WO2012041018A1 - 抗TNFα的人源化抗体及其抗原结合片段Fab和用途 - Google Patents

抗TNFα的人源化抗体及其抗原结合片段Fab和用途 Download PDF

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WO2012041018A1
WO2012041018A1 PCT/CN2011/001668 CN2011001668W WO2012041018A1 WO 2012041018 A1 WO2012041018 A1 WO 2012041018A1 CN 2011001668 W CN2011001668 W CN 2011001668W WO 2012041018 A1 WO2012041018 A1 WO 2012041018A1
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sequence
amino acid
variable region
light chain
chain variable
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PCT/CN2011/001668
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English (en)
French (fr)
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柯潇
高小平
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成都康弘生物科技有限公司
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Priority to US13/876,774 priority Critical patent/US9096669B2/en
Priority to KR1020137011174A priority patent/KR20130062366A/ko
Priority to AU2011307886A priority patent/AU2011307886C1/en
Priority to SG2013018601A priority patent/SG188987A1/en
Priority to CA2812430A priority patent/CA2812430C/en
Priority to KR1020157020565A priority patent/KR101712874B1/ko
Priority to JP2013530530A priority patent/JP5767708B2/ja
Priority to EP11827916.5A priority patent/EP2623515B1/en
Priority to BR112013007501-5A priority patent/BR112013007501B1/pt
Priority to RU2013109392/10A priority patent/RU2556815C2/ru
Publication of WO2012041018A1 publication Critical patent/WO2012041018A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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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
    • 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
    • C07K16/249—Interferons
    • 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
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61P19/00—Drugs for skeletal disorders
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    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A—HUMAN NECESSITIES
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    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/14—Decongestants or antiallergics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • C07K16/241—Tumor Necrosis Factors
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • 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
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    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to a humanized antibody against human tumor necrosis factor alpha and an antigen binding fragment thereof and use thereof.
  • autoimmune diseases are a complex process in which imbalances are regulated by many active cytokines.
  • TNFct has been shown to play an important role in immune regulation, but its excessive expression has been shown to be one of the major factors leading to autoimmune diseases. Therefore, biopharmaceuticals that inhibit TNFa activity are among the most successful therapies for the treatment of such diseases. Indications for approved treatment include rheumatoid arthritis, Crohn's Disease, and patches. Plaque Psoriasis, Psoriatic Arthritis, Ankylosing Spondylitis, Ulcerative Colitis, and Juvenile Idiopathic Arthritis There are many other related diseases that are undergoing clinical trials.
  • Remicade has a short half-life in the body of about 9 days.
  • Remicade has a good binding affinity, biological activity and clinical efficacy, but as a chimeric antibody containing 1/3 of the mouse sequence and 2/3 human sequence, about 10% - 47% of patients
  • HAMA human anti-mouse antibody
  • a common method of humanization of antibodies is to transplant the complementarity determining region (CDR) portion of the murine antibody variable region (VH, VK) into a previously selected human antibody framework, and the resulting antibody will have a majority of human origin.
  • CDR complementarity determining region
  • VH, VK murine antibody variable region
  • the humanized antibody or Fab against human tumor necrosis factor e provided by the invention comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is sequence 1 or sequence in the sequence listing 3.
  • the amino acid sequence of the light chain variable region is any one of sequence 15, sequence 9, sequence 11, sequence 7, sequence 13 and sequence 5 in the sequence listing; wherein the first amino acid residue of sequence 1 is Glu or Gln.
  • the antibody is specifically one of the following a-1:
  • KS10 the heavy chain variable region is SH01, the amino acid sequence is sequence 1, and the light chain variable region is
  • amino acid sequence is sequence 15 ;
  • the heavy chain variable region is SH01, the amino acid sequence is sequence 1, and the light chain variable region is SH05, the amino acid sequence is sequence 9;
  • the heavy chain variable region is SH01
  • the amino acid sequence is sequence 1
  • the light chain variable region is
  • KS12 the heavy chain variable region is SH02
  • amino acid sequence is sequence 3
  • the light chain variable region is
  • SH08 the amino acid sequence is sequence 15;
  • the heavy chain variable region is SH02
  • the amino acid sequence is sequence 3
  • the light chain variable region is
  • amino acid sequence is sequence 9;
  • the heavy chain variable region is SH02
  • the amino acid sequence is sequence 3
  • the light chain variable region is
  • the heavy chain variable region is SH02
  • the amino acid sequence is sequence 3
  • the light chain variable region is
  • amino acid sequence is sequence 5 ;
  • KS08 whose heavy chain variable region is SH02, the amino acid sequence is sequence 3, and the light chain variable region is
  • SH04 the amino acid sequence is sequence 7;
  • the heavy chain variable region is SH02
  • the amino acid sequence is sequence 3
  • the light chain variable region is
  • J- KS01 the heavy chain variable region is SH01, the amino acid sequence is sequence 1, and the light chain variable region is
  • amino acid sequence is sequence 5 ;
  • KS05 the heavy chain variable region is SH01
  • amino acid sequence is sequence 1
  • the light chain variable region is
  • SH04 the amino acid sequence is sequence 7;
  • KS09 whose heavy chain variable region is SH01, the amino acid sequence is sequence 1, and the light chain variable region is
  • the first amino acid residue of Sequence 1 is Glu or Gln.
  • a further object of the present invention is to provide an additional anti-human tumor necrosis factor (humanized antigen-binding fragment Fab.
  • the Fab comprises a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region Is the position 1-120 of SEQ ID NO: 27 or the 1-120th position of SEQ ID NO: 25, the amino acid sequence of the variable region of the light chain is the first to the ninth or the ninth 1-109.
  • the antibody provided by the invention consists of a heavy chain and a light chain, the constant region amino acid sequence of the heavy chain is identical to the amino acid sequence of the human antibody heavy chain constant region, and the constant region amino acid sequence of the light chain and the human antibody light chain constant region The amino acid sequence is the same.
  • the heavy chain constant region of the antibody may be a human constant region of any type (IgG, IgA, IgM, IgE, IgD) or subclass (IgG1, IgG2, IgG3, IgG4, IgM1, IgM2, IgAl, IgA2, );
  • the light chain constant region of the antibody may be of any type (kappa type or lambda type), subtype ( ⁇ 1, ⁇ 2, ⁇ 3, ⁇ 4), or an isotropic (Km (1), Km (2), Km (3) ))
  • the human constant region of the light chain may be of any type (kappa type or lambda type), subtype ( ⁇ 1, ⁇ 2, ⁇ 3, ⁇ 4), or an isotropic (Km (1), Km (2), Km (3) ))
  • the human constant region of the light chain may be of any type (kappa type or lambda type), subtype ( ⁇ 1, ⁇ 2, ⁇ 3, ⁇ 4), or an isotropic (Km (1), Km (2)
  • amino acid sequence of the heavy chain constant region of the antibody is specifically as shown in SEQ ID NO: 17 in the Sequence Listing; the amino acid sequence of the light chain constant region of the antibody is specifically as shown in SEQ ID NO: 19 in the Sequence Listing.
  • amino acid sequence of the heavy chain is SEQ ID NO: 21 in the Sequence Listing; the amino acid sequence of the light chain is SEQ ID NO: 23 in the Sequence Listing.
  • amino acid residues 1-120 of sequence 21 are the variable regions of the heavy chain,
  • amino acid residues 121-450 are the constant regions of the heavy chain;
  • amino acid residues 1 to 19 of the sequence 23 are the variable regions of the light chain, and
  • amino acid residues 1 to 214 are the light The constant region of the chain.
  • the first amino acid of sequence 21 is Glu or Gln.
  • the present invention provides a Fab heavy chain Fd fragment and the light chain; the heavy chain Fd fragment consisting of the V H and CH1, the light chain and the V K light chain constant region; the amino acid sequence of human CH1
  • the CH1 amino acid sequence of the antibody heavy chain constant region is identical, and the amino acid sequence of the light chain constant region is identical to the amino acid sequence of the human antibody light chain constant region.
  • the CH1 of the Fd fragment of the Fab may be a human constant region of any type (IgG, IgA, IgM, IgE, IgD) or subclass (IgG1, IgG2, IgG3, IgG4, IgM1, IgM2, IgAl, IgA2, ) CHI;
  • the light chain constant region of the Fab may be of any type (kappa type or lambda type), subtype ( ⁇ 1, ⁇ 2, ⁇ 3, ⁇ 4), or an isotropic (Km (1), Km (2), Km (3)) Human constant region of the light chain.
  • CHI amino acid sequence is shown in SEQ ID NO: 33 in the Sequence Listing; the light chain constant region amino acid sequence is shown as SEQ ID NO: 19 in the Sequence Listing.
  • the Fab is any one of the following bl) - b3 ):
  • KS-7F the amino acid sequence of the heavy chain fragment is sequence 27 in the sequence listing, and the amino acid sequence of the light chain is the sequence 31 in the sequence listing;
  • KS-7A the amino acid sequence of the heavy chain fragment is the sequence 25 in the sequence listing, and the amino acid sequence of the light chain is the sequence 31 in the sequence listing;
  • a further object of the present invention is to provide an antigen-binding fragment A or an antigen-binding fragment B, characterized in that: the antigen-binding fragment A is a Fab, Fab', F(ab') 2 , Fv (antibody) derived from the antibody a variable region fragment), a heavy chain variable region, a light chain variable region, a polypeptide fragment selected from the heavy chain variable region, or a polypeptide fragment selected from the light chain variable region; the antigen binding fragment B is the Fab Derived Fab', F(ab') 2 , Fv, heavy chain variable region, light chain variable region, polypeptide fragment selected from the heavy chain variable region, or polypeptide fragment selected from the light chain variable region.
  • F(ab') 2 consists of a pair of light chains and a pair of heavy chains slightly larger than Fd (called Fd'). Pepsin hydrolyzes IgG molecules to produce this fragment, which contains two Fabs that bind two epitopes. Bit.
  • Fd' contains approximately 235 amino acid residues including VH , CHI and hinge regions.
  • Fv consists of a light chain variable region (VL) and a heavy chain variable region ( VH ), which are joined together by non-covalent bonds and have a molecular weight of about 1/4 of the intact antibody molecule with a single antigen binding site. .
  • Fv includes ScFv (single-chain antibody), DsFv (disulfide-stabilized antibody), and the like.
  • ScFv is a combination of VH and VL with a suitable oligonucleotide (linker) to express it as a single peptide chain.
  • DsFv introduces a cysteine into the appropriate part of the light chain variable region and the heavy chain variable region to form a disulfide-fixed Fv segment, which has been proved to have better binding ability and stability than ScFv.
  • the coding sequences of the heavy chain variable region of the antibody and the antigen-binding fragment A are as shown in SEQ ID NO: 2 or SEQ ID NO: 4 in the Sequence Listing; the antibody and the light chain variable region coding sequence of the antigen-binding fragment A All are as shown in any one of Sequence 16, Sequence 10, Sequence 12, Sequence 8, Sequence 14 and Sequence 6 in the Sequence Listing; the coding sequences of the heavy chain variable regions of the Fab and the antigen-binding fragment B are both Is any one of Sequence 2, Sequence 4, Positions 1-360 of Sequence 28, and Positions 1-360 of Sequence 26; the Fab and the light chain variable region of the antigen-binding fragment B
  • the coding sequences are all of sequence 16, sequence 10, sequence 12, sequence 8, sequence 14, sequence 6, sequence 1-327 of sequence 32, and positions 1-327 of sequence 30 in the sequence listing.
  • sequence 2 and sequence 4 share 360 nucleotides; sequence 6, sequence 8, sequence 14, sequence 10, sequence 12 and sequence 16 share a total of 327 nucleotides.
  • the coding sequence for the heavy chain constant region of the antibody is shown as SEQ ID NO: 18 in the Sequence Listing; the coding sequence for the light chain constant region of the antibody is shown as SEQ ID NO: 20 in the Sequence Listing.
  • the coding sequence for CH1 of the Fab is shown in SEQ ID NO: 34 in the Sequence Listing; the light chain constant region coding sequence of the Fab is shown as SEQ ID NO: 20 in the Sequence Listing.
  • the heavy chain coding sequence of the antibody is specifically the sequence 22 in the sequence listing;
  • the light chain coding sequence of the antibody is specifically the sequence 24 in the sequence listing;
  • the coding sequence of the Fab is any one of the following cl) -c3):
  • KS-7F the coding sequence of the heavy chain fragment of the Fab is the sequence 28 in the sequence listing, and
  • the light chain coding sequence of the Fab is the sequence 32 in the sequence listing;
  • KS-7A the coding sequence of the heavy chain fragment of the Fab is the sequence 26 in the sequence listing, and the light chain coding sequence of the Fab is the sequence 32 in the sequence listing;
  • KS-2E The coding sequence of the heavy chain fragment of the Fab is the sequence 26 in the sequence listing, and the light chain coding sequence of the Fab is the sequence 30 in the sequence listing.
  • Still another object of the present invention is to provide a genetic material comprising: a recombinant vector, a recombinant strain, a recombinant cell line, a recombinant virus or an expression cassette containing the gene.
  • the recombinant vector is a prokaryotic expression vector or a eukaryotic expression vector expressing the antibody or Fab or antigen-binding fragment.
  • the recombinant strain is Escherichia coli carrying the gene.
  • the recombinant cell line may be a transgenic cell line or a fusion cell line, wherein the transgenic cell line may be a mammalian cell line transformed with the anti-human tumor necrosis factor alpha humanized antibody or Fab or antigen-binding fragment encoding gene of the present invention.
  • the CHO cell line, or 293 cells and sublines thereof; the fusion cell line may be a hybridoma cell secreting the anti-human tumor necrosis factor e humanized antibody of the present invention.
  • the recombinant virus is a recombinant adenovirus carrying a gene or a recombinant adeno-associated virus or the like.
  • the expression cassette is a DNA molecule, from upstream to downstream The following three fragments are: a promoter, the antibody or Fab which is transcribed by the promoter, or a gene encoding a gene and a terminator of the antigen-binding fragment.
  • the recombinant vector containing the antibody, or the Fab, or the antigen-binding fragment-encoding gene can be transfected or transformed into a host cell to express the corresponding protein to obtain the antibody or Fab, or the antigen-binding fragment.
  • the host cell may be a eukaryotic cell or a prokaryotic cell including, but not limited to, mammalian cells, bacteria, yeast, insect cells, and the like.
  • mammalian cells that can be used for large-scale expression of proteins, such as 293 cells, CHO cells, SP20 cells, NSO cells, COS cells, BHK cells, or PerC6 cells.
  • methods for transfecting cells including but not limited to: electroporation, liposome-mediated methods, calcium-mediated methods, and the like.
  • a preferred expression of the antibody or Fab, or the antigen-binding fragment is a gene amplification of a recombinant vector in a host cell that has been stably transfected to increase the expression level of the corresponding recombinant protein, for example, After the recombinant vector of dihydrofolate reductase (DHFR) is stably transfected into a host cell lacking DHFR, the concentration of methotrexate (MTX) can be added to the cell culture medium to amplify the number of copies of the recombinant vector in the host cell.
  • DHFR dihydrofolate reductase
  • MTX methotrexate
  • the protein concentration in the culture solution is determined by ELISA or other methods.
  • purification can be performed by Protein G affinity chromatography; IgG protein can be purified by Protein A affinity chromatography.
  • Dl use in the preparation of a medicament for the prevention and/or treatment of a human anti-TNF-related disease, or d2) in the preparation of a human anti-tumor necrosis factor ct product, or
  • the human tumor necrosis factor alpha-related disease is a disease caused by an increase in human tumor necrosis factor ct; preferably rheumatoid arthritis, autoimmune uveitis, Crohn's disease, patchy psoriasis, psoriasis Arthritis, ankylosing spondylitis, ulcerative colitis or juvenile idiopathic arthritis.
  • Still another object of the present invention is to provide a pharmaceutical composition
  • a pharmaceutical composition comprising an adjuvant and an active ingredient, the active ingredient comprising at least one of the following: the antibody, the Fab, the antigen-binding fragment, The gene, and the genetic material; the excipient is a pharmaceutically acceptable carrier or excipient.
  • the active ingredient of the pharmaceutical composition may also be any of the above Fabs or antibodies, or any of the above-described antigen-binding fragments, or any of the above-mentioned genes, or any of the above-mentioned genetic materials.
  • any of the following substances in the treatment of human tumor necrosis factor alpha-related diseases is also within the scope of the invention: the antibody, the Fab, the antigen-binding fragment, the gene, the genetic material And the pharmaceutical composition.
  • the disease associated with human tumor necrosis factor a is a disease caused by an increase in human tumor necrosis factor a, preferably autoimmune uveitis, rheumatoid arthritis, Crohn's disease, patchy psoriasis, silver shavings Arthritis, ankylosing spondylitis, ulcerative colitis or juvenile idiopathic arthritis.
  • DRAWINGS Figure 1 shows the biological activity of Elisa to detect humanized Fab inhibition of TNFa.
  • Figure 2 shows the biological activity of humanized Fab neutralizing TNFa by L929 cytotoxicity assay.
  • Figure 3 shows the binding of KS 10 to different species of antigen TNFct.
  • Figure 4 shows the results of treatment of KS 10 in experimental rheumatoid arthritis rats. From left to right, the normal group, the negative group, the model group, the KS 10 5 mg/kg group, the KS 10 10 mg/kg group, and the KS 10 20 mg/kg group.
  • Figure 5 shows the effect of KS 10 on synovial fluid/tissue IL- ⁇ levels in experimental rheumatoid arthritis rats. From left to right, the normal group, the negative group, the model group, the KS 10 5 mg/kg group, the KS10 10 mg/kg group, and the KS10 20 mg/kg group.
  • Figure 6 shows the effect of KS 10 on synovial fluid/tissue IL-6 levels in experimental rheumatoid arthritis rats. From left to right, the normal group, the negative group, the model group, the KS 10 5 mg/kg group, the KS10 10 mg/kg group, and the KS10 20 mg/kg group.
  • the following examples are provided to facilitate a better understanding of the invention but are not intended to limit the invention.
  • the experimental methods in the following examples are conventional methods unless otherwise specified.
  • the test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. In the quantitative tests in the following examples, three replicate experiments were set, and the results were averaged.
  • the CDR grafting of the heavy light chain of the humanized TNFa antibody described in the present invention, the PCR introduction site mutation and the screening of the mutant library are performed by conventional genetic recombination technology and immunological technology based on antigen-antibody interaction, and the specific experimental method steps are as follows. ⁇ Molecular Cloning>> Third Edition (Joseph Sambrook, Science Press) and similar experimental manuals.
  • the EC50 involved in the embodiment is obtained by inputting the measured OD value (OD450) into the graphp a d pri S m 5 software, and obtaining a correlation result graph.
  • This example relates to three humanized TNFa antibody Fabs (Fab consisting of the heavy chain Fd fragment of the antibody and the light chain of the antibody), which are KS-2E, KS-7A, KS-7F, respectively.
  • Hybridoma cells obtained by immunizing mice with human TNF- ⁇ were subjected to monoclonal screening to extract total RNA as a template, using universal primer P1 : 5-GCGAATTCAGGTSMARCTGCAGSAGTCWGG-3 , P2 :
  • 5-TGAGGAGACGGTGACCGTGGTCCCTTGGCCCCAG-3 guides PCR amplification of heavy chain variable region sequences
  • universal primer P3 5-GACATTCTGMTSACMCAGMCTCC-3
  • P4 5-GTTAGATCTCGAGCTTGGTCCC-3 guides PCR amplification of light chain variable region sequences
  • the purpose of gel extraction In the band, the products of the light chain and heavy chain of the amplified antibody were inserted into the pMD18-T vector (TaKaRa company number: D101C), and single colonies were picked respectively, and the antibody heavy chain and light chain were obtained by sequencing. Sequence of regions.
  • Each heavy chain fragment (Fd) DNA was inserted between restriction endonuclease 1 and 3 ⁇ 4 ⁇ 1 sites, and several Fab expression vectors were constructed (including three expressions of KS-2E, KS-7A and KS-7F, respectively). Fab expression vector).
  • the above different light chain and heavy chain fragment (Fd) combinations include three Fabs, KS-2E, KS-7F, and KS-7A.
  • the amino acid sequence of the heavy chain fragment of KS-2E is sequence 25 in the sequence listing, the nucleotide sequence is sequence 26, the light chain amino acid sequence is sequence 29 in the sequence listing, and the nucleotide sequence is sequence 30; the heavy chain fragment of KS-7A
  • the amino acid sequence is sequence 25 in the sequence listing, the nucleotide sequence is sequence 26, the light chain amino acid sequence is sequence 31 in the sequence listing, and the nucleotide sequence is 32; the amino acid sequence of the heavy chain fragment of KS-7F is sequence 27 in the sequence listing.
  • the nucleotide sequence is sequence 28, the light chain amino acid sequence is sequence 31 in the sequence listing, and the nucleotide sequence is sequence 32.
  • the specific method for transforming the pET22b(+) vector to obtain the above pTLR vector is as follows: First, a section containing a T7 promoter (T7 promoter), a lactose operon (lac operator), a ribosome binding site (RBS) sequence, and two a DNA fragment containing a Sa and ⁇ cleavage site (abbreviated as TLR DNA sequence, as shown in SEQ ID NO: 35 in the Sequence Listing), pET22b(+) vector (product of Novage, USA) and TLR DNA sequence using Sal I and After the double digestion of Not I, the T 4 ligase was ligated and transformed, and the conventional method was used to select the correctly modified vector by monoclonal sequencing.
  • T7 promoter T7 promoter
  • lac operator lactose operon
  • RBS ribosome binding site
  • the Fab expression vector constructed above (including three Fab expression vectors expressing KS-2E, KS-7A and KS-7F, respectively) was transformed into E. coli Top l O, and chloramphenicol resistant 2-YT plate (peptone 1.6). % , yeast extract 1%, NaC1 0.5%, agar powder 1.5%). The next day, the plate with the appropriate colony density was selected to pick up the monoclonal colonies. Each positive clone picked 8 monoclonal clones into 96-well deep well plates and induced expression by IPTG.
  • Each monoclonal colony was added to 6 ml of chloramphenicol-containing 2- YT liquid medium (peptone 1.6%, yeast extract 1%, NaCl 0.5%), shaken at 37 ° C, 250 rpm for 12 hours, 0.2 ⁇ l per tube
  • the bacterial solution was sterilized on chloramphenicol-resistant 2-YT plates.
  • 5 ml of the bacterial solution was inoculated into 500 ml of chloramphenicol-resistant 2-YT liquid medium, and cultured at 33 ° C, 300 rpm to an OD 6QQ of 0.6.
  • IPTG was added to the medium to a final concentration of 50 ⁇ M to induce expression of different Fab proteins with an induction time of 3 h.
  • the culture solution after the induction of expression was centrifuged at 5100 rpm, 10 ° C for 15 min, the supernatant was discarded, and the precipitate was fully resuspended with 40 ml of the pre-cooled TES solution; after resuspension, 66 ml of pre-cooled 1 was added.
  • /5 TES solution was incubated on the resuspended bacterial solution for 40 min on ice; after the end of the ice bath, centrifuged at 13,000 rpm, 4 ° C for 10 min; after centrifugation, the supernatant was collected, and the supernatant contained Fab protein (KS-2E). , KS-7A or KS-7F protein) periplasmic extract.
  • the Protein G (GE 17-0618-04) prepacked column was prepared, and the equilibration solution (20 mM phosphate buffer pH 6.5) was equilibrated. After the protein was loaded, the column was washed with the balance solution after the completion of the sample, and then directly eluted with the eluent (0.1 M Gly-HCl, pH 2.5), and the eluted sample was collected and buffered in advance according to Tris before collecting the sample. The liquid to sample volume ratio of 1:9 was added to 1.0M Tris-HCl (pH 9.0) in the sample collection tube, and the pH was quickly adjusted to 7.0. The collected liquid was the target protein. After the protein was analyzed by SDS-PAGE, the purity was The protein samples were assayed for concentration and finally stored at -80 °C after packing to obtain higher purity Fab proteins (including KS-2E, KS-7A and KS-7F).
  • the human TNFa (R&D Company, catalog number 210-TA-050) per well was used as a substrate to coat the plate, and then a series of 2-fold diluted 40 nM antibody Fabs were added (prepared in step 2).
  • Fab protein including KS-2E, KS-7A and KS-7F), after incubation with horseradish peroxidase-labeled goat anti-human C-Kappa secondary antibody (Sigma, catalog number K3502)
  • TMB was added to color, and 2M sulfuric acid was added to terminate the reaction, thereby detecting the Fab protein binding to TNFa.
  • KS-2E, KS-7F, KS-7A Contains 2 different VHs (VH01 and VH02, VH01 amino acid sequence such as sequence 25; nucleotide sequence such as sequence 26; VH02 amino acid sequence such as sequence 27; nucleotide sequence such as sequence 28) and 2 different VK (VK03 and VK05, the amino acid sequence of VK03 is as in sequence 29; the nucleotide sequence is as in sequence 30; the amino acid sequence of VK05 is as in sequence 31; the nucleotide sequence is as in sequence 32) (Table 1).
  • N/A indicates that the Fab fragment is less active and unnamed.
  • Table 2 Fab of humanized TNFa antibody binds EC 5 o of TNFa in Elisa assay
  • the CCK8 kit (Tongren Chemical Institute) was used to detect cell survival.
  • the above three humanized Fabs can effectively neutralize the biological activity of TNFa, wherein the biological activity of KS-7A neutralizing TNFa is superior to that of human mouse chimeric antibody Remicade.
  • the data in Figure 2 is the mean ⁇ standard deviation of 3 replicate experiments.
  • an affinity maturation library was constructed for the heavy chain VH02 CDR3 and the light chain VK05 CDR3 in the humanized TNF- ⁇ antibody Fab expression vector, respectively.
  • the most important region in which an antibody binds to an antigen is the CDR3 region of the heavy and light chains. Saturation point mutation screening of CDR3 may result in antibodies with higher affinity.
  • a saturation point mutation library of heavy chain and light chain CDR3 a series of point mutation primers were designed. After PCR reaction, these PCR products were mixed according to the degeneracy of the primer series and cloned into humanized TNF- The Fb expression plasmid of the ct antibody.
  • a point mutation library of the light chain CDR3 PCR was carried out by using a series of primer 5 and a light chain downstream primer 6 (5 '-CGGAATTCCGTACGTTTCACTTCCAGATTGG-3 ') with a light chain DNA as a template, and then the DNA product was cloned into a Fab expression plasmid. After electroporation, a point mutation library of the light chain CDR3 was obtained.
  • the primer 5 series contains the CDR3 mutation sites, and a total of 19 primers are listed in Table 3;
  • the heavy chain CDR3 point mutation library was constructed with a series of mutant primers 7 and a heavy chain downstream primer 8 (5 '-CCGCTCGAGGCGCTCACGGTCAGGGTGGTGCCCTG-3 ') PCR was carried out by using heavy-strand DNA as a template to obtain a DNA product, and then the DNA product was cloned into a Fab expression plasmid, and electroporation was carried out to obtain a point mutation library of the light chain CDR3.
  • the primer 7 series contains the CDR3
  • the mutation site a total of 22 primers are listed in Table 4, the final established light chain and heavy chain mutant library, ELISA results selected TNF-ct antibody Fab with high binding affinity to antigen TNF-ct (R&D), heavy chain mutation screening Two active higher heavy chain variable regions were obtained, designated as SH01 and SH02, respectively.
  • Light chain mutation screening resulted in four more active light chain variable regions, designated SH03, SH04, SH05, SH06, SH07 and SH08.
  • This example relates to two humanized TNFa antibody Fabs, the names being FA01, FA02, respectively.
  • the amino acid sequence of the light chain variable region of FA01 is the sequence 15 in the sequence listing (the nucleotide sequence is shown as the sequence 16 in the sequence listing), and the amino acid sequence of the constant region of the light chain is the sequence 19 in the sequence listing (nucleoside)
  • the acid sequence is as shown in SEQ ID NO: 20 in the sequence listing.
  • the heavy chain variable region amino acid sequence of the heavy chain Fd fragment is the sequence 1 in the sequence listing (the nucleotide sequence is shown in SEQ ID NO: 2 in the sequence listing), and the heavy chain is constant.
  • the CH1 amino acid sequence is the sequence 33 in the sequence listing (the nucleotide sequence is shown as the sequence 34 in the sequence listing); the amino acid sequence of the light chain variable region of FA02 is the sequence 9 in the sequence listing (nucleotide sequence such as sequence listing) In the sequence 10), the amino acid sequence of the constant region of the light chain is the sequence 19 in the sequence listing (the nucleotide sequence is shown in SEQ ID NO: 20 in the sequence listing), and the amino acid sequence of the heavy chain variable region of the heavy chain Fd fragment is Sequence 1 in the sequence listing (nucleotide sequence is shown in SEQ ID NO: 2 in the Sequence Listing), and the heavy chain constant region CH1 amino acid sequence is SEQ ID NO: 33 in the Sequence Listing (nucleotide sequence is shown as SEQ ID NO: 34 in the Sequence Listing).
  • the construction methods of the two humanized TNFa antibody Fabs were as described in Example 1.
  • the heavy chain coding DNA and the light chain coding DNA of the above two humanized TNFa antibody Fabs were inserted into the corresponding sites of the pTLR vector to obtain the expression of FA01, respectively.
  • Vector pFAO1 Fab, and expression vector pFA02 Fab of FA02. Further, the FA01 and FA02 proteins of higher purity were obtained by the method described in Example 1.
  • the above two humanized TNFa antibodies Fab were tested for biological activity by L929 cytotoxicity assay.
  • the specific method was the same as in Example 1, and the obtained OD450 value was input into graphpad prism 5 software to obtain EC50.
  • the results are shown in Table 5.
  • the activities of FA01 and FA02 were stronger than those of the Remicade antibody Fab fragment.
  • Example 1 and Example 2 The two heavy chain Fd fragments obtained in Example 1 and Example 2 (containing SH01 and SH02, respectively) and six light chains (including SH03, SH05, SH04, SH06, SH07 and SH08, respectively) are as shown in Table 6.
  • the respective combinations were spliced together with the overlap extension PCR and the IgG1 Fc constant region, and then recombined into the expression plasmid pcDNA3.1(+), and the constructed recombinant expression plasmid was transfected into CHO cells to express a full-length humanized antibody.
  • the specific procedures are as follows: (1) The light chain is directly recombined in the eukaryotic expression vector pcDNA3.1(+).
  • the primers were designed as follows: 5'-TGAAAGCTTATGGAAATTGTGCTGACTCAGTCTC-3* (Hindlll restriction site at the scribe); 5' AATCTCGAGTCAACACTCTCCCCTGTTGAAGCT-3* (Xho I restriction site at the scribe), PCR amplification, amplification product in enzyme Hindin (R0104L, NEB Co.), and the Xho I enzyme (R0146L, NEB Co.) was double digested ligase to T pcDNA3.1 double digested with the same 4 (+) is connected to the large fragment.
  • (2) The heavy chain was spliced together by overlap extension PCR and IgG1 Fc constant region, and then recombined into pcDNA3.1(+).
  • the heavy chain variable region coding DNAs of (Glu), KS05 (Glu), KS06 (Glu), KS09 (Glu), and KS10 (Glu) are shown in SEQ ID NO: 2 in the sequence listing (nucleotides 1-3) For GAG), the heavy chain variable region shown in SEQ ID NO: 1 (the first amino acid is Glu), KS01 (Gin), KS03 (Gin), KS05 (Gin), KS06 (Gin), KS09 (Gin), The heavy chain variable region coding DNA of KS10 (Gin) is replaced by CAG in nucleotide sequence 1-3 of sequence 2 in the sequence listing, and both encode the heavy chain variable region shown in SEQ ID NO: 1 (amino acid The heavy chain variable region coding DNA of Gin); KS02, KS04, KS07, KS08, KS11, and KS12 are all shown in sequence 4 in the sequence listing, and both encode the heavy chain variable region shown in SEQ ID NO: 3; KS01 (Glu) The coding DNA
  • the light chain variable region; the coding DNA of the light chain variable region of KS09 (Glu), KS09 (Gin) and KS1 1 are all sequence 14 of the sequence listing, each encoding the light chain variable region of sequence 13; KS10
  • the coding DNAs of the light chain variable regions of (Glu), KS10 (Gin) and KS12 are all sequence 16 in the sequence listing, and both encode the light chain variable region shown in SEQ ID NO: 15.
  • the DNA coding sequence of the heavy chain constant region of the 18 antibodies is a sequence listing The sequence of 18, the heavy chain constant region of the coding sequence 17; the DNA coding sequence of the light chain constant region of the 18 antibodies is the sequence 20 of the sequence listing, encoding the light chain constant region of the sequence 19.
  • the light chain recombinant plasmid and the heavy chain recombinant plasmid obtained in the first step were co-transfected into CHO cells to express a full length humanized antibody.
  • the corresponding plasmids as shown in Table 6 were stably transfected into CHO cells, respectively, and the corresponding 12 recombinant full-length antibodies were secreted into the cell culture supernatant, and the corresponding full-length IgG antibodies were purified.
  • the specific purification steps are as follows:
  • Affinity balance buffer PBS: 0.2M disodium hydrogen phosphate: 82.5mL / L ; 0.2M sodium dihydrogen phosphate: 17.5mL / L; 2M sodium chloride: 75mL / L; add ultrapure water, fully stirred Mix well and adjust the pH to 7.1-7.3 with 1 M sodium hydroxide or 1 M hydrochloric acid.
  • Affinity elution buffer NaCl 2.922 g/L, anhydrous sodium acetate 0.49 g/L, and 2.9 mL/L anhydrous acetic acid, pH 3.4-3.6.
  • Affinity regeneration buffer 5.8 mL/L anhydrous acetic acid pH 3.0.
  • Affinity CIP buffer 1M sodium hydroxide 100mL / L.
  • Centrifuge 5000-6000 rpm, 10-15 minutes, centrifuge the sample.
  • the AKTA explorer purification system and the Superdex 200 gel filtration column were installed.
  • the flow rate was adjusted to 5 ml/min, and 1 column volume was washed with ultrapure water, and 2 column volumes were washed with Superdex 200 column chromatography equilibration solution. Adjust the flow rate of 2.5 ml/min, and adjust the pH to directly load the affinity peak.
  • elution was performed with Superd eX 200 column chromatography equilibration solution, and the target peak at 280 nm was collected.
  • the 12 humanized TNFa antibody IgG obtained in the second step was subjected to L929 cytotoxicity test to detect the biological activity of TNFa, and the specific activity was as described in Example 1, to obtain the value of OD450, and the OD450 value was input into the graphpad prism 5 software.
  • Table 7 shows the EC50 of 12 humanized TNFa antibody IgGs inhibiting the biological activity of TNFa. The results showed that the biological activity of humanized TNFa antibody was greatly improved. Among them, KS10 activity was the highest, and the late study on antibody activity was mainly for KS10. ongoing.
  • hTNF-a R&D, 210-TA-050
  • NHS-LCLC-biotin Cat. No. 21338, purchased from Thermo-fisher
  • PD- 10 Desalting column 17.0851-01, purchased from GE
  • the remaining NHS-LCLC-biotin was removed to give a final tantalum product of 5 ( ⁇ g/ml biotin-hTNF-a).
  • the kinetic parameters of KS10 and hTNFa binding were detected by the Octet RED96 system (ForteBio) Kinetics Assays in the Octet technology platform.
  • the test procedure was set according to the instrument operating instructions. 5 ( ⁇ g/ml biotin-hTNFa was bound by Loading) On SA (Streptavidin biosensor); the appropriate concentration range of antibody was determined by pre-test, 6000 nM, 2000 nM, 666.7 nM, 222.2 nM 74.1 nM and 24.7 nM respectively.
  • Remicade was used as positive control and PBS (H 7.4 ) was used as blank control.
  • the experimental results show that the KD value of KS10 is lower than that of Remicade, indicating that its affinity with hTNFa is higher than that of human mouse chimeric antibody Remicade.
  • KS10 and Chimpanzee TNFa were detected (amino acid sequence as shown in SEQ ID NO: 36, nucleotide sequence as shown in SEQ ID NO: 37), and cynomolgus TNFa (Chimpanzee TNFa) (amino acid sequence as shown in SEQ ID NO: 38, nucleotide sequence As shown in SEQ ID NO: 39, mouse TNFa (mTNFa) (Cat. No.: CYT-252, purchased from PROSPEC) A TNFp (human TNFp) (Cat. No.: CYT-224, purchased from PROSPEC).
  • the optical wave displacement distance generated by the combination of antigen and antibody indirectly reacts to the affinity between the two, and the higher the light wave displacement distance, the higher the affinity.
  • the results (Fig. 3) showed that KS10 strongly binds to human TNFa, and also binds to chimpanzee TNFct, but weakly binds to rhesus TNFct, and hardly binds to mouse TNFct and human TNFP.
  • the specific binding ability of the antibody provided by the present invention after humanization was confirmed.
  • KD is the affinity constant
  • kon is the binding constant
  • kdis is the dissociation constant.
  • Example 6 Effect of KS10 (Glu) on human TNF- ⁇ -induced rheumatoid arthritis rats Sprague Dawley rats (animal license number: SCXK (chuan) 2008-24), 4-6 weeks old, SPF grade , 72, half male and half female, weighing 140-180g. Rats were adaptively incubated for one week and were randomly divided into 6 groups, 12 rats in each group, which were normal group, negative control group, model control group, KS10 5 mg/kg group, KS10 10 mg/kg group, KS10. 20 mg/kg group.
  • each group of rats was anesthetized with 10% chloral hydrate (350 mg/kg) except for the normal group.
  • the three KS10 dose groups (5 mg/kg, 10 mg/kg and 20 mg/kg) were tailed.
  • Intravenous administration, the negative control group and the model control group were injected with an equal volume of physiological saline via the tail vein.
  • 15 minutes after the injection 0.5 mg/ml hTNF- ⁇ (R&D, catalog number 210-TA-050) (dissolved with 1% BSA, injection volume 60 ⁇ l/only) was injected into the three KS10 in a 1 ml syringe.
  • joint synovial fluid is taken 20 hours after modeling, soft tissue around the joint is separated and homogenized After centrifugation, it was frozen at -70 °C, and the IL-i p was determined using a rat ELISA kit (IL- ⁇ purchased from R&D, article number: RLB00; IL-6 purchased from R&D, batch number: R6000B). , IL-6 levels.
  • the three doses of KS10 can significantly alleviate the joint redness caused by hTNF- ⁇ , and significantly antagonize the formation of rheumatoid arthritis induced by hTNF- ⁇ .
  • Cytokine assay showed that KS10 significantly reduced IL- ⁇ (Fig. 5) and IL-6 (Fig. 6) levels in synovial/articular soft tissues around the joint, indicating that KS10 completely blocked TNF- ⁇ .
  • the levels of IL- ⁇ and IL-6 directly confirmed the anti-human TNF- ⁇ effect of KS10.
  • KS10 (Glu) treatment of human TNF-ct-induced rat uveitis 40 healthy lewis rats were randomly divided into normal group, model group, negative control group and KS10 group. Rats were intraperitoneally injected with 10% chloral hydrate (dose 0.35 ml/kg). After anesthesia in rats, each of the model group and the negative control group was injected with vitreous flattening to the vitreous (using a 30G1/2 needle) to give ⁇ physiology.
  • Iris congestion of 0 to 2 points no congestion for 0 points, mild congestion for 1 point, severe congestion for 2 points
  • pupil reduction for 0 to 1 point normal pupil for 0 points, pupil reduction for 1 point
  • anterior chamber exudation 0 ⁇ 2 points (0 points in the anterior chamber, 1 point in mild exudation, 2 points in severe exudation)
  • 0 ⁇ 2 points in the pupil or iris post-adhesion no adhesion is 0 points
  • One adhesion is 1 point, and the two adhesions are 2 points).
  • KS 10 can significantly reduce the score of uvitis score, mainly to inhibit iris congestion, fiber exudation, pupil or iris post-adhesion, and increase intraocular transparency.
  • KS 10 has a significant antagonistic effect on hTNF- ⁇ -induced uveitis.
  • the present invention overcomes the conventional antibody humanization method by directly mutating the CDR-transplanted antibody (transplanting the complementarity determining region (CDR) of the murine antibody variable region (VH, VK) into the human antibody framework) This leads to the defect that the affinity of the antibody is impaired, and finally a humanized antibody similar to the starting antibody is obtained.
  • the Fab and IgG antibodies provided by the invention have greatly improved the degree of humanization (up to 95%), and have been proved to have similar affinity and biological activity to the human mouse chimeric antibody Remicade, and have human TNFct.
  • TNFct-related diseases preferably rheumatoid arthritis, autoimmune uveitis, Crohn's disease, patchy psoriasis, psoriatic arthritis, tonicity Spondylitis, ulcerative colitis or juvenile idiopathic arthritis.

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Description

抗 TNFa的人源化抗体及其抗原结合片段 Fab和用途 技术领域
本发明涉及一种抗人肿瘤坏死因子 α的人源化抗体及其抗原结合片段 Fab和用 途。
背景技术
自身免疫性疫病的发生发展是一个由许多有活性的细胞因子调控失衡的复 杂过程。 在众多因子中， TNFct被证实在免疫调节中发挥重要作用， 但其过量表 达被证实是导致自身免疫性等疾病主要因素之一。 因此， 抑制 TNFa活性的生物 药成为治疗该类疾病中最为成功的疗法之一， 已被批准治疗的适应症， 主要包 括类风湿性关节炎 （Rheumatoid Arthritis) 、 克隆病 （Crohn's Disease) 、 斑片 性银屑病 ( Plaque Psoriasis) 、 银屑病关节炎 （ Psoriatic Arthritis ) 、 强直性脊柱 炎 （Ankylosing Spondylitis) 、 溃疡性结肠炎 （Ulcerative Colitis) 和幼年特发性 关节炎 （Juvenile Idiopathic Arthritis) ， 同时还有许多其他相关疾病正在开展临 床试验。
欧美市场上已经有针对 TNFct的抗体药物和类似抗体的 Fc融合蛋白药物， 如 Remicade。 Remicade在体内的半衰期较短， 大约为 9天。 此外， Remicade虽 有良好的结合亲和性、 生物活性和临床疗效， 但作为一个包含 1/3 鼠源序列和 2/3人源序列的嵌合性抗体，约有 10% - 47%的患者在使用 Remicade后发生了免 疫反应， 通常产生人抗小鼠抗体 （HAMA) ， 影响该抗体的疗效和长期应用。 因此， 十分需要寻求一种人源化程度高的抗 TNFa抗体， 最大程度减少其中鼠源 序列比例， 以降低鼠源性并能安全用于治疗人体疾病。 一种常见的抗体人源化 方法是将鼠源抗体可变区 （VH、 VK) 的互补决定区 （CDR) 部分移植到事先选 择的人抗体框架之中， 所得抗体将具有大部分人源的序列， 同时又能够保留起 始鼠源抗体对相同抗原的选择性， 但这个过程通常导致抗体亲和力的损失。 发明公开
本发明的一个目的是提供抗人肿瘤坏死因子 e 人源化抗体或抗原结合片段 Fab, 该抗体或 Fab与人肿瘤坏死因子 α的亲和力与人鼠嵌合抗体 Remicade相 似， 甚至更高。
本发明所提供的抗人肿瘤坏死因子 e 的人源化抗体或 Fab， 含有重链可变 区和轻链可变区， 所述重链可变区的氨基酸序列为序列表中序列 1或序列 3， 所 述轻链可变区的氨基酸序列为序列表中序列 15、 序列 9、 序列 11、 序列 7、 序 列 13和序列 5中的任一种； 其中序列 1的第一位氨基酸残基为 Glu或 Gln。
所述抗体具体为下述 a-1中任一种：
a. KS10, 其重链可变区为 SH01， 氨基酸序列为序列 1， 轻链可变区为
SH08 , 氨基酸序列为序列 15 ;
b. KS03 , 其重链可变区为 SH01， 氨基酸序列为序列 1， 轻链可变区为 SH05 , 氨基酸序列为序列 9;
c. KS06, 其重链可变区为 SH01 , 氨基酸序列为序列 1， 轻链可变区为
SH06， 氨基酸序列为序列 11 ;
d. KS12, 其重链可变区为 SH02, 氨基酸序列为序列 3， 轻链可变区为
SH08， 氨基酸序列为为序列 15 ;
e. KS04, 其重链可变区为 SH02, 氨基酸序列为序列 3， 轻链可变区为
SH05 , 氨基酸序列为序列 9;
f. KS07, 其重链可变区为 SH02, 氨基酸序列为序列 3， 轻链可变区为
SH06， 氨基酸序列为序列 11 ;
g- KS02, 其重链可变区为 SH02, 氨基酸序列为序列 3， 轻链可变区为
SH03 , 氨基酸序列为序列 5 ;
h. KS08, 其重链可变区为 SH02, 氨基酸序列为序列 3， 轻链可变区为
SH04, 氨基酸序列为序列 7;
i. KS11 , 其重链可变区为 SH02, 氨基酸序列为序列 3， 轻链可变区为
SH07, 氨基酸序列为序列 13 ;
j- KS01 , 其重链可变区为 SH01 , 氨基酸序列为序列 1， 轻链可变区为
SH03 , 氨基酸序列为序列 5 ;
k. KS05 , 其重链可变区为 SH01 , 氨基酸序列为序列 1， 轻链可变区为
SH04, 氨基酸序列为序列 7;
1. KS09, 其重链可变区为 SH01 , 氨基酸序列为序列 1， 轻链可变区为
SH07, 氨基酸序列为序列 13 ;
所述序列 1的第一位氨基酸残基为 Glu或 Gln。
本发明的再一个目的是提供另外的抗人肿瘤坏死因子（ 人源化抗原结合片 段 Fab。 所述 Fab含有重链可变区和轻链可变区， 所述重链可变区的氨基酸序列 为序列表中序列 27的第 1-120位或序列 25的第 1-120位， 所述轻链可变区的氨 基酸序列为序列表中序列 31的的第 1-109位或序列 29的第 1-109位。
本发明所提供的抗体由重链和轻链组成， 所述重链的恒定区氨基酸序列与 人抗体重链恒定区氨基酸序列相同， 所述轻链的恒定区氨基酸序列与人抗体轻 链恒定区氨基酸序列相同。
所述抗体的重链恒定区可以是任意类型 （IgG、 IgA、 IgM、 IgE、 IgD) 或亚 类 （IgGl、 IgG2、 IgG3、 IgG4， IgMl、 IgM2， IgAl、 IgA2， ) 的人源恒定区； 所述抗体的轻链恒定区可为任意类型 （κ型或 λ型） 、 亚型 （λ1、 λ2、 λ3、 λ4) 、 或同种异性 （Km ( 1 ) 、 Km ( 2) 、 Km ( 3 ) ) 轻链的人源恒定区。
所述抗体的重链恒定区氨基酸序列具体如序列表中序列 17所示； 所述抗体 的轻链恒定区氨基酸序列具体如序列表中序列 19所示。
所述重链的氨基酸序列为序列表中序列 21 ; 所述轻链的氨基酸序列为序列 表中序列 23。 其中序列 21 的第 1-120位氨基酸残基为所述重链的可变区， 第 121-450位氨基酸残基为所述重链的恒定区；序列 23的第 1-109位氨基酸残基为 所述轻链的可变区， 第 1 10-214位氨基酸残基为所述轻链的恒定区。 序列 21的 第一位氨基酸为 Glu或 Gln。
本发明所提供的 Fab由重链 Fd片段和轻链组成；所述重链 Fd片段由 VH和 CH1组成， 所述轻链由 VK和轻链恒定区组成； 所述 CH1氨基酸序列与人抗体 重链恒定区的 CH1氨基酸序列相同， 所述轻链恒定区氨基酸序列与人抗体轻链 恒定区氨基酸序列相同。
所述 Fab的 Fd片段的 CH1可以是任意类型 （IgG、 IgA、 IgM、 IgE、 IgD ) 或亚类 （IgGl、 IgG2、 IgG3、 IgG4， IgMl、 IgM2, IgAl、 IgA2， ) 的人源恒定 区的 CHI ; 所述 Fab的轻链恒定区可为任意类型（κ型或 λ型）、 亚型（λ1、 λ2、 λ3、 λ4 ) 、 或同种异性 （Km ( 1 ) 、 Km ( 2 ) 、 Km ( 3 ) ) 轻链的人源恒定区。
所述 CHI氨基酸序列如序列表中序列 33所示；所述轻链恒定区氨基酸序列 如序列表中序列 19所示。
在本发明的具体实施例中， 所述 Fab为下述 bl ) -b3 ) 中任一种：
bl ) KS-7F: 所述重链片段的氨基酸序列为序列表中序列 27， 且所述轻链的 氨基酸序列为序列表中序列 31 ;
b2 ) KS-7A: 所述重链片段的氨基酸序列为序列表中序列 25， 且所述轻链 的氨基酸序列为序列表中序列 31 ;
b3 ) KS-2E: 所述重链片段的氨基酸序列为序列表中序列 25， 且所述轻链的 氨基酸序列为序列表中序列 29。 本发明的再一个目的是提供抗原结合片段 A或抗原结合片段 B， 其特征在 于： 所述抗原结合片段 A为由所述抗体衍生的 Fab、 Fab'、 F(ab')2、 Fv (抗体可 变区片段） 、 重链可变区、 轻链可变区、 选自重链可变区的多肽片段或选自轻 链可变区的多肽片段； 所述抗原结合片段 B为由所述 Fab衍生的 Fab'、 F(ab')2、 Fv、 重链可变区、 轻链可变区、 选自重链可变区的多肽片段或选自轻链可变区 的多肽片段。
上述 F(ab')2由一对轻链和一对略大于 Fd的重链 （称为 Fd' ) 组成， 胃蛋白 酶水解 IgG分子产生此片段， 它包含两个 Fab， 可结合 2个抗原表位。 Fd'约含 有 235个氨基酸残基， 包括 VH、 CHI和绞链区。 Fv由轻链可变区 （VL) 和重 链可变区 （VH ) 组成， 二者以非共价键结合在一起， 分子量约为完整抗体分子 的 1 / 6， 具有单一抗原结合位点。 Fv包括 ScFv (单链抗体） 、 DsFv (二硫键 稳定抗体） 等。 ScFv是将 VH和 VL用一段适当的寡聚核苷酸 （接头， linker) 连起来， 使之表达为单一的肽链。 DsFv是在轻链可变区和重链可变区适当部位 各引入一个半胱氨酸， 形成一个二硫键固定的 Fv段， 经证实其结合能力及稳定 性均优于 ScFv。
编码如下 A) -C ) 中任一种蛋白的基因也属于本发明的保护范围： A) 所述抗体； B ) 所述 Fab; C) 所述抗原结合片段 A或所述抗原结合片 段
所述抗体和所述抗原结合片段 A的重链可变区的编码序列均如序列表中序 列 2或序列 4所示； 所述抗体和所述抗原结合片段 A的轻链可变区编码序列均 如序列表中序列 16、 序列 10、 序列 12、 序列 8、 序列 14和序列 6中的任一种 所示； 所述 Fab和所述抗原结合片段 B的重链可变区的编码序列均为序列表中 序列 2、 序列 4、 序列 28的第 1-360位和序列 26的第 1-360位中的任一种； 所 述 Fab和所述抗原结合片段 B的轻链可变区的编码序列均为序列表中序列 16、 序列 10、 、 序列 12、 序列 8、 序列 14、 序列 6、 序列 32的第 1-327位和序列 30 的第 1-327位中的任一种。
其中， 序列 2和序列 4均共有 360个核苷酸； 序列 6、 序列 8、 序列 14、 序 列 10、 序列 12和序列 16均共有 327个核苷酸。
所述抗体的重链恒定区的编码序列如序列表中序列 18所示； 所述抗体的轻 链恒定区的编码序列如序列表中序列 20所示。
所述 Fab的 CH1的编码序列如序列表中序列 34所示；所述 Fab的轻链恒定 区编码序列如序列表中序列 20所示。
在本发明的实施例中， 所述抗体的重链编码序列具体为序列表中序列 22; 所述抗体的轻链编码序列具体为序列表中序列 24;
所述 Fab的编码序列为下述 cl ) -c3 ) 中任一种：
cl ) KS-7F: 所述 Fab 的重链片段的编码序列为序列表中序列 28， 且所述
Fab的轻链编码序列为序列表中序列 32;
c2) KS-7A: 所述 Fab 的重链片段的编码序列为序列表中序列 26， 且所述 Fab的轻链编码序列为序列表中序列 32;
c3 ) KS-2E: 所述 Fab 的重链片段的编码序列为序列表中序列 26， 且所述 Fab的轻链编码序列为序列表中序列 30。
其中序列 22的第 1-360位核苷酸为所述重链的可变区核苷酸， 第 360-1350 位核苷酸为所述重链的恒定区核苷酸； 其中序列 24的第 1-327位核苷酸为所述 轻链的可变区核苷酸， 第 328-642位核苷酸为所述轻链的恒定区核苷酸。
本发明的又一个目的是提供下述遗传材料： 含有所述基因的重组载体、 重 组菌、 重组细胞系、 重组病毒或表达盒。
所述重组载体为表达所述抗体或 Fab或抗原结合片段的原核表达载体或真 核表达载体。 所述重组菌为携带有所述基因的大肠杆菌。 所述重组细胞系可为 转基因细胞系或融合细胞系， 其中转基因细胞系可为转入本发明的抗人肿瘤坏 死因子 α人源化抗体或 Fab或抗原结合片段编码基因的哺乳动物细胞系， 优选 CHO细胞系， 或 293细胞及其亚系； 融合细胞系可为分泌本发明的抗人肿瘤坏 死因子 e 人源化抗体的杂交瘤细胞。 所述重组病毒为携带有所述基因的重组腺 病毒或重组腺相关病毒等。 所述表达盒是一个 DNA分子， 从上游至下游依次为 如下三个片段： 启动子、 由所述启动子启动转录的所述抗体或 Fab， 或所述抗原 结合片段的编码基因和终止子。
将含所述抗体， 或所述 Fab， 或所述抗原结合片段编码基因的重组载体转染 或转化宿主细胞， 即可表达相应的蛋白， 获得所述抗体或 Fab， 或所述抗原结合 片段。 所述宿主细胞可以是真核细胞， 也可以是原核细胞， 它们包含但不限于 哺乳动物细胞、 细菌、 酵母、 昆虫细胞等。 可用于蛋白质大规模表达的哺乳动 物细胞有多种， 如 293细胞、 CHO细胞、 SP20细胞、 NS0细胞、 COS细胞、 BHK细胞或 PerC6细胞等。 转染细胞的方法有多种， 其中包括但不限于： 电穿 孔， 脂质体介导法， 钙介导法等。
一种较佳的所述抗体或 Fab ,或所述抗原结合片段的表达方式是在已经稳定 转染的宿主细胞中对重组载体进行基因扩增， 以提高相应重组蛋白的表达量， 例如用含有二氢叶酸还原酶（DHFR)的重组载体稳定转染缺乏 DHFR的宿主细 胞后，可在细胞培养液中添加氨甲喋啶 (MTX)的浓度以扩增重组载体在宿主细胞 中的拷贝数量。
含有所述编码基因序列组合的 Fab 或 IgG 表达后， 可用酶联免疫吸附法
( ELISA)或其他方法测定培养液中的蛋白浓度。对于 Fab片段， 可用 Protein G 亲和层析法提纯； IgG蛋白可用 Protein A亲和层析法提纯。
所述抗体， 或所述 Fab， 或所述抗原结合片段， 或所述基因， 或所述遗传材 料的用途也属于本发明的保护范围：
dl ) 在制备预防和 /或治疗人抗肿瘤坏死因子 a相关疾病药物中的用途， 或 d2 ) 在制备中和人抗肿瘤坏死因子 ct产品中的用途， 或
d3 ) 在制备定性或定量检测人抗肿瘤坏死因子 ct的试剂盒中的用途。
所述人肿瘤坏死因子 α相关疾病为由人肿瘤坏死因子 ct升高引起的疾病； 优选为类风湿性关节炎、 自身免疫性葡萄膜炎、 克隆病、 斑片性银屑病、 银屑 病关节炎、 强直性脊柱炎、 溃疡性结肠炎或幼年特发性关节炎。
本发明的又一个目的是提供药物组合物， 该药物组合物含有辅料和活性成 分， 所述活性成分含有下述物质中的至少一种： 所述抗体， 所述 Fab， 所述抗原 结合片段， 所述基因， 和所述遗传材料； 所述辅料为药学上可接受的载体或赋 形剂。 该药物组合物的活性成分也可只为上述任一 Fab 或抗体， 或上述任一所 述抗原结合片段， 或上述任一所述基因， 或上述遗传材料中的任一种。
下述物质中的任一种在治疗人肿瘤坏死因子 α相关疾病中的应用也属于本 发明的保护范围： 所述抗体， 所述 Fab， 所述抗原结合片段， 所述基因， 所述遗 传材料， 和所述药物组合物。
所述与人肿瘤坏死因子 a相关疾病为由人肿瘤坏死因子 a升高引起的疾病， 优选为自身免疫性葡萄膜炎、 类风湿性关节炎、 克隆病、 斑片性银屑病、 银屑 病关节炎、 强直性脊柱炎、 溃疡性结肠炎或幼年特发性关节炎。
附图说明 图 1为 Elisa检测人源化 Fab抑制 TNFa的生物活性。
图 2为 L929细胞毒性实验检测人源化 Fab中和 TNFa的生物活性。
图 3为 KS 10与不同种属抗原 TNFct的结合情况。
图 4为 KS 10对实验性类风湿性关节炎大鼠的治疗评分结果。由左至右依次为 正常组、 阴性组、 模型组、 KS 10 5mg/kg组、 KS 10 10mg/kg组、 KS 10 20mg/kg 组。
图 5为 KS 10对实验性类风湿性关节炎大鼠关节滑液 /组织 IL-Ι β水平的影响。 由左至右依次为正常组、 阴性组、 模型组、 KS 10 5mg/kg组、 KS10 10mg/kg组、 KS10 20mg/kg组。
图 6为 KS 10对实验性类风湿性关节炎大鼠关节滑液 /组织 IL-6水平的影响。 由左至右依次为正常组、 阴性组、 模型组、 KS 10 5mg/kg组、 KS10 10mg/kg组、 KS10 20mg/kg组。
实施发明的最佳方式
以下的实施例便于更好地理解本发明， 但并不限定本发明。 下述实施例中 的实验方法， 如无特殊说明， 均为常规方法。 下述实施例中所用的试验材料， 如无特殊说明， 均为自常规生化试剂商店购买得到的。 以下实施例中的定量试 验， 均设置三次重复实验， 结果取平均值。
本发明描述的人源化 TNFa抗体重轻链的 CDR移植， PCR引入位点突变及 突变文库的筛选是通过常规基因重组技术及基于抗原抗体相互作用的免疫学技 术所完成， 具体实验方法步骤如 <<分子克隆>>第三版 (Joseph Sambrook, 科学出 版社)及类似实验手册所记载。 实施例中涉及到的 EC50 是将测得的 OD 值 ( OD450 ) 输入 graphpad priSm 5软件获得的， 同时获得相关结果图。
实施例 1、 人源化 TNFa抗体 Fab的表达及活性检测
本实施例涉及三种人源化 TNFa抗体 Fab( Fab由抗体的重链 Fd片段和抗体 的轻链组成） ， 分别是 KS-2E、 KS-7A、 KS-7F。
一、 KS-2E、 KS-7A、 KS-7F表达载体的构建
(一） 轻链、 重链的获得
以人 TNF-α ( R&D公司， 货号： 210-TA-050 ) 免疫鼠获得的杂交瘤细胞， 经过单克隆筛选后， 提取总 RNA 为模板， 利用通用引物 P1 ： 5-GCGAATTCAGGTSMARCTGCAGSAGTCWGG-3 ， P2 ：
5-TGAGGAGACGGTGACCGTGGTCCCTTGGCCCCAG-3引导进行 PCR扩增重 链可变区序列， 通用引物 P3 : 5-GACATTCTGMTSACMCAGMCTCC-3 , P4 : 5-GTTAGATCTCGAGCTTGGTCCC-3引导进行 PCR扩增轻链可变区序列，切胶 回收目的条带， 将扩增获得的抗体轻链和重链的产物分别插入到 pMD18-T载体 ( TaKaRa公司 货号： D101C ) 中， 分别挑取单菌落， 经过测序鉴定获得抗体重 链和轻链可变区序列。
(二） 人源化 Fab的构建 将轻链可变区与人源化抗体的轻链可变区进行氨基酸序列相似性比对， 将 重链可变区与人源化抗体的重链可变区进行氨基酸序列相似性比对， 通过 IgBLAST (http：〃 www.ncbi.nlm.nih.gov/igblast/)和 IMGT (免疫基因数据库 IMGT : http://www.imgt.org ) 中分别进行序列相似性搜索， 在搜索的结果中， 选择与轻、 重链均有较高序列相似性的抗体作为人源化抗体模板， 将获得的重链可变区 VH 移植到序列相似度较高的人抗体 IGHV3- 15 *07 ( Accession number: M99406 ) 的 框架之中， 对获得轻链可变区 VK 移植到相似度较高的人抗体 IGKV6-21 *01 ( Accession number: X63399 ) 的框架之中。
在经过对重链片段 （Fd ) 和轻链人源序列经过多轮的突变后， 将不同的轻 链和重链片段 （Fd) 组合插入到 pTLR载体 （经过改造的 pET22b(+)载体） 中。 具体操作为， 制备两端分别含有酶切位点 B纖 m和 EcoRl的各轻链 DNA， 以及 两端分别含有酶切位点 ΝοΛ和 Xhol的各重链片段 （Fd ) DNA, 将这两个 DNA 分别克隆入 pTLR载体 （经过改造的 pET22b(+)载体） 中的相应限制性内切酶位 点之间， 即各轻链 DNA分别插入到限制性内切酶 BamHl和 EcoRl位点之间， 各重链片段 （Fd ) DNA分别插入到限制性内切酶 1和¾^1位点之间， 构建 数个 Fab表达载体 （包括分别表达 KS-2E、 KS-7A和 KS-7F的三个 Fab表达载 体） 。
其中， 上述不同的轻链和重链片段（Fd)组合中包括 KS-2E、 KS-7F、 KS-7A 这三个 Fab。 KS-2E的重链片段氨基酸序列为序列表中序列 25，核苷酸序列为序 列 26， 轻链氨基酸序列为序列表中序列 29， 核苷酸序列为序列 30 ; KS-7A的重 链片段氨基酸序列为序列表中序列 25， 核苷酸序列为序列 26， 轻链氨基酸序列 为序列表中序列 31， 核苷酸序列为 32 ; KS-7F的重链片段氨基酸序列为序列表 中序列 27， 核苷酸序列为序列 28， 轻链氨基酸序列为序列表中序列 31， 核苷酸 序列为序列 32。
对 pET22b(+)载体进行改造获得上述 pTLR载体的具体方法如下： 首先通过 人工合成一段含有 T7 启动子 （T7 promoter) 、 乳糖操纵子 （ lac operator) 、 核 糖体结合位点（RBS )序列以及两端包含有 Sa 和 Νοή酶切位点的 DNA片段（简 称， T-L-R DNA序列，如序列表中序列 35所示）， pET22b(+)载体（美国 Novage 公司产品）和 T-L-R DNA序列分别用 Sal I和 Not I双酶切后， T4连接酶连接后 转化， 常规方法挑单克隆测序筛选出正确改造的载体。
二、 KS-2E、 KS-7A、 KS-7F的原核表达
将上述构建的 Fab表达载体 （包括分别表达 KS-2E、 KS-7A和 KS-7F的三 个 Fab表达载体） 分别转化大肠杆菌 Top l O , 涂氯霉素抗性 2-YT平板 （蛋白胨 1.6% , 酵母提取物 1%， NaC1 0.5% , 琼脂粉 1.5% ) 。 第二天选择合适菌落密度 的板挑取单克隆菌落， 每个阳性克隆挑 8个单克隆到 96孔深孔板里 IPTG诱导 表达， 每个单克隆菌落加入到 6ml含氯霉素的 2-YT液体培养基（蛋白胨 1.6%， 酵母提取物 1 %， NaCl 0.5% ) 中， 于 37 °C 250rpm摇 12小时， 每管中取 0.2μ1 菌液在氯霉素抗性的 2-YT平板上保菌。 将 5ml菌液接种到 500ml 氯霉素抗性 2-YT液体培养基， 33 °C， 300rmp培养至 OD6QQ为 0.6。 在培养基中加入 IPTG至 终浓度为 50μΜ， 诱导不同 Fab蛋白表达， 诱导时间为 3h。 将诱导表达结束后的 培养液在 5100 rpm， 10°C条件下，离心 15min，弃上清，沉淀用 40ml预冷的 TES 溶液充分重悬菌体沉淀； 重悬后再加入 66ml预冷的 1/5 TES溶液到重悬后的菌 液中， 冰上孵育 40min; 冰浴结束后， 13000rpm， 4°C， 离心 10 min; 离心后收 集上清， 上清即为含有 Fab蛋白 （KS-2E、 KS-7A或 KS-7F蛋白） 的细胞周质提 取液。周质提取液过 G-25 (GE公司， 17-0034-01) 柱脱盐处理后， 准备 Protein G ( GE公司 17-0618-04 ) 预装柱， 平衡液 （20mM 磷酸缓冲液 pH 6.5 ) 平衡后 蛋白上样， 上样结束后继续用平衡液清洗柱子， 然后用洗脱液 （0.1M Gly-HCl， pH2.5 )直接洗脱， 收集洗脱样品， 并预先在收集样品前按照 Tris 缓冲液与样品 体积比 1： 9加入 1.0M Tris-HCl ( pH9.0 )于样品收集管里， 快速调 pH7.0， 收集 到的液体即为目的蛋白， 蛋白经过 SDS-PAGE分析纯度后， 对蛋白样品进行浓 度测定，最后分装后保存于 -80°C，得到纯度较高的 Fab蛋白（包括 KS-2E、KS-7A 和 KS-7F这三个蛋白） 。
三、 KS-2E、 KS-7A、 KS-7F的活性检测
1、 ELISA法检测人源化 Fab与 TNFa的结合能力
以每孔 lOO ng的人源 TNFa ( R&D公司， 商品目录号为 210-TA-050 ) 为底 物包被酶标板， 然后分别加入一系列 2倍稀释的 40 nM抗体 Fab (步骤二制备的 Fab蛋白， 包括 KS-2E、 KS-7A和 KS-7F这三个蛋白）温育后， 再加辣根过氧化 酶标记的羊抗人 C-Kappa二抗 （Sigma公司， 产品目录号为 K3502 ) ， 最后加 TMB显色， 加入 2M 硫酸终止反应， 从而检测结合 TNFa的 Fab蛋白， 经过这 样的筛选方法获得了 3个活性较高的 Fab: KS-2E、 KS-7F、 KS-7A, 共包含了 2 个不同的 VH ( VH01和 VH02， VH01的氨基酸序列如序列 25 ; 核苷酸序列如序 列 26 ; VH02 的氨基酸序列如序列 27; 核苷酸序列如序列 28 ) 和 2个不同的 VK(VK03和 VK05， VK03的氨基酸序列如序列 29 ;核苷酸序列如序列 30; VK05 的氨基酸序列如序列 31 ; 核苷酸序列如序列 32) (表 1 ) 。
ELISA法检测人源化 Fab与 TNFa的结合能力的实验结果在图 1中显示。结 果表明上述获得的 3个人源化 Fab和人鼠嵌合抗体 Remicade的 Fab片段具有相 似的抗原亲和力， 其中 KS-7A和 KS-7F结合 TNFct的 EC5Q优于人鼠嵌合抗体 Remicade Fab (表 2 ) 。
表 1 人源化 TNF-a抗体的 Fab
Fab片段 VH VK
KS-2E VH01 VK03
N/A VH02 VK03
KS-7F VH01 VK05 S-7A VH02 V 05
注： N/A表示 Fab片段活性较差， 未命名。 表 2 人源化 TNFa抗体的 Fab在 Elisa实验中结合 TNFa的 EC5o
样品 EC50 ( nM )
Remicade Fab 3. 324
KS-2E 6. 659
KS-7A 3. 027
KS-7F 2. 740
注： 表 2中的数据为三次重复实验的平均值。
2、 L929细胞毒性实验检测人源化 Fab抑制 TNFa的生物活性
进一步通过细胞生物学实验， 即 L929细胞毒性实验验证了上述三种人源化 Fab ( KS-2E、 KS-7F和 KS-7A) 中和 TNFa的生物活性。 实验中， 取 l x lO4个对 数生长期 L929细胞在 DMEM ( 10%FBS , Gibco ) 培养液中接种于 96孔板， 24 小时后加入 0.5ng/ml 人源 TNFa ( R&D 公司， 商品目录号为 210-TA-050 ) 和 0.5μ§/ηι1放线菌素 D ( Fluka) ， 同时分四组分别加入上述三种人源化 Fab以及 对照抗体 Remicade的 Fab片段， 继续培养 24小时， 然后利用 CCK8试剂盒（同 仁化学研究所） 检测细胞存活情况。 结果如图 2所示， 上述三种人源化 Fab可 有效地中和 TNFa的生物活性， 其中 KS-7A中和 TNFa的生物活性优于人鼠嵌 合抗体 Remicade的 Fab片段。 图 2中数据为 3次重复实验的平均值 ±标准差。
实施例 2、 人源化 TNFa抗体 Fab CDR3亲和力成熟文库的构建
为了进一步提高上述人源化 TNF-α抗体 Fab片段对抗原的亲和力， 分别在 人源化 TNF-α抗体 Fab表达载体中对重链 VH02 CDR3和轻链 VK05 CDR3构建 了亲和力成熟文库。 抗体与抗原结合最重要的区域是重、 轻链的 CDR3 区域， 对 CDR3 进行饱和点突变筛选可能得到亲和力更高的抗体。 为了构建重链、 轻 链 CDR3 的饱和点突变文库， 设计一系列点突变引物， PCR反应后， 然后把这 些 PCR产物按照引物系列的简并度相同的比例混合起来再克隆到人源化 TNF-ct 抗体的 Fab表达质粒中。 轻链 CDR3的点突变文库的构建以系列引物 5和轻链 下游引物 6(5 '-CGGAATTCCGTACGTTTCACTTCCAGATTGG-3 ')以轻链 DNA 为模版进行 PCR得到 DNA产物， 然后将 DNA产物克隆到 Fab表达质粒， 经过 电转铺板， 即可得到轻链 CDR3 的点突变文库。 其中引物 5系列包含了 CDR3 的突变位点， 共有 19条引物列于表 3 ; 重链 CDR3的点突变文库的构建以系列 突 变 引 物 7 和 重 链 下 游 引 物 8(5 '-CCGCTCGAGGCGCTCACGGTCAGGGTGGTGCCCTG-3 ')以重链 DNA 为 模版进行 PCR得到 DNA产物， 然后将 DNA产物克隆到 Fab表达质粒， 经过电 转铺板， 既可得到轻链 CDR3的点突变文库。 其中引物 7系列包含了 CDR3的 突变位点， 共有 22条引物列于表 4， 最终建立的轻链和重链突变文库， ELISA 结果挑选与抗原 TNF-ct ( R&D公司） 结合力高的 TNF-ct抗体 Fab， 重链突变筛 选获得两个活性更高重链可变区， 分别命名为 SH01和 SH02; 轻链突变筛选获 得 4个活性更好的轻链可变区， 分别命名为 SH03、 SH04、 SH05 、 SH06、 SH07 和 SH08。
表 3 轻链 CDR3的点突变引物 5系列
名称 序列
引物 5-1Q1 GCAACCTACTACTGCNBKCAGAGCCATAGCTGG
引物 5-1Q2 GCAACCTACTACTGCDAKCAGAGCCATAGCTGG
引物 i-lQ3 GCAACCTACTACTGCCATCAGAGCCATAGCTGG
引物 i-2Ql GCAACCTACTACTGCCAGNBKAGCCATAGCTGGCCG 引物 i-2Q2 GCAACCTACTACTGCCAGDAKAGCCATAGCTGGCCG 引物 i-2Q3 GCAACCTACTACTGCCAGCATAGCCATAGCTGGCCG 引物 i-3Sl GCAACCTACTACTGCCAGCAGBDKCATAGCTGGCCGTTC 引物 i-3S2 GCAACCTACTACTGCCAGCAGVCTCATAGCTGGCCGTTC
GCAACCTACTACTGCCAGCAGAWKCATAGCTGGCCGTT
引物 5-3S3
C
GCAACCTACTACTGCCAGCAGAGCNBKAGCTGGCCGTTC
引物 -4H1
ACC
GCAACCTACTACTGCCAGCAGAGCDAKAGCTGGCCGTTC
引物 -4H2
ACC
GCAACCTACTACTGCCAGCAGAGCCAGAGCTGGCCGTTC
引物 -4H3
ACC
GCAACCTACTACTGCCAGCAGAGCCATBDKTGGCCGTTC
引物 -5S1
ACCTTC
GCAACCTACTACTGCCAGCAGAGCCATVCTTGGCCGTTC
引物 -5S2
ACCTTC
GCAACCTACTACTGCCAGCAGAGCCATAWKTGGCCGTT
引物 -5S3
CACCTTC
引 物 GCAACCTACTACTGCCAGCAGAGCCATAGCHNKCCGTTC
-6W1 ACCTTCGGC
弓 物
5-6W2 ACCTTCGGC
GCAACCTACTACTGCCAGCAGAGCCATAGCTGGNDKTTC
引物 5-6P1
ACCTTCGGCAGC
GCAACCTACTACTGCCAGCAGAGCCATAGCTGGDCTTTC
引物 5-6P2
ACCTTCGGCAGC 表 4 重链 CDR3的点突变引物 7系列
名称 序列
引物 ■1N1 GTATTACTGCAGCCGTNBKTACTACGGCAGCACC 引物 ■1N2 GTATTACTGCAGCCGTBAKTACTACGGCAGCACC 引物 ■1N3 GTATTACTGCAGCCGTAAATACTACGGCAGCACC 引物 ■2Y1 GTATTACTGCAGCCGTAATNBKTACGGCAGCACCTACG 引物 ■2Y2 GTATTACTGCAGCCGTAATVAKTACGGCAGCACCTACG
GTATTACTGCAGCCGTAATTACNBKGGCAGCACCTACGA
引物 ■3Y1
TTAC
GTATTACTGCAGCCGTAATTACVAKGGCAGCACCTACGA
引物 ■3Y2
TTAC
GTATTACTGCAGCCGTAATTACTACNHKAGCACCTACGA
引物 ■4G1
TTACTG
GTATTACTGCAGCCGTAATTACTACHGKAGCACCTACGA
引物 ■4G2
TTACTG
GTATTACTGCAGCCGTAATTACTACGGCBDKACCTACGA
引物 -5S1
TTACTGGGC 引物 ■5S2
TTACTGGGC
GTATTACTGCAGCCGTAATTACTACGGCAWKACCTACG
引物 ■5S3
ATTACTGGGC
GTATTACTGCAGCCGTAATTACTACGGCAGCNDKTACGA
引物 ■6T1
TTACTGGGCCC
GTATTACTGCAGCCGTAATTACTACGGCAGCBCTTACGA
引物 ■6T2
TTACTGGGCCC
GTATTACTGCAGCCGTAATTACTACGGCAGCACCNBKGA
引物 ■7Y1
TTACTGGGGCCAGG
GTATTACTGCAGCCGTAATTACTACGGCAGCACCVAKGA
引物 ■7Y2
TTACTGGGGCCAGG
GTATTACTGCAGCCGTAATTACTACGGCAGCACCTACNB
引物 ■8D1
KTACTGGGGCCAGGGC
GTATTACTGCAGCCGTAATTACTACGGCAGCACCTACHA
引物 ■8D2
KTACTGGGGCCAGGGC GTATTACTGCAGCCGTA
引物 ■8D3
ATACTGGGGCCAGGGC
引物 ■9Y1 TNBKTGGGGCCAGGGCACC
引物 7-9Y2
TVAKTGGGGCCAGGGCACC 实施例 3、 高亲和力的人源化 TNFa抗体 Fab的表达及活性检测
本实施例涉及两个人源化 TNFa抗体 Fab， 名称分别是 FA01、 FA02。其中， FA01的轻链可变区的氨基酸序列为序列表中序列 15 (核苷酸序列如序列表中的 序列 16所示） ， 轻链恒定区的氨基酸序列为序列表中序列 19 (核苷酸序列如序 列表中的序列 20所示） ， 重链 Fd片段的重链可变区氨基酸序列为序列表中序 列 1 (核苷酸序列如序列表中的序列 2所示） ， 重链恒定区 CH1氨基酸序列为 序列表中序列 33 (核苷酸序列如序列表中的序列 34所示） ； FA02的轻链可变 区的氨基酸序列为序列表中序列 9 (核苷酸序列如序列表中的序列 10所示） ， 轻链恒定区的氨基酸序列为序列表中序列 19 (核苷酸序列如序列表中的序列 20 所示） ， 重链 Fd片段的重链可变区氨基酸序列为序列表中序列 1 (核苷酸序列 如序列表中的序列 2所示），重链恒定区 CH1氨基酸序列为序列表中序列 33 (核 苷酸序列如序列表中的序列 34所示） 。
一、 高亲和力的人源化 TNFa抗体 Fab的构建和表达
这两个人源化 TNFa抗体 Fab的构建方法同实施例 1所述，将上述两个人源 化 TNFa抗体 Fab的重链编码 DNA和轻链编码 DNA插入 pTLR载体的相应位 点， 分别得到 FA01的表达载体 pFAOl Fab, 以及 FA02的表达载体 pFA02 Fab。 再通过实施例 1所述方法， 得到纯度较高的 FA01和 FA02蛋白。
二、 高亲和力的人源化 TNFct抗体 Fab的生物活性检测
通过 L929细胞毒性实验对上述两个人源化 TNFa抗体 Fab ( FA01和 FA02 ) 进行生物活性检测，具体方法同实施例 1，将得到的 OD450值输入 graphpad prism 5软件获得 EC50。 结果如表 5所示， FA01和 FA02的活性均比 Remicade抗体 Fab片段生物活性强。
表 5 人源化 Fab片段中和 TNFa的生物活性
组别 EC50 ( nM )
Remicade Fab 3. 184
FA01 2. 582
FA02 2. 870
注： 表 5中的数据为三次重复实验的平均值。
实施例 4、 人源化 TNFa抗体 IgG的表达及活性检测
一、 人源化 TNFa抗体 IgG重组表达载体的构建
将实施例 1和实施例 2所获得的 2种重链 Fd片段（分别含有 SH01和 SH02 ) 和 6种轻链 （分别含有 SH03、 SH05、 SH04、 SH06、 SH07和 SH08 ) 如表 6所 示分别组合， 以重叠延伸 PCR和 IgGl Fc恒定区拼接在一起， 再重组至表达质 粒 pcDNA3.1(+)中， 将构建好的重组表达质粒转染 CHO细胞， 表达全长人源化 抗体。
具体操作如下所述： （1 ) 轻链直接重组在真核表达载体 pcDNA3.1(+)中。 设计引物如下： 5' -TGAAAGCTTATGGAAATTGTGCTGACTCAGTCTC-3* (划 线 处 为 Hindlll 酶 切 位 点 ） ； 5' AATCTCGAGTCAACACTCTCCCCTGTTGAAGCT-3* (划线处为 Xho I酶切位 点），进行 PCR扩增，扩增产物以 Hindin酶（R0104L, NEB公司产品）和 Xho I 酶（R0146L，NEB公司产品）双酶切后，以 T4连接酶与同样双酶切的 pcDNA3.1(+) 大片段相连接。 （2)重链需以重叠延伸 PCR和 IgGl Fc恒定区拼接在一起， 再 重 组 至 pcDNA3.1(+) 。
5*-ACTGGTACCATGGAGGTGCAGCTGGTGGAGTCTGGGG-3* (戈 ij线处为 Kpn I 酶 切 位 点 ） ；
5 '- AATCTCGAGTC ATTT ACCCGG AGAC AGGGAGAGG-3 * (划线处为 Xho I酶 切位点） 。 将拼接 PCR产物以 Kpn I酶 （R0142L, NEB公司产品） 禾卩 Xho I 酶（R0146L，NEB公司产品）酶切后再重组至同样双酶切的表达质粒 pcDNA3.1(+) 中， 并经鉴酶切与测序鉴定正确。
表 7中 18个抗体按照上述方法制备表达载体。 其中， KS01 ( Glu) 、 KS03
( Glu) 、 KS05 ( Glu) 、 KS06 ( Glu) 、 KS09 ( Glu) 、 KS10 ( Glu) 的重链可 变区编码 DNA均为序列表中序列 2所示（第 1-3位核苷酸序列为 GAG) ， 均编 码序列 1所示的重链可变区（第 1为氨基酸为 Glu)， KS01 ( Gin)、 KS03 ( Gin)、 KS05 ( Gin) 、 KS06 ( Gin) 、 KS09 ( Gin) 、 KS10 ( Gin) 的重链可变区编码 DNA均为序列表中序列 2的第 1-3位核苷酸序列替换为 CAG， 均编码序列 1所 示的重链可变区 （第 1为氨基酸为 Gin) ； KS02、 KS04、 KS07、 KS08、 KS11、 KS12的重链可变区编码 DNA均为序列表中序列 4所示， 均编码序列 3所示的 重链可变区； KS01 ( Glu) 、 KS01 ( Gin)和 KS02的轻链可变区的编码 DNA均 为序列表中序列 6， 均编码序列 5所示的轻链可变区； KS03 ( Glu)、 KS03 ( Gin) 和 KS04的轻链可变区的编码 DNA均为序列表中序列 10， 均编码序列 9所示的 轻链可变区； KS05 ( Glu) 、 KS05 ( Gin)和 KS08的轻链可变区的编码 DNA均 为序列表中序列 8， 均编码序列 7所示的轻链可变区； KS06 ( Glu)、 KS06 ( Gin) 和 KS07的轻链可变区的编码 DNA均为序列表中序列 12， 均编码序列 11所示 的轻链可变区； KS09 ( Glu) 、 KS09 ( Gin) 和 KS1 1的轻链可变区的编码 DNA 均为序列表中序列 14， 均编码序列 13所示的轻链可变区； KS10 ( Glu) 、 KS10 ( Gin) 和 KS12的轻链可变区的编码 DNA均为序列表中序列 16， 均编码序列 15所示的轻链可变区。 所述 18种抗体的重链恒定区的 DNA编码序列为序列表 中序列 18， 编码序列 17所示重链恒定区； 所述 18种抗体的轻链恒定区的 DNA 编码序列为序列表中序列 20， 编码序列 19所示轻链恒定区。
二、 人源化 TNFa抗体 IgG全长抗体的表达及纯化
将步骤一所得的轻链重组质粒和重链重组质粒共转染至 CHO细胞中表达全 长人源化抗体。 常规方法， 将如表 6所示组合的相应质粒分别共稳定转染 CHO 细胞后， 所对应的 12种重组全长抗体分泌至细胞培养上清中， 以上清纯化获得 相应的全长 IgG抗体。 具体纯化步骤如下：
1)层析填料
Mabselect Sure(GE公司产品）、 Superdex 200(GE公司产品 GE)
2)缓冲液
亲和平衡缓冲液 (； PBS): 0.2M 磷酸氢二钠： 82.5mL/L; 0.2M 磷酸二氢钠: 17.5mL/L; 2M 氯化钠： 75mL/L; 加入超纯水， 充分搅拌混匀， 用 1M 氢氧化 钠或 1M盐酸调节 pH至 7.1-7.3。
亲和洗脱缓冲液： NaCl 2.922g/L， 无水乙酸钠 0.49 g/L， 加入 2.9mL/L无水 醋酸， pH3.4-3.6。
亲和再生缓冲液： 5.8mL/L无水醋酸 pH3.0。
亲和 CIP缓冲液： 1M氢氧化钠 100mL/L。
凝胶过滤层析溶液 (； PBS): 0.2M磷酸氢二钠： 82.5mL/L; 0.2M磷酸二氢钠: 17.5mL/L; 2M 氯化钠： 75mL/L; 加入超纯水， 充分搅拌混匀， 用 1M 氢氧化 钠或 1M盐酸调节 pH至 6.8。
3)样品准备 (澄清过滤）
离心： 5000— 6000rpm， 10— 15分钟， 离心样品。
过滤： 离心后样品上清液采用 H7 ( 0.45十 0.2um) 滤器过滤。
4)亲和层析
安装好 AKTA纯化系统及亲和层析柱 （Mabselect或 Mabselect Sure) 。 用 超纯水冲洗 2倍柱体积。 用亲和平衡缓冲液冲洗 5柱体积， 直至基线稳定， 上 样。 上样结束后， 用亲和平衡缓冲液冲洗 5〜10 柱体积。 用亲和洗脱缓冲液洗 柱， 收集 280nm处的主吸收峰， 继续流洗 1柱体积。 用亲和再生缓冲液流洗 3 柱体积。用亲和平衡缓冲液冲洗至 pH中性。用 Mabselect CIP缓冲液或 Mabselect Sure CIP缓冲液在位清洗 5柱体积。用亲和平衡缓冲液冲洗 3柱体积， 直至基线 稳定。 用超纯水冲洗 3柱体积， 直至基线稳定。 用 20%乙醇冲洗 3柱体积， 保 存亲和层析柱。
5)凝胶过滤层析
安装好 AKTA explorer纯化系统及 Superdex 200凝胶过滤层析柱。调节流速 5ml/min, 用超纯水冲洗 1柱体积， 用 Superdex200柱层析平衡液冲洗 2柱体积。 调节流速 2.5 ml/min, 取调节 pH 后亲和收集峰直接上样。 上样结束后， 用 SuperdeX200柱层析平衡液洗脱， 收集 280nm处目的峰。 继续冲洗 1柱体积。 用 O.OlM NaOH保存层析柱。
三、 人源化 TNFa抗体 IgG的活性检测
将步骤二所得的 12种人源化 TNFa抗体 IgG进行 L929细胞毒性实验检测 其中和 TNFa的生物活性， （具体操作如实施例 1所述） ， 得到 OD450的值， 将 OD450值输入 graphpad prism 5软件获得 EC50。表 7显示了 12种人源化 TNFa 抗体 IgG抑制 TNFa生物活性的 EC50, 结果表明大大提高了人源化 TNFa抗体 的生物活性， 其中， KS10 活性最高， 后期对抗体活性的深入研究主要是针对 KS10进行的。
表 6 高亲和力人源化 TNFa抗体
IgG抗体 VH VK
KS01 SH01 SH03
KS02 SH02 SH03
KS03 SH01 SH05
KS04 SH02 SH05
KS05 SH01 SH04
KS06 SH01 SH06
KS07 SH02 SH06
KS08 SH02 SH04
KS09 SH01 SH07
KS10 SH01 SH08
KS11 SH02 SH07
KS12 SH02 SH08 表 7人源化全长 TNFa抗体中和 TNFa的生物活性
组别 EC50 (nM)
KS01 ( Glu) 0.081
KS01 ( Gin) 0.087
KS02 0.109
KS03 ( Glu) 0.087
KS03 ( Gin) 0.093
KS04 0.126
KS05 ( Glu) 0.051
KS05 ( Gin) 0.064 KS06 ( Glu) 0.059
KS06 ( Gin) 0.068
KS07 0.100
KS08 0.113
KS09 ( Glu) 0.036
KS09 ( Gin) 0.032
KS10 ( Glu) 0.028
KS10 ( Gin) 0.025
KS11 0.059
KS12 0.136
Remicade 0.174
实施例 5、 KS10 ( Glu) 与 hTNFa结合动力学检测
将 hTNF-a ( R&D公司， 210-TA-050 ) 与 NHS-LCLC-biotin (货号: 21338, 购自 Thermo-fisher公司） 以摩尔比 1 :3混匀， 于室温放置 lh， 然后通过 PD-10 脱盐柱（17-0851-01，购自 GE公司）将剩余的 NHS-LCLC-biotin去除， 得到最终的 藕联产物为 5(^g/ml 生物素 -hTNF-a。
以 Octet技术平台中的 Octet RED96系统 (ForteBio公司） Kinetics Assays检 测 KS10与 hTNFa结合动力学参数检测， 其试验步骤设置按照仪器操作说明进 行， 通过 Loading将 5(^g/ml 生物素 -hTNFa结合于 SA ( Streptavidin biosensor) 上；通过预试验确定抗体的适宜浓度范围，分别为 6000 nM、 2000 nM、 666.7 nM、 222.2 nM 74.1 nM和 24.7nM。 以 Remicade 为阳性对照， PBS ( H 7.4 ) 为空 白对照。实验结果（表 8 )显示： KS10的 KD值低于 Remicade, 表明其与 hTNFa 的亲和力高于人鼠嵌合抗体 Remicade。
另外检测了 KS10与黑猩猩 TNFa ( Chimpanzee TNFa) (氨基酸序列如序 列 36所示， 核苷酸序列如序列 37所示） 、 猕猴 TNFa ( Chimpanzee TNFa) (氨 基酸序列如序列 38所示， 核苷酸序列如序列 39所示） 、 小鼠 TNFa (mTNFa) (货号： CYT-252, 购自 PROSPEC ) A TNFp ( human TNFp) (货号: CYT-224, 购自 PROSPEC ) 的结合亲和性。 根据 Octet RED96系统亲和性判定标准： 抗原 和抗体结合后产生的光波位移距离间接反应两者间的亲和性， 光波位移距离越 大， 亲和性越高。 结果 （图 3 ) 显示， KS10与人 TNFa有很强的结合， 与与黑 猩猩 TNFct 也有明显结合， 而与猕猴 TNFct 有较弱结合， 与小鼠 TNFct 和人 TNFP几乎不结合。 由此证实了本发明所提供的抗体经人源化之后的特异结合能 力。
表 8 KS10与 hTNFa结合动力学参数
抗体种类 KD (M) kon(l/Ms) kdis(l/s)
Remicade 8.12E-12 1.95E+04 1.58E-07
KS10 2.18E-12 4.69E+04 1.02E-07 注： KD为亲和力常数； kon为结合常数； kdis为解离常数。 实施例 6、 KS10 ( Glu) 对人 TNF-α诱导的类风湿性关节炎大鼠的作用 Sprague Dawley 大鼠 (动物许可号: SCXK (川） 2008-24) ， 4-6 周龄， SPF 级， 72只， 雌雄各半， 体重 140-180g。 大鼠适应性伺养一周后， 随机分为 6个 组别， 每组 12只大鼠， 分别为正常组、 阴性对照组， 模型对照组、 KS10 5mg/kg 组、 KS10 10mg/kg组、 KS10 20mg/kg组。 实验开始时， 除正常组以外， 每组大 鼠以 10%水合氯醛 (350mg/kg)麻醉，造模前，三种 KS10剂量组（5mg/kg、 10mg/kg 和 20mg/kg)采用尾静脉注射给药， 阴性对照组和模型对照组均经尾静脉注射等 体积的生理盐水。注射后 15分钟， 以 1毫升注射器将 0.5mg/ml hTNF-α ( R&D 公司， 商品目录号为 210-TA-050) (用 1% BSA溶解， 注射体积为 60μ1/只） 注 射入三种 KS10剂量组和模型对照组的大鼠左踝关节腔内造成急性关节炎 (单脚 造模)， 将相同体积 1% BSA 注射到阴性对照组大鼠左踝关节腔内。 以关节腔两 侧凹陷逐渐鼓起表明注射成功。 正常组大鼠不进行上述注射。
造模后 18小时， 将各组大鼠左踝关节肿胀程度按改进的 0-4级分类标准进 行相应评分： 0分： 无红肿； 1分： 仅有踝关节肿和 /或红； 2分： 踝关节红肿及 足底肿； 3分： 踝关节及足底红肿； 4分： 踝关节、 足底及脚面红肿 [参考文献： R 0 Williams, M Feldmann, and R N Maini. Anti-tumor necrosis factor ameliorates joint disease in murine collagen-induced arthritis. Proc Natl Acad Sci U S A.1992 October 15;89(20):9784-9788.] , 于造模后 20小时抽取关节滑液， 分离关节周边 软组织并匀浆， 离心后一并冻存于 -70°C， 采用大鼠 ELISA试剂盒 （IL-Ιβ购于 R&D公司，货号： RLB00; IL-6 购于 R&D公司，批号： R6000B )测定其中 IL-i p、 IL-6的水平。
根据评分结果 （图 4 ) 显示， KS10三个剂量均能明显减轻 hTNF-α引起的 大鼠关节红肿， 明显拮抗 hTNF-α诱导类风湿性关节炎的形成。 细胞因子检测显 示， KS10三个剂量均能明显降低关节滑液 /关节周边软组织中的 IL-Ιβ (图 5 ) 和 IL-6 (图 6) 水平， 表明 KS10能完全阻断 TNF-α所致的 IL-Ιβ和 IL-6水平， 直接证实了 KS10的抗人 TNF-α作用。
实施例 7、 KS10 ( Glu) 对人 TNF-ct诱导的大鼠葡萄膜炎的治疗作用 取健康 lewis大鼠 40只， 随机分为正常组、模型组、 阴性对照组、 KS10组。 大鼠腹腔注射 10%水合氯醛 （剂量 0.35ml/kg) ， 待大鼠麻醉后， 模型组与阴性 对照组每只由睫状肌扁平部向玻璃体注射（使用 30G1/2针头）给予 ΙΟμΙ生理盐 水， KS10组大鼠每只注射 ΙΟμΙ浓度为 4mg/ml的 KS10溶液 (； KS10用 PBS配制 30min后， 模型组与 KS10组每只玻璃体注射给予 ΙΟμΙ hTNF-α (约 0.5 mg/ml), 阴性对照组每只玻璃体给予 ΙΟμΙ 1% BSA。 在首次给药造模 24h、 48h将大鼠麻 醉置于裂隙灯下观察， 并进行评分， 评分指标分级参考文献 [Fleisher LN， Ferrell JB， Smith MG， McGahan MC. Lipid mediators of tumor necrosis factor-alpha-induced uveitis. Invest Ophthalmol Vis Sci. 1991 Jul;32(8):2393-9.]并 稍加修订： 虹膜充血 0〜2分 （无充血为 0分、 轻度充血为 1分、 重度充血为 2 分） ， 瞳孔縮小 0〜1分 （瞳孔正常为 0分、 瞳孔縮小为 1分） ， 前房渗出 0〜2 分 （前房无渗出为 0分、 轻度渗出为 1分、 重度渗出为 2分） ， 瞳孔或虹膜后 粘连 0〜2分 （无粘连为 0分、 一处粘连为 1分、 二者粘连为 2分） 。
评分结果如表 9所示， 这一结果显示， KS 10能明显降低葡萄膜炎评分指标 分数， 主要表现为能抑制虹膜充血， 纤维渗出， 瞳孔或虹膜后粘连， 增加眼内 透明度， 这表明 KS 10对 hTNF-α诱导的葡萄膜炎有明显的拮抗作用。
表 9 KS 10对 hTNF-α诱导的大鼠葡萄膜炎的治疗评分结果
~~ 首次给药造模后时间
24h 48h
正常 0. 88±0. 834 0. 38±0. 518
阴性 0. 90±0. 737 0. 38±0. 744
模型 3. 5±0. 971 3. 78±1. 202
KS 10 2. 5±0. 971* 1. 55±2. 422*
注： *表示与模型组相比， *Ρ<0. 05', ( £ 士 SD， η= 10 )
工业应用
本发明通过对 CDR移植后的抗体进行突变改造， 有力克服了常规抗体人源 化方法 （将鼠源抗体可变区 （VH、 VK) 的互补决定区 （CDR) 直接移植到人抗 体框架中） 导致抗体亲和力受损这一缺陷， 最终得到与起始抗体相近的人源化 抗体。 本发明所提供的 Fab及 IgG抗体的人源化程度大大提高 （达 95%以上） ， 且 经实验证明具有与人鼠嵌合抗体 Remicade相近， 甚至更高的亲和力及生物活性， 对人 TNFct具有更好的中和作用， 可更有效地治疗与 TNFct相关的疾病， 优选类风 湿性关节炎、 自身免疫性葡萄膜炎、 克隆病、 斑片性银屑病、 银屑病关节炎、 强直性脊柱炎、 溃疡性结肠炎或幼年特发性关节炎。

Claims

权利要求
1、 抗人肿瘤坏死因子 α的人源化抗体或抗原结合片段 Fab， 含有重链可变 区和轻链可变区， 其中所述重链可变区氨基酸序列为序列表中序列 1或序列 3; 所述轻链可变区氨基酸序列为序列表中序列 15、 序列 9、 序列 11、 序列 7、 序 列 13和序列 5中的任一种； 所述序列 1的第一位氨基酸残基为 Glu或 Gln。
2、 根据权利要求 1所述抗体， 其特征在于所述抗体为下述 a-1中任一种： a、KS10，其重链可变区为 SH01，氨基酸序列为序列 1，轻链可变区为 SH08, 氨基酸序列为序列 15;
b、KS03，其重链可变区为 SH01，氨基酸序列为序列 1，轻链可变区为 SH05, 氨基酸序列为序列 9;
c、 KS06，其重链可变区为 SH01，氨基酸序列为序列 1，轻链可变区为 SH06, 氨基酸序列为序列 11;
d、 KS12，其重链可变区为 SH02,氨基酸序列为序列 3，轻链可变区为 SH08, 氨基酸序列为为序列 15;
e、 KS04，其重链可变区为 SH02,氨基酸序列为序列 3，轻链可变区为 SH05, 氨基酸序列为序列 9;
f、 KS07，其重链可变区为 SH02,氨基酸序列为序列 3，轻链可变区为 SH06, 氨基酸序列为序列 11;
g、KS02，其重链可变区为 SH02,氨基酸序列为序列 3，轻链可变区为 SH03, 氨基酸序列为序列 5;
h、 KS08，其重链可变区为 SH02,氨基酸序列为序列 3，轻链可变区为 SH04, 氨基酸序列为序列 7;
i、 KSll，其重链可变区为 SH02,氨基酸序列为序列 3，轻链可变区为 SH07, 氨基酸序列为序列 13;
j、KS01，其重链可变区为 SH01，氨基酸序列为序列 1，轻链可变区为 SH03, 氨基酸序列为序列 5;
k、KS05，其重链可变区为 SH01，氨基酸序列为序列 1，轻链可变区为 SH04, 氨基酸序列为序列 7;
KKS09,其重链可变区为 SH01,氨基酸序列为序列 1，轻链可变区为 SH07, 氨基酸序列为序列 13;
所述序列 1的第一位氨基酸残基为 Glu或 Gln。
3、 一种抗人肿瘤坏死因子 ct的人源化抗原结合片段 Fab， 含有重链可变区 和轻链可变区，其中所述重链可变区的氨基酸序列为序列表中序列 27的第 1-120 位或序列 25 的第 1-120位， 所述轻链可变区的氨基酸序列为序列表中序列 31 的第 1-109位或序列 29的第 1-109位。
4、 根据权利要求 1或 2所述的抗体， 其特征在于： 所述抗体由重链和轻链 组成， 所述重链的恒定区氨基酸序列与人抗体重链恒定区氨基酸序列相同， 所 述轻链的恒定区氨基酸序列与人抗体轻链恒定区氨基酸序列相同；
5、 根据权利要求 1或 2或 4所述的抗体， 其特征在于： 所述重链的恒定区 氨基酸序列如序列表中序列 17所示； 所述轻链的恒定区氨基酸序列如序列表中 序列 19所示。
6、 根据权利要求 1、 2、 4和 5中任一所述的抗体， 其特征在于： 所述重链 的氨基酸序列为序列表中序列 21 ; 所述轻链的氨基酸序列为序列表中序列 23 ;
所述序列 21的第一位氨基酸为 Glu或 Gln。
7、 根据权利要求 1或 3所述的 Fab， 其特征在于： 所述 Fab由重链片段和 轻链组成， 所述重链片段由所述重链可变区和重链恒定区的 CH1组成， 所述轻 链由所述轻链可变区和轻链恒定区组成； 所述 CH1氨基酸序列与人抗体重链恒 定区的 CH1氨基酸序列相同， 所述轻链恒定区氨基酸序列与人抗体轻链恒定区 氨基酸序列相同；
8、 根据权利要求 1或 3或 7所述的 Fab， 其特征在于： 所述 CH1氨基酸序 列如序列表中序列 33所示； 所述轻链恒定区氨基酸序列如序列表中序列 19所 示。
9、根据权利要求 3或 7或 8所述的 Fab， 其特征在于： 所述 Fab为下述 M ) -b3 ) 中任一种：
b l ) KS-7F: 所述重链片段的氨基酸序列为序列表中序列 27， 且所述轻链的 氨基酸序列为序列表中序列 31 ;
b2 ) KS-7A: 所述重链片段的氨基酸序列为序列表中序列 25， 且所述轻链的 氨基酸序列为序列表中序列 31 ;
b3 ) KS-2E: 所述重链片段的氨基酸序列为序列表中序列 25， 且所述轻链的 氨基酸序列为序列表中序列 29。
10、 由权利要求 1、 2、 4、 5和 6中任一所述抗体衍生的抗原结合片段 A或 由权利要求 1、 3、 7、 8和 9中任一所述 Fab衍生的抗原结合片段 B， 其特征在 于： 所述抗原结合片段 A为 Fab、 Fab'、 F(ab')2、 Fv、 重链可变区、 轻链可变区、 选自重链可变区的多肽片段或选自轻链可变区的多肽片段；所述抗原结合片段 B 为 Fab'、 F(ab')2 Fv、 重链可变区、 轻链可变区、 选自重链可变区的多肽片段 或选自轻链可变区的多肽片段。
1 1、 编码如下 A) -C ) 中任一种蛋白的基因：
A) 权利要求 1、 2、 4、 5和 6中任一所述的抗体；
B ) 权利要求 1、 3、 7、 8和 9中任一所述的 Fab;
C ) 权利要求 10所述的抗原结合片段。
12、 根据权利要求 1 1所述的基因， 其特征在于： 权利要求 1、 2、 4、 5和 6 中任一所述抗体和权利要求 10所述抗原结合片段 A的重链可变区的编码序列均 如序列表中序列 2或序列 4所示； 权利要求 1、 2、 4、 5和 6中任一所述抗体和 权利要求 10所述抗原结合片段 A的轻链可变区编码序列均如序列表中序列 16、 序列 10、 序列 12、 序列 8、 序列 14和序列 6中的任一种所示；
权利要求 1、 3、 7、 8和 9中任一所述 Fab和权利要求 10所述抗原结合片 段 B的重链可变区的编码序列均为序列表中序列 2、 序列 4、序列 28的第 1-360 位和序列 26的第 1-360位中的任一种； 1、 3、 7、 8和 9中任一所述 Fab和权利 要求 10所述抗原结合片段 B的轻链可变区的编码序列均为序列表中序列 16、序 列 10、 序列 12、 序列 8、 序列 14、 序列 6、 序列 32的第 1-327位和序列 30的 第 1-327位中的任一种。
13、 根据权利要求 1 1 或 12所述的基因， 其特征在于： 所述抗体的重链恒 定区的编码序列如序列表中序列 18所示； 所述抗体的轻链恒定区的编码序列如 序列表中序列 20所示；
所述 Fab的 CH1的编码序列如序列表中序列 34所示；所述 Fab的轻链恒定 区编码序列如序列表中序列 20所示。
14、 根据权利要求 1 1-13中任一所述的基因， 其特征在于： 所述抗体的重链 编码序列为序列表中序列 22; 所述抗体的轻链编码序列为序列表中序列 24 ;
权利要求 3、 7、 8和 9中任一所述 Fab的编码序列为下述 cl ) -c3 ) 中任一 种：
c l ) KS-7F: 所述 Fab 的重链片段的编码序列为序列表中序列 28， 且所述 Fab的轻链编码序列为序列表中序列 32 ;
c2 ) KS-7A: 所述 Fab 的重链片段的编码序列为序列表中序列 26， 且所述
Fab的轻链编码序列为序列表中序列 32 ;
c3 ) KS-2E: 所述 Fab 的重链片段的编码序列为序列表中序列 26， 且所述 Fab的轻链编码序列为序列表中序列 30。
15、 下述遗传材料： 含有权利要求 1 1-14中任一所述基因的重组载体、 重组 菌、 重组细胞系、 重组病毒或表达盒。
16、 根据权利要求 15所述的遗传材料， 其特征在于： 所述重组载体为表达 所述 Fab 或抗体或抗原结合片段的原核表达载体或真核表达载体； 所述重组菌 为携带有所述基因的大肠杆菌； 所述重组细胞系为转入所述基因的哺乳动物细 胞系， 优选 CHO细胞系， 或 293细胞及其亚系； 所述重组病毒为携带有所述基 因的重组腺病毒或重组腺相关病毒。
17、 权利要求 1、 2、 4、 5和 6 中任一所述抗体， 或权利要求 1、 3、 7、 8 和 9中任一所述 Fab， 或权利要求 10所述抗原结合片段， 或权利要求 1 1-14中 任一所述基因， 或权利要求 15或 16所述遗传材料的用途：
dl ) 在制备预防和 /或治疗人抗肿瘤坏死因子 ct相关疾病药物中的用途， 或 d2 ) 在制备中和人抗肿瘤坏死因子 α产品中的用途， 或
d3 ) 在制备定性或定量检测人抗肿瘤坏死因子 ct的试剂盒中的用途。
18、 药物组合物， 含有辅料和活性成分， 所述活性成分含有下述物质中的 至少一种： 权利要求 1、 2、 4、 5和 6 中任一所述抗体， 权利要求 1、 3、 7、 8 和 9中任一所述 Fab， 权利要求 10所述抗原结合片段， 权利要求 1 1 - 14中任一 所述的基因， 和权利要求 15或 16所述遗传材料； 所述辅料为药学上可接受的 载体或赋形剂。
19、 下述物质中的任一种在治疗人肿瘤坏死因子（ 相关疾病中的应用： 权 利要求 1、 2、 4、 5和 6中任一所述抗体， 权利要求 1、 3、 7、 8和 9中任一所 述 Fab， 权利要求 10所述抗原结合片段， 权利要求 1 1 -14中任一所述的基因， 权利要求 15或 16所述遗传材料， 和权利要求 18所述的药物组合物。
20、 根据权利要求 17所述用途或权利要求 19所述应用， 其特征在于： 所 述人肿瘤坏死因子 a相关疾病为由人肿瘤坏死因子 a升高引起的疾病， 优选为 类风湿性关节炎、 自身免疫性葡萄膜炎、 克隆病、 斑片性银屑病、 银屑病关节 炎、 强直性脊柱炎、 溃疡性结肠炎或幼年特发性关节炎。
PCT/CN2011/001668 2010-09-30 2011-09-30 抗TNFα的人源化抗体及其抗原结合片段Fab和用途 WO2012041018A1 (zh)

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US13/876,774 US9096669B2 (en) 2010-09-30 2011-09-30 Humanized anti-TNF-α antibody and antigen-binding fragment (Fab) thereof and use of the same
KR1020137011174A KR20130062366A (ko) 2010-09-30 2011-09-30 인간화 항 TNF α 항체 및 그의 항원 결합 단편 (FAB) 및 용도
AU2011307886A AU2011307886C1 (en) 2010-09-30 2011-09-30 Anti TNF alpha humanized antibody and fragment antigen binding (Fab) and use thereof
SG2013018601A SG188987A1 (en) 2010-09-30 2011-09-30 HUMANIZED ANTI-a ANTIBODY AND ANTIGEN-BINDING FRAGMENT (FAB) THEREOF AND USE OF THE SAME
CA2812430A CA2812430C (en) 2010-09-30 2011-09-30 Humanized anti-tnf-.alpha. antibody and antigen-binding fragment (fab) thereof and use of the same
KR1020157020565A KR101712874B1 (ko) 2010-09-30 2011-09-30 인간화 항 TNF α 항체 및 그의 항원 결합 단편 (Fab) 및 용도
JP2013530530A JP5767708B2 (ja) 2010-09-30 2011-09-30 ヒト化抗ヒト腫瘍壊死因子α（ＴＮＦ−α）抗体およびその抗原結合性フラグメント（Ｆａｂ）およびその使用方法
EP11827916.5A EP2623515B1 (en) 2010-09-30 2011-09-30 ANTI TNFalpha HUMANIZED ANTIBODY AND FRAGMENT ANTIGEN BINDING (FAB) AND USE THEREOF
BR112013007501-5A BR112013007501B1 (pt) 2010-09-30 2011-09-30 Anticorpo humanizado anti-fator de necrose tumoral a, ou um fragmento de ligação ao antígeno do mesmo, uso e composição farmacêutica
RU2013109392/10A RU2556815C2 (ru) 2010-09-30 2011-09-30 ГУМАНИЗИРОВАННОЕ АНТИТЕЛО К ФНО-α, ЕГО АНТИГЕНСВЯЗЫВАЮЩИЙ ФРАГМЕНТ (Fab) И ИХ ПРИМЕНЕНИЕ

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