WO2013174264A1 - 抗BLyS抗体 - Google Patents

抗BLyS抗体 Download PDF

Info

Publication number
WO2013174264A1
WO2013174264A1 PCT/CN2013/076074 CN2013076074W WO2013174264A1 WO 2013174264 A1 WO2013174264 A1 WO 2013174264A1 CN 2013076074 W CN2013076074 W CN 2013076074W WO 2013174264 A1 WO2013174264 A1 WO 2013174264A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
amino acid
blys
acid sequence
light chain
Prior art date
Application number
PCT/CN2013/076074
Other languages
English (en)
French (fr)
Inventor
陈博
舒红兵
Original Assignee
武汉华鑫康源生物医药有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 武汉华鑫康源生物医药有限公司 filed Critical 武汉华鑫康源生物医药有限公司
Priority to JP2015511926A priority Critical patent/JP6006404B2/ja
Priority to BR112014025651-9A priority patent/BR112014025651B1/pt
Priority to EP20180342.6A priority patent/EP3792280A3/en
Priority to IN2367MUN2014 priority patent/IN2014MN02367A/en
Priority to RU2014133410A priority patent/RU2613422C2/ru
Priority to US14/401,288 priority patent/US9828423B2/en
Priority to EP13794561.4A priority patent/EP2853543B1/en
Publication of WO2013174264A1 publication Critical patent/WO2013174264A1/zh
Priority to ZA2014/08955A priority patent/ZA201408955B/en

Links

Classifications

    • 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
    • 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
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • 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
    • 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
    • A61P35/00—Antineoplastic agents
    • 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
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2875—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF/TNF superfamily, e.g. CD70, CD95L, CD153, CD154
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
    • C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • C07K2317/52—Constant or Fc region; Isotype
    • 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
    • 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
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C12N5/12—Fused cells, e.g. hybridomas
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Definitions

  • Systemic lupus erythematosus is a chronic progression involving multiple systems such as skin, joints, heart, lungs, kidneys, blood, and brain, often recurring with remission and recurrence. Immune disease. Systemic lupus erythematosus primarily affects African-Caribbean, Asian, and Hispanic people, while Caucasians (Caucasians) are less affected. The American Lupus Foundation believes that conservative predictions of systemic lupus erythematosus affect 300,000 people in the United States.
  • BLyS a co-stimulatory factor of B lymphocytes
  • IL-4 a co-stimulatory factor of B lymphocytes
  • BLyS In vitro experiments showed that after pre-activation of B cells with IgM, BLyS induced a large amount of proliferation and secreted a large amount of IgM and IgA, but this stimulation was not obvious for B cells during resting period. Further studies have shown that BLyS mainly acts on pre-B lymphocytes, immature B lymphocytes, and activation. Lymphocytes, but have no effect on plasma cells, lymphoid pluripotent stem cells. Like most cytokines, BLyS stimulates downstream signaling primarily through B cell surface receptors.
  • B cell activating factor receptor B cell activating factor receptor
  • BAFF-R B cell activating factor receptor
  • TACI transmembrane activator 1 and calcium modulator and cyclophi 1 in 1 igand interactor
  • BCMA B cell maturation Antigen
  • LymphoStat-B a human monoclonal antibody that antagonizes the bioact ivi t ies of B lymphocyte stimulator. Arthritis Rheum. 2003 Nov; 48 (11): 3253 -65) 0
  • the anti-BLyS antibody developed by American Human Genome has become the world's first new drug for the treatment of lupus erythematosus in the past 60 years. Benlysta only targets BLyS-stimulated B cells, which greatly reduces the side effects of the treatment compared with chemotherapy drugs, thus providing a safe and effective treatment for patients with systemic lupus erythematosus.
  • Benlysta is produced by a phage display library.
  • a disadvantage of phage display libraries is that the pairing of heavy and light chains is considered artificial without in vivo selection (Greg Winter, et al., Making Antibodies by Phage Display Technology. Annual Review of Immunology Vol. 12: 433- 455); The library was constructed from naive human PBMC, so the candidate antibody has low affinity ( Edwards BM, et al.. The remarkable flexibility of the human antibody repertoire; isolation of over one thousand different antibodies to a single protein, BLyS. J Mol Biol.
  • anti-BLyS antibodies that have the following properties: they are produced without the use of phage display libraries, but are produced, for example, using techniques such as humanized murine antibodies; are capable of binding to B lymphocyte stimulating factors with high affinity and are capable of inhibiting B lymphocytes
  • the binding of the cell stimulating factor to its receptor BR3-FC has high specificity; it has high immunogenicity; and/or has low affinity with MHC II factor, thereby minimizing the immune response while ensuring affinity.
  • the anti-BLyS antibodies of the invention are humanized.
  • amino acid sequence of the light chain variable region of the anti-BLyS antibody and the amino acid sequence of the heavy chain variable region are selected from the group consisting of:
  • the anti-BLyS antibody further comprises a human light chain constant region and a heavy chain constant region And the light chain variable region and the heavy chain variable region are linked to a human light chain constant region and a heavy chain constant region, respectively.
  • the human light chain constant region is a human light chain kappa constant region.
  • the human heavy chain constant region is a human heavy chain Fc fragment.
  • the invention also provides DNA molecules encoding the anti-BLyS antibodies of the invention.
  • the DNA molecule has a nucleotide sequence selected from the group consisting of SEQ ID NOS. 49-58.
  • the present invention provides a recombinant DNA vector comprising the DNA molecule of the anti-BLyS antibody of the present invention.
  • the invention also provides a host cell comprising the recombinant DNA vector of the invention.
  • the invention provides a method of preventing and/or treating a disease caused by an excess of B cell proliferation comprising administering an effective amount of the anti-BLyS antibody of any one of claims 1-7.
  • the disease caused by excessive proliferation of said B cells is selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, ankylosing arthritis, or B cell lymphoma.
  • the invention also provides a pharmaceutical composition comprising an effective amount of an antibody of the invention and a pharmaceutically acceptable carrier.
  • the invention also provides a method of making an anti-BLyS antibody comprising incubating a host cell of the invention, and obtaining the antibody. Detailed description of the invention
  • One of the objects of the present invention is to provide an anti-BLyS antibody which is capable of binding to a B lymphocyte stimulating factor and capable of inhibiting the binding of B lymphocyte stimulating factor to its receptor BR3-FC.
  • the invention extracts human peripheral blood RNA by genetic engineering technology, and obtains the cDNA of human BLyS by reverse transcription of human peripheral blood RNA peripheral RNA as a template, and then separately expands the cDNA of human BLyS as a template.
  • the human BLyS gene fragment was obtained, cloned into a eukaryotic expression system, and transfected into a host cell for expression and purification to obtain a human BLyS purified protein.
  • a total of 2000 different monoclonal hybridoma cells were obtained by immunizing mice with BLyS purified protein as human antigen.
  • a total of 211 secreted antibodies were cloned by binding to BLyS protein.
  • 5 secreted antibodies were obtained by detecting the ability to bind to BLYS receptor on BJAB.
  • Different ability to inhibit the binding of biotin-labeled BLyS to BLYS receptor-bound monoclonal hybridoma cells on BJAB respectively Named 1D12, 2B10, 2G3, 5A5 and 13G8.
  • the immunological characteristics of the antibodies secreted by the obtained five monoclonal hybridoma cell lines were identified by ELISA. The results showed that the antibodies secreted by the cloned hybridoma cell lines screened in the present invention were specific to BLyS, and to other TNF families. Antigens such as TNF- ⁇ , TNF- ⁇ are not reactive.
  • Blys stimulates B cell growth and maintains B cell survival
  • antibodies that inhibit, especially neutralize, Blys will inhibit B cell growth.
  • B cells are a special kind of cells in which organisms produce antibodies, it is difficult for B cells to produce antibodies against self-growth (Thomas Schirrmann, et al., Phage display for the generation of antibodies for proteome research, diagnostics and therapy, Molecules, 2011, 16, 412-426). Therefore, it is relatively difficult to produce anti-Blys antibodies by murine immunization.
  • the present invention obtains anti-Blys by the method of murine immunization, and maintains the advantage of the murine antibody.
  • the invention features an anti-BLyS antibody, the light chain CDR1, CDR2 and CDR3 and heavy chain thereof
  • the monoclonal hybridoma cell 1D12 secretes an anti-BLyS antibody comprising the light chain of the amino acid sequence set forth in SEQ ID NO.
  • the variable region and the heavy chain variable region of the amino acid sequence shown in SEQ ID NO. 32 are designated as monoclonal antibody 1D12.
  • the anti-BLyS antibody secreted by the monoclonal hybridoma cell 2B10 comprises a light chain variable region of the amino acid sequence set forth in SEQ ID NO. 33 and a heavy chain variable sequence of the amino acid sequence set forth in SEQ ID NO. Region, named monoclonal antibody 2B10.
  • the anti-BLyS antibody secreted by the monoclonal hybridoma cell 5A5 comprises a light chain variable region of the amino acid sequence set forth in SEQ ID NO. 37 and a heavy chain variable sequence of the amino acid sequence set forth in SEQ ID NO. Region, named monoclonal antibody 5A5.
  • the anti-BLyS antibody secreted by the monoclonal hybridoma cell 13G8 comprises a light chain variable region of the amino acid sequence set forth in SEQ ID NO. 39 and a heavy chain of the amino acid sequence set forth in SEQ ID NO.
  • the variable region was named monoclonal antibody 13G8.
  • amino acid sequences of the present invention can substitute, add and/or delete one or more of the amino acid sequences of the present invention without substantially affecting the activity of the antibody (eg 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more amino acids to obtain functionally equivalent amino acid sequences, ie functional variants of said amino acid sequences, which are all considered to be included in the scope of protection of the present invention .
  • Amino acids having similar properties are replaced as in the variable region.
  • a functional variant of an amino acid sequence according to the invention may be at least 95%, 96%, 97%, 98% or 99% identical to its source sequence.
  • the antibody sequence identity of the invention can be measured using sequence analysis software.
  • sequence analysis software for example, the computer program BLAST with default parameters, especially BLASTP or TBLASTN.
  • the prepared murine antibody can be humanized to obtain a humanized anti-BLyS antibody.
  • Methods of making humanized antibodies are well known to those skilled in the art.
  • a humanized anti-BLyS antibody of the invention can be prepared by transferring a CDR sequence of the invention into a variable region of a human antibody.
  • the humanized antibody does not produce an anti-antibody reaction (AAR) and a human anti-mouse antibody response (HAMA), is not rapidly cleared by neutralization by anti-antibody, and has immunological effector functions such as ADCC and CDC.
  • AAR anti-antibody reaction
  • HAMA human anti-mouse antibody response
  • amino acid sequence of the light chain variable region of the humanized anti-BLyS antibody of the invention and the amino S sequence of the heavy chain variable region are selected from the group consisting of:
  • the humanized anti-BLyS antibody of the present invention can inhibit not only B lymphocyte stimulating factor but also B lymphocyte stimulating factor It binds to its receptor BR3-Fc and has low affinity with MHC II factor, thus minimizing the immune response while ensuring affinity.
  • the human light chain constant region is a human light chain kappa constant region.
  • the human heavy chain constant region is a human heavy chain Fc fragment.
  • the human light chain kappa constant region and the human heavy chain Fc fragment are both derived from healthy human B lymphocytes.
  • the light and heavy chains of the intact humanized anti-BLyS antibody were obtained by genetic engineering techniques, overlapping extension PCR ligation of the variable and constant regions. molecule. Due to the degeneracy of the password ⁇ , 'may exist ⁇ [ ⁇ a variety of antibodies capable of encoding the invention
  • the invention provides a DNA molecule encoding a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO. 32, the nucleotide sequence of which is set forth in SEQ ID NO.
  • the invention provides a DNA molecule encoding a light chain variable region having the amino acid sequence set forth in SEQ ID NO. 33, the nucleotide sequence of which is set forth in SEQ ID NO.
  • the invention provides a DNA molecule encoding a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO. 34, the nucleotide sequence of which is set forth in SEQ ID NO.
  • the invention provides a DNA molecule encoding a light chain variable region having the amino acid sequence set forth in SEQ ID NO. 35, the nucleotide sequence of which is set forth in SEQ ID NO.
  • the invention provides a DNA molecule encoding a light chain variable region having the amino acid sequence set forth in SEQ ID NO. 37, the nucleotide sequence of which is set forth in SEQ ID NO. In one embodiment, the invention provides a DNA molecule encoding a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO. 38, the nucleotide sequence of which is set forth in SEQ ID NO.
  • the invention provides a DNA molecule encoding a light chain variable region having the amino acid sequence set forth in SEQ ID NO. 39, the nucleotide sequence of which is set forth in SEQ ID NO.
  • the invention provides a DNA molecule encoding a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO. 40, the nucleotide sequence of which is set forth in SEQ ID NO.
  • the recombinant DNA vector is an expression vector, and a person skilled in the art clones the DNA molecule of the antibody into an expression vector, transforms the host cell, and obtains a single-chain antibody by inducing expression.
  • the recombinant DNA vector of the invention comprises a DNA molecule encoding a humanized anti-BLyS antibody of the invention.
  • the DNA molecule encoding the humanized anti-BLyS antibody can be recombinantly constructed into a vector for mammalian transcriptional expression, and the expression vector of the present invention contains the heavy and light chain variable regions of the encoded human anti-BLyS monoclonal antibody and is constant. The DNA sequence of the region.
  • the expression vector further comprises a promoter and a DNA sequence encoding a secretion signal peptide, and at least one drug resistance gene for screening.
  • Methods used include DNA synthesis techniques and in vitro recombination techniques.
  • the invention also provides a host cell comprising the recombinant DNA vector of the invention.
  • the host cell of the present invention may be a prokaryotic host cell, a eukaryotic host cell or a bacteriophage.
  • the prokaryotic host cell may be Escherichia coli, Bacillus subtilis, Streptomyces or Proteus mirabilis.
  • the eukaryotic host cell may be a fungus such as Pichia pastoris, Saccharomyces cerevisiae, fission yeast, Trichoderma, such as grass armyworm, such as plant cells such as tobacco, such as BHK cells, CH0 cells, Mammalian cells such as COS cells and myeloma cells.
  • the host cell of the invention is preferably a mammalian cell, more preferably
  • BHK cells CH0 cells, NS0 cells or COS cells.
  • the present invention therefore provides a method for the prevention and treatment of B cell growth ⁇ 5 Excess Primer from a disease, an anti-BLyS antibody of any one of claims 1-7 which comprises administering an effective dose of the claims.
  • the present invention also provides a pharmaceutical application for the caused disease.
  • the diseases in which the B cells proliferate excessively include, but are not limited to, systemic lupus erythematosus, rheumatoid arthritis, ankylosing arthritis, or B cell lymphoma.
  • the invention also provides a pharmaceutical composition comprising an effective amount of any of the anti-BLyS antibodies or humanized anti-BLyS antibodies of the invention and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition can be formulated into a pharmaceutical preparation by a conventional method by mixing any one of the anti-BLyS antibody or the humanized anti-BLyS antibody of the present invention with one or more pharmaceutically acceptable adjuvants.
  • pharmaceutically acceptable carrier refers to one or more carriers known to those skilled in the art that are organic or inorganic, natural or synthetic, in combination with antibodies to promote their stability and clinical utility. Suitable carriers include pharmaceutically acceptable sterile saline and aqueous or anhydrous isotonic sterile solutions and sterile suspensions known to those skilled in the art.
  • the effective dosage and method of use of the present invention depend on a number of factors, including the patient's age, weight, sex, natural health status, nutritional status, strength of the compound's activity, time of administration, rate of metabolism, severity of the disease, and subjectivity of the treating physician. Judge. The effective dosage and method of use can be readily determined by those skilled in the art based on the above factors.
  • Figure 1 shows the SDS-PAGE detection of Example 1, wherein Lane A is the protein expressed by the recombinant plasmid of the present invention, Lane B blank vector recombinant plasmid, and Lane C is the protein molecular weight Marker;
  • Figure 2 is a flow chart showing the binding of different concentrations of biotin-labeled hi s-hBLyS recombinant protein to BJAB cells in Example 2.
  • the black line is 200 ng/mL biotin-labeled hi s-hBLyS recombinant protein group
  • the red line is 100 ng/ mL biotinylated hi s-hBLyS recombinant protein group
  • the green line is biotin-free labeled hi s-hBLyS recombinant protein only contains SA-APC group;
  • Figure 3 is a flow chart showing the binding of serum of the immunized mouse of Example 3 to BJAB cells
  • Figure 4 is a flow chart showing the binding of the antibody secreted by the monoclonal hybridoma cells of Example 4 to the BLyS receptor on BJAB cells, wherein The black line is the negative control I gG group, the red line is the biotin-labeled hi s-hBLyS recombinant protein contains only the SA-APC group, the purple line is the monoclonal hybridoma cell 3H9, and the blue line is the monoclonal hybridoma cell 1 3G8 ;
  • Figure 5 is a flow chart showing the binding of the antibody secreted by the monoclonal hybridoma cells of Example 4 to the BLyS receptor on BJAB cells, wherein the black line is the negative control group IgG, the red line is the monoclonal hybridoma cell group 1D12, purple The line is monoclonal hybridoma cell 2B10, and the blue line is monoclonal hybridoma cell 2G 3;
  • Figure 6 is a flow chart showing the binding of the antibody secreted by the monoclonal hybridoma cells of Example 4 to the BLyS receptor on BJAB cells, wherein the black line is the negative control I gG group, the red line is the monoclonal hybridoma cell 5A5 group, purple The line is a monoclonal hybridoma cell 1 D7 group, and the blue line is a monoclonal hybridoma fine 4D3 group;
  • Figure 4 is a graph showing the results of a protease-linked immunosorbent assay of the antibodies secreted by the monoclonal hybridoma cells 1 D12, 2B10, 2G 3, 5A5 and 1 3G8 and the hi s-hBLyS recombinant protein and the tumor necrosis factor (TNF) family;
  • Fig. 8 is a graph showing the results of inhibiting B cell proliferation in vitro by the humanized anti-BLyS antibody of Example 9, wherein the abscissa is the concentration of the humanized anti-BLyS antibody, and the ordinate is the fluorescence value.
  • Figure 9 shows the inhibitory effect of candidate antibody molecules on BLys-induced lymph node proliferation.
  • Figure 10 shows the inhibitory effect of candidate antibody molecules on BLys-induced elevation of serum IgA.
  • the present invention discloses anti-BLyS antibodies and uses thereof. Those skilled in the art can learn from the contents of this document and appropriately improve the process parameters. It is to be noted that all such alternatives and modifications will be apparent to those skilled in the art and are considered to be included in the present invention.
  • the products of the present invention have been described in terms of preferred embodiments, and it is obvious that those skilled in the art can make and modify and combine the methods described herein to implement and apply the present technology without departing from the scope of the present invention. .
  • Example 1 Cloning and expression of human BLyS gene
  • RNA Peripheral blood of healthy humans was isolated, and total RNA was extracted and purified using a commercial RNA kit (Qiagen). Reverse transcription synthesis of cDNA first-strand cDNA reaction system using purified total RNA as a template RNase free dH 2 0 9.5 ⁇
  • the reaction conditions were: 30 ° C lOmin, 42 ° C 30 min, 99 ° C 5 min 4 ° C 5 min. After the reaction was over, ice bath was taken for 5 min.
  • the cloned secreted BLyS primers PI and P2 were designed and synthesized, and the cDNA synthesized by reverse transcription was used as a template, and the upstream primer P1 and the downstream primer P2 were used for PCR amplification.
  • the nucleotide sequences of P1 and P2 are as follows:
  • P2 5'cgacgtcgac tcacagcagt ttcaatgcac caaaaaatgt gacatc.
  • the PCR amplification reaction system is:
  • the PCR reaction procedure is:
  • the PCR amplification product was recovered by gel electrophoresis, digested with Sa l I and Hind III, cloned into PCDNA3.1 eukaryotic expression plasmid system, and transfected into 293T cells with blank vector transformation plasmid as control (Chinese classical culture preservation) Center) After 3 days, the culture supernatant was collected and the resulting supernatant was passed through the Is.
  • the purified protein of his-hBLyS was purified by chromatography and detected by SDS-PAGE. The results are shown in Figure 1. As can be seen from the results of Fig. 1, the recombinant plasmid of the present invention has protein expression, but the blank vector recombinant plasmid has no obvious protein expression band.
  • the recombinant protein of Example 1 was purified to obtain the his-hBLyS recombinant protein and the biotin-XX-Li S dissolved in DMS0 was mixed in a weight ratio of 1:4 in ng/mL, left at room temperature for 1 hour, and the reaction mixture was passed through a gel. Column, biotinylated his-hBLyS and free biotin were isolated.
  • the isolated biotin-labeled his-hBLyS recombinant protein was divided into four different concentration groups of 25 ng/mL, 50 ng/mL, 100 ng/mL and 200 ng/mL, respectively, with 1 x 10 6 Burkitt lymphoma cells ( BJAB) Cells were mixed, incubated at 4 ° C for 15 min, washed 3 times with PBS, and added stre tavidin-allophycocyanin (SA-APC) to a final concentration of 0.2 g/mL at 4. C Incubate for 20 minutes. After washing 3 times with PBS, the cells were flow cytometer, and the results are shown in Fig. 2.
  • BJAB Burkitt lymphoma cells
  • the mixture was then incubated with BJAB cells for 15 minutes at 4 ° C, 3 times with physiological saline, and 0.2 g/mL of streptavidin- al lophycocyanin was added and incubated at 4 ° C for 15 minutes. After eluting 3 times with physiological saline, the sample was subjected to flow cytometry to detect whether the serum of the immunized mouse inhibited the binding of BLyS and its receptor BR3-Fc, and the results are shown in Fig. 3.
  • the lymph nodes at the roots of the thighs of the mice were taken, and the B-cell-rich suspension was taken after being ground in physiological saline, and electrofused with myeloma cells SP2/0 according to a conventional method.
  • the fused cells were distributed in 96-well plates and cultured in RPMI-1640 complete medium containing HAT at 5% CO 2 at 37 °C.
  • 211 secreted antibodies were screened for binding to the BLyS protein clone by enzyme-labeled reaction.
  • the antibodies produced by the 211 clones that bind to the BLyS protein were separately incubated with the biotin-labeled his-hBLyS recombinant protein for 20 minutes at room temperature. The mixture was then incubated with BJAB cells at 4 ° C for 15 minutes, and after 3 times with physiological saline, 0.2 ⁇ g/mL of streptavidin -allophycocyanin was added and incubated at 4 ° C for 15 minutes. After eluting 3 times with physiological saline, the sample was subjected to flow cytometry to select monoclonal hybridoma cells which inhibit the binding of biotin-labeled his-hBLyS recombinant protein and BJAB cells.
  • TNF tumor necrosis factor
  • TNF-a tumor necrosis factor-a
  • TNF- ⁇ tumor necrosis factor- ⁇
  • washing buffer After washing 3 times with washing buffer, add 1 OO L of the secreted antibodies of 1D12, 2B10, 2G3, 5A5 and 13G8 monoclonal hybridoma cells, incubate for 1 hour at room temperature, and rinse 3 times with washing buffer.
  • the washing buffer was diluted 1:100 times with horseradish peroxidase (HRP) to cross-link the goat anti-mouse antibody, incubate for 1 hour at room temperature, rinse 3 times with washing buffer, add 50 ⁇ 3, ' , 5, 5'-tetradecylbenzidine (3,3',5,5'-Tetramethylbenzidine, TMB) substrate solution color development, reaction at room temperature for 10 minutes, termination reaction with 25 0. 5M sulphuric acid solution The absorbance was read at 450 nm, and the statistical results are shown in Fig. 7.
  • the monoclonal hybridoma cells 1D12, 2B10, 2G3, 5A5 and 13G8 obtained by screening were cultured, and the cells were collected by centrifugation at 0.01 rpm, and the hybridized tumor cells extracted by the method described in Example 1 were reverse transcribed to synthesize the first strand of cDNA.
  • the variable region DNA sequence corresponding to the hybridoma cells was amplified by using the first strand of the synthetic cDNA as a template.
  • the primer sequences used in the amplification reaction are as follows:
  • Primer 1 5'atg g(ag)a tg(cg) agctg(tg) gt(ca) at(cg) etc tt;
  • Primer 2 5'ggg gatatc cacc atg (ag)ac ttc ggg (tc) tg age t(tg )g gtt tt;
  • Primer 3 5'ggg tatatc cacc atg get gtc ttg gggctg ctcttct;
  • Primer 1 5' atg gag aca gac aca ctcctgctat;
  • Primer 2 5'atg gattttcaa gtg cag a tt tic ag;
  • Primer 3 5'atg gag (ta)ca ca(gt)(ta)ct cag gtc ttt (ga)t a;
  • Primer 4 5'atg (gt) cc coat) (ga) ct cag (ct)t(ct) ct(tg)gt;
  • the amplification reaction system is
  • Heavy chain primer mix or light chain primer mix (100ng/L per primer)
  • the PCR reaction procedure is: 95 ° C pre-denaturation lOmin
  • the amino acid sequences of the light chain variable region and the heavy chain variable region are shown in Table 2.
  • Kaba t classification analysis based on the deduced amino acid sequence, determining the FR region and CDR region of the light chain and heavy chain of each hybridoma cell of 1D12, 2B10, 2G3, 5A5 and 1 3G8, wherein 1D12, 2B10, 2G 3, 5A5 and 1
  • the amino acid sequence of the light chain variable region of each hybridoma cell and the amino acid sequence of the heavy chain variable region are shown in Table 2:
  • the humanization was carried out according to the variable region sequence of the antibody secreted by each hybridoma cell.
  • the humanization transformation process mainly involves the following key steps:
  • the HLA-DR affinity is selected, and the human embryonic framework sequence with low affinity is selected;
  • the heavy chain constant region Fc fragment was amplified from human blood cells with upstream primer VH5 and downstream primer VH3, and the light chain k constant region was amplified from human blood cells with upstream primer VL5 and downstream primer VL3, and Xho I and Age were introduced in the heavy chain.
  • the I cleavage site and the light chain fragment introduce Sma I and Dra III cleavage sites.
  • the PCDNA3.1 plasmid was ligated and sequenced to determine the correct clone. Subsequent experimental materials were obtained by transfecting the cells with this series of plasmids.
  • VH5 5'gcggaattc(c/g)a ggtg(a/c)agct(g/t)ca(c/g)(c/g)a (a/g)tc(a/t)gg;
  • VH3 5'accgccggat ccaccaccgc ccg agccacc gccacctgcg gagacgatga cc(a/g)tgg tccc;
  • VL5 5 ' ggtggtggatccggcggtgg cggttccgacattgtgatgacccagtctcca;
  • VL3 5 ' ggatacagttggtgcagcctcgagctacc gttt.
  • B cells were extracted from human peripheral blood by CD19-labeled MACS magnetic beads, and seeded in a 96-well plate at 100,000 per dish.
  • Recombinant BLyS (10 ng/mL) was added to the complete medium, and the Fab fragment goat anti-human IgM (4 ⁇ ⁇ ) was used to stimulate sputum cell growth.
  • Fab fragment goat anti-human IgM (4 ⁇ ⁇ ) was used to stimulate sputum cell growth.
  • different concentrations of the different humanized anti-BLyS antibodies prepared in Example 8 were added, and after 6 days of culture, the number of B cells was tested using Cel lti ter Glo from Promega. Based on the fluorescence count reading (RLU), the statistical results are shown in Figure 13.
  • the four antibody humanized anti-BLyS antibodies BLyS-I, BLyS-I I, BLyS-I I I and BLyS-IV prepared in Example 8 all inhibited B cell growth to varying degrees.
  • Example 10 Humanized anti-BLyS antibody in vivo
  • lymph node weight can be used as a main pharmacodynamic evaluation index to measure BLyS-stimulated B cell proliferation. This is mainly due to the fact that about 50% of the lymph nodes are B cells, and the proportion of cells is higher after BLyS stimulation.
  • mouse 1D12, 2B10, 2G3, 5A5 and human Bly-I (13G8) can effectively inhibit the action of BLyS.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Rheumatology (AREA)
  • Endocrinology (AREA)
  • Mycology (AREA)
  • Epidemiology (AREA)
  • Transplantation (AREA)
  • Pain & Pain Management (AREA)
  • Physical Education & Sports Medicine (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

本发明属于生物医药领域，公开了抗BLyS抗体，所述抗BLyS抗体特异性针对BLyS，能够与B淋巴细胞刺激因子结合并且能够抑制B淋巴细胞刺激因子与其受体BR3-FC结合。本发明还提供了所述抗BLyS抗体在制备预防和/或治疗如系统性红斑狼疮等B细胞增殖过量引起的疾病的药物中的应用。

Description

抗 BLyS抗体
技术领域
本发明属于生物医药领域， 涉及抗 BLyS抗体及其应用， 所述抗 BLyS抗体 能够与 B淋巴细胞刺激因子结合并且能够抑制 B淋巴细胞刺激因子与其受体 BR3-Fc结合。 进一步涉及与 MHC 11因子亲和力低的人源化抗 BLyS抗体及其应 用。 背景技术
系统性红斑狼疫 ( sys temic lupus erythematosus , SLE )是一种累及全 身多个系统如皮肤、 关节、 心脏、 肺、 肾、 血液以及大脑的弥慢性进行性、 常以緩解和复发交替出现的自身免疫性疾病。 系统性红斑狼疮主要影响非洲- 加勒比人、 亚洲人和西班牙裔人， 而高加索人（白种人）受其影响的程度则较 小。 美国狼疮基金会认为， 保守预测系统性红斑狼疮在美国影响到 30万人。 而 Datamoni tor公司估计， 仅在中国、 印度和墨西哥三国就有 220万系统性红 斑狼疮患者， 其中中国系统性红斑狼疮患者超过 100万， 为全球之最。 由于系 统性红斑狼疮初期症状比较隐蔽， 因此实际病患人数可能远大于目前估计。 由此可见临床在系统性红斑狼疮的诊断和治疗方面均存在极大的客观需求。
系统性红斑狼疮的成因复杂且不明确， 并非单一因素引起， 可能与遗传、 环境、 性激素及免疫等多种因素有关。 目前， 世界科学界公认的系统性红斑 狼疮的病因是由于在细胞水平上， 自反应 B细胞在外周组织中存在时间过长， 产生人体自身抗原而造成自身免疫。 所以如果可以抑制早期 B细胞的生长增 殖， 则可以治疗红斑狼疮疾病。
B淋巴细胞刺激因子（B Lymphocyte St imulator , BLyS ), 又称 Ta l l-1 (TNF and Apol related leukocyte expres sed 1 igand 1)、 BAFF (B cel l ac t iva t ing factor belong ing to the TNF fami ly)、 THANK (TNF homologues tha t ac t ivate apoptos i s, NF- κ Β and JNK) , 属肿瘤坏死因子（TNF ) 家族， 是由舒红兵等 人于 1999年首先发现并克隆的一种新的细胞因子。 BLyS作为一种 B淋巴细胞 的共刺激因子， 在 ant i-I gM和 IL-4存在下， 它可以专一刺激 B细胞增殖和 分化， 在体液免疫中起着极其重要作用； 而其在体内的过量表达又与自身免 疫性疾病密切相关。
体外实验表明， 在用 IgM预活化 B细胞后， BLyS能诱导其大量增殖并分 泌大量 IgM和 IgA, 但是， 对于静息期间 B细胞， 这种刺激作用却不明显。 进 一步的研究表明， BLyS主要作用于前 B淋巴细胞、 未成熟 B淋巴细胞、 激活 淋巴细胞， 而对浆细胞、 淋巴多功能干细胞没有作用。 同大多数细胞因子相 同， BLyS主要是通过 B细胞表面受体而刺激下游信号传导。 多个研究小组证 实与 BLyS 结合的受体是： B 细胞活化因子受体（BR3、 BLyS receptor 3 或 BAFF-R ) 、 穿膜蛋白活化物 ( transmembrane activator 1 and calcium modulator and cyclophi 1 in 1 igand interactor , TACI)和 B细胞成熟抗原 (B cell maturation Antigen, BCMA ) 。 这种特异性决定了 BLyS是针对 B 细胞抗体介导的自身免疫疾病和淋巴癌中一个很好的标靶。 长、 IgA和 IgM分泌从而达到治疗系统性红斑狼疮的效果（Edwards BM等， The remarkable flexibility of the human antibody repertoire; isolation of over one thousand different antibodies to a single protein, BLyS. J Mol Biol. 2003 Nov 14; 334 (1): 103-18; Baker KP 等， Generation and characterization of LymphoStat-B, a human monoclonal antibody that antagonizes the bioact ivi t ies of B lymphocyte stimulator. Arthritis Rheum. 2003 Nov; 48 (11): 3253-65) 0 美国人类基因组公司开发的抗 BLyS抗 体 Benlysta成为过去 60年来世界首个治疗红斑狼疮的新型药物。 Benlysta 只针对 BLyS刺激的 B细胞， 与化疗药物相比， 大大降低了治疗过程中的副作 用， 因此为系统性红斑狼疮患者提供了一个安全有效的治疗方法。 近年来针 对 BLyS的靶向治疗研究与临床应用迅速发展， 除美国人类基因组公司外， 其 他公司均是采用在 BLyS 或其受体基础上改造的融合蛋白。 美国基因泰克 (Genentech)公司开发了 BR3-FC药物、 Zymogenet ics公司开发了 TACI-FC药 物、 安晋（AMGEN)公司开发了多肽 -FC药物。 与 Benlysta相比， 它们的特异性 差， 结合力弱， 疗效相对较差， 毒性较强。 这三种药物均停止或终止在临床 实验二期。 所以， 抗 BLyS的抗体药物才是针对此靶点的有效药物方法。
Benlysta通过噬菌体展示文库产生。 而噬菌体展示文库的缺点在于： 重 链和轻链的配对被认为是人工的而未经体内选择 ( Greg Winter, et al.， Making Antibodies by Phage Display Technology. Annual Review of Immunology Vol. 12: 433-455 ) ； 文库从幼稚人 PBMC构建， 因此候选抗体 亲和性低 ( Edwards BM, et al.. The remarkable flexibility of the human antibody repertoire; isolation of over one thousand different antibodies to a single protein, BLyS. J Mol Biol. 2003 Nov 14; 334 (1): 103-18 ) ； 大部分情况下， 来自噬菌体展示文库的候选药物具有 低产率、 低稳定性和体内药代动力学特性差的缺点 （Ponsel D, et al. , High affinity, developabi 1 i ty and functional size: the holy grail of comb ina tor ia l ant i body l ibrary genera t i on. Molecules. 2011 May 3; 16 (5) : 3675-700 ) 。
本领域中仍需要具有如下特性的抗 BLyS抗体： 其不使用噬菌体展示文库 产生， 而是例如使用人源化鼠抗体等技术产生； 能够以高亲和力与 B淋巴细 胞刺激因子结合并且能够抑制 B淋巴细胞刺激因子与其受体 BR3-FC的结合， 特异性高； 具有高的免疫原性； 和 /或与 MHC I I 因子亲和力低， 从而在保证 亲和力的前提下， 使免疫应答减低到最小。 发明概述
一方面， 本发明提供了抗 BLyS抗体， 其轻链 CDR1、 CDR2和 CDR3以及重
Figure imgf000004_0001
在一个实施方案中， 所述抗 BLyS抗体的轻链可变区的氨基酸序列和重链 可变区的氨基酸序列选自以下各组中的一组：
a: SEQ ID NO. 31和 SEQ ID NO. 32所示的氨基酸序列或其功能性变体； b: SEQ ID NO. 33和 SEQ ID NO. 34所示的氨基酸序列或其功能性变体； c: SEQ ID NO. 35和 SEQ ID NO. 36所示的氨基酸序列或其功能性变体； d: SEQ ID NO. 37和 SEQ ID NO. 38所示的氨基酸序列或其功能性变体； e: SEQ ID NO. 39和 SEQ ID NO. 40所示的氨基酸序列或其功能性变体。 在一个优选的实施方案中， 本发明所述的抗 BLyS抗体为人源化的。
在一个实施方案中， 所述抗 BLyS抗体的轻链可变区的氨基酸序列和重链 可变区的氨基酸序列选自以下各组中的一组：
I : SEQ ID NO. 41和 SEQ ID NO. 42所示的氨基酸序列或其功能性变体； I I： SEQ ID NO. 43和 SEQ ID NO. 44所示的氨基酸序列或其功能性变体； I I I : SEQ ID NO. 45和 SEQ ID NO. 46所示的氨基酸序列或其功能性变体； IV： SEQ ID NO. 47和 SEQ ID NO. 48所示的氨基酸序列或其功能性变体； 在一些优选的实施方案中， 所述抗 BLyS抗体还含有人类轻链恒定区和重 链恒定区， 且所述轻链可变区及重链可变区分别连接至人类轻链恒定区和重 链恒定区。 在一个实施方案中， 所述人类轻链恒定区为人类轻链 κ恒定区。 在一个实施方案中， 所述人类重链恒定区为人类重链 Fc片段。
本发明还提供了编码本发明所述抗 BLyS抗体的 DNA分子。 优选地， 所述 DNA分子具有选自 SEQ ID NO. 49—58的核苷酸序列。
本发明提供了一种重组 DNA载体，其含有本发明所述的抗 BLyS抗体的 DNA 分子。
本发明还提供了一种宿主细胞， 其含有本发明所述的重组 DNA载体。 另一方面， 本发明提供了预防和 /或治疗 B细胞增殖过量引起的疾病的方 法， 其包括施用有效剂量的权利要求 1—7任意一项所述的抗 BLyS抗体。 在一 些实施方案中， 所述 B 细胞增殖过量引起的疾病选自系统性红斑狼疮、 类风 湿性关节炎、 强直性关节炎或 B细胞淋巴癌。
本发明还提供了一种药物组合物， 其包含有效剂量的本发明所述抗体和 药学上可接受的载体。
本发明还提供了制备抗 BLyS抗体的方法， 其包括孵育本发明所述的宿主 细胞， 以及获得所述抗体。 发明详述
除非另有定义， 本文使用的所有科技术语具有本领域普通技术人员所理 解的相同含义。 关于本领域的定义及术语， 专业人员具体可参考 Current Protoco l s in Mo lecular Bio logy ( Ausubel )。 氨基酸残基的缩写是本领 i或 中所用的指代 20个常用 L-氨基酸之一的标准 3字母和 /或 1字母代码。
本发明的目的之一在于提供抗 BLyS抗体， 所述抗体能够与 B淋巴细胞刺 激因子结合并且能够抑制 B淋巴细胞刺激因子与其受体 BR3-FC的结合。
为实现本发明的该目的， 本发明采用如下技术方案：
本发明通过基因工程技术， 提取人外周血 RNA, 以人外周血 RNA外周血 RNA为模板反转录获得人 BLyS的 cDNA, 然后以人 BLyS的 cDNA为模板分别扩 增获得人的 BLyS基因片段， 克隆到真核表达系统中转染宿主细胞表达并纯化 获得人的 BLyS纯化蛋白。
以人的 BLyS纯化蛋白为抗原免疫小鼠获得 2000株不同单克隆杂交瘤细 胞， 通过酶标反应， 共选出 211株分泌的抗体可结合 BLyS蛋白的克隆。 在这 211株杂交瘤细胞中，通过检测与 BJAB上 BLyS受体结合能力获得 5株分泌的 抗体可以不同能力的抑制生物素标记的 BLyS与 BJAB上 BLyS受体结合的单克 隆杂交瘤细胞， 分别命名为 1D12、 2B10、 2G3、 5A5和 13G8。
用 ELISA对所获得的 5个单克隆杂交瘤细胞株分泌的抗体进行免疫学特 性进行鉴定， 结果显示本发明筛选得到的克隆杂交瘤细胞株分泌的抗体均特 异性针对 BLyS, 而对其他 TNF家族抗原如 TNF- α、 TNF- β , 则没有反应性。
进一步地， 本发明提取各杂交瘤细胞 RNA反转录获得 cDNA, 然后以 cDNA 为模板扩增各杂交瘤细胞所对应的可变区 DNA 序列， 测序后利用 www. expasy. ch 对所得的序列进行系列分析， 根据推导的氨基酸序列进行 Kabat分类分析， 确定各杂交瘤细胞分泌的抗体的轻链和重链的 FR区和 CDR 区。
由于 Blys具有刺激 B细胞生长， 维持 B细胞生存的作用， 所以抑制、 尤 其是中和 Blys作用的抗体将抑制 B细胞的生长。 而 B细胞是生物体产生抗体 的特殊种类细胞， 使 B 细胞产生抗自身生长的抗体是困难的 （ Thomas Schirrmann, et al. , Phage display for the generation of antibodies for proteome research, diagnostics and therapy, Molecules, 2011, 16, 412-426 )。 所以， 采用鼠免疫的方法产生抗 Blys抗体相对困难。 而本发明通 过鼠免疫的方法获得了抗 Blys, 保持了鼠源抗体的优势。
一方面， 本发明涉及抗 BLyS抗体， 其轻链 CDR1、 CDR2和 CDR3以及重链
Figure imgf000006_0001
NO. 25 NO. 26 NO. 27 NO. 28 NO. 29 NO. 30 在一个实施方案中， 所述单克隆杂交瘤细胞 1D12分泌的抗 BLyS抗体含 有 SEQ ID NO. 31所示氨基酸序列的轻链可变区和 SEQ ID NO. 32所示氨基酸 序列的重链可变区， 命名为单克隆抗体 1D12。
在一个实施方案中， 所述单克隆杂交瘤细胞 2B10分泌的抗 BLyS抗体含 有 SEQ ID NO. 33所示氨基酸序列的轻链可变区和 SEQ ID NO. 34所示氨基酸 序列的重链可变区， 命名为单克隆抗体 2B10。
在一个实施方案中， 所述单克隆杂交瘤细胞 2G3分泌的抗 BLyS抗体含有 SEQ ID NO. 35所示氨基酸序列的轻链可变区和 SEQ ID NO. 36所示氨基酸序列 的重链可变区， 命名为单克隆抗体 2G3。
在一个实施方案中， 所述单克隆杂交瘤细胞 5A5分泌的抗 BLyS抗体含有 SEQ ID NO. 37所示氨基酸序列的轻链可变区和 SEQ ID NO. 38所示氨基酸序列 的重链可变区， 命名为单克隆抗体 5A5。
在另一个实施方案中， 所述单克隆杂交瘤细胞 13G8分泌的抗 BLyS抗体 含有 SEQ ID NO. 39所示氨基酸序列的轻链可变区和 SEQ ID NO. 40所示氨基 酸序列的重链可变区， 命名为单克隆抗体 13G8。
当然， 在不实质性影响抗体活性的前提下， 本领域技术人员可以对本发 明的氨基酸序列进行取代、 添加和 /或缺失一个或更多个（例如 1、 2、 3、 4、 5、 6、 7、 8、 9或 10个或更多个）个氨基酸， 以获得具有功能等价的氨基酸 序列， 即所述氨基酸序列的功能性变体， 它们都被视为包括在本发明保护的 范围内。 如在可变区将具有类似性质的氨基酸进行替换。
本发明所述的氨基酸序列的功能性变体可以与其来源序列有至少有 95%、 96%、 97%、 98%或 99%的一致性。 本发明所述的抗体序列一致性可以使用序列 分析软件测量。 例如使用缺省参数的计算机程序 BLAST, 尤其是 BLASTP 或 TBLASTN。
为了进一步减低免疫原性， 可以将制备得到的鼠源抗体人源化， 获得人 源化抗 BLyS抗体。 制备人源化抗体的方法是本领域技术人员公知的。 例如， 可以通过将本发明的 CDR序列转移至人抗体可变区中来制备本发明的人源化 抗 BLyS抗体。 所述人源化抗体不会产生抗抗体反应 ( AAR )和人抗鼠抗体反 应 （HAMA ), 不会因被抗抗体中和而被快速清除， 并且具有免疫效应功能， 如 ADCC和 CDC作用。
在一个实施方案中， 本发明人源化抗 BLyS抗体的轻链可变区的氨基酸序 列和重链可变区的氨基 S史序列选自以下各组中的一组：
I : SEQ ID NO. 41和 SEQ ID NO. 42所示的氨基酸序列或其功能性变体； I I： SEQ ID NO. 43和 SEQ ID NO. 44所示的氨基酸序列或其功能性变体； I I I : SEQ ID NO. 45和 SEQ ID NO. 46所示的氨基酸序列或其功能性变体； IV： SEQ ID NO. 47和 SEQ ID NO. 48所示的氨基酸序列或其功能性变体； 本发明所述人源化抗 BLyS抗体不仅能够与 B淋巴细胞刺激因子结合抑制 B淋巴细胞刺激因子与其受体 BR3-Fc的结合， 而且与 MHC I I因子亲和力低， 从而在保证亲和力的前提下， 使免疫应答减低到最小。
在另一个优选的实施方案中， 本发明所述人源化抗 BLyS抗体还含有人类 轻链恒定区和重链恒定区， 且所述轻链可变区及重链可变区分别连接至人类 轻链恒定区和重链恒定区。 在一个实施方案中， 所述人源化抗 BLyS抗体含有 完整的轻链和完整的重链， 其中所述完整的轻链由抗 BLyS抗体所含有的轻链 可变区与人类轻链恒定区连接而成， 所述完整的重链由抗 BLyS抗体所含有的 重链可变区与人类重链恒定区连接而成。
优选地， 所述人类轻链恒定区为人类轻链 κ恒定区。
优选地， 所述人类重链恒定区为人类重链 Fc片段。
在一个实施方案中， 所述人类轻链 κ恒定区和人类重链 Fc片段均来源于 健康人 B淋巴细胞。 通过基因工程技术， 重叠延伸 PCR连接可变区和恒定区 得到完整的人源化抗 BLyS抗体的轻链和重链。 分子。 由于密码 ^的简并性,'可以存在 ^[艮多种能 编 本发明所述的抗体的
DNA分子。
在一个实施方案中， 本发明提供了编码具有 SEQ ID NO. 31所示的氨基酸 序列的轻链可变区的 DNA分子， 其核苷酸序列如 SEQ ID NO. 49。
在一个实施方案中， 本发明提供了编码具有 SEQ ID NO. 32所示的氨基酸 序列的重链可变区的 DNA分子， 其核苷酸序列如 SEQ ID NO. 50。
在一个实施方案中， 本发明提供了编码具有 SEQ ID NO. 33所示的氨基酸 序列的轻链可变区的 DNA分子， 其核苷酸序列如 SEQ ID NO. 51。
在一个实施方案中， 本发明提供了编码具有 SEQ ID NO. 34所示的氨基酸 序列的重链可变区的 DNA分子， 其核苷酸序列如 SEQ ID NO. 52。
在一个实施方案中， 本发明提供了编码具有 SEQ ID NO. 35所示的氨基酸 序列的轻链可变区的 DNA分子， 其核苷酸序列如 SEQ ID NO. 53。
在一个实施方案中， 本发明提供了编码具有 SEQ ID NO. 36所示的氨基酸 序列的重链可变区的 DNA分子， 其核苷酸序列如 SEQ ID NO. 54。
在一个实施方案中， 本发明提供了编码具有 SEQ ID NO. 37所示的氨基酸 序列的轻链可变区的 DNA分子， 其核苷酸序列如 SEQ ID NO. 55。 在一个实施方案中， 本发明提供了编码具有 SEQ ID NO. 38所示的氨基酸 序列的重链可变区的 DNA分子， 其核苷酸序列如 SEQ ID NO. 56。
在一个实施方案中， 本发明提供了编码具有 SEQ ID NO. 39所示的氨基酸 序列的轻链可变区的 DNA分子， 其核苷酸序列如 SEQ ID NO. 57。
在一个实施方案中， 本发明提供了编码具有 SEQ ID N0. 40所示的氨基酸 序列的重链可变区的 DNA分子， 其核苷酸序列如 SEQ ID NO. 58。
本文所使用的术语 "抗 BLyS抗体"包括任何同型体的抗体或免疫球蛋白， 或其保有与抗原特异结合能力的抗原结合片段， 包括但不限于 Fv、 s cFv ( s c 指单链）、 Fab、 F (ab， ) 2、 Fab' , scFv-Fc 片段、 双抗体（ diabody )、 嵌合 抗体、 单链抗体以及包含抗体的抗原结合部分和非抗体蛋白质的融合蛋白质。 抗体可以被标记和检测， 例如， 可以通过放射性同位素、 能产生可检测物的 酶、 荧光蛋白质、 生物素等等进行标记并被检测。 抗体还可以结合于固相载 体， 包括但不限于聚苯乙烯平板或珠粒等等。 子克隆到载体中， ^进^转化宿主11细 ^。 因此， 本发明还提供了一种重组 DNA 载体， 其含有编码本发明所述抗 BLyS抗体的 DNA分子。
优选地， 所述重组 DNA载体是一种表达载体， 本领域技术人员将所述抗 体的 DNA分子克隆到表达载体中， 转化宿主细胞， 通过诱导表达获得单链抗 体。
在一个实施方案中， 本发明所述重组 DNA载体含有编码本发明所述人源 化抗 BLyS抗体的 DNA分子。 编码的人源化抗 BLyS抗体的 DNA分子可被重组 构建用于哺乳动物转录表达的载体， 本发明的表达载体含有编码的人源抗 BLyS单克隆抗体的重链和轻链可变区和恒定区的 DNA序列。 然而， 也可有分 别构建两种表达载体， 一种含有重量可变区和恒定区， 另一种是含有轻链可 变区和恒定区的， 一同转染哺乳动物。
在一个优选的实施方案中， 表达载体进一步含有启动子和编码分泌信号 肽的 DNA序列， 以及至少一种用于筛选的抗药基因。 所用方法包括 DNA合成 技术和体外重组技术。
本发明还提供了一种宿主细胞， 其含有本发明所述的重组 DNA载体。 本 发明所述宿主细胞可以为其为原核宿主细胞、 真核宿主细胞或噬菌体。
其中， 所述原核宿主细胞可以为大肠杆菌、 枯草杆菌、 链霉菌或奇异变 形菌等。 所述真核宿主细胞， 可以为如巴斯德毕赤酵母、 酿酒酵母、 裂殖酵 母、 木霉等真菌， 如草地粘虫等昆虫细胞， 如烟草等植物细胞， 如 BHK细胞、 CH0细胞、 COS细胞、 骨髓瘤细胞等哺乳动物细胞。 在一些实施方案中， 本发明所述宿主细胞优选为哺乳动物细胞， 更优选
BHK细胞、 CH0细胞、 NS0细胞或 COS细胞。
因此本发明提供了预防和 治疗 B细胞增 ^过量引5起的疾病的方法， 其包括 施用有效剂量的权利要求 1—7任意一项所述的抗 BLyS抗体。 本发明还提供了 引起的疾病的药 中^应用。 所述 B 细胞增殖过量引 ^的疾病包括但不限于 系统性红斑狼疮、 类风湿性关节炎、 强直性关节炎或 B细胞淋巴癌。
本发明还提供了一种药物组合物， 其包含有效剂量的本发明所述任意一 种抗 BLyS抗体或人源化抗 BLyS抗体和药学上可接受的载体。 所述药物组合 物可以通过常规方法用本发明所述抗 BLyS抗体或人源化抗 BLyS抗体中的任 意一种与一种或多种药学上可接受的辅料混合制成药物制剂。 其中 "药学上 可接受的载体" 是指一种或多种本领域技术人员公知的有机或无机、 天然或 合成的、 与抗体联合可促进其稳定和临床应用的载体。 合适的载体包括本领 域技术人员已知的药学上可接受的无菌盐水及含水或无水等渗无菌液和无菌 混悬液。 本发明所述有效剂量和使用方法取决于诸多因素， 包括患者的年龄、 体重、 性别、 自然健康状况、 营养状况、 化合物的活性强度、 给药时间、 代 谢速率、 病程严重程度以及诊治医师的主观判断。 本领域的技术人员根据上 述因素可以容易地决定有效剂量和使用方法。
本发明所述的药物组合物可以用改善转移、 递送、 耐受性等的合适载体、 赋形剂和其他试剂配制。 所采用的剂型可以包括例如： 片剂、 粉剂、 糊剂、 膏剂、 凝胶剂、 蜡状物、 油、 脂质、 含脂质嚢泡、 DNA缀合物等。 根据本发明 的药物组合物可通过任何适宜的途径施用， 例如可口服、 鼻、 皮内、 皮下、 肌内或静脉内施用。 附图说明
图 1示实施例 1 SDS-PAGE检测图， 其中泳道 A为本发明所述重组质粒表 达的蛋白， 泳道 B空白载体重组质粒， 泳道 C为蛋白分子量 Marker ;
图 2示实施例 2不同浓度生物素标记 h i s-hBLyS重组蛋白与 BJAB细胞结 合的流式细胞图， 其中黑色线条为 200ng/mL生物素标记 hi s-hBLyS重组蛋白 组， 红色线条为 100ng/mL 生物素标记 hi s-hBLyS 重组蛋白组， 蓝色线条为 50ng/mL生物素标记 h i s-hBLyS重组蛋白组，紫色线条为 25ng/mL生物素标记 hi s-hBLyS重组蛋白组，绿色线条为不含生物素标记 hi s-hBLyS重组蛋白仅含 有 SA-APC组；
图 3示实施例 3免疫小鼠的血清与 BJAB细胞结合的流式细胞图； 图 4示实施例 4 单克隆杂交瘤细胞分泌的抗体与 BJAB细胞上 BLyS受体 结合的流式细胞图， 其中黑色线条为负对照 I gG组， 红色线条为不含生物素 标记 hi s-hBLyS 重组蛋白仅含有 SA-APC组， 紫色线条为单克隆杂交瘤细胞 3H9 , 蓝色线条为单克隆杂交瘤细胞 1 3G8 ;
图 5示实施例 4 单克隆杂交瘤细胞分泌的抗体与 BJAB细胞上 BLyS受体 结合的流式细胞图， 其中黑色线条为负对照 I gG组， 红色线条为单克隆杂交 瘤细胞 1D12组， 紫色线条为单克隆杂交瘤细胞 2B10 , 蓝色线条为单克隆杂交 瘤细胞 2G 3;
图 6示实施例 4 单克隆杂交瘤细胞分泌的抗体与 BJAB细胞上 BLyS受体 结合的流式细胞图， 其中黑色线条为负对照 I gG组， 红色线条为单克隆杂交 瘤细胞 5A5组， 紫色线条为单克隆杂交瘤细胞 1 D7组， 蓝色线条为单克隆杂 交瘤细 4D3组；
图 Ί示实施例 4单克隆杂交瘤细胞 1 D12、 2B10、 2G 3、 5A5和 1 3G8分泌 的抗体与 hi s-hBLyS重组蛋白和肿瘤坏死因子（TNF ) 家族蛋白酶联免疫吸附 试验结果图；
图 8示实施例 9人源化抗 BLyS抗体体外抑制 B细胞增殖结果图， 其中横 坐标为人源化抗 BLyS抗体浓度， 纵坐标为荧光值。
图 9示出了候选抗体分子对 BLys诱导的淋巴结增殖的抑制作用。
图 10示出了候选抗体分子对 BLys诱导的血清 I gA升高的抑制作用。 具体实施方式
本发明实施例公开了抗 BLyS抗体及其应用。 本领域技术人员可以借鉴本 文内容， 适当改进工艺参数实现。 特别需要指出的是， 所有类似的替换和改 动对本领域技术人员来说是显而易见的， 它们都被视为包括在本发明。 本发 明的产品已经通过较佳实施例进行了描述， 相关人员明显能在不脱离本发明 内容、 精神和范围内对本文所述的方法进行改动或适当变更与组合， 来实现 和应用本发明技术。
为了进一步理解本发明， 下面结合实施例对本发明提供进行详细说明。 实施例 1: 人 BLyS基因的克隆与表达
分离健康人外周血， 以商用 RNA试剂盒（ Q iagen公司）提取总 RNA并纯 化。 以纯化的总 RNA为模板， 进行逆转录合成 cDNA第一链 cDNA反应体系如 RNase free dH20 9.5μ
5 RT buffer(with 25mM Mg2+) 4μ
Figure imgf000012_0001
RNase Inhibitor(10U/ L)
Figure imgf000012_0002
总 RNA模板
ReverTra Ace
Figure imgf000012_0003
加入灭菌蒸馏水补足体系总量 20μΙ^。
反应条件为： 30°C lOmin, 42°C 30min, 99 °C 5min 4°C 5min。 反应结束 后冰浴 5min。
根据人的 BLyS的全长 DNA序列，设计合成克隆分泌型 BLyS引物 PI和 P2 , 以逆转录合成的 cDNA为模板， 以上游引物 P1和下游引物 P2进行 PCR扩增。 其中 P1和 P2为的核苷酸序列如下：
P1： 5 'tacgaagctt gcatcatcat catcatcatg gcggcggctc cggcggcggc tccccgttca gggtccagaa gaa;
P2: 5'cgacgtcgac tcacagcagt ttcaatgcac caaaaaatgt gacatc。
PCR扩增反应体系为：
10 xtaq buffer(with 1.5mM Mg2+)
dNTP(5mM)
上游引物（lOOng^L)
下游引物（lOOng^L)
模板 (5-50ng/ L)
Figure imgf000012_0004
Taq酶 (2U/ L) 0.5 L
加入灭菌蒸馏水补足体系总量 50μΙ^。
PCR反应程序为：
94°C预变性 300s
94°C变性 45s
55°C退火 45s 32个循环
72°C延伸 45s
72°C延伸 200s
将 PCR扩增产物凝胶电泳回收， 用 Sa l I和 Hind I I I 双酶切， 克隆到 PCDNA3. 1真核表达质粒系统，以空白载体转化质粒作对照，转染 293T细胞（中 国典型培养物保藏中心） 3天后， 收集培养基上清液， 所得上清通过 H i s亲和 层析纯化得到 his-hBLyS纯化蛋白， SDS-PAGE检测， 结果见图 1。 由图 1 结果可见， 本发明所述重组质粒有蛋白表达， 但是空白载体重组 质粒却没有明显蛋白表达条带。 本发明所述重组质粒蛋白大小约为 23Kb, 与 根据人 BLyS氨基酸序列推断的分子量 23Kb接近。 实施例 2: 检测与 BJAB细胞上受体结合的能力
1、 生物素标记 his-hBLyS重组蛋白
将实施例 1 纯化得到 his-hBLyS 重组蛋白与溶解于 DMS0 中的生物素 -XX-丽 S按 ng/mL计以 1: 4的重量体积比混合， 室温放置 1小时， 将反应混 合物通过凝胶柱， 分离生物素标记的 his-hBLyS和游离生物素。
2、 生物素标记 his-hBLyS重组蛋白与 BJAB细胞结合
将分离得到的生物素标记的 his-hBLyS重组蛋白分为 25ng/mL、 50ng/mL、 lOOng/mL和 200ng/mL四个不同浓度组， 分别与 1 x 106的伯基特淋巴瘤细胞 ( BJAB ) 细胞混合， 在 4 摄氏度孵育 15 分钟， PBS 洗涤 3 次， 加入 stre tavidin-allophycocyanin (SA-APC) 使其终浓度为 0.2 g/mL, 在 4。C 孵育 20分钟。 以 PBS洗涤 3次后上细胞流式仪检测， 检测结果见图 2。
由图 2结果可见， 生物素标记的重组人 BLyS蛋白在不同浓度下均可以和 BJAB细胞结合。 实施例 3: 免疫小鼠
将实施例 1纯化得到 his-hBLyS重组蛋白作为抗原与等量免疫佐剂 （福 氏佐剂 ) 混合， 取 4只 6周大雌性 FVB小鼠， 其中 3只进行免疫， 另一只进 行对照模拟实验。 在初次免疫以后， 每一周进行一次加强免疫。 在最后一针 加强免疫前，从免疫的小鼠心脏取血， 将其血清中与生物素标记的 his-hBLyS 重组蛋白 （浓度 50ng/mL)混合并在室温孵育 20分钟。 然后将混合物与 BJAB 细胞在 4°C孵育 15 分钟， 以生理盐水洗脱 3 次后， 加入 0.2 g/mL 的 streptavidin- al lophycocyanin并在 4°C孵育 15分钟。 以生理盐水洗脱 3 次后， 将样品上流式细胞仪检测所免疫小鼠的血清其是否可以抑制 BLyS和其 受体 BR3-Fc结合， 结果见图 3。
由图 3结果可见三只免疫小鼠中小鼠 1所取得血清可有效抑制生物素标 记的 his-hBLyS重组蛋白与 BJAB细胞结合， 所以选取小鼠 1作为后续实验个 体， 进行下一步融合实验。 实施例 4: 细胞融合与单克隆杂交瘤细胞筛选
在最后一针加强免疫后， 取小鼠大腿根处淋巴结， 在生理盐水中碾磨后 取富含 B细胞的悬浮液， 按常规方法电转与骨髓瘤细胞 SP2/0融合。 将融合 细胞分配于 96孔盘， 在含有 HAT的 RPMI-1640完全培养基中置于 5%C02、 37 °C条件下培养。 在不同单克隆杂交瘤细胞中， 通过酶标反应， 筛选出 211 个 分泌的抗体可结合 BLyS蛋白的克隆。
将可结合 BLyS 蛋白的 211 个克隆产生的抗体分别与生物素标记的 his-hBLyS重组蛋白混合室温孵育 20分钟。 然后将混合物与 BJAB细胞在 4°C 孵育 15分钟， 以生理盐水洗脱 3次后， 加入 0.2 μ g/mL的 streptavidin -allophycocyanin并在 4°C孵育 15分钟。 以生理盐水洗脱 3次后， 将样品上 流式细胞仪检测以筛选可以抑制生物素标记的 his-hBLyS重组蛋白和 BJAB细 胞结合的单克隆杂交瘤细胞。 结果共有 11个单克隆杂交瘤细胞分泌的抗体可 以不同程度的抑制生物素标记的 his-hBLyS重组蛋白与 BJAB上 BLyS受体结 合， 如表 1所示， 其中命名为 1D12、 2B10、 2G3、 5A5和 13G8的单克隆杂交 瘤细胞分泌的抗体比 3H9, 1D7, 4D3 的单克隆杂交瘤细胞分泌的抗体有更强 的抑制力。 1D12、 2B10、 2G3、 5A5和 13G8的检测结果见图 4-6。
表 1抑制生物素标记的 his-hBLyS重组蛋白 与 BJAB上 BLyS受体结合的细胞分泌的抗体亚型
Figure imgf000014_0001
实施例 5: 检验与其它肿瘤坏死因子 （TNF) 家族蛋白的结合
为进一步检验候选抗体的结合特异性，分别在 96孔酶标板加入上 1 μ g/mL 的 his-hBLyS 重组蛋白、 肿瘤坏死因子 -a (TNF-a )、 肿瘤坏死因子（ TNF- β )和 BSA , 在 0.05Μ ΡΗ9.0的碳酸盐包被緩冲液中， 4°C静置过夜。 次日， 弃去孔内溶液， 用洗涤緩冲液洗 3次。 然后加入含有 3%BSA的 PBS溶液封闭 20分钟。 用洗涤緩冲液洗 3次后加入 l OO L稀译后的 1D12、 2B10、 2G3、 5A5 和 13G8单克隆杂交瘤细胞分泌的抗体， 室温孵育 1小时后用洗涤緩冲液冲洗 3次，用洗涤緩冲液以 1 : 10000倍稀释辣根过氧化物酶 (Horseradish Peroxidase, HRP ) 交联羊抗小鼠抗体， 室温孵育 1 小时， 经洗涤緩冲液冲洗 3次后， 加 入 50μί 3, '， 5， 5' -四曱基联苯胺 ( 3,3',5,5'-Tetramethylbenzidine, TMB ) 底物溶液显色， 室温反应 10分钟后， 以 25 0. 5M的石克酸溶液终止反应并 在 450nm 处读出吸光度， 统计结果见图 7。
由图 7结果可见， 1D12、 2B10、 2G3、 5A5和 13G8单克隆杂交瘤细胞分泌 的抗体均识别结合 BLyS , 但它们均不识别 TNF- α或者 TNF- β。 实施例 6: 单克隆杂交瘤细胞分泌的抗体可变区序列的确定
将筛选获得的单克隆杂交瘤细胞 1D12、 2B10、 2G3、 5A5和 13G8进行培 养， l OOOrpm离心收集细胞，按实施例 1所述方法提取的各杂交瘤细胞 RNA反 转录合成 cDNA第一链。 以合成 cDNA第一链为模板扩增杂交瘤细胞所对应的 可变区 DNA序列。 扩增反应中所使用的引物序列如下：
重链扩增所需引物：
引物 1： 5'atg g(ag)a tg(cg) agctg(tg) gt(ca) at(cg) etc tt;
引物 2: 5'ggg gatatc cacc atg (ag)ac ttc ggg (tc) tg age t(tg )g gtt tt;
引物 3: 5'ggg tatatc cacc atg get gtc ttg gggctg ctcttct;
轻链扩增所需引物：
引物 1： 5' atg gag aca gac aca ctcctgctat;
引物 2: 5'atg gattttcaa gtg cag a tt tic ag;
引物 3 : 5'atg gag (ta)ca ca(gt)(ta)ct cag gtc ttt (ga)t a;
引物 4: 5'atg (gt)cc coat) (ga) ct cag (ct)t(ct) ct(tg)gt; 扩增反应体系为
lOxPCR buffer(with 25mM Mg2+)
dNTP(5mM)
重链引物混合物或轻链引物混合物 (每个引物 100ng/ L)
cDNA (5-50ng/ L)
Taq酶 (2U/ L)
加入灭菌蒸馏水补足体系总量 50μΙ^。
PCR反应程序为： 95°C预变性 lOmin
94°C变性 lmin
55°C退火 lmin 30个循环
72°C延伸 115min
72°C延伸 lOmin
将 PCR 扩增产物凝胶电泳回收， 送生物公司测序， 测序后利用 www. expasy. ch对所得的序列进行系列分析，其轻链可变区和重链可变区氨基 酸序列如表 2所示，根据推导的氨基酸序列进行 Kaba t分类分析， 确定 1D12、 2B10、 2G3、 5A5和 1 3G8各杂交瘤细胞轻链和重链的 FR区和 CDR区， 其中， 1D12、 2B10、 2G 3、 5A5和 1 3G8各杂交瘤细胞轻链可变区的氨基酸序列和重链 可变区的氨基酸序列见表 2：
表 2抗 BLyS抗体轻链可变区和重链可变区氨基酸序列
Figure imgf000016_0001
实施例 7: 抗 BLyS抗体的人源化
根据各杂交瘤细胞分泌的抗体的可变区序列， 进行人源化改造。
人源化改造过程主要涉及以下几个关键步骤：
A、 把各杂交瘤细胞分泌的抗体的基因序列与人胚胎系抗体基因序列进行 比对， 找出同源性高的序列；
B、 根据 in s i l i con分析考察 HLA-DR亲和性， 选出亲和力低的人胚胎系 框架序列；
C、 利用计算机模拟技术， 应用分子对接分析可变区及其周边的框架氨基 酸序列， 考察其空间立体结合方式。 通过计算静电力， 范德华力， 亲疏水性 和熵值， 分析各杂交瘤细胞分泌的抗体基因序列中可能与 BLyS作用以及维护 空间构架的关键氨基酸个体， 将其嫁接回已经选择的人胚胎系基因框架。 在 其基石出上， 获得 4个不同人源化抗 BLyS抗体， 其轻链可变区和重链可变区序 列如表 3所示。
表 3人源化抗 BLyS抗体轻链可变区和重链可变区序列
轻链可变区氨基酸序列 重链可变区氨基酸序列
I SEQ ID NO. 41 SEQ ID NO. 42 I I SEQ ID NO. 43 SEQ ID NO. 44
I I I SEQ ID NO. 45 SEQ ID NO. 46
IV SEQ ID NO. 47 SEQ ID NO. 48 实施例 8: 人源化抗 BLyS抗体表达栽体的构建
用上游引物 VH5和下游引物 VH3从人血细胞中扩增重链恒定区 Fc片段， 用上游引物 VL5和下游引物 VL3从人血细胞中扩增轻链 k恒定区， 并在重链 引入 Xho I和 Age I酶切位点， 轻链片段引入 Sma I和 Dra I I I酶切位点。 联入 PCDNA3. 1质粒， 并经测序确定正确克隆。 后续的实验材料均由此系列质 粒转染细胞后， 提存获得。 其中 VH5、 VH3、 VL5和 VL3的核苷酸序列如下： VH5:5'gcggaattc(c/g)a ggtg(a/c)agct(g/t)c a(c/g)(c/g)a(a/g)tc(a/t)gg；
VH3 : 5'accgccggat ccaccaccgc ccg agccacc gccacctgcg gagacgatga cc(a/g)tgg tccc;
VL5: 5 ' ggtggtggatccggcggtgg cggttccgacattgtgatgacccagtctcca;
VL3： 5 ' ggatacagttggtgcagcctcgagctacc gttt。
制备得到四个人源化抗体， 分别命名为 BLyS-I、 BLyS-I I、 BLyS-I I I 和 BLyS-IV0 实施例 9: 人源化抗 BLyS抗体体外抑制 B细胞增殖
将 CD19标记的 MACS磁珠从人外周血中提取 B细胞，以 100000/盘接种于 96孔盘中培养。 完全培养基中加入重组 BLyS ( 10ng/mL ), Fab片段羊抗人 IgM ( 4μ§ ) 以刺激 Β 细胞生长。 在此培养基石出上， 加入不同浓度的实施例 8 制备得到的不同人源化抗 BLyS抗体， 培养 6天以后， 用 Promega公司出品的 Cel l t i ter Glo测试 B细胞数量。 依据荧光计数所读数值 ( RLU ) , 统计结果见 图 13。
由图 8结果可见， 实施例 8制备的四个抗体人源化抗 BLyS抗体 BLyS-I、 BLyS-I I、 BLyS-I I I和 BLyS- IV均可在不同程度上抑制 B细胞增长。
以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。 应当 指出， 对于本技术领域的普通技术人员来说， 在不脱离本发明原理的前提下， 还可以对本发明进行若干改进和修饰， 这些改进和修饰也落入本发明权利要 求的保护范围内。
实施例 10: 人源化抗 BLyS抗体的体内
1 ) BLyS刺激 B细胞增殖剂量的确定 在小鼠尾静脉注射不同浓度重组 BLyS, —周以后测定小鼠体重（body wt gram)，脾脏重量（spleen wt mg ) , 淋巴结重量（LN wt mg)。 其结果如表 4 所示：
表 4
Figure imgf000018_0001
研究表明： 0.3mg/kg的自产 BLyS可以有效在体内刺激 B细胞增长， 而且 淋巴结重量可以作为衡量 BLyS刺激 B细胞增殖的主要药效学评价指标。 这主 要由于淋巴结中约 50%为 B细胞， 在 BLyS刺激后， 其细胞比例更高。
2 )抗 BLyS抗体对 BLyS作用的抑制的体内研究
在此基础上, 我们混合 0.3mg/kg BLyS与 0.05mg/kg鼠源 1D12, 2B10, 2G3, 5A5和人源 BLyS-I ( 13G8 ), 然后测试其淋巴节重量， 血清中 IgA含量 (见图 9和图 10)。
研究表明： 鼠源 1D12, 2B10, 2G3, 5A5 和人源 Bly-I (13G8)均可以有效抑制 BLyS作用。

Claims

权 利 要 求
1、抗 BLyS抗体，其轻链 CDR1、 CDR2和 CDR3以及重链 CDR1、 CDR2 和 CDR3的氨基酸序列选自以下各组中的一组或其功能性变体：
Figure imgf000019_0001
2、 根据权利要求 1所述抗 BLyS抗体， 其轻链可变区的氨基酸序列和重 链可变区的氨基酸序列选自以下各组中的一组：
a: SEQ ID N0.31和 SEQ ID N0.32所示的氨基酸序列或其功能性变体； b: SEQ ID N0.33和 SEQ ID N0.34所示的氨基酸序列或其功能性变体； c: SEQ ID N0.35和 SEQ ID N0.36所示的氨基酸序列或其功能性变体； d: SEQ ID N0.37和 SEQ ID N0.38所示的氨基酸序列或其功能性变体； e: SEQ ID N0.39和 SEQ ID NO.40所示的氨基酸序列或其功能性变体。
3、 根据权利要求 1或 2所述的抗 BLyS抗体， 其为人源化的。
4、 根据权利要求 1-3中任一项所述抗 BLyS抗体， 其轻链可变区的氨基 酸序列和重链可变区的氨基酸序列选自以下各组中的一组：
I: SEQ ID N0.41和 SEQ ID N0.42所示的氨基酸序列或其功能性变体； II： SEQ ID N0.43和 SEQ ID N0.44所示的氨基酸序列或其功能性变体； III: SEQ ID N0.45和 SEQ ID N0.46所示的氨基酸序列或其功能性变体； IV： SEQ ID N0.47和 SEQ ID N0.48所示的氨基酸序列或其功能性变体；
5、 根据权利要求 3或 4所述抗 BLyS抗体， 其特征在于， 还含有人类轻 链恒定区和重链恒定区， 且所述轻链可变区及重链可变区分别连接至人类轻 链恒定区和重链恒定区。
6、 根据权利要求 5所述抗 BLyS抗体， 其特征在于， 所述人类轻链恒定 区为人类轻链 K恒定区。
7、 根据权利要求 5或 6所述抗 BLyS抗体， 其特征在于， 所述人类重链 恒定区为人类重链 Fc片段。
8、 编码权利要求 1〜7任意一项所述抗 BLyS抗体的 DNA分子。
9、 权利要求 8所述的 DNA分子， 其具有选自 SEQ ID N0.49〜58的核苷 酸序列。
10、 一种重组 DNA载体， 其特征在于， 含有权利要求 8或 9所述 DNA 分子。
11、 一种宿主细胞， 其特征在于， 含有权利要求 10所述重组 DNA载体。
12、 预防和 /或治疗 B细胞增殖过量引起的疾病的方法， 其包括施用有效 剂量的权利要求 1〜7任意一项所述的抗 BLyS抗体。
13、 根据权利要求 12所述的方法， 其特征在于， 所述 B细胞增殖过量引 起的疾病选自系统性红斑狼疮、 类风湿性关节炎、 强直性关节炎或 B细胞淋 巴癌。
14、 一种药物组合物， 其特征在于， 包含有效剂量的权利要求 1〜7任意 一项所述抗体和药学上可接受的载体。
15、 一种制备抗 BLyS抗体的方法， 其包括：
孵育权利要求 11所述的宿主细胞， 以及
获得所述抗体。
+
PCT/CN2013/076074 2012-05-22 2013-05-22 抗BLyS抗体 WO2013174264A1 (zh)

Priority Applications (8)

Application Number Priority Date Filing Date Title
JP2015511926A JP6006404B2 (ja) 2012-05-22 2013-05-22 抗ＢＬｙＳ抗体
BR112014025651-9A BR112014025651B1 (pt) 2012-05-22 2013-05-22 Anticorpo anti-blys, seu método de preparação, composição farmacêutica, vetor de dna recombinante e molécula de dna
EP20180342.6A EP3792280A3 (en) 2012-05-22 2013-05-22 Anti-blys antibody
IN2367MUN2014 IN2014MN02367A (zh) 2012-05-22 2013-05-22
RU2014133410A RU2613422C2 (ru) 2012-05-22 2013-05-22 Антитело к blys
US14/401,288 US9828423B2 (en) 2012-05-22 2013-05-22 Anti-BLyS antibody
EP13794561.4A EP2853543B1 (en) 2012-05-22 2013-05-22 Anti-blys antibody
ZA2014/08955A ZA201408955B (en) 2012-05-22 2014-12-05 Anti-blys antibody

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201210160474.3A CN103421113B (zh) 2012-05-22 2012-05-22 抗BLyS抗体
CN201210160474.3 2012-05-22

Publications (1)

Publication Number Publication Date
WO2013174264A1 true WO2013174264A1 (zh) 2013-11-28

Family

ID=49623127

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2013/076074 WO2013174264A1 (zh) 2012-05-22 2013-05-22 抗BLyS抗体

Country Status (8)

Country Link
US (1) US9828423B2 (zh)
EP (2) EP3792280A3 (zh)
JP (1) JP6006404B2 (zh)
CN (1) CN103421113B (zh)
IN (1) IN2014MN02367A (zh)
RU (1) RU2613422C2 (zh)
WO (1) WO2013174264A1 (zh)
ZA (1) ZA201408955B (zh)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9758591B2 (en) 2012-08-24 2017-09-12 The Regents Of The University Of California Antibodies and vaccines for use in treating ROR1 cancers and inhibiting metastasis
EP3151857A4 (en) * 2014-06-03 2018-04-18 Merck Sharp & Dohme Corp. Anti-blys antibodies
US10688181B2 (en) 2016-06-27 2020-06-23 The Regents Of The University Of California Cancer treatment combinations

Families Citing this family (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104045713B (zh) * 2013-03-13 2019-02-12 江苏诺迈博生物医药科技有限公司 一种抗Blys的单克隆抗体及含有该抗体的药物组合物
CN104804092B (zh) * 2014-01-29 2018-03-27 天津胜发生物技术有限公司 一种抗b细胞生长刺激因子的纳米抗体及其用途
CN104804091B (zh) * 2014-01-29 2018-03-27 普瑞金（深圳）生物技术有限公司 一种抗b细胞生长刺激因子的纳米抗体及用途
CN104804090B (zh) * 2014-01-29 2018-03-27 普瑞金（深圳）生物技术有限公司 一种抗b细胞生长刺激因子的纳米抗体和用途
CN104628856A (zh) * 2015-01-23 2015-05-20 中国人民解放军军事医学科学院放射与辐射医学研究所 一种抗BLyS单链抗体及其制备方法和用途
CN105061596B (zh) * 2015-08-05 2019-01-29 江苏诺迈博生物医药科技有限公司 人b淋巴细胞刺激因子的单克隆抗体及其应用
CN106916224B (zh) * 2015-12-28 2021-03-23 中国人民解放军军事医学科学院放射与辐射医学研究所 抗b淋巴细胞刺激因子单克隆抗体及其制备方法和用途
EP3483181A4 (en) * 2016-07-06 2020-04-22 Shanghai Pharmaexplorer Co., Ltd. BLYS ANTIBODIES, PRODUCTION METHOD THEREFOR AND APPLICATION THEREOF
CA3092434A1 (en) * 2017-02-27 2018-08-30 Caerus Therapeutics, Inc. Compositions of a novel anti-human ceacam6 antibody and uses thereof
JP2022519534A (ja) * 2019-01-31 2022-03-24 シーメンス・ヘルスケア・ダイアグノスティックス・インコーポレイテッド 抗ナロキソンおよび抗ナルトレキソンモノクローナル抗体ならびにその作製方法および使用方法
CN112111461B (zh) * 2020-08-17 2022-08-30 浙江正熙生物医药有限公司 一种单细胞分选构建单克隆抗体高产株细胞工作库的方法
WO2022223028A1 (zh) * 2021-04-23 2022-10-27 上海君实生物医药科技股份有限公司 抗BLyS抗体、其药物组合物及其用途

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1401777A (zh) * 2002-05-24 2003-03-12 张志方 一种重组人b淋巴细胞刺激因子表达载体的构建、dna免疫制备单克隆抗体及其应用
CN1279055C (zh) * 2000-06-16 2006-10-11 人体基因组科学有限公司 免疫特异性结合BLyS的抗体
CN101851291A (zh) * 2010-02-09 2010-10-06 中国人民解放军第四军医大学 一种抗人baff单克隆抗体的重链和轻链可变区

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20000052796A (ko) * 1996-10-25 2000-08-25 벤슨 로버트 에이치. 뉴트로킨 알파
US6812327B1 (en) * 1996-10-25 2004-11-02 Human Genome Sciences, Inc. Neutrokine-alpha polypeptides
US7879328B2 (en) * 2000-06-16 2011-02-01 Human Genome Sciences, Inc. Antibodies that immunospecifically bind to B lymphocyte stimulator
WO2003055979A2 (en) * 2001-11-16 2003-07-10 Human Genome Sciences, Inc. ANTIBODIES THAT IMMUNOSPECIFICALLY BIND TO BLyS
US20020150579A1 (en) * 2002-01-10 2002-10-17 Kimberly Robert P. B lymphocyte stimulator (BLyS) as a marker in management of systemic lupus erythematosus
RU2005141541A (ru) * 2003-06-05 2006-06-27 Дженентек, Инк. (Us) Комбинированная терапия в-клеточных нарушений
CA2543093A1 (en) * 2003-10-20 2005-05-12 Biogen Idec Ma Inc. Therapeutic regimens for baff antagonists

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1279055C (zh) * 2000-06-16 2006-10-11 人体基因组科学有限公司 免疫特异性结合BLyS的抗体
CN1401777A (zh) * 2002-05-24 2003-03-12 张志方 一种重组人b淋巴细胞刺激因子表达载体的构建、dna免疫制备单克隆抗体及其应用
CN101851291A (zh) * 2010-02-09 2010-10-06 中国人民解放军第四军医大学 一种抗人baff单克隆抗体的重链和轻链可变区

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
BAKER KP: "Generation and characterization of LymphoStat-B, a human monoclonal antibody that antagonizes the bioactivities of B lymphocyte stimulator", ARTHRITIS RHEUM., vol. 48, no. 11, November 2003 (2003-11-01), pages 3253 - 65, XP002422474, DOI: doi:10.1002/art.11299
EDWARDS BM ET AL.: "The remarkable flexibility of the human antibody repertoire; isolation of over one thousand different antibodies to a single protein, BLyS", J MOL BIOL., vol. 334, no. 1, 14 November 2003 (2003-11-14), pages 103 - 18, XP004469368, DOI: doi:10.1016/j.jmb.2003.09.054
GREG WINTER ET AL.: "Making Antibodies by Phage Display Technology", ANNUAL REVIEW OF IMMUNOLOGY, vol. 12, pages 433 - 455, XP000564245, DOI: doi:10.1146/annurev.iy.12.040194.002245
PONSEL D ET AL.: "High affinity, developability and functional size: the holy grail of combinatorial antibody library generation", MOLECULES, vol. 16, no. 5, 3 May 2011 (2011-05-03), pages 3675 - 700, XP009168724, DOI: doi:10.3390/molecules16053675
See also references of EP2853543A4
THOMAS SCHIRRMANN ET AL.: "Phage display for the generation of antibodies for proteome research, diagnostics and therapy", MOLECULES, vol. 16, 2011, pages 412 - 426

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9758591B2 (en) 2012-08-24 2017-09-12 The Regents Of The University Of California Antibodies and vaccines for use in treating ROR1 cancers and inhibiting metastasis
US10344096B2 (en) 2012-08-24 2019-07-09 The Regents Of The University Of California Antibodies and vaccines for use in treating ROR1 cancers and inhibiting metastasis
US11312787B2 (en) 2012-08-24 2022-04-26 The Regents Of The University Of California Antibodies and vaccines for use in treating ROR1 cancers and inhibiting metastasis
EP3151857A4 (en) * 2014-06-03 2018-04-18 Merck Sharp & Dohme Corp. Anti-blys antibodies
US10688181B2 (en) 2016-06-27 2020-06-23 The Regents Of The University Of California Cancer treatment combinations
US11654193B2 (en) 2016-06-27 2023-05-23 The Regents Of The University Of California Cancer treatment combinations

Also Published As

Publication number Publication date
EP2853543B1 (en) 2020-09-02
JP6006404B2 (ja) 2016-10-12
CN103421113A (zh) 2013-12-04
EP2853543A4 (en) 2016-03-30
ZA201408955B (en) 2015-12-23
RU2613422C2 (ru) 2017-03-16
JP2015517505A (ja) 2015-06-22
EP3792280A2 (en) 2021-03-17
CN103421113B (zh) 2018-01-19
EP2853543A8 (en) 2015-05-13
BR112014025651A2 (pt) 2017-07-04
RU2014133410A (ru) 2016-07-10
EP2853543A1 (en) 2015-04-01
US9828423B2 (en) 2017-11-28
EP3792280A3 (en) 2021-07-21
IN2014MN02367A (zh) 2015-08-14
US20150259409A1 (en) 2015-09-17

Similar Documents

Publication Publication Date Title
WO2013174264A1 (zh) 抗BLyS抗体
AU2014361473B2 (en) PD-1 antibody, antigen-binding fragment thereof, and medical application thereof
WO2018024237A1 (zh) 抗pd-l1纳米抗体及其应用
TWI701259B (zh) ４﹘１ｂｂ抗體及其製備方法和應用
ES2738731T3 (es) Anticuerpos frente a la IL-22 humana y sus usos
WO2018233574A1 (zh) 一种抗pd-l1人源化纳米抗体及其应用
WO2020094071A1 (zh) Nkg2a抗体及其制备方法和应用
NZ542306A (en) Antibodies against human IL-21 receptor and uses therefor
CN114560938B (zh) 抗gdf15中和性单克隆抗体及其应用
CN110790839A (zh) 抗pd-1抗体、其抗原结合片段及医药用途
WO2022199590A1 (zh) 一种靶向bcma的纳米抗体及其应用
CN109627340B (zh) Cd3和prlr双特异性抗体及其构建与应用
CN108635579B (zh) 抗人bFGF纳米抗体在制备治疗黑色素瘤药物中的应用
WO2019096219A1 (zh) 一种人源化抗il-13抗体及其制备方法和应用
WO2021088904A1 (zh) 抗人程序性死亡配体-1(pd-l1)的抗体及其用途
WO2019192493A1 (zh) 抗人lag-3单克隆抗体及其应用
WO2019238074A1 (zh) 一种高亲和力高生物活性的lag-3抗体及其应用
WO2022228183A1 (zh) 抗siglec15抗体及其制备方法和用途
CN113754770B (zh) 一种特异性结合人ctla4的抗体及包含其的药物和试剂盒
CN111333732B (zh) 一种靶向人bcma且激活nk细胞的双特异性抗体的制备及其应用
CN110461875B (zh) 结合light的抗原结合分子
TWI816616B (zh) 抗人程序性死亡配體-1 (pd-l1) 的抗體及其用途
WO2023051656A1 (zh) 双特异性抗体及其应用
CN115947855B (zh) 抗cd24抗体的制备及其用途
WO2024120526A1 (zh) 抗ilt4抗体及其制备方法和应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 13794561

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 14401288

Country of ref document: US

ENP Entry into the national phase

Ref document number: 2015511926

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

REG Reference to national code

Ref country code: BR

Ref legal event code: B01A

Ref document number: 112014025651

Country of ref document: BR

WWE Wipo information: entry into national phase

Ref document number: IDP00201407820

Country of ref document: ID

ENP Entry into the national phase

Ref document number: 2014133410

Country of ref document: RU

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 112014025651

Country of ref document: BR

Kind code of ref document: A2

Effective date: 20141015