WO2000010384A1 - Animaux non humains presentant une disruption au niveau du gene de cmp-n-acetyl-neuraminate hydroxylase - Google Patents

Animaux non humains presentant une disruption au niveau du gene de cmp-n-acetyl-neuraminate hydroxylase Download PDF

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WO2000010384A1
WO2000010384A1 PCT/JP1999/004561 JP9904561W WO0010384A1 WO 2000010384 A1 WO2000010384 A1 WO 2000010384A1 JP 9904561 W JP9904561 W JP 9904561W WO 0010384 A1 WO0010384 A1 WO 0010384A1
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human
acid
gene
glycoprotein
cnhase
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PCT/JP1999/004561
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English (en)
Japanese (ja)
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Toshisuke Kawasaki
Yasunori Kozutsumi
Isao Ishida
Kazuma Tomizuka
Hitoshi Yoshida
Noboru Inoue
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Kirin Beer Kabushiki Kaisha
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out

Definitions

  • the present invention relates to a non-human mammal deficient in the cytidine-phosphate-N-acetylneuraminic acid hydroxylase gene.
  • Sialic acid is a generic name for the acyl derivative of neuraminic acid added to the terminal of the hetero-oligosaccharide chain contained in gandriside glycoprotein in the living body.
  • N-Acetylneuraminic acid (hereinafter abbreviated as Neu5Ac) is the most ubiquitous in nature, followed by N-glycolylneuraminic acid (abbreviated as Neu5Gc). ).
  • Neu5Gc is produced by hydroxylation of Neu5Ac, and its biosynthetic enzyme is cytidinemonophosphate-N-acetylneuraminic acid (hereinafter abbreviated as CMP-NeuAc).
  • Cytidine-phosphate mono-N-acetylneulaminate hydroxylase CMP-NeuAc hydroxylase; hereinafter abbreviated as CNHase. Shaw and Schauer, Biol. Chem. Hoppe-Sey 1 er, 369, 477- 486, 1988
  • CNHase Cytidine-phosphate mono-N-acetylneulaminate hydroxylase
  • Shaw and Schauer Biol. Chem. Hoppe-Sey 1 er, 369, 477- 486, 1988
  • the enzyme activity of hydroxylating free Neu5Ac Mukuria et al., Biochem. J., 305, 459-46 4, 1995.
  • Neu5Gc is not found in healthy humans found in various mammals and has antigenicity against humans.
  • An antibody against glycosphingolipid having Neu5Gc is known as a Hanganatziu-Deicher antibody (HD antibody) (Biochemical Dictionary 2nd edition, Iwanami Shoten, 1990
  • the CNHase gene (cDNA) can be obtained from mouse liver (Kawano et al., J. Biol. Chem., 270, 16458-16463, 1995; JP-A-6-113838), and pig submandibular gland (Schlen zka et al. , FEBS Lett., 385, 197-200, 1996; W097 / 03200). Although these known technical documents refer to the possibility of CNHase gene disruption in animals or animal cells, they do not disclose genomic DNA essential for disruption of the CNHase gene by homologous recombination.
  • the biosynthetic pathway of Neu5Gc is reported to include not only a CNHase-mediated pathway but also an enzyme activity that hydroxylates free Ne5Ac. (Mukuria et al, Biochem. J., 305, 459-464, 1995), it is a biochemical component in non-human mammals and cells that lack the CNHase gene. Therefore, it could not be easily predicted that Neu5Gc would not be contained.
  • An object of the present invention is to provide a non-human mammal deficient in the CNHase gene and a non-human mammal cell deficient in the CNHase gene. Disclosure of the invention
  • the present inventors have conducted intensive studies to achieve the above object, and as a result, succeeded in producing a non-human mammal deficient in the CNHase gene by the target gene homologous recombination method.
  • the present invention provides a non-human mammal deficient in the CNHase gene.
  • the non-human mammal include a mouse.
  • the present invention also provides a non-human mammal cell deficient in the CNHase gene.
  • non-human mammalian cells include cells derived from mice. These non-human animals and non-human animal cells are useful because they can be used particularly for the production of glycoproteins administered to humans.
  • Deletion of the CNHase gene may be due to a target gene homologous recombination method for the CNHase gene in the genomic DNA of a non-human mammal or non-human mammal cells.
  • the non-human mammal is preferably an ungulate or a rodent.
  • the non-human mammal cell may be derived from a non-human mammal deficient in the above CNHase gene, and may be obtained by subjecting a cell derived from a non-human mammal to a treatment in which the CNHase gene is deficient. It may be something.
  • the present invention provides a method for producing a Neu5Gc-free glycoprotein in a non-human mammal or a non-human mammal cell deficient in the CNHase gene.
  • a method for producing a glycoprotein containing no G c are often useful proteins, preferably for human consumption or medical use, and more preferably for erythropoietin, G—CSF, TP. o or site immunoglobulins (antibodies).
  • the present invention are often useful proteins, preferably for human consumption or medical use, and more preferably for erythropoietin, G—CSF, TP. o or site immunoglobulins (antibodies).
  • glycoproteins that do not contain Neu5Gc and that are produced in non-human mammals or non-human mammalian cells deficient in the CNHase gene.
  • glycoproteins include human antibodies.
  • the present invention provides a non-human animal that produces a glycoprotein that does not contain Neu5Gc.
  • glycoproteins include human antibodies.
  • the present invention also provides a non-human mammal deficient in the CNHase gene and a non-human animal that produces a protein to produce a glycoprotein that does not contain Neu5Gc. And a method for producing a non-human animal.
  • the glycoprotein is preferably a useful protein as a gene product of a foreign gene, and is preferably used for human edible or medical use, and is more preferably used for erythropoietin, G — Cytokines such as CSF and TPO or human immunoglobulins (antibodies).
  • non-human mammals and non-human mammalian cells deficient in the CNHase gene of the present invention do not contain Neu5Gc as a biochemical component
  • the non-human mammals and non-human mammals do not contain Neu5Gc as a biochemical component.
  • Mammalian cells are useful as hosts that produce glycoproteins that do not contain Neu5Gc in their sugar chains.
  • the CNHase gene deficiency treatment of a non-human mammal or a non-human mammal cell is preferably performed by a homologous recombination method of a target gene for the CNHase gene in the genomic DNA of the animal or the cell.
  • the basic technique of the target gene homologous recombination method is well known to those skilled in the art (Experimental Medicine, voll4, No. 20, extra edition, 1996 "Geneta Getting File ⁇ '97"Youtosha; Shinichi Aizawa, Biomanual Series 8, Generating, Yodosha, 1995).
  • the cDNA of the CNHase gene is derived from mouse liver (Kawano et al., J. Biol.
  • genomic DNA fragment of NHase a genomic DNA fragment suitable for producing a targeting vector.
  • a genomic library may be further screened using a part of the obtained genomic DNA fragment as a probe.
  • mice CDNA and pig DNA of CNHase are registered in the GenBank Database.
  • the accession number of mouse cDNA is D21826, and the accession number of pig cDNA is Y15010. Both sequences downloaded from the database were analyzed using DNA sequence analysis software (DNAS ver. 3.7). When the homology of the two DNA sequences was compared by the homology plot method, a region with high homology was found over about 1.3 kb, and the sequence homology reached 86%.
  • the sequence conservation of the CNHase gene in mammals is high, and the genomic DNA fragments of CNHase from non-human mammals other than mice and pigs (such as horses, higgies, and Chinese hamsters) are also
  • the sequence as a probe or using the cDNA of the CNHase of the animal obtained using these cDNA sequences as a probe as a probe the hybridization conditions can be adjusted. It can be obtained by adjusting it appropriately.
  • a targeting vector for gene deletion is constructed.
  • a marker gene for disrupting the gene structure and selecting cells into which the vector has been introduced can be introduced.
  • Neomycin resistance gene, hygromycin resistance gene, puromycin resistance gene and the like can be suitably used.
  • negative genes such as the thymidine kinase gene of the viral virus, the diphtheria toxin A gene (DT-A), etc. good.
  • a genomic DNA fragment containing the third and fourth exons of the CNHase obtained from the genome library was used to construct the fourth exon.
  • a neomycin resistance gene was inserted thereinto, and a targeting vector using DT-A as a negative car was prepared.
  • Gene targeting The targeting vector obtained above is introduced into cells.
  • ES cells derived from the non-human mammal are used.
  • CNHase gene-deficient non-human mammal cell a primary cultured cell derived from the non-human animal or an established cell line is used.
  • the established cells are preferably those known to be suitable for producing useful proteins. Specific examples include CHO cells, BHK cells, C127 cells, and X6 3.6.5.3 cells.
  • DNA transfection methods can be used to introduce the targeting vector into cells, but the method using an electrophoretic method (Shinichi Aizawa, Biomanual Series 8, Gene Targeting, Yodosha) , 195) are simple and suitable.
  • the DNA introduction efficiency can be optimized by appropriately adjusting the applied voltage, the resistance value, the temperature, the concentration of the suspended cells, and the amount of vector DNA to be added depending on the cells used.
  • the targeting vector is selected by a selection method according to the marker gene incorporated in the vector (for example, in a vector containing a neomycin resistance gene, selection in a medium supplemented with G418).
  • Select cells integrated in the genome ⁇ Analyze genomic DNA extracted from each selected cell colony by PCR or Southern blotting, and target vector to homologous region in the genome Select integrated colonies.
  • the colony cells are purified by limiting dilution, etc., and used for subsequent experiments.
  • a non-human mammal or a non-human mammal cell in which both alleles on the genome of CNHase are deficient is obtained by the following method.
  • chimeric animals are prepared.
  • the obtained chimera is crossed with a wild species to obtain F 1.
  • individuals that are homozygous for the CNHase gene deficiency are selected. W 00
  • homologous recombinant homozygotes by culturing the cells in a medium with an increased drug concentration as a selection marker for the homologous recombinant (one-sided deletion) obtained above (Shinichi Aizawa, Bio Manual Series 8, Generating, Yodosha, 1995).
  • the obtained homologous recombination homozygotes can be used as host cells for producing useful proteins in the case of established cells.
  • Homologous Recombination When the homozygote is an ES cell, a chimeric mouse is prepared using the cell. In the organs and tissues of the born chimeric mice, there are cells homozygous for the CNHase gene deficiency.
  • a targeting vector having a different gene from the marker gene used in the first targeting was used. Target alleles that are not deleted.
  • the above-mentioned targeting gene in the targeting vector is constructed by intervening the LoxP sequence during the construction of the targeting vector, so that the Cre enzyme is temporarily Expression allows removal by site-specific recombination (Sauer et al., Pro Natl. Acad. Sci. USA, 85, 5166-, 1988). By using this method, it is possible to obtain homologous recombination homozygotes using one kind of DNA.
  • homologous recombination homozygotes (animals or cells) selected by PCR and the Southern blot method, measure the CNHase activity and measure the Neu5Gc content.
  • Neu5Gc content of a protein having a sugar chain produced by the homologous recombination homozygote (animal or cell) is measured.
  • the CNHase activity is measured by the method described in JP-A-6-113838, and the Neu5Gc content is measured by the method of Hara et al. (Anal. Biochera., 179, 162-166, 1989). Do better.
  • the CNHase activity is below the measurement limit.
  • Neu5Gc is the sum of Neu5Ac and Neu5Gc sialic acids for the recombinant homozygote (animal or cell) and the protein having sugar chains produced by the recombinant homozygote (animal or cell). 2.2% or less, preferably 1.1% or less, and more preferably the measurement limit or less.
  • Non-human animals have been developed that retain part or all of the human antibody gene and produce human antibodies by sensitization with the antigen (Tomizuka et al., Nat Genet. 16, 133-143). , 1997; Mendez et al., Nat Genet 15, 146-156, 1997; Lonberg et al., Nature, 368, 856-859, 1994)).
  • the nucleotide sequence of the CNHase gene is highly conserved among different species. That is, the CNHase gene cDNA and genomic DNA of the above-mentioned animal species can be easily obtained using the previously reported cDNA derived from mouse (Kawano et al., Supra) or pig derived (Schlenzka et al., Supra). Conceivable. Gene targeting in these ES or ES-like cells was performed using the targeting vector prepared from the genomic DNA, and CNHase residues were used in the same manner as in the mouse. Gene-deficient non-human mammals, or preferably non-human mammals deficient in the CNHase gene, can be produced.
  • transgenic mice Schot al., Supra
  • transgenic cells can be used. (Cibe 11i et al., Science 280: 1256, 1998) is also possible.
  • the method of the present invention is achieved through the following steps (1) to (5).
  • the targeting vector of the CNHase gene produced in (2) was introduced into a fetal cell derived from a fetal embryo (Cibelli et al., Science 280: 1256, 1998). The cells into which the vector has been introduced for the expression of are selected.
  • the fibroblasts derived from the drug-resistant fetal embryo are analyzed, for example, by Southern analysis, and clones in which homologous recombination has occurred at the CNHase locus are selected.
  • target homologous recombination in normal somatic cells and fibroblasts derived from mammals (Zheng et al., Proc. Natl. Acad. Sci. USA, 88: 8067, 1991, Arbones et al. Nature Genet., 6:90, 1994, Yanezb, Gene Ther., 5: 149, 1998, Russell et al., Nature Genet., 18: 325, 1998).
  • the homozygous recombinant obtained in (4) above which is
  • one of the CNHase gene loci is deleted (heterozygous CNHase gene deletion). Individuals and their progeny are produced (Cibelli et al.). Science 280: 1256, 1998).
  • a homozygous individual deficient in the CNHase gene deficient in both CNHase loci is produced.
  • Neu5Gc of the glycoprotein produced by the pest individual is 1158 (; and ⁇ 1156 (; not more than 2.2%, preferably not more than 1.1%, based on the sum of both sialic acids. It is preferably below the measurement limit.
  • FIG. 1 shows the partial structure of mouse CNHase genomic DNA.
  • FIG. 2 shows a mouse CNHase genomic DNA fragment used in the construction of a targeting vector for the CNase gene knockout of E14 ES cells.
  • FIG. 3 shows the construction of a targeting vector for knockout of the CNHase gene of E14 ES cells.
  • Figure 4 shows the results of Southern blot analysis of mouse ES cell CNHase gene disrupted clones 15, 17, 20, 41, 42, 43, 69, 70, 74, 77, 108, 152, 185 and 129 wild-type mice. Is shown.
  • FIG. 5 shows the results of Southern blot analysis of wild-type, heterozygous knockout, and honeymoon mice for the CNHase gene.
  • FIG. 6 shows the results of analyzing the blood of the mouse in which the CNHase gene was disrupted by HPLC chromatography.
  • FIG. 7 shows the results of HPLC chromatographic analysis of Neu5Gc added to the blood of mice in which the CNHase gene was disrupted.
  • FIG. 8 shows the results of sialic acid analysis of the antibody purified from the serum of a mouse that produces an antibody containing a human immunoglobulin / c chain by HPLC chromatography.
  • Figure 9 shows the production of antibodies that lack the CNHase gene and contain the human immunoglobulin ⁇ chain.
  • the results of sialic acid analysis of the antibody purified from live mouse serum are shown by HPLC chromatography.
  • FIG. 10 shows the results of sialic acid analysis of an antibody purified from serum of a mouse deficient in the CNHase gene by HPLC close-up chromatography.
  • Restriction enzymes Bg: Bgl 1, H: Hindlll, E: EcoRI, Nh: Nhel, B: BamHI, Ns: NspV, S: Sal I, KprKpnl, Cla: Clal, Xb: Xbal, N: Notl
  • the dotted line indicates the phage vector or vector plasmid pBluescriptSK (+) DNA sequence
  • the solid line indicates the CN Hase gene DNA sequence
  • the white box indicates the third exon
  • the shaded box indicates the fourth exon.
  • ATG indicates the translation start codon.
  • E14 ES cells were obtained from Martin L. Hooper (Nature, 326: 292, 1987).
  • E14 ES cells are derived from 129 mice. It was decided to prepare a CNHase gene knockout vector using genomic DNA clones derived from 129 mice.
  • genomic DNA library a lambda DNA library derived from Stratagene 129SVJ, liver, female, 4-8 weeks was used.
  • a mouse CNHase cDNA (Kawano et al., J. Biol. Chem., 270, 16458-16463, 1995) was used as a probe for screening.
  • the purified plasmid DNA was cleaved at a site with a restriction enzyme Kpnl and used for transfection into E14 ES cells.
  • BamHI-Sall, 600 bp fragment (Fig. 3) was used as a probe to detect homologous recombinants in which the Neo resistance gene was inserted into the fourth exon of the CNHase gene from among the transformant E14 ES cells.
  • Sail is an array in the adapter.
  • the genomic DNA of the transformant E14 ES cells is digested with the restriction enzyme Bg11 and subjected to southern blotting. Alleles that have undergone homologous recombination give a 7 kb band. On the other hand, a heterologous recombinant gives a band of 10 kb or more.
  • the CNHase gene targeting vector prepared in Example 1 was linearized with the restriction enzyme Kpnl (Takara Shuzo). (Shinichi Aizawa, Biomanual Series 8, Geneta Igeti) The method was introduced into mouse E14 ES cells according to the method of Ning, Yodosha, 1995). E14 ES cells are treated with trypsin, suspended in HBS at 2.5 x 10 7 cells / ml, and 5 g DNA is added. Use Gene Pulser (without connecting a resistor or resistor unit). The electric robot went.
  • a voltage of 960 F and a voltage of 250 V was applied at room temperature using a 4 mm-length election port displacement cell.
  • the electroporated cells are suspended in 20 ml of ES medium (Shinichi Aizawa, Biomanual Series 8, Gene Targeting, Yodosha, 1995), and a 100 mm tissue culture plastic dish (previously seeded with feeder cells) is used. The seeding was performed on two sheets. Similarly, an experiment using 10, I5 gDNA was performed. 1 day later 3
  • the medium was replaced with a medium containing 00 ⁇ g / ml G418 (Geneticin, Sigma). Pick up a total of 192 colonies formed 7-9 days later, grow each to confluence in a 12-well plate, and use 4/5 of this to 0.2 ml of storage medium (ES medium + 10% DMS0 (Sigma )) And stored frozen at -80 ° C. The remaining 1/5 was seeded on a 12-well gelatin-coated plate, cultured for 2 days, and genomic MA was prepared from 10 6 -10 7 cells using a Puregene DNA Isolation Kit (Gentra System).
  • CNHase gene homologous recombinant E14 ES cell line clone 108 was set up in a frozen cell stock, and blastocysts obtained by mating C57BL / 6 male and female mice were used to transfer embryos. Each was injected. Approximately 10 injected embryos were transplanted to the uterus of a foster parent ICR mouse (Clea Japan, Inc.) 2.5 days after the pseudopregnancy treatment. Chimera individuals are judged by whether or not wild color (dark brown) derived from E14 cells is found in white from the host embryo (ICR) in coat color. Of the three mice that were born and survived, two had apparently wild-colored, ie, ES cell-contributing individuals (male-chimera rate 91%).
  • Example 4 Transmission of offspring of CNHase gene knockout traits from Chimera mice The chimera mice of Example 3 were bred to C57BL / 6 female mice to produce offspring F1 having E14 ES cell traits. DNA was prepared from the tails of these mice (Shinichi Aizawa, Bio Manual Series 8, Gene Targeting, Yodosha, 1995). Southern blot analysis was performed as in Example 2, and one of the CNHase genes was analyzed. Knockouts (Bgl1 digestion yielded bands of 10 kb or more and 7 kb) and wild-type (knockouts of 10 kb or more) could be identified.
  • mice we crossed mice (hetero) in which one allele was knocked out and succeeded in obtaining a mouse (homo) in which both alleles were knocked out.
  • Figure 5 shows some of the results. In the homozygous mouse, only the 7 kb band is seen.
  • a mouse that produces non-human glycans ie, N-glycolylneuraminic acid (Neu5Gc)
  • N-glycolylneuraminic acid ie, N-glycolylneuraminic acid (Neu5Gc)
  • N-dalicholylneuraminic acid ie, N-dalicholylneuraminic acid (Neu5Gc) due to gene disruption. I confirmed that it was gone.
  • N-glycolylneuraminic acid (Neu5Gc) was not detected. That is, sialic acid in the blood of mice before disruption of the CNHase gene includes 72% N-glycolylneuraminic acid (NeuSGc) and 28% N-acetylnuraminic acid (Neu 5A c) was detected, but N-glycolyllanamic acid (Neu5Gc) was not detected in the blood sialic acid of mice in which the CNHase gene was disrupted. Only N-acetyl neuraminic acid (Neu5Ac) was detected.
  • FIG. 7 shows the result of HP LC chromatography of a sample obtained by adding Neu5Gc to a blood-derived sample of a mouse in which the CNHase gene was disrupted.
  • a peak of Neu5Gc is seen at 19.29 minutes.
  • N-glycolylneuraminic acid (Neu5Gc) and N-acetylneuraminic acid (Ne Quantitative analysis of u5Ac) was performed according to the method of Hara et al. (Anai. Biochem. 179 162-166 1989), using DMB (1,2-Diamino-4,5-methyl-1-enedioxybenzene, dihydroch 1). or i de) (The sialic acid labeling method was used. The detection limit of sialic acid by this method is 57 to 192 fmo 1 e per injection of 10 ⁇ l of sample.
  • DMB reagent 70 m DMB solution 200 [mu] 1 and 7.5 M / 3 - mercaptoethanol Ichiru solution 200 1 and 180 mM Na 2 S 2 0 4 ( Hyde port Sarufuai Tonato Li um) solution 200 [mu] 1 and 4 ⁇ acetate solution 700 microns 1
  • Sialic acid in serum samples reacted with DMB reagent was analyzed under the following HPLC conditions.
  • the HPLC column used was 0DS80TM 4.6 x 250 mm.
  • the eluent used was a mixture of acetonitrile: methanol: water (9: 7: 84), the flow rate was 0.5 ml / min, and the detection was The measurement is performed using a fluorescence detector set to 373 nm excitation light and 448 nm emission light.
  • Example 6 Production of a mouse having a CNHase gene deficiency and producing an antibody containing human immunoglobulin (method by mating)
  • a mouse strain that retains the human chromosome 2 fragment prepared by the method described in [W097 / 07671 and W098 / 37757 (Example 42 or 43) and transmits offspring is defined as a CNHase gene-deficient mouse strain.
  • Antibody molecules containing human / c chains produced in this mouse strain may not contain Neu5Gc. It is confirmed by the method described in (Example 5).
  • a mouse strain capable of producing a complete human antibody molecule containing a human heavy chain and / or a chain and not adding Neu5Gc to a protein can be produced. And can be. It is confirmed by the method described in Example 5 that the complete human antibody molecule comprising the human heavy chain and the K chain produced in this mouse strain does not contain Neu5Gc.
  • a mouse strain that retains the human chromosome 22 fragment + the chromosome 14 fragment prepared by the method described in [W097 / 07671 and W098 / 37757 (Example 71)] and transmits offspring is a CNHase gene-deficient mouse.
  • a mouse strain that can produce a complete human antibody molecule containing a human heavy chain and a heavy chain and cannot add Neu5Gc to a protein can be produced. It is confirmed by the method described in Example 5 that the complete human antibody molecule comprising the human heavy chain and the ⁇ chain produced in this mouse strain does not contain Neu5Gc.
  • the complete human antibody molecule consisting of the human heavy chain and the K chain or the L chain produced in this mouse strain does not contain Neu5Gc, as confirmed by the method shown in Example 5 (Example 5).
  • You. (Example 7) Production of a mouse having a CNHase gene deficiency and producing an antibody containing human immunoglobulin (a method using a chimeric mouse)
  • a C N Hase gene bilateral allele-disrupted strain is obtained by the method described in [W097 / 07671 and W098 / 37757 (Example 51 or 59) and the like].
  • the marker gene is removed from the obtained strain by the method described in [W097 / 07671 and W098 / 37757 (Example 52 or 60)].
  • a mouse ES cell line in which the endogenous antibody heavy chain, light chain, and alleles on both sides of the CNHase gene are destroyed and which does not contain the marker gene is established.
  • CNHase-deficient animal cells can be obtained from the CNHase-deficient animal of the present invention or from the animal after crossing with an appropriate strain.
  • cancer can be generated and the cancer cells can be used by injecting an appropriate carcinogen into BALB / c (Horibata et al., Expl Cell Res, 60, 61-, 1970).
  • the myeoma cells obtained by such a method can be fused with B cells such as mouse spleen to produce a hybridoma and produce a monoclonal antibody (Tamie Ando, Takeshi Chiba, Introduction to Monoclonal Antibody Experimental Procedures, Kodansha, 1991).
  • recombinant proteins such as immunoglobulins by genetic recombination (Bebbington et al., Bio / Techno 1 ogy, 10, 169—, 1992).
  • CNHase-deficient mouse of the present invention it is possible to produce a monoclonal antibody or a recombinant protein that is substantially free of Neu5Gc because it lacks CNHase.
  • CNHase-deficient animal of the present invention By crossing the CNHase-deficient animal of the present invention with an animal into which a human chromosome has been introduced, a CNHase-deficient non-human animal having a human chromosome can be obtained.
  • CNHase-deficient animal cells can be obtained from such an animal or from the animal after crossing with an appropriate strain. For example, BALB / C mice are injected with mineral oil or the like to generate cancer, and the cancer cells can be used for producing hybridomas (Horibata et al., Expl Cell Res, 60, 61-). , 1970).
  • the myeloid cell obtained by such a method produces immunoglobulin, so that the target monoclonal antibody can be used for hybridoma production or recombinant antibody production. And a complex is formed or mixed with it, reducing productivity or making purification difficult.
  • myeloma cells that do not substantially produce endogenous immunoglobulin are desired (Shulma et al., Nature, 276, 269-, 1978).
  • the human chromosome can be expected to drop out of the myeloma cell with a certain probability (Tomizuka et al., Nature Genet., 16, 133-, 1997). That is, if a myeloma cell that efficiently produces human immunoglobulin is obtained and a cell from which the human chromosome has been eliminated can be cloned, the cell no longer produces immunoglobulin.
  • hybridoma which is used to produce a monoclonal antibody, or used to produce recombinant proteins such as immunoglobulin by genetic recombination. be able to.
  • B cells such as mouse spleen, which retains the human chromosome of the present invention and lacks CNHase
  • the hybridoma cells lack CNHase.
  • recombinant protein such as a human antibody substantially free of Neu5Gc can be produced. Can be produced.
  • Example 6-2 a mouse individual having the human chromosome 2 fragment and having a homozygous deletion of the CNHase gene was prepared.
  • This mouse strain was crossed with a mouse individual homozygous for the endogenous antibody heavy and light chain gene deletions produced by the methods described in W097 / 07671 and W098 / 37757.
  • an individual (A) having the human chromosome 2 fragment, heterozygous CNHase gene deletion, endogenous antibody heavy chain gene deletion heterogeneity, and antibody light chain / c gene deletion heterology was obtained.
  • D a mouse individual that does not retain human chromosome 2 and is homozygous for deletion of the CNHase gene, wild type deficient in endogenous antibody heavy chain, and wild type deficient in antibody light chain / c was obtained. Most of the antibody molecules expressed in D mouse individuals are thought to consist of mouse heavy chains and mouse / c chains.
  • Antibodies were purified from serum of B mouse individuals (5-week-old to 8-week-old) using protein G. That is, mouse serum was applied to a protein G column (manufactured by Amersham Pharmacia Biotech), the column was washed with PBS, and the antibody was eluted with a 0.1 M glycine solution. This antibody was immediately neutralized and concentrated. When the concentration of human antibody / chain and the concentration of mouse antibody chain in the antibody solution derived from an individual B mouse were measured by the ELISA method described in W097 / 07671 and W098 / 37757, the human antibody chain was the average. Showed a high concentration. Furthermore, sialic acid was analyzed for these antibodies by the method described in (Example 5). As a result, two types of sialic acids, Neu5Gc and Neu5Ac, were detected in the antibody derived from the B mouse individual, and Neu5Gc was the main component (FIG. 8).
  • Antibodies were purified from serum of C mouse individuals (5-week to 8-week old) prepared in (Example 10) using protein G. That is, mouse serum was applied to a protein G column, the column was washed with PBS, and the antibody was eluted with a 0.1 M glycine solution. This antibody was immediately neutralized and concentrated. According to the ELISA method described in W097 / 07671 and W098 / 37757, when the concentration of the human antibody / c chain and the concentration of the mouse antibody chain in the antibody solution derived from the individual C mouse were measured, the human antibody / c chain On average it showed a high concentration. Furthermore, these antibodies were analyzed for sialic acid by the method described in (Example 5).
  • Neu5Ac was detected and Neu5Gc was hardly detected in the antibody derived from the C mouse individual (FIG. 9).
  • Neu5Gc peak slightly observed (approximately 1.1% of both Neu5Ac and Neu5Gc sialic acids.
  • about 2.2% of the peak of Neu5Gc was observed in a different individual from the example shown in Fig. 9) ) Is from mother mice that are heterozygous for CNHase gene deletion. It is thought to be coming.
  • Antibodies were purified from serum of mouse individuals (12 to 20 weeks old) using protein G. That is, mouse serum was applied to a protein G column, the column was washed with PBS, and the antibody was eluted with a 0.1 M glycine solution. This antibody was immediately neutralized and concentrated.
  • mouse antibody chains were detected by the ELISA method described in W097 / 07671 and W098 / 37757.
  • these antibodies were analyzed for sialic acid by the method described in (Example 5). As a result, in the antibody derived from the D mouse individual, Neu5Ac was detected, but Neu5Gc was not detected (FIG. 10).
  • non-human mammals and non-human mammalian cells deficient in the CNHase gene have been provided. Since the non-human mammal and the non-human mammalian cell of the present invention do not contain Neu5Gc as a biochemical component, the non-human mammal and the non-human mammalian cell It is useful as a host for producing glycoproteins that do not contain Neu5Gc in the sugar chain.

Abstract

L'invention concerne des mammifères non humains et des cellules mammaliennes non humaines, chez lesquels le gène de cytidine monophosphate-N-acétylneuraminate hydroxylase a subi une disruption.
PCT/JP1999/004561 1998-08-24 1999-08-24 Animaux non humains presentant une disruption au niveau du gene de cmp-n-acetyl-neuraminate hydroxylase WO2000010384A1 (fr)

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AU53060/99A AU5306099A (en) 1998-08-24 1999-08-24 Nonhuman animals with disruption of cmp-n-acetylneuraminate hydroxylase gene

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JP23778498 1998-08-24
JP10/237784 1998-08-24

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Cited By (1)

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Publication number Priority date Publication date Assignee Title
EP1393746A1 (fr) * 2001-04-17 2004-03-03 Juridical Foundation, The Chemo-Sero-Therapeutic Research Institute Compositions d'anticorps polyclonaux humains

Citations (1)

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JPH07203958A (ja) * 1994-01-27 1995-08-08 Kao Corp チャイニーズハムスター卵巣細胞変異株

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JPH07203958A (ja) * 1994-01-27 1995-08-08 Kao Corp チャイニーズハムスター卵巣細胞変異株

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1393746A1 (fr) * 2001-04-17 2004-03-03 Juridical Foundation, The Chemo-Sero-Therapeutic Research Institute Compositions d'anticorps polyclonaux humains
EP1393746A4 (fr) * 2001-04-17 2008-09-10 Chemo Sero Therapeut Res Inst Compositions d'anticorps polyclonaux humains

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