WO2019029713A1 - 一种由基因组被编辑的cho宿主细胞产生的具有独特糖谱的重组抗体及其制备方法 - Google Patents

一种由基因组被编辑的cho宿主细胞产生的具有独特糖谱的重组抗体及其制备方法 Download PDF

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WO2019029713A1
WO2019029713A1 PCT/CN2018/100008 CN2018100008W WO2019029713A1 WO 2019029713 A1 WO2019029713 A1 WO 2019029713A1 CN 2018100008 W CN2018100008 W CN 2018100008W WO 2019029713 A1 WO2019029713 A1 WO 2019029713A1
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antibody
seq
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秦超
周远清
萧翠珍
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百奥泰生物科技(广州)有限公司
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Priority to CA3070741A priority Critical patent/CA3070741A1/en
Priority to AU2018315371A priority patent/AU2018315371B2/en
Priority to EP18842885.8A priority patent/EP3666891A4/en
Priority to SG11202000679XA priority patent/SG11202000679XA/en
Priority to US16/337,371 priority patent/US11505609B2/en
Priority to JP2020529801A priority patent/JP2020534862A/ja
Priority to NZ760974A priority patent/NZ760974A/en
Publication of WO2019029713A1 publication Critical patent/WO2019029713A1/zh
Priority to US18/050,970 priority patent/US20230203169A1/en
Priority to AU2024203006A priority patent/AU2024203006A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
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    • C07—ORGANIC CHEMISTRY
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2887—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • C—CHEMISTRY; METALLURGY
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • C—CHEMISTRY; METALLURGY
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    • C12P21/00—Preparation of peptides or proteins
    • C12P21/005—Glycopeptides, glycoproteins
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    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/01—Hexosyltransferases (2.4.1)
    • C12Y204/01068—Glycoprotein 6-alpha-L-fucosyltransferase (2.4.1.68), i.e. FUT8
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
    • C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
    • C07K2317/41—Glycosylation, sialylation, or fucosylation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention belongs to the field of bioengineering and technology, and relates to a recombinant antibody having a unique glycoform which is produced by a genome-edited CHO host cell and a preparation method thereof.
  • CHO cells are Chinese Hamster Ovary (CHO). In 1957, the University of Colorado, Dr. Theodore T. Puck was isolated from the ovary of a female hamster. It is an epithelial adherent cell and is widely used in bioengineering.
  • the ATCC preserves the CHO-K1 cell line, numbered CCL-61, and is widely used for expression of recombinant DNA proteins.
  • the initial cells are adherent cells, which can also be suspended in growth after multiple passages. CHO cells are prone to gene mutations and are more susceptible to gene transfection. Early studies have also shown that antibodies produced by CHO cells are closest to human serum antibody glycoforms compared to other engineered cell lines, and thus CHO cells are good mammalian gene expression host cells.
  • the mechanism of action of a therapeutic antibody is to form a complex with the target molecule, causing neutralization of the target antigen or clearance of the antigen or pathogen by the immune effect of the Fc portion of the antibody.
  • the specific binding and activity of antibody drugs to target molecules depends on their complex multi-level structure and post-translational modifications, while glycosylation is the most important post-translational modification of antibodies, for the biological activity, in vivo metabolism and immunogen of antibodies. Sex has an important role.
  • the glycosylated form of the antibody drug is mainly N-glycosylation, and the monosaccharides involved are mainly: glucose, galactose, mannose, N-acetylglucosamine, N-acetylgalactosamine, fucose, sialic acid. (NANA, NGNA).
  • NANA N-acetylglucosamine
  • N-acetylgalactosamine fucose
  • sialic acid sialic acid.
  • NANA NGNA
  • the two-branched or multi-branched biantennary complex oligosaccharide linked by the Fc segment Asn297 of the antibody molecule can be classified into G0, G1 (1, 3), G1 (1, 6) and G2, etc.
  • One type is further classified into 16 types according to whether or not there is fucose (F) or galactose (B) (Glycobiology, August 2015, 1-10. DOI: 10.1093/glycob/cwv065). Therefore, even if the sialylation and high mannose type are not considered at the end, the antibody heavy chain has at least 36 oligosaccharide types, and with the random combination of the two heavy chains of the antibody, there may be more than 400 types of glycoforms. The antibody is rendered highly heterogeneous.
  • G0F promotes the role of the complement pathway and accelerates the clearance rate.
  • G2F is increased in the umbilical cord of pregnant women and newborns.
  • Sialic acid modification has a significant effect on the inflammatory effects of intravenous immunoglobulin. Decreased fucose resulted in a significant increase in ADCC activity (JBC (2003) Chemistry 278, 3466-3473). Therefore, it is necessary to design and optimize the sugar chain of an antibody depending on the main mechanism of action of the therapeutic antibody and the drug use.
  • glycosylation of antibodies is not followed by templates, and the type of glycosylation and the ratio of each oligosaccharide component are affected by host cell type and culture conditions.
  • Methods for engineering oligosaccharide components of monoclonal antibodies to enhance their Fc-mediated effects by engineering host cells are disclosed in various literatures and patent reports.
  • an antibody prepared using CHO cells overexpressed with ⁇ (1,4)-N-acetylglucosaminyltransferase III (GnTIII) exhibits higher ADCC activity than the antibody expressed in the parental cell, and the difference in activity is approximately 10 to 20 times (Biotechnol Bioeng.
  • GnTIII GnTIII
  • overexpression of GnTIII is toxic to CHO cells and because exogenous expression tends to increase with the number of passages during culture.
  • the expression level of GnTIII is decreased, and the fucose content of the antibody produced by the host cell is changed, thereby affecting the homogeneity of the antibody drug.
  • Examples of cell lines that produce defucosylated antibodies also include Lec13 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249: 533-545 (1986), but due to their extremely low protein yield Suitable as a host cell for the production of therapeutic antibodies (Yutaka Kanda et al Biotechnol Bioeng.
  • this method has the same disadvantages as the above-mentioned GnTIII overexpressed CHO cell line.
  • the host cell needs to introduce a foreign sequence, and the efficiency of the siRNA inhibiting the target gene can only be up to 70%.
  • the stability of siRNA expression can be Affect the quality of the properties of antibody drugs.
  • the invention provides a pair of polypeptides having the amino acid sequence set forth in SEQ ID NO. 10 and SEQ ID NO. 11 or at least 90% of the sequence set forth in SEQ ID NO. 10 and SEQ ID NO. At least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity.
  • the pair of polypeptides set forth in SEQ.NO.10 and SEQ.NO.11 are the amino acid sequences of the DNA binding domains upstream and downstream of TALEN, respectively, which can specifically bind to specific base regions of the gene. .
  • the invention provides a pair of polynucleotides encoding a pair of polypeptides as set forth in SEQ.NO.10 and SEQ.NO.11, respectively.
  • the one pair of polynucleotides has the nucleic acid sequence set forth in SEQ ID NO. 12 and SEQ ID NO. 13 or has at least the sequence set forth in SEQ ID NO. 12 and SEQ ID NO. 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity.
  • the invention provides a pair of fusion proteins which are formed by fusing a pair of polypeptides described above to the amino acid sequence of a DNA cleavage domain of a transcriptional activator-like effector (FokI).
  • the amino acid sequence of the DNA cleavage domain of the transcriptional activator-like effector (FokI) is native or engineered.
  • the pair of fusion proteins have an amino acid sequence as set forth in SEQ ID NO. 14 and SEQ ID NO. 16, or at least 90 with the sequence set forth in SEQ ID NO. 14 and SEQ ID NO.
  • the pair of fusion proteins can specifically recognize two nucleotide sequences of the Fut8 gene of CHO.
  • the two nucleotide sequences of the Fut8 gene are two nucleotide sequences on exon 1 (Exon1, SEQ ID NO. 7) of the Fut8 gene.
  • the two nucleotide sequences of the Fut8 gene have the nucleotide sequences set forth in SEQ ID NO. 3 and SEQ ID NO. 4, respectively.
  • the invention also provides a pair of nucleotides, each of which encodes a pair of fusion proteins as described above.
  • the pair of nucleotides has the nucleic acid sequence set forth in SEQ ID NO. 15 and SEQ ID NO. 17, or has the sequence set forth in SEQ ID NO. 15 and SEQ ID NO. At least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity.
  • the present invention provides a vector of at least any one of any one of the above-described polynucleotides.
  • the vector is a plasmid.
  • the invention also provides a host cell transformed with the vector described above.
  • the transformed cells of the vector described above are genome-edited CHO host cells, the parental cells of which are derived from the CHO-K1 cell line.
  • the one genome is edited in a CHO host cell, the parental cells of which are adapted to a serum-free suspension culture; the parental cell is designated CHO-BAT.
  • the one genome-edited CHO host cell is a subcloning of CHO-K1 selected to satisfy one or more of the following characteristics:
  • the cells have high transfection efficiency
  • the cells have a short exponential growth time
  • the cells have the ability to achieve high cell densities in CD-CHO cultures.
  • the one genome is a CHO host cell that is edited, and the cell has endogenous ⁇ 1 due to base deletion, insertion, and nonsense mutation in certain regions of the FUT8 gene relative to the parent cell.
  • 6 fucosyltransferase (Fut8) loses enzymatic activity;
  • the cell does not contain a foreign DNA sequence
  • the recombinant antibody expressed by the cell as a host cell has unique glycan profile characteristics.
  • the one genome-edited CHO host cell is characterized in that the genome of exon 1 of the cell FUT8 gene is edited, resulting in loss of enzymatic activity of endogenous Fut8 of the cell;
  • the cell does not comprise a DNA sequence of an expression vector introduced during the process of generating a base deletion or an unintentional mutation of the FUT8 gene;
  • Recombinant antibodies expressed by the cells as host cells have unique glycan profile characteristics and are characterized by having non-fucosylated N-linked oligosaccharide chains, as well as other glycan profile characteristics of the antibodies.
  • the one genome of the edited CHO host cell, the FUT8 gene is knocked out; the cell is negative for binding to lectin LCA; the cell is designated CHO-BAT-KF.
  • the present invention provides a kit comprising at least one of the above-mentioned pair of polypeptides; or at least one of the pair of polynucleotides; or at least any one of a pair of fusion proteins One; or a vector containing the above; or a host cell as described above.
  • the invention provides the use of a pair of polypeptides/one pair of polynucleotides/a pair of fusion proteins/vectors described above for editing Fut8 genes in CHO cells.
  • the present invention provides the use of the above-described pair of polypeptides/one pair of polynucleotides/a pair of fusion proteins/vectors/host cells for producing antibodies, particularly antibodies having a unique glycan profile, or providing the above A pair of polypeptides/one pair of polynucleotides/one pair of fusion proteins/vectors/host cells produced antibodies.
  • the present invention provides a method for editing Fut8 gene of CHO, comprising the steps of: transferring said pair of fusion proteins or a pair of polynucleotides or vectors into CHO cells, and culturing at 37 ° C for 14 days; The Cut cells in which the Fut8 gene was knocked out were obtained by pressure screening and limiting dilution.
  • a method for editing Fut8 gene of CHO comprising the steps of: transferring said pair of fusion proteins or a pair of polynucleotides or vectors into CHO cells, and culturing at 37 ° C for 14 days; The Cut cells in which the Fut8 gene was knocked out were obtained by pressure screening and limiting dilution.
  • the present invention provides a method of producing a recombinant antibody having a unique glycan profile produced by a genome-edited CHO host cell or an antibody produced by the method, comprising the steps of:
  • the vector is transfected into wild-type CHO cells in step (1); more preferably, the plasmid is stably transfected into wild-type CHO cells;
  • the CHO cell is CHO-K1; more preferably, the CHO-K1 is adapted to serum-free culture.
  • the antibody is an anti-CD20 antibody; more preferably, the antibody is a humanized or fully human anti-CD20 antibody; more preferably, the antibody is BAT4306F; more preferably, The antibody BAT4306F has two light chains of SEQ ID NO. 20 and two heavy chains of SEQ ID NO.
  • the inventors have employed the methods, cells, polypeptides and the like of the present invention for preparing various types of antibodies, and have found that different types of antibodies prepared exhibit highly consistent glycoforms with low degree of heterogeneity of glycoforms. . It is indicated that the methods, cells, and the like of the present invention are suitable for the preparation of all types of antibodies.
  • the antibody binds to CD20.
  • the CD20 binding antibody is a humanized antibody.
  • the humanized antibody BAT4306F is the heavy chain variable region B-HH6 amino acid sequence and the light chain variable region B-KV1 amino acid sequence from the B-Lyl antibody sequence of patent WO2005044859.
  • the BAT4306F antibody comprises a pair of light and heavy chains of the sequence: SEQ ID NO. 20 and SEQ ID NO.
  • the CD20 binding antibody is the fully human antibody BAT4406F comprising a pair of light and heavy chains of the sequence: SEQ ID NO. 22 and SEQ ID NO.
  • the antibody is BAT1206F and the BAT1206F antibody has two light chains of SEQ ID NO.
  • the antibody is BAT0206F and BAT0206F binds to EGFR, the antibody having two light chains of SEQ ID NO. 24 and two heavy chains of SEQ ID NO.
  • the antibody is BAT0808, which binds to Trop2, which has two light chains of SEQ ID NO. 26 and two heavy chains of SEQ ID NO.
  • the modified glycoprotein is secreted by a host cell. In some embodiments, the modified glycoprotein is an antibody.
  • the preparation method of the recombinant antibody produced by the genome-edited CHO host cell of the present invention having a unique glycoform or the antibody produced by the method comprises the following steps:
  • the above pair of fusion proteins or a pair of polynucleotides or vectors are transferred into wild-type CHO cells, and the cells are transfected with CD CHO (Sigma) + 10% FBS (fetal calf serum) containing phytohemagglutinin (LCA). After 15 days, the viable cells were inoculated into 96-well cell culture plates at 0.5 cells/well, and the serum concentration was reduced to 5%. After 7 days, the cells were transferred to 24-well cell culture plates and returned to the CO2 incubator. After 7 days, the cells were taken. Part of the cells were centrifuged at 1000 rpm for 5 min, and PBS was changed once.
  • CD CHO Sigma
  • FBS fetal calf serum
  • LCA phytohemagglutinin
  • the CHO genome was extracted with a plasmid extraction kit (Omega), and the polymerase chain reaction (PCR) was carried out using primers L130for (SEQ ID NO.1), L130rev (SEQ ID NO.2), and taq enzyme using the genome as a template.
  • the PCR product was ligated with the T vector (Promega) to transform the coated plate.
  • T7 primers single colonies were picked and sequenced with T7 primers, and the sequence was analyzed by DNASTAR analysis software. Baseline-deleted CHO cells were compared with the wild-type CHO genome sequence.
  • Expanded culture named CHO-BAT-KF, established cell bank in CHO-BAT-KF in logarithmic growth phase, frozen cells in CD CHO cryopreservation solution containing 7.5% DMSO, and transferred to liquid nitrogen tank for long-term storage. ; taken plasmid was linearized gene encoding the antibody, measurement of OD260, taken with 107 50 ⁇ g of plasmid CHO-BAT-KF mixing rotor in the electrical, power transfer device (Biorad) transfection, cells were plated in 96-well culture plate. after 48h Methionine sulfoximine (MSX) was added, and after 14 days, it was blocked with anti-FC polyclonal antibody-coated ELISA plate and 3% BSA.
  • MSX Methionine sulfoximine
  • the supernatant was added to the plate and incubated at 37 ° C for 2 h, washed with PBST 5 times, and added with anti-horse root.
  • peroxidase-labeled goat anti-human kappa / lambda light chain, 2M H 2 SO 4 read on a microplate reader OD450 value.
  • the clone with high titer was expanded and the cell supernatant was collected by centrifugation to obtain an antibody protein for knocking out fucose.
  • the invention also provides a cell which is a CHO host cell whose genome is edited.
  • the invention also provides a nucleic acid comprising the sequence set forth in SEQ ID NO. 28, or comprising at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least the sequence set forth in SEQ ID NO. Nucleic acids of 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.8% identity.
  • the invention also provides a CHO host cell, which is preserved in the China Center for Type Culture Collection, the preservation number is CCTCC NO: C2017127, the preservation date is 2017.8.10, and the deposit address is: China, Wuhan, Wuhan University; Chinese hamster ovary cells CHO-BAT-KF fut8 (-/-).
  • the host cell is maintained in serum-free medium. In some embodiments, the host cell is maintained in suspension culture.
  • the invention also relates to a medium comprising the host cell, and a culture fermentor comprising a plurality of said host cells in a medium. In some embodiments, the medium is serum free.
  • the present invention provides an antibody produced by a genome-edited CHO host cell having a unique glycoform, the antibody being a humanized or fully human antibody having a unique glycosyl group In this way, the degree of N-glycosylation heterogeneity is low and the ADCC effect is significantly increased.
  • the recombinant antibody having a unique glycan profile produced by a genome-edited CHO host cell is a humanized antibody that binds to CD20 on the cell membrane surface.
  • the recombinant antibody having a unique glycan profile produced by a genome-edited CHO host cell has a unique glycosylation pattern characterized by the antibody
  • the level of one or more sugar moieties in the N-linked polysaccharide is changed, wherein the sugar moiety is selected from the group consisting of glucose (Glc), fucose (Fuc), galactose (Gal), mannose (Man), high Mannose, Glucosamine, G0 and acetylglucosamine (GlcNAc) have a unique glycosylation pattern.
  • the feature of the glycosylation pattern satisfies one or more of the following preferred conditions:
  • the antibody has a low fucose content; (0-5%)
  • the antibody contains a low level of galactose; ( ⁇ 30%)
  • the antibody has a low mannose level; ( ⁇ 5%)
  • the antibody has a high high mannose level; ( ⁇ 5%)
  • the antibody G0 level is higher. ( ⁇ 60%)
  • the antibody contains a lower level of galactose, ⁇ 5%.
  • the antibody has a higher G0 level, ⁇ 80%.
  • the recombinant antibody having a unique glycan profile produced by a genome-edited CHO host cell satisfies a preferred condition: a fucose content of zero.
  • the recombinant antibody produced by the genome-edited CHO host cell has a unique glycan profile, the antibody N-polysaccharide having a very low degree of heterogeneity and a uniform sugar chain.
  • the recombinant antibody having a unique glycan profile produced by a genome-edited CHO host cell has a stronger ADCC effect on Fc.
  • the antibody has a glycoprotein as set forth in BAT 4306F above Figure 10 of the specification.
  • the BAT4306F has two light chains of SEQ ID NO. 20 and two heavy chains of SEQ ID NO. 21; however, it is not excluded that the sequence is mutated as long as these Mutation does not affect the effect of the antibody.
  • the present invention provides a CHO host cell in which the Fut8 gene is knocked out, wherein the first exon of the Fut8 gene in such a CHO host cell contains an inactivating mutation.
  • This mutation can be one or more amino acid substitutions or deletions, or a frameshift mutation, such as the mutation shown in Figure 6.
  • the present invention also provides a pharmaceutical composition comprising the above antibody.
  • the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
  • the invention also provides a method of preventing or treating a disease comprising administering to a subject in need thereof an effective amount of an antibody/antibody fragment of the invention disclosed.
  • the disease is selected from the group consisting of cancer, allergy, cardiovascular disease, inflammatory disease, metabolic disease, neurological disease, viral infection, and/or bacterial infection.
  • the disease can be cancer or an allergy.
  • the subject is a mammal, such as a human.
  • the marketed antibody drugs are basically limited to the single N-glycosylation modification of Fc, but because of its inconsistent and variable glycoform composition and content, there is still some complexity, especially for production stability.
  • the invention provides a recombinant antibody with a unique glycoform produced by a genome-edited CHO host cell, which has low N-glycosylation heterogeneity and good sugar chain homogeneity; and the ADCC effect is enhanced, greatly The quality properties and pharmaceutical properties of the antibody drug are improved.
  • the binding affinity of the antibody to the FcyRIIIA receptor is increased as compared to the corresponding antibody produced by the unmodified CHO-K1 (ATCC #CCL-61) or the suspension-adapted parental cell CHO-BAT.
  • the modified host cell produces an antibody that has increased affinity for Fc[gamma]RIIIA compared to the corresponding antibody produced by the corresponding unmodified host cell.
  • Figure 1 shows adherently grown CHO-K1 (ATCC #CCL-61) and cell line CHO-BAT adapted to suspension growth in serum-free medium.
  • Fig. 3 Functional verification of TLEN protein.
  • the left side of the electrophoresis map is wild type.
  • the cell genomic PCR product shows two bands of 500 bp and 750 bp, while the wild type has only a single band of 750 bp, which is in line with the expected result, which proves that the Talen protein pair has Functional; where Lane1: 100bp marker; Lane2: wt; Lane3: pool.
  • Figure 4 FACS analysis of cells grown in 24-well plates, cells with edited cells cloned FITC-labeled LCA-negative cells, and wild-type cells were positively bound by FITC-labeled LCA binding.
  • Figure 5 shows a glycan-chain microarray analysis showing that the gene-edited 41,43 cloned fucose content was reduced to 0-10%, and the wild-type antibody 1206 had a fucose content of 80%.
  • FIG. 6 The targeting sequence of the TALEN protein was sequenced by PCR amplification and the results were aligned using lasergeneMegAlign sequence analysis software.
  • 191-1, 191-2, 217-1, and 217-3 are four selected cell clones whose genomes have been adjusted, and the genomes are extracted as DNA templates, and the PCR products are reacted with primers L130for and L130rev. CEL-1 base mismatch analysis.
  • the results showed that the cell clones 191-1 and 191-2 were heterozygous, and the cell clones 217-1 and 217-3 were homozygous.
  • CHO-2G8 was selected as a host cell for subsequent experiments, and the host cell was named CHO-BAT-KF.
  • Figure 7 Comparison of CHO-BAT-KF and parental cell CHO-BAT growth density.
  • Figure 8 Comparison of CHO-BAT-KF and parental cells CHO-BAT growth viability.
  • Figure 9 is a MALDI-TOF MS analysis of N-polysaccharides of BAT4306F and 4306 antibody molecules using a MALDI-TOF MS mass spectrometer.
  • Each N-glycan from BAT4306F has one less algae than the N-polysaccharide from 4306.
  • Sugar the left picture shows the antibody molecule 4306 produced by the parental cell, and the right side is the BAT4306F antibody molecule.
  • BAT4306F has a lower fucose content than GAZYVA (Obinutuzumab), with a lower degree of heterogeneity in the sugar chain and better product homogeneity.
  • Figure 11 uses Raji as a target cell and PBMC as an effector cell to compare the ADCC effect of BAT4306 wild type, sugar chain modified BAT4306F, Obinutuzumab, Rituximab and other anti-CD20 antibodies.
  • Figure 12 compares the ability of the sugar chain modified BAT4306F, Obinutuzumab, and rituximab to eliminate B cells in whole blood at 50, 25, 10 ng/mL.
  • Figure 13 compares the glycan profiles of anti-CD20 antibodies BAT4306F, BAT4406F, anti-EGFR antibody BAT0206F, anti-Trop2 antibody BAT0808 produced with CHO-BAT-KF cells.
  • the edited CHO host cell of the present invention is deposited in the China Center for Type Culture Collection (CCTCC) under the accession number CCTCC NO: C2017127, and the deposit date is 2017.8.10.
  • the deposit address is: China, Wuhan, Wuhan University; : Chinese hamster ovary cells CHO-BAT-KF fut8 (-/-).
  • the "level" or “content” of the sugar moiety of the antibody means the same meaning, and indicates the mass ratio of a certain sugar moiety in all the sugar fractions of the antibody.
  • amino acid refers to a carboxy alpha-amino acid which may be encoded by a nucleic acid either directly or in a precursor form.
  • a single amino acid is encoded by a nucleic acid consisting of three nucleotides (so-called codons or base triplets). Each amino acid is encoded by at least one codon.
  • amino acid refers to a naturally occurring carboxy a-amino acid which includes alanine (three letter code: ala, one letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine (cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G) , histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), styrene (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y) And proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W
  • polynucleotide or “nucleic acid” or “nucleic acid sequence” are used interchangeably to mean a single nucleotide (also referred to as a base) a, c, g and t (or in RNA).
  • the polynucleotide molecule can be a naturally occurring polynucleotide molecule, or a synthetic polynucleotide molecule, or a combination of one or more naturally occurring polynucleotide molecules and one or more synthetic polynucleotide molecules.
  • the definition also includes naturally occurring polynucleotide molecules in which one or more nucleotides are altered (eg, by mutagenesis), deleted or added.
  • the nucleic acid can be isolated or integrated into the chromosome of another nucleic acid such as an expression cassette, plasmid or host cell.
  • a nucleic acid is characterized by a nucleic acid sequence consisting of a single nucleotide. It is well known to those skilled in the art to convert, for example, the amino acid sequence of a polypeptide to the corresponding nucleic acid sequence encoding the amino acid sequence.
  • a nucleic acid can be characterized by its nucleic acid sequence consisting of a single nucleotide, or by the amino acid sequence of the polypeptide encoded thereby.
  • polynucleotide or “nucleic acid” or “nucleic acid sequence” may comprise modified nucleotides, as a percentage of the total number of nucleotides present in the nucleic acid molecule, for example at least about 5%, 10%, 15 %, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% modified nucleotide.
  • polypeptide in the present invention is a polymer composed of amino acids linked by peptide bonds, which may be naturally or synthetically produced.
  • a polypeptide having less than about 20 amino acid residues may be referred to as a "peptide", however, a molecule composed of two or more peptides or a molecule containing one polypeptide having more than 100 amino acid residues may be referred to as a "protein.”
  • the polypeptide may also contain non-amino acid components such as sugar groups, metal ions or carboxylic acid esters. Non-amino acid components can be added by cells expressing the polypeptide and can vary with the type of cell.
  • a polypeptide is defined herein according to its amino acid backbone structure or the nucleic acid encoding it.
  • polypeptide may comprise a modified amino acid, as a percentage of the total number of amino acids present in the amino acid molecule, such as at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% modified amino acids.
  • host cell refers to a microorganism or eukaryotic cell or cell line cultured in a mononuclear entity, which may be or has been used as a recipient of a recombinant vector or other transfer polynucleotide, and includes a transfected original cell. Descendants.
  • the host cell is a non-lymphocyte, and the host cell produces a unique glycan profile that exhibits the same.
  • the host cell is a NSO cell, a simian COS cell, a Chinese hamster ovary (CHO) cell, and the like.
  • the host cell is selected from the group consisting of Chinese hamster ovary (CHO) cells.
  • the host cell is selected from the group consisting of CHO-K1, CHO-S, DUXB11, CHO-1E5, CHO3F, CHO/DG44, CHO-BAT, and CHO-2.6 cells.
  • the host cell produces an antibody that exhibits a unique glycan profile.
  • the genomically altered CHO host cells of the invention such as CHO-BAT-KF fut8 (-/-), can be grown in cultures, as well as in devices (including fermenters) that can be used to grow cultures. They can grow into a single layer or adhere to a surface. Alternatively, the host cell can be grown in suspension.
  • the cells can be grown in serum-free medium.
  • the medium may be a commercially available medium such as, but not limited to, DMEM/F12.
  • the edited CHO host cells maintain their specific unique glycan profile in the culture medium for many generations. For example, an edited CHO host cell retains its specific unique glycan profile after at least about 20, 30, 40 or 50 passages. In some embodiments, the modified CHO host cell retains its specific unique glycan profile after at least about 60 passages. In yet other embodiments, the modified CHO host cell retains its specific unique glycan profile after a generation of at least about 100, 150, 200, 500 or more.
  • the glycosylation pattern of the host cell may be N- or O-glycosylation of any protein moiety, wherein it may be at the amide nitrogen of asparagine, or hydroxylysine, hydroxyproline, serine or threonine, respectively.
  • One or more sugar molecules are added to the hydroxyl oxygen of the acid.
  • the glycosylation pattern is characterized by a change in the levels of at least two or more sugar molecules or sugars such as monosaccharides, disaccharides, polysaccharides or oligosaccharides.
  • the sugar molecule may be a trisaccharide, a tetrasaccharide, a pentasaccharide, a hexose, a heptasaccharide, an octasaccharide, a pentose, or a derivative thereof, such as a deoxysaccharide (such as deoxyhexaose); an N- or O-substituted a derivative such as sialic acid; or a sugar having an amino group.
  • a deoxysaccharide such as deoxyhexaose
  • an N- or O-substituted a derivative such as sialic acid
  • a sugar having an amino group such as a sugar having an amino group.
  • Sugar molecules can include, but are not limited to, galactose (Gal), glucose (Glc), mannose (Man), N-acetylneuraminic acid (NeuAc), fucose (Fuc), N-acetylgalactosamine ( GalNAc), N-acetylglucosamine (GlcNAc); and xylose (Xyl).
  • Gal galactose
  • Man mannose
  • NeuAc N-acetylneuraminic acid
  • Fuc fucose
  • GalNAc N-acetylgalactosamine
  • GlcNAc N-acetylglucosamine
  • Xylose xylose
  • the "antibody” of the present invention includes all forms of antibodies such as recombinant antibodies, humanized antibodies, chimeric antibodies, single chain antibodies, fusion antibodies, monoclonal antibodies, polyclonal antibodies and the like.
  • the antibody can also be a fragment.
  • the antibody can also be combined with a drug, toxin or therapeutic radioisotope.
  • Host cells of the invention can also produce bispecific antibody fusion proteins, including hybrid antibodies that bind to more than one antigen.
  • antibodies include naked and binding antibodies as well as antibody fragments, which may be monospecific or multispecific.
  • the antibody or antibody fragment is not particularly limited and may be selected from the group consisting of anti-HER2, anti-CD20, anti-EGF, anti-VEGF, anti-PDGF, anti-EpCam, anti-CD3, anti-CD4, anti-CD19, anti-CD30. , anti-CD33, anti-CD40, anti-CD51, anti-CD55, anti-CD80, anti-CD95, anti-CCR2, anti-CCR3, anti-CCR4, anti-CCR5, anti-folate, anti-CXCR4, anti-EGFR or Trop2 antibodies, and the like.
  • the antibody is a humanized or fully human antibody.
  • an antibody having the desired degree of purity is optionally combined with a physiologically acceptable carrier, excipient or stabilizer (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980) ())
  • a physiologically acceptable carrier excipient or stabilizer
  • Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the dosages and concentrations employed, including: buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine Preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethylammonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl parabens; Acid esters such as methyl or propyl parabens; catechol; resorcinol; cyclohexanol; 3-propanol; and m-cresol; low molecular weight (less than about 10 residues) a polypeptide; a protein such as serum albumin, gelatin, or an immunoglobulin; a hydrophilic polymer such as polyvinylpyrrolidone; an amino acid such as glycine
  • the antibodies, pharmaceutical compositions, pharmaceutical preparations of the invention may be administered by any suitable means, including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal routes, and, if desired, for local immunosuppressive therapy. It can be administered intralesionalally.
  • Parenteral infusion methods include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration.
  • the antibodies of the invention may be suitably administered by pulsed perfusion (in particular, dose gradient changes of the antibodies of the invention).
  • the administration time is short or long, preferably by injection, and most preferably by intravenous or subcutaneous injection.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • FcR-expressing effector cells eg, natural killer (NK) cells, neutrophils, and macrophages
  • Primary cells used to mediate ADCC include NK cells, monocytes, and macrophages.
  • NK cells typically express Fc ⁇ RIII predominantly, while monocytes express Fc ⁇ RI, Fc ⁇ RII, and Fc ⁇ RIII.
  • the maternal CHO cell line is edited to produce a CHO cell line with a unique glycan profile. The edited CHO cell line can then produce antibodies with higher ADCC activity than the antibody produced by the parental CHO cells.
  • CHO-K1 cultured in DMEM/F12 medium containing 10% FBS when the cell confluence reached 80%-90%, washed with PBS, trypsinized, 5% FBS DMEM/F12 medium was terminated, counted and Centrifugation.
  • the cells were resuspended in DMEM/F12 medium containing 5% FBS, and the cells were seeded at a density of 1 ⁇ 10 5 cells/ml.
  • washing with PBS, trypsinization, 2% FBS medium DMEM/F12 was terminated, counted and centrifuged.
  • the cells were resuspended in medium DMEM/F12 containing 2% FBS, and the cells were seeded at a density of 1 ⁇ 10 5 cells/ml. After the above cell confluence reached 80%-90%, the above cells were trypsinized according to the previous steps, and terminated with DMEM/F12 medium containing 1% FBS, and passaged for 3-4 passages.
  • the CD CHO medium was mixed with DMEM/F12 at a ratio of 1:1 (V/V), and the final concentration was adjusted to 6 mM glutamine, and the serum content was adjusted to 1%.
  • the low serum-adapted CHO-K1 cells obtained above were inoculated into a T25 square bottle at a density of 3 ⁇ 10 5 cells/mL, and cultured at 37 ° C in a 5% CO 2 incubator. When the cell confluence reached 80-90%, trypsinize digestion, stop with DMEM/F12 containing 1% FBS and CD CHO medium mixed medium (volume ratio 1:2), count and centrifuge to 3 ⁇ 10 5
  • the cells/mL density were inoculated into T25 square flasks and cultured in a 37 ° C, 5% CO 2 incubator. Gradually reduce the ratio of DMEM/F12 in the mixed medium to (1:8).
  • CD CHO medium contains 1% serum cultured CHO-K1 cells.
  • CHO-K1 cultured in CD CHO medium defined by chemical composition of 1% FBS, when the cell confluence reached 80%-90%, washed with PBS, trypsinized, and CD CHO medium containing 0.5% FBS Stop, count and centrifuge. The cells were resuspended in CD CHO medium containing 0.5% FBS, and inoculated into a T25 square bottle at a density of 1 ⁇ 10 5 cells/ml.
  • the clones with larger cell size were transferred to 24-well plates. After 1 week, microscopic examination showed that the cell clones with higher growth density and cell size were transferred to the 6-well plate for further culture. After 1 week, microscopic examination showed that the markers were completely suspended and agglomerated. The clones with less cell density were transferred to a 100 mL flask and the culture volume was 10 mL. The cell density and viability of each cell were recorded. The domesticated, serum-free cultured CHO-K1 cells were renamed CHO-BAT.
  • L130P forms a fusion protein L130-FokI (SEQ ID NO. 14) with FokI endonuclease, and this fusion protein recognizes exon 1 left-wing base L130PTN (SEQ ID NO. 3), and the corresponding nucleic acid sequence L130 of fusion protein L130-FokI -FokIN is shown in SEQ ID NO. 15, and the length is 19 bp.
  • R184P forms a fusion protein R184P-FokI (SEQ ID NO. 16) with FokI endonuclease, and this fusion protein recognizes the exon 1 right-wing base R184PTN (SEQ ID NO.
  • nucleic acid sequence R184P of the fusion protein R184P-FokI -FokIN is shown in SEQ ID NO. 17, and the length is 17 bp.
  • a plasmid vector (see Figure 2) containing the TALEN protein encoding exon 1 left-wing L130PTN and right-wing R184PTN was constructed as described by Tomas Cermak et al. (2011). The restriction endonucleases NcoI and XbaI restriction sites were added to both ends of L130-FokIN and R184P-FokIN, and the two sequences were synthesized and cloned into pCS2-peas-T vector using NcoI and XbaI (Fig. 2).
  • the CEL-I enzyme is a nuclease that recognizes mismatched bases in double-stranded DNA and cleaves double-stranded DNA from mismatches. If the targeting sequence is edited by Fut8 TALEN, then the region containing the targeting sequence amplified from the maternal genome is mixed with the region containing the targeting sequence amplified from the genome of the transformed cell, denatured, annealed The annealed double-stranded DNA has a base mismatch, so that the CEL-I enzyme can cleave the annealed double-stranded DNA, and the agarose electrophoresis results in two bands.
  • CHO-BAT cells 5 ⁇ 10 5 CHO-BAT cells were seeded in 6-well plates one day before transfection, and the medium was DMEM/F12 containing 10% fetal calf serum. Plasmids L130N and R184N were transiently transfected into cells as described in the reagent instructions. Three days after transfection, the cells were harvested by centrifugation and the genome was extracted using a genomic extraction kit. Using this as a template, PCR reaction was carried out using primer L130 for (SEQ ID NO. 1) and primer L130 rev (SEQ ID NO. 2). The PCR amplification of the region of the parental cell containing the targeting sequence is the same as above.
  • the L130N and R184N plasmids were transiently transformed into previously established cell lines stably expressing anti-CD20 antibodies. Methods The methods provided by the lipofectamine 2000 (Invitrogen) reagent description were briefly described as follows; 24 ⁇ L of liposomes packed with 4 ⁇ g of plasmid DNA of each of L130N and R184N were added to 1 ⁇ 10 6 cells in a 10 cm cell culture dish.
  • DMEM/F12 medium containing 10% (v/v) FBS (GBICO) and 400 ⁇ g/mL LCA (Vector). After 1 week, most of the cells were rounded and suspended in the medium, and some of the cells grew normally adherently. The supernatant was discarded, and LCA-resistant cells were digested with trypsin 0.25% (v/v), resuspended in DMEM/F12 medium containing 10% (v/v) FBS after centrifugation. The 96-well plates were seeded at a density of 0.5 per well. After 2 weeks of growth, the monoclonal cells were picked and transferred to 24-well plates.
  • FITC-labeled LCA-negative cells (Fig. 4) were expanded to produce antibodies.
  • the oligosaccharide content of the antibodies produced by the two cells was determined by Beijing Ednor Biotech, and the results are shown in Fig. 5.
  • the L130 and R184 plasmids were transiently transformed into the CHO-K1 cell line. Screening of monoclonal cells resistant to LCA As described in Example 3, the genomes of candidate cell clones were extracted, respectively, using primers L130 for (see Table 1, SEQ ID NO. 1) and primer L130 rev (see Table 1, SEQ). ID NO. 2) A PCR reaction was carried out, and a candidate cell clone was subjected to CEL-1 base mismatch analysis of a PCR amplification product of a region containing a targeting sequence.
  • the agarose electrophoresis after CEL-1 digestion is two bands. If it is homozygous, CEL-1 digests the annealed fragment and the agarose electrophoresis is a band.
  • This PCR fragment was directly cloned into a T vector (pGEM-T Easy Vector) and sequenced. The sequencing results were compared with the sequence of the fragment of the parent cell in this region. As shown in Fig. 6, according to the comparison results, two homozygous edited genomes were selected and recorded as CHO-2G8, CHO-1D6.
  • the clone CHO-2G8 in which the fut8 gene was knocked out was selected as a host cell and renamed as CHO-BAT-KF.
  • the cell growth density is shown in Fig. 7.
  • the cell growth activity is shown in Fig. 8.
  • the growth density and activity of CHO-BAT-KF with the fut8 gene knocked out and CHO-BAT cells without the fut8 gene knocked out were not significant. The difference.
  • BAT4306F produced by purifying CHO-2G8 cells from the culture medium through a protein A affinity column is used. 4306 produced by CHO-K1 cells and quantified by UV UV280. The desalted monoclonal antibody (1 mg) was incubated with PNGaseF overnight at 37 ° C to release the N-glycan from the antibody. The released N-glycans were separated from the antibody by 30K Amicon ultrafiltration, flow-through lyophilized and resuspended in 200 ⁇ l of deionized water.
  • MALDI-TOF MS analysis of N-glycans from both antibody molecules as previously described was performed using a MALDI-TOF MS mass spectrometer.
  • the oligosaccharide from antibody BAT4306F produced by CHO-2G8 is present as a single peak and is substantially in the same population, which is different from the profile of the antibody 4306 oligosaccharide produced by the parental host cell (Fig. 9).
  • purified antibodies targeting CD20 were used to determine their ADCC activity in vitro (LDH method promega).
  • the antibody was purified from the culture medium by BAT4306F produced by CHO-2G8 cells through a Protein A affinity column and quantified by UVUV280.
  • Female parental unmodified 4306 was expressed in wild-type CHO cells and purified in the same manner.
  • wil2-S cells were cultured well with RPMI-1640 medium containing 10% FBS (4-7 days). The cells in the logarithmic growth phase were centrifuged at 1000 rpm for 10 minutes to discard the supernatant.
  • Collect PBMCs of effector cells add B solution (RPMI-1640 medium without serum red phenol red), wash twice with the same as above, count, adjust the cells with B solution to 3 ⁇ 10 5 /ml, mix and add the above U - 96 well cell culture plates, 50 ul per well. Incubate for 3 hours at 37 ° C in a 5% CO 2 incubator. At 45 min from the incubation time of 3 h, 20 ⁇ l of lysate was added to the maximum release well of the target cells, and incubation was continued for 45 min at 37 ° C in a 5% CO 2 incubator. The U-96 well cell culture plate was placed in a centrifuge and centrifuged at 250 g for 4 min.
  • B solution RPMI-1640 medium without serum red phenol red
  • BAT4306F, 4306 was determined by the FASC method with reference to the method of Klervi Even-Desrumeaux et al (2012).
  • the affinity of Rituximab for different cell surface CD20 was compared. Briefly described as follows: Wil2-s in logarithmic growth phase was collected, centrifuged at 800 rpm for 5 min, and the supernatant was discarded. The cells were washed once with PBS, the density was calculated, resuspended in PBS, and dispensed into a 1.5 mL centrifuge tube to make 500,000 cells per tube.
  • the antibody concentrations were set to 30, 3.33, 1.11, 0.37, 0.1, 0.04, 0.014, 0.0046 ⁇ g/mL, and 200 ul of antibody was sequentially added to the above cells, and the cells were resuspended and mixed. At the same time, the same volume of PBS was added as a negative control. 4 ° C, placed in the dark for 2h. Centrifuge, 1200 rpm, 5 min, discard the supernatant and wash once with PBS.
  • the mechanism of action of anti-CD20 antibody in B lymphoma patients is ADCC, CDC, and directly induced B cell apoptosis
  • the effect of an anti-CD20 antibody is ultimately reflected in the B cells of this antibody-clearing patient. Capabilities, not just an increase in a mechanism of action.
  • the biological function of BAT4306F to clear B cells in whole blood of different NHL patients was evaluated in vitro. Briefly described as follows: The heparin sodium anticoagulant tube is used to take about 3 mL of blood from a newly diagnosed NHL patient. Store at room temperature and wait for the experimenter to take it.
  • Biotin-labeled Fc ⁇ RIIIa 158V, Fc ⁇ RIIIa 158F was diluted to 2.5 ⁇ g/mL with AB; Loading: Biotin-labeled Fc ⁇ RIIIa 158V dilution was loaded for 10 min (to about 1.3 nM signal); 3.6.3 affinity detection with Fc ⁇ RIIIa 158V: The drug to be tested, BAT4306F and Obinutuzumab, was diluted to 500 nM with AB, and Rituximab was diluted to 3000 nM with AB, followed by a 2-fold gradient dilution of 7 points with the same buffer.
  • the SA sensor runs the following steps: Baseline: AB detects the baseline, 150 s; Association: drug dilution at the gradient concentration Liquid sample and blank (AB), combined with 90s; Dissociation: dissociation in AB for 120s; Regeneration: regeneration for 5s in NaOH at pH 10.5; Neutralization: AB for 5s. The regeneration and neutralization cycles were performed 3 times. After collecting the data, the data was analyzed by the instrument's data analysis software Acquisition 8.2. The data collected by Baseline was used as the baseline, and the reference signal was deducted (for sample blank and sensor blank double subtraction), and the obtained data was analyzed by group and performed. Fitting.
  • the glycosidase was used to excise the sugar chain of the sample from the glycosylation site, and the procaine amide hydrochloride fluorescein was removed. After coupling the label, it was separated on a HILIC column and eluted with 100 mM ammonium formate with a mobile phase A of pH 4.5 and acetonitrile with mobile phase B. The elution gradient was 0-36 minutes from 28% A- 38% A, detected with a fluorescence detector. In the system-adapted solution, the separation of glycoforms G1 and G1' must not be less than 1.0. The results are shown in Fig. 13.
  • Table 4 shows that the glycoforms of the four antibodies are highly uniform and the homogeneity of the sugar chains is good. It shows that the method or the cell of the present invention has universal applicability, and is not only suitable for producing anti-CD20 antibody, but also for producing antibodies at other sites of action, so that the target antibody has homogeneity and enhanced ADCC activity.

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Abstract

提供了一种由基因组被编辑的CHO宿主细胞用于产生的具有独特糖谱的重组抗体的方法。该方法为采用TALEN技术，将已经适应无血清悬浮生长的CHO细胞的fut8基因进行编辑，被编辑的CHO宿主细胞能够产生具有独特糖谱的重组抗体，该独特糖谱主要体现在抗体具有非岩藻糖基化N-连接寡糖链，N-糖基化异质性程度低，有均一的糖链。由该方法制备得到的抗体ADCC效应和抗体稳定性增加。

Description

一种由基因组被编辑的CHO宿主细胞产生的具有独特糖谱的重组抗体及其制备方法 技术领域
本发明属于生物工程与技术领域，涉及到一种由基因组被编辑的CHO宿主细胞用于产生的具有独特糖谱的重组抗体及其该宿主细胞和抗体的制备方法。
背景技术
CHO细胞是中国仓鼠卵巢细胞(Chinese Hamster Ovary，CHO)，1957年美国科罗拉多大学Dr.Theodore T.Puck从一成年雌性仓鼠卵巢分离获得，为上皮贴壁型细胞，是目前生物工程上广泛使用的细胞系。工业生产上应用较多的是CHO-K1细胞，为转化细胞系，细胞染色体分布频率是2n＝22，系亚二倍体细胞。ATCC保存CHO-K1细胞株，编号为CCL-61，被广泛地用于重组DNA蛋白的表达。最初细胞为贴壁型细胞，经多次传代筛选后，也可悬浮生长。CHO细胞容易发生基因突变，也较易进行基因转染。早期的研究也证明，与其他工程细胞株比较，用CHO细胞产生的抗体与人类血清抗体糖型最为接近，因此CHO细胞是良好的哺乳动物基因表达宿主细胞。
治疗性抗体的作用机制是与靶分子形成复合物，引起靶标抗原中和或者通过抗体Fc段的免疫效应清除抗原或者病原体。抗体药物与靶分子的特异性结合和活性作用的发挥依赖其复杂的多级结构和翻译后修饰，而糖基化作为抗体最重要的翻译后修饰，对于抗体的生物活性、体内代谢和免疫原性有着重要的作用。抗体药物的糖基化形式主要为N-糖基化，涉及的单糖主要有：葡萄糖、半乳糖、甘露糖、N-乙酰葡糖胺、N-乙酰半乳糖胺、岩藻糖、唾液酸(NANA、NGNA)。根据末端半乳糖数量，抗体分子Fc段Asn297连接的二分支或多分枝双触角复合型寡糖可分为G0、G1(1，3)、G1(1，6)和G2等多种类型，每一种类型又根据是否具有岩藻糖(F)或者平分型半乳糖(B)分为16种(Glycobiology,August 2015,1-10.DOI:10.1093/glycob/cwv065)。因此，即使不考虑末端是否存在唾液酸化和高甘露糖型，抗体重链的寡糖类型至少有36种，且随着抗体两条重链的随机组合，可能存在的糖型达400多种，使抗体呈现出高度的异质性。
不同糖型对治疗性抗体的药学性质有不同的影响。高甘露糖糖型(Man5)含量，导致抗体在血液中的快速清除，半衰期缩短(MAbs，2012，4(4):509－520)。G0F促进补体通路作用，加快清除速率。G2F在孕妇和新生儿脐带中含量增加。唾液酸修饰对静脉注射免疫球蛋白的炎症作用影响明显。岩藻糖的降低导致ADCC活性明显增强(JBC(2003)Chemistry 278,3466-3473)。因此，根据治疗性抗体的主要作用机制和药物用途，设计和优 化抗体的糖链是有必要的。
与蛋白合成不同，抗体的糖基化并没有模板可遵循，其糖基化类型和各寡糖组分的比例受到宿主细胞类型及培养条件的影响。通过工程化宿主细胞来改造单克隆抗体的寡糖组分增强其Fc介导的效应的方法散见于不同的文献、专利报道。例如用β(1,4)-N-乙酰葡糖胺转移酶III(GnTIII)过表达的CHO细胞制备的抗体表现出比亲本细胞中表达的抗体更高的ADCC活性，而且活性的差异大约为10到20倍(Biotechnol Bioeng.(2001)Aug 20；74(4):288-94)，但GnTIII的过度表达对于CHO细胞有毒性并且由于是外源表达往往随着培养过程中传代次数增加，GnTIII表达量会下降，用此作为宿主细胞产生的抗体的岩藻糖含量会变化，从而影响抗体药物的均一性。产生脱岩藻糖化抗体的细胞系的例子还包括蛋白质岩藻糖化缺陷的Lec13CHO细胞(Ripka et al.Arch.Biochem.Biophys.249:533-545(1986)，但由于其极低的蛋白产量不适合作为治疗性抗体生产的宿主细胞(Yutaka Kanda et al Biotechnol Bioeng.(2006)Jul 5；94(4):680-8)。α-1-6岩藻糖基转移酶基因FUT8敲除的CHO细胞(Yamane-Ohnuki et al.(2004),Biotech.Bioeng.87:614)也导致生产的抗体岩藻糖含量降低。在如Yamane-Ohnuki和Kyowa Hakko专利所述的FUT8敲除细胞系中，公开了一种控制抗体岩藻糖化水平和提高ADCC(抗体依赖性细胞毒性)效应的方法，该方法就是用特异的siRNA抑制宿主细胞中fut8基因的表达从而降低该宿主细胞所生产抗体的岩藻糖水平，该方法与前述GnTIII过度表达的CHO细胞系有一样的缺点，首先宿主细胞需要引入外源序列，其次siRNA抑制目的基因的效率最多只能到70％左右，最后siRNA表达的稳定性可能影响抗体药物的质量属性。
最近，利用新兴的基因组编辑技术对宿主细胞目的基因进行编辑，失活胞内的FUT8酶，降低抗体的岩藻糖水平在不同的文献、专利中屡有报道。如Malphettes et al(2010)报道了采用锌指酶(ZFN)技术敲除亲本细胞DG44，得到fut8基因纯合敲除的DG44衍射生克隆，用该细胞系生产的抗体完全不含岩藻糖。Beurdeley et al(2012)报道了采用TALEN技术对CHO-K1细胞的fut8基因进行编辑，使宿主细胞丧失FUT8酶活性；再如Sun et al(2015)报道了用CRISPR/Cas9技术对fut8基因的10号外显子编辑，使CHO-K1细胞丧失FUT8酶活性。
发明内容
基于此，有必要针对现有抗体药物基本限于Fc的单一N-糖基化修饰，但因糖型组分和含量不一致且易变化而影响生产稳定性等问题，提供一种由基因组被编辑的CHO宿主细胞产生的具有独特糖谱的抗体以及该抗体的制备方法。本发明的上述目的通过以下的技术手段实现：
第一方面，本发明提供一对多肽，具有SEQ ID NO.10和SEQ ID NO.11所示的氨基酸序列，或者与SEQ ID NO.10和SEQ ID NO.11所示序列具有至少90％，至少91％，至少92％，至少93％，至少94％，至少95％，至少96％，至少97％，至少98％或至少99％同一性。在一些实施例中，SEQ.NO.10和SEQ.NO.11所示的一对多肽分别为TALEN的上游和下游的DNA结合域的氨基酸序列，其可以特异地与基因的特定碱基区域结合。
第二方面，本发明提供了一对多核苷酸，该一对核苷酸分别编码SEQ.NO.10和SEQ.NO.11所示的一对多肽。在一些实施例中，所述的一对多核苷酸具有SEQ ID NO.12和SEQ ID NO.13所示的核酸序列，或者与SEQ ID NO.12和SEQ ID NO.13所示序列具有至少90％，至少91％，至少92％，至少93％，至少94％，至少95％，至少96％，至少97％，至少98％或至少99％同一性。
第三方面，本发明提供了一对融合蛋白，该融合蛋白由上述的一对多肽与转录激活子样效应因子(FokI)的DNA切割域的氨基酸序列融合而成。在一些实施例中，转录激活子样效应因子(FokI)的DNA切割域的氨基酸序列为天然的或者经过人工改造的。在一些实施例中，所述的一对融合蛋白具有如SEQ ID NO.14和SEQ ID NO.16所示氨基酸序列，或者与SEQ ID NO.14和SEQ ID NO.16所示序列具有至少90％，至少91％，至少92％，至少93％，至少94％，至少95％，至少96％，至少97％，至少98％或至少99％同一性。在一些实施例中，该对融合蛋白可以特异性识别CHO的Fut8基因的两段核苷酸序列。在一些实施例中，所述的Fut8基因的两段核苷酸序列为Fut8基因的外显子1(Exon1，SEQ ID NO.7)上的两段核苷酸序列。在一些实施例中，所述的Fut8基因的两段核苷酸序列分别具有SEQ ID NO.3和SEQ ID NO.4所示的核苷酸序列。在一些实施例中，SEQ ID NO.3和SEQ ID NO.4所示的核苷酸序列之间，有一段Space，其具有SEQ ID NO.5所示的序列。
第四方面，本发明还提供了一对核苷酸，所述的一对核苷酸分别编码上述的一对融合蛋白。在一些优选的实施例中，所述一对核苷酸具有SEQ ID NO.15和SEQ ID NO.17所示的核酸序列，或者与SEQ ID NO.15和SEQ ID NO.17所示序列具有至少90％，至少91％，至少92％，至少93％，至少94％，至少95％，至少96％，至少97％，至少98％或至少99％同一性。
第五方面，本发明还提供了上述任意一对多核苷酸中的至少任意一条多核苷酸的载体。在一些实施例中，所述的载体为质粒。
第六方面，本发明还提供了一种利用上述载体转化的宿主细胞。
在其中一些实施例中，这些被上述载体转化细胞为基因组被编辑的CHO宿主细胞，其亲本细胞来源于CHO-K1细胞系。
在其中一些实施例中，所述一种基因组被编辑的CHO宿主细胞，其亲本细胞适应无血 清悬浮培养；所述亲本细胞命名为CHO-BAT。
在其中一些实施例中，所述一种基因组被编辑的CHO宿主细胞，其亲本细胞CHO-BAT是满足如下一项或者多项特征而选择的CHO-K1的亚克隆：
所述细胞具有高转染效率；
所述细胞具有短指数生长时间；
所述细胞具有在CD-CHO培养中达到高细胞密度的能力。
在其中一些实施例中，所述一种基因组被编辑的CHO宿主细胞，该细胞相对于亲本细胞由于FUT8基因的某些区域存在碱基缺失、插入、无义突变导致该细胞内源性α1,6—fucosyltransferase(Fut8)失去酶活性；
所述细胞不含有外源DNA序列；
所述细胞作为宿主细胞表达的重组抗体具有独特的糖谱特征。
在其中一些实施例中，所述一种基因组被编辑的CHO宿主细胞，其特征在于：所述细胞FUT8基因的外显子1的基因组被编辑，导致该细胞内源性Fut8失去酶活性；所述细胞不包含使FUT8基因产生碱基缺失、无意突变的过程中引入的表达载体的DNA序列；
所述细胞作为宿主细胞表达的重组抗体具有独特的糖谱特征，其特征主要包括具有非岩藻糖基化N-连接寡糖链，同时包括抗体的其它糖谱特征。
在其中一些实施例中，所述一种基因组被编辑的CHO宿主细胞，其FUT8基因被敲除；所述细胞与凝集素LCA结合呈阴性；所述细胞命名为CHO-BAT-KF。
第七方面，本发明提供了一种试剂盒，其含有上述的一对多肽中的至少任意一条；或者含有上述一对多核苷酸中的至少任意一条；或者含有一对融合蛋白中的至少任意一个；或者含有上述的载体；或者含有上述的宿主细胞。
第八方面，本发明提供了上述的一对多肽/一对多核苷酸/一对融合蛋白/载体在对CHO细胞的Fut8基因编辑的应用。
第九方面，本发明提供了上述的一对多肽/一对多核苷酸/一对融合蛋白/载体/宿主细胞在生产抗体，尤其是具有独特糖谱的抗体中的应用，或者提供了上述的一对多肽/一对多核苷酸/一对融合蛋白/载体/宿主细胞生产的抗体。
第十方面，本发明提供了一种CHO的Fut8基因编辑的方法，其包含以下步骤：将上述一对融合蛋白或者一对多核苷酸或者载体转入CHO细胞，于37℃条件下培养14天，经加压筛选、有限稀释得到Fut8基因被敲除的CHO细胞，示例性的方法可参照Wood et al.,J Immunol.145:3011(1990)。
第十一方面，本发明提供了由基因组被编辑的CHO宿主细胞产生具有独特糖谱的重组抗体的制备方法或由该方法产生的抗体，其包含以下步骤：
(1)将上述的一对融合蛋白或者一对多核苷酸或者载体转染CHO细胞(例如野生型CHO细胞)，经加压筛选、有限稀释得到Fut8基因被敲除的CHO细胞；
(2)将编码抗体基因表达盒的质粒电转染Fut8基因被敲除的CHO细胞，加压筛选、有限稀释得到分泌抗体的稳定CHO细胞株；
作为优选的实施方式，步骤(1)中将载体转染野生型CHO细胞；更优选地，将质粒稳定转染野生型CHO细胞；
作为优选的实施方式，所述的CHO细胞为CHO-K1；更优选地，所述的CHO-K1适应无血清培养。
作为优选的实施方式，所述的抗体为抗CD20抗体；更优选地，所述的抗体为人源化或者全人源抗CD20抗体；更优选地，所述的抗体为BAT4306F；更优选地，所述的抗体BAT4306F具有两条SEQ ID NO.20所示的轻链和两条SEQ ID NO.21所示的重链。发明人采用了本发明的方法、细胞、多肽等用于制备多种类型的抗体，经研究发现，制备出的不同类型的抗体均表现出高度一致的糖型，且糖型异质化程度低。表明本发明方法、细胞等适用于所有类型的抗体的制备。在一个实施方案中，所述抗体结合CD20。在一个实施方案中，CD20结合抗体是人源化抗体。在优选的实施方案中，所述人源化抗体BAT4306F是来自专利WO2005044859里B-Ly1抗体序列的重链可变区B-HH6氨基酸序列和轻链可变区B-KV1氨基酸序列。BAT4306F抗体包含以下序列的一对轻链和重链:SEQ ID NO.20和SEQ ID NO.21。在一个实施方案中，CD20结合抗体是全人源抗体BAT4406F，其包含以下序列的一对轻链和重链：SEQ ID NO.22和SEQ ID NO.23。在一个实施例方案中，所述的抗体是BAT1206F，BAT1206F抗体具有两条SEQ ID NO.18所示的轻链和两条SEQ ID NO.19所示的重链。在一个实施例方案中，所述的抗体是BAT0206F，BAT0206F结合EGFR，抗体具有两条SEQ ID NO.24所示的轻链和两条SEQ ID NO.25所示的重链。在一个实施方案中，所述的抗体是BAT0808，其结合Trop2，BAT0808具有两条SEQ ID NO.26所示的轻链和两条SEQ ID NO.27所示的重链。在一些实施方案中，所述经修饰的糖蛋白由宿主细胞分泌。在一些实施方案中，所述经修饰的糖蛋白为抗体。
作为一个示范性的具体的实施方式，本发明的由基因组被编辑的CHO宿主细胞产生具有独特糖谱的重组抗体的制备方法或由该方法产生的抗体具体的步骤为：
将上述的一对融合蛋白或者一对多核苷酸或者载体转入野生型CHO细胞，转染后细胞加入含植物凝集素(LCA)的CD CHO(Sigma)+10％FBS(胎牛血清)进行加压筛选、14天后将存活细胞按0.5个细胞/孔接种96孔细胞培养板，血清浓度降到5％，7天后将细胞转入 24孔细胞培养板，放回CO2培养箱，7天后取部分细胞，1000rpm，5min离心，PBS换液一次，取2μl荧光素标记的LCA与细胞混合，冰上孵育30min，PBS洗一次，在流式细胞仪(BD，C6)上读取荧光，用未经转染的野生型CHO细胞作为阴性对照，阳性细胞转到6孔细胞培养板，血清浓度降为1％，7天后将细胞转到小摇瓶，培养基为无血清的CD CHO，完成驯化过程。取部分细胞用质粒提取试剂盒(Omega)提取CHO基因组，以基因组为模板，用引物L130for(SEQ ID NO.1)、L130rev(SEQ ID NO.2)、taq酶进行聚合酶链式反应(PCR)，PCR产物与T载体(Promega)连接转化涂板，次日，挑取单菌落用T7引物测序，用DNASTAR分析软件分析序列，与野生型CHO基因组序列相比，出现碱基缺失的CHO细胞扩大培养，命名为CHO-BAT-KF，将处在对数生长期的CHO-BAT-KF建立细胞库，用含7.5％DMSO的CD CHO冻存液冻存细胞，转入液氮罐长期保存；取编码抗体基因的质粒线性化，测定OD260，取50μg质粒与10 7个CHO-BAT-KF在电转杯混匀，用电转仪(Biorad)转染，铺96孔细胞培养板，48h后加入蛋氨酸亚氨基代砜(methionine sulfoximine,MSX)，14天后用抗FC多抗包ELISA板，3％BSA封闭后，取上清加入板中37℃孵育2h，PBST洗涤5次，加入抗辣根过氧化物酶标记羊抗人kappa/lambda轻链，2M H 2SO 4，在酶标仪上读取OD450值。滴度高者克隆扩大培养，离心收集细胞上清，得到敲除岩藻糖的抗体蛋白。
本发明同时提供了一种细胞，其为基因组被编辑的CHO宿主细胞。
所述的基因组被编辑的CHO宿主细胞，其被编辑的Fut8基因具有SEQ ID NO.28所示的序列，或者与SEQ ID NO.28所示序列具有至少90％，至少91％，至少92％，至少93％，至少94％，至少95％，至少96％，至少97％，至少98％，至少99％，至少99.5％，或至少99.8％同一性的序列。
本发明同时提供了含有如SEQ ID NO.28所示序列的核酸，或者含有SEQ ID NO.28所示序列具有至少90％，至少91％，至少92％，至少93％，至少94％，至少95％，至少96％，至少97％，至少98％，至少99％，至少99.5％，或至少99.8％同一性的序列的核酸。
本发明还提供了一种CHO宿主细胞，其保藏于中国典型培养物保藏中心，保藏编号为CCTCC NO：C2017127，保藏日期2017.8.10，保藏地址为：中国，武汉，武汉大学；分类命名为：中国仓鼠卵巢细胞CHO-BAT-KF fut8(-/-)。
在一些实施方案中，所述宿主细胞保持在无血清培养基中。在一些实施方案中，所述宿主细胞保持在悬浮液培养物中。本发明还涉及包含所述宿主细胞的培养基、以及在培养基包含多个所述宿主细胞的培养发酵罐。在一些实施方案中，所述培养基无血清。
第十二方面，本发明提供一种抗体，该抗体由基因组被编辑的CHO宿主细胞产生的具 有独特糖谱的重组抗体，所述抗体为人源化或者全人源抗体，其具有独特的糖基化方式，N-糖基化异质性程度低，且ADCC效应显著增加。
在其中一些实施例中，所述一种由基因组被编辑的CHO宿主细胞产生的具有独特糖谱的重组抗体，是结合细胞膜表面CD20的人源化抗体。
在其中一些实施例中，所述一种由基因组被编辑的CHO宿主细胞产生的具有独特糖谱的重组抗体，具有独特的糖基化方式，所述糖基化方式的特征在于，所述抗体N-连接的多糖中的一个或多个糖部分的水平发生改变，其中所述糖部分选自由葡萄糖(Glc)、岩藻糖(Fuc)、半乳糖(Gal)、甘露糖(Man)、高甘露糖、葡萄糖胺(Glucosamine)、G0和乙酰葡萄糖胺(GlcNAc)，具有独特的糖基化方式。所述糖基化方式的特征满足如下优选条件中的一种或者多种：
所述抗体岩藻糖含量很低；(0-5％)
所述抗体含半乳糖水平较低；(≤30％)
所述抗体甘露糖水平较低；(≤5％)
所述抗体高甘露糖水平较低；(≤5％)
所述抗体G0水平较高。(≥60％)
在其中的一些实施例中，所述抗体含半乳糖水平更低，≤5％。
在其中的一些实施例中，所述抗体含G0水平较高，≥80％。
在其中一些实施例中，所述一种由基因组被编辑的CHO宿主细胞产生的具有独特糖谱的重组抗体，满足优选条件：岩藻糖含量为0。
在其中一些实施例中，所述一种由基因组被编辑的CHO宿主细胞产生的具有独特糖谱的重组抗体，所述抗体N-多糖异质程度极低，有均一的糖链。
在其中一些实施例中，所述一种由基因组被编辑的CHO宿主细胞产生的具有独特糖谱的重组抗体，其Fc的ADCC效应较强。
在其中的一些实施例中，所述的抗体具有如说明书附图10上方的BAT4306F所示的糖谱。
在其中的一些实施例中，所述BAT4306F具有两条SEQ ID NO.20所示的轻链和两条SEQ ID NO.21所示的重链；但也不排除该序列发生了突变，只要这些突变并不影响该抗体的作用。
第十三方面，本发明提供了Fut8基因被敲除的CHO宿主细胞，这样的CHO宿主细胞里面的Fut8基因的第一个外显子含有一个失活性突变。这个突变可以是一个或者多个氨基酸替换或者缺失，或者是移码突变，比如图6中显示的突变。
本发明还提供了一种药物组合物，所述的药物组合物含有上述的抗体。作为优选的实施方式，所述的药物组合物还含有药物上可接受的载体。
本发明还提供一种预防或治疗疾病的方法，该方法包括给需要的对象施用有效量的本发明公开所述抗体/抗体片段。在一些实施方案中，所述疾病选自由癌症、过敏症、心血管疾病、炎症性疾病、代谢性疾病、神经性疾病、病毒感染和/或细菌感染所组成的组。例如，所述疾病可以为癌症或过敏症。在一些实施方案中，所述对象为哺乳动物，例如人。
与现有抗体药物相比，本发明具有以下优势：
目前，上市的抗体药物基本限于Fc的单一N-糖基化修饰，但因其糖型组分和含量不一致且易变化，仍有一定复杂性，尤其给生产稳定性带来了挑战。本发明提供的一种由基因组被编辑的CHO宿主细胞产生的具有独特糖谱的重组抗体，一方面N-糖基化异质性程度低，糖链均一性好；同时ADCC效应增强，极大地改善了抗体药物的质量属性和药学特性。
与对应的由未修饰的CHO-K1(ATCC#CCL-61)或悬浮适应的亲本细胞CHO-BAT产生的抗体相比，所述抗体对FcγRIIIA受体的结合亲和性增加。
所述经修饰的宿主细胞产生抗体，与由对应的未修饰的宿主细胞产生的对应的抗体相比，该抗体与FcγRIIIA亲和力增强。
附图说明
图1显示的是贴壁生长的CHO-K1(ATCC#CCL-61)和适应在无血清培养基悬浮生长的细胞株CHO-BAT。
图2 TALEN表达质粒pCS2-Fok1的图谱。
图3 TLEN蛋白的功能验证，电泳图左边为野生型，基因编辑后细胞基因组PCR产物显示为500bp及750bp两条带，而野生型只有750bp单一条带，符合预期结果，证明Talen蛋白对是有功能的；其中，Lane1:100bp marker；Lane2:wt；Lane3:pool。
图4 FACS分析了24孔板生长的细胞，基因被编辑的细胞克隆FITC标记的LCA结合呈阴性的细胞，野生型细胞FITC标记的LCA结合呈阳性结合。
图5用糖链芯片分析显示，送检的被基因编辑的41，43号克隆岩藻糖含量降低到0-10％，野生型抗体1206的岩藻糖含量为80％。
图6 TALEN蛋白的靶向序列通过PCR扩增之后测序，结果用lasergeneMegAlign序列分析软件比对。191-1、191-2、217-1、217-3是四个被挑选的基因组被调整的细胞克隆，分别提取基因组作为DNA模板，用引物L130for和L130rev进行PCR反应后，将扩增产物进行CEL-1碱基错配分析。结果显示，细胞克隆191-1、191-2为杂合子，细胞克隆217-1、217-3 为纯合子。根据比对结果，挑选基因组被编辑的纯合子217-1、217-3，记为CHO-2G8、CHO-1D6。最后选定CHO-2G8作为宿主细胞进行后续实验，并将该宿主细胞命名为CHO-BAT-KF。
图7 CHO-BAT-KF与亲本细胞CHO-BAT生长密度的比较。
图8 CHO-BAT-KF与亲本细胞CHO-BAT生长活力的比较。
图9采用MALDI-TOF MS质谱仪对BAT4306F和4306抗体分子的N-多糖进行MALDI-TOF MS分析，每个来自于BAT4306F的N-多糖都比来自于4306的N-多糖少了1个岩藻糖，左图是亲本细胞生产的抗体分子4306，右面图是BAT4306F抗体分子
图10.BAT4306F比GAZYVA(Obinutuzumab)有更低的岩藻糖含量，糖链的异质性程度更低，产品的均一性更好。
图11用Raji作为靶细胞，PBMC作为效应细胞，比较了BAT4306野生型，糖链修饰的BAT4306F，Obinutuzumab，Rituximab等抗CD20抗体的ADCC效应。
图12比较了糖链修饰的BAT4306F，Obinutuzumab,，rituximab三种抗体，在50，25，10ng/mL浓度下清除体外全血中B细胞的能力。
图13比较了用CHO-BAT-KF细胞生产的抗CD20抗体BAT4306F、BAT4406F，抗EGFR的抗体BAT0206F，抗Trop2的抗体BAT0808的糖谱。
本发明基因组被编辑的CHO宿主细胞，保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO：C2017127，保藏日2017.8.10，保藏地址为：中国，武汉，武汉大学；分类命名为：中国仓鼠卵巢细胞CHO-BAT-KF fut8(-/-)。
具体实施方式
以下通过具体的实施例进一步说明本发明的技术方案，具体实施例不代表对本发明保护范围的限制。其他人根据本发明理念所做出的一些非本质的修改和调整仍属于本发明的保护范围。
需要说明的是，本发明中，抗体糖部分的“水平”或“含量”表述同样的意思，表示某种糖部分在抗体所有糖部分中所占的质量比。
如发明中，“氨基酸”是指羧基α-氨基酸，其可直接或以前体的形式由核酸编码。单个氨基酸由三个核苷酸(所谓的密码子或碱基三联体)组成的核酸编码。每一个氨基酸由至少一个密码子编码。相同氨基酸由不同密码子编码称为“遗传密码的简并性”。本申请中所用的术语“氨基酸”是指天然发生的羧基α-氨基酸，其包括丙氨酸(三字母代码：ala，一字母代码：A)、精氨酸(arg，R)、天冬酰胺(asn，N)、天冬氨酸(asp，D)、半胱氨酸(cys，C)、谷氨酰胺(gln，Q)、谷氨酸(glu，E)、甘氨酸(gly，G)、组氨酸(his，H)、异亮氨酸(ile，I)、亮氨 酸(leu，L)、赖氨酸(lys，K)、甲硫氨酸(met，M)、苯丙氨酸(phe，F)、脯氨酸(pro，P)、丝氨酸(ser，S)、苏氨酸(thr，T)、色氨酸(trp，W)、酪氨酸(tyr，Y)和缬氨酸(val，V)。
本发明中，可互换使用的术语“多核苷酸”或“核酸”或“核酸序列”是指，由单核苷酸(也称为碱基)a、c、g和t(或在RNA中u)组成的多聚体分子，例如DNA、RNA或其修饰形式。该多核苷酸分子可以是天然发生的多核苷酸分子、或合成的多核苷酸分子、或一个或多个天然发生的多核苷酸分子与一个或多个合成的多核苷酸分子的组合。该定义还包括其中一个或多个核苷酸被改变(例如，通过诱变)、缺失或添加的天然发生的多核苷酸分子。核酸可以是分离的，或整合在另一个核酸例如表达盒、质粒或宿主细胞的染色体中。以由单核苷酸组成的核酸序列来表征核酸。对于本领域技术人员来说，将例如多肽的氨基酸序列转化成编码该氨基酸序列的相应核酸序列的操作和方法是熟知的。因此，核酸可以以其由单核苷酸组成的核酸序列来表征，也可以用由其编码的多肽的氨基酸序列来表征。
并且，所述的“多核苷酸”或“核酸”或“核酸序列”可包含修饰的核苷酸，占核酸分子中存在的核苷酸总数的百分比，例如至少约5％，10％，15％，20％，25％，30％，35％，40％，45％，50％，55％，60％，65％，70％，75％，80％，85％，90％，95％或100％修饰的核苷酸。
本发明中“多肽”是由通过肽键连接的氨基酸组成的聚合物，其可以是天然或合成产生的。少于大约20个氨基酸残基的多肽可称为“肽”，然而，由2个或多个肽组成的分子或含有具有100个以上氨基酸残基的一个多肽的分子可称为“蛋白质”。多肽也可含有非氨基酸成分，例如糖基、金属离子或羧酸酯类。非氨基酸成分可通过表达该多肽的细胞加入，并且可随细胞的类型变化而变化。在本文中根据其氨基酸主链结构或编码它的核酸来定义多肽。添加例如糖基通常不是规定的，但可存在。”并且，所述的“多肽”可包含修饰的氨基酸，占氨基酸分子中存在的氨基酸总数的百分比，例如至少约5％，10％，15％，20％，25％，30％，35％，40％，45％，50％，55％，60％，65％，70％，75％，80％，85％，90％，95％或100％修饰的氨基酸。
本发明中，“宿主细胞”指以单核实体培养的微生物或真核细胞或细胞系，它们可以是或已用作重组载体或其它转移多核苷酸的受者，并且包括受转染原始细胞的后代。在一些实施例中，所述宿主细胞为非淋巴细胞，所述的宿主细胞产生表现出相同的独特糖谱。在一些实施方式中，所述的宿主细胞如NS0细胞、猿猴COS细胞、中国仓鼠卵巢(CHO)细胞等等。在一些实施方式，所述宿主细胞选自中华仓鼠卵巢(CHO)细胞。在一些实施方式中，所述的宿主细胞选自CHO-K1、CHO-S、DUXB11、CHO-1E5、CHO3F、CHO/DG44、CHO-BAT和CHO-2.6细胞。在一些实施方案中，所述宿主细胞产生抗体，该抗体表现出独特的糖谱。本发明的经基因组变编辑的CHO宿主细胞，如CHO-BAT-KF fut8(-/-)可以在培养物、以及可以用来使培养物生长的装置(包括发酵罐)中生长。它们可以生长成为单层或附着 在表面上。或者，宿主细胞可以在悬浮液中生长。该细胞可以在不含血清的培养基中生长。所述培养基可以是市售的培养基，例如但不限于DMEM/F12。经编辑的CHO宿主细胞在培养基中可以很多代地保持其特定的独特糖谱。例如，经编辑的CHO宿主细胞在至少约20、30、40或50代后仍保持其特定的独特糖谱。在一些实施方案中，经修饰的CHO宿主细胞在至少约60代后仍保持其特定的独特糖谱。在另一个其它实施方案中，经修饰的CHO宿主细胞在至少约100、150、200、500或1000以上的代之后仍保持其特定的独特糖谱。
宿主细胞的糖基化方式可以是任何蛋白质部分发生N-或O-糖基化，其中，可以分别在天冬酰胺的酰胺氮处、或者羟基赖氨酸、羟基脯氨酸、丝氨酸或苏氨酸的羟基氧处添加一个或多个糖分子。糖基化方式的特征在于，至少两个或多个糖分子或糖类(如单糖、二糖、多糖或寡糖)的水平发生改变。例如，糖分子可以是三糖、四糖、五糖、六糖、七糖、八糖、九糖、或它们的衍生物，如脱氧糖(如脱氧六糖)；N-或O-取代的衍生物，如唾液酸；或者具有氨基的糖。糖分子可以包括但不限于半乳糖(Gal)、葡萄糖(Glc)、甘露糖(Man)、N-乙酰基神经氨酸(NeuAc)、岩藻糖(Fuc)、N-乙酰基半乳糖胺(GalNAc)、N-乙酰基葡萄糖胺(GlcNAc)；以及木糖(Xyl)。糖分子可以通过α或β连接而与其它糖分子连接。
本发明“抗体”包括所有形式的抗体，如重组抗体、人源化抗体、嵌合体抗体、单链抗体、融合抗体、单克隆抗体、多克隆抗体等。该抗体还可为片段。该抗体还可以与药物、毒素或治疗用放射性同位素结合。本发明的宿主细胞也可产生双特异抗体融合蛋白质，包括与一个以上抗原结合的杂化抗体。因此，抗体包括裸抗体和结合抗体以及抗体片段，它们可以是单特异性或多特异性。
作为可选的实施方式，所述的抗体或抗体片段无特别限制，可以选自抗HER2、抗CD20、抗EGF、抗VEGF、抗PDGF、抗EpCam、抗CD3、抗CD4、抗CD19、抗CD30、抗CD33、抗CD40、抗CD51、抗CD55、抗CD80、抗CD95、抗CCR2、抗CCR3、抗CCR4、抗CCR5、抗叶酸、抗CXCR4、抗EGFR或Trop2抗体等等。作为优选的实施方式，所述的抗体为人源化或者全人源抗体。
本发明的药物组合物中，通过将具有所需程度的纯度的抗体与可选的生理学上可接受的载体、赋型剂或稳定剂(Remington’s Pharmaceutical Sciences 16th edition,Osol,A.Ed.(1980))混合，以冻干制剂或水性溶液的形式，制得用于贮存的本发明抗体的药物制剂。可接受的载体、赋型剂或稳定剂在所应用的剂量和浓度下对受者无毒，包括：缓冲液，如磷酸盐、柠檬酸盐以及其它的有机酸；抗氧化剂，包括抗坏血酸和蛋氨酸；防腐剂(如氯化十八烷基二甲基苄基铵；六甲基氯化铵；苯扎氯铵、苄索氯铵；苯酚、丁醇或苄基醇；烷基对羟苯甲酸酯，如甲基或丙基对羟苯甲酸酯；儿茶酚；间苯二酚；环己醇；3-丙醇；以及间甲酚)；低分子量(少于约10个残基)多肽；蛋白质，如血清白蛋白、明胶、或免疫球蛋白；亲水性聚合物，如聚乙烯吡咯烷酮；氨基酸，如甘氨酸、谷氨酰胺、天冬酰胺、组氨酸、精氨酸或赖氨 酸；单糖、双糖类以及其它碳水化合物，包括葡萄糖、甘露糖或糊精；螯合剂，如EDTA；糖，如蔗糖、甘露糖醇、海藻糖或山梨糖醇；成盐反离子，如钠；金属络合物(如Zn-蛋白质络合物)；和/或非离子表面活性剂，如Tween、PluronicsTM或聚乙二醇(PEG)。
本发明的抗体、药物组合物、药物制剂可通过任何合适的方式来施用，包括非肠道方式、皮下、腹膜内、肺内和鼻内方式，并且，如果需要的话，为了进行局部免疫抑制治疗，可以采用病灶内施用方式。非肠道灌注方式包括肌肉内、静脉内、动脉内、腹膜内或皮下施用方式。另外，本发明抗体可通过脉冲式灌注(特别是，本发明抗体的剂量梯度变化)来合适地施用。根据施用时间短或长，优选通过注射来给药，最优选通过静脉内或皮下注射来给药。ADCC(抗体依赖性细胞介导的细胞毒性)是指细胞介导的反应，其中，表达FcR的效应细胞(如，自然杀伤(NK)细胞、嗜中性粒细胞和巨噬细胞)识别靶细胞上结合的抗体，并且接下来使靶细胞裂解。用于介导ADCC的原代细胞包括NK细胞、单核细胞和巨噬细胞。NK细胞通常主要表达FcγRIII，而单核细胞表达FcγRI、FcγRII和FcγRIII。本发明中，母本CHO细胞系被编辑以产生具有独特糖谱的CHO细胞系。然后该经被编辑的CHO细胞系可以产生ADCC活性比由母本CHO细胞产生的抗体的ADCC活性高的抗体。
实施例1 适应无血清悬浮培养的亲本细胞的筛选
用含10％FBS的DMEM/F12培养基培养的CHO-K1，当细胞汇合度达到80％-90％时，用PBS洗涤，胰酶消化，5％FBS的DMEM/F12培养基终止，计数并离心。用含5％FBS的DMEM/F12培养基重悬细胞，以1×10 5个细胞/毫升的密度接种细胞。当细胞的汇合度达到80％-90％，用PBS洗涤，胰酶消化，2％FBS的培养基DMEM/F12终止，计数并离心。用含2％FBS的培养基DMEM/F12重悬细胞，以1×10 5个细胞/毫升的密度接种细胞。待上述细胞汇合度达到80％-90％，按照之前步骤把上述细胞胰酶消化，用含1％FBS的DMEM/F12培养基终止，传代3-4代。CD CHO培养基与DMEM/F12按1：1(V/V)的比例进行混合，终浓度调整为6mM谷氨酰胺，血清含量调整为1％。把上述获得的低血清适应了的CHO-K1细胞以3×10 5cells/mL密度接种到T25方瓶，在37℃、5％CO 2培养箱进行培养。当细胞汇合度达到80-90％，胰酶消化，用含1％FBS的DMEM/F12与CD CHO培养基混合培养基(体积比为1:2)终止，计数并离心，以3×10 5cells/mL密度接种到T25方瓶，在37℃、5％CO 2培养箱进行培养。逐步降低混合液培养基中DMEM/F12比例至(1:8)，当细胞存活率大于90％，此时可将细胞培养基中的DMEM/F12成分完全剔除，建立起了适应化学成分限定的CD CHO培养基包含1％血清培养的CHO-K1细胞。用含1％FBS的化学成分限定的CD CHO培养基培养的CHO-K1，当细胞汇合度达到80％-90％时，用PBS洗涤，胰酶消化，用含0.5％FBS的CD CHO培养基终止，计数并离心。用含0.5％FBS的CD CHO培养基重悬细胞，以1×10 5个细胞/毫升的密度接种于T25方瓶，当细胞的活力达到80％-90％，用PBS洗涤，胰酶消化，0.25％FBS的CD  CHO培养基终止，计数并离心。用含0.25％FBS的CD CHO培养基重悬细胞，以1×10 5个细胞/毫升的密度接种细胞。直到细胞在该阶段生长健康才开始下一阶段降低血清浓度。把适应了无血清的CD CHO培养的CHO-K1细胞，进行有限稀释，接种到30个96孔板，调整细胞密度到1个细胞/孔。2周后镜检观察，标记出长出单克隆的细胞。把细胞面积较大的克隆转到24孔板，1周后镜检观察，标记出生长密度较高，细胞大小一致的细胞克隆转到6孔板继续培养，1周后镜检观察，标记完全悬浮，结团不严重的，细胞密度较大的克隆转到100mL三角瓶，培养体积10mL。记录每株细胞的细胞密度，活力。把经过驯化适应无血清培养的CHO-K1细胞重新命名为CHO-BAT。
实施例2 FUT8 TALEN重组质粒构建
分析中国仓鼠卵巢癌细胞CHO-K1的基因组全序列(NW-003613860)，由此得到Fut8基因组序列(Gene ID:100751648)和它的cDNA(见表1，SEQ ID NO.8)序列，Fut8基因组由9个外显子及11个内含子组成，因为Fut8酶活性中心是由外显子1(SEQ ID NO.7)所编码的氨基酸(SEQ ID NO.9下划线氨基酸序列)组成，设计Fut8基因外显子1左右两翼作为Talen的靶向序列。根据TALEN设计指导原则及编辑基因作用机制，设计了Fut8 TALEN蛋白L130P(SEQ ID NO.10)，R184P(SEQ ID NO.11)。L130P与FokI内切酶形成融合蛋白L130-FokI(SEQ ID NO.14)，此融合蛋白识别外显子1左翼碱基L130PTN(SEQ ID NO.3)，融合蛋白L130-FokI相应的核酸序列L130-FokIN见SEQ ID NO.15，长度是19bp。R184P与FokI内切酶形成融合蛋白R184P-FokI(SEQ ID NO.16)，此融合蛋白识别外显子1右翼碱基R184PTN(SEQ ID NO.4)，融合蛋白R184P-FokI相应的核酸序列R184P-FokIN见SEQ ID NO.17，长度是17bp。含有编码针对外显子1左翼L130PTN和右翼R184PTN的TALEN蛋白的质粒载体(见图2)构建如Tomas Cermak et al.(2011)所述。在L130-FokIN、R184P-FokIN两端加入限制性核酸内切酶NcoI和XbaI酶切位点，合成这两段序列，采用NcoI和XbaI克隆进pCS2-peas-T载体(图2)。左翼结合序列和右翼结合序列中间有个19bp长度的间隔序列(Space，SEQ ID NO.5)。Fut8 TALEN的两个质粒L130N和R184N的DNA测序结果见表1，SEQ ID NO.12、SEQNO.13，L130N、R184N核酸序列翻译成氨基酸，相应序列氨基酸序列L130P、R184P见表1，SEQ ID NO.10、SEQ ID NO.11。
表1序列表
Figure PCTCN2018100008-appb-000001
Figure PCTCN2018100008-appb-000002
Figure PCTCN2018100008-appb-000003
Figure PCTCN2018100008-appb-000004
Figure PCTCN2018100008-appb-000005
Figure PCTCN2018100008-appb-000006
Figure PCTCN2018100008-appb-000007
Figure PCTCN2018100008-appb-000008
Figure PCTCN2018100008-appb-000009
Figure PCTCN2018100008-appb-000010
Figure PCTCN2018100008-appb-000011
Figure PCTCN2018100008-appb-000012
实施例3 FUT8 TALEN蛋白的功能有效性分析
CEL-I酶是一种核酸酶，它能识别双链DNA中错配的碱基并从错配处剪断双链DNA。 如果靶向序列被Fut8 TALEN编辑了，那么把从母本基因组扩增出的包含靶向序列的区域与从被转化的细胞的基因组扩增的包含靶向序列的区域混合在一起变性、退火后，退火的双链DNA存在碱基错配，这样CEL-I酶能把退火后的双链DNA切断，表现为琼脂糖电泳结果为两条带。取5×10 5个CHO-BAT细胞于转染前一天接种于6孔板，培养基为含10％胎牛血清的DMEM/F12。质粒L130N和R184N被按试剂说明提供的方法瞬时转染进入细胞。转染3天后，离心收获细胞，用基因组抽提试剂盒提取基因组。以此为模板，用引物L130 for(SEQ ID NO.1)和引物L130 rev(SEQ ID NO.2)进行PCR反应。亲本细胞包含靶向序列的区域的PCR扩增同上。两个PCR产物各取20μl混合在一起，加热到94℃然后自然冷却至室温。向200ng退火的DNA加入0.5μl CEL-I酶42℃孵育30分钟，PCR反应产物跑琼脂糖凝胶电泳。该反应产物用琼脂糖电泳分析，结果见图3。
结果表明：与野生型相比，基因编辑PCR产物能看到500bp及750bp两条带，而野生型只有750bp单一条带，符合预期结果，证明Talen蛋白对是有功能的。
实施例4 FUT8 TALEN蛋白对抗体岩藻糖含量的影响
为了确定设计的FUT8 TALEN蛋白对宿主基因组的调整是否影响到生产的抗体的糖链(岩藻糖含量是否变化)。L130N和R184N质粒瞬时转化到先前已经建立的稳定表达抗CD20抗体的细胞系。方法参考lipofectamine 2000(Invitrogen)试剂说明提供的方法，简单描述如下；在10cm的细胞培养皿内，向1×10 6细胞加入24μL包装有L130N和R184N各4μg质粒DNA的脂质体。转染两天后，更换为包含有10％(v/v)FBS(GBICO)和400μg/mL LCA(Vector)的DMEM/F12培养基。1周后，大部分细胞变圆并悬浮于培养基，另一些细胞正常贴壁生长。弃去上清，用胰酶0.25％(v/v)消化LCA抗性的细胞，离心后重悬于含10％(v/v)FBS的DMEM/F12培养基。以每孔0.5个的密度接种于96孔板。生长2周后，单克隆的细胞被挑选转入24孔板。FACS分析了24孔板生长的细胞，FITC标记的LCA结合呈阴性的细胞(图4)被扩大培养生产抗体。两种细胞生产的抗体的寡糖含量由北京爱德诺生物公司测定，结果如图5所示。
结果表明：瞬时转化L130和R184质粒到抗体生产细胞降低了抗体岩藻糖含量。
实施例5 基因组被调整的宿主细胞的建立
为了建立一个基因组调整了的CHO-K1细胞，以便用它作为宿主细胞生产的蛋白、抗体无岩藻糖，L130和R184质粒瞬时转化到CHO-K1的细胞系。抗LCA的单克隆细胞的筛选同实施例3所述，候选的细胞克隆的基因组分别被提取，用引物L130 for(见表1，SEQ ID NO.1)和引物L130 rev(见表1，SEQ ID NO.2)进行PCR反应，对候选的细胞克隆包含靶 向序列的区域的PCR扩增产物进行CEL-1碱基错配分析。如果候选克隆是杂合子，那么CEL-1酶切后琼脂糖电泳是两条带，如果是纯合子，那么CEL-1酶切该退火片段切不动，琼脂糖电泳是一条带。该PCR片段直接克隆至T载体(pGEM-T Easy Vector)然后测序。测序结果与亲本细胞该区域的片段序列对比如图6，根据比对结果，挑选两个基因组被编辑的纯合子，记为CHO-2G8,CHO-1D6。
实施例6 宿主细胞生长特性评价
选定fut8基因被敲除的克隆CHO-2G8为宿主细胞，重新命名为CHO-BAT-KF。分别取30mL含终浓度为6mM Gln的CD CHO AGT TM，接种细胞密度为30万/mL的3株CHO-BAT-KF与1株CHO-BAT于125mL小摇瓶，在d0、d3、d6、d7分别取0.5mL细胞计数，测定细胞密度及细胞活率，评价敲除Fut8基因后细胞生长特性变化情况。细胞生长密度如图7，细胞生长活力如图8，由图7和图8可知，被敲除fut8基因的CHO-BAT-KF与没有敲除fut8基因的CHO-BAT细胞生长密度和活性没有显著的差别。
实施例7 宿主细胞生产抗体的糖基化谱图分析
为了确定用本发明所述的基因组调整了的CHO-2G8细胞系生产的抗体的糖链具有变异了的N-多糖修饰，通过蛋白质A亲和柱从培养基纯化CHO-2G8细胞所产生的BAT4306F和CHO-K1细胞所产生的4306，并通过紫外UV280进行定量。脱盐的单克隆抗体(1毫克)与PNGaseF在37℃孵育过夜，让N-聚糖从抗体中释放。被释放的N-聚糖通过30K的Amicon超滤与抗体分离，流穿液冷冻干燥并重悬于200μl去离子水。采用MALDI-TOF MS质谱仪对如前所述来自两个抗体分子的N-多糖进行MALDI-TOF MS分析。来自CHO-2G8所产生的抗体BAT4306F的寡糖以单峰存在，并且基本上同种群，该群与由母本宿主细胞产生的抗体4306寡糖的谱图不同(图9)。
结果表明：4306的N-多糖的三个峰分别是G0F、G1F、G2F。基于BAT4306F的N-多糖的出峰时间和分子量，推断N-多糖的3个峰分别是G0、G1、G2，即每个来自于BAT4306F的N-多糖都比来自于4306的N-多糖少了1个岩藻糖。
同时，对市售的Gazyva与BAT4306F的糖链进行对比，分析其糖链的异质性程度，如图10所示。结果说明：BAT4306F的N-多糖异质程度更低，有更均一的糖链。对不同抗体在CHO-BAT-KF细胞表达的糖型进行分析，如表4所示。
实施例8 宿主细胞生产抗体的ADCC活性分析
为了确定具有本发明N-多糖的抗体的修饰是否能够提高其生物功能(如ADCC活性)，使用靶向CD20的经纯化的抗体以测定它们在体外的ADCC活性(LDH法promega)。抗体通过蛋白质A亲和柱从培养基纯化CHO-2G8细胞所产生的BAT4306F，并通过紫外UV280进行定量。母本未修饰的4306在野生型CHO细胞中表达，并以同样方式纯化。为了进行ADCC检测，用含10％FBS的RPMI-1640培养液培养wil2-S细胞状态良好(4-7天)。取对数生长期的细胞，1000转离心10分钟弃上清。加入A液(含10％FBS的无酚红的RPMI-1640培养液)混匀，同上离心洗两次，计数，用A液调整细胞为3×10 5个/mL，加入U-96孔细胞培养板中，每孔100μl。调节抗体的终浓度孔依次为1.2、0.24、0.048、0.0096、0.00192、0.000384、0.0000768、0.00001536(μg/mL)。置于37℃、5％CO 2培养箱孵育30min。收集效应细胞PBMC，加入B液(不含血清的无酚红的RPMI-1640培养液)同上离心洗两次，计数，用B液调整细胞为3×10 5个/ml，混匀加入上述U-96孔细胞培养板，每孔50ul。置于37℃、5％CO 2培养箱孵育3小时。离3h的孵育时间还有45min时，在靶细胞最大释放孔中加入20μl裂解液，继续置于37℃、5％CO 2培养箱孵育45min。将U-96孔细胞培养板置于离心机中，250g离心4min。取50μl/孔上清至另一平底96孔板中，加入已经配好的显色底液50μl/孔，轻轻震荡混匀，于室温避光反应30min。加入终止液50μl/孔，轻轻震荡混匀。在酶标仪OD490处读取结果。
结果表明：与母本CHO细胞产生的未修饰的4306相比，具有在无血清培养基中CHO-2G8细胞克隆所产生的N-多糖的BAT4306F对Raji细胞和wil2-S细胞的ADCC活性显著提高(图11)。
实施例9 宿主细胞生产抗体对CD20的亲和力分析
为了确定如本发明所述细胞产生的N-多糖经过修饰的抗体是否对结合CD20阳性的细胞的能力有影响，参考Klervi Even-Desrumeaux et al(2012)方法，通过FASC方法检定了BAT4306F、4306，对照Rituximab对不同细胞表面CD20的亲和力。简单描述如下：收集对数生长期细胞Wil2-s，离心800rpm，5min，弃上清。用PBS洗一遍，计算密度，用PBS重悬，分装到1.5mL离心管中，使每管50万细胞。离心，1200rpm，5min，弃上清。配置抗体浓度分别为30、3.33、1.11、0.37、0.1、0.04、0.014、0.0046μg/mL，依次加入200ul抗体到上述细胞中，重悬细胞并混匀。同时加相同体积的PBS作为阴性对照。4℃，避光放置2h。离心，1200rpm，5min，弃上清，用PBS洗一遍。加入100μl PBS重悬细胞，加入2μl FITC-羊抗人IgG1 Fab的二抗，4℃，避光放置30min。离心，1200rpm，5min，弃上清，用PBS洗一遍。上流式细胞仪C6检测。结果计算公式：Kd＝[Ab]*{Fmax/(F-Fback)-1}，结果如下表所示。
表2 抗体对细胞结合实验的IC 50值及Kd值结果统计
Figure PCTCN2018100008-appb-000013
结果表明：N-多糖经过修饰的抗体没有影响抗体结合CD20的亲和力。
实施例10 体外评价BAT4306F在不同NHL病人全血中清除B细胞的能力
尽管抗CD20抗体在B淋巴瘤患者体内的作用机制有ADCC、CDC、直接诱导的B细胞凋亡等多种机制，但一个抗CD20抗体的效果如何，最终反映在这个抗体清除患者体内的B细胞能力，而不是单单在于提高了某一种作用机制。为了确定具有本发明N-多糖的抗体的修饰是否能够提高其清除B细胞的能力，体外评价了BAT4306F在不同NHL病人全血中清除B细胞的生物功能。简单描述如下：用肝素钠抗凝管取新诊断为NHL病人的血3mL左右。室温静止保存，待实验人员来取。分别取待测血样90μL分装到新的FACS管，向各管样品中加入10μL不同浓度的BAT4306F抗体稀释液，使抗体在各管待测样品中的终浓度为10nM、1nM、0.1nM、0.01nM、0.001nM。37℃培养箱静止放置3-4小时，然后从各管取50uL血样加入BD TruCount tubes，向血样中加入BD的B细胞计数抗体混合物(抗CD45(lymphocyte population),抗CD3(T cells),和抗CD19(B cells))。室温下暗处静放15min,加入BD FACS裂解液，然后上机测量(BD C6)，结果如图12所示。
结果表明：在被测试的三个浓度水平中，BAT4306F抗体清除B细胞的能力都强于N-多糖未经修饰的抗体Rituximab。
实施例11 BAT4306F与FcγRIIIa分子的亲和力增强
为了验证基因组被编辑的CHO-BAT-KF细胞产生的具有独特糖谱的重组抗体与FcγRIIIA亲和力增强，分别测定了BAT4306F、市售GAZYVA和Rituximab与FcγRIIIA的亲和力。传感器置PBS中预湿10min。将生物素标记的FcγRIIIa 158V，FcγRIIIa 158F用AB稀释至2.5μg/mL；Loading：生物素标记的FcγRIIIa 158V稀释液中Loading 10min(至信号约1.3nM)；3.6.3与FcγRIIIa 158V亲和力检测：将待测药物BAT4306F、Obinutuzumab用AB稀释至500nM，Rituximab用AB稀释至3000nM，之后用同样缓冲液做2倍梯度稀释7个 点。将AB、FcγRIIa V158、再生缓冲液、药物稀释液、中和缓冲液依次加入96孔板相应列中，SA传感器运行如下步骤：Baseline：AB中检测基线，150s；Association：在梯度浓度的药物稀释液样品及空白(AB)，结合90s；Dissociation：在AB中解离120s；Regeneration：在pH 10.5的NaOH中再生5s；Neutralization：AB中和5s。再生、中和循环进行3次。采集数据后，用仪器的数据分析软件Acquisition 8.2对数据进行分析，以Baseline采集所得信号为基线、扣减参比信号(进行样品空白和传感器空白双扣除)，对所得数据进行群组分析并进行拟合。
表3 BAT4306F与对FcγRIIIa 158F的亲和力结果统计
Figure PCTCN2018100008-appb-000014
结果表明：在被测试的三个抗体中，CHO-BAT-KF细胞产生的具有独特糖谱的重组抗体与FcγRIIIA亲和力最强。
实施例12
为了验证其他抗体序列在所述CHO-BAT-KF宿主细胞表达生产的抗体的糖谱是稳定的，一致的，我们在CHO-BAT-KF细胞分别表达了另外几种抗体，一种是具有两条SEQ ID NO.22所示的轻链和两条SEQ ID NO.23所示的重链的BAT4406F抗体，一种是具有两条SEQ ID NO.24所示的轻链和两条SEQ ID NO.25所示的重链的抗EGFR抗体BAT0206F，一种是具有两条SEQ ID NO.26所示的轻链和两条SEQ ID NO.27所示的重链的抗Trop2抗体BAT0808。具体实验参照产品说明书进行(LudgerTagTM PROC(procainamide)Glycan Labeling Kit)，对样品进行变性还原后利用糖苷酶把样品的糖链从糖基化位点切除，经过普鲁卡因酰胺盐酸盐荧光素偶联标记后，然后上HILIC色谱柱进行分离，用流动相A为pH4.5的100mM的甲酸铵和流动相B为乙腈进行洗脱分离，洗脱梯度为0-36分钟从28％A-38％A，用荧光检测器进行检测。系统适应性溶液中，糖型G1与G1’的分离度不得低于1.0。结果如图13，表4显示，4种抗体的糖型高度一致，糖链的均一性良好。表明本发明的方法或者细胞具有普遍适用性，不仅适用于生产抗CD20抗体，也可以用于生产其他作用位点的抗体，使目标抗体具有均一性和增强的ADCC活性。
表4.用CHO-BAT-KF细胞生产四种抗体的糖型比例(％)
  G0-GN G0 Man5 G1 G1' G2 其他
BAT4306F 0.36 71.32 0.40 16.04 8.14 1.96 1.78
BAT4406F 0.42 72.31 0.45 15.51 7.88 1.83 1.60
BAT0808 0.52 79.11 0.51 11.36 6.11 1.03 1.36
BAT0206F 0.45 76.15 0.50 12.90 6.90 1.36 1.74

Claims (19)

  1. 一对多肽，其特征在于，具有SEQ ID NO.10和SEQ ID NO.11所示的氨基酸序列，或者与SEQ ID NO.10和SEQ ID NO.11所示序列具有至少90％，至少91％，至少92％，至少93％，至少94％，至少95％，至少96％，至少97％，至少98％或至少99％同一性。
  2. 一对多核苷酸，其特征在于，所述的一对多核苷酸分别编码如权利要求1所述的一对多肽；优选地，所述的一对多核苷酸序列分别如SEQ ID NO.12和SEQ ID NO.13所示，或者与SEQ ID NO.12和SEQ ID NO.13所示序列具有至少90％，至少91％，至少92％，至少93％，至少94％，至少95％，至少96％，至少97％，至少98％或至少99％同一性。
  3. 一对融合蛋白，其特征在于，所述的一对融合蛋白由权利要求1所述的一对多肽分别与天然的或经过人工改造的Fok I的DNA切割域融合而成；优选地，所述的一对融合蛋白氨基酸序列分别如SEQ ID NO.14和SEQ ID NO.16所示，或者与SEQ ID NO.14和SEQ ID NO.16所示序列具有至少90％，至少91％，至少92％，至少93％，至少94％，至少95％，至少96％，至少97％，至少98％或至少99％同一性。
  4. 一对多核苷酸，其特征在于，分别编码如权利要求3所述的一对融合蛋白；优选地，所述的多核苷酸序列分别如SEQ ID NO.15和SEQ ID NO.17所示，或者与SEQ ID NO.15和SEQ ID NO.17所示序列具有至少90％，至少91％，至少92％，至少93％，至少94％，至少95％，至少96％，至少97％，至少98％或至少99％同一性。
  5. 一种包含权利要求2、4任一所述的一对多核苷酸中的至少任意一条多核苷酸的载体；优选地，所述的载体为质粒。
  6. 一种用权利要求5所述载体转染的宿主细胞；优选地，所述的宿主细胞为CHO；更优选地，所述的CHO细胞为CHO-K1；更优选地，所述的CHO-K1适应无血清培养；
    优选地，所述的宿主细胞用于表达抗体；更优选的，所述的宿主细胞用于表达具有独特糖谱的抗体；优选地，所述的独特糖谱与野生型糖谱比较，至少一种糖部分的水平发生变化；
    优选地，所述的糖部分选自葡萄糖、岩藻糖、半乳糖、甘露糖、高甘露糖、葡萄糖胺、G0或乙酰葡萄糖胺；
    优选地，所述的抗体的糖部分满足以下条件中的一种或多种：
    a.所述的抗体岩藻糖的水平降低；优选地，所述的岩藻糖水平小于等于5％；更优选地，所述的岩藻糖水平为0；
    b.所述的抗体甘露糖的水平降低；优选地，所述的甘露糖含量小于等于5％；
    c.所述的抗体高甘露糖的水平降低；优选地，所述的高甘露糖含量小于等于5％；
    d.所述的抗体半乳糖的水平降低；优选地，所述的半乳糖的水平小于等于30％；更优选地，半乳糖水平小于等于5％；
    e.所述的抗体G0含量升高；优选地，所述的G0含量大于等于60％；更优选地，所述的G0水平大于等于80％；
    优选地，所述的G0含量大于等于60％，不含岩藻糖；更优选地，G0含量大于等于60％，不含岩藻糖，同时甘露糖含量小于等于5％或/和含高甘露糖含量小于等于5％；
    优选地，所述的抗体具有如说明书附图10中所示的糖谱；
    优选地，所述的抗体为抗EGFR或CD20或Trop2抗体；更优选地，所述的抗体为人源化或者全人源抗CD20抗体；
    优选地，所述的抗体为BAT1206F；更优选地，所述的抗体BAT1206F具有两条SEQ ID NO.18所示的轻链和两条SEQ ID NO.19所示的重链；
    优选地，所述的抗体为BAT4406F；更优选地，所述的抗体BAT4406F具有两条SEQ ID NO.22所示的轻链和两条SEQ ID NO.23所示的重链；
    优选地，所述的抗体为BAT4306F；更优选地，所述的抗体具有两条SEQ ID NO.20所示的轻链和两条SEQ ID NO.21所示的重链；
    优选地，所述的抗体为BAT0206F；更优选地，所述的抗体BAT0206F具有两条SEQ ID NO.24所示的轻链和两条SEQ ID NO.25所示的重链；
    优选地，所述的抗体为BAT0808；更优选地，所述的抗体BAT0808具有两条SEQ ID NO.26所示的轻链和两条SEQ ID NO.27所示的重链。
  7. 一种试剂盒，其特征在于，含有权利要求1所述的一对多肽中的至少任意一条；或者优选地，含有权利要求2所述的一对多核苷酸中的至少任意一条；或者优选地，含有权利要求3中所述的一对融合蛋白中的至少任意一个；或者优选地，含有权利要求4中所述的一对多核苷酸中的至少任意一条；或者优选地，含有权利要求5中所述的载体；或者优选地，含有权利要求6中所述的宿主细胞。
  8. 权利要求1-5任一所述的一对多肽/一对多核苷酸/一对融合蛋白/一对多核苷酸/载体在CHO细胞的Fut8基因编辑中的应用。
  9. 权利要求1-6任一所述的一对多肽/一对多核苷酸/一对融合蛋白/一对多核苷酸/载体/宿主细胞在生产抗体中的应用或其所生产的抗体；
    优选地，所述的抗体具有独特糖谱；更优选地，所述的独特糖谱为与野生型糖谱比较，至少一种糖部分的水平发生变化；
    优选地，所述的糖部分选自葡萄糖、岩藻糖、半乳糖、甘露糖、高甘露糖、葡萄糖胺、G0或乙酰葡萄糖胺；
    优选地，所述的抗体的糖部分满足以下条件中的一种或多种：
    a.所述的抗体岩藻糖的水平降低；优选地，所述的岩藻糖水平小于等于5％；更优选地，所述的抗体岩藻糖水平为0；
    b.所述的抗体甘露糖的水平降低；优选地，所述的甘露糖含量小于等于5％；
    c.所述的抗体高甘露糖的水平降低；优选地，所述的高甘露糖含量小于等于5％；
    d.所述的抗体半乳糖的水平降低；优选地，所述的半乳糖的水平小于等于30％；更优选地，半乳糖水平小于等于5％；
    e.所述的抗体G0含量升高；优选地，所述的G0含量大于等于60％；更优选地，所述的G0水平大于等于80％；
    优选地，所述的抗体G0含量大于等于60％，不含岩藻糖；更优选地，G0含量大于等于60％，不含岩藻糖，同时甘露糖含量小于等于5％或/和含高甘露糖含量小于等于5％：
    优选地，所述的抗体具有如说明书附图10中所示的糖谱；
    优选地，所述的抗体结合EGFR或CD20或Trop2；更优选地，所述的抗体结合CD20；
    更优选地，所述的抗体为人源化或者全人源抗CD20抗体；
    优选地，所述的抗体为BAT1206F；更优选地，所述BAT1206F抗体具有两条SEQ ID NO.18所示的轻链和两条SEQ ID NO.19所示的重链；
    优选地，所述的抗体为BAT4406F；更优选地，所述的抗体BAT4406F具有两条SEQ ID NO.22所示的轻链和两条SEQ ID NO.23所示的重链；
    优选地，所述的抗体为BAT4306F；更优选地，所述的抗体BAT4306F具有两条SEQ ID NO.20所示的轻链和两条SEQ ID NO.21所示的重链；
    优选地，所述的抗体为BAT0206F；更优选地，所述的抗体BAT0206F具有两条SEQ ID NO.24所示的轻链和两条SEQ ID NO.25所示的重链；
    优选地，所述的抗体为BAT0808；更优选地，所述的抗体BAT0808具有两条SEQ ID NO.26所示的轻链和两条SEQ ID NO.27所示的重链。
  10. 一种CHO的Fut8基因编辑的方法，其特征在于，包含以下步骤：
    将权利要求3所述的一对融合蛋白或者权利要求4所述的一对多核苷酸或者权利要求5 所述载体转入CHO细胞，培养得到Fut8基因被敲除的CHO细胞。
  11. 一种抗体的生产方法或由该方法产生的抗体，其特征在于，所述方法包含以下步骤：
    (1)将权利要求3所述的一对融合蛋白或者权利要求4所述的一对多核苷酸或者权利要求5所述载体转染CHO细胞，通过凝集素加压筛选、有限稀释得到CHO基因组中α-1-6岩藻糖基转移酶基因FUT8敲除的CHO细胞株CHO-BAT-KF；
    (2)将含抗体基因表达盒的真核表达载体质粒电转染CHO-BAT-KF，通过加压筛选得到去除岩藻糖的抗体蛋白。
  12. 根据权利要求11所述的生产方法或由该方法产生的抗体，其特征在于，步骤(1)中将权利要求5所述载体转染野生型CHO细胞；更优选地，将权利要求5所述的质粒稳定转染野生型CHO细胞；
    优选地，所述的抗体具有独特糖谱；更优选地，所述的独特糖谱与野生型糖谱比较，至少一种糖部分的水平发生变化；
    优选地，所述的糖部分选自葡萄糖、岩藻糖、半乳糖、甘露糖、高甘露糖、葡萄糖胺、G0或乙酰葡萄糖胺；
    优选地，所述的抗体的糖部分满足以下条件中的一种或多种：
    a.所述的抗体岩藻糖的水平降低；优选地，所述的岩藻糖水平小于等于5％；更优选地，所述的抗体岩藻糖水平为0；
    b.所述的抗体甘露糖的水平降低；优选地，所述的甘露糖含量小于等于5％；
    c.所述的抗体高甘露糖的水平降低；优选地，所述的高甘露糖含量小于等于5％；
    d.所述的抗体半乳糖的水平降低；优选地，所述的半乳糖的水平小于等于30％；更优选地，半乳糖水平小于等于5％；
    e.所述的抗体G0含量升高；优选地，所述的G0含量大于等于60％；更优选地，所述的G0水平大于等于80％；
    优选地，所述的抗体G0含量大于等于60％，不含岩藻糖；更优选地，G0含量大于等于60％，不含岩藻糖，同时甘露糖含量小于等于5％或/和含高甘露糖含量小于等于5％：
    优选地，所述的抗体具有如说明书附图10中所示的糖谱；
    优选地，所述的抗体结合EGFR或CD20或Trop2；更优选地，所述的抗体结合CD20；
    更优选地，所述的抗体为人源化或者全人源抗CD20抗体；
    优选地，所述的抗体为BAT1206F；更优选地，所述BAT1206F抗体具有两条SEQ ID NO.18所示的轻链和两条SEQ ID NO.19所示的重链；
    优选地，所述的抗体为BAT4406F；更优选地，所述的抗体BAT4406F具有两条SEQ ID NO.22所示的轻链和两条SEQ ID NO.23所示的重链；
    优选地，所述的抗体为BAT4306F；更优选地，所述的抗体BAT4306F具有两条SEQ ID NO.20所示的轻链和两条SEQ ID NO.21所示的重链；
    优选地，所述的抗体为BAT0206F；更优选地，所述的抗体BAT0206F具有两条SEQ ID NO.24所示的轻链和两条SEQ ID NO.25所示的重链；
    优选地，所述的抗体为BAT0808；更优选地，所述的抗体BAT0808具有两条SEQ ID NO.26所示的轻链和两条SEQ ID NO.27所示的重链。
    优选地，所述的CHO细胞为CHO-K1；更优选地，所述的CHO-K1适应无血清培养。
  13. 一种抗体，其特征在于，所述的抗体为BAT4306F抗体，BAT4306F抗体G0含量大于等于60％，不含岩藻糖，更优选地，BAT4306F抗体G0含量大于等于60％，不含岩藻糖，同时甘露糖含量小于等于5％或/和含高甘露糖糖型含量小于等于5％；更优选地，所述的抗体具有如说明书附图10中BAT4306F所示的糖谱；
    优选地，所述抗体BAT4306F具有两条SEQ ID NO.20所示的轻链和两条SEQ ID NO.21所示的重链；
    优选地，所述的抗体采用权利要求11所述的方法生产。
  14. 一种细胞，其特征在于，保藏编号为CCTCC NO：C2017127，于2017年8月10日保藏于中国典型培养物保藏中心；
    优选地，所述细胞的Fut8基因具有SEQ ID NO.28所示的序列。
  15. 根据权利要求14所述的细胞，其特征在于，所述的细胞表达抗体；
    优选地，所述的宿主细胞表达抗体；
    优选地，所述的宿主细胞表达具有独特糖谱的抗体；更优选地，所述的独特糖谱与野生型糖谱比较，至少一种糖部分的水平发生变化；
    优选地，所述的糖部分选自葡萄糖、岩藻糖、半乳糖、甘露糖、高甘露糖、葡萄糖胺、G0或乙酰葡萄糖胺；
    优选地，所述的抗体的糖部分满足以下条件中的一种或多种：
    a.所述的抗体岩藻糖的水平降低；优选地，所述的岩藻糖水平小于等于5％；更优选地，所述的岩藻糖水平为0；
    b.所述的抗体甘露糖的水平降低；优选地，所述的甘露糖含量小于等于5％；
    c.所述的抗体高甘露糖的水平降低；优选地，所述的高甘露糖含量小于等于5％；
    d.所述的抗体半乳糖的水平降低；优选地，所述的半乳糖的水平小于等于30％；更优选地，抗体半乳糖水平小于等于5％；
    e.所述的抗体G0含量升高；优选地，所述的G0含量大于等于60％；更优选地，所述的G0水平大于等于80％；
    优选地，所述的抗体G0含量大于等于60％，不含岩藻糖；更优选地，G0含量大于等于60％，不含岩藻糖，同时甘露糖含量小于等于5％或/和含高甘露糖含量小于等于5％；
    优选地，所述的抗体具有如说明书附图10中所示的糖谱。
  16. 如权利要求14所述的细胞在抗体方面的应用或其生产的抗体；
    优选地，所述的抗体具有独特糖谱；优选地，所述的独特糖谱与野生型糖谱比较，至少一种糖部分的水平发生变化；
    优选地，所述的糖部分选自葡萄糖、岩藻糖、半乳糖、甘露糖、高甘露糖、葡萄糖胺、G0或乙酰葡萄糖胺；
    优选地，所述的抗体的糖部分满足以下条件中的一种或多种：
    a.所述的抗体岩藻糖的水平降低；优选地，所述的岩藻糖水平小于等于5％；更优选地，所述的抗体岩藻糖水平为0；
    b.所述的抗体甘露糖的水平降低；优选地，所述的甘露糖含量小于等于5％；
    c.所述的抗体高甘露糖的水平降低；优选地，所述的高甘露糖含量小于等于5％；
    d.所述的抗体半乳糖的水平降低；优选地，所述的半乳糖的水平小于等于30％；更优选地，半乳糖水平小于等于5％；
    e.所述的抗体G0含量升高；优选地，所述的G0含量大于等于60％；更优选地，所述的G0水平大于等于80％；
    优选地，所述的抗体G0含量大于等于60％，不含岩藻糖；更优选地，G0含量大于等于60％，不含岩藻糖，同时甘露糖含量小于等于5％或/和含高甘露糖含量小于等于5％；
    优选地，所述的抗体具有如说明书附图10中所示的糖谱。
  17. 一种药物组合物，其特征在于，含有权利要求9、11、13或16任一所述的抗体；优选地，所述的药物组合物还含有药物上可接受的载体。
  18. 一种疾病的预防/治疗方法，其特征在于，给予需要的对象施用有效量的权利要求9、11、13或16所述的抗体。
  19. 一种核酸序列或含有该序列的CHO细胞，其特征在于，所述核酸序列含有如SEQ ID NO.28所示序列，或者与SEQ ID NO.28所示序列具有至少90％，至少91％，至 少92％，至少93％，至少94％，至少95％，至少96％，至少97％，至少98％，至少99％，至少99.5％，或至少99.8％同一性的序列。
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