WO2001064249A1 - Inhibiteur de decomposition de tissu - Google Patents

Inhibiteur de decomposition de tissu Download PDF

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Publication number
WO2001064249A1
WO2001064249A1 PCT/JP2000/005886 JP0005886W WO0164249A1 WO 2001064249 A1 WO2001064249 A1 WO 2001064249A1 JP 0005886 W JP0005886 W JP 0005886W WO 0164249 A1 WO0164249 A1 WO 0164249A1
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antibody
chain
plasmid
dna
region
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PCT/JP2000/005886
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English (en)
Japanese (ja)
Inventor
Hidemi Saito
Toshiaki Tsunenari
Etsuro Onuma
Koh Sato
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Chugai Seiyaku Kabushiki Kaisha
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Priority to CA002401357A priority Critical patent/CA2401357A1/fr
Publication of WO2001064249A1 publication Critical patent/WO2001064249A1/fr

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    • 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
    • C07K16/26—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A61P21/04—Drugs for disorders of the muscular or neuromuscular system for myasthenia gravis
    • 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
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/02—Non-specific cardiovascular stimulants, e.g. drugs for syncope, antihypotensives
    • 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
    • C07K2317/56—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
    • C07K2317/50—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
    • C07K2317/567—Framework region [FR]
    • 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

Definitions

  • the present invention relates to a tissue degradation inhibitor comprising a substance that inhibits binding between parathyroid hormone-related peptide (PTHrP) and its receptor.
  • PTHrP parathyroid hormone-related peptide
  • Parathyroid hormone-related peptide is a protein produced by tumors, the main causative agent of hypercalcemia, and is associated with bone resorption and renal tubules. It induces tumor-producing hypercalcemia (Humoral hypercalcemia of mal ig cy, hereinafter referred to as “wake”) by promoting calcium reabsorption of calcium.
  • the treatment of HHM uses a calcitonin-bisphosphonate preparation that has a bone resorption inhibitory effect, but the progression of HHM is rapid and significantly worsens the quality of life (QOL) of terminal cancer patients.
  • QOL quality of life
  • Antibodies against parathyroid hormone-related peptides have an effect on ⁇ immediately after administration, and are therefore superior to bisphosphonate preparations that require days to develop the effect. ing. Furthermore, it is also useful as a therapeutic drug for cachexia found in terminal cancer patients (Japanese Patent Application Laid-Open No. 11-80025).
  • Cancer cachexia is one of the paraneoplastic syndromes characterized by rapid weight loss and is found in many advanced cancer patients. This weight loss was conventionally thought to be due to a decrease in food intake due to decreased appetite. The expression of cancer cachexia not only has a significant effect on the survival time, but also significantly impairs the patient's Q0L and adversely affects treatment with chemotherapeutics. Thus, controlling cachexia is important as part of cancer treatment.
  • An object of the present invention is to provide a tissue degradation inhibitor containing, as an active ingredient, a substance that inhibits the binding of PTHrP to its receptor.
  • the present inventors have conducted intensive studies to solve the above problems, and as a result, have found that a substance that inhibits the binding of a parathyroid hormone-related peptide to its receptor can suppress tissue degradation. It was completed.
  • the present invention is a tissue degradation inhibitor containing, as an active ingredient, a substance that inhibits binding between a parathyroid hormone-related peptide and its receptor.
  • a substance that inhibits binding between a parathyroid hormone-related peptide and its receptor.
  • the substance include an antagonist to a parathyroid hormone-related peptide receptor, an anti-parathyroid hormone-related peptide antibody (for example, a humanized or chimerized monoclonal antibody), a fragment of the antibody, and ⁇ or Modifications and the like can be given.
  • examples of the humanized antibody include a humanized # 23-57-137-1 antibody and the like.
  • the tissue include a muscle tissue and an adipose tissue. Such tissue degradation includes those caused by cancer cachexia, sepsis, severe trauma or muscular dystrophy.
  • the inhibitor may be, for example, a patient whose blood concentration of cytodynamics (eg, at least one selected from the group consisting of IL-6, G-CSF, IL-11 and LIF) is higher than normal. It is also effective in In particular, although it is said that inflammatory cytokines such as IL-6 and IL-1K LIF are involved in tissue degradation, even in the presence of such cytokines, Inhibitors are effective.
  • cytodynamics eg, at least one selected from the group consisting of IL-6, G-CSF, IL-11 and LIF
  • the present invention provides a substance that inhibits the binding between parathyroid hormone-related peptide (PTHrP) and its receptor (PTHrP receptor). It is a tissue decomposition inhibitor contained as a component.
  • the present inventors presumed that the anti-PTHrP antibody plays a role in increasing or decreasing body weight based on the knowledge that the anti-PTHrP antibody has a pharmaceutical effect of restoring body weight in addition to a pharmaceutical effect of correcting calcium concentration, We thought that the weight loss caused by cachexia was caused by tissue destruction. Then, they found that an anti-PTHrP antibody suppressed tissue destruction, and completed the present invention.
  • PTHrP receptor refers to, for example, a receptor that binds to PTHrP described in Japanese Patent Application Laid-Open No. 6-506598, and indicates PTHrP present on a target organ (eg, bone or kidney). It does not matter whether it is a receptor or not.
  • a substance that inhibits the binding of ⁇ to the PTHrP receptor means (1) a substance that binds to PTHrP, thereby inhibiting the binding of PTHrP to the PTHrP receptor (for example, an anti-PTHrP antibody); and (2) A substance that inhibits the binding of PTHrP to the PTHrP receptor by binding to the PTHrP receptor (for example, an antagonist to the PTHrP receptor (also referred to as a PTHrP antagonist), specifically, at least a PTHrP peptide. Substitution or deletion of one amino acid, partial sequence of PTHrP peptide, etc.).
  • the origin, type (monoclonal, polyclonal) and shape of the anti-PTHrP antibody used in the present invention are not limited as long as they have an effect of suppressing tissue degradation by binding to PTHrP.
  • anti-PTHrP antibody examples include a humanized antibody, a human antibody (W096 / 33735), a chimeric antibody (JP-A-4-228089), a mouse antibody (for example, Hachiburi Doma # 23-57-137-1).
  • Antibody # 23-57-137-1 antibody).
  • the antibody may be a polyclonal antibody, but is preferably a monoclonal antibody.
  • Examples of the PTHrP antenna include, but are not limited to, polypeptides and small molecules.
  • polypeptides and small molecules For example, as a substance that binds to a PTHrP receptor antagonistically to PTHrP, JP-A-7-165790, JP-T-5-509098 or Peptides (UNITED STATES) 1995, 16 (6) 1031-1037, Has the PTHrP antagonist activity described in B iochemi stry (UNITED STATES) Apr. 281992, 31 (16) 4026-4033 Polypeptides.
  • polypeptides in which at least one amino acid has been deleted, substituted, added, or inserted and which have the same PTHrP antagonist activity are also the PTHrP antagonists of the present invention. include.
  • an anti-PTHrP antibody will be described as an example of "a substance that inhibits the binding between PTHrP and a PTHrP receptor".
  • the anti-PTHrP antibody used in the present invention can be obtained as a polyclonal or monoclonal antibody using known means.
  • a mammal-derived monoclonal antibody is particularly preferable.
  • Monoclonal antibodies derived from mammals include those produced in hybridomas and those produced in hosts transformed with expression vectors containing antibody genes by genetic engineering techniques. This antibody is an antibody that binds to PTHrP, inhibits PTHrP from binding to ⁇ receptors, blocks PTHrP signaling, and inhibits the biological activity of PTHrP.
  • Such antibodies include, for example, the # 23-57-137-1 antibody produced by the hybridoma clone # 23-57-137-1 and the like.
  • Hybridoma clone # 23-57-137-1 is designated as mouse-mouse hybridoma # 23-57-137-1 by the National Institute of Bioscience and Human-Technology, National Institute of Advanced Industrial Science and Technology (1-3-1 Higashi, Tsukuba, Ibaraki, Japan). On August 15, 1996, it was deposited internationally under the Budapest Treaty as FERM BP-5631.
  • a monoclonal antibody-producing hybridoma can be prepared as follows. That is, using PTHrP as a sensitizing antigen, immunizing the immunized antigen according to a normal immunization method, fusing the obtained immune cells with a known parent cell by a normal cell fusion method, It can be produced by screening monoclonal antibody-producing cells.
  • human PTHrP used as a sensitizing antigen for obtaining antibodies was identified by Suva, LJ et al. It is obtained by expressing the amino acid sequence of the PTHrP gene disclosed in Science (1987) 237, 893. That is, after inserting a gene sequence encoding PTHrP into a known expression vector system to transform a suitable host cell, the desired PTHrP protein is purified from the host cell or culture supernatant by a known method. I do.
  • this purified PTHrP protein is used as a sensitizing antigen.
  • the N-terminal 34 peptides of PTHrP can be prepared by chemical synthesis and used as a sensitizing antigen.
  • the mammal to be immunized with the sensitizing antigen is not particularly limited, but is preferably selected in consideration of compatibility with the parent cell used for cell fusion.
  • rodents Animals eg, mice, rats, hamsters, etc.
  • egrets, monkeys, etc. are used.
  • Immunization of an animal with a sensitizing antigen is performed according to a known method.
  • the sensitizing antigen is injected intraperitoneally or subcutaneously into a mammal.
  • the sensitizing antigen is diluted and suspended in an appropriate amount with PBS (Phosphate-Buffered Saline), physiological saline, or the like, and then mixed with a normal adjuvant, for example, Freund's complete adjuvant, if desired, and then emulsified.
  • a normal adjuvant for example, Freund's complete adjuvant, if desired, and then emulsified.
  • an appropriate carrier can be used during immunization of the sensitizing antigen.
  • immunocytes are collected from the mammal and subjected to cell fusion.
  • Preferred immunocytes are splenocytes, in particular. No.
  • Mammalian myeloma cells are used as the other parent cells to be fused with the immune cells.
  • This myeloma cell can be selected from various known cell lines, for example, P3 (P3x63Ag8.653) (J.Immol. (1979) 123, 1548-1550), P3x63Ag8U.1 (Current Topics in Microbiology and Immunology u978) 81, 1 -7), NS-1 (Kohler.G. And Milstein, C. Eur.J. Immunol. (1976) 6, 511-519), MPC-11 (Margulies.DH et al., Cell (1976) 8, 405-415), SP2 / 0 (Shu 1 man, M.
  • Cell fusion between the immune cells and myeloma cells is basically performed by a known method, for example, the method of Milstein et al. (Kohler. G. and Milstein, Methods Enzymol. (1981) 73, 3-46). And so on.
  • the cell fusion is carried out in a normal nutrient medium, for example, in the presence of a cell fusion promoter.
  • a cell fusion promoter for example, polyethylene glycol (PEG), Sendai virus (HVJ) and the like are used, and if necessary, an auxiliary agent such as dimethyl sulfoxide can be added to enhance the fusion efficiency.
  • the ratio of the immune cells to the myeloma cells can be arbitrarily set. For example, it is preferable that the number of immune cells be 10 times larger than that of myeloma cells.
  • the culture medium used for the cell fusion for example, RPMI 1640 culture medium, MEM culture medium suitable for the growth of the myeloma cell line, and other ordinary culture medium used for this type of cell culture can be used. Yes, and serum supplements such as fetal calf serum (FCS) can be used in combination.
  • FCS fetal calf serum
  • a predetermined amount of the immune cells and myeloma cells are mixed well in the culture solution, and a PEG solution (for example, an average molecular weight of about 1000 to 6000), which has been heated to about 37 in advance, is usually 30 to 60% ( (w / v) and mix to form the desired fused cells (hybridomas).
  • a PEG solution for example, an average molecular weight of about 1000 to 6000
  • a culture solution is sequentially added, and the operation of centrifuging and removing the supernatant is repeated to remove cell fusion agents and the like that are unfavorable for the growth of hybridomas.
  • hybridomas are selected by culturing them in a normal selective culture medium, for example, a HAT medium (a culture medium containing hypoxanthine, aminopterin and thymidine).
  • a HAT medium a culture medium containing hypoxanthine, aminopterin and thymidine.
  • the culturing in the HAT culture solution is continued for a time (usually several days to several weeks) sufficient for cells other than the target hybridoma (non-fused cells) to die.
  • the usual limiting dilution method is performed, and screening and single cloning of hybridomas producing the desired antibody are performed.
  • human lymphocytes are sensitized to PTHrP in vitro, and the sensitized lymphocytes are derived from humans and have myeloma cells with permanent division ability.
  • a desired human antibody having PTHrP binding activity See Japanese Patent Publication No. 59878.
  • transgenic animals having the entire repertoire of human antibody genes are administered with PTHrP as an antigen to obtain anti-PTHrP antibody-producing cells, and human antibodies to PTHrP are obtained from immortalized cells. Good (see International Publication Nos. WO94 / 25585, WO93 / 12227, WO92 / 03918, WO94 / 02602).
  • the hybridoma producing the monoclonal antibody thus produced can be subcultured in a normal culture solution, and can be stored for a long time in liquid nitrogen.
  • the hybridoma is cultured according to an ordinary method, and the culture is obtained as a culture supernatant.
  • the hybridoma is administered to a mammal compatible with the hybridoma.
  • the former method is suitable for obtaining high-purity antibodies, while the latter method is suitable for mass production of antibodies.
  • a recombinant antibody produced by cloning an antibody gene from a hybridoma, inserting the antibody gene into an appropriate vector, introducing the antibody into a host, and producing the recombinant antibody using a gene recombination technique is used.
  • a gene recombination technique See, for example, Vandamme, AM et al., Eur. J. Biocliem. (1990) 192, 767-775, 1990).
  • V variable
  • mRNA can be isolated by known methods, for example, guanidine ultracentrifugation (Chirgwin, JM et al., Biochemistry (1979) 18, 5294-5299), AGPC method (Chomczynski, P. et al., Anal. Biochem. (1987) 162, 156-159) to prepare total RNA, and then use mRNA Purification Kit (Pharmacia) to prepare the target mRNA. Also, mRNA can be directly prepared by using the QuickPrep mRNA Purification Kit (Pharmacia).
  • cDNA for the antibody V region is synthesized using reverse transcriptase.
  • Synthesis of cDNA is performed using AMV Reverse Transcriptase First-strand c-band Synthesis Kit (manufactured by Iidaigaku Kogyo).
  • AMV Reverse Transcriptase First-strand c-band Synthesis Kit manufactured by Iidaigaku Kogyo.
  • 5'-Ampli 5'-RACE method using FINDER RACE Kit (Clontech) and PCR (Frohman, MA et al., Proc. Natl. Acad. Sci. USA (1988) 85, 8998-9002, Belyavsky, A. et al., Nucleic Acids Res. (1989) 17, 2919-2932), etc.
  • the antibody gene is incorporated into an expression vector so as to be expressed under the control of an expression control region, for example, an enhancer or a promoter.
  • host cells are transformed with the expression vector to express antibodies.
  • Expression of the antibody gene can be performed by separately transforming the DNA encoding the antibody heavy chain (H chain) or light chain (L chain) into an expression vector and co-transforming the host cell, or The host cell may be transformed by incorporating the DNA encoding the L chain into a single expression vector (see WO94 / 11523).
  • transgenic animals can be used for the production of recombinant antibodies.
  • an antibody gene is inserted into a gene encoding a protein (eg, goat casein) that is specifically produced in milk to prepare a fusion gene.
  • a DNA fragment containing the fusion gene into which the antibody gene has been inserted is injected into a goat embryo, and the embryo is introduced into a female goat.
  • the desired antibody is obtained from the milk produced by the transgenic goat born from the goat that has received the embryo or its progeny.
  • Hormones may also be used in transgeneic goats as appropriate to increase the amount of milk containing the desired antibodies produced from transgenic goats (Ebert, K.. Et al., Bio / Technology (1994) 12, 699-702).
  • Modified antibody in addition to the above-mentioned antibodies, genetically modified antibodies artificially modified for the purpose of reducing heterologous antigenicity to humans and the like, for example, chimeric antibodies and humanized antibodies can be used. These modified antibodies can be produced using the following method.
  • the chimeric antibody useful in the present invention is produced by ligating the DNA encoding the antibody V region obtained as described above with the DNA encoding the human antibody C region, incorporating the DNA into an expression vector, and introducing the resultant into a host.
  • Humanized antibodies are also referred to as reshaped human antibodies, which are used to replace the complementarity determining regions (CDRs) of non-human mammals, such as mouse antibodies, with those of human antibodies. It is also transplanted into a plant, and its general genetic recombination technique is also known (see European Patent Application Publication No. EP 125023, WO 96/02576).
  • a DNA sequence designed to link the CDR of a mouse antibody to the framework region (FR) of a human antibody is modified so that it has a portion that overlaps both terminal regions of the CDR and FR. Amplify by PCR using several prepared oligonucleotides as primers. The obtained DNA is ligated to DNA encoding the human antibody C region, and then inserted into an expression vector, which is then introduced into a host to produce a humanized antibody (EP 239400, WO No. 96/02576).
  • the framework region of the human antibody to be linked via CDR is selected so that the complementarity determining region forms a favorable antigen-binding site. If necessary, the amino acids of the framework region in the variable region of the antibody may be substituted so that the complementarity-determining region of the reshaped human antibody forms an appropriate antigen-binding site (Sato, K. et al. , Cancer Res. (1993) 53, 851-856).
  • human antibodies are used for the C region of chimeric and humanized antibodies.
  • the human antibody C region may be modified in order to improve the stability of the antibody or its production.
  • Chimeric antibodies consist of the variable region of a non-human mammal-derived antibody and the constant region of a human antibody-derived constant. Area.
  • a humanized antibody is composed of a complementarity determining region of an antibody derived from a mammal other than human, a framework region and a C region derived from a human antibody. Since the humanized antibody has reduced antigenicity in the human body, it is useful as an active ingredient of the inhibitor of the present invention.
  • Humanized antibodies that can be used in the present invention include humanized # 23-57-137-1 antibody.
  • the humanized # 23-57-137-1 antibody has the complementarity determining region of the mouse-derived # 23-57-137-1 antibody, and the human antibody HSU03868 (GEN-BANK, Deitos M et al. Scand. J. Immunol., 39, 95-103, 1994) linked to three FR fragments (FR1, FR2 and FR3) and FR fragment (FR4) derived from human antibody S25755 (NBRF-PDB).
  • the H chain is linked to the framework region of the human antibody S31679 (NBRF-PDB, Cuisinier AM et al., Eur. J. Immunol., 23, 110-118, 1993), and is designed to have antigen-binding activity.
  • the amino acid residues in the work region are partially substituted.
  • Escherichia coli harboring a plasmid containing DNA encoding the L-chain or ⁇ -chain of the humanized # 23-57-137-1 antibody was purchased from the Institute of Biotechnology, Institute of Bio-Industrial Technology (1-1-1, Higashi, Tsukuba, Ibaraki, Japan). No. 3), as of August 15, 1996, for Escherichia coli JM109 (hMBClHcDNA / pUC19), a large intestinal bacterium having a plasmid containing DNA encoding the ⁇ chain.
  • Escherichia coli JM109 (IIMBC ILQ A / PUC 19), which is Escherichia coli having a plasmid containing DNA encoding an L chain, has been deposited internationally as FERM BP-5629 as FERMBP-5630 under the Budapest Treaty. .
  • the antibody used in the present invention may be an antibody fragment or a modified product thereof, as long as it binds to PTHrP and inhibits the activity of PTHrP.
  • antibody fragments include Fab, F (ab ') 2, Fv, and single-chain Fv (scFv) in which an Fv of an H chain or an L chain is linked with an appropriate linker.
  • an antibody is treated with an enzyme such as papine or pepsin to generate an antibody fragment, or a gene encoding these antibody fragments is constructed, and the gene is introduced into an expression vector. Expressed in cells (eg, Co, MS et al., J. Immunol. (1994) 152, 2968-2976, Better, M.
  • scFv can be obtained by linking the H chain V region and L chain V region of the antibody.
  • the H chain V region and L chain V region are linked via a linker, preferably a peptide linker (Huston, JS et al., Pro Natl. Acad. ScI. USA (1988) ) 85, 5879-5883).
  • the H chain V region and L chain V region in the scFv may be from any of the antibodies described herein.
  • the peptide linker connecting the V regions for example, any single-chain peptide consisting of 12 to 19 amino acid residues is used.
  • the scFv-encoding DNA is a DNA encoding the H chain or H chain V region of the antibody and a DNA encoding the L chain or L chain V region, all or a desired amino acid sequence thereof. Is amplified by PCR using a pair of primers that define both ends of the DNA, and then the DNA encoding the peptide linker and both ends are ligated to the H and L chains, respectively. It is obtained by combining and amplifying a pair of primers defined as follows.
  • the expression vector containing them and the host transformed with the expression vector can be obtained according to a conventional method.
  • scFv can be obtained according to a conventional method.
  • the fragments of these antibodies can be obtained and expressed in the same manner as described above, and produced by a host.
  • the “antibody” in the present invention also includes fragments of these antibodies.
  • an anti-PTHrP antibody conjugated with various molecules such as polyethylene glycol (PEG) can also be used.
  • PEG polyethylene glycol
  • the “antibody” in the present invention also includes these modified antibodies.
  • modified antibodies can be used to chemically modify the resulting antibodies. It can be obtained by decorating. Methods for modifying antibodies have already been established in this field.
  • the antibody gene constructed as described above can be expressed and obtained by a known method.
  • a useful promoter commonly used an antibody gene to be expressed, and a polyA signal operably linked to the 3 ′ downstream thereof can be expressed.
  • the promoter enhancer human cytomegalovirus immediate early promoter / enhancer can be mentioned.
  • viral promoters / enhancers such as retrovirus, poliovirus, adenovirus, simian virus 40 (SV40), and human promoters. Promoters derived from mammalian cells such as Long Factor Factor la (HEF1 ⁇ ).
  • a useful promoter commonly used, a signal sequence for antibody secretion, and an antibody gene to be expressed can be functionally linked to express the gene.
  • the promoter include lacz promoter and araB promoter. The method of Ward et al. (Nature (1098) 341, 544-546; FASEB J. (1992) 6, 2422-2427) when using the lacz promoter, or the method of Better et al. when using the araB promoter (Science (1988) 240, 1041-1043).
  • a pelB signal sequence (Lei, SP et al J. Bacteriol. (1987) 169, 4379) may be used for production in the periplasm of E. coli. Then, the antibody produced in the periplasm was separated Later, the antibody structure is appropriately refolded and used.
  • markers can include aminoglycoside transferase (APH) gene, thymidine kinase (TK) gene, Escherichia coli xanthinguanine phospholiposyltransferase (Ecogpt) gene, dihydrofolate reductase (dhir) gene and the like.
  • Eukaryotic cells include, for example, established mammalian cell lines, insect cell lines, animal cells such as eukaryotic fungal cells and yeast cells, and prokaryotic cells include, for example, bacterial cells such as Escherichia coli cells.
  • Eukaryotic cells include, for example, established mammalian cell lines, insect cell lines, animal cells such as eukaryotic fungal cells and yeast cells
  • prokaryotic cells include, for example, bacterial cells such as Escherichia coli cells.
  • the antibodies used in the present invention are expressed in mammalian cells, such as CH0, COS, myeloma, BHK, Vero, HeLa cells.
  • the transformed host cells are cultured in vitro or in vivo to produce the desired antibody.
  • Culture of the host cell is performed according to a known method.
  • DMEM, MEM, RPMI1640, IMDM can be used as a culture solution
  • a serum replacement solution such as fetal calf serum (FCS) can be used in combination.
  • FCS fetal calf serum
  • Antibodies expressed and produced as described above can be separated from cells and host animals and purified to homogeneity. Separation and purification of the antibody used in the present invention can be carried out using an affinity column.
  • affinity column For example, columns using a protein A column include Hyper D, P0R0S, Sepharose FF (Pharmacia) and the like.
  • separation and purification methods used for ordinary proteins may be used, and there is no limitation.
  • antibodies can be separated and purified by appropriately selecting and combining chromatographic columns, filters, ultrafiltration, salting out, dialysis, etc. other than the above affinity columns (Antibodies A Laboratory) Manual. Ed Harlow, David Lane, Cold Spring Harbor Laboratory, 1988). 8. Confirmation of antibody activity
  • Antigen binding activity of the antibodies used in the present invention (Antibodies A Laboratory Manual. Ed Harlow, David Lane, Cold Spring Harbor Laboratory, 1988), and recombinant receptor binding inhibitory activity (Harada, A. et al. , International Immunology (1993) 5, 681-690) can be measured by a known method.
  • Enzyme-linked immunosorbent assay ⁇ (enzyme immunoassay), RIA (radioimmunoassay) or fluorescent antibody method.
  • enzyme immunoassay a sample containing an anti-PTHrP antibody, for example, a culture supernatant of an anti-PTHrP antibody-producing cell or a purified antibody is added to a plate coated with PTHrP (1 to 34). After adding a secondary antibody labeled with an enzyme such as alkaline phosphatase, incubating and washing the plate, adding an enzyme substrate such as P-nitrophenyl phosphate and measuring the absorbance, the antigen binding activity Can be evaluated.
  • an enzyme substrate such as P-nitrophenyl phosphate
  • the neutralizing activity of the anti-PTHrP antibody is measured.
  • the tissue degradation inhibitor containing the anti-PTHrP antibody of the present invention as an active ingredient can be administered either orally or parenterally, but is preferably administered parenterally, specifically, in a pulmonary drug form (eg, nephrizer).
  • the injection form include systemic or local administration by intravenous injection such as infusion, intramuscular injection, intraperitoneal injection, subcutaneous injection and the like.
  • the administration method can be appropriately selected depending on the age and symptoms of the patient.
  • the effective dose is selected in the range of lOOmg to lOOmg / kg body weight at a time. Alternatively, a dosage of 0.01 to 100 mg / body per patient can be chosen.
  • the dose of the inhibitor containing the anti-PTHrP antibody of the present invention is not limited to these doses.
  • Diseases to which the inhibitor of the present invention is administered include cancer cachexia, pulmonary blood, trauma (severe, Moderate), muscular dystrophy, and the like, but are not limited thereto, and include those that have multiple onsets of the above-mentioned disease, or those that have been combined with other diseases.
  • Blood levels of at least one of leukin-6 (IL-6), granulocyte colony stimulating factor (G-CSF), IL-1K leukocyte inhibitory factor (LIF) and acid glycoprotein (a, -AG) It can be administered to patients with higher than normal values.
  • the “normal value” means an average value of each blood concentration in 10 to 100 healthy persons, or a generally published clinical test value.
  • laboratory values are 0.2-4.6 pg / inL for IL-6, 7.7-38.9 pg / mL for G-CSF, and 31.3 pg / mL for IL-11 It is 42-93 mg / dl for ⁇ -Guang AG.
  • the inhibitor of the present invention may be administered before or after the clinical symptoms of the above-mentioned disease occur, or may be administered when weight loss is predicted.
  • the inhibitor containing the anti-PTHrP antibody of the present invention as an active ingredient can be formulated by a conventional method (Remington's Pharmaceuticals Science, latest establishment, Mark Publ. shing Company, Easton, USA), and pharmaceutically acceptable carriers and additives.
  • Such carriers and pharmaceutical additives include water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium carboxymethylcellulose, sodium polyacrylate, and alginic acid.
  • HSA human serum albumin
  • the actual additive is selected from the above alone or in an appropriate combination depending on the dosage form of the inhibitor of the present invention, but is not limited thereto.
  • a purified anti-PTHrP antibody is dissolved in a solvent, for example, a physiological saline solution, a buffer solution, a glucose solution, etc., and an anti-adsorption agent, for example, Tween80, Tween 20, gelatin, human serum What added albumin etc. can be used.
  • a solvent for example, a physiological saline solution, a buffer solution, a glucose solution, etc.
  • an anti-adsorption agent for example, Tween80, Tween 20, gelatin, human serum What added albumin etc.
  • FIG. 1 is a graph showing the effects of humanized antibodies on body weight and blood ionized calcium concentration in mice transplanted with 0CC-1-JCK.
  • FIG. 2 is a graph showing the effects of humanized antibodies on body weight and blood ionized calcium concentration in Colon 26-transplanted mice.
  • FIG. 3 is a graph showing the effects of humanized antibodies on body weight and blood ionized calcium concentration in rats transplanted with LC-6-JCK.
  • FIG. 4 is a graph showing changes in white blood cell count in rats transplanted with LC-6-JCK. BEST MODE FOR CARRYING OUT THE INVENTION
  • a humanized anti-PTHrP antibody against PTHrP (1-34) was used as an example of the anti-PTHrP antibody.
  • the humanized anti-PTHrP antibody was prepared by a genetic recombination technique as described in Reference Example 4 (L chain version Q).
  • the obtained humanized anti-PTHrP antibody (hereinafter referred to as “humanized antibody”) was adjusted to pH 6.6 by adding lmol / L Tris solution to 20 Mol / L citrate solution (to a concentration of 6). 34 mg / mL), -7 (stored at or below TC.)
  • the antibody used was determined to be of interest by SDS-PAGE for molecular weight and by binding assay using ELISA for binding. confirmed.
  • mice Mouse colon cancer cell line Colon26 or human oral floor cancer cell line 0CC- mouse This is a mouse to which JCK can be transplanted, and is commonly used BALB / cAnNCrj mouse (for transplantation of Colon26) or BALB / cAJc-nu mouse (0CC- -For JCK transplantation), 30 males were used for each experiment. Mice were selected to be 6 weeks old at the time of tumor cell transplantation.
  • BALB / cAnNC rj was purchased from Nippon Charsriver and BALB / cAJc nu was purchased from Clea Japan.
  • mice After acclimating the purchased 30 mice for about one week, 7 mice were selected at random and a group without tumor transplantation was prepared as a normal group. The remaining 23 animals were implanted with tumors, and after 7 days from the implantation, tumor engraftment was confirmed. Twenty animals with tumor engraftment were selected. The mice were divided into a group to which PBS was administered and a group to which the humanized antibody of the present invention was administered, with each group being adjusted to be equal to the body weight as an index.
  • the breeding environment of the mice is as follows.
  • Rearing cage M-4 cage (215x320x130 marauder, made of polycarbonate, with flooring) Rearing density: 5 to 7 animals Z cage
  • Drinking and water supply method tap water, free water supply with 200 mL water bottle
  • Tumor strains used Colon26 (mouse colorectal cancer strain), 0CC-JCK (Human roental floor cancer strain) Origin of tumor strains: Colon26 (Cancer Research Institute Chemical Treatment Center), 0CC-JCK (Laboratory Animal Central Research Institute)
  • Tumor strain passage Colon26 was passaged every two weeks in vivo using BALB / cAnNCrj.
  • 0CC-ICK iCK was passaged every three weeks in vivo using BALB / cAJc-nu.
  • Colon26, 0CC-KK-transplanted mouse model was prepared as follows. In other words, the mouse used for in vivo passage on the day of tumor cell transplantation was dislocated to the cervical vertebra and the tumor was removed. Then, a three thigh angle block was prepared and implanted subcutaneously in a mouse. The mouse that survived the tumor after the tumor transplantation was used as a model established mouse.
  • Serum biochemical values (glucose, triglyceride, cholesterol) were measured using an autoanalyzer (Hitachi, Model 7170 automatic analyzer).
  • the humanized antibody was administered intravenously (i.v.) at a dose (0.1 mgZ mouse) (humanized antibody administration group).
  • Dulbecco's PBS (-) was administered as a PBS administration group.
  • Administration was performed twice on the 7th and 10th days after transplantation. No treatment was administered to the normal group without tumor transplantation.
  • Table 1 summarizes the group composition of mice and the dosing schedule.
  • Test items body weight, adipose tissue (around testis) weight, muscle tissue (gastrocnemius muscle) weight, serum glucose, serum triglyceride, serum cholesterol, blood iCa concentration, general condition
  • mice transplanted with 0CC-1-JCK body weight was measured on days 7, 10, and 13 after tumor implantation. Blood iCa levels were measured on days 7 and 13. For other items, day 13 was measured.
  • Efficacy evaluation The effect of humanized antibody administration on the reduction of body weight, adipose tissue and muscle tissue weight was examined. The effect of restoring blood iCa, serum glucose, serum triglyceride, and serum cholesterol to near normal levels was also examined.
  • Control group PBS administration group and humanized antibody administration group
  • the body weight, adipose tissue weight, muscle tissue weight, blood iCa concentration, serum glucose level, serum triglyceride level, and serum cholesterol level changed in the humanized antibody group compared to the PBS group. I have.
  • Body weight, adipose tissue weight, muscle tissue weight, blood iCa concentration, serum glucose level, serum triglyceride level, and serum cholesterol level were measured by t-test in normal group and PBS administration group. Tests were performed, and for those with significant differences, the significance of the PBS administration group and the humanized antibody administration group was examined by t-tes determination. The significance level was 5% on both sides. SAS was used for these analyses.
  • Body weight The body weight was significantly reduced in the PBS-administered group compared to the normal group (12 and 13 days after tumor transplantation. *: Tested by 0.05, t-test in comparison with the normal group). .
  • the weight reduction was significantly suppressed in the humanized antibody-administered group compared to the PBS-administered group (#: p-0.05 compared to the PBS-administered group, and tested by t-test), which was the same as the normal group.
  • Blood iCa concentration A significant increase was observed in the PBS-administered group compared to the normal group (13 days after tumor transplantation (*)).
  • the humanized antibody group significantly suppressed the increase in blood iCa concentration compared to the PBS group (#) (blood iCa concentration on day 13 of transplantation (thigh o / L); normal group 1.36 ⁇ 0.01, PBS administration group 2.73 ⁇ 0.10, humanized antibody administration group 1.62 ⁇ 0.01).
  • Fat thread and tissue weight a significant decrease was observed in the PBS-administered group compared to the normal group (*) ⁇ The decrease was significantly suppressed in the humanized antibody-administered group compared to the PBS-administered group ( #).
  • Muscle tissue weight A significant decrease was observed in the PBS-administered group compared to the normal group (*) ⁇ The decrease was significantly suppressed in the humanized antibody-administered group compared to the PBS-administered group (#) .
  • Body weight The body weight was significantly reduced in the PBS-administered group as compared with the normal group (* 10 days after tumor transplantation, *: p-0.05 compared to the normal group, and tested by t-test). The weight reduction was significantly suppressed in the humanized antibody administration group as compared with the PBS administration group (#: compared with the PBS administration group!). ). The body weight (g) on the 14th day after transplantation was 26.78 ⁇ 0.38 in the normal group, 19.04 ⁇ 0.35 in the PBS group, and 21.51 ⁇ 0.32 in the humanized antibody group.
  • Blood iCa concentration In the PBS-administered group, a significant increase was observed compared to the normal group (14 days after tumor transplantation (*)). However, the humanized antibody-administered group compared to the PBS-administered group The rise was significantly suppressed (#). The blood iCa concentration (thigh ol / L) on day 14 after transplantation was 1.34 ⁇ 0.01 in the normal group, 1.94 ⁇ 0.09 in the PBS group, and 1.54 ⁇ 0.03 in the human antibody group.
  • Adipose tissue weight A significant decrease was observed in the PBS-administered group compared to the normal group (*). The decrease was significantly suppressed in the humanized antibody administration group compared to the PBS administration group (#).
  • Muscle tissue weight A significant decrease was observed in the PBS-administered group compared to the normal group (*). The decrease was significantly suppressed in the humanized antibody administration group compared to the PBS administration group (#).
  • the serum glucose concentration was significantly lower in the PBS-administered group than in the normal group (*), but no improvement was observed by administration of the humanized antibody.
  • Serum triglyceride concentration was significantly lower in the PBS-administered group than in the normal group, and the decrease was suppressed, though not significant, in the humanized antibody-administered group.
  • Serum cholesterol concentration was unchanged in the PBS-administered and normal groups, and did not change in the humanized antibody-administered group.
  • Cancer cachexia is a clinical syndrome in which cancer-bearing patients have significant weight loss due to anorexia, increased metabolism, and the like. In addition, hypoglycemia and hyperlipidemia are mentioned as its serum biochemical characteristics.
  • tumors that induce cancer cachexia in tumor transplantation models are known. Representative tumor lines include SEKI melanoma (human melanoma), Colon 26 (mouse colon cancer cell line), and OCC-1-JCK (human cell line). Cavity floor cancer cell line). When these tumors are transplanted into nude mice, significant weight loss is observed.
  • HHM is PTHrP have been identified as the causative agent (LJ Suva, eta l., Sc i ence 237, 893-896, 1987) 0
  • the present inventor created a cancer cachexia model in which two types of tumor lines, a human oral floor cancer cell line 0CC-JCK and a mouse colon cancer cell line Colon 26, were transplanted, and examined the effects of the humanized antibody. investigated.
  • mice transplanted with CC-KK have significantly reduced body weight (more fat and muscle tissue weight) since 10 days after tumor transplantation, and colon-transplanted mice since 7 days after tumor transplantation, and have low glucose status. Characteristic of cancer cachexia in clinical practice Was. In addition, these models exhibited hypercalcemia, indicating that HHM was associated with cancer cachexia.
  • mice implanted with 0CC-JCK an improvement effect of low triglyceride was observed, and although not significant, a tendency of improvement in low glucose was observed.
  • the humanized anti-PTHrP antibody since the humanized anti-PTHrP antibody has an inhibitory effect on tissue degradation for adipose tissue and muscle tissue, it is useful as a therapeutic or preventive drug for tissue degradation associated with various diseases such as cancer cachexia. is there.
  • the humanized anti-PTHrP antibody used was prepared in Example 1.
  • a nude rat F344 / N Jcl-rnu (CLEA Japan) was used as a rat capable of transplanting a human tumor cell line and producing a stable HHM model. Eighty rats purchased at the age of 5 weeks were acclimated for about one week, and tumor transplantation was performed on 55 randomly selected rats. Euthanasia was performed by ether inhalation.
  • the breeding environment of the rat is as follows.
  • Rearing cages and breeding density From the habituation period and from the time of tumor transplantation to the start of the experiment, the animals are reared in stainless steel wire mesh hanging cages (dimensions: 660 x 310 x 200 mm) with 8 to 11 animals per cage. 265 x 425 x 160 mm).
  • Feed and feeding method CE-2 (CLEA Japan), free feeding Drinking and water supply method: tap water, free water supply
  • Tumor strain to be used LC-6-JCK (human large cell lung cancer strain)
  • a tumor block was implanted subcutaneously in a mouse (BALB / cA Jcl-nu; CLEA Japan, Inc.) and subcultured as an in vivo tumor line. Passaging was performed every 3-4 weeks.
  • Model preparation The mouse that had passed the tumor line was dislocated from the cervical vertebra and the tumor was excised, and this was cut into a three-thigh angle block. This tumor block was prepared by implanting it subcutaneously in a rat. Transplantation was performed on 55 rats randomly extracted from 80 rats. The remaining 25 animals were left untreated.
  • HHM model was used in which the transplanted tumor was subcutaneously growing grossly and the blood iCa concentration was higher than that in the normal group. In this test, individuals whose iCa concentration was 1.80 ol / L or more were used.
  • Grouping method Tumor transplantation On day 49, blood iCa concentration was measured to confirm the establishment of the HHM model. In the HHM model, stratification was performed using this blood iCa concentration as an index. Twenty-four animals were randomly sampled, so that there was no difference between the groups, saline saline administration group, humanized antibody administration group, The tumors were categorized into 3 groups (8 / group). Eight animals were randomly taken from 25 untreated rats and used as a normal group.
  • Serum was collected under anesthesia using blood from the descending aorta in a separapit tube (Sekisui Chemical Co., Ltd.). And then allowed to stand at room temperature for at least 30 minutes. The serum was separated by centrifugation (3,000 rpm, 20 min, HITACHI, 05PR-22), and stored frozen at -2 (TC or lower).
  • Serum biochemical values (glucose, triglycerides, cholesterol, free fatty acids) were measured using an autoanalyzer (Hitachi, Model 7170 automatic analyzer).
  • Adipose tissue (around the testis) and muscle tissue (gastrocnemius) were removed and weighed using a SHIMAZU LIBROR EB-330H equipped with a SHIMAZU EP-50 printer.
  • the humanized antibody was administered intravenously (iv) at a dose of 3.0 mg / kg (humanized antibody administration group).
  • physiological saline was administered at L v. (Saline administration group).
  • the solution was administered in a volume of 0.1 mL per 100 g body weight. No treatment was administered to the normal group without tumor transplantation.
  • a plastic strap (tie band: band for sterilization bag) was used to wrap around the skin at the base of the tumor (tumor extirpation group). A few hours later and the next day, they were re-barbed and tied up again. The next day, it was confirmed that the tumor was necrotic.
  • body weight, blood iCa concentration and white blood cell count were measured, and the results were divided into groups.
  • saline and humanized antibody were administered to the saline-administered group and humanized antibody-administered group, respectively, and the tumor was removed for the tumor removal group.
  • Body weight, blood iCa concentration and leukocyte count were measured on days 1, 4, 7, and 10 after administration. I got it.
  • adipose tissue and muscle tissue weight were measured, blood was collected, and serum was collected. Serum biochemical values and cytokines were measured.
  • the difference between the mean values on each measurement day was tested using an unpaired t-test.
  • the variance is subjected to the F test, and when the F value is 5% or more, the Student's test is regarded as equal variance. The level was set at 53 ⁇ 4 on both sides.
  • the difference between the mean values for each measurement day between the Saline administration group and the humanized antibody administration group or the tumor excision group was tested by Dimnett's multiple comparison, and the significance level was set to 5 on both sides. SAS Ver.6.12 was used for these statistical analyses.
  • Body weight (Fig. 3, upper panel): The body weight of the saline administration group (one stroke) was significantly lower than that of the normal group (1-1) on all measurement days (p compared to the normal group). 0.05, and tested by ⁇ ⁇ -test.)
  • the body weight significantly increased from day 4 after administration and was significantly different from day 7 to day 10 compared to the saline-administered group (#: saline-administered group).
  • Blood iCa concentration (Fig. 3, lower panel): In the saline administration group, the value was significantly higher on all measurement days than in the normal group. In the humanized antibody administration group, the increase in blood iCa concentration was significantly suppressed on days 1, 4, 7, and 10 after administration compared to the saline administration group (#). In the tumor extirpation group as well, the increase in blood iCa concentration was suppressed on days 1, 4, 7, and 10 after administration compared to the saline administration group (#).
  • Adipose tissue weight A significant decrease was observed in the saline-administered group compared to the normal group (*). The reduction was significantly suppressed in the humanized antibody-administered group and the tumor extirpated group compared to the saline-administered group (#). Muscle tissue weight: significant decrease in saline administration group compared to normal group
  • Table 5 (mean ⁇ SE) group human IL-6 human G-Ci> F human IL-11 human LIF rat ⁇ , -AG
  • the leukocyte count significantly increased on all measurement days compared to the normal group (tested with p-0.05 vs. / -test with respect to the normal group).
  • the leukocyte count of the humanized antibody group was significantly reduced on day 4 after administration compared to the saline group (#, significance level: 5%, tested by multiple comparison of Dimne tt) t, other measurements There was no difference between the days.
  • a significant decrease was observed on days 1, 4, 7, and 10 after administration (#).
  • the present inventors prepared a cancer cachexia model in which a human large cell lung cancer strain LC-6-JCK was transplanted into a rat, and examined the effect of the humanized antibody in comparison with the case where a tumor was excised.
  • the LC-6-JCK transplanted rat developed hypercalcemia with significant weight loss after tumor transplantation, and a model was established in which HHM was associated with cancer cachexia. At the same time, adipose tissue and muscle tissue weights were reduced, and glucose was low, indicating the characteristics of clinical cancer cachexia. In addition, production of tumor-derived site-in, production of its inducible protein, and increased white blood cell count were observed.
  • the humanized anti-PTHrP antibody since the humanized anti-PTHrP antibody has an effect of suppressing tissue degradation on adipose tissue and muscle tissue, it is useful as a therapeutic drug for tissue degradation associated with various diseases such as cancer cachexia.
  • various diseases such as cancer cachexia.
  • the production of tumor-derived cytokines was not suppressed, the site potential of IL-6, G-CSF, IL-11, LIF, -AG, etc. It has been found that, even in a state where the blood concentration of quinine is high, administration of the substance that inhibits the binding of PTHrP of the present invention to its receptor can suppress tissue degradation associated with various diseases.
  • inflammatory site-inducing factors such as IL-6 and IL-1K LIF are said to be related to tissue degradation (see, for example, ClinicaI Science, 89, 431-439, 1995). Nevertheless, even if such site force-in is present at a high concentration, administration of the substance that inhibits the binding of PTHrP of the present invention to its receptor can lead to tissue associated with various diseases. It has been found that decomposition can be suppressed.
  • Hybrid antibody-producing hybridomas # 23-57-154 and # 23-57-137-1 against human PTHrP were prepared as follows (Sato, K. et al., J. Bone Miner. Res. 8, 849-860, 1993).
  • the amino acid sequence of human PTHrP (group 34) is shown in SEQ ID NO: 75.
  • PTHrP (l-34) (manufactured by Peninsula) and thyroglobulin, a carrier protein, were conjugated using carbodiimide (Dojinn).
  • PTHrP (34) bound to thyroglobulin is dialyzed, adjusted to a protein concentration of 2 ⁇ g / inl, mixed 1: 1 with Freund's adjuvant (Di ico), and made into 16 emulsions.
  • Female BALB / C mice were immunized subcutaneously on the back or intraperitoneally 100 times per animal 11 times. For the first immunization, Freund's complete adjuvant was used, and for the second and subsequent boosters, Freund's incomplete adjuvant was used.
  • the antibody titer in the serum of the immunized mice was measured by the following method. That is, blood was collected from the mouse tail vein, and after separation of serum, antiserum diluted with RIA buffer and 1251-labeled PTHrP (1-34) were mixed, and the binding activity was measured. Mice with increased antibody titers were finally immunized with 50 g / animal of PTHrP (1-34) without carrier protein in the peritoneal cavity.
  • mice On the third day of the final immunization, the mice were sacrificed, the spleens were removed, and the spleen cells were fused with the mouse myeloma cell line P3x63Ag8U.1 according to a standard method using 50% polyethylene glycol 4000. Cell fusion with 2 x 10 4 wells with 85 96 wells Plated on the plate. Selection of hybridomas was performed using HAT medium.
  • Hybridoma screening was carried out by measuring the presence or absence of the PTHrP-recognizing antibody by the solid phase RIA method and selecting the culture supernatant in the wells where growth was observed in the HAT medium.
  • the hybridomas were collected from the wells in which the antibody binding ability was observed, suspended in RPMI-1640 medium containing 15% FCS, and supplemented with OPI-supplement (Sigma). Unification was implemented. Clones # 23-57-154 and # 23-57-137-1 having strong binding ability to PTHrP (1-34) were obtained.
  • Hybrid Mouse Clone # 23-57-137-1 is designated as mouse-mouse hybridoma # 23-57-137-1 by the Institute of Bioscience and Biotechnology, Institute of Industrial Science and Technology (1-3 1-3 Higashi, Tsukuba City, Ibaraki Prefecture) On August 15, 1996, it was deposited internationally under the Budapest Convention as FERMBP-5631.
  • MRNA from hybridoma # 23-57-137-1 was prepared using Quick Prep mRNA Purification Kit (Pharmacia Biotech). The cells of Hypri-Doma # 23-57-137-1 were completely homogenized with extraction buffer, and the mRNA was purified using oligo (dT) -Cellulose Spun Column according to the instructions provided with the kit, followed by ethanol precipitation. . The mRNA precipitate was dissolved in the elution buffer.
  • Ampli FINDER Anchor (SEQ ID NO: 42) was ligated to the 5 'end of the synthesized cDNA by reacting with T4 RNA ligase at 37 for 6 hours and at room temperature for 16 hours.
  • Anchor primer SEQ ID NO: 2
  • MHC-G1 primer SEQ ID NO: 3
  • lOmM Tris-HCl pH 8.3
  • 50 mM KCI 0.25 mM dNTPs (dATP, dGTP, dCTP, dTTP)> 1.5 mM MgCl 2
  • 2.5 units of TaKaRa Taq Takara Shuzo
  • the solution was overlaid with 1 mineral oil.
  • PCR was performed 30 times using a Thermal Cycler Model 480J (Perkin Elmer) with a temperature cycle of 94 at 45 seconds, 60 at 45 seconds, and 72 ° C for 2 minutes.
  • Cloning of the gene encoding the L chain V region of the mouse monoclonal antibody against human PTHrP was performed by the 5'-RACE method (Frohman, MA et al., Pro Natl. Acad. Sci. USA 85, 8998-9002, 1988; Belyavsky, A. et al., Nucleic Acids Res. 17, 2919-2932, 1989).
  • 5'-RACE method 5'-Ampli FinderRACE Kit (ClonetecW was used, and the operation was performed according to the attached instructions.
  • the primer used for cDNA synthesis was oligo-dT primer. Prepare as above.
  • a PCR primer MLC (SEQ ID NO: 4) was designed from the conserved sequence of the mouse L chain ⁇ chain constant region, and synthesized using a 394 DNA / RNA Synthesizer (ABI).
  • the PCR solution contains 10 mM Tris-HCl (pH 8.3), 50 mM KCK 0.25 mM dNTPs (dATP, dGTP, dCTP, dTTP), 1.5 Mm MgCl 2 , and 2.5 units of AmpliTaQ (PERK IN ELMER) , 50 pmole Anchor primer (SEQ ID NO: 2), and a reaction mixture 1 n1 of MLC (SEQ ID NO: 4) and cDNA ligated to Amli FINDER Anchor.
  • the solution was overlaid with 501 mineral oil.
  • PCR was performed 35 times using a Thermal Cycler Model 480J (Perkin Elmer) with a temperature cycle of 94 for 45 seconds, 6 (TC for 45 seconds, and 72 for 2 minutes).
  • the DNA fragment amplified by the PCR method as described above was separated by agarose gel electrophoresis using 3% Nu Sieve GTG agarose (FMC Bio. Products). A piece of agarose containing a DNA fragment of about 550 bp as the H chain V region and about 550 bp as the L chain V region was cut out, and the DNA fragment was purified using the GENECLEAN II Kit (BI0101) according to the instructions attached to the kit. . After the purified DNA was precipitated with ethanol, it was dissolved in 10 mM Tris-HCl (pH 7.4) and ImM EDTA solution 1.
  • the resulting DNA solution 1a1 was digested with the restriction enzyme Xmal (New England Biolabs) for 1 hour at 37, and then digested with the restriction enzyme EcoRI (Takara Shuzo) at 37 for 1 hour. The digestion mixture was extracted with phenol and black form, and the DNA was recovered by ethanol precipitation.
  • a DNA fragment containing a gene encoding a mouse H chain V region and an L chain V region having an EcoRI recognition sequence at the 5′-end and an Xmal recognition sequence at the 3′-end was obtained.
  • the EcoRI-Xmal DNA fragment containing the genes encoding the mouse H chain V region and L chain V region prepared as described above, and the PUC19 vector prepared by digestion with EcoRI and Xmal were ligated with DNA Using Syon Kit ver.2 (Takara Shuzo), the reaction was carried out at 16 ° C for 1 hour in accordance with the attached instructions, and they were connected. Next, 10 1 of the above ligation mixture was added to E.
  • coli is spread on an LB agar medium or a 2xYT agar medium (Molecular Cloning: A Labgoratory Manual, Sambrook, et al., Cold Spring Harbor Laboratory Press, 1989), and incubated at 37 overnight to transform the E. coli. I got
  • This transformant was cultured overnight in LB medium or 2XYT medium 2 ml containing 100 g / ml or 50 g / ml ampicillin at 37 at 37, and a plasmid extractor PI-100 ⁇ ( Plasmid DNA was prepared using Kurabo Industries or QIAprep Spin Plasmid Kit (QIAGEN), and the nucleotide sequence was determined.
  • the nucleotide sequence of the cDNA coding region in the above plasmid was determined using Dye Terminator Cycle Sequencing kit (Perkin-Elmer) by DNA Sequencer 373A (ABI Perkin-Elmer). Using M13 Primer M4 (Takara Shuzo) (SEQ ID NO: 5) and M13 Primer RV (Takara Shuzo) (SEQ ID NO: 6) as sequencing primers, the sequence was determined by confirming the nucleotide sequence in both directions.
  • the plasmid containing the gene encoding the mouse H chain V region derived from the hybridoma # 23-57-137-1 thus obtained was named MBC1H04, and the plasmid containing the gene encoding the L chain V region was named MBC1L24. did.
  • the nucleotide sequence (including the corresponding amino acid sequence) of the gene encoding the H chain V region and L chain V region of mouse # 23-57-137-1 antibody contained in plasmids MBC1H04 and MBC1L24 is shown in SEQ ID NO: 57, respectively. See Figure 65. These amino acid sequences are shown in SEQ ID NO: 46 for the fragment of the H chain V region and SEQ ID NO: 45 for the fragment of the L chain V region.
  • Escherichia coli having the plasmids MBC1H04 and MBC1L24 was referred to as Escherichia coli JM109 (MBC1H04) and Escherichia coli JM109 (MBC1L24), respectively.
  • Escherichia coli hired 09 (MBC1H04) was referred to as FERM BP-5628
  • Escherichia coli JM109 (MBC1L24) was referred to as FERM BP-5627. It has been internationally deposited on an approximately basis.
  • the overall structures of the ⁇ -chain V region and the L-chain V region are similar to each other, and each of the four framework parts is linked by three hypervariable regions, that is, complementarity determining regions (CDRs).
  • CDRs complementarity determining regions
  • the amino acid sequence of the framework is relatively well conserved, while the amino acid sequence of the CDR region is extremely variable (Kabat, EA et al., "SeQuence of Proteins of Immunological Interest tj US Dept. Heal th and Human Services, 1983).
  • the CDR region was identified in Table 6 by applying the amino acid sequence of the variable region of the mouse monoclonal antibody against human PTHrP to the database of the amino acid sequence of the antibody created by Kabat et al. And examining homology. The decision was made as shown.
  • amino acid sequences of CDRs 1-3 of the L chain V region are shown in SEQ ID NOs: 59-61, respectively, and the amino acid sequences of CDRs 1-3 of the H chain V region are shown in SEQ ID NOs: 62-64, respectively.
  • the cloned mouse H chain V region was modified by PCR to ligate it to an expression vector containing the human H chain C region Cr1 genomic DM.
  • the rear primer ⁇ -S 1 hybridizes to DNA encoding the 5′-side of the V region leader sequence and has a Kozak consensus sequence (Kozak, M. et al., J. MoI. Biol., 196, 947-950, 1987) and the restriction enzyme Hind III.
  • the primer MBCl-a (SEQ ID NO: 8) was designed so as to hybridize to the DNA sequence encoding the 3'-side of the J region and to have a splice donor sequence and a recognition sequence for the restriction enzyme BamHI.
  • TaKaRa Ex Taq (Takara Shuzo) was used.
  • a reaction mixture of 501 0.07 g of plasmid MBC1H04 as ⁇ -type DNA, pm-a and MBC SI as primers were 50 pmole and 2.5 U of TaKaRa Ex Taq, respectively.
  • the mineral oil was overlaid under the conditions containing 0.25 mM dNTP and subjected to 30 temperature cycles of 94 for 1 minute, 55 for 1 minute, and 72 for 2 minutes.
  • the MA fragment amplified by the PCR method was separated by agarose gel electrophoresis using 3% Nu Sieve GTG agarose (FMC Bio. Products).
  • a piece of agarose containing the DNA fragment of 437b length was cut out, and the DNA fragment was purified using GENECLEAN 11 Kit (BI0101) according to the instructions attached to the kit. After the purified DM was collected by ethanol precipitation, it was dissolved in 10 mM Tris-HCl (pH 7.4) and 20 ⁇ l of a 1 mM EDTA solution. The obtained DNA solution 1 ⁇ 1 was digested with restriction enzymes BamHI and HindIII (Takara Shuzo) at 37 for 1 hour. The digested mixture was extracted with phenol and black form, and the DNA was recovered by ethanol precipitation.
  • the HindIII-BamHI DNA fragment containing the gene encoding the mouse H chain V region prepared as described above was subcloned into the PUC19 vector prepared by digesting with HindIII and BamHI.
  • primers M13 Primer M4 and M13 Primer RV as primers, use Dye Terminator Cycle Seauencing kit (Perk in-Elmer), and base with DNA Seauencer 373A (Perkin-Elmer). The sequence was determined.
  • the plasmid having the BamHI recognition sequence was named MBC1H / PUC19.
  • H chain V region for production of cDNA-type mouse-human chimeric H chain Mouse H chain constructed as described above to be ligated to cDNA of human H chain C region C ⁇ 1
  • the V region was modified by PCR.
  • the rear primer MBC1HVS2 (SEQ ID NO: 9) for the H chain V region converts the second asparagine of the sequence encoding the beginning of the leader region of the V region to glycine, and the Kozak consensus sequence (Kozak, M., et al. et al., J. Mol. Biol., 196, 947-950, 1987) and were designed to have Hind III and EcoRI recognition sequences.
  • the forward primer MBC1HVR2 (SEQ ID NO: 10) for the V region of the H chain hybridizes to the DNA sequence encoding the 3'-side of the J region, and encodes the sequence 5'-side of the C region and Apa I And designed to have a Smal recognition sequence.
  • TaKaRa Ex Taa (Takara Shuzo) was used. Using a buffer attached, 501 mineral oil was superimposed under the conditions containing dNTPs, and the cycle was performed 30 times with a temperature cycle of 94 1 minute, 55 1 minute, and 72 1 minute. DNA fragments amplified by the PCR method were separated by agarose gel electrophoresis using 1% Sea Kem GTG agarose (FMC Bio. Products). A piece of agarose containing a 456 bp long DNA fragment was cut out, and the DNA fragment was purified using GENECLEAN II Kit (BI0101) according to the instructions attached to the kit. After the purified DNA was precipitated with ethanol, it was dissolved in 10 mM Tris-HCl (pH 7.4) and 1 mM EDTA solution1.
  • the obtained DNA solution 11 was digested with restriction enzymes EcoRI and Smal (Takara Shuzo) at 37 for 1 hour. The digestion mixture was extracted with phenol and black form, and the DNA was recovered by ethanol precipitation. The EcoRI-Smal DNA fragment containing the gene encoding the mouse H chain V region prepared as described above was subcloned into the PUC19 vector prepared by digesting with EcoRI and Smal. To confirm the nucleotide sequence of this plasmid, use Primer M13 Primer M4 and M13 Primer RV as primers, and use DNA Seauencer 373A (Perkin-Elmer) with Dye TerminatorCycle Sequencing kit (Perkin-Elmer). It was determined.
  • CDNA containing the human antibody H chain C region C ⁇ 1 was prepared as follows. That is, humanized PM1 antibody H chain V region and human antibody H chain C region IgGl genomic DNA (N. Takahashi, et al., Cell 29, 671-679 1982), an expression vector DHFR- ⁇ -RVh-PM-lf (see W092 / 19759), a humanized PM1 antibody L chain V region and a human antibody L MRNA was prepared from CHO cells transfected with an expression vector encoding a genomic DNA in the chain / chain C region (see PMla OV092 / 19759), and the humanized PM1 antibody H chain V was prepared by RT-PCR.
  • the cDNA containing the region and the human antibody C region Cr1 was cloned and subcloned into HindIII and BamHI sites of pUC19. After confirming the nucleotide sequence, the plasmid having the correct sequence was named pRVh-PMli-cDNA.
  • Hind III-BamHI blunted fragment was ligated to an expression vector prepared by digesting DHFR- ⁇ -RVh-PMl-f lacking the Hind III site and EcoRI site with Hind III and Smal, and An expression vector RVh-PMli-cDNA was constructed containing cDNAs encoding the type V PM1 antibody H chain V region and the human antibody C region Cr1.
  • Expression vector RVh-PMlf containing cDNA encoding humanized PM1 antibody H chain V region and human antibody C region Cr1 RVh-PMlf— After digesting cDNA with Apal and BamHI, DNA fragment containing H chain C region Harvested and introduced into MBClHv / pUC19 prepared by digestion with Apal and BamHI.
  • the plasmid thus prepared was named MBClHcDM ZPUC19. This plasmid contains cDNAs encoding the H chain V region of mouse antibody and the C region of human antibody C region, and has EcoRI and HindIII recognition sequences at the 5'-end and BamHI recognition sequences at the 3'-end.
  • Plasmid MBClHcDNA / pUC19 was digested with EcoRI and BamHI, and the obtained DNA fragment containing the nucleotide sequence encoding the H chain of the chimeric antibody was introduced into an expression vector pCOSl prepared by digesting with EcoRI and BamHI.
  • the expression plasmid for the chimeric antibody thus obtained was named MBClHcDNA / pCOSl.
  • the expression vector pCOSl was derived from HEF-PMh-gr1 (see W092 / 19759) by digestion with EcoRI and Smal. The gene was deleted and EcoRI-Not I-BamHI adapter (Takara Shuzo) was ligated to construct.
  • the plasmid MBClHcDNA / pUC19 is digested with EcoRI and BamHI, and the resulting DNA fragment containing the chimeric antibody H chain sequence is digested with EcoRI and BamHI.
  • the resulting plasmid was introduced into the thus prepared expression plasmid pCHOl.
  • the expression plasmid of the chimeric antibody thus obtained was named MBClHcDNA / pCHOl.
  • the expression vector pCHOl was prepared by deleting the antibody gene from DHFR-AE-rvH-PMl-f (see W092 / 19759) by digestion with EcoRI and Smal and ligating EcoRI-Not I-BamHI Adapter (Takara Shuzo). It was constructed.
  • a Hind III site deleted PUC19 vector was constructed.
  • the digested mixture was extracted with phenol and chloroform, and the DNA was recovered by ethanol precipitation.
  • the recovered DNA was placed in a reaction mixture containing 50 mM Tris-HCl ( ⁇ (7.5), 10 mM MgCl 2 , 1 mM DTT, 100 mM NaCl, 0.5 mM dNTP, and 6 U Klenow fragment (GIBCO BRL). The reaction was performed at room temperature for 20 minutes to blunt the ends. The reaction mixture was extracted with phenol and chloroform, and vector DNA was recovered by ethanol precipitation.
  • Transformants were cultured in 20 ml of 2XYT medium containing 50; ug / nil ampicillin at 37T: overnight, and plasmid DNA was purified from the cell fraction using Plasniid Mini Kit (QIAGEN) according to the attached protocol did.
  • the purified plasmid was digested with Hind III, and the plasmid confirmed to have deleted the Hind III site was named PUC19 AHind III.
  • At least four isotypes of human antibody L chain and ⁇ chain C region are known: Mcg + Ke + Oz-, Meg- Ke- Oz-, Meg- Ke-0z +, and Meg- Ke + Oz- (P. Dariavach, et. al., Pro Natl. Acad. Sci. USA, 84, 9074-9078, 1987).
  • # 23— 57-137-1 The EMBL database was used to search for the human antibody L chain ⁇ chain C region having homology to the mouse L chain ⁇ chain C region, and the isotype was Mcg + Ke + Oz- (accession No. X57819) (P. Dariavach, et al., Pro Natl. Acad. Sci.
  • HLAMB1 SEQ ID NO: 11
  • HLAMB3 SEQ ID NO: 13
  • HLAMB2 SEQ ID NO: 12
  • HLAMB4 SEQ ID NO: 14
  • the external primers HLAMBS (SEQ ID NO: 15) and HLAMBR (SEQ ID NO: 16) have sequences homologous to HLAMB1 and HLAMB4, respectively, and HLAMBS has EcoRI, HindIII, and Blnl recognition sequences, and HLAMBR has EcoRI recognition sequences. Contains. HLAMB1-HLAMB2 and HLAMB3-HLAMB4 were reacted in the first PCR. After the reaction, they were mixed in equal amounts and assembled by the second PCR. Further, external primers HLAMBS and HLAMBR were added, and the full-length DNA was amplified by the third PCR.
  • PCR was performed using TaKaRa Ex Taa (Takara Shuzo) according to the attached prescription.
  • a reaction mixture of 100 1 containing pmole of H MB4 and 5 U of TaKaRa Ex Taa overlay the mineral oil of 1 on 94 for 1 min at 60 min for 1 min at 60 min Performed 5 times with a 1 minute temperature cycle.
  • reaction solutions were mixed one by one, and 50 ⁇ 1 of mineral oil was layered on the upper layer and subjected to three temperature cycles at 94 for 1 minute, 60 at 1 minute, and 72 at 1 minute.
  • the third PCR is performed by adding 50 pmole each of the external primers HLAMBS and HLAMBR to the reaction mixture, and performing the cycle 30 times at 94 ° C for 1 minute, at 60 ° C for 1 minute, and at 72 ° C for 1 minute for 30 minutes.
  • the cycle 30 times at 94 ° C for 1 minute, at 60 ° C for 1 minute, and at 72 ° C for 1 minute for 30 minutes.
  • the DM fragment of the third PCR product was electrophoresed on a 3% low-melting point agarose gel (NuSieve GTG Agarose, FMC), and then recovered and purified from the gel using the GENECLEANII Kit (BI0101) according to the attached instructions.
  • the obtained DNA fragment was placed in a reaction mixture of 20 1 containing 50 mM Tris-HC1 (pH 7.5), 10 mM MgCl 2 , 1 ⁇ DTT, 100 mM NaCl, and 8 U EcoRI (Takara Shuzo) at 37 for 1 hour at 37 ° C. Digested. The digested mixture was extracted with phenol and black-mouthed form, DNA was recovered by ethanol precipitation, and dissolved in lOmM Tris-HCl (PH7.4), 1 mM EDTA solution 81.
  • Plasmid PUC19 AHind III 0.8 xg was similarly digested with EcoRI, extracted with phenol and black form, and recovered by ethanol precipitation. Reaction of digested plasmid PUC19 AHind III in a reaction mixture containing 50 mM Tris-HC1 (pH 9.0), 1 mM MgCl 2 alkaline phosphatase (E. coli C75, Takara Shuzo) for 37 minutes for 30 minutes Then, dephosphorization treatment (BAP treatment) was performed. The reaction solution was extracted with phenol and black form, and the DNA was recovered by ethanol precipitation, and then dissolved in 10 mM Tris-HCl (pH 7.4) and 1 mM EDTA solution 101.
  • Tris-HC1 pH 9.0
  • MgCl 2 alkaline phosphatase E. coli C75, Takara Shuzo
  • BAP treatment dephosphorization treatment
  • the BAP-treated plasmid pUC19AHindIII1n1 and the PCR product 4n1 were ligated using DNA Ligation Kit Ver.2 (Takara Shuzo) and transformed into Escherichia coli JM109 competent cells.
  • the obtained transformant was cultured overnight in 2 ⁇ 1 to medium 2] 11 containing 50 ⁇ g / ml ampicillin] 11, and the plasmid was purified from the cell fraction using a QIAprep Spin Plasmid Kit (QIAGEN).
  • plasmid C ⁇ AZpUCig containing the deleted DNA was converted into type I, and reacted with primers HLAMBS and HCLMR, and HCLMS and HLAMB4.
  • Each PCR product was purified and assembled in a second PCR. Further, external primers HLAMBS and HLAMB4 were added, and the full-length DNA was amplified by the third PCR.
  • a 100 a1 reaction mixture containing 0.1 g of C ⁇ AZpUC19, 50 pmole of each of the primers HLAMBS and HCLMR, 50 pmole of each of HCLMS and HLAMB4, and 5 U of TaKaRa Ex Taa (Takara Shuzo) was used for the first PCR.
  • the mineral oil of No. 1 was subjected to 30 temperature cycles of 94 ° C for 1 minute, 60 ° C for 1 minute and 72t: 1 minute for 1 minute.
  • PCR products HLAMBS-HCLMR (236bp) and HCLMS-HLAMB4 (147bp) were each electrophoresed on a 3% low melting point agarose gel, and then recovered and purified from the gel using the GENECLEANII Kit (BI0101).
  • a reaction mixture of 201 containing 40 ng of each purified DNA fragment and 1 U of TaKaRa Ex TaQ (Takara Shuzo) was used. The temperature was cycled 5 times for 1 minute at 72 and for 1 minute at 72.
  • the second? ⁇ Reaction liquid 2 Using a 100 1 reaction mixture containing 50 pmole of each of the external primers HLAMBS and HLAMB4 and 5 U of TaKaRa Ex TaQ (Takara Shuzo), 500 1 of mineral oil was overlaid. PCR was performed 30 times at a temperature cycle of 94 ° C for 1 minute, 60 ° C for 1 minute, and 72 ° C for 1 minute. The third PCR product, a 357 bp DNA fragment, was electrophoresed on a 3% low-melting point agarose gel, and then recovered and purified from the gel using the GENECLEANII Kit (BI0101).
  • the obtained DNA fragment (0.0 g) was digested with EcoRI and subcloned into BAP-treated plasmid pUC19AHindIII. Transformed into E. coli JM109 competent cells, cultured overnight in 2 ml of 2 XYT medium containing 50 ⁇ g / ml ampicillin, Plasmid was purified from the cell fraction using QIAprep Spin Plasmid Kit (QIAGEN). The nucleotide sequence of the purified plasmid was determined using M13 Primer M4 and M13 Primer RV (Takara Shuzo) using a 373A DNAsequencer (ABI). The plasmid confirmed to have the correct base sequence without deletion was designated as CAZpUC19.
  • a DNA fragment encoding the L chain / chain C region was cloned from the plasmid HEF-PMlk-gk (W092 / 19759) by PCR. 394 Designed so that the forward primer HKAPS (SEQ ID NO: 19) synthesized using the DNA / RNA synthesizer (ABI) has EcoRI, Hind III and Blnl recognition sequences, and the rear primer HKAPA (SEQ ID NO: 20) has an EcoRI recognition sequence. did.
  • Plasmid HEF-PMlk-gk (0.1 g), which forms a ⁇ -form, primers HKAPS, HKAPA, 50 pmole each, and 100 U of a reaction mixture containing 5 U of TaKaRaEx Taa (Takara Shuzo) were used, and 501 mineral oil was overlaid.
  • the reaction was performed at 94 for 1 minute, 60 at 1 minute, and 72 at 1 minute for 30 cycles.
  • the 360 bp PCR product was electrophoresed on a 3% low-melting-point agarose gel, and recovered and purified from the gel using the GENECLEANII Kit (BI0101).
  • the obtained DNA fragment was digested with EcoRI and cloned into BAP-treated plasmid pUC19 ⁇ Hindd.
  • E. coli JM109 competent cells were transformed, cultured overnight in 2nd XYT medium containing 50 g / ml ampicillin, and the plasmid was purified from the cell fraction using a QIAprep Spin Plasmid Kit (QIAGEN).
  • the nucleotide sequence of the purified plasmid was determined using 373A DNA sequencer (ABI) using M13 Primer M4 and M13 Primer RV (Takara Shuzo). Plasmid confirmed to have the correct nucleotide sequence was designated as CK-pUC19.
  • a chimeric # 23-57-137-1 antibody light chain expression vector was constructed. By linking the gene encoding the # 23-57-137-1 L chain V region to the Hind III and Blnl sites just before the human antibody constant region of plasmids CAZ PUC19 and CK / PUC19, respectively. A PUC19 vector encoding the chimeric # 23-57-137-1 antibody L chain V region and L chain ⁇ chain or L chain ⁇ chain constant region was prepared. Chimeric antibody L chain residue by EcoRI digestion The gene was cut out and subcloned into a HEF expression vector.
  • the V region of the # 23-57-137-1 antibody L chain from plasmid MBC1L24 was cloned using the PCR method.
  • the synthesis of each primer was performed using 394 DNA / RNA synthesizer (ABI).
  • the backward primer MBCCHLl (SEQ ID NO: 21) contains the Hind III recognition sequence and Kozak sequence (Kozak, M. et al., J. Mol. Biol. 196, 947-950, 1987), and the forward primer MBCCHL3 (sequence No. 22) was designed to have a Bglll, EcoRI recognition sequence.
  • PCR is, 10mM Tris-HCl (pH8.3) , 50mM KCK 1.5mM MgCl 2, 0.2mM d NT P, of 0.1 xg MBC1L24, containing AmpliTa PERKIN ELMER) of the 50 pmoles, 1 1 a MBCCHLl and MBCCHL3 as primers one Using 100 1 of the reaction mixture, 50; 1 mineral oil was applied to the upper layer 30 times at a temperature cycle of 94 at 45 seconds, 60 at 45 seconds, and 72 at 2 minutes for 30 times.
  • Plasmid PUC19 l_ig was similarly digested with Hind III and EcoRI, extracted with phenol and chloroform, recovered by ethanol precipitation, and BAP-treated with alkaline phosphatase (E. coli C75, Takara Shuzo). The reaction solution was extracted with phenol and chloroform, the DNA was recovered by ethanol precipitation, and then dissolved in 10 mM Tris-HCl (pH 7.4) and 101 of 1 mM EDTA solution.
  • the BAP-treated plasmid pUC19 1a1 and the above PCR product 41 were ligated using DNA Ligation Kit Ver.2 (Takara Shuzo) and transformed into E. coli JM109 competent cells (Futatsu gene) in the same manner as described above. .
  • This was spread on 2 XYT agar medium containing 50 / g / ml ampicillin and cultured overnight at 37 ° C.
  • the obtained transformant was cultured overnight at 37 ° C in 2 ml of 2XYT medium containing 50 ⁇ g / ml ampicillin. From the cell fraction
  • the plasmid was purified using QIAprep Spin Plasmid Kit (QIAGEN). After determining the nucleotide sequence, the plasmid having the correct nucleotide sequence was designated as CHL / PUC19.
  • Plasmids C ⁇ / pUC19 and C ⁇ / pUC19 were each added in 1 g each to 20 mM Tris-HC1 (pH 8.5) lOmM MgCl 2 , 1 m DTT lOOmM KCK 8 U Hind III (Takara Shuzo) and 2 U Blnl (Takara Shuzo) ) was quenched in a reaction mixture 20 1 containing at 37 ° C for 1 hour.
  • the digestion mixture was extracted with phenol and black form, and the DNA was recovered by ethanol precipitation, followed by BAP treatment at 37 for 30 minutes.
  • the reaction solution was extracted with phenol and black form, DNA was recovered by ethanol precipitation, and dissolved in lOmM Tris-HCl (pH 7.4), 1 mM EDTA solution 101.
  • This L chain V region DNA41 was subcloned into BAP-treated plasmids CAZpUC19 or C // pUC19, respectively, and transformed into E. coli JM109 competent cells. 50 8/1111 Anpishirin incubated 2 ⁇ 1> Medium 31111 De night containing, the plasmid was purified from the cell fraction using QIAprep Spin Plasmid Kit (QIAGEN). These were designated as plasmids MBClL (A) / pUC19 and BC1L ( ⁇ ) / pUC19, respectively.
  • Plasmids MBClL (A) / pUC19 and MBC1L ( ⁇ ) / pUC19 are each digested with EcoRI, electrophoresed on a 3% low melting point agarose gel, and then a 743 bp DNA fragment is recovered from the gel using the GENECLEANII Kit (BI0101). It was purified and dissolved in 10 mM Tris-HCl (pH 7.4) and ImM EDTA solution 101.
  • plasmid HEF-PMlk-gk was digested with EcoRI as an expression vector, extracted with phenol and black form, and the DNA was recovered by ethanol precipitation. After BAP treatment of the recovered DNA fragment, it was electrophoresed on a 1% low-melting-point agarose gel. The 6561 bp DM fragment was recovered from the gel using the GENECLEANII Kit (BI0101), purified, and then purified with 10 mM Tris-HCl (pH 7.4). It was dissolved in ImM EDTA solution 101.
  • the BAP-treated HEF vector 21 was ligated to each of the above plasmids MBC1L ( ⁇ ) or MBCIL (K) EcoRI fragment 31 and transformed into E. coli JM109 competent cells.
  • the cells were cultured in 2 ml of 2XYT medium containing 50 g / ml ampicillin, and plasmid was purified from the cell fraction using a QIAprep Spin Plasniid Kit (QIAGEN).
  • the purified plasmid was added to a reaction mixture containing 20 mM Tris-HCl (pH 8.5), 10 mM MgCl 2 , 1 mM DTT, 10 mM KC1, 8 U Hindlll (Takara Shuzo) and 2 U Pvul (Ma Shuzo). Digested for 1 hour at 37 ° C in 1.
  • the fragment is inserted in the correct direction, a digested fragment of 5104/2195 bp will be generated, and if the fragment is inserted in the reverse direction, a digested fragment of 4378/2926 bp will be generated.Therefore, the plasmid inserted in the correct direction will be converted to MBClL (A) / neo and MBClL (/ c) / neo.
  • the expression plasmid was transiently expressed in C0S-7 cells.
  • the transient expression of chimeric antibodies is performed using a Gene Pulser device (Bio Rad) with a combination of plasmids MBClHcDNA / pCOSl and MBCIL (A) / neo or MBC1HcDNA / pCOS1 and MBC1L ( ⁇ ) / neo.
  • a Gene Pulser device Bio Rad
  • MBClHcDNA / pCOSl and MBCIL A
  • MBC1HcDNA / pCOS1 and MBC1L ⁇
  • the cells subjected to electoral poration were resuspended in DMEM medium (GIBC0) containing 2% Ultra Low IgG fetal serum (GIBC0), and used in a 10 cm culture dish. They were cultured in a C0 2 incubator scratch. After culturing for 72 hours, the culture supernatant was collected, cell debris was removed by centrifugation, and used for ELISA samples.
  • DMEM medium GIBC0
  • GIBC0 Ultra Low IgG fetal serum
  • An ELISA plate for antibody concentration measurement was prepared as follows.
  • a 96-well ELISA plate (Maxisorp, NUNC) each hole a solid-phased buffer (0.1M NaHC0 3, 0.02% Immobilized with goat anti-human IgG antibody (TAGO) 100/1 adjusted to a concentration of lg / ml with NaN 3 ), diluted with 200 1 of dilution buffer (50 mM Tris-HC1, lmM MgCl 2 , 0.1 M NaCK 0.05% Tween20 , 0.02% NaN 3, 1% bovine serum albumin (BSA), pH 7.2) after blocking, the culture supernatant or the purified chimeric antibody COS cells expressing the chimeric antibodies were diluted stage was added to each well Was.
  • BSA bovine serum albumin
  • an anti-human IgG antibody (TAG0) 100 conjugated with Ariri phosphatase was added.
  • TAG0 anti-human IgG antibody
  • Hu IgG Purified (The Binding Site) was used as a standard for concentration measurement.
  • An E1ISA plate for measuring antigen binding was prepared as follows. Each well of a 96-well ELISA plate was immobilized with 100 I of human PTHrP (tri-34) (Peptide Laboratories) adjusted to a concentration of 1 g / ml with a solid phase immobilization buffer. After blocking with a 200 ⁇ 1 dilution buffer, the culture supernatant of the COS cells expressing the chimeric antibody or the purified chimeric antibody was serially diluted and added to each well. After incubating at room temperature and washing with PBS-Tween 20, 100 l of alkaline phosphatase-conjugated goat anti-human IgG antibody (TAG0) was added.
  • TAG0 alkaline phosphatase-conjugated goat anti-human IgG antibody
  • the chimeric antibody had the ability to bind to human PTHrP (1-34) and had the correct structure of the cloned mouse antibody V region.
  • the ability of the chimeric antibody to bind to PTHrP (1-34) does not change whether the L chain C region is a ⁇ chain or a ⁇ chain
  • the humanized antibody L chain C region It was constructed using the typed antibody L chain and ⁇ chain.
  • the expression plasmid was introduced into CH0 cells (DXB11) to establish a cell line stably producing chimeric antibodies.
  • a stable cell line for producing chimeric antibodies was established using a Gene Pulser device (Bio Rad) with the combination of expression plasmids MBClHcDNA / pCHOl and MBC1L ( ⁇ ) / neo or MBClHcDNA / pCHOl and MBCIL ( ⁇ ) Zneo for CH0 cells. Then, CH0 cells were co-transduced by electoral poration.
  • Each expression vector was cleaved with the restriction enzyme Pvul to obtain linear DNA, and after extraction with phenol and chloroform, the DNA was recovered by ethanol precipitation and used for elect-mouth poration.
  • a pulse was applied with a capacitance of 1,500V.
  • the electroporated cells were suspended in ⁇ - ⁇ medium (GIBC0) supplemented with 10% ⁇ fetal serum (GIBC0) and three 96-well plates ( Falcon) and cultured in a C0 2 ink Yube Isseki one using.
  • GIBC0 ⁇ - ⁇ medium
  • GIBC0 ⁇ - ⁇ medium
  • GENETICIN G418Suliate, GIBC0
  • cells into which the antibody gene was introduced were selected.
  • cells were observed under a microscope about two weeks, and after successful cell growth was observed, the antibody production was measured by the above antibody concentration measurement ELISA, and cells with high antibody production were selected.
  • the CDR-grafting primers MBC1HGP1 (SEQ ID NO: 23) and MBC1HGP3 (SEQ ID NO: 24) have a sense DNA sequence
  • the CDR-grafting primers MBC1HGP2 (SEQ ID NO: 25) and MBC1HGP4 (SEQ ID NO: 26) It has a sense DNA sequence and has a complementary sequence of 15 to 21 bp at each end of the primer.
  • External primers MBC1HVS1 (SEQ ID NO: 27) and MBC1HV1 (SEQ ID NO: 28) have homology with CDR grafting primers MBC1HGP1 and MBC1HGP4.
  • CDR-grafting primers MBC1HGP1, MBC1HGP2, MBC1HGP3 and MBC1HGP4 were separated using a urea-denatured polyacrylamide gel (Molecular Cloning: A Laboratory Manual, Sambrook II, Cold Spring Harbor Laboratory Press, 1989) and extracted from the gel. The extraction was performed by the crush and soak method (Molecular Cloning: A Laboratory Manual, Sambrook II, Cold Spring Harbor Laboratory Press, 1989).
  • each CDR-grafting primer was separated on a 6% denaturing polyacrylamide gel, and DNA fragments of the desired size were identified by irradiating ultraviolet rays on a silica gel thin plate. It was recovered from the gel by the method and dissolved in 201 1 of 10 mM Tris-HCl (pH 7.4) and ImM EDTA solution. PCR was performed by using TaKaRa Ex Taq (Takara Shuzo) and adding the CDR-grafting primers MBC1HGP1, MBC1HGP2, MBC1HGP3 and MBC1HGP4 prepared as described above to 11 and 0.25 mM dNTPs in 100 1 reaction mixture, respectively.
  • a piece of agarose containing a 421 bp long DNA fragment was cut out, and the DNA fragment was purified using GENECLEANII Kit (BI0101) according to the instructions attached to the kit.
  • Purified DM Was precipitated with ethanol, and then dissolved in 10 mM Tris-HCl (pH 7.4) and 201 EDTA solution 201.
  • the obtained PCR reaction mixture was subcloned into PUC19 prepared by digesting with BamHI and Hindlll, and the nucleotide sequence was determined.
  • the plasmid having the correct sequence was named hMBCHv / pUC19.
  • the humanized H chain V region constructed as described above was modified by PCR.
  • the rear primer MBC1HVS2 hybridized with the sequence encoding the 5'-side of the V region leader sequence, and had a Kozak consensus sequence (Kozak, M, et al., J. Mol. Biol. 196, 947-950, 1987), designed to have Hindlll and EcoRI recognition sequences.
  • the forward primer MBC1HVR2 for the V region of the H chain hybridizes to the DNA sequence encoding the 3 'side of the J region, and encodes the sequence 5'-side of the C region and has Apa I and Sma I recognition sequences. Designed to be.
  • PCR was performed using TaKaRa Ex Tad (Takara Shuzo), 0.4 g of hMBCHv / pl) C19 as ⁇ type DNA, MBC1HVS2 and MBC1HVR2 as primers, each containing 50 pmole, 2.5 U TaKaRa Ex TaQ, and 0.25 mM dNTP. Using the attached buffer solution, the temperature was cycled 30 times at 94 for 1 minute, at 55 for 1 minute, and at 72T: for 1 minute. DNA fragments amplified by the PCR method were separated by agarose gel electrophoresis using 3% Nu Sieve GTG agarose (FMC Bio. Products).
  • a piece of agarose containing a DNA fragment of 456 bp was cut out, and the DNA fragment was purified using GENECLEANI I Ki BIOlOl) according to the instructions attached to the kit. After the purified DNA was precipitated with ethanol, it was dissolved in 10 mM Tris-HCl (pH 7.4) and 201 of ImM EDTA solution. The obtained PCR reaction mixture was subcloned into PUC19 prepared by digesting with EcoRI and Smal, and the nucleotide sequence was determined.
  • the plasmid having the Apal and Smal recognition sequences was designated as hMBClHv / pUC19.
  • Plasmid RVh-PMli-cDNA containing the sequence of hPMl antibody H chain cDNA was converted to Apal and BamHI
  • the DNA fragment containing the H chain C region was recovered and introduced into hMBClHv / pUC19 prepared by digestion with Apal and BamHI.
  • the plasmid thus prepared was named hMBClHcDM / pUC19.
  • This plasmid contains the H chain V region of humanized # 23-57-137-1 antibody and the human H chain C region Cr1, with EcoRI and Hindlll recognition sequences at the 5'-end, and BamHI recognition at the 3'-end. Has an array.
  • SEQ ID NO: 58 shows the nucleotide sequence and the corresponding amino acid sequence of humanized H chain version "a" contained in plasmid hMB HcDNA / pUC19.
  • the amino acid sequence of version a is shown in SEQ ID NO: 56.
  • hMBClHcDNA / pUC19 was digested with EcoRI and BamHI, and the obtained DNA fragment containing the H chain sequence was introduced into expression plasmid pCOS1 prepared by digesting with EcoRI and BamHI.
  • the expression plasmid for the humanized antibody thus obtained was named hMBClHcDNA / pCOSl.
  • hMBC IHC DNA / PUC 19 is digested with EcoRI and BamHI to prepare a plasmid for expression in CH0 cells, and the obtained DNA fragment containing the H chain sequence is digested with EcoRI and BamHI.
  • the resulting plasmid was introduced into the thus prepared expression plasmid pCHO1.
  • the expression plasmid for the humanized antibody thus obtained was named hMBClHcDNA / pCHOl.
  • An L chain gene was constructed in which the FR regions of the humanized antibody and the mouse (chimeric) antibody were recombined, and each region for humanization was evaluated.
  • a hybrid antibody was prepared in which FR1 and 2 were derived from a human antibody, and FR3 and 4 were derived from a mouse antibody.
  • Plasmids MBClL (A) / neo and hMBCIL (in) / neo are each 10 mM Tris-HCl (pH 7.5), 10 mM MgCl 2 l ImM DTT, 50 mM NaCl, 0.01% (w / v) BSA, Aflll (Takara Shuzo) 10 U Digestion was carried out at 37 for 1 hour in a reaction mixture 1001 containing The reaction mixture was electrophoresed on a 2% low-melting point agarose gel, and a 6282 bp fragment (referred to as cl) and a 1022 bp fragment (referred to as c2) from plasmid MBClL (A) / neo, and plasmid hMBUL (A) / neo A 6282 bp fragment (hi) and a 1022 bp fragment (h2) were obtained using GENECLEANII Kit (BI0101). Recovered
  • BAP treatment was performed on 1 g of each of the collected c1 and h1 fragments.
  • the DNA was extracted with phenol and chloroform, recovered by ethanol precipitation, and dissolved in 10 mM Tris-HCl (pH 7.4) and 101 of an lmM EDTA solution.
  • the BAP-treated c1 and h1 fragment 11 were ligated to h2 and c2 fragment 4 ⁇ 1, respectively (4 ° C., overnight) and transformed into E. coli JM109 competent cells.
  • the cells were cultured in 2 ml of 2XYT medium containing 50 / g / ml 7 ampicillin, and the plasmid was purified from the cell fraction using a QIAprep Spin Plasmid Kit (QIAGEN).
  • the purified plasmid was used in a reaction mixture 201 containing 10 mM Tris-HCl (pH 7.5), 10 mM MgCl 2 , lmM DTT, ApaLI (Takara Shuzo) 2U, or BamHI (Takara Shuzo) 8U, Hindlll (Takara Shuzo) 8U.
  • a reaction mixture 201 containing 10 mM Tris-HCl (pH 7.5), 10 mM MgCl 2 , lmM DTT, ApaLI (Takara Shuzo) 2U, or BamHI (Takara Shuzo) 8U, Hindlll (Takara Shuzo) 8U.
  • the expression vector encoding the L chain of the human FR1, 2Z mouse FR3, 4 hybrid antibody was designated as h / mMBClLU) / neo.
  • h / mMBClLU The expression vector encoding the L chain of the human FR1, 2Z mouse FR3, 4 hybrid antibody was designated as h / mMBClLU) / neo.
  • mouse c2 since a clone of the mouse c2 was not obtained, it was cloned into the HEF vector after recombination on the pUC vector.
  • Plasmid MBClL (A) / pUC19, liMBClLaA / P UC19 and hMBClLc / pUC19 each 3 ⁇ 4 10 mM Tris-HCl of (pH7.5), lOmM MgCl 2, lmM DTT, 50mM NaCl, 0.013 ⁇ 4 (w / v) BSA, Hindlll 16U , Aflll 4U was digested for 1 hour at 37 1 in a reaction mixture 30 1.
  • the reaction mixture was electrophoresed on a 2% low-melting point agarose gel, and the DNA of plasmids MBClLU) / pUC19 to 215 bp (c2 '), and the plasmids hMBClLaA / pUC19 and hMBClL (/ pUC19 of 3218 bp (hal', hdl '), respectively) Fragments were collected and purified from the gel using the GENECLEANII Kit (BIOIOl).
  • the hal 'and hdl' fragments were each ligated to the c2 'fragment and transformed into E. coli JM109 competent cells.
  • the obtained plasmid Hm / hMBClLaA / pUC19 and m / hMBCILd ⁇ / pUC19 were digested with EcoRI. Each 743 bp DNA fragment was electrophoresed on a 2% low-melting-point agarose gel, recovered from the gel using GENECLEANII KiBIOlO1), purified, and dissolved in lOmM Tris-HC1 (pH 7.4) and ImM EDTA solution 201. .
  • each DNA fragment was ligated to the above-mentioned BAP-treated HEF vector 1 11 and transformed into E. coli JM109 competent cells.
  • the cells were cultured in 2 ml of 2XYT medium containing 50 g / ml ampicillin, and the plasmid was purified from the cell fraction using QIAprep Spin Plasmid Kit (QIAGEN).
  • Each purified plasmid was mixed with 20 mM Tris-HCl (pH 8.5), lOmM MgCl 2 , ImM DTT, lOOmM KC1, Hindlll (Takara Shuzo) 8U, Pvul (Takara Shuzo) 2U in a reaction mixture 201 at 37. For 1 hour. Plasmid was confirmed by the fact that a digested fragment of 5104/2195 bp was generated if the fragment was inserted in the correct direction, and a digested fragment of 4378/2926 bp if inserted in the reverse direction.
  • the expression vectors encoding the mouse FR1, 2-human FR3, 4-hybrid antibody L chain were m / hMBC1La ⁇ / neo and m / hMBCILd ⁇ / neo, respectively.
  • Plasmids MBC1L (A) / neo and h / mMBClU / ne were each 10 g of lOmM Tris-HC1 (pH7.9), lOmM MgCl 2 , ImM DTT, 50 mM NaCl, 0.013 ⁇ 4 (w / v) BSA , SnaBI (Takara Shuzo) was digested for 1 hour at 37 in 37 in a reaction mixture containing 6 U.
  • the cells were cultured in 2 ml of 2XYT medium containing 50 / ig / ml ampicillin, and the plasmid was purified from the cell fraction using a QIAprep Spin Plasmid Kit (QIAGEN).
  • the purified plasmids are mixed at 37 in a reaction mixture containing 10 mM Tris-HCl (pH 7.5), 10 mM MgCl 2 , ImM DTT, 8 U of Apal (Takara Shuzo) or 2 U of ApaLI (Takara Shuzo). Digested for 1 hour.
  • the humanized # 23-57-137-1 antibody L chain was produced by CDR-grafting by PCR.
  • FR1, FR2 and FR3 from human antibody HSU03868 (GEN-BANK, Def tos M et al., Scand. J. Immunol., 39, 95-103, 1994), and FR4 from human antibody S25755 (NBRF-PDB).
  • Six PCR primers were used to generate a humanized # 23-57-137-1 antibody light chain (version "a").
  • CDR-grafting primers MBC1LGP1 (SEQ ID NO: 29) and MBC1LGP3 (SEQ ID NO: 30) have a sense DNA sequence
  • CDR-grafting primers MBC1LGP2 (SEQ ID NO: 31) and MBC1LGP4 (SEQ ID NO: 32) have an antisense DNA sequence.
  • each has a complementary sequence of 15 to 21 bp at both ends of the primer.
  • the external primers MBC1LVS1 (SEQ ID NO: 33) and MBC1LVR1 (SEQ ID NO: 34) have homology with the CDR grafting primers MBC1LGP1 and MBC1LGP4.
  • CDR-grafting primers MBC1LGP1, MBC1LGP2, MBC1LGP3 and MBC1LGP4 were separated using urea-denatured polyacrylamide gel (Molecular Cloning: A Laboratory Manual, Sambrook3 ⁇ 4, Cold Spring Harbor Laboratory Press, 1989). The extraction of the gel force and the like was performed by the crush and soak method (Molecular Cloning: A Laboratory Manual, Sambrook et al., Cold Spring Harbor Laboratory Press, 1989). That is, each lnmole of the CDR-grafting primer was separated on a 6% denaturing polyacrylamide gel, and the DNA fragment of the desired size was identified by irradiating it with ultraviolet light on a silica gel thin plate. The gel was recovered from the gel and dissolved in 20 1 of 10 mM Tris-HCl (pH 7.4) and lmM EDTA solution.
  • PCR was performed using TaKaRa Ex Tad (Takara Shuzo) and the CDR-grafting primers MBC 1LGP1, BC 1LGP2, MBC 1LGP3, and MBC1LGP4 prepared in 100 1 reaction mixture as described above at 11 and 0.25 mM, respectively. Perform the test 5 times at 94 ° C for 1 minute, at 55 for 1 minute, and at 72 for 1 minute using the attached buffer under conditions including dNTP and 2.5 U TaKaRa Ex TaQ. 50 pmole of the external primers MBC 1LVS1 and MBC 1LVR1 were added to the reaction mixture, and the mixture was further reacted 30 times at the same temperature cycle. DNA fragments amplified by the PCR method were separated by agarose gel electrophoresis using 3% Nu Sieve GTG agarose (FMC Bio. Products).
  • TaKaRa TaQ (Takara Shuzo)
  • MBC1LVS1 and MBC1LGP10R as primers, 50 pmole each
  • 2.5 U TaKaRa Ex TaQ (Takara Shuzo) 0.21 Mineral oil in the upper layer using the attached buffer under conditions containing 0.25mM dNTPs. 1 min at 94 C, 1 min at 55 ° C, 72 min at 72 ° C Performed 30 times with 1 minute temperature cycle. DNA fragments amplified by the PCR method were separated by agarose gel electrophoresis using 3% Nu Sieve GTG agarose (FMC Bio. Products).
  • a piece of agarose containing DNA fragment of 421b length was cut out and GENECLEANI I DNA fragments were purified using the Kit (BIOIOI) according to the instructions attached to the kit.
  • the resulting PCR reaction mixture was subcloned into PUC19 prepared by digestion with BamHI and Hindlll.
  • the nucleotide sequence was determined using the 13 Primer M4 primer and the M13 Primer RV primer.As a result, the correct sequence was obtained.
  • This plasmid was digested with Hindlll and Blnl, and the 416 bp fragment was digested with 1% agarose gel. Separated by electrophoresis. The DNA fragment was purified using GENECLEANII Kit (BI0101) according to the instructions attached to the kit.
  • the resulting PCR reaction mixture was introduced into a plasmid CAZpUC19 prepared by digesting with Hindlll and Blnl, and named plasmid hMBClLaA / pUC19.
  • This plasmid was digested with EcoRI, and the sequence containing the sequence encoding the humanized L chain was introduced into the plasmid pCOSl so that the start codon of the humanized L chain was located downstream of the EF1 ⁇ promoter. .
  • the plasmid thus obtained was named hMBClLaA / pCOSl.
  • the nucleotide sequence (including the corresponding amino acid) of the humanized L chain version "a" is shown in SEQ ID NO: 66.
  • the amino acid sequence of version a is shown in SEQ ID NO: 47.
  • Version "b” was created using mutagenesis by PCR. Version “b” was designed to change glycine at position 43 (amino acid number 43 according to Kabat's definition) to proline and lysine at position 49 (amino acid number 49 according to Kabat's definition) to aspartic acid.
  • PCR was performed using the mutagenic primer MBC1LGP5R (SEQ ID NO: 36) and the primer MBC1LVS1 as a plasmid, and the resulting DNA fragment was digested with BamHI and Hindi II and subcloned into the BamHI and Hindlll sites of pUC19. . After the nucleotide sequence was determined, it was digested with restriction enzymes Hindlll and ⁇ and ligated with hMBClLaA / pUC19 digested with Hindlll and ⁇ .
  • the plasmid thus obtained was designated as hMBClL! / PUC19, this plasmid was digested with EcoRI, the fragment containing the DNA encoding the humanized L chain was introduced into the plasmid pCOSl, and the fragment was downstream of the EF1 ⁇ promoter. The start codon of the humanized L chain was located.
  • the plasmid thus obtained was named hMBClLtU / pCOSl.
  • Version "c” was prepared using mutagenesis by PCR. Version “c” was designed to replace serine at position 84 (amino acid number 80 according to Kabat's rules) with proline. Mutagenic primer MBC1LGP6S (SEQ ID NO: 37) and primer M13 PCR was performed with Primer RV using plasmid hMBClLa ⁇ / pUC19 as a rust type, and the obtained DNA fragment was digested with BamHI and Hindlll, and subcloned into PUC19 prepared by digesting with BamHI and Hindlll.
  • hMBClLc A / pl Cl9 The plasmid thus obtained was named hMBClLcA / pCOSl.
  • Versions “d”, “e” and “” were prepared by mutagenesis by PCR method. ”Each of the versions has the 91st position of the“ a ”,“ b ”and“ c ”versions in order (amino acid as defined by Kabat). No.
  • mutagenic primer MBC1LGP11R SEQ ID NO: 38
  • primer M-S1 SEQ ID NO: 44
  • PCR was performed using hMBClLbA / pCOSl and hMBClLc ⁇ / pCOSl as type I, and the obtained DNA fragment was digested with BamHI and Hindlll, and subcloned into PUC19 prepared by digestion with BamHI and HindiII. It was digested with Hindlll and Blnl and ligated with C ⁇ no pUC19 prepared by digestion with Hindlll and Blnl.
  • the plasmids thus obtained were designated as hMBClLdA / pUC19, hMBClLe ⁇ / pUC19, and hMBClLfi / pUC19, respectively. These plasmids are digested with EcoRI, and the sequence containing the sequence encoding the humanized L chain is introduced into the EcoRI site of the plasmid pCOSl to initiate the humanized L chain downstream of EF1 Hypromo overnight. Codons are located.
  • the plasmids thus obtained were named hMBClLc / pCOSl and MBClLe ⁇ / pCOSK hMBClLf ⁇ / pCOSl, respectively.
  • Versions "g” and “h” were made using mutagenesis by PCR. Each version was designed so that the histidine at position 36 (amino acid number 36 according to Kabat's definition) of the "a” and “d” versions was changed to tyrosine in order.
  • PCR was performed using hMBClLaA / pUC19 as type I, and the resulting PCR product and M13 Primer M4 were purified. Further PCR was carried out using the plasmid hMBClLaA / pUC19 as type III, as a primer.
  • the obtained DNA fragment was digested with Hindlll and Blnl, and subcloned into a plasmid CAZpUC19 prepared by digestion with Hindlll and Blnl.
  • PCR was performed using primers MBC1LGP13R (SEQ ID NO: 40) and MBC1LVS1 as primers.
  • the obtained PCR fragment was digested with Apal and Hindll, and introduced into plasmids MBClLa ⁇ / pUC19 and hMBCILd ⁇ / pUC19 digested with Apal and Hindi11.
  • the plasmid was subcloned into pUC19, the nucleotide sequence was determined, and the clone in which the mutation corresponding to each version was introduced was selected.
  • the bases of version "j", “1", “m” and “o” The sequences (including the corresponding amino acids) are shown in SEQ ID NOs: 67, 68, 69, and 70.
  • the amino acid sequences of these purgeons are shown in SEQ ID NOs: 48, 49, 50, and 51, respectively.
  • Plasmid hMBClLQA / pCOSl was digested with Hindlll and EcoRI, subcloned into plasmid PUC19 digested with Hindlll and EcoRI, and named plasmid hMBClLdA / PUC19.
  • Table 7 shows the position of the substituted amino acid in each version of the humanized L chain.
  • Y indicates tyrosine
  • ⁇ indicates proline
  • ⁇ indicates lysine
  • V indicates valine
  • D indicates aspartic acid
  • I indicates isoleucine.
  • E. coli JM109 (hMBClHcDNA / pUC19) and Escherichia were used as the above-mentioned plasmids 113 ⁇ 4 ⁇ (: 11 ⁇ 0/11 (: 191118 (: 11 (1 ⁇ / ⁇ 1) (19).
  • Escherichia coli JM109 (hMBClHcDNA / pUC19) on August 15, 1996, at the Institute of Biotechnology and Industrial Technology (Ibaraki Pref. FERM BP-5629 and Escherichia coli JM109 (hMBClLqA / pUC19) have been deposited internationally as FERM BP-5630 based on the Budapest Treaty.
  • the above expression plasmid was transiently expressed in COS-7 cells. That is, in the transient expression of the L chain hybrid antibody, the plasmids hMBClHcDM / pCOSl and h / mMBClLU) / neo, hMBClHcDNA / pCOSl and m / hMBClLa ⁇ / neo, Combination of hMBClHcDNA / pCOSl and in / hMBClLdA / neo, hMBClHcDNA / pCOSl and hmmMBCIL ( ⁇ ) / neo, or combination of hMBClHcDNA / pCOSl and mhmMBClL (A) / neo using an electroporator using a Gene Pulser
  • PBS (-) to 1 X10 7 cells / ⁇ 1 are suspended at a cell concentration COS- 7 cells 0.8ml in each of plasmid DNAIO g added pulses at 1, 500V, 25 F in capacitance Gave.
  • the electroporated cells were suspended in a DMEM culture medium (GIBC0) containing 2% Ultra Low IgG fetal serum (GIBC0), and the 10 cm culture dishes were removed. They were cultured in a C0 2 incubator scratch using. After culturing for 72 hours, the culture supernatant was collected, cell debris was removed by centrifugation, and used for ELISA samples.
  • GIBC0 DMEM culture medium
  • GIBC0 Ultra Low IgG fetal serum
  • An ELISA plate for antibody concentration measurement was prepared as follows.
  • TAG0 concentration of 100 xl
  • 1 dilution buffer 50 mM Tris-HCl, ImM MgCl 2 , 0.1 M NaCl 0.05% Tween20, 0.02% NaN 3 , 1% bovine serum albumin (BSA), pH 7.2
  • the culture supernatant of C0S-7 cells expressing the hybrid antibody or humanized antibody or the purified hybrid antibody or humanized antibody was serially diluted and added to each well.
  • ELISA plates for antigen binding measurement were prepared as follows. Each well of the 96-well plate for ELISA was immobilized with human PTHrPU-34) 100 1 adjusted to a concentration of 1 xg / ml with an immobilization buffer. After blocking with 200 zl of the dilution buffer, the culture supernatant of C0S-7 cells expressing the hybrid antibody or humanized antibody or the purified hybrid antibody or humanized antibody was serially diluted and added to each well. After incubating at room temperature and washing with PBS-Tween20, 100 zzl of alkaline phosphatase-conjugated goat anti-human IgG antibody (TAG0) was added.
  • TAG0 alkaline phosphatase-conjugated goat anti-human IgG antibody
  • the antibody combining the humanized H chain version “a” and the chimeric L chain had the same PTHrP binding ability as the chimeric antibody. This result indicates that version "a” is sufficient for humanization of the heavy chain V region.
  • the humanized H chain version "a” was used as the H chain of the humanized antibody.
  • the antigen-binding activity of the humanized antibody using one of each of the versions "a” to “t” as the L chain was measured.
  • the humanized antibody having the L chain versions “j”, “1”, “m”, “o”, “q”, “r”, “s”, and “t” has the same PTH as the chimeric antibody. It showed rP binding ability.
  • the expression plasmid was introduced into CH0 cells (DXB11) in order to establish a stable humanized antibody cell line.
  • the stable production of humanized antibody cell lines was established using the expression plasmids for CH0 cells, hMBC IHcDNA / pCHO 1 and hMBC 1 Lm ⁇ / pCOS 1 or hMBC IHcDNA / pCHO 1, and hMBC ILQ ⁇ / pCOS 1 or hMBClHcDNA / pCHOl.
  • CHO cells were co-transduced with the combination of hMBClLrA / pCOSl by electroporation using a Gene Pulser instrument (Bio Rad). Each expression vector was cleaved with the restriction enzyme Pvul to obtain linear DNA.
  • the DNA was recovered by ethanol precipitation and used for elect-portion poration.
  • PBS PBS
  • 10 ⁇ g of each plasmid DNA and pulse at 1,500 V 25 F capacitance was added.
  • the cells treated with Elect-orifice were suspended in 10% fetal serum (GIBC0), suspended in ⁇ - ⁇ medium (GIBC0), and used in a 96-well plate (Falcon). They were cultured in a C0 2 incubator one Te.
  • GIBC0 fetal calf serum
  • GENETICIN G418 Sulfate, GIBC0
  • MEM-free medium containing no ribonucleosides and deoxyribonucleosides
  • the neutralizing activity of the mouse antibody, chimeric antibody and humanized antibody was measured using rat osteosarcoma cell line R0S17 / 2.8-5 cells. That, R0S17 / 2.8- to 5 cells, with 10% fetal bovine Ham containing serum (GIBC0)'S F-12 medium (GIBC0) in, were cultured in C0 2 incubator scratch. R0S17 / 2.8-5 cells are seeded in a 96-well plate at 104 cells / ⁇ 00 1Z well, cultured for 1 day, and replaced with Ham'S F-12 medium (GIBC0) containing 4 mM Hydrocortisone and 10% fetal bovine serum.
  • GIBC0 Ham'S F-12 medium
  • the plate was washed with 2601 Ham'S F-12 medium (GIBC0), lniM isobutyl-trimethylxanthine (IBMX, SIGMA) and 10% fetal bovine serum and 10 mM HEPES. Of Ham's F-12 was added thereto and incubated at 37 ° C for 30 minutes.
  • a mouse antibody, a chimeric antibody, or a humanized antibody whose neutralizing activity is to be measured was previously prepared in groups of 10 g / ml, 3.3 g / mK1, 1 / zg / ml and 0.337 g / ml, 10 zg / ml.
  • the present invention provides a tissue degradation inhibitor containing, as an active ingredient, a substance that inhibits the binding of a parathyroid hormone-related peptide to its receptor.
  • Inhibitor of the present invention c sequence Listing Free one text Bok is useful as a pharmaceutical composition for suppressing weight loss associated with such cancer cachexia
  • SEQ ID NO: 16 synthetic DNA SEQ ID NO: 17: synthetic DNA SEQ ID NO: 18: synthetic DNA SEQ ID NO: 19: synthetic DNA SEQ ID NO: 20: synthetic MA SEQ ID NO: 21: synthetic DNA SEQ ID NO: 22: synthetic DNA SEQ ID NO: 23: synthetic DNA SEQ ID NO: 24: synthetic thigh SEQ ID NO: 25: Synthetic DNA SEQ ID NO: 26: Synthetic DNA SEQ ID NO: Synthetic DNA SEQ ID NO: 28: Synthetic DNA SEQ ID NO: 29: Synthetic DM SEQ ID NO: 31: Synthetic DNA SEQ ID NO: 32: Synthetic DNA SEQ ID NO: 33: Synthetic DNA SEQ ID NO: 34: Synthetic DNA SEQ ID NO: 35: Synthetic DNA SEQ ID NO: 36: Synthetic DNA SEQ ID NO: 37: Synthetic MA SEQ ID NO: 38: Synthetic DNA SEQ ID NO: 39: Synthetic DNA SEQ ID NO: 40: Synthetic DNA SEQ ID NO: 41: Synthetic DNA SEQ ID NO: 41 42: Synthetic marauder

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Abstract

L'invention concerne un inhibiteur de décomposition de tissu contenant une substance qui inhibe la liaison entre un peptide associé à une hormone parathyroïdienne et son récepteur.
PCT/JP2000/005886 2000-02-28 2000-08-30 Inhibiteur de decomposition de tissu WO2001064249A1 (fr)

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WO2004072286A1 (fr) * 2003-01-23 2004-08-26 Ono Pharmaceutical Co., Ltd. Substance specifique a pd-1 humain
US11091550B2 (en) 2018-02-09 2021-08-17 Ono Pharmaceutical Co., Ltd. Bispecific antibody

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WO2004072286A1 (fr) * 2003-01-23 2004-08-26 Ono Pharmaceutical Co., Ltd. Substance specifique a pd-1 humain
US7563869B2 (en) 2003-01-23 2009-07-21 Ono Pharmaceutical Co., Ltd. Substance specific to human PD-1
US7998479B2 (en) 2003-01-23 2011-08-16 Ono Pharmaceutical Co., Ltd. Substance specific to human PD-1
US8246955B2 (en) 2003-01-23 2012-08-21 Ono Pharmaceutical Co., Ltd. Substance specific to human PD-1
US8951518B2 (en) 2003-01-23 2015-02-10 Ono Pharmaceutical Co., Ltd. Substance specific to human PD-1
US9783609B2 (en) 2003-01-23 2017-10-10 Ono Pharmaceutical Co., Ltd. Substance specific to human PD-1
US11091550B2 (en) 2018-02-09 2021-08-17 Ono Pharmaceutical Co., Ltd. Bispecific antibody

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