WO2020093957A1 - 结合人IL-1β的抗体、其制备方法和用途 - Google Patents

结合人IL-1β的抗体、其制备方法和用途 Download PDF

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WO2020093957A1
WO2020093957A1 PCT/CN2019/115230 CN2019115230W WO2020093957A1 WO 2020093957 A1 WO2020093957 A1 WO 2020093957A1 CN 2019115230 W CN2019115230 W CN 2019115230W WO 2020093957 A1 WO2020093957 A1 WO 2020093957A1
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antibody
seq
antigen
human
binding fragment
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PCT/CN2019/115230
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French (fr)
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夏瑜
***
张鹏
李百勇
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泽达生物医药有限公司
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Priority to CN201980064671.5A priority Critical patent/CN113383016B/zh
Priority to EP19882764.4A priority patent/EP3878867A4/en
Priority to JP2021523978A priority patent/JP7256266B2/ja
Priority to US17/292,384 priority patent/US11976115B2/en
Publication of WO2020093957A1 publication Critical patent/WO2020093957A1/zh

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    • C07—ORGANIC CHEMISTRY
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    • 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
    • C07K16/244—Interleukins [IL]
    • C07K16/245—IL-1
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
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    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
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    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
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Definitions

  • the present invention relates to the field of antibodies. More specifically, the present invention discloses an antibody that binds to human IL-1 ⁇ ;
  • the invention also relates to the preparation method and use of the above antibody.
  • Interleukin 1 protein includes interleukin 1 ⁇ (IL-1 ⁇ ) and interleukin 1 ⁇ (IL-1 ⁇ ).
  • IL-1 ⁇ is a multifunctional cytokine that mediates the growth and differentiation of various lymphocytes, and participates in and regulates various inflammatory response processes.
  • the secretion level of IL-1 ⁇ in the body is regulated by the IL-1 family such as IL-1 ⁇ and IL-1R inhibitor (IL-1Ra).
  • IL-1 ⁇ receptors include IL-1I type receptor (IL-1RI) and IL-1 type II receptor (IL-1RII).
  • IL-1RI is expressed in almost all nucleated cells, and IL-1 ⁇ binds to IL-1RI to cause the accumulation of IL-1 receptor (IL-1R) accessory protein (IL-1RAcP) and form a complex, thereby activating the signal Pathway;
  • IL-1RII exists in the body in the form of cell membrane surface expression and soluble form.
  • IL-1 ⁇ will downregulate IL-1 ⁇ activity after binding to IL-1RII.
  • IL-1 ⁇ is the main cause of various immune diseases such as Cryopyrin protein-related syndrome, tumor necrosis factor receptor-related periodic syndrome, systemic juvenile idiopathic arthritis, and high immunity Globulin D syndrome (HIDS) / mevalonate kinase deficiency (MKD), osteoporosis, osteoarthritis, and other inflammatory arthritis.
  • Cryopyrin protein-related syndrome tumor necrosis factor receptor-related periodic syndrome
  • systemic juvenile idiopathic arthritis systemic juvenile idiopathic arthritis
  • HIDS high immunity Globulin D syndrome
  • MKD mevalonate kinase deficiency
  • osteoporosis osteoarthritis
  • osteoarthritis and other inflammatory arthritis.
  • IL-1 ⁇ interleukin-1
  • IL-1 ⁇ plays an important role in the differentiation and maturation of TH17 cells (de Jong E, Suddason T, Lord GM.
  • IL-1 ⁇ inhibitors can block the IL-1 signaling pathway and play an important role in the treatment of osteoporosis, inflammatory arthritis and other immune diseases.
  • the inventors of the present invention have undergone a large number of tests, from antigen immunization, hybridoma screening, antibody expression purification to biological activity identification, and obtained a series of antibodies that bind to human IL-1 ⁇ .
  • the present invention has carried out in vivo pharmacological studies on candidate murine antibodies and humanized antibodies. The results show that murine antibodies 19E4, 18H11, and 9D5 can significantly reduce IL-1 ⁇ -induced mouse arthritis, and humanized antibody 18H11 At a dose of 10 mg / kg, H1L1 can significantly improve the walking behavior of the affected limb and significantly reduce the swelling area of the knee joint of the affected limb. Therefore, the antibody with novel structure and binding to human IL-1 ⁇ disclosed in the present invention is expected to be a potential therapeutic drug for the treatment of arthritis, osteoporosis, and other immune diseases.
  • the first object of the present invention is to provide an antibody or antigen-binding fragment thereof that binds to human IL-1 ⁇ .
  • the amino acid sequences of the heavy chain complementarity determining regions H-CDR1, H-CDR2, and H-CDR3 of the antibody are: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3; the amino acid sequences of the light chain complementarity determining regions L-CDR1, L-CDR2, L-CDR3 are SEQ ID NO: 4, SEQ ID NO: 5. SEQ ID NO: 6.
  • the antibody binding to human IL-1 ⁇ disclosed in the present invention is a murine antibody 18H11, the amino acid sequence of the heavy chain variable region is SEQ ID NO: 7, and the amino acid sequence of the light chain variable region is SEQ ID NO: 8.
  • the antibody binding to human IL-1 ⁇ disclosed in the present invention is the humanized antibody 18H11H1L1, the amino acid sequence of the heavy chain variable region is SEQ ID NO: 9, and the amino acid sequence of the light chain variable region is SEQ ID NO: 10.
  • the antibody binding to human IL-1 ⁇ disclosed in the present invention is humanized antibody 18H11H2L2, the amino acid sequence of the heavy chain variable region is SEQ ID NO: 11, and the amino acid sequence of the light chain variable region is SEQ ID NO: 12.
  • the antibody binding to human IL-1 ⁇ disclosed in the present invention is humanized antibody 18H11H3L3, the amino acid sequence of the heavy chain variable region is SEQ ID NO: 13, and the amino acid sequence of the light chain variable region is SEQ ID NO: 14.
  • the antigen-binding fragments of the present invention include Fab fragments, F (ab ') 2 fragments, Fv fragments, single chain antibodies or single domain antibodies.
  • the second object of the present invention is to provide a nucleotide molecule encoding the antibody or antigen-binding fragment thereof that binds to human IL-1 ⁇ .
  • the nucleotide sequence encoding the heavy chain variable region of the mouse antibody 18H11 is SEQ ID NO: 15, and the nucleotide sequence encoding the light chain variable region is SEQ ID NO: 16.
  • nucleotide sequence encoding the heavy chain variable region of the humanized antibody 18H11H1L1 is SEQ ID NO: 17, and the nucleotide sequence encoding the light chain variable region is SEQ ID NO: 18.
  • nucleotide sequence encoding the heavy chain variable region of the humanized antibody 18H11H2L2 is SEQ ID NO: 19
  • nucleotide sequence encoding the light chain variable region is SEQ ID NO: 20.
  • nucleotide sequence encoding the humanized antibody 18H11H3L3 heavy chain variable region is SEQ ID NO: 21
  • nucleotide sequence encoding the light chain variable region is SEQ ID NO: 22.
  • the third object of the present invention is to provide an expression vector containing the nucleotide molecule.
  • the fourth object of the present invention is to provide a host cell containing the expression vector.
  • the fifth object of the present invention is to provide a method for preparing the human IL-1 ⁇ -binding antibody or antigen-binding fragment thereof, which includes the following steps:
  • the sixth object of the present invention is to provide a pharmaceutical composition containing the human IL-1 ⁇ -binding antibody or antigen-binding fragment thereof.
  • the pharmaceutical composition contains the antibody or antigen-binding fragment thereof that binds to human IL-1 ⁇ as described in any one of the above and a pharmaceutically acceptable carrier.
  • the seventh object of the present invention is to provide the use of the human IL-1 ⁇ -binding antibody or antigen-binding fragment thereof or the pharmaceutical composition.
  • the use is for preparing medicines for treating various immune diseases caused by overexpression of IL-1 ⁇ , such as arthritis, osteoporosis or psoriasis.
  • the use is for preparing a medicine for treating arthritis.
  • An eighth object of the present invention is to provide an antibody or antigen-binding fragment thereof that binds to human IL-1 ⁇ , the heavy chain complementarity determining region H-CDR2 of the antibody or antigen-binding fragment thereof includes a C53A mutation site, and the antibody
  • the heavy chain complementarity determining region H-CDR2 (IMGT coding mode) of 18H11-Hu-C53A: ISAYNGDT, and its amino acid sequence is shown in SEQ ID NO: 38.
  • the present invention also provides binding epitopes of the above antibodies to human IL-1 ⁇ : the main binding epitopes are tryptophan W at position 120 and isoleucine I at position 122 of SEQ ID NO: 23, followed by Phenylalanine F at position 112, serine S at position 123, and threonine T at position 124.
  • the antibody binding to human IL-1 ⁇ obtained by the screening of the present invention can specifically bind to human IL-1 ⁇ .
  • ELISA test results show that the EC 50 of the mouse antibody 18H11 is equivalent to the positive control antibody, and the EC of the humanized antibody 18H11H1L1 50 is better than the positive control antibody.
  • the antibody binding to human IL-1 ⁇ obtained by the screening of the present invention can effectively competitively block the binding of IL-1 ⁇ to its related receptors.
  • the EC 50 of murine antibody 18H11 and humanized antibody 18H11H1L1 are both positive control The antibody is equivalent.
  • the antibody binding to human IL-1 ⁇ obtained by the screening of the present invention can effectively inhibit the secretion of IL-6 by MRC-5 cells induced by IL1 ⁇ .
  • the murine antibody 18H11 and the humanized antibody 18H11H1L1 inhibit IL-6 secretion of cells induced by IL-1 ⁇ better than the positive control antibody.
  • the antibody 18H11H1L1 that binds to human IL-1 ⁇ obtained by screening in the present invention is analyzed by Fortebio Kinetics, and its antibody affinity is comparable to that of the positive control antibody.
  • the antibody binding to human IL-1 ⁇ obtained by screening in the present invention can significantly reduce IL-1 ⁇ -induced arthritis in mice.
  • the therapeutic effects of mouse-derived antibody 18H11 and humanized antibody 18H11H1L1 on mouse behavioral scores, joint swelling effects / knee joint area, and body weight effects are comparable to positive control antibodies.
  • mutant antibody 18H11-Hu-C53A obtained in the present invention using the heavy chain of 18H11 and H1L1 as the template and containing the C53A site in H-CDR2 has better thermal stability than the wild-type antibody.
  • the terms "antibody (Ab)” and “immunoglobulin G (IgG)” are heterotetrameric glycoproteins of about 150,000 daltons with the same structural characteristics, which are composed of two identical light chains (L) It consists of two identical heavy chains (H). Each light chain is connected to the heavy chain through a covalent disulfide bond, and the number of disulfide bonds between heavy chains of different immunoglobulin isotypes is different. Each heavy and light chain also has regularly spaced disulfide bonds in the chain. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions.
  • VH variable region
  • Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain .
  • the antibody of the present invention may be a monoclonal antibody, a polyclonal antibody, a multispecific antibody (for example, a bispecific antibody) formed from at least two antibodies, an antigen-binding fragment of the antibody, or the like.
  • the antibodies of the present invention include murine antibodies, chimeric antibodies, humanized antibodies, and the like.
  • the term "monoclonal antibody (mAb)” refers to an antibody obtained from a substantially homogeneous population, that is, the individual antibodies contained in the population are the same, except for a few possible naturally occurring mutations. Monoclonal antibodies are highly specific against a single antigenic site. Moreover, unlike conventional polyclonal antibody preparations (usually with different antibodies directed against different determinants), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they are synthesized by hybridoma culture and will not be contaminated by other immunoglobulins. The modifier "monoclonal" indicates the characteristics of antibodies and is obtained from a substantially uniform antibody population. This should not be interpreted as requiring any special method to produce antibodies.
  • the term "murine antibody” refers to an antibody derived from a rat or a mouse, preferably a mouse.
  • the murine antibody of the present invention is obtained by immunizing mice with human IL-1 ⁇ as an antigen and screening for hybridoma cells. More preferably, the murine antibodies of the present invention include 19E4, 18H11, 9D5. Most preferably, the murine antibody of the invention is 18H11.
  • humanized antibody refers to an antibody obtained by replacing all or part of the CDR regions of a human immunoglobulin (acceptor antibody) with the CDR regions of a non-human antibody (donor antibody).
  • An antibody fragment, in which the donor antibody may be a non-human (eg, mouse, rat, or rabbit) antibody with the desired specificity, affinity, or reactivity.
  • some amino acid residues in the framework region (FR) of the acceptor antibody can also be replaced with corresponding non-human antibody amino acid residues, or with other antibody amino acid residues, to further improve or optimize antibody performance.
  • the humanized antibody of the present invention is recombined from the CDR region of the murine antibody 18H11 and the non-CDR region derived from the human antibody. More preferably, the humanized antibodies of the present invention include 18H11, H1L1, 18H11, H2L2, and 18H11, H3L3. Most preferably, the humanized antibody of the present invention is 18H11 and H1L1.
  • variable means that some parts of the variable region of an antibody are different in sequence, and it forms the binding and specificity of various specific antibodies to its specific antigen.
  • variability is not evenly distributed throughout the variable region of the antibody. It is concentrated in three segments called the complementarity determining region (CDR) or hypervariable region in the light chain and heavy chain variable regions.
  • CDR complementarity determining region
  • FR framework region
  • the variable regions of the natural heavy and light chains each contain four FR regions, which are roughly in a ⁇ -sheet configuration, connected by three CDRs that form a connecting loop, and in some cases may form part of the ⁇ -sheet structure.
  • the CDRs in each chain are closely together through the FR region and together with the CDRs of the other chain form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Volume I, pages 647-669) (1991)).
  • the constant regions are not directly involved in the binding of antibodies and antigens, but they exhibit different effector functions, such as participation in antibody-dependent cell-mediated cytotoxicity (ADCC).
  • the term "antigen-binding fragment” refers to a fragment of an antibody that can specifically bind to human IL-1 ⁇ .
  • the antigen-binding fragments of the present invention include Fab fragments, F (ab ') 2 fragments, Fv fragments, single chain antibodies (scFv), single domain antibodies (sdAb), and the like.
  • Fab fragments are fragments produced by digesting antibodies with papain.
  • the F (ab ') 2 fragment is a fragment produced by digesting the antibody with pepsin.
  • the Fv fragment is composed of dimers in which the heavy chain variable region and the light chain variable region of an antibody are closely and non-covalently associated.
  • a single chain antibody is an antibody composed of an antibody heavy chain variable region and a light chain variable region connected by a short peptide of 15 to 20 amino acids (linker).
  • Single-domain antibodies also known as nanobodies (nanobodies) or heavy-chain antibodies, are composed only of heavy chains, and their antigen-binding region is only a single domain connected to the Fc region through a hinge region.
  • binding and specific binding refer to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen to which it targets.
  • antibodies bind the antigen with an equilibrium dissociation constant (KD) of less than about 10 -7 M, such as less than about 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M or less.
  • KD refers to the equilibrium dissociation constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen.
  • the smaller the equilibrium dissociation constant the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen.
  • SPR Surface Plasmon Resonance
  • ELISA ELISA
  • epitope and “human IL-1 ⁇ epitope” refer to regions located on human IL-1 ⁇ that are specifically associated with antibody binding.
  • expression vector may be pTT5, pSECtag series, pCGS3 series, pCDNA series vectors, etc., as well as other vectors used in mammalian expression systems, etc.
  • the expression vector includes those connected with appropriate transcription and translation regulatory sequences Fusion DNA sequence.
  • the term "host cell” refers to a cell suitable for expressing the above expression vector, and may be a eukaryotic cell, such as a mammalian or insect host cell culture system, which can be used for the expression of the fusion protein of the present invention, CHO (Chinese Hamster) Ovary, Chinese Hamster (Ovary), HEK293, COS, BHK, etc. and derived cells of the above-mentioned cells are all applicable to the present invention.
  • the term "pharmaceutical composition” means that the antibody that binds to human IL-1 ⁇ of the present invention can be combined with a pharmaceutically acceptable carrier to form a pharmaceutical preparation composition to more effectively exert therapeutic effects, and these preparations can guarantee the disclosure of the present invention
  • the term "disease in which IL-1 ⁇ is overexpressed” means that the expression level of IL-1 ⁇ in cells in an abnormal disease state is higher than that in normal cells of the same tissue type.
  • the diseases overexpressing IL-1 ⁇ of the present invention include, but are not limited to, osteoporosis, osteoarthritis, other inflammatory arthritis, psoriasis and other immune diseases.
  • IL-1 ⁇ / IL-1b Interleukin-1 beta Interleukin 1 beta IL-1R Interleukin-1 receptor Interleukin 1 receptor IL-1RI Interleukin-1 receptor, type I Interleukin 1 receptor, type I IL-1RII Interleukin-1 receptor, type II Interleukin 1 receptor, type II IL-1Ra Interleukin-1 receptorceptantagonist Interleukin-1 type I receptor antagonist IL-1RAcP Interleukin 1 receptor accessory protein Interleukin 1 type I receptor accessory protein IL-6 Interleukin-6 Interleukin-6 PcAb Positive controlsantibody Positive control antibody PBS Phosphate buffer saline Phosphate buffer cDNA Complementary deoxyribonucleic acid Complementary deoxyribonucleic acid SDS-PAGE Polyacrylamide gelelectrophoresis Polyacrylamide gel electrophoresis ELISA Enzyme-linked immunosorbent assay Enzyme-linked immunosorbent assay EC 50 Median effective concentration Half effective concentration Half effective
  • Figure 1 is a graph of IL1 ⁇ -hFc purification results, where M: protein molecular weight standard, 1: before purification column, 2: penetration after purification column, 3-9: purification column elution.
  • M protein molecular weight standard
  • 1 before purification column
  • 2 penetration after purification column
  • 3-9 purification column elution.
  • the theoretical size of IL1 ⁇ -hFc protein is 43.7kDa, and the molecular weight of the protein dimer is about 87kDa.
  • Figure 2 shows the purification result of IL1RI (1-332) -HIS culture supernatant, M: protein molecular weight standard, 1: before purification column, 2: penetration after purification column, 3-4: purification column elution.
  • Figure 3 shows the purification result of IL1b-His
  • M protein molecular weight standard
  • FT penetration after loading on the purification column
  • load before loading on the purification column
  • 1-7 elution from the purification column.
  • the theoretical size of IL1b-His protein is 18.2kDa, and there is no dimer molecule in the protein.
  • Figure 4 is a graph of PcAb purification results, M: protein molecular weight standard, 1: non-reduced antibody, 2: reduced antibody, 3: BSA control.
  • the theoretical size of PcAb protein is 150kDa, the molecular weight of antibody heavy chain is about 45kDa, and the molecular weight of light chain is about 30kDa.
  • Figure 5 shows the results of measuring the binding affinity of murine antibodies to the antigen IL1 ⁇ by ELISA.
  • Fig. 6 is the result of measuring the affinity of the murine antibody and IL1RI for binding antigen IL1 ⁇ by ELISA.
  • FIG. 7 is a graph showing that the murine antibody inhibits IL-6 secretion of MRC5 cells induced by IL1 ⁇ by ELISA.
  • Fig. 9 shows the effect of mouse-derived antibodies 19E4, 18H11 and 9D5 on the swelling of the knee joint of mice.
  • Fig. 10 shows the results of measuring the binding affinity of humanized antibody to antigen IL1 ⁇ by ELISA.
  • FIG. 11 is the result of measuring the affinity of humanized antibody to compete with IL1RI for binding antigen IL1 ⁇ by ELISA.
  • Fig. 12 is a graph showing the results of measuring the inhibition of IL-6 secretion by MRC5 cells induced by IL1 ⁇ by humanized antibodies by ELISA.
  • Figure 13 shows the results of the effects of binding human IL-1 ⁇ antibodies 18H11, H1L1 and 18H11 on the pathological behavior of mice with knee arthritis induced by Lenti-IL-1 ⁇ -NIH / 3T3.
  • Figure 14 shows the results of the effect of binding human IL-1 ⁇ antibodies 18H11, H1L1 and 18H11 on the knee joint area of a mouse knee arthritis model mouse induced by Lenti-IL-1 ⁇ -NIH / 3T3.
  • Figure 15 shows the results of SDS-PAGE protein electrophoresis of each mutant antibody.
  • Fig. 17 shows the determination of the IL-6 secretion activity of MRC-5 cells induced by IL-1 ⁇ by antibodies.
  • Fig. 18 is a measurement of the binding affinity of each sample to IL-1 ⁇ .
  • Fig. 19 is a measurement of the activity of each sample in inhibiting IL-6 secretion of MRC-5 cells induced by IL-1 ⁇ .
  • Figure 20 shows the detection of the affinity of 18H11-Hu-C53A to compete with IL1RI for the binding antigen IL1 ⁇ .
  • Figure 21 shows the inhibitory effect of different doses of antibody on murine IL-6 secretion.
  • Fig. 22 shows the cross-species immunoreactivity of 18H11-Hu-C53A by ELISA.
  • Figure 23 shows the determination of the selectivity of 18H11-Hu-C53A for the family member proteins IL-1, alpha-1, IL-1 R2 and IL-1 RA.
  • Figure 24-27 shows the determination of the binding epitope of 18H11-Hu-C53A to IL-1 ⁇ .
  • Figure 24 shows the affinity of 18H11-Hu-C53A with IL-1 ⁇ -A1-F99-His and IL-1 ⁇ -A1-W120-His;
  • Figure 25 shows the determination of the binding ability of 18H11-Hu-C53A to amino acids 111-115;
  • Figure 26 shows the determination of the binding ability of 18H11-Hu-C53A to amino acids 116-120;
  • Figure 27 shows the determination of the binding ability of 18H11-Hu-C53A to amino acids 120-124.
  • IL1-beta NCBI Reference: Sequence: NP_000567.1
  • TEV-hIgG1Fc hFc: Ig gamma-1 chain, C region, ACCESSION: P01857, 106-330
  • IL1b- TEV-hIgG1Fc TEV-hIgG1Fc
  • ENLYFQG is the recognition site of TEV digestion.
  • IL1RI (1-332) NCBI Reference: Sequence: NP_000868.1 amino acid sequence and His X6 tag are fused and designed as follows (IL1RI (1-332) -His):
  • Figure 1 shows the purification result of IL1 ⁇ -hFc (IL1b-TEV-hIgG1Fc), showing that the IL1 ⁇ -hFc protein was enriched and purified by Protein A column (HiTrapProtein A, HP, GE) and eluted, and the resulting protein was partially aggregated and recovered The eluted sample was concentrated and changed, and stored at -80 °C.
  • Figure 2 is the IL1RI (1-332) -HIS culture supernatant purification result diagram. IL1RI (1-332) -HIS protein was purified by Ni Sepharose excel column (GE Healthcare Life Sciences) and eluted, and the eluted sample was recovered and concentrated and exchanged. Store at -80 °C.
  • IL-1 ⁇ -His the molecular weight is 18572.14g / mol, the concentration is 1.068mg / ml, the volume is 1000 ⁇ L, and the total amount is 1068 ⁇ g.
  • the preparation method see Example 1.2.
  • IL1RI (1-332) -His molecular weight is 36859g / mol, concentration is 1.22mg / ml, volume is 1065 ⁇ L, total amount is 1.3mg, preparation method see example 1.1.
  • the volume of biotin required (ng number of protein to be labeled / molecular weight of protein to be labeled) * 20 * 557 / 5560ng / ml.
  • PcAb heavy chain variable region amino acid sequence (SEQ ID NO: 26)
  • PcAb heavy chain constant region amino acid sequence (SEQ ID NO: 27)
  • Emulsify the immunogen IL1 ⁇ -his according to the ratio of antigen and adjuvant volume of 1: 1, and use Freund's complete adjuvant (FCA, Sigma, F5881-10 ⁇ for the first immunization) 10ml) emulsified antigen; start the second immunization 2 weeks apart, use Freund's incomplete adjuvant (FIA, Sigma, F5881-10 ⁇ 10ml) emulsified antigen, subcutaneous injection at 5 o'clock, each mouse (BALB / C small Mouse, SPF grade, female, 6 weeks old) The amount of antigen injected was 50 ⁇ g, and the volume injected at each injection point was 50 ⁇ L.
  • the target positive clones obtained by cell fusion screening were subjected to two rounds of subcloning by the limiting dilution method, and each round of subcloning was screened by indirect ELISA method, and finally a stable cell line was obtained.
  • the obtained stable cell line is cultured with IMDM medium containing low IgG fetal bovine serum, and finally the cell culture medium supernatant is purified to obtain monoclonal antibodies.
  • Example (4) Dilute the antibody (see Example 1.5 for the preparation method of murine antibody, see Example 1.4 for the preparation method of the control antibody PcAb) to 1 ⁇ g / ml, and then dilute 3 times down to a total of 7 concentration gradients, and the dilution is the zero-point control. 50 ⁇ L / well, 37 ° C for 30min. Wash three times with PBST.
  • the information of the five murine antibodies is as follows:
  • HRP Conjugated Anti-Mouse IgG (1: 5000) (enzyme-labeled secondary antibody preparation solution: use a pipette to take Goat Anti-Mouse IgG (H + L), HRP secondary antibody mother liquor 1 ⁇ L, add to 5ml In 1% BSA buffer, mix by shaking. That is, 1: 5000 dilution of secondary antibody, which is now used.)
  • HRP conjugated Goat AntiHuman IgG (1: 5000) Take 1 ⁇ L of Goat AntiHuman IgG and HRP secondary antibody mother liquor, add it to 5 ml of 1% BSA buffer, and mix by shaking. That is, 1: 5000 dilution of secondary antibody, which is now ready for use.), 50 ⁇ L / well, 37 °C 30min . Wash 4 times with PBST.
  • KF021ZP4 9D5, KF021ZP4 18B1, KF021ZP4 18E1, KF021ZP4 18H11, KF021ZP4 19E4 can effectively block the binding of IL-1 ⁇ -hFc and IL1RI (1-332) -his.
  • MRC-5 cells (cell center of Chinese Academy of Sciences) with good growth status were digested with trypsin (Gibco), counted, inoculated into 96-well plates, and grown overnight.
  • a negative control group (MRC-5 cells + IL-1 ⁇ + mIgG or MRC-5 cells + IL-1 ⁇ + hIgG), a positive control group (MRC-5 cells + IL-1 ⁇ + PcAb),
  • MRC-5 cells + IL-1 ⁇ + antibodies of different concentrations In the experimental group (MRC-5 cells + IL-1 ⁇ + antibodies of different concentrations), cells were placed in a 37-degree cell incubator for 24 hours.
  • IL-1 ⁇ (Sino, concentration 588nM);
  • Antibody 9D5 (see Example 1.5 for preparation method, concentration 2.17 mg / mL);
  • Antibody 18E1 (see Example 1.5 for preparation method, concentration 1.8 mg / mL);
  • Antibody 18B1 (see Example 1.5 for the preparation method, concentration 1.56 mg / mL);
  • Antibody 18H11 (see Example 1.5 for preparation method, concentration 0.4 mg / mL);
  • Antibody 19E4 (see Example 1.5 for preparation method, concentration 0.14 mg / mL);
  • the detection results of IL-6 are shown in Figure 7.
  • the experimental results show that IL-1 ⁇ can induce MRC-5 cells to secrete IL-6, and antibodies 9D5, 18E1, 18B1, 18H11, and 19E4 can inhibit IL-1 ⁇ , effectively blocking IL-1 ⁇ and stimulate MRC-5 cells to secrete IL-6 This process.
  • 9D5, 18H11 and 19E4 are more effective in blocking IL-6 secretion induced by IL-1 ⁇ .
  • Balb / c mice SPF grade, female, 4-6 weeks old, 29, source: Beijing Vital Lihua Experimental Animal Technology Co., Ltd., animal quality certificate: 11400700113776.
  • Balb / c mice SPF grade, female, 6-8 weeks old, 18, source: Guangdong Medical Laboratory Animal Center, animal quality certificate: 44007200023548.
  • NIH / 3T3 ATCC, cell number is 15th generation
  • Antibody Anti-HEL2mg / ml Take 0.984ml (4.8mg) of the original solution (concentration: 4.88mg / ml) and add it to 1.416ml sodium chloride injection, a total of 2.4ml.
  • Antibody PcAb 2mg / ml Take the original solution (concentration: 7.65mg / ml) 0.627ml (4.8mg) and add it to 1.773ml sodium chloride injection for a total of 2.4ml.
  • Antibody 9D5 2mg / ml Take the original solution (concentration: 5.70mg / ml) 0.842ml (4.8mg) and add it to 1.558ml sodium chloride injection for a total of 2.4ml.
  • Antibody 18H11 2mg / ml Take the original solution (concentration: 3.31mg / ml) 1.450ml (4.8mg) was added to 0.950ml sodium chloride injection, a total of 2.4ml.
  • Antibody 19E4 2mg / ml Take 0.767ml (4.8mg) of the original solution (concentration: 6.26mg / ml) and add it to 1.633ml sodium chloride injection for a total of 2.4ml.
  • Balb / c mice were anesthetized by intraperitoneal injection of chloral hydrate, and the corresponding cell suspension was inoculated in the left knee joint of the mice.
  • the normal group was inoculated with NIH / 3T3 (the number of cells inoculated was 50,000 per mouse).
  • Others Groups (model group, positive drug group, 19E4 group, 18H11 group, and 9D5 group) were inoculated with Lenti-IL-1 ⁇ -NIH / 3T3 (the number of cells inoculated was 50,000 per cell).
  • mice On the 5th day after the inoculation, the mice were subjected to behavioral scoring, while the mice were euthanized, dissected, and the length and width of the white membrane of the knee joint were measured with a vernier caliper.
  • mice move normally and can move bilaterally.
  • the experimental results are shown in Table 3.
  • the body weight of the model group was significantly lower than that of the normal group (P ⁇ 0.01).
  • the positive drug had no obvious effect on the body weight of mice (P ⁇ 0.01); 19E4 reduced the body weight of mice slightly weaker than the model group; 18H11 and 9D5 had no obvious effect on the body weight of mice, which was equivalent to the positive drug (P> 0.05); 18H11 The effects of 9D5 and 9D5 on mouse body weight are equivalent (P> 0.05).
  • sequence information of the candidate antibodies 18H11, 19E4 and 9D5 are as follows:
  • 18H11 heavy chain variable region amino acid sequence is shown in SEQ ID NO: 7
  • nucleotide sequence is shown in SEQ ID NO: 15
  • light chain variable region amino acid sequence is shown in SEQ ID NO: 8
  • nucleotide sequence As shown in SEQ ID NO: 16.
  • amino acid sequence of 19E4 heavy chain variable region is shown in SEQ ID NO: 30, the nucleotide sequence is shown in SEQ ID NO: 31; the amino acid sequence of light chain variable region is shown in SEQ ID NO: 32, and the nucleotide sequence As shown in SEQ ID NO: 33.
  • 9D5 heavy chain variable region amino acid sequence is shown in SEQ ID NO: 34, nucleotide sequence is shown in SEQ ID NO: 35; light chain variable region amino acid sequence is shown in SEQ ID NO: 36, nucleotide sequence As shown in SEQ ID NO: 37.
  • the 18H11, 19E4, 9D5 heavy chain cDNA sequence (the heavy chain variable region sequence is shown in SEQ ID NO: 15, SEQ ID NO: 31, SEQ ID NO: 35; the constant region sequence is immunoglobulin gamma 2b heavy chain precursor [ Musmusculus] 140-475, ACCESSION: ACX70084.1) and light chain cDNA sequence (the light chain variable region sequence is shown in SEQ ID NO: 16, SEQ ID NO: 33, SEQ ID NO: 37; the constant region is an antibody kappa light chain, partial [Mus musculus], 106-213 GenBank: BAB33404.1) were cloned into pUC57simple (provided by Kingsray) vectors, and obtained pUC57simple-18H11H / 19E4H / 9D5.12H and pUC57simple-18H11L / 19E4L respectively /9D5.12L plasmid.
  • the plasmids pUC57simple-18H11H / 19E4H / 9D5.12H and pUC57simple-18H11L / 19E4L / 9D5.12L were digested (HindIII & EcoRI), and the heavy chain light chains recovered by electrophoresis were subcloned into pcDNA3.1 vectors respectively, and the recombinant plasmids were extracted. A total of 293F cells were transfected.
  • the culture solution was filtered by high-speed centrifugation and microporous filter vacuum, and then loaded onto the HiTrap MabSelectSuRe column, eluting protein with Elution Buffer in one step, recovering the target sample and changing the solution to PBS with HiTrap Desalting.
  • the test results show that: the recombinant antibodies 18H11 (Re), 19E4 (Re) and 9D5 (Re) all bind to IL1 ⁇ , and they have binding activity comparable to the mouse-derived antibodies 18H11, 19E4 and 9D5 and the positive control antibody PCAb; the recombinant antibody 18H11 ( Re), 19E4 (Re) and 9D5 (Re) can effectively block the binding of IL1 ⁇ and IL1RI.
  • the affinity of 18H11 (Re) and IL1RI for binding antigen IL1 ⁇ is comparable to that of mouse antibody 18H11 and positive control antibody PCAb.
  • the present invention divides the heavy chain and light chain variable regions into 14 structurally meaningful peptide segments, and compares them with the corresponding segments of known antibodies in the PDB database.
  • the corresponding segment with the highest sequence homology is selected from multiple sequence alignments to simulate the structure of this segment.
  • all the simulated structural segments are combined to construct the structure of the variable region.
  • antibody constant region sequence from the NCBI database, heavy chain constant region is Ig gamma-1chain, C region, ACCESSION: P01857, light chain constant The region is Ig kappa chain region, ACCESSION: P01834).
  • amino acid sequence of the heavy chain variable region of the humanized antibody 18H11 and H1L1 is shown in SEQ ID NO: 9, the nucleotide sequence is shown in SEQ ID NO: 17; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 10. The nucleotide sequence is shown in SEQ ID NO: 18.
  • the amino acid sequence of the heavy chain variable region of the humanized antibody 18H11 and H2L2 is shown in SEQ ID NO: 11, the nucleotide sequence is shown in SEQ ID NO: 19; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 12. The nucleotide sequence is shown in SEQ ID NO: 20.
  • amino acid sequence of the heavy chain variable region of the humanized antibody 18H11 and H3L3 is shown in SEQ ID NO: 13; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 14. The nucleotide sequence is shown in SEQ ID NO: 22.
  • the amino acid sequence of the heavy chain complementarity determining region is HCDR1: GYLFTGYY (SEQ ID NO: 1), HCDR2: ISCYNGDT (SEQ ID NO: 2) and HCDR3: SRSDYYGTSDY (SEQ ID NO: 3), the light chain complementarity determining region
  • the amino acid sequence is LCDR1: SSVSY (SEQ ID NO: 4), LCDR2: TTS (SEQ ID NO: 5) and LCDR3: QQRIIYPPT (SEQ ID NO: 6).
  • IL-1 ⁇ -his was coated with CBS dilution and incubated at 4 ° C overnight. Wash once with PBST. Add 1% BSA in PBS for blocking, 37 °C for 30min. Wash three times with PBST. The antibody (Table 4) was serially diluted at 37 ° C for 30 min. Wash three times with PBST. Add secondary antibody HRP conjugated Goat Anti Human IgG (1: 5000) (enzyme-labeled secondary antibody preparation solution: use a pipette to take Goat Anti Human Human IgG, HRP secondary antibody mother liquor 1 ⁇ L, add to 5ml of 1% BSA buffer solution, Shake to mix.
  • the secondary antibody is diluted 1: 5000, and it is now used.), 37 °C for 30min. Wash 4 times with PBST. Add 50 ⁇ L of TMB to each well for color development, stop the reaction with a stop solution after shading at room temperature for 5 min, and read at 450 nm.
  • IL-1 ⁇ -hFc was diluted and coated with CBS and incubated at 4 ° C overnight. Wash once with PBST. Add 1% BSA in PBS for blocking, 37 °C for 30min. Wash three times with PBST. The antibody (Table 4) was gradient diluted and incubated at room temperature for 10 minutes. IL1RI (1-332) -his was added, mixed well with the antibody, and then incubated at 37 ° C for 30 minutes. Wash three times with PBST. Add Mouse Anti-His, HRP conjugated (cwbio), 37 °C 30min. Wash 4 times with PBST. Add 50 ⁇ L of TMB to each well for color development, stop the reaction with a stop solution after shading at room temperature for 5 min, and read at 450 nm.
  • MRC-5 cells (cell center of Chinese Academy of Sciences) with good growth status were digested with trypsin (Gibco), counted, inoculated into 96-well plates, and grown overnight.
  • IL-1 ⁇ and PcAb and the antibody to be tested were incubated at 37 degrees for 20 minutes, and added to the cells for 24 hours. among them:
  • IL-1 ⁇ (Sino, concentration 588nM);
  • Test antibody 18H11H1L1 (concentration 5mg / mL)
  • Test antibody 18H11H2L2 (concentration 1.5mg / ml);
  • the detection results of IL-6 are shown in Figure 12.
  • the experimental results show that IL-1 ⁇ can induce MRC-5 cells to secrete IL-6, and antibodies 18H11H1L1 and 18H11H2L2 can inhibit IL-1 ⁇ , effectively blocking the process of IL-1 ⁇ stimulating MRC-5 cells to secrete IL-6.
  • the antibody 18H11H1L1 is more effective than the positive control antibody in inhibiting IL6 secretion induced by IL1 ⁇ , and the antibody 18H11H2L2 is equivalent to the positive control antibody.
  • a Fortebio Octet Qke molecular interaction instrument was used to detect the affinity of the antibody to IL1 ⁇ , and the AR2G sensor was activated using EDC / sulfo-NHS for 300 seconds.
  • 5 ⁇ g / mL antibody (10 mM sodium acetate, pH 6.0 dilution) was fixed on the sensor surface for 300 s.
  • the sensor uses 1M ethanolamine, pH 8.5 blocking, time 300s.
  • the sensor is equilibrated in PBST buffer for 300s.
  • the antibody immobilized on the sensor binds to IL1 ⁇ -his, the concentration of IL1 ⁇ -his is 1.56-100nM (using PBST two-fold gradient dilution), time 300s, the antigen antibody dissociates in PBST buffer, time 600s.
  • the data was fitted and analyzed with a 1: 1 model to obtain the affinity constant.
  • KD (M) kon (1 / Ms) kon Error kdis (1 / s) kdis Error Rmax (nM) KF021ZP4 18H11 H1L1 1.42E-10 3.36E + 05 3.79E + 03 4.78E-05 5.44E-06 0.1380-0.1905 KF021ZP4PCAB 1.79E-10 5.89E + 05 7.84E + 03 1.05E-04 5.97E-06 0.1289-0.1756
  • the purpose of this experiment is to examine the therapeutic effect of humanized antibody 18H11 H1L1 and mouse antibody 18H11 on the mouse knee arthritis model induced by Lenti-IL-1 ⁇ -NIH / 3T3.
  • Balb / c mice Balb / c mice; grade: SPF grade; age: 5-7 weeks; gender: female; body weight: 15-20g; number: 40; source: Guangdong Medical Laboratory Animal Center; animal quality certificate number: 44007200032490.
  • NIH / 3T3 source: ATCC; cell generation: 24th generation.
  • Lenti-IL-1 ⁇ -NIH / 3T3 source: Akesobio; cell generation: 21st generation.
  • NIH / 3T3 (P19) and Lenti-IL-1 ⁇ -NIH / 3T3 (P16) cells from liquid nitrogen, thaw quickly in a 37 ° C water bath, and add the cell suspension to 10% FBS (Gibco) and 1 % Pen / Strep (Gibco) in DMEM complete medium (Gibco), cultured at 37 °C, 5% CO 2 incubator; then NIH / 3T3, Lenti-IL-1 ⁇ -NIH / 3T3 was subcultured, and Lenti-IL-1 ⁇ -NIH / 3T3 cells were supplemented with Blasticidin S HCl (Gibco) for screening.
  • the IL-1 ⁇ content of the supernatant of Lenti-IL-1 ⁇ -NIH / 3T3 cells was detected according to the instructions of Human IL-1 ⁇ Precoated ELISA kit (Shenzhen Dake Biotechnology Co., Ltd.).
  • mice 40 Balb / c mice were randomly divided into 5 groups after weighing, namely Normal group, Isotype Control group, PcAb group, 18H11 H1L1 group, 18H11 group, 8 mice per group.
  • Dose design administration dose: 10mg / kg; administration volume: 10ml / kg; administration concentration: 1mg / ml; administration route: tail vein injection; administration frequency: once, administration before inoculation.
  • Model group Accurately measure 0.490ml of Anti-HEL (4.9mg / ml), dilute with 1.91ml of normal saline, and set aside;
  • PcAb group Accurately measure 0.505ml of KF021ZP4 PcAb (4.75mg / ml), dilute with 1.895ml normal saline, and set aside;
  • 18H11 Group H1L1 Accurately measure 0.759ml of KF021ZP4 18H11 H1L1 (3.16mg / ml), dilute with 1.641ml normal saline, and set aside;
  • Group 18H11 Accurately measure 0.712ml of KF021ZP4 18H11 (3.37mg / ml), dilute with 1.688ml normal saline, and set aside;
  • the model group was injected with Anti-HEL in the tail vein
  • the PcAb group was injected with the tail vein PcAb
  • the 18H11 H1L1 group was injected with the tail vein 18H11
  • the HH group was injected with the 18H11 tail vein
  • the normal group was given the tail vein Inject an equal volume of saline.
  • NIH / 3T3 and Lenti-IL-1 ⁇ -NIH / 3T3 cells reach the required number of inoculations, start collecting cells (the cell density should not exceed 80% of the culture flask area).
  • start collecting cells the cell density should not exceed 80% of the culture flask area.
  • aspirate the old culture medium wash once with PBS, add an appropriate amount of 0.05% Trypsin-EDTA (1x) (Gibco) for 1 minute at room temperature, and then add DMEM complete medium containing 10% FBS to stop the digestion.
  • the cell suspension was centrifuged at 1200 rpm / min for 4 minutes, the supernatant was removed, resuspended and counted using serum-free DMEM medium, the cell concentration was adjusted to 2 million / ml, and placed on ice for use.
  • mice After the mice were anesthetized, the normal group was inoculated with NIH / 3T3 cells in the right knee joint cavity of the mouse, 25 ⁇ l / mouse, that is, 50,000 cells / mouse. In the right knee joint cavity of the mouse, 25 ⁇ l / mouse was inoculated with 50,000 cells / mouse. After inoculation, the wound of the knee joint was sutured, and penicillin diluted 20 times was applied to prevent wound infection.
  • mice On the 5th day after cell inoculation, conduct behavioral scoring for each group of mice.
  • the behavioral scoring standard is: 0 points, the mice are normally active and can move bilaterally; 1 point, the mice walk abnormally and can move bilaterally; 2 Points, the affected limb of the mouse touched the ground briefly, but could move bilaterally; 3 points, the affected limb of the mouse could not touch the ground, and moved unilaterally; after scoring, the mice in each group were euthanized and the length of the knee synovium of the affected limb of the mouse was measured with a vernier caliper (mm) and width (mm), and calculate the knee joint area (mm 2 ).
  • the model group's knee joint area increased significantly (P ⁇ 0.01), the knee joint swelling.
  • the positive drug group PcAb group
  • 18H11 H1L1 group and 18H11 group can effectively inhibit the swelling area of the knee joint of the affected limbs in mice (P ⁇ 0.01); 18H11 H1L1 and 18H11 have the same efficacy as the positive drugs.
  • Example 6.1 According to the method of Example 6.1, the binding affinity test of each mutant antibody to the antigen IL1 ⁇ was carried out.
  • Example 10 Mutant antibody inhibits IL-6 secretion induced by IL-1 ⁇
  • the experimental method refers to Example 6.3. Based on the experimental results of Example 9, we selected antibodies with a good binding affinity to IL-1 ⁇ , and measured their inhibitory activity against IL-6 secretion induced by IL-1 ⁇ in MRC-5 cells.
  • IC 50 is 2.416nM and 2.323nM respectively.
  • Example 6.1 and 6.3 Determine the binding affinity of each sample to IL-1 ⁇ and inhibit IL-1 ⁇ -induced activity of IL-6 secretion from MRC-5 cells.
  • the results show that 18H11-Hu-C53I, 18H11-Hu-C53A and 18H11 H1L1 (that is, 18H11-Hu-WT) were bathed at 40 °C from 0d to 28d.
  • 18H11 H1L1 inhibited IL-1 ⁇ -induced MRC-5
  • the activity of cells secreting IL-6 decreased by approximately 2.9 times
  • 18H11-Hu-C53I decreased by approximately 6.2 times
  • 18H11-Hu-C53A decreased by approximately 1.5 times.
  • the IC 50 of each sample is shown in the table.
  • the relative thermal stability of 18H11-Hu-C53A is better than that of 18H11 H1L1 and 18H11-Hu-C53I.
  • Example 12 Affinity test of 18H11-Hu-C53A competing with IL1RI for binding antigen IL1 ⁇
  • Example 6.2 According to the method of Example 6.2, the affinity test of the mutant antibody 18H11-Hu-C53A and IL1RI for binding to the antigen IL1 ⁇ was carried out.
  • 18H11 H1L1,18H11-Hu-C53A and PCAb and IL1RI block 20 IL1 ⁇ binding IC 50 are 0.028nM, 0.028nM and 0.030nM, indicate 18H11-Hu-C53A can be effectively blocked with ILl [beta]
  • the binding of IL1RI is comparable to that of 18H11 H1L1 and the positive control antibody PCAb.
  • the binding kinetic parameters of 18H11, H1L1, 18H11-Hu-C53A, and PCAb and IL1 ⁇ were measured by Biacore.
  • the method is as follows: capture method, Protein A chip (purchased from GE, batch number 10261132) to capture 0.5 ⁇ g / mL antibody, and set contact time (Contact time) is 75s, flow rate is 10 ⁇ L / min, and regeneration contact time is 30s.
  • Antigen is used as an analyte.
  • the contact time is 180s
  • the dissociation time is 900s
  • the flow rate is 30 ⁇ L / min
  • the regeneration contact time is 30s.
  • the kinetic parameters shown in the test results are shown in Table 8.
  • mice Take adaptively cultured Balb / c female mice (5-7 weeks, 15-20g), divided into 7 groups according to body weight, 8 mice in each group; intraperitoneal injection of antibody 18H11-Hu-C53A, the dose is as follows: 2.5mg / kg, 0.5mg / kg, 0.1mg / kg, the control group was injected with PBS or the same subtype control IgG.
  • each mouse was injected subcutaneously with 5 ⁇ g of recombinant human IL-1 ⁇ -His; and blood was collected from each group of mice 4 hours after injection of IL-1 ⁇ ; serum was separated at 4 ° C and serum IL- ⁇ was determined by ELISA 6 expression level to determine the inhibitory effect of different doses of antibody on murine IL-6 secretion.
  • the cross-species immunoreactivity of 18H11-Hu-C53A was determined by ELISA.
  • the cynomolgus monkey and rat IL-1 ⁇ proteins namely Macaca-IL-1 ⁇ (NCBI Reference Sequence: NP_001270498.1) and Rat-IL-1 ⁇ (NCBI Reference Sequence: NP_113700.2) were coated in 96-well ELISA at 0.2 ⁇ g / well Plate to determine the cross-reaction of 18H11-Hu-C53A to these two species IL-1 ⁇ .
  • Macaca-IL-1 ⁇ NCBI Reference Sequence: NP_001270498.1
  • Rat-IL-1 ⁇ NCBI Reference Sequence: NP_113700.2
  • the preparation methods of Macaca-IL-1 ⁇ and Rat-IL-1 ⁇ refer to Example 1.1.
  • Example 16 Determination of the selectivity of 18H11-Hu-C53A for the family member proteins IL-1, alpha-1, IL-1 R2 and IL-1 RA
  • the human IL-1 alpha, IL-1 R2 and IL-1 RA proteins (all purchased from Sino Biological Company, the article numbers are 10128-HNCH-20, 10111-H08H-50 and 10123-HNAE-100), according to 0.2
  • the 96-well ELISA plate was coated with ⁇ g / well to determine the selectivity of 18H11-Hu-C53A to these proteins.
  • Example 6.1 For other specific experimental methods, refer to Example 6.1.
  • IL-1 ⁇ According to the spatial structure of IL-1 ⁇ (SEQ ID NO: 23, positions 1-153), it is divided into two protein fragments for expression and purification, namely IL-1 ⁇ -A1-F99-His (position 1 alanine A to Phenylalanine F at position 99, with 6 ⁇ His tag at C-terminus) and IL-1 ⁇ -A1-W120-His (Alanine A at position 1 to tryptophan W at position 120, at C-terminus There is a 6 ⁇ His tag).
  • IL-1 ⁇ -A1-F99-His Dilute IL-1 ⁇ -A1-F99-His, IL-1 ⁇ -A1-W120-His and IL-1 ⁇ -WT-His (that is, IL-1 ⁇ -his) to 0.5 ⁇ g / mL with coating solution, and coat ELISA plate to determine the affinity of 18H11-Hu-C53A with each protein.
  • IL-1 ⁇ -his Dilute IL-1 ⁇ -A1-F99-His, IL-1 ⁇ -A1-W120-His and IL-1 ⁇ -WT-His (that is, IL-1 ⁇ -his) to 0.5 ⁇ g / mL with coating solution, and coat ELISA plate to determine the affinity of 18H11-Hu-C53A with each protein.
  • IL-1 ⁇ -his Dilute IL-1 ⁇ -A1-F99-His, IL-1 ⁇ -A1-W120-His and IL-1 ⁇ -WT-His (that is, IL-1
  • the results show that 18H11-Hu-C53A does not bind to IL-1 ⁇ -A1-F99-His at all, but it still has a certain affinity for IL-1 ⁇ -A1-W120-His, but it is lower than IL- 1 ⁇ -WT-His. Therefore, the primers were designed to carry out single point mutations by alanine scanning (Alanine scanning) between the 99th amino acid F and the 120th amino acid W of IL-1 ⁇ and other amino acids in the vicinity of the latter, and carry out expression purification , Refer to the above method to determine the binding epitope of 18H11-Hu-C53A.

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Abstract

本发明公开了一种全新结构的可结合人IL-1β的抗体或其抗原结合片段，其具有良好的阻断IL-1β与其受体相结合，从而下调IL-1β活性的生物学功能，可应用于制备治疗IL-1β过表达介导的免疫类疾病(例如关节炎、骨质疏松、肿瘤坏死因子受体相关周期性综合症等)的药物，具有良好的临床应用前景。

Description

结合人IL-1β的抗体、其制备方法和用途 技术领域
本发明涉及抗体领域，更具体地，本发明公开了一种结合人IL-1β的抗体；
本发明还涉及上述抗体的制备方法和用途。
背景技术
白介素1蛋白包括白介素1α(IL-1α)和白介素1β(IL-1β)。IL-1β是一种多功能性细胞因子，介导多种淋巴细胞的生长和分化，并参与与调控多种炎症反应过程。IL-1β在体内的分泌水平受IL-1家族如IL-1α及IL-1R抑制剂(IL-1Ra)等的调控。
多种免疫细胞如巨噬细胞表达IL-1α和分泌IL-1β。IL-1β受体有IL-1I型受体(IL-1RI)和IL-1 II型受体(IL-1RII)。其中，IL-1RI几乎所有有核细胞均有表达，IL-1β结合IL-1RI进而引起IL-1受体(IL-1R)辅助蛋白(IL-1RAcP)的聚集并形成复合物，从而激活信号通路；IL-1RII同时以细胞膜表面表达和可溶性形式存在于体内，IL-1β结合IL-1RII后将下调IL-1β的活性。
多种研究表明，IL-1β的过表达是引起多种免疫类疾病的主要原因如Cryopyrin蛋白相关综合征、肿瘤坏死因子受体相关周期性综合症、全身型幼年特发性关节炎、高免疫球蛋白D综合证(HIDS)/甲羟戊酸激酶缺乏症(MKD)、骨质疏松、骨关节炎以及其他炎症性关节炎。在多种炎症及自发免疫类疾病中，血清中IL-1β的含量随着病情的恶化及严重程度而升高(Pascual V,Allantaz F,et al.,Role of interleukin-1(IL-1)in the pathogenesis of systemic onset juvenile idiopathic arthritis and clinical response to IL-1 blockade.J Exp Med 2005；201:1479-86)。另外有研究表明，IL-1β在TH17细胞分化成熟中起重要促进作用(de Jong E,Suddason T,Lord GM.Translational mini-review series on Th17 cells:development of mouse and human T helper 17 cells.Clin Exp Immunol.2010 Feb,159(2):148-58)。成熟的TH17细胞能分泌IL-17，促进多种免疫类疾病如银屑病的发生。因此IL-1β抑制剂能阻断IL-1信号通路，在治疗骨质疏松、炎症性关节炎及其他免疫类疾病治疗中具有重要作用。
因此研发出有效的抗IL-1β抗体来满足患者的用药需求一直是本领域的人员着力解决的问题。
发明内容
为了解决上述问题，本发明的发明人经过了大量试验，从抗原免疫、杂交瘤筛选、抗体表达纯化到生物活性鉴定，筛选获得到了一系列结合人IL-1β的抗体。本发明对候选鼠源抗体和人源化抗体开展了体内药理学研究，结果显示，鼠源抗体19E4、18H11、9D5都可以明显降低IL-1β诱导的小鼠关节炎病变，人源化抗体18H11 H1L1在剂量10mg/kg下,能明显改善小鼠患肢的行走行为以及明显减少患肢膝关节的肿胀面积。因此，本发明公开的具有全新 结构的结合人IL-1β的抗体有望成为治疗关节炎、骨质疏松、及其他免疫类疾病的潜在治疗药物。
因此，本发明的第一个目的在于提供一种结合人IL-1β的抗体或其抗原结合片段，该抗体的重链互补决定区H-CDR1、H-CDR2、H-CDR3的氨基酸序列分别为SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3；其轻链互补决定区L-CDR1、L-CDR2、L-CDR3的氨基酸序列分别为SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6。
其中，本发明公开的结合人IL-1β的抗体为鼠源抗体18H11，其重链可变区氨基酸序列为SEQ ID NO：7，轻链可变区氨基酸序列为SEQ ID NO：8。
其中，本发明公开的结合人IL-1β的抗体为人源化抗体18H11H1L1，其重链可变区氨基酸序列为SEQ ID NO：9，轻链可变区氨基酸序列为SEQ ID NO：10。
其中，本发明公开的结合人IL-1β的抗体为人源化抗体18H11H2L2，其重链可变区氨基酸序列为SEQ ID NO：11，轻链可变区氨基酸序列为SEQ ID NO：12。
其中，本发明公开的结合人IL-1β的抗体为人源化抗体18H11H3L3，其重链可变区氨基酸序列为SEQ ID NO：13，轻链可变区氨基酸序列为SEQ ID NO：14。
其中，本发明所述的抗原结合片段包括Fab片段、F(ab’)2片段、Fv片段、单链抗体或单域抗体。
本发明的第二个目的在于提供编码所述结合人IL-1β的抗体或其抗原结合片段的核苷酸分子。
其中，编码鼠源抗体18H11重链可变区的核苷酸序列为SEQ ID NO：15，编码轻链可变区的核苷酸序列为SEQ ID NO：16。
其中，编码人源化抗体18H11H1L1重链可变区的核苷酸序列为SEQ ID NO：17，编码轻链可变区的核苷酸序列为SEQ ID NO：18。
其中，编码人源化抗体18H11H2L2重链可变区的核苷酸序列为SEQ ID NO：19，编码轻链可变区的核苷酸序列为SEQ ID NO：20。
其中，编码人源化抗体18H11H3L3重链可变区的核苷酸序列为SEQ ID NO：21，编码轻链可变区的核苷酸序列为SEQ ID NO：22。
本发明的第三个目的在于提供含有所述核苷酸分子的表达载体。
本发明的第四个目的在于提供含有所述表达载体的宿主细胞。
本发明的第五个目的在于提供所述的结合人IL-1β的抗体或其抗原结合片段的制备方法，包括以下步骤：
(a)在表达条件下，培养如上所述的宿主细胞，从而得到所述的结合人IL-1β的抗体或其抗原结合片段；
(b)分离并纯化(a)所述的结合人IL-1β的抗体或其抗原结合片段。
本发明的第六个目的在于提供含有所述的结合人IL-1β的抗体或其抗原结合片段的药物组合物。所述药物组合物含有如上任一项所述的结合人IL-1β的抗体或其抗原结合片段和药 学上可接受的载体。
本发明的第七个目的在于提供所述的结合人IL-1β的抗体或其抗原结合片段或所述的药物组合物的用途。所述用途是用于制备治疗由于IL-1β的过表达引起的多种免疫类疾病的药物，如关节炎、骨质疏松或银屑病等。根据本发明的优选实施例，所述用途是用于制备治疗关节炎的药物。
本发明的第八个目的在于提供一种结合人IL-1β的抗体或其抗原结合片段，所述抗体或其抗原结合片段的重链互补决定区H-CDR2包括C53A突变位点，所述抗体18H11-Hu-C53A的重链互补决定区H-CDR2(IMGT编码方式)：ISAYNGDT，其氨基酸序列如SEQ ID NO：38所示。
本发明也提供上述抗体与人IL-1β的结合表位：主要结合表位分别为SEQ ID NO：23的第120位的色氨酸W和第122位的异亮氨酸I，其次为第112位的苯丙氨酸F、第123位的丝氨酸S和第124位的苏氨酸T。
有益效果：
1、本发明筛选获得的结合人IL-1β的抗体能够特异性与人IL-1β结合，ELISA测试结果表明，其中鼠源抗体18H11的EC 50与阳性对照抗体相当，人源化抗体18H11H1L1的EC 50优于阳性对照抗体。
2、本发明筛选获得的结合人IL-1β的抗体能够有效地竞争性阻断IL-1β与其相关受体的结合，其中，鼠源抗体18H11和人源化抗体18H11H1L1的EC 50均与阳性对照抗体相当。
3、本发明筛选获得的结合人IL-1β的抗体能够有效地抑制由IL1β诱导的MRC-5细胞分泌IL-6。其中，鼠源抗体18H11和人源化抗体18H11H1L1抑制由IL-1β诱导细胞的IL-6分泌效果较阳性对照抗体更优。
4、本发明筛选获得的结合人IL-1β的抗体18H11H1L1，经Fortebio Kinetics分析，其抗体亲和力与阳性对照抗体相当。
5、本发明筛选获得的结合人IL-1β的抗体能够明显降低IL-1β诱导的小鼠关节炎病变。其中，鼠源抗体18H11和人源化抗体18H11H1L1在小鼠行为学评分、关节肿胀影响/膝关节面积、体重影响等方面的治疗效果与阳性对照抗体相当。
6、本发明获得的以18H11 H1L1的重链为模板，在H-CDR2包含C53A位点的突变抗体18H11-Hu-C53A，其比野生型抗体具有更好的热稳定性。
本发明中，术语“抗体(Ab)”和“免疫球蛋白G(IgG)”是有相同结构特征的约150000道尔顿的异四聚糖蛋白，其由两个相同的轻链(L)和两个相同的重链(H)组成。每条轻链通过一个共价二硫键与重链相连，而不同免疫球蛋白同种型的重链间的二硫键数目不同。每条重链和轻链也有规则间隔的链内二硫键。每条重链的一端有可变区(VH)，其后是多个恒定区。每条轻链的一端有可变区(VL)，另一端有恒定区；轻链的恒定区与重链的第一个恒 定区相对，轻链的可变区与重链的可变区相对。本发明的抗体可以是单克隆抗体、多克隆抗体、由至少两种抗体形成的多特异性抗体(例如双特异性抗体)、抗体的抗原结合片段等。本发明的抗体包括鼠源抗体、嵌合抗体、人源化抗体等。
本发明中，术语“单克隆抗体(单抗)”指从一类基本均一的群体获得的抗体，即该群体中包含的单个抗体是相同的，除少数可能存在的天然发生的突变外。单克隆抗体高特异性地针对单个抗原位点。而且，与常规多克隆抗体制剂(通常是具有针对不同决定簇的不同抗体)不同，各单克隆抗体是针对抗原上的单个决定簇。除了它们的特异性外，单克隆抗体的好处还在于它们是通过杂交瘤培养来合成的，不会被其它免疫球蛋白污染。修饰语“单克隆”表示了抗体的特性，是从基本均一的抗体群中获得的，这不应被解释成需要用任何特殊方法来生产抗体。
本发明中，术语“鼠源抗体”是指来源于大鼠或小鼠的抗体，优选小鼠。本发明的鼠源抗体为使用人IL-1β为抗原免疫小鼠并进行杂交瘤细胞筛选获得。更优选的，本发明的鼠源抗体包括19E4、18H11、9D5。最优选的，本发明的鼠源抗体为18H11。
本发明中，术语“人源化抗体”是指，人源免疫球蛋白(受体抗体)的全部或部分CDR区被一非人源抗体(供体抗体)的CDR区替换后得到的抗体或抗体片段，其中的供体抗体可以是具有预期特异性、亲和性或反应性的非人源(例如，小鼠、大鼠或兔)抗体。此外，受体抗体的构架区(FR)的一些氨基酸残基也可被相应的非人源抗体的氨基酸残基替换，或被其他抗体的氨基酸残基替换，以进一步完善或优化抗体的性能。关于人源化抗体的更多详细内容，可参见例如，Jones et al.,Nature,321:522 525(1986)；Reichmann et al.,Nature,332:323 329(1988)；Presta,Curr.Op.Struct.Biol.,2:593 596(1992)；和Clark,Immunol.Today 21:397 402(2000)。优选的，本发明的人源化抗体由鼠源抗体18H11的CDR区和来源自人抗体的非CDR区重组。更优选的，本发明的人源化抗体包括18H11 H1L1、18H11 H2L2和18H11 H3L3。最优选的，本发明的人源化抗体为18H11 H1L1。
本发明中，术语“可变”表示抗体中可变区的某些部分在序列上有所不同，它形成各种特定抗体对其特定抗原的结合和特异性。然而，可变性并不均匀地分布在整个抗体可变区中。它集中于轻链和重链可变区中称为互补决定区(CDR)或超变区中的三个片段中。可变区中较保守的部分称为框架区(FR)。天然重链和轻链的可变区中各自包含四个FR区，它们大致上呈β-折叠构型，由形成连接环的三个CDR相连，在某些情况下可形成部分β折叠结构。每条链中的CDR通过FR区紧密地靠在一起并与另一链的CDR一起形成了抗体的抗原结合部位(参见Kabat等，NIH Publ.No.91-3242，卷I，647-669页(1991))。恒定区不直接参与抗体与抗原的结合，但是它们表现出不同的效应功能，例如参与抗体依赖的细胞介导的细胞毒性作用(ADCC，antibody-dependent cell-mediated cytotoxicity)等。
本发明中，术语“抗原结合片段”是指能够与人IL-1β特异性结合的抗体的片段。本发明的抗原结合片段的例子包括Fab片段、F(ab’)2片段、Fv片段、单链抗体(scFv)、单域抗体(sdAb)等。Fab片段是用木瓜蛋白酶消化抗体产生的片段。F(ab’)2片段是用胃蛋白酶消化 抗体产生的片段。Fv片段是由抗体的重链可变区和轻链可变区紧密非共价关联的二聚物组成。单链抗体(scFv)，是由抗体重链可变区和轻链可变区通过15～20个氨基酸的短肽(linker)连接而成的抗体。单域抗体(sdAb)又称为纳米抗体(nanobody)或重链抗体，仅由重链构成，其抗原结合区仅是一个通过铰链区与Fc区连接的单结构域。
本发明中，术语“结合”、“特异性结合”是指两分子间的非随机的结合反应，如抗体和其所针对的抗原之间的反应。通常，抗体以小于大约10 -7M，例如小于大约10 -8M、10 -9M、10 -10M、10 -11M或更小的平衡解离常数(KD)结合该抗原。本发明中，术语“KD”是指特定抗体-抗原相互作用的平衡解离常数，其用于描述抗体与抗原之间的结合亲和力。平衡解离常数越小，抗体-抗原结合越紧密，抗体与抗原之间的亲和力越高。例如，使用表面等离子体共振术(Surface Plasmon Resonance，缩写SPR)在BIACORE仪中测定抗体与抗原的结合亲和力或使用ELISA测定抗体与抗原结合的相对亲和力。
本发明中，术语“表位”和“人IL-1β表位”是指位于人IL-1β上并与抗体特异性结合相关的区域。
本发明中，术语“表达载体”可以为pTT5，pSECtag系列，pCGS3系列，pCDNA系列载体等，以及其它用于哺乳动物表达***的载体等，表达载体中包括连接有合适的转录和翻译调节序列的融合DNA序列。
本发明中，术语“宿主细胞”是指适用于表达上述表达载体的细胞，可以是真核细胞，如哺乳动物或昆虫宿主细胞培养***均可用于本发明的融合蛋白的表达，CHO(中国仓鼠卵巢，Chinese Hamster Ovary)，HEK293，COS、BHK等及上述细胞的衍生细胞均可适用于本发明。
本发明中，术语“细胞”和“细胞系”可互换使用。
本发明中，术语“药物组合物”是指本发明的结合人IL-1β的抗体可以和药学上可以接受的载体一起组成药物制剂组合物从而更稳定地发挥疗效，这些制剂可以保证本发明公开的结合人IL-1β的抗体的氨基酸核心序列的构像完整性，同时还保护蛋白质的多官能团防止其降解(包括但不限于凝聚、脱氨或氧化)。
本发明中，术语“IL-1β过表达的疾病”是指处于异常疾病状态的细胞中IL-1β的表达水平高于相同组织类型的正常细胞中的IL-1β表达水平。本发明的IL-1β过表达的疾病包括但不限于骨质疏松、骨关节炎以及其他炎症性关节炎、银屑病等多种免疫类疾病。
以下实施例是对本发明进行进一步的说明，不应理解为对本发明的限制。实施例不包括对传统方法的详细描述，如那些用于构建载体和质粒的方法，将编码蛋白的基因***到这样的载体和质粒的方法或将质粒引入宿主细胞的方法。这样的方法对于本领域中具有普通技术的人员是众所周知的，并且在许多出版物中都有所描述，包括Sambrook,J.,Fritsch,E.F.and Maniais，T.(1989)Molecular Cloning：A Laboratory Manual，2nd edition，Cold spring Harbor Laboratory Press。
本说明书中，缩写的术语含义说明如下：
IL-1α Interleukin-1 alpha 白介素1α
IL-1β/IL-1b Interleukin-1 beta 白介素1 beta
IL-1R Interleukin-1 receptor 白介素1受体
IL-1RI Interleukin-1 receptor,type I 白介素1受体，I型
IL-1RII Interleukin-1 receptor,type II 白介素1受体，II型
IL-1Ra Interleukin-1 receptorantagonist 白介素1 I型受体拮抗剂
IL-1RAcP Interleukin 1 receptoraccessory protein 白介素1 I型受体辅助蛋白
IL-6 Interleukin-6 白细胞介素-6
PcAb Positive control antibody 阳性对照抗体
PBS Phosphate buffer saline 磷酸盐缓冲液
cDNA Complementary deoxyribonucleic acid 互补脱氧核糖核酸
SDS-PAGE Polyacrylamide gelelectrophoresis 聚丙烯酰胺凝胶电泳
ELISA Enzyme-linked immunosorbent assay 酶联免疫吸附试验
EC 50 Median effective concentration 半数有效浓度
附图说明
图1为IL1β-hFc纯化结果图，其中，M：蛋白质分子量标准，1：上纯化柱前，2：上纯化柱后穿透，3-9：纯化柱洗脱。IL1β-hFc蛋白理论大小43.7kDa，蛋白二聚体分子量约为87kDa。
图2为IL1RI(1-332)-HIS培养上清纯化结果图，M：蛋白质分子量标准，1：上纯化柱前，2：上纯化柱后穿透，3-4：纯化柱洗脱。
图3为IL1b-His纯化结果图，M：蛋白质分子量标准，FT：上纯化柱后穿透，load：上纯化柱前，1-7：纯化柱洗脱。IL1b-His蛋白理论大小18.2kDa，蛋白不存在二聚体分子。
图4为PcAb纯化结果图，M：蛋白质分子量标准，1：非还原的抗体，2：还原的抗体，3：BSA对照。PcAb蛋白理论大小150kDa，抗体重链分子量约为45kDa，轻链分子量约为30kDa。
图5为通过ELISA的方法测定鼠源抗体与抗原IL1β结合亲和力的结果。
图6为通过ELISA的方法测定鼠源抗体与IL1RI竞争结合抗原IL1β的亲和力结果。
图7为通过ELISA的方法测定鼠源抗体抑制由IL1β诱导的MRC5细胞分泌IL-6的结果图。
图8为鼠源抗体19E4、18H11以及9D5对小鼠的行为学的影响结果。与正常组比较， **P<0.01；与模型组比较， ##P<0.01；与阳性药组比较， ΔP<0.01；n＝8(正常组n＝7)。
图9为鼠源抗体19E4、18H11以及9D5对小鼠的膝关节肿胀的影响结果。与正常组比较， **P<0.01；与模型组比较， ##P<0.01；与阳性药组比较， ΔP<0.01；与18H11组比较， ▲P<0.05；n＝8(正常组n＝7)。
图10为通过ELISA的方法测定人源化抗体与抗原IL1β结合亲和力的结果。
图11为通过ELISA的方法测定人源化抗体与IL1RI竞争结合抗原IL1β的亲和力结果。
图12为通过ELISA的方法测定人源化抗体抑制由IL1β诱导的MRC5细胞分泌IL-6的结果图。
图13为结合人IL-1β抗体18H11 H1L1和18H11对Lenti-IL-1β-NIH/3T3诱导的小鼠膝关节炎模型小鼠病理性行为的影响结果。
图14为结合人IL-1β抗体18H11 H1L1和18H11对Lenti-IL-1β-NIH/3T3诱导的小鼠膝关节炎模型小鼠膝关节面积的影响结果。
图15为各突变抗体SDS-PAGE蛋白电泳结果。
图16为各突变抗体及18H11 H1L1(WT)与IL1β结合的EC 50。
图17为测定抗体抑制由IL-1β诱导MRC-5细胞的IL-6分泌活性。
图18为测定各样品与IL-1β结合亲和力。
图19为测定各样品抑制由IL-1β诱导MRC-5细胞分泌IL-6的活性。
图20为18H11-Hu-C53A与IL1RI竞争结合抗原IL1β的亲和力检测。
图21为各不同剂量抗体对鼠IL-6分泌的抑制作用。
图22为ELISA的方法测定18H11-Hu-C53A的跨种属免疫反应性。
图23为18H11-Hu-C53A对家族成员蛋白IL-1 alpha、IL-1 R2和IL-1 RA的选择性的测定。
图24-图27为18H11-Hu-C53A对IL-1β的结合表位确定。
图24为18H11-Hu-C53A与IL-1β-A1-F99-His和IL-1β-A1-W120-His的亲和力；
图25显示18H11-Hu-C53A与第111-115位氨基酸的结合力测定；
图26显示18H11-Hu-C53A与第116-120位氨基酸的结合力测定；
图27显示18H11-Hu-C53A与第120-124位氨基酸的结合力测定。
具体实施方式
实施例1 抗原及候选抗体的制备
1.1 IL1β-hFc和IL1RI-his的制备
(1)将人源IL1-beta(NCBI Reference Sequence:NP_000567.1)氨基酸序列与TEV-hIgG1Fc(hFc:Ig gamma-1 chain C region，ACCESSION：P01857，106-330)进行融合设计如下(IL1b-TEV-hIgG1Fc)：
氨基酸序列：(SEQ ID NO：23)
Figure PCTCN2019115230-appb-000001
Figure PCTCN2019115230-appb-000002
其中， ENLYFQG为TEV酶切识别位点。
(2)将人源IL1RI(1-332)(NCBI Reference Sequence:NP_000868.1)氨基酸序列与His X6 tag进行融合设计如下(IL1RI(1-332)-His)：
氨基酸序列：(SEQ ID NO：24)
Figure PCTCN2019115230-appb-000003
(3)将上述设计的融合蛋白(IL1b-TEV-hIgG1Fc和IL1RI(1-332)-His)所对应的氨基酸序列进行密码子人工优化(IL1b-TEV-hIgG1Fc N端需要加信号肽)，并委托金斯瑞公司合成优化后的DNA，克隆到pUC57simple(金斯瑞公司提供)载体中，获得pUC57simple-IL1b-TEV-hIgG1Fc和pUC57simple-IL1RI(1-332)-His质粒。
(4)将上述两个质粒进行酶切后(Xba I和BamH I)，电泳回收得到基因片段IL1b-TEV-hIgG1Fc和IL1RI(1-332)-His分别与pcDNA3.1线性化载体(XbaI&BamHI)进行连接反应重组构建获得pcDNA3.1-IL1b-TEV-hIgG1Fc和pcDNA3.1-IL1RI(1-332)-His质粒。
(5)将上面构建的两个重组质粒分别转染FreeStyle TM 293-F Cells细胞(Invitrogen)7天后，将培养液进行高速离心(4000rpm 20min)、微孔滤膜抽真空过滤(0.45um微孔滤膜)，根据厂家提供的操作方法分别采用Protein A柱和Ni柱(蛋白纯化液相色谱***/AKTA Purifier 10,GE)进行纯化，得到纯化的IL1b-TEV-hIgG1Fc和IL1RI(1-332)-His融合蛋白。
实验结果如图1和图2。图1是IL1β-hFc(IL1b-TEV-hIgG1Fc)纯化结果图，显示IL1β-hFc蛋白用Protein A柱(HiTrap Protein A HP，GE)富集纯化后洗脱，得到的蛋白发生部分聚体，回收洗脱样品浓缩换液，-80℃保存。图2是IL1RI(1-332)-HIS培养上清纯化结果图，IL1RI(1-332)-HIS蛋白用Ni Sepharose excel柱(GE HealthcareLife Sciences)纯化后洗脱，回收洗脱样品浓缩换液，-80℃保存。
1.2 IL1β-his的制备
(1)将人源IL1-beta(NCBI Reference Sequence:NP_000567.1)氨基酸序列与His X6 tag进行融合设计如下(IL1b-His)：
氨基酸序列：(SEQ ID NO：25)
Figure PCTCN2019115230-appb-000004
Figure PCTCN2019115230-appb-000005
(2)将上述设计的融合蛋白所对应的氨基酸序列进行密码子人工优化，并委托金斯瑞公司合成优化后的DNA，克隆到pUC57simple(金斯瑞公司提供)载体中，获得pUC57simple-IL1b-His质粒。
(3)将上述质粒进行酶切后(Nco I和Xho I)，电泳回收得到基因片段并与pET28a线性化载体(NcoI&XhoI)进行连接反应重组构建获得pET28a-IL1b-His质粒。
(4)将上面构建好的质粒转化BL21(DE3)感受态获得表达菌株，挑克隆鉴定正确后扩大培养。OD值达到0.8-1.0时，加入终浓度1mM IPTG(Amresco)16度诱导20h；离心收菌，用buffer A(50mM Tris-HCl,300mM NaCl,10mM Imidazole,5mM B-ME,10％glycerol,pH 8.0(控内毒素))重悬菌体再高压破碎，高速离心1h，0.45μm微孔滤膜抽真空过滤获得上清。根据厂家提供的操作方法采用Ni亲和层析柱HisTrap FF(蛋白纯化液相色谱***/AKTA Purifier 10,GE)进行纯化，得到纯化的IL1b-His融合蛋白。
实验结果如图3IL1b-His纯化结果图。IL1b-His蛋白用Ni亲和层析柱富集纯化后洗脱，回收洗脱样品浓缩换液，-80℃保存，后续QC检测结果显示没有聚体出现。
1.3 IL1β-his-bio和IL1RI-his-bio的制备
(1)准备好待标记的蛋白：
IL-1β-His:分子量为18572.14g/mol，浓度1.068mg/ml，体积1000μL，总量为1068μg,制备方法见实施例1.2。
IL1RI(1-332)-His：分子量为36859g/mol，浓度1.22mg/ml，体积1065μL，总量为1.3mg,制备方法见实施例1.1。
(2)将商购的生物素(分子量为557g/mol，Thermo)粉末放置室温平衡约15min，准确称量3mg，溶解于540μL的去内毒素的水中，浓度为5560ng/ml。混匀备用。
(3)计算需要加入到蛋白中的生物素的体积(μL)：
需要的生物素的体积＝(待标记的蛋白的ng数/待标记的蛋白的分子量)*20*557/5560ng/ml。
(4)分别加入相应体积的5560ng/ml的生物素溶液到待标记的2种蛋白的溶液中，冰上静置1h。
(5)通过HiTrap Desalting柱(GE)，在蛋白纯化仪AKTA Purifier UPC 100(GE)中进行换液成PBS，并去掉游离的生物素，将蛋白分装，液氮速冻后进入-80℃保存。
1.4 阳性对照抗体PcAb的制备
(1)根据已有的人源IL1-beta(NCBI Reference Sequence:NP_000567.1)蛋白序列及参照专利US8273350B2抗人IL1-beta的抗体序列，PcAb抗体序列如下：
PcAb的重链可变区氨基酸序列：(SEQ ID NO：26)
Figure PCTCN2019115230-appb-000006
PcAb的重链恒定区氨基酸序列：(SEQ ID NO：27)
Figure PCTCN2019115230-appb-000007
PcAb的轻链可变区氨基酸序列：(SEQ ID NO：28)
Figure PCTCN2019115230-appb-000008
PcAb的轻链恒定区氨基酸序列：(SEQ ID NO：29)
Figure PCTCN2019115230-appb-000009
(2)将上述的抗体序列所对应的氨基酸序列进行密码子人工优化，并委托金斯瑞公司合成优化后的DNA，克隆到pUC57simple(金斯瑞公司提供)载体中，获得pUC57simple-PCABH，pUC57simple-PCABL质粒。
(3)将质粒pUC57simple-PCABH，pUC57simple-PCABL进行酶切后(Xba I和BamH I)，电泳回收得到的基因片段PCABH，PCABL并与pcDNA3.1载体进行连接反应重组构建获得pcDNA3.1-PCABH，pcDNA3.1-PCABL。
(4)将上面构建的重组质粒pcDNA3.1-PCABH，pcDNA3.1-PCABL转染FreeStyle TM293-F细胞7天后，将培养液进行高速离心、微孔滤膜抽真空过滤，根据厂家提供的操作方法采用Protein A柱(蛋白纯化液相色谱***/AKTA Purifier 10,GE)进行纯化，得到纯化后的抗体PCAB。
实验结果如图4PcAb纯化结果图。PcAb蛋白用Protein A柱富集纯化后洗脱，回收洗脱样品浓缩换液，-80℃保存。
1.5 鼠源抗体的制备
(1)根据抗原与佐剂的体积为1:1的比例对免疫原IL1β-his(制备方法见实施例1.2)进行乳化，首次免疫使用弗氏完全佐剂(FCA，Sigma，F5881-10×10ml)进行乳化抗原；相隔2周开始第二次免疫，使用弗氏不完全佐剂(FIA，Sigma，F5881-10×10ml)乳化抗原，皮下5点注射，每只小鼠(BALB/C小鼠，SPF级，雌性，6周龄)注射的抗原的量为50μg，每个注射点注射的体积为50μL。
(2)在第二次免疫后10天，对小鼠剪尾采集少量血液样本进行血清效价检测，经间接ELISA检测血清效价滴度达到1:81000或者以上，小鼠进行加强免疫。
(3)处死经过加强免疫的小鼠，解剖分离出脾脏并在细胞筛网上研磨制备成单细胞悬液，与骨髓瘤细胞SP2/0-14Ag以5:1的比例混合，离心敲散细胞团后在37℃水浴中缓慢滴加PEG/DMSO SOLUTION(Sigma)作用完成细胞融合，常规离心(1500rpm)处理后用含1×HAT(50×，Sigma)，15％胎牛血清(Gibco)，1×青链霉素的IMDM培养基(Hyclone)进行重悬并且铺到96孔细胞培养板中培养。
(4)用间接ELISA直接对96孔细胞培养板进行筛选，初筛得到的阳性克隆孔用间接ELISA方法进行第二轮三点稀释筛选，以及第三轮用原液进行竞争ELISA筛选，最终筛选得到目的阳性克隆。
(5)细胞融合筛选得到的目的阳性克隆用有限稀释法进行两轮亚克隆，每一轮亚克隆都用间接ELISA方法进行筛选，最后得到稳定细胞株。
(6)获得的稳定细胞株用含低IgG胎牛血清的IMDM培养基进行培养，最后细胞培养基上清经过纯化得到单克隆抗体。
实施例2 鼠源抗体的测试
2.1 鼠源抗体与抗原IL1β结合亲和力测试(ELISA)
(1)将Streptavidin(1.0mg/ml,生工)用CBS(0.05M包被碳酸缓冲盐溶液)稀释成2μg/ml，50μL/孔进行包被，4℃过夜孵育。PBST洗涤1次。
(2)加入0.2μg/ml的IL-1β-his-bio(制备方法见实施例1.3)，50μL/孔，37℃30min。PBST(洗板液)洗涤3次。
(3)加入1％BSA in PBS(BSA干粉1g，加入PBS将溶液定容到100ml)进行封闭，300μL/孔，37℃30min。PBST洗涤3次。
(4)将抗体(鼠源抗体制备方法见实施例1.5，对照抗体PcAb制备方法见实施例1.4)稀释成1μg/ml，再3倍往下稀释共7个浓度梯度，稀释液为零点对照。50μL/孔，37℃30min。PBST洗涤3次。
5个鼠源抗体信息如下：
抗体名称 浓度(μg/ml)
KF021ZP4 9D5 2.17
KF021ZP4 18B1 1.56
KF021ZP4 18E1 1.5
KF021ZP4 18H11 0.4
KF021ZP4 19E4 0.14
(5)加入二抗HRP conjugated Goat Anti Mouse IgG(1:5000)(酶标二抗使用液配制：使用移液器取Goat Anti Mouse IgG(H+L),HRP二抗母液1μL，加入到5ml的1％BSA缓冲液中， 振荡混匀。即1:5000稀释二抗，现配现用。)和HRP conjugated Goat Anti Human IgG(1：5000)(酶标二抗使用液配制：使用移液器取Goat Anti Human IgG，HRP二抗母液1μL，加入到5ml的1％BSA缓冲液中，振荡混匀。即1:5000稀释二抗，现配现用。)，50μL/孔，37℃30min。PBST洗涤4次。
(6)每孔加入50μL TMB(Neogen)进行显色，室温遮光反应5min后用终止液终止反应，450nm处读数。
实验结果见表1和图5。结果表明：KF021ZP4 9D5，KF021ZP4 18B1，KF021ZP4 18E1，KF021ZP4 18H11，KF021ZP4 19E4与IL-1β-his-bio均有结合。
表1 鼠源抗体与抗原IL1β结合亲和力测试结果
抗体名称 结合EC 50(nM)
KF021ZP4 9D5 0.036
KF021ZP4 18B1 1.013
KF021ZP4 18E1 0.833
KF021ZP4 18H11 0.174
KF021ZP4 19E4 0.726
KF021ZP4 PcAb 0.062
2.2 鼠源抗体与IL1RI竞争结合抗原IL1β的亲和力测试(ELISA)
(1)将IL-1β-hFc(制备方法见实施例1.1)稀释成4μg/ml，50μL/孔，4℃孵育过夜。PBST洗涤1次。
(2)加入1％BSA in PBS进行封闭，300μL/孔，37℃30min。PBST洗涤3次。
(3)将抗体(同实施例2.1)稀释成2μg/ml(终浓度为1μg/ml)，再3倍往下稀释共7个浓度梯度，稀释液为零点对照。50μL/孔，室温孵育10min。加入0.08μg/ml的IL1RI(1-332)-his(终浓度为0.04μg/ml，制备方法见实施例1.1)，50μL/孔，与抗体充分混匀后放入37℃孵育30min。PBST洗涤3次。
(4)加入二抗Mouse anti His,HRP conjugated(1:8000)，50μL/孔，37℃30min。PBST洗涤4次。
(5)每孔加入50μL TMB进行显色，室温遮光反应5min后用终止液终止反应，450nm处读数。
实验结果见表2和图6。结果表明：KF021ZP4 9D5，KF021ZP4 18B1，KF021ZP4 18E1，KF021ZP4 18H11，KF021ZP4 19E4能有效地阻断IL-1β-hFc与IL1RI(1-332)-his的结合。
表2 鼠源抗体与IL1RI竞争结合抗原IL1β的亲和力测试结果
Figure PCTCN2019115230-appb-000010
Figure PCTCN2019115230-appb-000011
2.3 鼠源抗体抑制由IL1β诱导细胞的IL6分泌检测
(1)将生长状态良好的MRC-5细胞(中国科学院细胞中心)用胰酶(Gibco)消化后，计数，接种至96孔板中，生长过夜。
(2)按照试验要求设计阴性对照组(MRC-5细胞+IL-1β+mIgG或MRC-5细胞+IL-1β+hIgG)、阳性对照组(MRC-5细胞+IL-1β+PcAb)、试验组(MRC-5细胞+IL-1β+不同浓度的抗体)，将细胞置于37度细胞培养箱培养24h。
其中：
IL-1β(Sino，浓度588nM)；
阳性抗体PcAb(制备方法见实施例1.4，浓度3.4mg/mL)；
抗体9D5(制备方法见实施例1.5，浓度2.17mg/mL)；
抗体18E1(制备方法见实施例1.5，浓度1.8mg/mL)；
抗体18B1(制备方法见实施例1.5，浓度1.56mg/mL)；
抗体18H11(制备方法见实施例1.5，浓度0.4mg/mL)；
抗体19E4(制备方法见实施例1.5，浓度0.14mg/mL)；
(3)24h后取细胞上清进行IL-6检测，用ELISA试剂盒(达科为)进行定量检测，具体操作按照试剂盒说明书进行。
IL-6的检测结果见图7。实验结果表明：IL-1β能够诱导MRC-5细胞分泌IL-6，抗体9D5、18E1、18B1、18H11和19E4能够抑制IL-1β，有效地阻断IL-1β刺激MRC-5细胞分泌IL-6这一过程。其中，9D5、18H11和19E4在阻断由IL-1β诱导的IL-6分泌的效果更优。
选择亲和力更优的9D5、18H11和19E4三个鼠源抗体进行体内药理研究。
实施例3 鼠源抗体体内药理研究
主要材料：
1)实验动物
Balb/c小鼠：SPF级，雌性，4-6周龄，29只，来源：北京维通利华实验动物技术有限公司，动物质量合格证：11400700113776。
Balb/c小鼠：SPF级，雌性，6-8周龄，18只，来源：广东省医学实验动物中心，动物质量合格证：44007200023548。
2)细胞
NIH/3T3(ATCC，细胞代数为第15代)
Lenti-IL-1β-NIH/3T3(本实验室转染，细胞代数为第11代)
3)抗体
阴性对照：Anti-HEL(Akesobio)
阳性对照：PcAb
候选抗体：9D5、18H11、19E4
氯化钠注射液(浙江天瑞药业有限公司)
实验步骤：
1)药物配制
抗体Anti-HEL2mg/ml(用于模型组)：取原液(浓度：4.88mg/ml)0.984ml(4.8mg)加入到1.416ml氯化钠注射液中，共2.4ml。抗体PcAb 2mg/ml(用于阳性药组)：取原液(浓度：7.65mg/ml)0.627ml(4.8mg)加入到1.773ml氯化钠注射液中，共2.4ml。抗体9D5 2mg/ml(用于9D5组)：取原液(浓度：5.70mg/ml)0.842ml(4.8mg)加入到1.558ml氯化钠注射液中，共2.4ml。抗体18H11 2mg/ml(用于18H11组)：取原液(浓度：3.31mg/ml)1.450ml(4.8mg)加入到0.950ml氯化钠注射液中，共2.4ml。抗体19E4 2mg/ml(用于19E4组)：取原液(浓度：6.26mg/ml)0.767ml(4.8mg)加入到1.633ml氯化钠注射液中，共2.4ml。
2)动物分组
将47只小鼠根据体重随机分成5组，分别为：正常组(氯化钠注射液，10ml/kg，n＝7)，模型组(Anti-HEL，20mg/kg，n＝8)，阳性药组(PcAb，20mg/kg，n＝8)，19E4组(19E4，20mg/kg，n＝8)，18H11组(18H11，20mg/kg，n＝8)，9D5组(9D5，20mg/kg，n＝8)。
3)给药：尾静脉注射给药。
4)细胞接种
将Balb/c小鼠经腹腔注射水合氯醛麻醉后，在小鼠左侧膝关节部位接种相应的细胞悬液，其中正常组接种NIH/3T3(细胞接种数量为5万个/只)，其他组别(模型组、阳性药组、19E4组、18H11组以及9D5组)接种Lenti-IL-1β-NIH/3T3(细胞接种数量为5万个/只)。
5)体重测量：接种后第3天和第5天对小鼠进行体重测量。
6)行为学评分和膝关节测量
接种后第5天，对小鼠进行行为学评分，同时安乐死处理小鼠，进行解剖，用游标卡尺测量膝关节白膜的长度与宽度。
行为学评分标准：
0分：小鼠活动正常，可双侧活动。
1分：小鼠行走异常，可双侧活动。
2分：小鼠患肢短暂触地，可双侧活动。
3分：小鼠患肢不能触地，单侧活动。
实验结果
1)抗体19E4、18H11以及9D5对小鼠的行为学的影响
实验结果如图8。模型组相对于正常组小鼠有明显的行为异常(P<0.01)。给药后，与模型组比较，阳性药可明显降低小鼠行为异常(P<0.01)，3个候选抗体(19E4、18H11、9D5)同时明显降低小鼠行为异常现象(P<0.01)。与阳性药组相比，19E4降低小鼠行为异常的药效劣于阳性药组(P<0.05)，18H11和9D5降低小鼠行为异常的药效与阳性药相当(P>0.05)；18H11和9D5降低小鼠行为异常药效相当(P>0.05)。
2)抗体19E4、18H11以及9D5对小鼠的膝关节肿胀的影响
实验结果如图9。模型组引起小鼠膝关节肿胀面积明显高于正常组(P<0.01)。给药后，阳性药明显降低小鼠吸膝关节肿胀面积(P<0.01)，3个候选抗体(19E4、18H11、9D5)同时明显降低小鼠膝关节肿胀面积(P<0.01)。与阳性药药效相比，19E4降低小鼠膝关节肿胀面积比阳性药差(P<0.01)，18H11降低小鼠膝关节肿胀面积与阳性药等效(P>0.05)，9D5降低小鼠膝关节肿胀面积比阳性药弱(P<0.05)。18H11降低小鼠关节肿胀程度优于9D5(P<0.05)。
3)抗体19E4、18H11以及9D5对小鼠的体重的影响
实验结果见表3，模型组小鼠体重比正常组明显下降(P<0.01)。阳性药对小鼠体重影响不明显，(P<0.01)；19E4降低小鼠体重比模型组稍弱；18H11及9D5对小鼠体重影响不明显，与阳性药等效(P>0.05)；18H11和9D5对小鼠体重的影响等效(P>0.05)。
表3 19E4、18H11以及9D5对小鼠的体重的影响(±SD，n＝8)
Figure PCTCN2019115230-appb-000012
与正常组比较， **P<0.01， *P<0.05；与模型组比较， ##P<0.01， #P<0.05；与阳性组比较， ΔΔP<0.01；n＝8(正常组n＝7)。
本实验结果表明，候选抗体19E4、18H11、9D5都可以明显降低IL-1β诱导的小鼠关节炎病变，其中候选抗体18H11和9D5与阳性对照抗体PcAb药效相当。综合小鼠行为学评分和膝关节肿胀的实验数据，在本实验条件下18H11比9D5优效。
经测序，候选抗体18H11、19E4、9D5的序列信息如下：
18H11重链可变区氨基酸序列如SEQ ID NO：7所示，核苷酸序列如SEQ ID NO：15所示；轻链可变区氨基酸序列如SEQ ID NO：8所示，核苷酸序列如SEQ ID NO：16所示。
19E4重链可变区氨基酸序列如SEQ ID NO：30所示，核苷酸序列如SEQ ID NO：31所示；轻链可变区氨基酸序列如SEQ ID NO：32所示，核苷酸序列如SEQ ID NO：33所示。
9D5重链可变区氨基酸序列如SEQ ID NO：34所示，核苷酸序列如SEQ ID NO：35所示；轻链可变区氨基酸序列如SEQ ID NO：36所示，核苷酸序列如SEQ ID NO：37所示。
实施例4 重组抗体的制备和亲和力测试
4.1 重组抗体18H11(RE)、19E4(RE)、9D5(RE)的制备
将18H11、19E4、9D5的重链cDNA序列(重链可变区序列如SEQ ID NO:15、SEQ ID NO:31、SEQ ID NO:35所示；恒定区序列为immunoglobulin gamma 2b heavy chain precursor[Mus musculus]140-475，ACCESSION:ACX70084.1)和轻链cDNA序列(轻链可变区序列如SEQ ID NO:16、SEQ ID NO:33、SEQ ID NO:37所示；恒定区为antibody kappa light chain,partial[Mus musculus],106-213 GenBank:BAB33404.1)分别克隆到pUC57simple(金斯瑞公司提供)载体中，分别获得pUC57simple-18H11H/19E4H/9D5.12H和pUC57simple-18H11L/19E4L/9D5.12L质粒。
分别将质粒pUC57simple-18H11H/19E4H/9D5.12H和pUC57simple-18H11L/19E4L/9D5.12L进行酶切(HindIII&EcoRI)，电泳回收得到的重链轻链分别亚克隆到pcDNA3.1载体中，提取重组质粒共转染293F细胞。细胞培养7天后，将培养液通过高速离心、微孔滤膜抽真空过滤后，上样至HiTrap MabSelectSuRe柱，用Elution Buffer一步洗脱蛋白，回收目标样品并用HiTrap Desalting换液至PBS。
4.2 重组抗体的亲和力测试
参照实施例2.1和2.2的方法，开展重组抗体18H11(RE)、19E4(RE)、9D5(RE)与抗原IL1β结合亲和力测试(ELISA)，以及重组抗体与IL1RI竞争结合抗原IL1β的亲和力测试(ELISA)。
测试结果表明：重组抗体18H11(Re)，19E4(Re)和9D5(Re)与IL1β均有结合，具有与鼠源抗体18H11，19E4和9D5及阳性对照抗体PCAb相当的结合活性；重组抗体18H11(Re)，19E4(Re)和9D5(Re)均能有效地阻断IL1β与IL1RI的结合，其中18H11(Re)与IL1RI竞争结合抗原IL1β的亲和力与鼠源抗体18H11及阳性对照抗体PCAb相当。
实施例5 人源化抗体的构建
本发明根据鼠源抗体18H11的序列，将重链和轻链可变区被分为14个结构上有意义的肽段，并与已知的结构在PDB数据库的抗体对应段相比较。从多个序列比对中选定出具有最高的序列同源性的那个对应的段来模拟这一段的结构。然后将所有模拟的结构段组合来构建可变区的结构。通过对该模型进行多轮的能量最小化来获得一个可靠的抗体结构模型。
在建立结构模型的同时，用鼠源的VH和VL区氨基酸序列在数据库中与人源的种系序 列进行比较，选择一个具有最高同源性的序列。通过在上述获得的三维立体结构模型中对每个存异的氨基酸进行了非常仔细地检查，确定其是否对结构完整性和CDR区有潜在影响。人源序列中的一致氨基酸也被考虑进去以确保序列的最大人源化。在确定最终序列前，还搜寻了潜在糖基化位点并在不影响抗体结合力的情况下去除。最终人源化的基因命名为18H11 H1、18H11 H2、18H11 L1、18H11 L2等(抗体恒定区序列，来自NCBI的数据库，重链恒定区为Ig gamma-1chain C region，ACCESSION:P01857，轻链恒定区为Ig kappa chain C region，ACCESSION:P01834)。
人源化抗体18H11 H1L1的重链可变区氨基酸序列如SEQ ID NO：9所示，核苷酸序列如SEQ ID NO：17所示；轻链可变区氨基酸序列如SEQ ID NO：10所示，核苷酸序列如SEQ ID NO：18所示。
人源化抗体18H11 H2L2的重链可变区氨基酸序列如SEQ ID NO：11所示，核苷酸序列如SEQ ID NO：19所示；轻链可变区氨基酸序列如SEQ ID NO：12所示，核苷酸序列如SEQ ID NO：20所示。
人源化抗体18H11 H3L3的重链可变区氨基酸序列如SEQ ID NO：13所示，核苷酸序列如SEQ ID NO：21所示；轻链可变区氨基酸序列如SEQ ID NO：14所示，核苷酸序列如SEQ ID NO：22所示。
其中，重链互补决定区的氨基酸序列为HCDR1：GYLFTGYY(SEQ ID NO：1)、HCDR2：ISCYNGDT(SEQ ID NO：2)和HCDR3：SRSDYYGTSDY(SEQ ID NO：3)，轻链互补决定区的氨基酸序列为LCDR1：SSVSY(SEQ ID NO：4)、LCDR2：TTS(SEQ ID NO：5)和LCDR3：QQRIIYPPT(SEQ ID NO：6)。
实施例6 人源化抗体的测试
6.1 人源化抗体与抗原IL1β结合亲和力测试(ELISA)
将IL-1β-his用CBS稀释包被，4℃过夜孵育。PBST洗涤1次。加入1％BSA in PBS进行封闭，37℃30min。PBST洗涤3次。将抗体(表4)进行梯度稀释，37℃30min。PBST洗涤3次。加入二抗HRP conjugated Goat Anti Human IgG(1：5000)(酶标二抗使用液配制：使用移液器取Goat Anti Human IgG，HRP二抗母液1μL，加入到5ml的1％BSA缓冲液中，振荡混匀。即1:5000稀释二抗，现配现用。)，37℃30min。PBST洗涤4次。每孔加入50μL TMB进行显色，室温遮光反应5min后用终止液终止反应，450nm处读数。
表4 三个人源化抗体信息
抗体名称 浓度(mg/ml)
KF021ZP4 18H11 H1L1 5.0
KF021 ZP4 18H11 H2L2 1.5
KF021 ZP4 18H11 H3L3 5.24
实验结果见表5和图10。结果表明：18H11 H1L1，18H11 H2L2，18H11 H3L3与IL-1β-his均有结合。
表5 人源化抗体与抗原IL1β结合亲和力测试结果
抗体名称 结合EC 50(nM)
18H11 H1L1 0.046
18H11 H2L2 0.074
18H11 H3L3 0.191
KF021ZP4 PCAb 0.169
6.2 人源化抗体与IL1RI竞争结合抗原IL1β的亲和力测试(ELISA)
将IL-1β-hFc用CBS稀释包被，4℃孵育过夜。PBST洗涤1次。加入1％BSA in PBS进行封闭，37℃30min。PBST洗涤3次。将抗体(表4)进行梯度稀释室温孵育10min，加入IL1RI(1-332)-his，与抗体充分混匀后放入37℃孵育30min。PBST洗涤3次。加入Mouse anti His,HRP conjugated(cwbio)，37℃30min。PBST洗涤4次。每孔加入50μL TMB进行显色，室温遮光反应5min后用终止液终止反应，450nm处读数。
实验结果见表6和图11。结果表明：18H11 H1L1，18H11 H2L2，18H11 H3L3能有效地阻断IL-1β-hFc与IL1RI(1-332)-his的结合。
表6 人源化抗体与IL1RI竞争结合抗原IL1β的亲和力测试结果
抗体名称 结合EC 50(nM)
18H11 H1L1 0.520
18H11 H2L2 0.683
18H11 H3L3 1.251
KF021ZP4 PCAb 0.516
6.3 人源化抗体抑制由IL1β诱导细胞的IL6分泌检测
(1)将生长状态良好的MRC-5细胞(中国科学院细胞中心)用胰酶(Gibco)消化后，计数，接种至96孔板中，生长过夜。
(2)IL-1β和PcAb以及待检测的抗体37度孵育20min，加到细胞作用24小时。其中：
IL-1β(Sino，浓度588nM)；
阳性抗体PcAb(浓度3.4mg/mL)；
对照抗体hIgG(浓度4.88mg/mL)
待测抗体18H11H1L1(浓度5mg/mL)
待测抗体18H11H2L2(浓度1.5mg/ml)；
(3)24h后取细胞上清进行IL-6检测，用ELISA试剂盒(达科为)进行定量检测，具体操作按照试剂盒说明书进行。
IL-6的检测结果见图12。实验结果表明：IL-1β能够诱导MRC-5细胞分泌IL-6，抗体18H11H1L1和18H11H2L2能够抑制IL-1β，有效地阻断IL-1β刺激MRC-5细胞分泌IL-6这一过程。其中，抗体18H11H1L1抑制由IL1β诱导细胞的IL6分泌效果较阳性对照抗体更优，抗体18H11H2L2与阳性对照抗体相当。
6.4 人源化抗体18H11 H1L1的亲和力检测(Fortebio Kinetics)
采用Fortebio Octet Qke分子相互作用仪检测抗体与IL1β的亲和力，使用EDC/sulfo-NHS激活AR2G传感器，时间300s。5μg/mL抗体(10mM乙酸钠，pH6.0稀释)固定在传感器表面，时间300s。传感器使用1M乙醇胺，pH8.5封闭，时间300s。传感器在PBST缓冲液中平衡，时间300s。固定在传感器上的抗体与IL1β-his结合，IL1β-his浓度为1.56-100nM(使用PBST两倍梯度稀释)，时间300s，抗原抗体在PBST缓冲液中解离，时间600s。数据以1:1模型拟合分析，得到亲和力常数。
实验结果见表7。结果表明：人源化抗体18H11H1L1的亲和力结果优于阳性对照抗体。
表7 人源化抗体18H11 H1L1的亲和力结果
抗体名称 KD(M) kon(1/Ms) kon Error kdis(1/s) kdis Error Rmax(nM)
KF021ZP4 18H11 H1L1 1.42E-10 3.36E+05 3.79E+03 4.78E-05 5.44E-06 0.1380-0.1905
KF021ZP4 PCAB 1.79E-10 5.89E+05 7.84E+03 1.05E-04 5.97E-06 0.1289-0.1756
实施例7 人源化抗体的体内药理研究
本实验目的是检测人源化抗体18H11 H1L1、鼠源抗体18H11对Lenti-IL-1β-NIH/3T3诱导的小鼠膝关节炎模型的治疗作用。
主要材料
1)实验动物
Balb/c小鼠；级别：SPF级；年龄：5-7周；性别：雌性；体重：15-20g；数量：40只；来源：广东省医学实验动物中心；动物质量合格证号：44007200032490。
2)细胞
NIH/3T3：来源：ATCC；细胞代数：第24代。
Lenti-IL-1β-NIH/3T3：来源：Akesobio；细胞代数：第21代。
3)抗体
阴性对照：Anti-HEL(Akesobio)
阳性对照：PcAb
候选抗体：18H11 H1L1、18H11
氯化钠注射液(广东利泰制药股份有限公司)
实验方法
1)细胞培养
从液氮中取出NIH/3T3(P19)、Lenti-IL-1β-NIH/3T3(P16)细胞，在37℃水浴中快速融化复苏，将细胞悬液加入到含有10％FBS(Gibco)和1％Pen/Strep(Gibco)的DMEM完全培养基(Gibco)中，在37℃，5％CO 2培养箱中培养；接着按细胞的常规培养方法对NIH/3T3、Lenti-IL-1β-NIH/3T3进行传代培养，每次传代对Lenti-IL-1β-NIH/3T3细胞添加Blasticidin S HCl(Gibco)进行筛选。
2)IL-1β检测
根据Human IL-1β Precoated ELISA kit(深圳市达科为生物工程有限公司)的说明书检测Lenti-IL-1β-NIH/3T3细胞上清液的IL-1β含量。
3)分组和给药
动物分组：40只Balb/c小鼠称重后随机分为5组，即Normal组、Isotype Control组、PcAb组、18H11 H1L1组、18H11组，每组8只。
剂量设计：给药剂量：10mg/kg；给药容量：10ml/kg；给药浓度：1mg/ml；给药途径：尾静脉注射；给药频率：一次，接种前给药。
药物配制：
模型组：精确量取Anti-HEL(4.9mg/ml)0.490ml，用1.91ml生理盐水稀释，备用；
PcAb组:精确量取KF021ZP4 PcAb(4.75mg/ml)0.505ml，用1.895ml生理盐水稀释，备用；
18H11 H1L1组:精确量取KF021ZP4 18H11 H1L1(3.16mg/ml)0.759ml，用1.641ml生理盐水稀释，备用；
18H11组:精确量取KF021ZP4 18H11(3.37mg/ml)0.712ml，用1.688ml生理盐水稀释，备用；
动物给药：细胞接种前，根据小鼠的体重，模型组尾静脉注射Anti-HEL，PcAb组尾静脉注射PcAb，18H11 H1L1组尾静脉注射18H11 H1L1，18H11组尾静脉注射18H11，正常组尾静脉注射等体积生理盐水。
4)细胞收集
当NIH/3T3、Lenti-IL-1β-NIH/3T3细胞达到所需接种数量时，开始收集细胞(细胞密度不应超过培养瓶面积的80％)。在生物安全柜内，吸掉旧的培养基，PBS清洗一次后加入适量的0.05％Trypsin-EDTA(1x)(Gibco)室温消化1分钟，然后加入含有10％FBS的DMEM完全培养基终止消化，细胞悬液于1200rpm/min离心4分钟，去上清，使用无血清的DMEM培养基重悬并计数，将细胞浓度调整至200万/ml，置于冰上备用。
5)造模(细胞接种)
小鼠麻醉后，正常组接种NIH/3T3细胞于小鼠右侧膝关节腔内，25μl/只，即接种细胞数5万/只，其余各组接种Lenti-IL-1β-NIH/3T3细胞于小鼠右侧膝关节腔内，25μl/只，即接种细 胞数5万/只，接种后对膝关节处伤口进行缝合，并涂拭稀释20倍的青霉素，防止伤口感染。
6)行为学评分以及膝关节面积的测量
细胞接种后第5天，对各组小鼠进行行为学评分，行为学评分标准为：0分，小鼠活动正常，可双侧活动；1分，小鼠行走异常，可双侧活动；2分，小鼠患肢短暂触地，可双侧活动；3分，小鼠患肢不能触地，单侧活动；评分后各组小鼠安乐死并用游标卡尺测量小鼠患肢膝关节滑膜的长(mm)和宽(mm)，并计算出膝关节面积(mm 2)。
7)实验统计分析方法
数据以均数±标准差(X±S)表示，组间比较采用GraphPad统计学处理软件处理后，进行单因素方差分析评价结果，P<0.05有显著性差异，P<0.01有非常显著性差异。
实验结果
1)抗体18H11 H1L1、18H11对小鼠行为学评分
如图13。与正常组相比，模型组小鼠病理性的行为异常明显增加(P<0.01)。给药后，阳性药组(PcAb组)、18H11 H1L1组和18H11组均能够有效抑制小鼠患肢的异常行走行为(P<0.01)；18H11 H1L1、18H11药效与阳性药相当。
2)抗体18H11 H1L1、18H11对小鼠膝关节面积的影响
如图14。与正常组相比，模型组小鼠膝关节面积明显增加(P<0.01)，膝关节肿胀。给药后，阳性药组(PcAb组)、18H11 H1L1组和18H11组均能够有效抑制小鼠患肢膝关节的肿胀面积(P<0.01)；18H11 H1L1、18H11药效与阳性药相当。
本实验结果表明，在Lenti-IL-1β-NIH/3T3建立的膝关节炎模型中，PcAb、18H11 H1L1、18H11三个抗体在剂量10mg/kg下,均能明显改善小鼠患肢的行走行为以及明显减少患肢膝关节的肿胀面积；18H11 H1L1和18H11抗体的药效与阳性药相当。
实施例8 突变抗体的制备
以18H11 H1L1的重链为模板，设计引物将重链的第53位半胱氨酸(C，位于重链互补决定区H-CDR2内)进行PCR分别突变成丙氨酸(A)、缬氨酸(V)、亮氨酸(L)、异亮氨酸(I)、甲硫氨酸(M)、苯丙氨酸(F)、天冬酰胺(N)、谷氨酸(E)、谷氨酰胺(Q)、组氨酸(H)、赖氨酸(K)、精氨酸(R)、天冬氨酸(D)、甘氨酸(G)、丝氨酸(S)、苏氨酸(T)、酪氨酸(Y)、色氨酸(W)、脯氨酸(P)。向PCR结束后的反应体系中加入0.5μLDpn I酶(New England BioLabs,Cat#R0176L)，置于37℃中反应消化30min，反应完成后在冰上放置5min，开始转化，次日挑菌、测序，并选取正确突变的克隆，分别与18H11 H1L1的轻链共转染293-F细胞，7天后，将培养液进行高速离心、微孔滤膜抽真空过滤，根据厂家提供的操作方法采用Protein A柱进行纯化，获得各突变抗体。各突变抗体SDS-PAGE蛋白电泳结果如图15所示，WT即18H11 H1L1。
实施例9 突变抗体的亲和力测试(ELISA)
参照实施例6.1的方法，开展各突变抗体与抗原IL1β结合亲和力测试。
各突变抗体及18H11 H1L1(WT)与IL1β结合的EC 50分别如图16和对应表所示。
实施例10 突变抗体抑制由IL-1β诱导细胞的IL-6分泌检测
实验方法参照实施例6.3，我们根据实施例9的实验结果选取与IL-1β结合亲和力较好的抗体，测定其抑制由IL-1β诱导MRC-5细胞的IL-6分泌活性。
如图17所示，结果表明相对于其他突变抗体，18H11-Hu-C53I(即C53I)和18H11-Hu-C53A(即C53A)抑制IL-1β诱导MRC-5细胞分泌IL-6的活性较好，IC 50分别为2.416nM和2.323nM。
实施例11 突变抗体的热稳定性检测
将18H11-Hu-C53I、18H11-Hu-C53A和18H11 H1L1样品置于40℃水浴锅作用28d，并分别于不同的时间点取样，在取完最后一个时间点的样品后，参照实施例6.1和6.3分别测定各样品与IL-1β结合亲和力及抑制由IL-1β诱导MRC-5细胞分泌IL-6的活性。
结果如图18所示，各样品的EC 50见图中表格，我们可知样品于40℃水浴，从0d至28d，18H11 H1L1与IL-1β结合亲和力衰减约2.6倍，18H11-Hu-C53A衰减约1.4倍，18H11-Hu-C53I衰减约2.7倍。
如图19所示，结果表明18H11-Hu-C53I、18H11-Hu-C53A和18H11 H1L1(即18H11-Hu-WT)经40℃水浴，从0d至28d，18H11 H1L1抑制IL-1β诱导MRC-5细胞分泌IL-6的活性下降约2.9倍，18H11-Hu-C53I下降约6.2倍，18H11-Hu-C53A下降约1.5倍。各样品的IC 50见图中表格。
综上可知，18H11-Hu-C53A的相对热稳定性优于18H11 H1L1和18H11-Hu-C53I。
实施例12 18H11-Hu-C53A与IL1RI竞争结合抗原IL1β的亲和力检测
参照实施例6.2的方法，开展突变抗体18H11-Hu-C53A与IL1RI竞争结合抗原IL1β的亲和力测试。
如图20所示，18H11 H1L1、18H11-Hu-C53A及PCAb阻断IL1β与IL1RI结合的IC 50分别为0.028nM、0.028nM和0.030nM，表明18H11-Hu-C53A也能有效地阻断IL1β与IL1RI的结合，其活性与18H11 H1L1及阳性对照抗体PCAb相当。
实施例13 18H11-Hu-C53A的亲和力测试(Biacore)
通过Biacore测定18H11 H1L1、18H11-Hu-C53A及PCAb与IL1β的结合动力学参数，方法如下：采用捕获法，Protein A芯片(购自GE公司，批号10261132)捕获0.5μg/mL抗体， 设置contact time(接触时间)为75s，flow rate(流速)为10μL/min，regeneration contact time(再接触时间)为30s。抗原作为分析物，设置contact time(接触时间)为180s，dissociation time(解离时间)为900s，flow rate(流速)为30μL/min，regeneration contact time(再接触时间)为30s。测试结果显示的各动力学参数如表8所示。
表8 突变抗体18H11-Hu-C53A的亲和力结果
样品 ka(1/Ms) kd(1/s) KD(M)
PcAb 1.69E+06 1.04E-04 6.16E-11
18H11 H1L1 8.18E+05 8.12E-05 9.93E-11
18H11-Hu-C53A 5.59E+05 9.81E-05 1.75E-10
实施例14 18H11-Hu-C53A的体内药效测定
取适应性培养一周的Balb/c雌性小鼠(5-7周，15-20g)，按照体重平均分成7组，每组8只；腹腔注射抗体18H11-Hu-C53A，剂量如下：2.5mg/kg，0.5mg/kg，0.1mg/kg，对照组注射PBS或同亚型对照IgG。注射抗体24h后，对各小鼠皮下注射5μg的重组人IL-1β-His；并于注射IL-1β后的4h对各组小鼠采血；4℃分离血清，通过ELISA测定血清中鼠IL-6的表达水平，以确定各不同剂量抗体对鼠IL-6分泌的抑制作用。
结果如图21所示，与PBS组相比(阴性对照组，PBS作为对照，实际值为0)，18H11-Hu-C53A在2.5mg/kg剂量下对鼠IL-6分泌的抑制率高达91.06％，0.5mg/kg剂量下对鼠IL-6分泌的抑制率为73.11％，0.1mg/kg剂量下对鼠IL-6分泌的抑制率为29.46％。
实施例15 18H11-Hu-C53A的跨种属免疫反应性的测定
本实施例通过ELISA的方法测定了18H11-Hu-C53A的跨种属免疫反应性。
将猕猴和大鼠的IL-1β蛋白即Macaca-IL-1β(NCBI Reference Sequence：NP_001270498.1)和Rat-IL-1β(NCBI Reference Sequence：NP_113700.2)按照0.2μg/孔包被96孔ELISA板，测定18H11-Hu-C53A对这两个种属IL-1β的交叉反应，其它具体实验方法参照实施例6.1。Macaca-IL-1β、Rat-IL-1β的制备方法参照实施例1.1。
结果如图22所示，表明18H11-Hu-C53A可以很好的识别猕猴的IL-1β蛋白，EC 50为0.025nM而不能识别大鼠的IL-1β蛋白。
实施例16 18H11-Hu-C53A对家族成员蛋白IL-1 alpha、IL-1 R2和IL-1 RA的选择性的测定
将人的IL-1 alpha、IL-1 R2和IL-1 RA蛋白(均购自Sino Biological公司，货号分别为10128-HNCH-20、10111-H08H-50和10123-HNAE-100)，按照0.2μg/孔包被96孔ELISA板， 测定18H11-Hu-C53A对这些蛋白的选择性，其它具体实验方法参照实施例6.1。
结果如图23所示，表明18H11-Hu-C53A对IL-1 R2和IL-1 RA均无交叉反应，对IL-1α(IL-1 alpha)的EC 50为0.652nM，对IL-1β(IL-1beta)的EC 50为0.026nM。这些结果表明18H11-Hu-C53A能特异性的识别人IL-1β。
实施例17 18H11-Hu-C53A对IL-1β的结合表位确定
根据IL-1β(SEQ ID NO：23的第1-153位)的空间结构将其分成两个蛋白片段进行表达纯化，即IL-1β-A1-F99-His(第1位丙氨酸A至第99位苯丙氨酸F，C-端带有6×His标签)和IL-1β-A1-W120-His(第1位丙氨酸A至第120位色氨酸W，C-端带有6×His标签)。用包被液将IL-1β-A1-F99-His、IL-1β-A1-W120-His和IL-1β-WT-His(即IL-1β-his)分别稀释成0.5μg/mL，包被ELISA板，测定18H11-Hu-C53A与各蛋白的亲和力，具体方法参照实施例6.1。
如图24所示，结果表明18H11-Hu-C53A与IL-1β-A1-F99-His完全不结合，而对IL-1β-A1-W120-His还有一定的亲和力，但要低于IL-1β-WT-His。因此，接下来设计引物对IL-1β的第99位氨基酸F和第120位氨基酸W之间及后者附近的其他氨基酸通过丙氨酸扫描(Alanine scanning)进行逐个单点突变，并进行表达纯化，参照上述方法以确定18H11-Hu-C53A的结合表位。
代表性的实验结果分别如图25、图26和图27所示，可以得知对18H11-Hu-C53A与IL-1β结合影响最大的氨基酸，即主要结合表位分别为第120位的色氨酸W和第122位的异亮氨酸I，其次为第112位的苯丙氨酸F、第123位的丝氨酸S和第124位的苏氨酸T。

Claims (16)

  1. 结合人IL-1β的抗体或其抗原结合片段，其特征在于，所述的抗体或其抗原结合片段包含：
    (a)重链互补决定区H-CDR1、H-CDR2、H-CDR3，所述的H-CDR1的氨基酸序列如SEQ ID NO：1所示，所述的H-CDR2的氨基酸序列如SEQ ID NO：2所示，所述的H-CDR3的氨基酸序列如SEQ ID NO：3所示，和
    (b)轻链互补决定区L-CDR1、L-CDR2、L-CDR3，所述的L-CDR1的氨基酸序列如SEQ ID NO：4所示，所述的L-CDR2的氨基酸序列如SEQ ID NO：5所示，所述的L-CDR3的氨基酸序列如SEQ ID NO：6所示。
  2. 如权利要求1所述的结合人IL-1β的抗体或其抗原结合片段，其特征在于，所述的抗体是鼠源抗体或人源化抗体。
  3. 如权利要求2所述的结合人IL-1β的抗体或其抗原结合片段，其特征在于，所述的抗体是鼠源抗体，其重链可变区的氨基酸序列如SEQ ID NO：7所示，轻链可变区的氨基酸序列如SEQ ID NO：8所示。
  4. 如权利要求2所述的结合人IL-1β的抗体或其抗原结合片段，其特征在于，所述的抗体是人源化抗体，其重链可变区的氨基酸序列如SEQ ID NO：9、SEQ ID NO：11或SEQ ID NO：13之一所示，轻链可变区的氨基酸序列如SEQ ID NO：10、SEQ ID NO：12或SEQ ID NO：14之一所示。
  5. 如权利要求1所述的结合人IL-1β的抗体或其抗原结合片段，其特征在于，所述的抗原结合片段包括Fab片段、F(ab’)2片段、Fv片段、单链抗体或单域抗体。
  6. 如权利要求1-5中任一项所述的结合人IL-1β的抗体或其抗原结合片段，其特征在于，所述抗体或其抗原结合片段的重链互补决定区H-CDR2包括C53A突变位点，其H-CDR2的氨基酸序列如SEQ ID NO：38所示。
  7. 核苷酸分子，其特征在于，所述的核苷酸分子编码如权利要求1-6中任一项所述的结合人IL-1β的抗体或其抗原结合片段。
  8. 如权利要求7所述的核苷酸分子，其特征在于，所述的核苷酸分子编码重链可变区的核苷酸序列如SEQ ID NO：15、SEQ ID NO：17、SEQ ID NO：19或SEQ ID NO：21之一所示，编码轻链可变区的核苷酸序列如SEQ ID NO：16、SEQ ID NO：18、SEQ ID NO：20或SEQ ID NO：22之一所示。
  9. 表达载体，其特征在于，所述表达载体含有如权利要求7-8中任一项所述的核苷酸分子。
  10. 宿主细胞，其特征在于，所述宿主细胞含有如权利要求9所述的表达载体。
  11. 如权利要求1-6中任一项所述的结合人IL-1β的抗体或其抗原结合片段的制备方法，其特征在于，所述方法包括以下步骤：
    (a)在表达条件下，培养如权利要求10所述的宿主细胞，从而表达所述的结合人IL-1β 的抗体或其抗原结合片段；
    (b)分离并纯化(a)所述的结合人IL-1β的抗体或其抗原结合片段。
  12. 药物组合物，其特征在于，所述药物组合物含有如权利要求1-6中任一项所述的结合人IL-1β的抗体或其抗原结合片段和药学上可接受的载体。
  13. 如权利要求1-6中任一项所述的结合人IL-1β的抗体或其抗原结合片段或如权利要求12所述的药物组合物在制备治疗IL-1β过表达引起的免疫类疾病的药物中的用途。
  14. 如权利要求13所述的用途，其特征在于，所述IL-1β过表达引起的免疫类疾病为关节炎、骨质疏松或银屑病。
  15. 结合人IL-1β的抗体或其抗原结合片段，其特征在于，所述抗体或其抗原结合片段与人IL-1β的主要结合表位为：SEQ ID NO：23的第120位的色氨酸和第122位的异亮氨酸。
  16. 如权利要求15所述的结合人IL-1β的抗体或其抗原结合片段，其特征在于，所述结合表位进一步包括SEQ ID NO：23的第112位的苯丙氨酸、第123位的丝氨酸和第124位的苏氨酸。
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