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Definitions

• the present invention relates to the use of a bispecific antibody comprising an antibody having specificity for human PD-1, an antibody against a human T cell receptor complex or a human B cell receptor complex.
• the immune system has acquired a mechanism that can respond to various foreign antigens.
• the mechanism is to bring about diversity of antigen reception by recombination of V (D) J fragments in T cells and B cells.
• V (D) J fragments in T cells and B cells.
• This mechanism resulted in the production of autoreactive lymphocytes at the same time, but these autoreactive lymphocytes were removed by negative selection in the thymus and bone marrow, and also in the periphery, clonal removal was the autoimmune tolerance It is controlled by the mechanism.
• mice that develop autoimmune disease due to single gene deficiency is extremely important for the etiological consideration from the viewpoint of molecular biology.
• CTLA 4 Z—mice that cause lethal systemic lymphocyte infiltration (Science, 1995, 270, 5238, ⁇ ⁇ 985-988, Immunity, 1995, III, 5, p.541-547), SHP—1 deficient mothaten mice (Cell, 1993, 73rd, No.
• PD-1 is a 5 50 type 1 membrane protein belonging to the immunoglobulin family.
• Human PD-1 is composed of 2 8 8 amino acids like mouse PD-1, and has an N-terminal signal peptide (20 amino acids) and a hydrophobic region that is a transmembrane region in the middle (Zambo).
• PD-1 expression is observed in thymocytes upon differentiation from CD 4— CD 8— to CD 4 + CD 8 + cells (Internal Immunology, 1996, 8th). ⁇ , No. 5, pp. 773-780, Journal of Experimental Medicin, 2000, Vol. 191, No. 5, pp. 891-898).
• PD-1 expression in the periphery is activated by stimulation from antigen receptor T cells and B cells (International Immunology, 1996, 8th, 5th, p.765-772) and active bone marrow cells including macrophages.
• PD-1 has ITIM (Immunoreceptor Tyrosine-based Inhibitory Motif) in its intracellular region and is therefore considered a negative regulator of immune responses.
• Lubus-like autoimmune diseases such as glomerulonephritis and arthritis in PD-1-deficient mice (C 5 7 BL / 6 gene background) (international 'immunology' (International Immunology), 1998, No.10, No.10, p.1563 ⁇ : 1572, Immunity, 1999, No.11, No.2, p.141 ⁇ : 151) and PD-1 is autoimmune because it develops dilated cardiomyopathy-like disease (BALB / c gene background) (Science, 2001, Vol.291, No.5502, p.319-322).
• PD-1 is a regulator of various autoimmune diseases and is considered to be one of the causative genes of autoimmune diseases.
• the inventors of the present invention have conducted extensive studies on the assumption that controlling the function of PD-1 can reduce or enhance the immune function, infectious diseases, rejection at the time of transplantation, and tumors. Has arrived at the present invention relating to a substance that controls the function of PD-1.
• TCR T cell receptor
• BCR B cell receptor
• PD-1 negatively regulates various immunocompetent cells such as lymphocytes and myeloid cells in the immune system, and ITIM (immunoreceptor Tyrosine-based Inhibitory Motif) 3 ⁇ 4 in the intracellular region of PD-1
• ITIM immunocompetent cells
• ITIM immunocompetent cell
• ITIM immunocompetent cell
• the present inventors considered that the molecular mechanism of PD-1 inhibitory signaling is the recruitment of phosphatases. Therefore, we came to think that the function of PD-1 can be expressed by positioning PD-1 in the vicinity of TCR and BCR.
• CD3 is a membrane protein expressed in T cells and is one of the complexes that constitute TCR.
• the cDNA encoding the anti-human PD-1 antibody and the cDNA encoding the anti-human TCR antibody were isolated, and the expression vector expressing the fusion protein consisting of the antigen recognition sites of both antibodies.
• the present invention is a.
• a part that recognizes human PD-1 a part that recognizes a membrane protein present in the cell membrane where human PD_1 is expressed, and a substance that has specificity for human PD-1 consisting of a linker,
• the substance recognizing human PD-1 is a substance having specificity for human PD-1 as described in 1 above, wherein the part recognizing human PD-1 is an antibody against human PD-1 or a partial fragment thereof.
• the human PD-1 as described in 1 above comprising an antibody against human PD-1 or a partial fragment thereof, and an antibody against a membrane protein present in a cell membrane expressing human PD-1 or a partial fragment thereof and a linker.
• Antibodies against human PD-1 or partial fragments thereof and T cell receptor complex A bispecific antibody consisting of an antibody against the union or a partial fragment thereof and a linker,
• Bispecific antibody consisting of an antibody against human PD-1 or a partial fragment thereof, an antibody against a B cell receptor complex or a partial fragment thereof and a linker,
• a polypeptide constituting an antibody against human PD-1 which is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 in a substantially pure form, or a homologue thereof, a fragment thereof or a fragment thereof.
• a polypeptide that constitutes an antibody against human PD-1 and consists of the amino acid sequence shown in SEQ ID NO: 4 in a substantially pure form, a homologue thereof, a fragment thereof, or a homologue thereof. Or a polypeptide comprising an amino acid sequence in which 1 to 10 amino acids of the polypeptide are deleted, substituted and / or added,
• a polypeptide complex comprising the polypeptides described in 1 0 and 1 1 above,
• a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 11 in substantially pure form, or a homologue thereof, a homologue thereof, a fragment thereof or a fragment thereof, or 1 to 10 amino acids of the polypeptide A polypeptide consisting of an amino acid sequence in which is deleted, substituted and / or added,
• a bispecific antibody according to any one of 7 to 9 above, which is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 11 in a substantially pure form or a homologue thereof, a fragment thereof, or Homozygous of the fragment A log, or a polypeptide consisting of an amino acid sequence in which 1 to 10 amino acids of the polypeptide are deleted, substituted and / or added,
• a polynucleotide comprising the base sequence shown in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 9, a homologue thereof or a complementary strand polynucleotide thereof, a fragment thereof or a homologue of the fragment thereof,
• a replication or expression vector comprising the polynucleotide according to 15 or 16 above,
• a method for producing a substance comprising culturing the host cell according to 18 above under conditions for expressing the substance according to any one of 1 to 6 above,
• a method for producing a bispecific antibody comprising culturing the host cell according to 18 above under the conditions for expressing the bispecific antibody according to any of 7 to 9 above,
• a method for producing a polypeptide comprising culturing the host cell according to 18 above under conditions for expressing the polypeptide according to any one of 10 to 14 above,
• composition comprising an amount of the polypeptide described in claim 1 effective in treating and / or preventing a disease given by human PD-1.
• Diseases involving human PD-1 are selected from the group consisting of neurodegenerative diseases, autoimmune diseases, collagen diseases, organ transplant rejection, tumors and infectious diseases 23.
• Neurodegenerative diseases include senile dementia, Al ⁇ heimer's disease, Down's syndrome, Parkinson's disease, Croy's felt'Jakob disease, amyotrophic spinal sclerosis, diabetic neuropathy, Parkinson's syndrome, Huntington's disease, Machadje Cef 23.
• Autoimmune diseases include glomerulonephritis, arthritis, dilated cardiomyopathy-like disease, ulcerative colitis, Sjogren's syndrome, Crohn's disease, systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, dryness, Allergic contact dermatitis, polymyositis, scleroderma, periarteritis nodosa, rheumatic fever, vitiligo vulgaris, insulin-dependent diabetes mellitus, Behcet's disease, Hashimoto's disease, Addison's disease, dermatomyositis, myasthenia gravis , Reiter syndrome, Graves' disease, pernicious anemia, Good Pastaia syndrome, infertility, chronic active hepatitis, pemphigus, autoimmune thrombocytopenic purpura, autoimmune hemolytic anemia and vasculitis
• the part that recognizes human PD-1 in the present invention may be any substance that recognizes human PD_1, such as anti-human PD-1 antibody, anti-human PD-1 antibody fragment, human PD-1 As such, fragments of human PD-1, ligands of human PD-1 (human P DL 1 (Freeman, GJ. 18 others, Journal Experimental Medicine, 2000, 19) No. 7, ⁇ ⁇ 1027 to 1034), human PD-L2, human PD-H3, etc.), fragments thereof, and low-molecular organic compounds.
• human P DL 1 Freeman, GJ. 18 others, Journal Experimental Medicine, 2000, 19
• human PD-L2, human PD-H3, etc. fragments thereof, and low-molecular organic compounds.
• An antibody against human PD_1 or a partial fragment thereof includes a fragment of an anti-human PD-1 antibody or anti-human PD-1 antibody, and is a complete polyclonal or monoclonal antibody, a humanized antibody, a fully human antibody or a shortened version thereof.
• Type eg F (ab) 2 , Fab ', Fab, Fv
• F (ab ′) 2 , Fab ′, Fab, and Fv antibody fragments can be obtained by treating a complete antibody with a protease enzyme and optionally reducing it.
• the cDNA is isolated from the hyperprideoma that produces the antibody, and the antibody or its antibody fragment or antibody fragment is fused with another protein using an expression vector prepared by genetic modification. It can be produced as a protein in the evening.
• the membrane protein present in the cell membrane expressing human PD-1 means the membrane protein expressed on the cell membrane of the same cell as the cell expressing human PD-1, and human PD-1 Any of the membrane proteins present in the cell membranes derived from human-derived primary cultured cells or human cell lines expressing the phenotype are also included. For example, a T cell receptor complex or a B cell receptor complex is preferred.
• the part that recognizes the membrane protein present in the cell membrane expressing human PD-1 is a substance that recognizes the membrane protein expressed on the cell membrane of the same cell as the cell expressing human PD_1.
• antibodies against membrane proteins, fragments of the antibodies, membrane proteins themselves, membrane protein fragments, ligands of membrane proteins, fragments of the ligands, low molecular weight organic compounds that bind to membrane proteins, etc. Can be mentioned.
• the partial fragment is an antibody against a membrane protein expressed on the cell membrane of the same cell as human PD-1 expressing cells or a partial fragment of the antibody, and is a complete polyclonal or monoclonal antibody Any form such as a humanized antibody, a fully human antibody or a truncated form thereof (eg, F (ab ′) 2 , Fab 1 , Fa b, Fv) antibody may be used.
• a T cell receptor complex is a complex composed of at least an ⁇ subunit and a subunit, and a CD 3 composed of an subunit, 5 subunits, a subunit and a subunit. is there.
• the B cell receptor complex is a complex composed of at least membrane-bound immunoglobulin and CD 79 / CD 79/5 subunit.
• the antibody against the T cell receptor complex or a partial fragment thereof may be any antibody that recognizes the T cell receptor complex or a partial fragment thereof, and constitutes a T cell receptor complex. It also includes antibodies to T cell receptors and their respective CD3 subunits, or partial fragments thereof, fully polyclonal or monoclonal antibodies, humanized antibodies, fully human antibodies or their truncated forms (eg F (ab) 2 , Fab ′, Fab, Fv) Any form such as an antibody may be used.
• a monoclonal antibody against CD3 can be produced using an already established hyperpridoma.
• a commercially available anti-CD3 antibody Hiichi CD3 emAb, manufactured by Pharmingen) is also available.
• the antibody against the B cell receptor complex or a partial fragment thereof may be any antibody that recognizes the B cell receptor complex or a partial fragment thereof, and constitutes the B cell receptor complex.
• Antibodies against membrane-bound immunoglobulins and CD 79 subunits or partial fragmentation of the antibodies Any fragment, including fully polyclonal or monoclonal antibodies, humanized antibodies, fully human antibodies or truncated forms thereof (eg, F (ab) 2 , Fab ′, Fab, Fv) antibodies, etc. It may be a form.
• a commercially available anti-BCR antibody can be used.
• an anti-IgG (H + L) polyclonal antibody manufactured by Zymed
• a substance consisting of a part that recognizes human PD-1 and a part that recognizes a membrane protein present in the cell membrane in which human PD_1 is expressed is human PD-1 that is expressed on the cell membrane of the same cell. Means a substance that can bind simultaneously to the extracellular domain of and the extracellular domain of membrane proteins. Specifically, a bispecific protein consisting of an antibody or antibody fragment specific for human PD-1 and an antibody or antibody fragment specific for a membrane protein present in the cell membrane expressing human PD-1. An antibody is mentioned.
• a vice-specific antibody is an asymmetric antibody that has two independent antigen recognition sites with two different antigen specificities.
• the manufacturing method of the bispecific antibody is based on a known chemical method (Nisonoff, A. et al., 1 person, Bios Chemistry and Biophysics.) 1961, volume 90, pp.460-462, Brennan, M. et al., Science (Science), 1985, 299, pp.81-83) are well known.
• two kinds of antibodies are first hydrolyzed by enzymes, then the disulfide bond of the antibody H chain is cleaved with a reducing agent, followed by mixing and reoxidizing dissimilar antibodies.
• a reactive antibody is obtained.
• a crosslinking agent such as glutaraldehyde Ya Karubojiimido been disclosed (JP-2 -1556 JP).
• the peptide linker is preferably 3-12 amino acid residues, but the sequence is not particularly limited (Hudson, PJ., 1 other, Journal of Immunology Medicine, Journal of Immunology Medicine, 1999, No.231, No. 1-2, ⁇ ⁇ 177-: 189).
• a bispecific antibody consisting of an antibody against human PD-1 or a partial fragment thereof and an antibody against a membrane protein present on the cell membrane where human PD-1 is expressed or a partial fragment thereof is expressed on the cell membrane of the same cell. It is an antibody that can bind to the extracellular domain of human PD-1 and the extracellular domain of membrane protein simultaneously.
• a noisy specific antibody can be produced by the following production method.
• Human PD-1 or human-derived membrane protein is used as an immunizing antigen to sensitize animals
• sensitized spleen cells were fusion of sensitized spleen cells with sensitized myeloma cells.
• sensitized antigen human PD-1 or human derived membrane protein
• the obtained anti-human PD-1 antibody and anti-human membrane protein antibody can be prepared by crosslinking with a linker.
• the prepared Fab SH can be prepared by crosslinking with a linker.
• the linker can connect the above-mentioned part that recognizes human PD-1 and the part that recognizes the membrane protein present in the cell membrane where human PD-1 is expressed, maintaining an appropriate distance.
• the linker can connect the above-mentioned part that recognizes human PD-1 and the part that recognizes the membrane protein present in the cell membrane where human PD-1 is expressed, maintaining an appropriate distance.
• peptides, amides and the like can be mentioned.
• linker a commercially available one can be used, and, for example, phenol dimaleimide (manufactured by Aldrich) is available. When both or one of them is a low-molecular-weight organic compound as a substance that recognizes membrane proteins present in the cell membrane where human PD-1 and human PD-1 are expressed,
• human PD-1 or human-derived membrane protein is preferably administered intraperitoneally or in a fat pad to the sensitized animal.
• the sensitized animal is not particularly limited as long as it is an animal from which a monoclonal antibody is generally obtained, such as a mouse or rat. For example, in the case of mice, it is sufficient to administer 10 to 20 antigens at a time.
• Cell fusion in (2) is performed by removing the spleen from the sensitized immunized product immunized in (1) whose antibody titer has increased sufficiently, and suspending the spleen cell suspension according to a conventional method. And then adding polyethylene glycol (preferably PEG4000) at 37 ° C. to the mixture of the obtained splenocytes and myeoma cells derived from the sensitized product.
• polyethylene glycol preferably PEG4000
• Several types of mouse myeloma cells are known, such as P 3 X 63 Ag 8, P 3 / NS 1 / 1—Ag4-1, and SP-2 / 0-Ag-14, all of which are readily available.
• Myeloma cells are useful in HGPRT (hypoxanthine.guanine 'phosphoribosyl' transferase) deficient cell lines that cannot survive in HAT medium (medium containing hypoxanthine, aminopterin and thymidine). Desirably, the cell line itself does not secrete antibodies.
• HGPRT hyperxanthine.guanine 'phosphoribosyl' transferase
• the cell line itself does not secrete antibodies.
• SP-2Z0-Ag-14 is used.
• the resulting cell fusion mixture is then dispensed into a 96 microwell plate at low cell density and cultured in HAT medium. Unfused myeloma cells, myeloma cell-hyperdoma, and unfused spleen cells, and splenocyte-hyperdoma, die in 1 to 2 weeks of culture because the survival conditions are not satisfied. Only hyperidoma with mamma cells grows.
• the screening in (3) is performed by reacting the hybridoma culture supernatant with the immobilized antigen, and quantifying the antibody in the supernatant specifically adsorbed to the antigen using the labeled second antibody. Determine whether it is a hyperprideoma producing antibodies against human PD-1 or human-derived membrane proteins.
• the step (4) is performed by cloning the antibody-producing hyperpridoma according to the soft agar culture method (Monoclonal Antibodies, 1980, 372). In this case, a limiting dilution method can be used.
• the step (5) can be obtained by culturing the cloned hyperpridoma in a normal medium and separating and purifying it from the culture supernatant. To obtain a larger amount of antibody efficiently, the hyperidoma is obtained from a mouse. A method of intraperitoneal administration, growth, and separation and purification from the ascites is used.
• the step (6) can be purified by conventional methods such as salting out, ion exchange chromatography, gel filtration, hydrophobic chromatography, affinity chromatography, etc., but more effectively protein A-Sepharose CL- Affinity chromatography using 4 B (Amersham Biosciences) is used.
• the bispecific antibody of the present invention specifically recognizes human PD-1, it can be used for purification and concentration of human PD-1, such as affinity chromatography.
• the step (7) can be cross-linked by binding, for example, sulfo EMCS (N- (6-maleimidocaproxy) sulfosuccinimide sodium salt) to the amide group or SH (mercapto) group of the antibody as a cross-linking agent.
• sulfo EMCS N- (6-maleimidocaproxy) sulfosuccinimide sodium salt
• SH mercapto
• Pepsin digested Fiab) 2 is the usual method, For example, it can be purified by salting out, ion exchange chromatography, gel filtration, hydrophobic chromatography, affinity chromatography, etc., but more effectively gel using Cefacryl S-200 (Amersham Biosciences) Filtration is used.
• step (9) 2-mercaptoethanol is added to F (ab) 2 and reduced at 30 ° C for 30 minutes.
• Reduced Fab SH can be purified by conventional methods such as salting out, ion exchange chromatography, gel filtration, hydrophobic chromatography, affinity chromatography, etc. More effectively, The gel filtration used is used.
• a linker is bound to the Fab SH fraction of one antibody.
• Any crosslinking agent can be used as long as it can bind to the mercapto (SH) group of Fab SH .
• phenylene maleimide is added and allowed to react at room temperature for 30 minutes.
• the resulting bivalent specific substance can be purified by conventional methods such as salting out, ion exchange chromatography, gel filtration, hydrophobic chromatography, affinity chromatography, etc. For this, gel filtration using Cefacryl S-2200 is used.
• the antibody obtained in the step (6) can be used as it is, or can be appropriately labeled by a conventional method (for example, pyotinylated label, FITC label, etc.).
• a conventional method for example, pyotinylated label, FITC label, etc.
• an enzyme-labeled secondary antibody or a piotinylated antibody the enzyme-labeled streptavidin is added, and then the specific binding between the antibody and the antigen is measured in the presence of a chromophore product. Measure with By using this accessory system, small molecules that specifically recognize PD-1 or membrane proteins can be obtained.
• the process of (12) is the step of obtaining a low molecule obtained when one is an antibody or Fab.
• an appropriate functional group into the antibody, it can be bound to an antibody or Fab.
• a maleimide group is introduced, it can be bound to an antibody or Fab mercapto (SH) group.
• SH Fab mercapto
• each antibody cDNA is isolated from the hybridoma producing monoclonal antibodies against each antigen, and both cDNAs or their cDNA fragments are fused by gene recombination.
• a bispecific antibody can be produced from the host cell.
• a bispecific antibody comprising an antibody against human PD-1 or a partial fragment thereof and an antibody against a membrane protein present in the cell membrane expressing human PD_1 or a partial fragment thereof
• Anti-human PD_1 monoclonal antibody, anti-membrane protein monoclonal antibody antibody gene is isolated from each hyperpridoma producing antibody
• the produced protein can be separated and purified.
• the step (1) comprises a step of isolating RNA from hyperidoma cells and isolating cDNA encoding the antibody gene or a partial peptide thereof.
• total NA total RNA
• mRNA total RNA
• the process of isolating total NA (total RNA) or mRNA from a hyperidoma cell is a known method (hereinafter, unless otherwise specified, Sambrook, J. et al., 2 authors, Molecular Cloning (Molecular Cloning), 1989, Cold Spring Can be performed according to 2 methods from Harbor Laboratory or FMAusubel, Current Protocol (Method described in Current Protocol in Molecular Biology).
• a synthetic DNA primer having a partial base sequence of the antibody protein of the present invention is used as a means for cloning the cDNA encoding the antibody gene of the present invention or a partial peptide thereof.
• Polymerase Chain Reaction Polymerase Chain Reaction; In the following, it is abbreviated as PCR method.
• a DNA fragment that is amplified by or incorporated into an appropriate vector is a DNA fragment or synthetic DNA that encodes part or all of the antibody protein of the present invention. It can be sorted by high pre-labeling with the ones used and labeled.
• the hybridization method can be performed according to a known method.
• the antibody gene can also be amplified directly by using reverse RNA polymerase chain reaction (hereinafter abbreviated as RT-PCR method) using total RNA or mRNA.
• Examples of expression systems (host-vector system) for producing peptides using genetic recombination techniques include expression systems for bacteria, yeast, insect cells and mammalian cells.
• vector systems include plasmids derived from E. coli (eg, PBR322, pBR325, pUC 12, pUC 13), plasmids derived from Bacillus subtilis (eg, pUB 110, pTP 5, pC 194), yeast-derived plasmids (eg, p SH 19, pSH 1 5), bacteriophage such as human phage, retrovirus, vaccinia virus, animal virus such as baculovirus, etc.
• plasmids derived from E. coli eg, PBR322, pBR325, pUC 12, pUC 13
• Bacillus subtilis eg, pUB 110, pTP 5, pC 194
• yeast-derived plasmids eg, p SH 19, pSH 1 5
• bacteriophage such as human phage, retrovirus, vaccinia virus, animal virus such as baculovirus, etc.
• the promoter used in the present invention may be any promoter as long as it is suitable for the host used for gene expression.
• SR promoter, SV40 promoter, LTR promoter, CMV promoter, HSV-TK promoter and the like can be mentioned.
• CMV (cytomegalovirus) promoter, SR promoter and the like are preferable.
• the host is Escherichia coli, trp promoter overnight, lac promoter overnight, recA promoter, human PL promoter overnight, lpp promoter, T7 promoter, etc. are preferable, and the host is Bacillus. If it is a fungus, SP 01 promoter, SP02 promoter, pen nP promoter, etc. are preferred. If the host is yeast, PH05 promoter, PGK promoter, GAP promoter, ADH A promoter is preferred. When the host is an insect cell, a polyhedrin promoter, a P10 promoter, etc. are preferable.
• the expression vector contains an enhancer, a splicing signal, a poly A addition signal, a selection marker, an SV40 replication origin (hereinafter sometimes abbreviated as SV40 ori), and the like.
• a selection marker include dihydrofolate reductase (hereinafter abbreviated as dhfr) gene (methotrexate (MTX) resistance), ampicillin resistance gene (hereinafter abbreviated as Amp r). ), Neomycin resistance gene (hereinafter sometimes abbreviated as Ne or G418 resistance), and the like.
• a signal sequence suitable for the host is added to the N-terminal side of the protein of the present invention.
• the host is Escherichia, PhoA ⁇ signal sequence, OmpA ⁇ signal sequence, etc.
• the host is Bacillus
• the host is yeast, signal sequence, subtilisin ⁇ signal sequence, etc.
• MFa signal sequence, SUC2 signal sequence, etc. if the host is an animal cell, the insulin signal sequence, the signal sequence, antibody signal sequence, antibody molecule signal sequence, etc. It is available.
• a transformant can be produced using the thus constructed vector containing DNA encoding the protein of the present invention.
• the Escherichia bacterium transformed with the expression vector is cultured in an appropriate medium, and the desired peptide can be obtained from the microbial cells.
• a bacterial signal peptide for example, a signal peptide of pe 1B
• the peptide of interest can be secreted during periplasm.
• it can produce fusion proteins with other peptides.
• a cDNA encoding the target protein is used to transform a suitable mammalian cell as a host cell using an appropriate expression vector, and a transformant is appropriately used. By culturing in a suitable medium, the desired peptide is secreted into the culture medium.
• Examples of host cells that can be used include Escherichia bacteria, Bacillus bacteria, yeast, insect cells, insects, and animal cells.
• Specific examples of the genus Escherichia include, for example, Escherichia coli K12, DH1, JM103, JA221, HB101, C600, JM109, DH5, and DH5.
• Examples of Bacillus bacteria include Bacillus subtilis Mill 4.
• yeast include Saccharomyces cerevisiae AH22, AH22R—, NA87-1 1 A DKD-5 D 20B-12, (Saccharomyces pombe) NCYC 19 13, NCYC 2036, Pichiapastoris KM 71, etc. are used.
• Insect cells for example, when the virus is Ac NPV, a larval cell line derived from a night stealer (Spodoptera fragiperdaCell; S f cell), MG 1 cell derived from the midgut of Trichoplusia ni, derived from eggs of Trichoplusia ni High Five TM cells, cells derived from Mamestra brassicae or cells derived from Estigmene acrea are used.
• S f cells include S f 9 cells (ATCCCRL1711), Sf 21 cells (Vaughn, JL, In Vivo, 1977, No.
• CHO cell Chinese hamster cell CHO
• CHO cell dhfr gene-deficient Chinese hamster cell CHO
• CHO (dhf r-) cells Mouse L cells, mouse At T-20, mouse myeloma cells, rat GH3, HEK 293 T cells, human FL cells, etc.
• Insect cells or insects can be transformed, for example, according to the method described in Bio / Technology, 1988, Vol. 6, ⁇ ⁇ 47-55.
• transforming animal cells for example, "Cell engineering separate volume 8 new cell engineering experiment protocol", Shujunsha, 1995, No. 263, Virology, 1973, No. 52, This can be done according to the method described in No. 456.
• the peptide obtained as described above can be purified by usual methods such as salting out, ion exchange chromatography, gel filtration, hydrophobic chromatography, affinity chromatography and the like.
• polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 11 which is a substantially pure form of the present invention generally means 90% or more of the polypeptide at the time of production, for example, 9 A polypeptide having an amino acid sequence which is 5, 98 or 99% homologous.
• the homologue of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 11 in substantially pure form is generally at least 20 and preferably at least 30 An amino acid sequence that is at least 70%, preferably at least 80% or 90%, more preferably 95% or more homologous in, for example, 40, 60 or 100 consecutive amino acid regions. It has a polypeptide.
• a fragment of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 11 in substantially pure form is at least 10 amino acids, preferably at least 15 amino acids
• polypeptides comprising 20, 25, 30, 40, 50 or 60 amino acid moieties, and their homologues are generally at least 10 amino acids, preferably at least 15 amino acids, for example 20 , 2 5, 3 0, 4 0, 5 0 or 60 series
• a peptide is one or more amino acids in the amino acid sequence (preferably about 1 to 25, preferably about 1 to 10, more preferably a number (1 to 5)).
• a polypeptide consisting of an amino acid sequence in which one or several amino acids of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 11 shown in substantially pure form are Includes an amino acid sequence in which one or more amino acids are substituted in the amino acid sequence (preferably about 1 to 25, preferably about 1 to 10, more preferably several (1 to 5)) It is a polypeptide.
• Peptides are those in which one or more amino acids are inserted into the amino acid sequence (preferably about 1 to 25, preferably about 1 to 10, more preferably a number (1 to 5)).
• the homologue of the polynucleotide consisting of the DNA base sequence shown in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 9 is generally at least 20, preferably at least 30, for example 40, 60 or 1
• a fragment of a polynucleotide comprising the DNA base sequence shown in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 9 is at least 20 bases, preferably at least 30 bases, such as 40, 50, 60 or 10 It includes a polynucleotide consisting of a 0 base moiety.
• the homologue of the fragment is generally at least 20, preferably at least 30, for example 40, 60 or 100 consecutive base sequence regions, at least 70%, preferably Is a polynucleotide that is at least 80 or 90% homologous, more preferably 95% or more homologous.
• DNA that hybridizes with DNA containing the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 9 under highly stringent conditions include SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 9, respectively.
• Hybridization can be performed by a known method or a method similar thereto, for example, by J. Sambrook, Molecular Cloning 2nd edition, Cold Spring Harbor Labratry (ColdSpring Harbor Labratry). It can be carried out according to the method described.
• the high stringent conditions are, for example, a sodium concentration of about 19 to 40 mM, preferably about 19 to 20 mM, and a temperature of about 50 to 70 ° (preferably about 60 to Indicates conditions at 65 ° C, especially when the sodium concentration is about 19 mM and the temperature is about 65 ° C.
• the substance having specificity for human PD-1 of the present invention is used for the treatment and Z or prevention of the following diseases.
• Substances having specificity for human PD-1 of the present invention include, for example, neurodegenerative diseases (senile dementia, Alzheimer's disease, Down's syndrome, Parkinson's disease, Croy's felt 'Jakob disease, amyotrophic spinal sclerosis, It is useful for the treatment and / or prevention of diseases such as diabetic neuropathy, Parkinson's syndrome, Huntington's disease, Machadojesef's disease, amyotrophic lateral sclerosis, Croy's felt-Jakob disease).
• neurodegenerative diseases senile dementia, Alzheimer's disease, Down's syndrome, Parkinson's disease, Croy's felt 'Jakob disease, amyotrophic spinal sclerosis.
• the substance having specificity for human PD-1 of the present invention is associated with PD-1 and a disease caused by an enhanced immune response, such as an autoimmune disease (glomerulonephritis, arthritis, dilated myocardium).
• an autoimmune disease glomerulonephritis, arthritis, dilated myocardium.
• Symptom-like disease ulcerative colitis, Siegren's syndrome, Crohn's disease, systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, dryness, allergic contact dermatitis, multiple myositis, scleroderma, nodular periarterial Inflammation, rheumatic fever, vitiligo vulgaris, insulin-dependent diabetes mellitus, Behcet's disease, Hashimoto's disease, Addison's disease, dermatomyositis, myasthenia gravis, Reiter's syndrome, Dale's disease, pernicious anemia, Good
• the substance having specificity for human PD-1 of the present invention is collagen disease (systemic eritematodes, rheumatoid arthritis, generalized scleroderma, generalized progressive sclerosis, dermatomyositis, It is useful for the treatment and / or prevention of diseases such as polymyositis, Siegren's syndrome, mixed connective tissue disease, nodular polyarteritis, and rheumatic fever.
• collagen disease systemic eritematodes, rheumatoid arthritis, generalized scleroderma, generalized progressive sclerosis, dermatomyositis.
• the substance having specificity for human PD-1 of the present invention includes organ transplant rejection reaction, allergic disease, and further, PD-1 is involved, and diseases caused by decreased immune reaction, for example, Treatment of diseases such as tumors and infections And / or useful for prevention.
• the substance having specificity for human PD-1 of the present invention is used for the above purpose, it is usually administered systemically or locally in an oral or parenteral form.
• Dosage varies depending on age, weight, symptoms, therapeutic effect, administration method, treatment time, etc., but is usually in the range of O.lm g to 10 O mg per adult per day. Orally administered several times to several times, or parenterally (preferably intravenously administered) once to several times a day in the range of O.Olm g to 3 O mg per adult Or administered intravenously in the range of 1 to 24 hours per day.
• the dosage varies depending on various conditions, and therefore, an amount smaller than the above dosage may be sufficient, or may be necessary beyond the range.
• the compound of the present invention When the compound of the present invention is administered, it is used as a solid preparation for internal use for oral administration, a solution for internal use, and an injection, external preparation, suppository for parenteral administration and the like.
• solid preparations for internal use for oral administration include tablets, pills, capsules, powders, and condyles.
• Capsules include hard capsules and soft capsules.
• one or more active substances are left as they are or excipients (lactose, mannitol, glucose, microcrystalline cellulose, starch, etc.), binders (hydroxypropylcellulose, Polyvinylpyrrolidone, magnesium aluminate metasilicate, etc.), disintegrants (calcium glycol glycolate, etc.), lubricants (magnesium stearate, etc.), stabilizers, solubilizers (glutamic acid, aspartic acid, etc.), etc. They are mixed and formulated according to conventional methods.
• coating agent sucrose, gelatin, hydroxypropylcellulose, hydroxypropylmethyl cell It may be covered with roast sulfate, etc., or may be covered with two or more layers. Also included are capsules of absorbable substances such as gelatin.
• Liquid preparations for internal use for oral administration include pharmaceutically acceptable solutions, suspensions, emulsions, syrups, elixirs and the like.
• a solution one or more active substances are dissolved, suspended or emulsified in a commonly used diluent (purified water, ethanol or a mixture thereof).
• this liquid preparation may contain a wetting agent, a suspending agent, an emulsifier, a sweetening agent, a flavoring agent, a fragrance, a preservative, a buffering agent and the like.
• injections for parenteral administration include solutions, suspensions, emulsions, and solid injections that are used by dissolving or suspending in solutions for use.
• injectables are used by dissolving, suspending or emulsifying one or more active substances in a solvent.
• the solvent include distilled water for injection, physiological saline, vegetable oil, propylene glycol, polyethylene glycol, alcohols such as ethanol, and combinations thereof.
• this injection contains a stabilizer, a solubilizer (glutamate, aspartate, polysorbate 80 (registered trademark), etc.), a suspending agent, an emulsifier, a soothing agent, a buffer, a preservative, etc. You may go out. They are sterilized in the final step or prepared by aseptic manipulation.
• aseptic solid preparations such as lyophilized products can be produced and used by dissolving them in sterilized or sterile distilled water for injection or other solvents before use.
• compositions for parenteral administration include one or more active substances, externally formulated liquids, ointments, coatings, inhalants, sprays, suppositories and vagina Pessari etc. for internal administration are included.
• sprays may contain a buffer that provides isotonicity with stabilizers such as sodium bisulfite, for example, isotonic agents such as sodium chloride, sodium citrate, or citrate. You may contain.
• stabilizers such as sodium bisulfite
• isotonic agents such as sodium chloride, sodium citrate, or citrate. You may contain.
• the production method of the prescription agent is described in detail, for example, in US Pat. Nos. 2868691 and 3095355.
• PD-1 Since PD-1 is involved in the immune reaction, it is involved in the immune reaction by measuring the expression of human PD-1 using the substance having specificity for human PD-1 of the present invention. It can be used for screening of substances to be used.
• the substance having specificity for human PD-1 of the present invention includes a part that recognizes human PD-1, a part that recognizes a membrane protein present in a cell membrane expressing human PD-1, and a linker. Therefore, it is an excellent substance that can specifically recognize human PD-1 and membrane proteins and transmit human PD-1 signals.
• FIG. 1 is a diagram showing the structure of a vice-specific antibody expression vector.
• Figure 2 shows that A and B are X 63 cell surface antigens and (CD 3 (+) / PD) forcibly expressing (CD 3 (—) / PD-1 (+) cells), respectively, of bispecific antibodies.
• -1 (1) Reactivity against human peripheral blood mononuclear cell (PBMC) surface antigens.
• PBMC peripheral blood mononuclear cell
• the filled area of the histogram shows control IgG, and the open area is a graph showing the distribution of P D-1 or CD 3 positive cells.
• FIG. 3 is a graph showing the effect of a vice-specific antibody on the proliferation response of activated human peripheral blood T cells.
• “one” indicates the control IgG addition group
• “BsAb” indicates the bispecific antibody addition group
• the vertical axis indicates the measured absorbance, and indicates P ⁇ 0.05 in the significant difference test.
• J 1 10 High Pridoma cDNA Library and CD 3 Antibody Hyper Pridoma cDNA Library were prepared using Ready-To-Go You-Prime First-Strand Beads (trade name: purchased from Amersham Falmacia).
• cDNA was prepared from (total RNA) by the oligo dT prime method. The operation and procedure were in accordance with the attachment.
• a DNA encoding a bispecific antibody was prepared by ligating the cDNA isolated in Example 1.
• the ligation of the anti-human PD-1 antibody IG heavy chain cDNA (SEQ ID NO: 1) and the anti-human CD3 antibody Ig g light chain cDNA (SEQ ID NO: 7) was linked to the linker No. 1 (SEQ ID NO: 19) and No. 2 ( Fragment 1 was prepared by PCR reaction using SEQ ID NO: 20) and primers No. 1 (SEQ ID NO: 14) and No. 2 (SEQ ID NO: 15) (FIG. 1).
• fragment 1 was prepared by PCR reaction using No. 3 (SEQ ID NO: 16) and No. 4 (SEQ ID NO: 17) (Fig. 1).
• fragment 1 and fragment 2 are linked by linker No. 5, 5 (SEQ ID NO: 23) and ⁇ ⁇ 6 (SEQ ID NO: 24) and primer No. 5 (SEQ ID NO: 18) and ⁇ ⁇ 4 (SEQ ID NO: 22). ) was used to produce fragment 3 (Fig. 1), and its nucleotide sequence was determined (SEQ ID NO: 9).
• Each PCR reaction was performed in two steps. The first PCR reaction was repeated 20 times at 94 ° C for 30 seconds, 40 ° C for 30 seconds, and 72 ° C for 50 seconds. The second PCR reaction was the first PCR reaction. Using the reaction solution as a vertical sample, the temperature operation was repeated 30 times at 94 ° C for 30 seconds, at 50 ° C for 30 seconds, and at 72 ° C for 50 seconds.
• bispecific antibody is IipofectAMNE-plus (trade name: Invito
• the human kidney cell line 293T (ATCC Number: CRL-1 1268) into which J110-CD3scDb-pSec / hygroA was introduced was purchased. After culturing for 4 days after the introduction of the expression vector gene, the culture supernatant was recovered. The culture supernatant was sterilized by filtration through a 0.22 II1 PVDF filter, dialyzed in a 40% PEG 20000 solution, and concentrated. The concentrated supernatant was purified using HiTrapChelatingHP column (trade name: purchased from Amersham Falmacia).
• HiTrapChelatingHP column (trade name: purchased from Amersham Falmacia).
• the confirmation of the activity of the bispecific antibody was evaluated as an effect on the proliferation response of activated human peripheral blood T cells.
• PBMC peripheral blood of healthy humans using a Lymphoprep Tube (trade name: purchased from HYCOMED PHARMA). The operation and procedure were in accordance with the attachment. The separated cells are then lysed with hemolysis buffer (0.8% NH 4 C 1, 0.1% KC0 3 , ImM EDTA), and erythrocytes were hemolyzed. Subsequently, T cells purified using Nylon Fiber Column T (trade name: purchased from Roche) were suspended in a medium (RPMI 1640 medium containing 10% urine fetal serum).
• a medium RPMI 1640 medium containing 10% urine fetal serum
• the bispecific antibody significantly reduced the proliferation activity of activated human peripheral blood T cells.

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