WO2001077362A1 - Dosage immunologique d'anticorps anti hm1 . 24 - Google Patents

Dosage immunologique d'anticorps anti hm1 . 24 Download PDF

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Publication number
WO2001077362A1
WO2001077362A1 PCT/JP2001/002964 JP0102964W WO0177362A1 WO 2001077362 A1 WO2001077362 A1 WO 2001077362A1 JP 0102964 W JP0102964 W JP 0102964W WO 0177362 A1 WO0177362 A1 WO 0177362A1
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Prior art keywords
antibody
soluble
antigen
protein
antigen protein
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PCT/JP2001/002964
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English (en)
Japanese (ja)
Inventor
Yasuko Kinoshita
Yuji Ishikawa
Original Assignee
Chugai Seiyaku Kabushiki Kaisha
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Priority to AU2001246846A priority Critical patent/AU2001246846A1/en
Publication of WO2001077362A1 publication Critical patent/WO2001077362A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

Definitions

  • the present invention also provides (A3) the above method, wherein the soluble HM1.24 antigen has an amino acid sequence shown in SEQ ID NO: 7 or 17.
  • FIG. 1 is a schematic diagram showing a sandwich ELISA system using an HA-tagged soluble antigen.
  • FIG. 2 is a diagram comparing the amount of soluble antigen produced by the HA-tagged soluble HM1.24 antigen-producing cell line in the culture supernatant. The higher the dilution ratio, the higher the production.
  • Figure 4 shows the standard curve of humanized anti-HM1.24 antibody in a Sandwich ELISA system using purified antigens diluted 5 'from 750-fold to 3-fold. This is a graph.
  • FIG. 6 shows the nucleotide sequence of the cDNA encoding the HM1.24 antigen protein and the corresponding amino acid sequence.
  • FIG. 7 shows the nucleotide sequence of the cDNA encoding the HM1.24 antigen protein and the corresponding amino acid sequence.
  • Figure 10 is a drawing substitute showing the results of immunoprecipitation using anti-HM1.24 antibody and detection of HM1.24 antigen in various cell lines and HM1.24 expressing CH0 cells by Western blotting. It is a photograph. After immunoprecipitation using Sepharose 4B (lanes 1 to 6) or unbound Sepharose 4B (lanes 7 and 8) bound to the anti-HM1.24 antibody, Western blotting was performed using the anti-HM1.24 antibody. HM1.24 antigen was detected (displayed on the right). (*; Anti-HM1.24 antibody heavy chain)
  • the amino acid sequence shown in SEQ ID NO: 5 has an amino acid in which 1 or more, preferably 1 or 42 or less, more preferably 1 or 17 or less amino acid residues have been deleted. You may. Alternatively, in the amino acid sequence shown in SEQ ID NO: 5, one or more, preferably 1 or 50 or less, more preferably 1 or 14 or less amino acids are added to the amino acid sequence. You may.
  • the soluble HM1.24 antigen protein used in the present invention may be simultaneously modified by the above amino acid substitution, deletion and / or addition.
  • Soluble bandage 1. 24 antigen protein can exhibit its biological activity if it has an amino acid sequence from amino acid Asn at position 1 to amino acid Arg at position 90 in SEQ ID NO: 5. It's clear. Therefore, the soluble HM1.24 antigen protein of the present invention comprises an amino acid sequence from amino acid Asn at position 1 to amino acid Arg at position 90 in SEQ ID NO: 5. Sequence, or substitution, deletion and / or replacement of one or more amino acid residues with respect to the amino acid sequence from amino acid Asn at position 1 to Arg at position 90. It may be a soluble HM1.24 antigen protein having an amino acid sequence modified by addition.
  • Soluble HM1.24 antigen protein has the amino acid sequence from amino acid Arg at position 90 to amino acid Gin at position 132 in SEQ ID NO: 5 as long as it has the biological activity, or this amino acid It may be a soluble band 1.24 antigen protein having an amino acid sequence modified by substitution, deletion and / or addition of one or more amino acid residues to the sequence.
  • a soluble HM1.24 antigen protein having an amino acid sequence modified by substitution, deletion and / or addition of one or more amino acid residues to the amino acid sequence shown in SEQ ID NO: 5; And a soluble HM1.24 antigen protein having the amino acid sequence shown in SEQ ID NO: 7 or 17, 10 or 18, or 11 or 19.
  • the soluble marauder 1.24 antigen protein of the present invention may have an amino acid sequence, molecular weight, isoelectric point, presence or absence of glycosylation, location of glycosylation, sugar, depending on the species of origin, the host producing them and / or the purification method.
  • the chain structure, phosphorylation state, and the presence or absence of Z or disulfide bonds are different.
  • the protein may have any structure as long as it can be suitably used in the present invention. Human is preferred as the species from which the protein is derived.
  • Examples of the DNA encoding the soluble HM1.24 antigen protein of the present invention include a base sequence consisting of base adenine at position 1 to base guanine at position 396 of the base sequence shown in SEQ ID NO: 5.
  • the DNA encoding the soluble HM1.24 antigen protein of the present invention may be any DNA as long as it has the nucleotide sequence shown in SEQ ID NO: 5.
  • Examples of such DNA include dienomic DNA, cDNA, and synthetic DNA. These may be cDNA libraries obtained from various cells, tissues or organs, or species other than human, DNA obtained from a dienomic library, or may be a commercially available DNA library. Good.
  • the vector used for these libraries may be any vector such as plasmid, bacterio phage, YAC vector and the like.
  • the DNA encoding the soluble HM1.24 antigen protein of the present invention also includes a polypeptide that hybridizes to the base sequence shown in SEQ ID NO: 5 and has the biological activity of the soluble HM1.24 antigen protein. May be DNA to be loaded.
  • Examples of the conditions under which DNA encoding the soluble HM1.24 antigen protein hybridizes include DNA that hybridizes under appropriate stringency conditions. Examples of such hybridization conditions include low DNA.
  • stringency conditions include, for example, cleaning conditions provided by 42 ° C., 5 ⁇ SSC, 0.1% sodium dodecyl sulfate, and 50% formamide. More preferably, high stringency conditions can be mentioned.
  • Conditions of high stringency include, for example, 60 ° (: 0.1 SSC, 0.1% sodium dodecyl sulfate).
  • the cleaning conditions provided by the camera It is already known that a protein encoded by a DNA that hybridizes under appropriate conditions to a nucleotide sequence encoding a protein has the same biological activity as the protein.
  • the soluble HM1.24 antigen protein of the present invention is encoded by the above-mentioned “hybridizing DNA”, and also includes a protein having the biological activity of the soluble HM1.24 antigen protein.
  • peptides include FLAG (Hopp, TP et al., BioTechnology (1988) 6, 1204-1210), 6 XHis consisting of 6 His (histidine) residues, lOXHis, and influenza agglutinin. (HA), human c-myc fragment, VSV-GP fragment, pl8HIV fragment, T7_tag, HSV-tag, E-tag, SV40T antigen fragment, lck tag, a-tubulin fragment, B-tag A known peptide such as a fragment of Protein in C or the like is used.
  • polypeptides include GST (daltathione-S-transferase), HA, immunoglobulin constant region, b-galactosidase, MBP (maltose binding protein) and the like. These can be used commercially available ones.
  • the DNA encoding the protein of the present invention can be constructed from the above-described DNA by a commercially available kit known in the art. For example, it can be constructed by digestion with a restriction enzyme, addition of a linker, insertion of an initiation codon (ATG) and a no or termination codon (ATT, TGA or TAG), and the like.
  • the protein expression vector of the present invention may be any expression vector that is suitably used in the present invention.
  • expression vectors include mammalian-derived expression vectors, such as pEF, pCDM8, insect cell-derived expression vectors, such as pBacPAK8, plant-derived expression vectors, such as ⁇ 1, pMH2, and animal virus-derived expression vectors.
  • P HSV mammalian-derived expression vectors
  • pMV insect cell-derived expression vectors
  • pBacPAK8 insect cell-derived expression vectors
  • plant-derived expression vectors such as ⁇ 1, pMH2, and animal virus-derived expression vectors.
  • P HSV pMV
  • expression vectors derived from yeast e.g. PNVl l
  • expression vectors derived from Bacillus subtilis e.g. pPL608, Rokappatauitaderutaomikuron
  • expression vectors derived from Escherichia coli for example pGEX, pGEMEX, include
  • the expression vector of the protein of the present invention may be produced, for example, by ligating DNA coding for the soluble HM1.24 antigen protein downstream of the promoter and introducing it into the expression vector.
  • a Z motor a mammalian promoter
  • Enhancers such as EF1-a promoter / enhancer, gamma actin promoter / enhancer, promoters derived from insect vinoles, such as polynuclear (polyhedrin) virus mouth motors, and plant-derived motorhead enhancers.
  • Tano Komozyk Winores promoter Jenhanser Promoters / enhancers derived from animal vinoles, e.g., SV40 motor / enhancer, human CMV promoter Z enhancer, promoters / enhancers derived from yeast, e.g., alcohol dehydrogenase promoter no enhancer, E. coli-derived promoter Z enhancer, e.g., Lac promoter Z Enhancer, Trp Promoter / Enhancer, Tac Promoter / Enhancer.
  • the protein used in the present invention can be obtained as a recombinant protein produced using a gene recombination technique as described above.
  • a recombinant protein is produced by cloning the nucleotide sequence of the gene described herein from a cell, tissue, or organ that expresses them, incorporating the nucleotide sequence into an appropriate vector, and introducing the vector into a host. This recombinant protein can be used in the present invention.
  • mRNA encoding the gene is isolated from cells, tissues, or organs expressing the protein used in the present invention.
  • mRNA Can be isolated by known methods, for example, guanidine ultracentrifugation (Chirgwin, J. M. et al., Biochemistry (1979) 18, 5294-5299), AGPC method (Chomczynski, P. and Sacchi, N. et al. , Anal. Biochem., 1987) lb2, 156-159).
  • mRNA Purification Kit Pharmacia
  • mRNA can be directly prepared by using the QuickPrep mRNA Purification Kit (Pharmacia).
  • CDNA of the gene is synthesized from the obtained mRNA using reverse transcriptase.
  • cDNA can also be synthesized using AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (Seikagaku Corporation) or the like.
  • AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (Seikagaku Corporation) or the like.
  • PCR polymerase chain reaction
  • promoters / enhancers that can be used for protein expression include virus promoters / enhancers such as retrovirus, poliovirus, adenovirus, and simian virus 40 (SV40).
  • Nfa click ter 1 a may be used promoter Z Enhansa from mammalian cells (HEF1 a).
  • HEF1 a mammalian cells
  • expression can be achieved by operably linking a useful promoter commonly used, a signal sequence for protein secretion, and a gene to be expressed.
  • the promoter include a lacZ promoter and an araB promoter.
  • the lacZ promoter the method of Ward et al. (Nature (1098) 341, 544-546; FASEB J. (1992) 6, 2422-2427), and when the araB promoter is used, the method of Better et al. (Science (1988) 240, 1041-1043).
  • any production system can be used for protein production.
  • Production systems for protein production include in vitro and in vivo production systems.
  • Examples of in vitro production systems include production systems using eukaryotic cells and production systems using prokaryotic cells.
  • CH0 cells include dhfr-CHO (Proc. Natl. Acad. Sci. USA (1980) 77, 4216-4220) and CHO Kl (Proc. Natl. Acad. Sci. USA (1968) 60, 1275) can be suitably used.
  • yeast such as the genus Saccharomyces, for example, Saccharomyces' Saccharomyces cerevisiae
  • filamentous fungi such as the genus Aspergillus, such as Aspernogillus niger
  • prokaryotic cells there are production systems using bacterial cells.
  • Bacteria As cells Escherichia coli (E. coli) and Bacillus subtilis are known.
  • Proteins can be obtained by transforming these cells with the desired DNA and culturing the transformed cells in vitro. Culture is performed according to a known method. For example, DMEM, MEM, RPMI1640, IMDM can be used as a culture solution. At that time, a serum replacement solution such as fetal calf serum (FCS) may be used in combination, or serum-free culture may be performed. The pH during culturing is preferably about 6-8. Culture is usually performed at about 30 to 40 ° C. for about 15 to 200 hours, and if necessary, the medium is replaced, aerated, and agitated.
  • FCS fetal calf serum
  • examples of in vivo production systems include production systems using animals and production systems using plants.
  • the desired DNA is introduced into these animals or plants, and the protein is produced and recovered in the animals or plants.
  • mice When using animals, there are production systems using mammals and insects. Goats, pigs, sheep, mice, and mice can be used as mammals (Vicki Glaser, SPECTRUM Biotechnology Applications, 1993). When a mammal is used, a transgenic animal can be used.
  • a silkworm When a silkworm is used, a paculovirus into which a target DNA has been inserted is used to infect a chick, and a desired protein is obtained from the body fluid of the silkworm (Susumu, M. et al., Nature (1985) 315, 592-594).
  • tobacco When using a plant, for example, tobacco can be used.
  • a plant expression vector for example, pMON530, and this vector is introduced into a nocteria such as Agrobacterium tumefaciens.
  • tobacco for example, Nicotiana tabacum
  • the desired protein is obtained from the leaves of the tobacco (Julian, K.-C. Ma et al., Eur. J. Immunol. 1 994) 24, 131-138).
  • Known methods for introducing an expression vector into a host include, for example, the calcium phosphate method (Virology (1973) 52, 456-467) and the electroporation method (EMBO J. (1982) 1 , 841-845).
  • sequences with higher expression efficiency can be designed in consideration of the codon usage of the host used for expression (Grantham, R. et al., Nucleic Acids Research (1981) 9, r43-r74). .
  • the gene is introduced into these animals or plants as described above, and proteins are produced and recovered in the animals or plants.
  • the protein expressed and produced as described above can be separated from the host inside and outside the cell and from the host and purified to homogeneity.
  • the separation and purification of the protein used in the present invention may be carried out by using the separation and purification methods used for ordinary proteins, and is not limited at all.
  • the concentration of the protein can be measured using a known method. For example, measurement of absorbance or Bradford method may be used.
  • the present invention includes a step of reacting a soluble HM1.24 antigen protein with an anti-HM1.24 antibody contained in a test sample to detect or measure an anti-HM1.24 antibody bound to the soluble HM1.24 antigen protein.
  • the present invention relates to a method for immunochemical measurement of a soluble HM1.24 antigen protein.
  • the immunochemical measurement method provided in the present invention is performed as an in vitro assay system.
  • One example of this method is schematically shown in FIG.
  • In vitro Atsushi systems are performed in non-cellular systems. Specifically, the soluble HM1.24 antigen protein was bound to the support, a test sample containing the anti-band 1.24 antibody was added to this protein, and the mixture was incubated, washed, and then washed to remove the soluble HM1.24 antigen bound to the support. What is necessary is just to detect or measure the binding of the anti-HM1.24 antibody to the protein. Or, specifically, The anti-HM1.24 antibody was bound to the support, and a test sample containing the soluble HM1.24 antigen protein was added to this protein. The sample was incubated, washed, and washed to dissolve the anti-HM1.24 antibody bound to the support. The binding of the HM1.24 antigen protein may be detected or measured.
  • Soluble HM1.24 antigen protein or anti-HM1.24 antibody is a protein produced from cells that express them uniquely, cells transfected with the DNA encoding them, and animals or plants transfected with the DNA encoding them. Can be used in a purified state or in a partially purified state.
  • Either purified or partially purified soluble HM1,24 antigen protein or anti-HM1.24 antibody protein is bound to the support.
  • the protein can be immobilized on the support by standard methods.
  • the support to which the protein is bound include insoluble polysaccharides such as agarose, dextran, cellulose, and synthetic resins such as polystyrene, polyacrylamide, and silicon.
  • beads and plates manufactured using them as raw materials are used.
  • a column filled with these may be used.
  • a multiwell plate eg, a 96-well multiwell plate
  • a biosensor chip can be used.
  • the binding between the protein and the support may be performed by a commonly used method such as chemical bonding or physical adsorption.
  • an antibody that specifically recognizes a protein can be bound to a support in advance by the above-described method, and the antibody can be bound to the protein to bind.
  • the binding between soluble HM1.24 antigen protein and anti-HM1.24 antibody, which can be bound via avidin / biotin is usually buffered. Performed in liquid.
  • the buffer for example, a phosphate buffer, a Tris buffer, or the like is used. Incubation is performed under conditions that are already commonly used, such as incubation at 4 ° C to room temperature for 1 hour to 24 hours.
  • the post-incubation washing may be any as long as it does not hinder the binding between the soluble HM1.24 antigen protein and the anti-HM1.24 antibody.
  • a buffer containing a surfactant is used.
  • the surfactant for example, 0.05% Tween 20 is used.
  • test sample containing the soluble HM1.24 antigen protein or anti-HM1.24 antibody measured in the present invention includes human body fluids (blood, serum, urine, joint fluid, etc.), cell culture supernatant, Animal secretions (milk, etc.), pharmaceutical preparations, and the like.
  • a control group may be provided together with a group in which a test sample is brought into contact with a protein.
  • a negative control group containing no test sample and / or a positive control group containing a purified soluble HM1.24 antigen protein or anti-HM1.24 antibody standard were used. Can be kept.
  • the bound protein can be detected by the immunochemical measurement method of the present invention. Alternatively, the bound protein can be quantitatively measured. In these cases, the results obtained in the negative control group without the test sample, the results obtained in the group with the test sample and / or the purified soluble HM1.24 antigen protein or anti-band 1.24 antibody Including goods By comparing the results obtained with the positive control group, the binding between the soluble HM1.24 antigen protein and the anti-HM1.24 antibody can be detected.
  • the results of their detection are obtained as numerical values, and by comparing those numerical values, the soluble HM1.24 antigen protein or anti-HM1.24 antibody contained in the test sample can be quantitatively measured. Can also. When measuring quantitatively, compare the values obtained in the negative control group without the test sample with the values obtained in the group to which the test sample containing the soluble HM1.24 antigen protein or anti-HM1.24 antibody was applied. As a result, the amount of binding between the soluble HM1.24 antigen protein and the anti-bandwidth antibodies 124 can be quantified. If soluble HM1.24 antigen protein or anti-HM1.24 antibody is contained in the test sample, soluble HM1.24 antigen protein or anti-HM1.24 antibody is detected or measured by the presence of the bound protein. be able to.
  • a biosensor utilizing the surface plasmon resonance phenomenon can be used as a means for detecting or measuring a soluble HM1.24 antigen protein or an anti-HM1.24 antibody in a test sample.
  • a biosensor that utilizes the surface plasmon resonance phenomenon allows real-time observation of the interaction between protein and protein as a surface plasmon resonance signal without labeling using a small amount of protein. It is possible (for example, BIAcore; manufactured by Pharmacia). Therefore, it is possible to detect or measure the binding between the soluble HM1.24 antigen protein and the anti-HM1.24 antibody by using a biosensor such as BIAcore.
  • a test sample containing an anti-HM1.24 antibody or a soluble HM1.24 antigen protein is brought into contact with a sensor chip on which a soluble HM1.24 antigen protein or an anti-HM1.24 antibody is immobilized, and the soluble HM1.
  • Anti-HM1.24 antibody or soluble HM1.24 antigen protein that binds to .24 antigen protein or anti-HM1.24 antibody can be detected or measured as a change in resonance signal.
  • the sensor chip CM5 (Biosensor) is activated to immobilize the soluble HM1.24 antigen protein or anti-HM1.24 antibody on the sensor chip. That is, the sensor chip is activated by an aqueous solution of ED CI NHS (200 mM EDC (N-ethyl_ ⁇ '-(3-dimethylaminopropyl pill) carbonate hydrochloride), 50 mM NHS (N-hydroxysuccinimide)). After that, the sensor chip is washed with HBS buffer (10 mM HEPES pH7, 150 mM NaCl, 3.4 mM MEDTA, 0.05% Tween20).
  • HBS buffer 10 mM HEPES pH7, 150 mM NaCl, 3.4 mM MEDTA, 0.05% Tween20.
  • a test sample containing an appropriate amount of an anti-HM1.24 antibody or a soluble HM1.24 antigen protein dissolved in an HBS buffer is brought into contact with a sensor chip to be immobilized.
  • the remaining active groups on the sensor chip are blocked with an ethanolamine solution (1M ethanolamine hydrochloride, pH 8.5). Wash the sensor chip again with HBS buffer and use it for binding evaluation.
  • a test sample containing an appropriate amount of an anti-HM1.24 antibody or soluble HM1.24 antigen protein dissolved in an HBS buffer is injected.
  • the amount of the anti-HM1.24 antibody or soluble HM1.24 antigen protein in the test sample bound to the soluble HM1.24 antigen protein or anti-HM1.24 antibody immobilized on one sensor chip was determined by the resonance signal value. Is observed as an increase in
  • a control group may be set up together with the group containing the test sample.
  • a negative control containing no test sample was used.
  • the bound protein can be quantitatively measured as a change in the resonance signal value.
  • the results obtained in the negative control group containing no test sample, the results obtained in the group containing the test sample, and / or a known amount of soluble HM1.24 antigen protein or anti-HM1.24 antibody were used. By comparing the results obtained with the positive control group containing the target protein, the target protein in the test sample can be detected or measured.
  • a primary antibody that specifically recognizes a soluble HM1.24 antigen protein or an anti-HM1.24 antibody is used as a means for detecting or measuring the protein in the bound test sample. Can be used.
  • a test sample is brought into contact with a soluble HM1.24 antigen protein or an anti-1.224 antibody, washed, and the bound protein is detected or measured with a primary antibody that specifically recognizes the protein. That is, preferably, one protein bound to the support is brought into contact with a test sample containing the other protein. After incubating and washing, the bound protein may be detected or measured with a primary antibody that specifically recognizes the protein.
  • the primary antibody is preferably labeled with a labeling substance.
  • the soluble HM1.24 antigen protein may be fused to another peptide or polypeptide. Therefore, anti-band 1.25 antibody can be used to detect soluble HM1.24 antigen protein contained in the test sample, and other peptides or polypeptides fused with soluble HM1.24 antigen protein can be used. Antibodies to the peptides can be used. Further, in order to detect the anti-HM1.24 antibody contained in the test sample, an antibody that specifically recognizes the anti-HM1.24 antibody can be used. Anti-HM1.24 antibody In the case of a mouse antibody, an anti-mouse immunoglobulin antibody can be used as an antibody that specifically recognizes the anti-HM1.24 antibody. When the anti-HM1.24 antibody is a chimeric antibody or a humanized antibody, an anti-immunoglobulin antibody can be used as an antibody that specifically recognizes the anti-HM1.24 antibody.
  • the protein can be labeled by a commonly known method.
  • the labeling substance include a radioisotope, an enzyme, a fluorescent substance, biotin / avidin and the like.
  • labeling substances commercially available labeling substances can be used. Is to radioisotopes, for example 3 2?, 3 3 P, 1 3 1 I, 1 2 5 I, 3 H, 1 C, 3 5 S and the like.
  • the enzyme include alkaline phosphatase, horseradish peroxidase, ⁇ -galactosidase, ⁇ -darcosidase and the like.
  • the fluorescent substance include fluorescein isothiocyanate (FITC) and rhodamine. These can be obtained commercially and labeled by a known method.
  • a solution containing the soluble bandage 1.24 antigen protein or anti-HM1.24 antibody is added to the plate, and left overnight to fix it on the plate.
  • the antibody for each is immobilized on a plate in advance, and the immobilized antibody is treated with soluble HM1.24 antigen protein or anti-HM1.24 antibody. They may be combined. After washing the plate, block it with, for example, BSA to prevent nonspecific binding of the protein. Wash again and add a test sample containing anti-HM1.24 antibody or soluble HM1.24 antigen protein to the plate.
  • a group containing no test sample (negative control) and / or a group to which a known concentration of anti-HM1.24 antibody or soluble ⁇ 1.24 antigen protein was added were placed and incubated. I do. After incubation, wash and add antibodies to the test sample. After a suitable incubation, the plate is washed and the protein is detected or measured with a primary antibody that specifically recognizes the protein. For detection or measurement, in the case of a radioisotope, detection or measurement is performed by liquid scintillation. In the case of an enzyme, the substrate is added, and the enzymatic change of the substrate, for example, color development is detected or measured by an absorptiometer. For fluorescent substances, detect or measure with a fluorometer. By comparing these results with the values obtained for the control group, the test sample containing the inhibitor can be determined.
  • the soluble HM1.24 antigen protein or the anti-HM1.24 antibody in the test sample is detected or measured by using a soluble HM1.24 antigen protein or an anti-HM1.24 antibody.
  • a primary antibody that specifically recognizes and a secondary antibody that specifically recognizes the primary antibody can be used.
  • a test sample is brought into contact with a soluble HM1.24 antigen protein or an anti-HM1.24 antibody, incubated, and then washed to bind the bound protein to the protein.
  • Detection or measurement is performed with a primary antibody that recognizes the primary antibody and a secondary antibody that specifically recognizes the primary antibody. That is, specifically, a soluble HM1.24 antigen protein or an anti-HM1.24 antibody is immobilized on a support, and a test sample is contacted. After the incubation, washing is performed, and the bound protein may be detected or measured with a primary antibody that specifically recognizes the protein and a secondary antibody that specifically recognizes the primary antibody.
  • the secondary antibody is preferably labeled with a labeling substance.
  • the antibody can be labeled by the above-mentioned method generally known.
  • the test sample containing the inhibitor can be determined.
  • the soluble HM1.24 antigen protein may be fused to another peptide or polypeptide. Therefore, an anti-HM1.24 antibody can be used as a temporary antibody for detecting the soluble HM1.24 antigen protein contained in the test sample, and other antibodies fused with the soluble HM1.24 antigen protein can be used. Antibodies to the peptide or polypeptide can also be used. In addition, in order to detect the anti-HM1.24 antibody contained in the test sample, the anti-HM1.24 antibody was specifically identified. Differentially recognizing antibodies can be used.
  • an anti-mouse immunoglobulin antibody can be used as a primary antibody that specifically recognizes the anti-HM1.24 antibody.
  • an anti-band 1.24 antibody is a chimera antibody or a humanized antibody
  • an anti-immunoglobulin antibody can be used as a primary antibody that specifically recognizes the anti-HM1.24 antibody.
  • an antibody that specifically recognizes the primary antibody can be appropriately selected as the secondary antibody. For example, if the primary antibody is a hidge antibody, an anti-hidgymno glopurin antibody can be used. In addition, when the primary antibody is a rabbit antibody, an anti-rabbit antibody can be used.
  • the present invention is particularly preferably carried out using ELISA (Enzyme-linked Immunosorbent Assay) as follows. That is, soluble HM1. 24 antigen protein and fused HA antibody immobilized buffer against (Flu Enza agglutinin) (0. 1 M NaHC0 3, 0. 02% NaN 3, pH9. 6) Ri is diluted by the . An appropriate amount of the diluted aqueous solution is added to each well of a 96-well imnoplate (manufactured by Nunc), and the mixture is immobilized at 4 ° C to solidify.
  • ELISA Enzyme-linked Immunosorbent Assay
  • soluble HM1.24 antigen protein fused with HA diluted in dilution buffer 1% ⁇ SA, 0.5% Tween20, PBS
  • dilution buffer 1% ⁇ SA, 0.5% Tween20, PBS
  • a certain amount of a test sample containing a chimeric anti-HM1.24 antibody having a human IgG antibody constant region (C region) is added, and incubated at room temperature for 1 hour.
  • C region human IgG antibody constant region
  • Each well is washed three times with wash buffer, 5000-fold alkaline phosphatase diluted labeled catcher formic anti human I g G antibody (manufactured by IB I) 100 mu 1 was added to each well at a dilution buffer, for 1 hour at room temperature Incubate.
  • Each well is washed five times with wash puffer developing solution; the (substrate puffer. 50 mM NaHC0 3, 10mM MgCl 2, pH9 8 to Sigma 104 dissolved in a concentration of 1 mg / ml) 100 ⁇ 1 to each well
  • the absorbance at 405 ⁇ m is measured using a microplate reader (Model 3550, manufactured by BIO-RAD).
  • the chimeric anti-HM1.24 antibody can be detected or measured by comparing these results with the values obtained in the negative control group and the no control group or the positive control group.
  • a soluble HM1.24 antigen protein can be detected or measured by the same method.
  • the screening method of the present invention can also be used for High Throughput Screening (HTS). Specifically, carried out until Bro Kkingu manually, subsequent reaction is O over Bok Meshiyo Ni spoon by made by a mouth pots, it can be force s to achieve a High Throughput screening.
  • HTS High Throughput Screening
  • the antibody-immobilized buffer against HA (0. 1M NaHC0 3, 0. 02% NaN 3, pH9. 6) Ri is diluted by the.
  • An appropriate amount of the diluted aqueous solution is added to each well of a 96-well immunoplate (manufactured by Nunc), and the mixture is incubated at 4 ° C for solid phase immobilization.
  • washing buffer prepared with 0.05% Tween20 in PBS
  • BSA manufactured by SI GMA
  • soluble HMl.24 antigen protein fused with HA diluted with dilution buffer (1% BSi, 0.5% Tween20, PBS). Incubate overnight at 4 ° C to bind the anti-HA antibody and soluble HM1.24 antigen protein fused with HA.
  • iiB iomek2000 HTS system manufactured by Beckman
  • ⁇ Set the immunoplate here, and test sample containing chimeric anti-HM1.24 antibody, primary antibody against chimeric anti-HM1.24 antibody
  • the dispenser as the Bi omek 2000 dispensing machine there have the Mul t ipi P et t e96 Penetration dispenser of the solution Imuno plates to each well by using the (Sagian Ltd.) Dispensing and solution removal can be performed.
  • EL404 microplate washers BioTek
  • a SPECTRAmax250 plate reader (Molecular Devices) can be used to measure the absorbance.
  • the program is set to perform the following operations. That is, wash each well three times with a washing buffer, and add a fixed amount of the test sample and the test sample containing the chimeric anti-HM1.24 antibody diluted with dilution buffer (1% BSA, 0.5% Tween20, PBS). At the same time, place a group containing no test sample (negative control) and a group to which a known concentration of chimera anti-HM1.24 antibody was added (positive control), and incubate them at room temperature for 1 hour.
  • dilution buffer 1% BSA, 0.5% Tween20, PBS
  • wash each well three times with wash buffer add 100 ⁇ l of egret anti-human IgG antiserum (New England Biolabs) diluted 5000 times with dilution buffer to each well and incubate at room temperature for 1 hour .
  • wash each well three times with the washing buffer add 100 ⁇ l of an alga phosphatase-labeled goat anti-money IgG antibody (manufactured by TAG0) diluted 5,000-fold with a dilution buffer to each well, and incubate for 1 hour at room temperature.
  • an alga phosphatase-labeled goat anti-money IgG antibody manufactured by TAG0
  • each well is washed five times with washing buffer, color development solution (substrate buffer;. 50 mM NaHC0 3, 10 mM MgCl 2, pH9 of 8 to lmg / ml concentration this ⁇ Army P-Nitrophenyl phosphate (manufactured by Sigma)) was added to each well, and allowed to react at room temperature. Then, the absorbance at 405 nm was measured using a microplate plate, Biomek plate. ) Measurement is carried out using a probe (manufactured by Beckman / Molecular Devices). By comparing these results with the values obtained for the control group, the chimeric anti-HM1.24 antibody contained in the test sample can be detected or measured. In addition, a soluble HM1.24 antigen protein can be detected or measured by the same method.
  • the immunochemical assay method provided by the present invention can measure soluble HM1.24 antigen protein or anti-HM1.24 antibody up to a concentration of 500 pg / ml.
  • the antibody used in the present invention may be a commercially available antibody or an antibody contained in a commercially available kit, or may be obtained as a monoclonal antibody or a polyclonal antibody using known means. .
  • the monoclonal antibody is immunized with a desired sensitizing antigen using a conventional immunization method, and the obtained immune cells are fused with a known parent cell by a normal cell fusion method. Thus, it can be produced by screening the monoclonal antibody-producing cells.
  • a monoclonal or polyclonal antibody may be prepared as follows.
  • the sensitizing antigen from which the antibody is obtained is not limited to the animal species from which it is derived, but the mammal, eg, human, mouse or rat, from which the peptide or polypeptide used in the present invention actually originates. Origins are preferred. Of these, human-derived sensitizing antigens are particularly preferred.
  • a human soluble HM1.24 antigen protein when used as a sensitizing antigen, its nucleotide sequence and amino acid sequence are as disclosed in this specification. It can be obtained using gene sequences.
  • those peptides or polypeptides to be fused with the soluble protein 1.24 antigen protein are used as the sensitizing antigen, those peptides or polypeptides may be chemically synthesized, It can be obtained by genetic engineering techniques.
  • the protein, peptide or polypeptide used as a sensitizing antigen its full length may be used, or a fragment thereof may also be used. Examples of the fragment include a C-terminal fragment and an N-terminal fragment.
  • cells expressing a protein, peptide or polypeptide used as a sensitizing antigen can be used as the 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. Teeth, magpies, and primates are used.
  • mice for example, mice, rats, hamsters and the like are used.
  • Egrets are used as egrets.
  • monkeys are used as primates.
  • monkeys of the lower nose for example, cynomolgus monkeys, macaques, baboons, and chimpanzees 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 to an appropriate amount with PBS (Phosphat e-Buffe red Saline), physiological saline, or the like, and the suspension is mixed with an ordinary adjuvant, for example, an appropriate amount of Freund's complete adjuvant, if desired.
  • PBS Phosphat e-Buffe red Saline
  • physiological saline or the like
  • an ordinary adjuvant for example, an appropriate amount of Freund's complete adjuvant, if desired.
  • a suitable carrier can be used during immunization of the sensitizing antigen. Immunize in this way and have the desired An increase in the antibody level is confirmed by a conventional method.
  • the blood of the mammal sensitized with the antigen is taken out.
  • the serum is separated from the blood by a known method.
  • a serum containing the polyclonal antibody may be used as the polyclonal antibody, and if necessary, a fraction containing the polyclonal antibody may be further isolated from the serum.
  • immune cells may be removed from the mammal and subjected to cell fusion.
  • preferred immune cells used for cell fusion include splenocytes, in particular.
  • Mammalian myeloma cells as the other parent cells to be fused with the immune cells have already been described.
  • Various known cell lines for example, P3 (P3x63Ag8.653) (Kearney, JF et al., J. Immunol. (1979) 123, 154 8-1550), P3x63Ag8.
  • the cell fusion of the immune cells and myeoma cells is basically performed by a known method, for example, the method of Milstein et al. (Galfre, G. and Milstein, Methods Enzymol. (1981) 73, 3-46. ) Etc. You.
  • the cell fusion is performed, for example, in a normal nutrient culture in the presence of a cell fusion promoter.
  • a fusion promoter for example,
  • Polyethylene glycol (PEG), Sendai virus (HVJ) and the like can be used, and if necessary, an auxiliary agent such as dimethyl sulfoxide can be added to increase the fusion efficiency.
  • the ratio of the use of the immune cells to the myeloma cells is preferably, for example, 1 to 10 times the number of the immune cells to the myeloma cells.
  • the culture medium used for the cell fusion for example, an RPMI 1640 culture medium, a MEM culture medium suitable for the culture of the myeloma cell line, and other ordinary culture mediums used for cell culture of this type are used. It is possible, and serum supplement such as fetal calf serum (FCS) can be used in combination.
  • FCS fetal calf serum
  • a predetermined amount of the immune cell and the myeloma cell are mixed well in the culture medium, and a PEG solution previously heated to about 37 ° C., for example, a PEG solution having an average molecular weight of about 1000 to 6000 is prepared.
  • a PEG solution previously heated to about 37 ° C.
  • a PEG solution having an average molecular weight of about 1000 to 6000 is prepared.
  • the desired fused cells are formed by adding and mixing at a concentration of 30 to & 0% (w / v). Subsequently, by repeatedly adding an appropriate culture solution and centrifuging to remove the supernatant, a cell fusion agent or the like unfavorable for the growth of the hybridoma can be removed.
  • the hybridoma is selected by culturing it in a normal selective culture medium, for example, a HAT culture medium (a culture medium containing hypoxanthine, aminopterin and thymidine).
  • a HAT culture medium a culture medium containing hypoxanthine, aminopterin and thymidine.
  • the culture in the HAT culture solution is continued for a time sufficient for the death of cells other than the target hybridoma (non-fused cells), usually several days to several weeks.
  • screening and cloning of hybridomas producing the desired antibody are performed by the usual limiting dilution method.
  • human lymphocytes such as those infected with Epstein-Barr virus, are sensitized in vitro with peptides or polypeptides, their expressing cells or their lysates, and sensitized. It is also possible to fuse a lymphocyte with a myeloma cell having permanent division ability derived from human, for example, U266, to obtain a hybridoma producing a desired human antibody having a peptide or polypeptide binding activity ( JP-A-63-17688).
  • a transgenic animal having a human antibody gene repertoire is immunized with a peptide or polypeptide serving as an antigen, an expression cell thereof, or a lysate thereof to obtain antibody-producing cells.
  • a human antibody to the peptide or polypeptide used in the present invention may be obtained using a hybridoma fused to a single cell (see International Patent Application Publication Nos. 2-03918, W093-2227, and simplified version 4). -02602, W094-25585, Figures 6-33735 and 6-34096;).
  • the hybridomas producing monoclonal antibodies produced in this way can be subcultured in ordinary culture medium and can be stored for a long time in liquid nitrogen. .
  • a method of culturing the hybridoma according to a usual method and obtaining a culture supernatant thereof, or transferring the hybridoma to a mammal compatible therewith A method of transplanting, growing, and obtaining ascites is used.
  • the former method is suitable for obtaining high-purity antibodies, while the latter method is suitable for mass production of antibodies.
  • cells in which immune cells such as sensitized lymphocytes that produce antibodies are immortalized with oncogenes may be used.
  • the thus obtained monoclonal antibody can also be obtained as a recombinant antibody produced using a gene recombination technique.
  • a recombinant antibody is produced by cloning an antibody gene from an immune cell such as a hybridoma or a sensitized lymphocyte producing the antibody, incorporating the clone into an appropriate solid, introducing the clone into a host, and producing the recombinant antibody.
  • the recombinant antibody can be used (see, for example, Borrebaeck, CA K. and Larrick, JW, THERAPEUTIC MONOCLONAL ANTIBODIES, Published in the United Kingdom by MACMILLAN PUBLISHERS LTD, 1990). .
  • the antibody used in the present invention may be an antibody fragment or a modified antibody as long as it has a desired binding activity.
  • the antibody fragment include Fab, F (ab ') 2, Fv, or a single chain Fv (scFv) in which an Fv of an H chain and an L chain are linked by an appropriate linker.
  • an antibody is treated with an enzyme, for example, 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.
  • a suitable host cell eg, Co, MS et al., J. Immunol. (1994) 152, 2968-2976; Better, M.
  • the antibodies detected or measured by the immunochemical measurement method of the present invention include the above-mentioned antibodies, for example, antibodies produced by hybridomas, recombinant antibodies, chimeric antibodies, and humanized antibodies. Either may be used.
  • the antibody expressed and produced as described above can be separated from the host inside and outside the cell and from the host and purified to homogeneity.
  • the amount of the antibody used in the present invention Separation and purification may be performed by using the separation and purification methods used in ordinary proteins, and are not limited at all.
  • Columns used for affinity chromatography include a protein tin A column and a protein G column.
  • columns using Protein A column include Hyper D, POROS, Separose FF (Pharmacia), and the like.
  • Chromatography other than affinity chromatography includes, for example, ion-exchange chromatography, hydrophobic chromatography, gel filtration, reversed-phase chromatography, and adsorption chromatography. Power S (Strategies for Protein Purification and Character Characterization: A Laboratory Course Manual. Ed Daniel. Marshak et al., Cold Spring Harbor Laboratory Press, 1996). These chromatographies can be performed using liquid chromatography such as HPLC and FPLC.
  • ELISA Enzyme immunoassay
  • RIA radioimmunoassay
  • fluorescent antibody method for example, ELISA, EIA (enzyme immunoassay), RIA (radioimmunoassay) or fluorescent antibody method.
  • Hypri-Doma HM1.24 which produces anti-HM1.24 antibody, was submitted to the National Institute of Advanced Industrial Science and Technology (1-3-1 Higashi, Tsukuba, Ibaraki Prefecture) by the National Institute of Advanced Industrial Science and Technology (FERM) on September 14, 1995. Deposited internationally as BP-5233 under the Pastoral Treaty.
  • Another embodiment of the present invention is a method of measuring the antigen-binding activity of an anti-HM1.24 antibody contained in a test sample and controlling the quality of the anti-HM1.24 antibody using the same. In a drug containing an antibody as an active ingredient, it is important that not only the amount of the antibody but also the biological activity of the antibody be appropriately maintained.
  • the biological activity of an antibody is the antigen-binding activity
  • confirming the level of an antibody that retains the antigen-binding activity in a pharmaceutical composition is a matter of determining the height of the active ingredient. It is indispensable for quality control of pharmaceuticals contained as a product.
  • the method of the present invention relates to a method for appropriately controlling the quality of a drug containing an anti-bandit 1.24 antibody as an active ingredient, and an anti-HM1.24 antibody and an anti-HM1.24 antibody whose quality is appropriately controlled.
  • a pharmaceutical composition containing an antibody as an active ingredient is provided.
  • the method of the present invention provides a method for producing an anti-HM1.24 antibody and a method for producing a pharmaceutical composition containing an anti-HM1.24 antibody as an active ingredient by providing an appropriate quality control method. Things.
  • HEF expression vector International Patent Application Publication No.W092-19759 containing the EF1a promoter prepared by digestion with EcoR I (Takara Shuzo) and Notl (Takara Shuzo), and an Ig leader sequence. Coat the HA tag dos genetic Napea the (Ame r sham Pharmac ia Co.), 50 mmo l / L Tr is -.
  • the Escherichia coli transformant was cultured overnight at 37 ° C in LB medium containing 50 / xg / mL ampicillin. From this culture, the alkaline method (Molecular loning: A Laboratory Manual, Sambrook et al.) Plasmid DNA was prepared according to the Old Ipring Harbor Laboratory Press, (1989)).
  • the gene in the extracellular region of the HM1.24 antigen was amplified by PCR using a Thermal Cycler (Perk in Elmer Cetus).
  • a Thermal Cycler Perk in Elmer Cetus
  • 100 pmol of the primers shown in SEQ ID NOs: 3 and 4 10 mmol / L Tris-HC1, pH 8.3, 50 mmol / L KC1,
  • a mixture containing 0.1 mmol / L dNTPs (dATP, dGTP, dCTP, dTTP), 1.5 mmol / LM gCl 2 and 5-unit DNA polymerase Ampli Taq (Perkin Elmer Cetus) was first mixed at 94 ° C. After the first denaturation at The cycle was performed 30 times at 94 ° C for 1 minute, at 55 ° C for 1 minute, and at 72 ° C for 1 minute, and finally, incubation was performed at 72 ° C for 10 minutes.
  • This PCR product was used as the gene for the extracellular region of HM1.24 antigen (SEQ ID NO: 5), and the plasmid DNA digested with Kpnl and BamHI was combined with 50 mmol / L Tris_HCl, pH 7.6, 10 mmol / L MgCl 2 , Incubate for 3 hours at 16 ° C in a reaction mixture containing 10 mM dithiothreitol, 1 mmol / L ATP, 50 mg / mL polyethylene glycol and 1 unit T4 DNA ligase (T0Y0B0). Connected. In the same manner as described above, the ligation reaction mixture was added to E. coli DH5o! Competent cells to obtain an E. coli transformant, from which plasmid DNA was prepared. This plasmid DNA was designated as HA-tagged soluble antigen expression plasmid, psHM.
  • the base sequence determination of psHM and psHM164 was performed using an automatic DNA sequencer (Applied Biosystem Inc.) and an aq Dye terminator / ycle Sequencing kit (.Applied Biosystem Inc.). Was performed according to The primers shown in SEQ ID NOs: 8 and 9 (manufactured by Certainly Technology) were used. As a result, it was confirmed that the fusion protein (SEQ ID NO: 10 and 11) in which the HA tag peptide was linked to the soluble antigen was expressed.
  • Example 2 Establishment of cells expressing high levels of soluble human HM1.24 antigen
  • the linearized expression vectors (psHM and psHM164) obtained by digestion with Pvul (GIBC0-BRL) were prepared by the electroporation method. CH0 cell M The gene was introduced into the Bll strain (provided by the Medical Research Council collaboration Center).
  • the electroporated cells were transferred to 100 mL of 10% FCS (GIBC0-BRL), 1% penicillin-streptomycin (GIBC0-BRL). ) Suspended in -MEM (without nucleotides) selection medium (GIBC0-BRL), 96-well flat bottom plate (FALCON) at 100 ⁇ L / well (1 ⁇ 10 4 cells / well) The seeds were sown. After incubation overnight at 37 ° C, 5% C0 2 incubator one further 100 ⁇ ⁇ 7 Ueru added selection culture areas, was carried out selector tion.
  • FCS GIBC0-BRL
  • GIBC0-BRL penicillin-streptomycin
  • an assay was performed by a sandwich ELISA (see the section on cell line selection), and 24 clones that highly expressed HA-sHM or HA-sHM164 were selected and expanded on a 24-well plate. (LmL / well). After confirming stable growth of the clones selected in the nucleic acid-free medium, the clones were further subjected to atssay and squeezed to 10 clones each.
  • the ELISA for soluble human HM1.24 described below was performed as follows. In order to select high-producing strains, the production of soluble antigen was determined using anti-HA antibody (Boehringer Mannheim) and humanized anti-HM1.24 antibody (Koichiro Ono et al. The 20th Annual Meeting of the Molecular Biology Society of Japan, etc. The cell lines were selected by comparison using sandwich ELISA with abstract 3-501-P-478). Since the purified antigen was not obtained, the antigen concentration was not known, so the concentration was compared taking into account the number of cells when ELISA was performed.
  • E. coli having a plasmid containing light chain purge ion a was designated as Escherichia coli DH5a ( P UC19-VLa-AHM-gK) by the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology, and on August 29, 1997 as FERM BP-5645. ⁇ Deposited internationally under the EST.
  • Escherichia coli having a plasmid containing the heavy chain purgion s of a humanized anti-HM1.24 antibody is referred to as Escherichia coli DH5a (pUC19-RVHs-AHM-gy1).
  • Anti-HA antibody (Boehringer Mannheim) prepared at 1 ⁇ g / mL in Coating Buffer (CB: 0.1 mol / L sodium bicarbonate buffer, pH 9.6, 0.02% sodium azide) was added to a flat-bottomed 96-well plate (manufactured by Nunc) at 100 L / well, and coated at 4 ° C.
  • CB Coating Buffer
  • dilute buffer (50 mmol / well) to the anti-HA antibody-coated plate washed three times with PBS (-) containing 300 ⁇ L / well 0.05% Tween 20 using a plate washer.
  • CGM / sHM As a positive control, CGM / sHM (Kyoko Oyoro et al., Japanese Society of Hematology 60 times, General title 690) was used. Next, a humanized anti-HM1.24 antibody (Koichiro Ono et al. 20th Annual Meeting of the Molecular Biology Society of Japan 3-501-P-478) was diluted to 1 ⁇ g / mL in the same washed plate. 100 ⁇ L / mL of the solution prepared with the buffer was added and reacted at room temperature for 1 hour.
  • alkaline phosphatase-labeled hidge anti-human IgG antibody (manufactured by BI0S0URCE) diluted 5,000-fold with a dilution buffer was added in 100 ⁇ l / 7 ⁇ l portions, and the mixture was added For 1 hour.
  • SIGMA104 p-nitrophenyl phosphate sodium salt hexahydrate: manufactured by SIGMA
  • SB 0.05 mol / L sodium bicarbonate
  • Buffer solution pH 9.8, 10 mmol / L MgCl 2
  • absorbance of 405 nm-655 nm with MICR0PLATE READER (BIO-RAD) was measured.
  • DXB11 cells transfected with soluble HM1.24 antigen (sHM) lacking the transmembrane region of HM1.24 antigen and sHM164 expression vector lacking the C-terminus of sHM with HA tag added 10 strains each ( sHM producing strain::!-1, 8-2, 9-3, 11-4, 14-5,-16, -17, -22, -23, -24, sHM164 producing strain: 164-1, -2 , 13, 15, -6, 17, 18, 1, 10, 1, 13, -16) (Continued, 10 nmol / L Methotrexate in a 25 cm 2 flask) M TX) containing medium (a-MEM (GIBC0-BRL), 10% FCS (GIBC0-BRL, 1% penicillin-streptomycin (GIBC0-BRL), 100 nmol / L) MTX (manufactured by SIGMA)).
  • a-MEM GIBC0-BRL
  • FCS GIBC0-BRL
  • the amount of antigen produced in the culture supernatant (3 day culture) was measured by ELISA.
  • Gene amplification was performed with 100 nmol / L MTX for 11-4, a sHM-producing strain, and sHM164-producing strains, 164-2 and 164-1-13, which had high expression levels and had sufficiently increased cells (post-group B). .).
  • the remaining strains were not well adapted to 10 nmol / L MTX, so we continued to culture in 10 nmol / L MTX medium.
  • the amount of antigen produced in the culture supernatant was measured by ELISA, and the sHM-producing strains 8-2, 9-3, 14-16 and 14-24 and the SHM164-producing strain 164 with high expression levels were measured.
  • — 1, 164—5 and 164—8 Gene amplification was performed using 100 nmol / L MTX (see Subsection B.). At this point, 164-13, which had the highest production, was converted to CGM / sHM (Kyoko Oyoro et al. The antigen production was about 10 times that of the general abstract 690).
  • ⁇ -MEM GIBC0-BRL
  • FCS GIBC0-BRL
  • SIGMA penicillin-strain Tomycin
  • sHM-producing strains 11-4 and sHM164-producing strains 164-2 and 164-13 were sHM-producing strains 8-2, 9-3, 14-16 and 14-24 and sHM164-producing strains 164-1 and 164.
  • the amount of antigen production in the culture supernatant (2 day culture) was measured by ELISA.
  • sHM-producing strains 8-2 and sHM164-producing strains 164-2 and 164-13 which have higher or possibly higher yields.
  • the amount of antigen produced in the supernatant was measured again by ELISA.
  • the 164-2 strain which produced the highest amount, produced more than 5 times more antigen than CGM / sHM (Kyoko Oyoro et al., General Presentation 690 of the 60th Annual Meeting of the Japanese Society of Hematology).
  • the 164-2 strain which produced the highest amount, showed slightly lower antigen production than CGM / sHM, and the production tended to decrease. For this reason, we decided to carry out syndial cloning by the limiting dilution method.
  • the cells were passaged from the wells producing the highest yield (8-2: 6 wells, 164-2: 15 wells, 164-13: 9 wells) to 24-well plates. Prepare two plates for subculture and assay.When the assay plate becomes confluent, change the medium, culture for 3 days, and measure the amount of antigen production in the culture supernatant by ELISA. .
  • TBS Tris buffer
  • FBS FBS
  • TBS-T Tris buffer
  • Teen 20 50 ⁇ g / mL mouse anti-HM1.24 antibody (Blood (1994) 84, 1922-1930) and shake at 25 ° C. For 1 hour. TBS-T was added and the buffer was changed 6 times at 10 minute intervals while shaking at room temperature to wash the membrane.
  • the alkaline phosphatase-labeled goat anti-mouse IgG antibody (manufactured by Zymed)), which was diluted 2000-fold with TBS-T, was used as a secondary antibody while shaking at 25 ° C in the same manner. The reaction was performed for 30 minutes.
  • the membrane was washed by adding TBS-T and shaking at 25 ° C for 10 minutes six times.
  • This membrane was prepared using BCIP / NBT chromogenic substrate (manufactured by Promega) with 33 ⁇ L of Nitrobrute Trazolium ( ⁇ ⁇ ) and 16.5 ⁇ L of 5-Mouth-4 -Immerse the membrane in a western detection buffer (0.1 mol / L NaCl, 0.1 mmol / L Tris-HCl buffer containing 5 mmol / L MgCl 2 ; pH 9.5) containing indolinole phosphate (BCIP) to develop color I let it.
  • BCIP indolinole phosphate
  • the plate was washed with distilled water and HM1.24 antigen was detected.
  • the four clones (164-2-1, 164-2-13, 164-2-17, and 164-2-31) obtained were both in a reduced state, and were heterologous by glycosylation. Soluble antigen was detected as a broad band of 23-28 kDa, which is considered genetic. However, 18 kDa
  • Soluble human HM1.24 antigen was purified from the culture supernatant of CH0 cells expressing soluble human HM1.24 antigen. Culture solution of soluble human HM1.24 antigen-expressing CH0 cells
  • the buffer was replaced with PBS using a Fast Desalting HR10 / 10 column after diluting 5-fold with PBS. From the absorption at 280 nm, the concentration of the soluble human HM1.24 antigen obtained was estimated to be about 0.382 mg / mL, and a total of 42 mL of purified product was obtained. Purity was 95% or more based on the peak area ratio of reversed phase chromatography.
  • Example 4 Construction of an ELISA system using purified soluble human HM1.24 antigen
  • An anti-HA antibody (Boehringer Mannheim) was coated with a coating buffer (CB: 0.1 mol / L sodium bicarbonate buffer, pH 9.6). , 0.02% sodium azide) at a concentration of 1 ⁇ g / mL was added to a flat-bottom 96-well plate (manufactured by Nunc) at 100 ⁇ ! 7-well and coated at 4 ° C.
  • CB 0.1 mol / L sodium bicarbonate buffer, pH 9.6
  • 0.02% sodium azide at a concentration of 1 ⁇ g / mL was added to a flat-bottom 96-well plate (manufactured by Nunc) at 100 ⁇ ! 7-well and coated at 4 ° C.
  • the purified HM1.24 antigen diluted 750-fold, 2250-fold, 6750-fold, 20250-fold, or 60750-fold with the dilution buffer is 100 ⁇ l /
  • the reaction mixture was added and reacted at room temperature for 1 hour.
  • human-type anti-HM1.24 antibody (Koichiro Ono et al. 20th Annual Meeting of the Molecular Biology Society of Japan 3-501-P-478) was diluted to 1 g / mL in the washed plate.
  • the solution prepared with the buffer solution was added at 100 / well, and reacted at room temperature for 1 hour.
  • an alkaline phosphatase-labeled sheep anti-human IgG antibody (manufactured by BI0S0URCE) diluted 5,000-fold with a dilution buffer was added at 100 / xL / well, and reacted at room temperature for 1 hour. .
  • the plate is washed 5 times, and the substrate buffer (SB: 0.05 mol / L sodium bicarbonate buffer, pH 9.8, 10 mmol / L MgCl 2 ) is used with SIGMA104 (manufactured by SIGMA) as the substrate.
  • SIGMA104 manufactured by SIGMA
  • 100 mg / well of 1 mg / mU was added to each well to develop color, and the absorbance at 405 nm-655 nm was measured with a microplate reader (manufactured by BI0-RAD).
  • a standard curve of the anti-HM1.24 antibody was obtained, and it was judged that use at a 5000-fold dilution was appropriate.
  • Fig. 5 shows the standard curve of the humanized anti-HM1.24 antibody in the ELISA system when the purified antigen was used at 5000-fold dilution. The measurement limit was about 500 pg / mL. .
  • a mouse anti-HM1.24 monoclonal antibody-producing hybridoma was prepared according to the method described in Goto, T. et al., Blood (1994) 84, 1992-1930.
  • KPC-32 Screening of the antibodies in the hybridoma culture supernatant was performed by Cell ELISA using KPC-32 (Posner, MR et al., J. Immunol. Methods (1982) 48, 23). 5Xl0 4 pieces of KPC-32 were suspended in 50 ml of PBS, 96-well plates (U-bottomed, Corning, Iwaki, Ltd.) was dried dispensed 37 ° C De ⁇ to. After blocking with PBS containing 1% serum albumin (BSA), the hybridoma culture supernatant was added and incubated at 4 ° C for 2 hours.
  • BSA serum albumin
  • the reaction was stopped with 1 mol / L sulfuric acid, and the absorbance at 492 nm was measured using an ELISA reader (manufactured by Bio-Rad).
  • the culture supernatant of the positive hybridoma was adsorbed to human serum in advance, and the reactivity to other cell lines was screened by ELISA. Positive hybridomas were selected and their reactivity to various cells was determined by flow cytometry.
  • the selected hybridoma clone was cloned twice and injected into the abdominal cavity of a pristane-treated BALB / C mouse to obtain ascites.
  • Monoclonal antibodies were purified from mouse ascites by precipitation with ammonium sulfate and protein A affinity chromatography kit (Ampure PA, manufactured by Amersham). Purified antibodies were obtained using a Quick Tag FITC binding kit (Boehringer Mannheim). F ITC labeled.
  • HM1.24 a hybridoma clone most useful for flow cytometry analysis and having CDC activity against RPMI 8226 was selected and named HM1.24.
  • the subclass of the monoclonal antibody produced by this hybridoma was determined by ELISA using a subclass-specific anti-mouse porcupine antibody (Zymed).
  • Anti HM1. 24 antibody had a subclass of I g G2a kappa.
  • Hypri-Doma HM1.24 which produces anti-HM1.24 antibody, was submitted to the Institute of Biotechnology, Institute of Industrial Science and Technology (1-3 1-3, Higashi, Tsukuba, Ibaraki Prefecture) on September 14, 1995, as FERM BP 5233. Was deposited internationally under the Budapest Treaty.
  • a humanized anti-HM1.24 antibody was obtained by the following method.
  • Total RNA was prepared from the hybridoma HM1.24 prepared in Reference Example 1 by a conventional method. From this, cDNA encoding the mouse antibody V region was synthesized and amplified by the polymerase chain reaction (PCR) method and the 5'-RACE method. A DNA fragment containing the gene encoding the mouse V region was obtained, and each of these DNA fragments was ligated to a plasmid pUC-based cloning vector and introduced into E. coli competent cells to obtain an E. coli transformant.
  • PCR polymerase chain reaction
  • the above plasmid was obtained from this transformant, the nucleotide sequence of the cDNA coding region in the plasmid was determined according to a conventional method, and the complementarity determining region (CDR) of each V region was further determined.
  • CDR complementarity determining region
  • the human antibody REI chain is used as the human antibody L chain, and the framework region (FR) 1-3 is used as the human antibody H chain using FR1-3 of human antibody HG3 and FR4 is used as the human antibody H chain.
  • FR4 of human antibody JH6 Used FR4 of human antibody JH6. The amino acid of FR in the V region of the H chain was substituted so that the CDR-grafted antibody formed an appropriate antigen-binding site.
  • each of the genes was separately introduced into a HEF vector, and the humanized anti-HM1.24 antibody was humanized.
  • a vector for expressing the L chain or H chain of the HM1.24 antibody was prepared.
  • a cell line producing a humanized anti-HM1.24 antibody was established.
  • the antigen-binding activity and the binding inhibitory activity of the humanized anti-HM1.24 antibody obtained by culturing this cell line on the human amniotic membrane-derived cell line WISH were examined by Cell ELISA.
  • the humanized anti-l.24 antibody has the same antigen-binding activity as the chimeric antibody, and furthermore, the binding inhibition activity using the biotinylated mouse anti-HM1.24 antibody was confirmed by the chimeric antibody or the mouse antibody. It had the same activity as.
  • Escherichia coli having a plasmid containing DNA encoding the L chain V region and H chain V region of the chimeric anti-HM1.24 antibody was Escherichia coli DH5a (pUC19-l.24L-g ⁇ ) and Escherichia coli, respectively.
  • E. coli DH5a (pUC19-1.24H-gy1) was submitted to the Institute of Biotechnology and Industrial Technology, Institute of Industrial Science and Technology (1-1-3 Higashi, Tsukuba, Ibaraki Prefecture) on August 29, 1996. Each FERM BP-5646 * 5 jt / FERM BP-5644 based on Budapest International deposit.
  • E. coli having a plasmid containing DNA encoding the L chain V region a purge ion (SEQ ID NO: 12) and the H chain V region version (SEQ ID NO: 13) of a humanized anti-HM1.24 antibody each Escherichia co li DH5a (pUC19- RVLa- AHM- g k) and Escherichia coli DH5 a (PUC19- RVHr- a ⁇ - gyl) as, Agency of industrial Science technology Research Institute (Higashi, Tsukuba, Ibaraki, 1 On October 29, 1996, FERM BP-5645 and FERM BP-5643 were deposited internationally under the Budapest Treaty.
  • Escherichia coli having a plasmid containing DNA encoding the H chain V region s purge ion (SEQ ID NO: 14) of a humanized anti-HM1.24 antibody is cherichis coli DH5a (pUC19-RVHs-AHM- g ⁇ 1) was reported to the Institute of Biotechnology and Industrial Technology (I 1-3, Higashi 1-3-1, Tsukuba, Ibaraki Prefecture) as FERM BP-6127 on September 29, 1997. And deposited internationally under the Budapest Treaty.
  • the human myeloma cell lines RPMI8226 and U266 were cultured in RPMI1640 medium (GIBC0-BRL) supplemented with 10% fetal calf serum (FBS), and the human myeloma cell line KPMM2 (JP-A-7-236475) was Culture was performed in RPMI1640 medium supplemented with 20% fetal bovine serum.
  • RPMI1640 medium GIBC0-BRL
  • FBS fetal calf serum
  • KPMM2 JP-A-7-236475
  • the pCOSl expression vector was constructed by deleting the genes contained by digestion with EcoRI and Yale from HEF-PMh-gyl (see W092-19759) and ligating EcoRI-Notl-BamHI Adapter (Takara Shuzo). did.
  • the cells are washed with a phosphate buffer (PBS), and the cells are washed with PBS containing 5 mM EDTA, and the cells are washed with 1% S, 5% FBS, 0.02% NaN 3 with 1-2% caro PBS.
  • PBS phosphate buffer
  • the cell suspension was prepared at X10 6 cells / ml, and the cells were incubated on a panning plate (described later) coated with anti-HM1.24 antibody for 2 hours, and the plate was washed with 5% FBS and 0.02%. The plate was washed gently three times with 3 ml of PBS containing 3% NaN 3. After washing, a Hirt solution (Hitt J., Mol. Biol.
  • the panning plate was prepared as follows. Add 3 ml 3 ⁇ 4HMl. 24 antibody solution (lOii g / ml in 50 mM Tris-HCl, pH 9.5) to a 60 mm dish (Falcon), clean for 2 hours at room temperature, and wash three times with 0.15 M NaCl After that, 3 ml of 5% FBS, 1 mM EDTA, and 0.02% NaN 3 were added, and the cells were settled at room temperature for 2 hours to perform blocking. After removing the blocking solution, the panning plate was stored at -20 ° C until use.
  • plasmid DNA having a cDNA of about 0.9 kb P as an insert was enriched.
  • the nucleotide sequence was determined using the Dye Terminator Cycle Sequencing Kit (Applied Biosystems) using the 373A or 377 DNA Sequencer (Applied Biosystems).
  • clone P3.19 was composed of 1,012 bp cDNA, It was found to have an open reading frame (23-549) encoding an amino acid (FIGS. 6 and 7) (SEQ ID NO: 15).
  • the amino acid sequence predicted from this cDNA showed a structure characteristic of the type II membrane protein, and had two N-type sugar chain binding sites.
  • Library B was subjected to immunoscreening using anti-HM1.24 antibody. That is, a phage library containing 1.5 ⁇ 10 5 independent clones was overlaid on agar together with E. coli NM522 (Pharmacia Biotech), and cultured at 42 for 3.5 hours. After the cultivation, a nitrosenorrelose finole letter (Schleicher & Schue 11) pretreated with 10 mM IPTG was overlaid on the plate, and further cultured at 37 ° C for 3 hours.
  • anti-HM1.24 antibody solution (10 ⁇ g / ml blocking buffer), incubate at room temperature for 1 hour, wash and dilute 5,000-fold diluted alkaline phosphatase-conjugated anti-mouse Ig antibody Serum (picoBlue Immunoscreening kit; Stratagene) was used!]; ⁇ , And incubated at room temperature for 1 hour.
  • the spot that reacted with antibody 0 3 m g / ml- preparative Robusurete Torazori ⁇ beam, 0.15mg / ml 5 -.
  • Bromo-4 Ichiku B row 3 Lee emission chromogenic solution containing indolyl phosphate (lOOmM Tris-HCl , PH 9.5, 100 mM NaCl, 5 mM MgCl 2 ).
  • Escherichia coli DH5 is a plasmid containing the plasmid pRS38-pUC19 in which DNA encoding a human protein having the same sequence as the human HM1.24 antigen protein was inserted between the Xbal cleavage sites of pUC beta.
  • a P RS38-pUC19
  • FERM BP-4434 It has been deposited internationally under the Budapest Treaty.
  • a CH0 transformed cell line transfected with P3.19 was established.
  • P3.19 clone After being introduced into CHO cells by the ratioon method, the cells were cultured in the presence of 500 g / ml G418 (GIBC0-BRL) to obtain a HM1.24 antigen-expressing CH0 cell line.
  • FACS buffer PBS ( ⁇ ) / 2% FCS / 0.1% NaN 3
  • HM1.24 antibody was added, and the mixture was reacted on ice for 30 minutes.
  • FACS buffer PBS ( ⁇ ) / 2% FCS / 0.1% NaN 3
  • HM1.24 antibody was added, and the mixture was reacted on ice for 30 minutes.
  • GAM-FITC solution 25 / zg / ml in FACS buffer; Becton Dickinson
  • the cells were resuspended in 600 ⁇ l of FACS buffer and measured by FACScan (Becton Dickinson).
  • UPC10 was used as a negative control antibody.
  • the cell lysis buffer method 50 mM sodium folate, 150 mM NaCl, 0.5% sodium deoxycholate, 1% Nonidet P_40, 0.1 mg / ml phenylenomethine
  • Ultrasonic crushing was performed in lesnorefoninolefnoreolide and protease inhibitor IJ Cactenyl [Boehringer Mannheim]) to obtain a solubilized fraction.
  • the solubilized fraction was added to Sepharose 4B beads conjugated with anti-HM1.24 antibody. After centrifugation, the precipitate was separated by SDS-PAGE (12% gel) and transferred to a PVDF membrane.
  • the PVDF membrane was reacted with an anti-HM 1.24 antibody, followed by POD-anti-mouse IgG, and then detected using an ECL kit (Amersham).
  • P3.19 encodes a protein consisting of 180 amino acids with an estimated molecular weight of 19.8 kDa, and has two N-type sugar chain binding motifs (Fig. 6). Therefore, it was considered that the presence of those having different molecular weights observed by immunoprecipitation was due to differences in N-glycan modification. In fact, immunoprecipitates have been found to bind to several lectins. Industrial applicability

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Abstract

Cette invention a trait à un procédé permettant de produire, avec un rendement élevé, une protéine d'antigène HM1 . 24, hautement purifiée et soluble. Elle porte, plus précisément, sur un procédé de production de domaine extracellulaire d'antigène HM1 . 24 soluble, lequel procédé se caractérise par la culture de cellules animales transformées par un vecteur d'expression contenant un promoteur (A1)EF1α et un gène codant un antigène HM1 . 24 soluble dépourvu du domaine intracellulaire ligaturé en aval du promoteur. On procède ensuite à l'isolation dans la culture de l'antigène HM1 . 24 soluble et à sa purification.
PCT/JP2001/002964 2000-04-06 2001-04-05 Dosage immunologique d'anticorps anti hm1 . 24 WO2001077362A1 (fr)

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Cited By (21)

* Cited by examiner, † Cited by third party
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WO2004039398A1 (fr) * 2002-10-30 2004-05-13 Chugai Seiyaku Kabushiki Kaisha Vaccins contre le cancer utilisant le hm1.24
JP2010503397A (ja) * 2006-09-13 2010-02-04 アボット・ラボラトリーズ 細胞培養の改善
US8911964B2 (en) 2006-09-13 2014-12-16 Abbvie Inc. Fed-batch method of making human anti-TNF-alpha antibody
US8921526B2 (en) 2013-03-14 2014-12-30 Abbvie, Inc. Mutated anti-TNFα antibodies and methods of their use
US8946395B1 (en) 2013-10-18 2015-02-03 Abbvie Inc. Purification of proteins using hydrophobic interaction chromatography
US9017687B1 (en) 2013-10-18 2015-04-28 Abbvie, Inc. Low acidic species compositions and methods for producing and using the same using displacement chromatography
US9062106B2 (en) 2011-04-27 2015-06-23 Abbvie Inc. Methods for controlling the galactosylation profile of recombinantly-expressed proteins
US9067990B2 (en) 2013-03-14 2015-06-30 Abbvie, Inc. Protein purification using displacement chromatography
US9085618B2 (en) 2013-10-18 2015-07-21 Abbvie, Inc. Low acidic species compositions and methods for producing and using the same
US9150645B2 (en) 2012-04-20 2015-10-06 Abbvie, Inc. Cell culture methods to reduce acidic species
US9181337B2 (en) 2013-10-18 2015-11-10 Abbvie, Inc. Modulated lysine variant species compositions and methods for producing and using the same
US9181572B2 (en) 2012-04-20 2015-11-10 Abbvie, Inc. Methods to modulate lysine variant distribution
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US9206390B2 (en) 2012-09-02 2015-12-08 Abbvie, Inc. Methods to control protein heterogeneity
US9234033B2 (en) 2012-09-02 2016-01-12 Abbvie, Inc. Methods to control protein heterogeneity
US9249182B2 (en) 2012-05-24 2016-02-02 Abbvie, Inc. Purification of antibodies using hydrophobic interaction chromatography
US9499614B2 (en) 2013-03-14 2016-11-22 Abbvie Inc. Methods for modulating protein glycosylation profiles of recombinant protein therapeutics using monosaccharides and oligosaccharides
US9550826B2 (en) 2013-11-15 2017-01-24 Abbvie Inc. Glycoengineered binding protein compositions
US9598667B2 (en) 2013-10-04 2017-03-21 Abbvie Inc. Use of metal ions for modulation of protein glycosylation profiles of recombinant proteins
CN108295242A (zh) * 2016-06-28 2018-07-20 深圳先进技术研究院 用于预防和/或治疗银屑病药物组合物、cd317胞外段蛋白的应用
CN112462071A (zh) * 2020-11-13 2021-03-09 北京元恩生物技术有限公司 一种利巴韦林专用酶联免疫检测试剂盒

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US8652839B2 (en) 2002-10-30 2014-02-18 Chugai Seiyaku Kabushiki Kaisha HM1.24—utilizing cancer vaccines
JP2006508094A (ja) * 2002-10-30 2006-03-09 中外製薬株式会社 Ｈｍ１．２４を応用した癌ワクチン
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