example 6 was subjected to the following assay (the procedure is the same as in Example 4).
  • ELISA enzyme-linked immunosorbent assay
  • Example 4 The specific procedure is the same as that of Example 4, Test A. Some experimental results are shown in Figures 13-15 and Tables 18-20. Tables 18-20 illustrate that the chimeric antibodies were conjugated to recombinant human IL-13 protein, recombinant human IL-13 R130Q variant, and recombinant monkey IL-13 at the ELISA level.
  • the IgG control was murine IgG and the data in the table is the OD 450 nm value.
  • Tables 21-22 illustrate that chimeric antibodies bind to human IL-13 and block human IL-13 and cell surface receptor IL-13Ra1/hIL. -4Ra heterodimer binding, or blocking the binding of IL-13 to the cell surface human IL-13Ra2 receptor.
  • the IgG control was murine IgG and the data in the table is the mean fluorescence intensity.
  • TARC thymic activation regulating chemokine
  • Table 23 shows that chimeric antibodies can neutralize the secretion of TARC by A549 cells induced by IL-13 and TNF ⁇ co-stimulation by binding to human IL-13.
  • the data in Table 23 is the concentration (pg/ml) of TARC in the culture supernatant, wherein the IgG control is murine IgG.
  • MRC5 cells (purchased from ATCC) were cultured in 10% (w/w) fetal bovine serum EMEM medium (purchased from Gibco) and cultured at 37 ° C under 5% (v/v) CO 2 . After being expanded to 75-90% confluence in a T-175 cell culture flask, the medium was discarded, washed 1-2 times with PBS buffer, and then trypsin-EDTA (purchased from Life Technology). Digest and collect cells. After counting, the cells were diluted to 1 ⁇ 10 5 cells/mL with a culture solution, and 100 ⁇ L of each well was added to a 96-well cell culture plate (1 ⁇ 10 4 cells per well), and the plate was placed at 37 ° C.
  • the 5% (v/v) CO 2 incubator was cultured overnight; the next day, the culture supernatant was discarded, and the gradient-diluted chimeric antibody prepared in Example 6 was mixed with the immunogen A prepared in Example 1 to obtain a mixture C ( The final concentration of immunogen A in mixture C was 5 ng/mL), mixture C was added to the culture plate, and the cell culture plate was incubated overnight at 37 ° C in a 5% (v/v) CO 2 incubator. After 20 hours, the concentration of periostin in the supernatant was determined using a periostin ELISA kit (purchased from RnD systems). The experimental procedure was carried out in accordance with the instructions of the kit. Some experimental results are shown in Figures 19 and 24.
  • Table 24 shows that the chimeric antibody binds to human IL-13 and is capable of neutralizing the secretion of periostin from MRC5 cells induced by human IL-13.
  • the data in Table 24 is the concentration of periostin (pg/ml) in the culture supernatant, and the IgG control is murine IgG.
  • VCAM-1 vascular cell adhesion molecule-1
  • Human umbilical vein endothelial cells (HUVEC) (purchased from AllCells) were cultured in HUVEC complete medium and cultured at 37 ° C under 5% (v/v) CO 2 . After it was expanded to T-175 to 75-90% confluence, the medium was discarded, washed 1-2 times with PBS buffer, and then digested and collected with trypsin-EDTA (purchased from Life Technology). . After counting, the cells were diluted to 1.5 ⁇ 10 5 cells/mL with the culture medium, and 3000 cells (20 ⁇ L) per HUVEC cells were added to the 384-well cell culture plate; the antibody prepared in Example 6 was diluted with the implementation.
  • HUVEC Human umbilical vein endothelial cells
  • the immunogen A prepared in Example 1 and recombinant human TNF ⁇ were mixed at a volume ratio of 1:1:2 to obtain a mixture D.
  • the mixture D was added to the culture plate in an amount of 20 ⁇ L per well to obtain a mixture D such that the volume ratio of the mixture D to the HUVEC cells in the culture plate was 1:1.
  • the recombinant human TNF ⁇ accounted for a final concentration of 25 ng/mL of the mixture D'
  • the immunogen A accounted for 0.5 ng/mL of the final concentration of the mixture D'.
  • the cell culture plates were incubated overnight at 37 ° C in a 5% (v/v) CO 2 incubator. After 20 hours, the culture supernatant was discarded, and the mouse anti-human VCAM-1 (CD106) antibody (purchased from Biolegend) was diluted with the medium to a final concentration of 2 ⁇ g/mL, and added to the plate at 20 ⁇ L per well on ice. Incubate for 2 hours. Wash 3 times with FACS buffer [PBS buffer containing 2% (w/w) BSA], add 20 ⁇ L of fluorescent (Alexa 488)-labeled donkey anti-mouse secondary antibody (purchased from invitrogen) per well, and incubate 0.5-on ice. 1.0 hour.
  • FACS buffer PBS buffer containing 2% (w/w) BSA
  • Table 25 shows that the chimeric antibody binds to human IL-13 and is capable of neutralizing the expression of vascular adhesion factor-1 on the surface of HUVEC cells induced by co-stimulation of human IL-13 and TNF ⁇ .
  • the data in Table 25 is the mean fluorescence intensity; the IgG control is murine IgG; TNF ⁇ is the background value stimulated with TNF ⁇ alone, without IL-13.
  • Example 8 IL-13 chimeric antibody inhibits human IL-13-induced respiratory inflammation in mice
  • mice Female Balb/c mice (8-12 weeks old, purchased from Shanghai Lingchang Biotechnology Co., Ltd.) were received at the SPF level after receiving, and the experiment was started after 1 week of adaptation. Mice were injected intraperitoneally with the chimeric antibodies prepared in Example 6 on days 1 and 3, clone numbers c29D9H8, c28A2E11 and c35E2C3, respectively, 200 ⁇ L per animal (3 mg antibody per kg body weight). On the second and fourth days, the immunogen A prepared in Example 1 was used for stimulation induction at a dose of 1 mg/mL, and each animal was given an airway spray of 25 ⁇ L. On the fifth day, all animals were treated with the FinePointe Whole Body Plethysmography System (DSI).
  • DSI FinePointe Whole Body Plethysmography System
  • FinePointe WBP system purchased from DSI, was tested for lung function. Animals were given methyl acetylcholine by aerosolization in a conscious, unconstrained state, and the airway narrowing index Penh (enhanced expiratory interval) was recorded by the instrument's own software. (The specific operation steps of the test are carried out according to the method described in the instruction manual of the instrument)
  • Table 26 shows that mice stimulated high airway responsiveness under stimulation with human IL-13, or immunogen A, making them more sensitive to methyl acetylcholine stimulation and increasing penh readings.
  • the chimeric antibody binds to human IL-13 and neutralizes airway hyperresponsiveness induced by human IL-13.
  • the data in the table is the airway narrowing index Penh, which represents airway hyperresponsiveness; the IgG control is human IgG.
  • Anti-human Fc IgG (purchased from Geneway) was coupled to the surface of a CM5 chip (purchased from GE) by an amino coupling method to 6000-10000 RU, and FC1 was used as a reference channel.
  • the coupling fixation procedure was as follows: 50 mM N-hydroxysuccinimide (NHS) and 200 mM 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide in a freshly prepared molar ratio of 1:1 The mixture of hydrochloride (EDC) activated the chip for 7 minutes. Then, 10-50 ⁇ g/mL of anti-human Fc IgG diluted in 10 mM sodium acetate buffer (pH 5.0) was injected.
  • NHS N-hydroxysuccinimide
  • EDC 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
  • the chimeric antibody prepared in Example 6 was diluted to 5 ⁇ g/mL with HBS-EP-containing buffer (which can be appropriately adjusted depending on the capture level).
  • the chip was captured at a flow rate of 10 ⁇ L/min to obtain a response value of approximately 100 to 300 RU.
  • the purified immunogen A prepared in Example 1 was then diluted to 100 nM (i.e., the highest concentration tentatively 100 nM) and flowed through the surface of the chip at a flow rate of 30 ⁇ L/min. If sufficient signal values are obtained, the purified immunogen A prepared in Example 1 is diluted by several concentration gradients and flowed through the surface of the chip, respectively.
  • the human germline antibody heavy and light chain variable region templates that best match the non-CDR regions of the chimeric antibodies c29D9H8, c28A2E11 and c35E2C3 described above were selected in the Germline database.
  • c28A2E11 template antibody heavy chain variable region of the outer V H human germline antibody heavy chain exon VH1-F, an outer J H exon J H -1, the template for the light chain variable region of human germline antibody outer light chain V L exon 7B, J L exon J L -1.
  • c35E2C3 template antibody heavy chain variable region of an antibody heavy chain human germline outer V H exon VH4-59, template peripheral J H exon J H -6, light chain variable region of human germline antibody Light chain V K exon O12, J K exon J K -4.
  • c begins with a chimeric antibody
  • Hu begins with a humanized antibody
  • donor framework residues and the back mutations are shown: for example, each humanized anti-IL-13 antibody Hu-29D9H8 heavy chain "T73K” in the variable region Hu-29D9H8.VH1 and its variants indicates that the 73rd amino acid shown in Figure 22 is mutated from "T" glutamate to "K” arginine, and the site of the back mutation is located.
  • the framework area as an example, will not be explained one by one.
  • cDNA was synthesized based on the amino acid sequences of the light chain variable region and the heavy chain variable region of each humanized antibody (ie, SEQ ID NO. 86, 87, 88, 89, 90, 91, 92, 93 in the sequence listing, respectively).
  • the sequence of the heavy chain cDNA was digested with FspAI and AfeI.
  • the cDNA fragment was inserted into the containing signal by FspAI/AfeI, FspAI/BsiwI or FspAI/HindIII cleavage sites, respectively.
  • An expression vector for the IgG1 constant region of the peptide and human heavy chain antibody and an expression vector comprising the signal peptide and the human antibody light chain kappa/Lambda constant region (in which the expression vector is purchased from Invitrogen), the recombinant plasmid is verified by sequencing, and used The alkaline lysis kit (purchased from MACHEREY-NAGEL) was used to extract a recombinant plasmid having a purity of 500 ⁇ g or more, and was filtered through a 0.22 ⁇ m filter (purchased from Millopore) for transfection.
  • 293E cells Prior to transfection, 293E cells (purchased from Invitrogen) were cultured in medium Freestyle 293 expression medium (purchased from Invitrogen). When transfected, 10% (v/v) F68 (purchased from Invitrogen) was added to Freestyle 293 expression medium to a final concentration of 0.1% (v/v) of F68 to obtain Freestyle 293 expression containing 0.1% (v/v) F68.
  • the shaker was set to 37 ° C, 130 RPM and 8% CO 2 (v/v).
  • Peptone was added the next day to a final concentration of 0.5% (w/v).
  • the antibody titer of the culture solution was measured.
  • the supernatant was collected by centrifugation (3500 RPM, 30 minutes), and filtered through a 0.22 ⁇ m filter to obtain a filtered cell supernatant for purification.
  • the humanized anti-IL-13 antibodies prepared in Example 10 were subjected to the following assays (the procedure is the same as in Examples 4 and 7).
  • ELISA enzyme-linked immunosorbent assay
  • Tables 31-33 illustrate that the above humanized anti-IL-13 antibody binds to recombinant human IL-13 protein at the ELISA level.
  • the IgG control was human IgG and the data in the table is the OD 450 nm value.
  • Tables 34-44 show that some humanized anti-IL-13 antibodies bind to human IL-13 and block human IL-13 and cell surface receptors. Binding of human IL-13Ra1/hIL-4Ra heterodimer, or blocking the binding of IL-13 to the cell surface human IL-13Ra2 receptor.
  • the IgG control was human IgG and the data in the table was the mean fluorescence intensity.
  • Table 34-2 FACS detection of humanized anti-IL-13 antibody blocking IL-13 and cell surface receptor IL-13Ra2
  • Table 40-1 FACS detection of humanized anti-IL-13 antibody blocks IL-13 binding to cell surface receptor IL-13Ra1/IL-4Ra heterodimer
  • Table 43-1 FACS detection of humanized anti-IL-13 antibody blocking IL-13 binding to cell surface receptor IL-13Ra1/IL-4Ra heterodimer
  • Table 44-1 FACS detection of humanized anti-IL-13 antibody blocking IL-13 binding to cell surface receptor IL-13Ra1/IL-4Ra heterodimer
  • TARC thymic activation regulating chemokine
  • Tables 45-51 illustrate that humanized anti-IL-13 antibodies are capable of neutralizing co-stimulation conditions by human IL-13/human IL-13R130Q variant and TNF ⁇ by binding to human IL-13/human IL-13R130Q variant.
  • the under-induced A549 cells secrete TARC.
  • the data in Tables 45-51 are the concentration of TARC (pg/ml) in the culture supernatant, where the IgG control is human IgG.
  • Example 12 Characterization analysis of humanized anti-IL-13 antibody (Biacore)
  • Biacore analysis was performed using human IL-13 antigen immobilized on a CM5 chip.
  • the Biacore technique utilizes a change in the refractive index of the surface layer of the antibody after binding to the IL-13 antigen immobilized on the surface layer.
  • the detection is performed in combination with surface plasmon resonance (SPR) of a laser refracted from the surface. Analysis of signal kinetics binding rate and dissociation rate allows for the discrimination of non-specific and specific interactions.
  • SPR surface plasmon resonance
  • Table 52 shows that the affinity of the humanized anti-IL-13 antibody prepared in Example 10 to human IL-13 was KD ⁇ 9 x 10 -8 M.
  • Example 13 IL-13 humanized antibody inhibits human IL-13-induced lung goblet cell metaplasia and mucus hypersecretion in mice
  • mice Female Balb/c mice (8-12 weeks old, purchased from Shanghai Lingchang Biotechnology Co., Ltd.) were received at the SPF level after receiving, and the experiment was started after 1 week of adaptation.
  • the humanized antibodies prepared by intraperitoneal injection of mice on the first day and the third day were: IgG control, Tab2 (completed according to the amino acid sequence of clone 228B/C in US Pat. No. 8,088,618B2), Tab3 (according to patent In US7935343B2, the cloned BAK0502G9 amino acid sequence was expressed and purified), Hu-35E2C3-19, 200 ⁇ L per animal (3 mg or 0.3 mg antibody per kg body weight).
  • Example 1 On the second and fourth days, the immunogen A prepared in Example 1 was used for stimulation induction at a dose of 1 mg/mL, and each animal was given an airway spray of 25 ⁇ L. Another group of mice did not give antibodies, nor immunogen A, the most negative control group.
  • euthanasia was performed and lung tissue was taken and placed in 10% (v/v) formalin. The fixed lung tissue was paraffin-embedded, sectioned, stained by PAS staining, and the goblet cell metaplasia (mucus secretion) was observed.
  • Table 53 shows that mice stimulated goblet cell metaplasia and excessive mucus secretion in lung tissue under the stimulation of human IL-13, or immunogen A, and increased the proportion of PAS staining positive regions.
  • Humanized antibodies bind to human IL-13 and neutralize goblet cell metaplasia and mucus hypersecretion induced by human IL-13.
  • the data in the table is the average of the proportion of PAS staining positive areas in the lung tissue of different mice in each group; the IgG control is human IgG.
  • sequence information related to the present invention is as follows:

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Abstract

La présente invention concerne un anticorps anti-IL13 humanisé et son procédé de préparation. L'anticorps anti-IL-13 a une affinité élevée, inhibe la sécrétion d'une chimiokine régulée par activation du thymus induite par IL-13 et d'une périostine et l'expression de la molécule-1 d'adhésion cellulaire vasculaire, inhibe l'hyperréactivité des voies respiratoires induite par IL-13 chez une souris, et peut être utilisé dans la préparation d'un médicament pour la prévention ou le traitement de maladies liées à l'expression ou à la fonction anormale de l'IL-13.
PCT/CN2018/115694 2017-11-15 2018-11-15 Anticorps anti-il13 humanisé, son procédé de préparation et son utilisation WO2019096219A1 (fr)

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CN101287760A (zh) * 2005-10-21 2008-10-15 诺瓦提斯公司 抗il13的人抗体和治疗用途
WO2010103274A1 (fr) * 2009-03-11 2010-09-16 Ucb Pharma S.A. Molécules d'anticorps ayant une spécificité de liaison pour il-13 humain
CN102993302A (zh) * 2003-12-23 2013-03-27 遗传技术研究公司 新的抗il-13抗体及其应用

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CN102993302A (zh) * 2003-12-23 2013-03-27 遗传技术研究公司 新的抗il-13抗体及其应用
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WO2010103274A1 (fr) * 2009-03-11 2010-09-16 Ucb Pharma S.A. Molécules d'anticorps ayant une spécificité de liaison pour il-13 humain

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