Recombinant antibody with unique glycan profile produced from genome-edited CHO host cell, and preparation method therefor

Abstract

To provide a method for producing a recombinant antibody having a unique sugar chain spectrum from a genome-edited CHO host cell.SOLUTION: The method of the present invention is a method for editing fut8 genes in CHO cells suitable for suspension culturing in a serum free medium by TALEN technology, so that the edited CHO host cells can produce recombinant antibodies with unique glycan profiles. The unique glycan profile is characterized by having mainly non-fucosylated N-linked oligosaccharide chains, a low degree of heterogeneity of N-glycosylation, and uniform glycan chains. The antibodies prepared by the methods of the invention have increased ADCC activity and antibody stability.SELECTED DRAWING: Figure 10

Description

[Technical Field]

[0001] The present invention relates to the field of biotechnology, and is directed to the production of unique CHO cells from genome-edited CHO host cells. Recombinant antibody with a unique glycosylation spectrum, the host cell and a method for producing the antibody - Patents.com It is something. [Background technology]

[0002] CHO cells were developed in 1957 by Dr. Theodore T. Pucc of the University of Colorado in the United States. Chinese hamster ovary cells (Chi) isolated from the ovaries of adult female hamsters CHO (Chinese Hamster Ovary) belongs to the epithelial adherent culture type cells. Among them, CHO-K1 cells are widely used in industry. CHO-K1 cells are a transformed cell line, and their cellular chromosome distribution frequency The number of cells is 2n=22 and belongs to subdiploid cells. The CHO-K1 cell line used is widely used for the expression of recombinant DNA proteins. The cells are initially cultured as adherent cells, but after several generations of subculture and screening, they are transformed into suspension cells. The genes in CHO cells are prone to mutation, and gene transfer is difficult. Early studies have also shown that CHO is more efficient than other process cell lines. It was demonstrated that the antibodies produced in the cells have the closest glycosylation type to human serum antibodies. Therefore, CHO cells are good host cells for mammalian gene expression. The mechanism of action of therapeutic antibodies is to form a complex with the target molecule, thereby neutralizing the target antigen. Initiates or eliminates antigens or pathogens through an immune response mediated by the antibody Fc segment The specific binding and activity of antibody drugs to target molecules depend on their complex higher-order structure and translation. Among them, glycosylation is the most important post-translational modification for antibodies. have important effects on antibody biological activity, in vivo metabolism, and immunogenicity. The glycosylation form of the drug is mainly N-glycosylated, and the monosaccharide involved is mainly glucose. , galactose, mannose, N-acetylglucosamine, N-acetylgalactosamine , fucose, and sialic acid (NANA, NGNA). The antibody molecule Fc segment Asn297 is linked to the bi- or multi-branched two-chain (bian Tennary complex oligosaccharides consist of G0, G1(1,3), G1(1,6) and G2 complexes. There are several types. Among them, each type contains fucose (F) or bisecting glass. They are divided into 16 types depending on whether they contain lactose (B) (Glycobiology, August 2015,1-10.DOI:10.1093 / glycob / cwv0 65) Therefore, the oligosaccharide type of the antibody heavy chain has terminal sialylation and high mannose type. There are at least 36 types, regardless of whether they exist or not. Because of random combinations, there are over 400 possible glycan types. However, antibodies exhibit a high degree of heterogeneity.

[0003] Different glycan types have different effects on the pharmaceutical properties of therapeutic antibodies. The content of 5-amino-5-glycan (Man5) contributes to rapid clearance of antibodies from the blood and shortens their half-life. (MAbs, 2012, 4(4):509-520). G0F acts on the complement pathway. G2F contributes to the elimination rate and accelerates the rate of elimination. Sialic acid modification significantly affects the inflammatory effects of intravenous immunoglobulin administration. The reduction of fucose leads to a significant increase in ADCC activity (JBC (2003) Chemistry Therefore, the main mechanism of action and clinical efficacy of therapeutic antibodies are unclear. It is necessary to design and improve the glycosylation of antibodies depending on their pharmaceutical applications.

[0004] Unlike protein synthesis, antibody glycosylation does not have a template to follow. The glycosylation type and proportion of each oligosaccharide component are influenced by the host cell type and culture requirements. The oligosaccharide content of monoclonal antibodies was improved by engineering host cells. The methods for enhancing Fc-mediated activity are scattered across different literature and patent reports. For example, β(1,4)-N-acetylglucosamine transferase III ( Antibodies prepared in CHO cells overexpressing GnTIII were significantly more potent than antibodies expressed in parental cells. All showed higher ADCC activity, with the difference in activity being approximately 10-20 times (Biotech ol Bioeng.(2001)Aug 20;74(4):288-94). However, overexpression of GnTIII itself is toxic to CHO cells, and its expression Since GnTIII is exogenous, the expression of GnTIII decreases with increasing passage number during the culture process. Therefore, when used as a host cell, the fucose content of the produced antibody is The homogeneity of the antibody drug is affected by the modification. Examples of cell lines that produce defucosylated antibodies include For this purpose, we used protein fucosylation-deficient Lec13 CHO cells (Ripka et al. Research. Biochem. Biophys. 249:533-545 (1986) However, the protein yield is extremely low, making it unsuitable as a host cell for therapeutic antibody production. Not suitable (Yutaka Kanda et al. Biotechnol Bioeng. (20 06)Jul 5;94(4):680-8). α-1-6 fucosyltransferase It was also found in CHO cells in which the gene FUT8 was knocked out (Yamane-Ohnuk i et al.(2004), Biotech. Bioeng.87:614) This leads to a reduction in the fucose content of antibodies. a The fucosylation process of antibodies was investigated in the FUT8 knockout cell line described in the Hakko patent. We have disclosed a method for controlling the level of cytotoxicity and enhancing ADCC (antibody-dependent cellular cytotoxicity) activity. The method involves suppressing the expression of the fut8 gene in a host cell using a specific siRNA. The method reduces the level of fucose in the antibodies produced by the host cells. has the same drawbacks as the CHO cell line overexpressing GnTIII mentioned above. It is necessary to introduce a foreign sequence into the target gene, and then to silence the target gene by siRNA. The efficiency is at most around 70%, and finally, the stability of siRNA expression is a key factor in the quality of antibody drugs. It may affect the attributes.

[0005] Recently, emerging genome editing technologies have been utilized to edit target genes in host cells and There are different methods for inactivating the FUT8 enzyme and reducing the fucose levels in antibodies, both in the literature and in patents. For example, Malphettes et al. (2010) reported that zinc finger Using ZFN technology, the gene was knocked out from the parent DG44 cells, resulting in homozygous A fut8 gene knockout DG44 derived clone was obtained and produced from the cell line. It has been reported that the antibody does not contain any fucose. ) employed TALEN technology to edit the fut8 gene in CHO-K1 cells to induce the growth of host cells. It has been reported that FUT8 enzyme activity in the cells is lost. ) edited the 10th exon of the fut8 gene using CRISPR / Cas9 technology. It was reported that this product abolished FUT8 enzyme activity in CHO-K1 cells. Summary of the Invention

[0006] Based on this, existing antibody drugs are almost entirely composed of a single N-glycosylation modification on the Fc. Although it is a limitation, the glycan type components and content rates are not consistent and are prone to fluctuation, and Regarding the issues of genome-edited CHO, such as the impact on manufacturing stability, Antibodies with unique glycosylation spectra produced from host cells and production of said antibodies The above-mentioned objects of the present invention are achieved by the following means: The first aspect of the present invention is the application of the sequences shown in SEQ ID NO. 10 and SEQ ID NO. 11. The amino acid sequences of SEQ ID NO. 10 and SEQ ID NO. 11 are also shown. and at least 90%, at least 91%, at least 92%, at least 9 3%, at least 94%, at least 95%, at least 96%, at least 97%, A pair of polypeptides having sequences with at least 98% or at least 99% identity. In some embodiments, SEQ ID NO. 10 and SEQ ID NO. 1 The pair of polypeptides shown in Figure 1 represent the upstream and downstream DNA binding domains of the TALEN. They are amino acid sequences, each capable of binding to a specific base region of a specific gene.

[0007] The second aspect of the present invention is shown in SEQ ID NO. 10 and SEQ ID NO. 11, respectively. In some embodiments, a pair of polynucleotides encoding a pair of polypeptides is provided. wherein the pair of polynucleotides is SEQ ID NO. 12 and SEQ ID NO. 13, or having the nucleic acid sequence shown in SEQ ID NO. 12 and SEQ ID NO. At least 90%, at least 91%, at least 92% identical to the sequence shown in No. 13 , at least 93%, at least 94%, at least 95%, at least 96%, at least The sequences are those that have 97%, at least 98% or at least 99% identity.

[0008] The third aspect of the present invention is a method for treating a transcription activator-like effector (Fok) I) to the amino acid sequence of the DNA cleavage domain of the present invention. In some embodiments, the DNA cleavage domain of a transcription activator-like effector (FokI) The amino acid sequence of the amino acid may be naturally occurring or artificially modified. The pair of fusion proteins is SEQ ID NO. 14 and SEQ ID NO. 16 or having the amino acid sequence shown in SEQ ID NO. 14 and SEQ ID NO. 1 6. Identifies at least 90%, at least 91%, at least 92%, or at least 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 fusion proteins is composed of two nucleotide sequences of the Fut8 gene of CHO. In some embodiments, the two nucleotide fragments of the Fut8 gene can be specifically recognized. The sequence fragment is located in exon 1 (Exon 1, SEQ ID NO. 7) of the Fut8 gene. In some embodiments, the two nucleotide sequence fragments of the Fut8 gene are The nucleotide sequence fragments are set forth in SEQ ID NO. 3 and SEQ ID NO. 4, respectively. In some embodiments, the nucleotide sequence of SEQ ID N is Between the nucleotide sequence shown in SEQ ID NO. 0.3 and SEQ ID NO. 4, There is one spacer with the sequence shown in No. 5.

[0009] The fourth aspect of the present invention is a method for producing a nucleotide sequence encoding each of the pair of fusion proteins. In some preferred embodiments, the nucleotide pair is SEQ having the nucleic acid sequence shown in SEQ ID NO. 15 and SEQ ID NO. 17, or Identification of at least 90%, at least 10%, of the sequences shown in SEQ ID NO. 15 and SEQ ID NO. 17 at least 91%, at least 92%, at least 93%, at least 94%, at least 9 5%, at least 96%, at least 97%, at least 98% or at least 99% It has the same identity as the

[0010] The fifth aspect of the present invention is a method for detecting at least one of the pair of polynucleotides. In some embodiments, a vector is provided that comprises any one of the polynucleotides. The vector is a plasmid.

[0011] A sixth aspect of the present invention also provides a host cell transformed with the vector.

[0012] In some embodiments, the cells transformed with these vectors are The genome is the edited CHO host cell, the parent cell of which is derived from the CHO-K1 cell line.

[0013] In some embodiments, the genome-edited CHO host cell is a CHO host cell derived from a parent cell. The parent cells were adapted for serum suspension culture and were designated CHO-BAT.

[0014] Among these, in some embodiments, the parent CHO cell of the genome-edited CHO host cell - The BAT is a CHO-K1 selected to meet one or more of the following characteristics: The cells have high transfection efficiency, The cells have a short logarithmic growth time, The above cells have the ability to reach high cell densities in CD-CHO culture.

[0015] In some embodiments, the genome-edited CHO host cells are In all cases, the FUT8 gene region contains base deletions, insertions, and nonsense mutations, so the cells The endogenous α1,6-fucosyltransferase (Fut8) loses its enzymatic activity. It was.

[0016] The cells are free of exogenous DNA sequences. The recombinant antibodies expressed in the above cells as host cells have unique glycan spectrum characteristics. Have.

[0017] In some embodiments, the genome-edited CHO host cell is a CHO host cell that expresses the FUT8 gene. By editing the genome of exon 1 of the gene, the endogenous Fut8 of the cell loses its enzymatic activity. In addition, the above cells produce base deletions and nonsense mutations in the FUT8 gene. It is characterized by not containing any DNA sequences from the expression vector incorporated during the process.

[0018] These cells are designed to host recombinant antibodies that express unique glycospectral characteristics. Its characteristics include having predominantly nonfucosylated N-linked oligosaccharide chains, and , including other glycospectral features of antibodies.

[0019] In some embodiments, the genome-edited CHO host cell contains the FUT8 gene. The gene was knocked out, and the cells were negative for binding to the agglutinin LCA. The cells were designated CHO-BAT-KF.

[0020] The seventh aspect of the present invention is a method for producing a polypeptide comprising at least one of the pair of polypeptides described above, or a combination of the pair of polypeptides described above. At least one of the polynucleotides of the pair of fusion proteins is and a kit comprising at least one of the vectors or the host cells. do.

[0021] The eighth aspect of the present invention is a method for producing a pair of polypeptides / a pair of polynucleotides / a pair of fusion proteins. The present invention provides the use of proteins / vectors to edit the Fut8 gene in CHO cells.

[0022] The ninth aspect of the present invention is a method for producing a pair of polypeptides / a pair of polynucleotides / a pair of fusion proteins. Protein / vector / host cell-mediated antibody production, especially antibodies with unique glycosylation spectra or the pair of polypeptides / pair of polynucleotides / Antibodies prepared by the fusion protein / vector / host cell pair are provided.

[0023] The tenth aspect of the present invention is a method for producing a fusion protein comprising the above-mentioned pair of fusion proteins, or the above-mentioned pair of polynucleotides, or the above-mentioned vector. The vector was introduced into CHO cells and cultured at 37°C for 14 days. This includes obtaining CHO cells in which the Fut8 gene has been knocked out by limiting dilution.

[0004] The present invention provides a method for editing the Fut8 gene in CHO. An exemplary method is described in Wood et al. See, e.g., J Immunol. 145:3011 (1990).

[0024] A tenth aspect of the present invention is a method for producing a unicellular mutant of a genome-edited CHO host cell, the method comprising the steps of: A method for producing a recombinant antibody having a unique glycan spectrum, or a product produced using the same Provide antibodies that:

[0025] (1) The pair of fusion proteins, the pair of polynucleotides, or the vector is C Transfection into HO cells (e.g., wild-type CHO cells) and pressure screening A process in which Fut8 gene knockout CHO cells are obtained by limiting dilution. , (2) The Fut8 gene is knocked into a plasmid encoding an expression cassette for the antibody gene. Electroporation into isolated CHO cells, pressure screening, and limiting dilution A process for obtaining a stable antibody-secreting CHO cell line by

[0026] In a preferred embodiment, in step (1), the vector is transfected into wild-type CHO cells. and more preferably, the plasmid is transferred into stable wild-type CHO cells. To perform injection. In a preferred embodiment, the CHO cells are CHO-K1, more preferably The aforementioned CHO-K1 is suitable for serum-free culture. In a preferred embodiment, the antibody is an anti-CD20 antibody, and more preferably, the antibody is an anti-CD20 antibody. The aforementioned antibodies are humanized or fully human anti-CD20 antibodies, and more preferably, the aforementioned The antibody is BAT4306F, and more preferably, said BAT4306F antibody is SEQ Both light chains shown in SEQ ID NO. 20 and heavy chains shown in SEQ ID NO. 21 The inventors have employed the methods, cells, polypeptides, etc. of the present invention to produce multiple types of After further investigation, it was found that the prepared antibodies were These all express highly consistent glycan types, and the degree of heterogeneity of these glycan types is low. It was then discovered that the method, cells, etc. of the present invention are suitable for preparing all types of antibodies. In one embodiment, the antibody binds to CD20. The 20-binding antibody is a humanized antibody. In a preferred embodiment, the BAT4306F humanized antibody The antibody is derived from the B-Ly1 antibody sequence of the heavy chain variable region of the B-Ly1 antibody from patent WO2005044859. The HH6 amino acid sequence and the B-KV1 amino acid sequence of the light chain variable region. The antibody has the sequences shown in SEQ ID NO. 20 and SEQ ID NO. 21. In one embodiment, the CD20 binding antibody is BAT44 of fully human origin. 06F antibody, the sequences of which are shown in SEQ ID NO. 22 and SEQ ID NO. 23. In one embodiment, the antibody is BAT1206F. The BAT1206F antibody has both light chains shown in SEQ ID NO. 18 and SEQ ID NO. In one embodiment, the antibody has both heavy chains as shown in BAT020. 6F, BAT0206F binds to EGFR, and the antibody has SEQ ID NO. 24 and both heavy chains as set forth in SEQ ID NO. 25. wherein the antibody is BAT0808, and the antibody binds to Trop2 and is Both light chains shown in SEQ ID NO. 26 and both heavy chains shown in SEQ ID NO. 27 In some embodiments, the modified glycoprotein is secreted from a host cell. In some embodiments, the modified glycoprotein is an antibody.

[0027] In one specific embodiment, the genome of a CHO host cell of the present invention is transfected with a virion. A method for producing a recombinant antibody having a unique glycan spectrum, or a product produced using the method The antibody produced includes the following specific steps: The pair of fusion proteins, the pair of polynucleotides, or the vector is a wild-type C After transfection into HO cells, the cells were incubated with C containing phytohemagglutinin (LCA). D CHO (Sigma) + 10% FBS (fetal bovine serum) was added and pressure screening was performed. After 14 days, viable cells were seeded at 0.5 cells / well in a 96-well cell culture plate. The serum concentration was then reduced to 5%, and after 7 days, the cells were transferred to 24-well plates for cell culture. After 7 days, some cells were removed and incubated at 1000 rpm. The mixture was centrifuged for 5 min, exchanged once with PBS, and then labeled with 2 μl fluorescein. The LCA was taken and mixed with the cells, and the mixture was incubated on ice for 30 minutes. The mixture was then washed once with PBS. The fluorescence value was read using a flow cytometer (BD, C6) and used as a negative control. Untransfected wild-type CHO cells were used, and positive cells were cultured for The cells were transferred to a 6-well plate, the serum concentration was reduced to 1%, and after 7 days, the cells were transferred to a small roller. The cells were transferred to a bottle, where the medium was serum-free CD CHO, completing the acclimation process. A portion of the cells was taken, and the CHO genome was extracted using a plasmid extraction kit (Omega). The primers L130for (SEQ ID NO. 1), L130r ev (SEQ ID NO. 2), polymerase chain reaction (PCR) using taq enzyme The PCR product was ligated into a T vector (Promega), transformed, and plated. The next day, single colonies were sequenced with T7 primers and DNA was analyzed. The sequence was analyzed using STAR analysis software, and base deletions were identified compared to the wild-type CHO genome sequence. The CHO cells were cultured on a scale-up scale and named CHO-BAT-KF. Establish a cell library from CHO-BAT-KF cells and culture in CD40 containing 7.5% DMSO. The cells were cryopreserved in a freezing medium, transferred to a liquid nitrogen can, and stored for a long period of time. The plasmid to be loaded is taken and digested, its OD260 is measured, and the result is electroporation. Add 50 μg of plasmid to the 7 CHO-BAT-KF and electroporation were performed. Transfection was performed using a transfection system (Biorad) and placed in a 96-well plate for cell culture. After 48 hours, the mice were inoculated with methionine imino sulfone. After 14 days, the cells were coated with anti-FC polyclonal antibody. After blocking the ELISA plate with 3% BSA, the supernatant was added to the plate and incubated at 37°C. After 2 hours of incubation, the cells were washed five times with PBST and then incubated with anti-horseradish peroxidase-labeled The antibody was added to a sheep anti-human kappa / lambda light chain antibody (2M H2SO4) and microplate The OD450 value was read using a rate reader. The strains with high titers were cloned and expanded. The cell supernatant was collected to obtain the fucose-knocked out antibody protein.

[0028] The present invention also provides a cell, which is a genome-edited CHO host cell. The genome-edited CHO host cell contains an edited Fut8 gene having SEQ ID NO: The sequence shown in SEQ ID NO. 28 or at least the sequence shown in SEQ ID NO. 28 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%, at least sequences with at least 99%, at least 99.5%, or at least 99.8% identity Has.

[0029] The present invention also relates to a nucleic acid comprising the sequence shown in SEQ ID NO. 28, or SEQ Identifies at least 90%, at least 91%, or at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or Nucleic acids containing sequences with at least 99.8% identity are provided.

[0030] The present invention has also been deposited in the China Typical Culture Collection Center under the accession number: CCTCC N O:C2017127, Entrustment date: August 10, 2017, Address: Wuhan University, Wuhan, China, Min. Class and name: Chinese hamster ovary cell CHO-BAT-KF fut8(- / -) The present invention provides a CHO host cell, In some embodiments, the host cells are maintained in serum-free medium. The host cells are maintained in suspension culture. The present invention further provides a medium containing the host cells. and a culture fermenter containing a plurality of the host cells in a medium. The above medium is serum-free.

[0031] The twelfth aspect of the present invention is a unique glycoprotein produced from genome-edited CHO host cells. A recombinant antibody with a chain spectrum, said antibody being humanized or of fully human origin. It has a unique glycosylation pattern and a low degree of N-glycosylation heterogeneity. The present invention provides an antibody having a significantly increased ADCC activity.

[0032] In some embodiments, the genome is produced from an edited CHO host cell. The recombinant antibody with a unique glycosylation spectrum binds to CD20 on the cell membrane surface. It is a humanized antibody.

[0033] In some embodiments, the genome is produced from an edited CHO host cell. Recombinant antibodies with unique glycosylation patterns have unique glycosylation spectra. The glycosylation pattern may be one or more of the N-linked glycosaccharides in the antibody. Glucose (Glc), fucose (Fuc), galactose (Gal), mannose (Man ), high mannose, glucosamine, G0 and acetoglucosamine These proteins are characterized by varying levels of sugar moieties selected from the group consisting of GlcNAc and GlcNAc. The glycosylation pattern characteristics satisfy one or more of the following preferred requirements: The above antibodies have a low fucose content (0-5%). The antibodies contain relatively low levels of galactose (≦30%) The level of mannose in the above antibodies is relatively low (≦5%). The level of high mannose in the above antibodies is relatively low (≦5%). The G0 level of the above antibodies is relatively high (≥60%).

[0034] Among these, in some embodiments, the antibody contains a lower level of galactose, ≦5%. In some embodiments, the antibody contains a relatively high level of G0, ≥ 80 %.

[0035] In some embodiments, the genome is produced from an edited CHO host cell. The recombinant antibody with a unique glycan spectrum has a fucose content of zero. Meet the preferred requirements. In some embodiments, the genome is produced from an edited CHO host cell. Recombinant antibodies, which have a unique glycosylation spectrum, exhibit a very high degree of N-glycan heterogeneity. It has low and uniform sugar chains. In some embodiments, the genome is produced from an edited CHO host cell. Recombinant antibodies with unique glycosylation spectrum have relatively strong ADCC activity of Fc. . In some embodiments, the antibody is BAT4306F, which is shown at the top of FIG. The sugar chain spectrum shown in

[0036] In some embodiments, the BAT4306F is as set forth in SEQ ID NO. 20. and both heavy chains as shown in SEQ ID NO. 21. In this case, it does not mean that mutations may occur in the sequence, and these mutations may Any amount may be used as long as it does not affect the action of the antibody.

[0037] The thirteenth aspect of the present invention is a method for detecting a nucleotide sequence in the first exon of the Fut8 gene in a CHO host cell. A CHO host cell is provided in which the Fut8 gene is knocked out, the Fut8 gene containing an inactivating mutation. The mutation may be a substitution or deletion of one or more amino acids, or a frameshift mutation, e.g. It may be a mutation as shown in FIG.

[0038] The present invention further provides a pharmaceutical composition comprising the above-described antibody. The pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0039] The present invention further provides a method for administering an effective amount of the antibody / antibody fragment disclosed in the present invention to a subject in need thereof. In some embodiments, a method for preventing or treating a disease is provided, comprising administering to The diseases include cancer, allergic diseases, cardiovascular diseases, inflammatory diseases, metabolic diseases, neurological diseases, For example, the disease is selected from the group consisting of a viral infection and / or a fungal infection. or an allergic disease. In some embodiments, the subject is a mammal, e.g. Human.

[0040] Compared with conventional antibody drugs, the present invention has the following advantages: Conventionally, commercially available antibody drugs are almost exclusively limited to a single N-glycosylation modification of the Fc. However, the sugar chain type components and content are not consistent and are prone to fluctuation, so it is difficult to determine a specific There are complexities, especially in terms of production stability. Recombinant proteins with unique glycan spectrum produced from edited CHO host cells On the other hand, antibodies have low heterogeneity in N-glycosylation and good glycan uniformity, ADCC activity has been strengthened, and the quality attributes and pharmaceutical characteristics of antibody drugs have been greatly improved. Ta. The antibodies were either their corresponding unmodified CHO-K1 (ATCC#CCL-61) or Compared with antibodies produced from the suspension-appropriate parental CHO-BAT cells, Increased binding affinity to receptors.

[0041] The antibodies produced by the modified host cells are similar to the antibodies produced by the corresponding unmodified host cells. These antibodies have increased affinity for FcγRIIIA compared to the corresponding antibodies produced by cells. [Brief explanation of the drawings]

[0042] [Figure 1] Figure 1 shows the cell lines CHO-K1 (ATCC #CCL-61) growing in suspension and CHO-BAT, a cell line adapted for suspension growth in serum-free medium. [Figure 2] Figure 2 is a schematic diagram of the TALEN expression plasmid pCS2-Fok1. [Figure 3] Figure 3 shows the functional validation of the Talen protein. The left side of the electropherogram shows the wild-type genome. The PCR product of the gene-edited cell genome showed two bands at 500 bp and 750 bp, while the wild-type showed only a single band at 750 bp. This is consistent with the expected results, demonstrating the functionality of the Talen protein pair. Lane 1: 100 bp marker, Lane 2: wt, Lane 3: pool. [Figure 4] Figure 4 shows the analysis of cells grown in 24-well plates by FACS. Gene-edited cell clones are negative for FITC-labeled LCA binding, while wild-type cells are positive for FITC-labeled LCA binding. [Figure 5] Figure 5 shows that the analysis using a glycochip showed that the fucose content of the tested gene-edited clones 41 and 43 was reduced to 0-10%, while the fucose content of the wild-type antibody 1206 was 80%. [Figure 6]Figure 6 shows the results of PCR amplification of the TALEN protein targeting sequence and sequencing, followed by alignment using LasergeneMegAlign sequence analysis software. 191-1, 191-2, 217-1, and 217-3 represent four genome-edited cell clones. Their genomes were extracted and used as DNA templates for PCR reactions using primers L130 for and L130 rev. The amplified products were then subjected to CEL-1 mismatch analysis. The results indicated that cell clones 191-1 and 191-2 were heterozygous, while cell clones 217-1 and 217-3 were homozygous. Based on the alignment results, genome-edited homozygous clones 217-1 and 217-3 were selected and designated CHO-2G8 and CHO-1D6. Finally, CHO-2G8 was selected as the host cell for further testing, and the host cell was designated CHO-BAT-KF. [Figure 7] FIG. 7 is a graph comparing the growth density of CHO-BAT-KF with that of the parental CHO-BAT cells. [Figure 8] FIG. 8 is a graph comparing the proliferation ability of CHO-BAT-KF with that of the parent CHO-BAT cells. [Figure 9] Figure 9 shows MALDI-TOF MS analysis of the N-polysaccharides of the BAT4306F and 4306 antibody molecules using a MALDI-TOF MS mass spectrometer. The N-polysaccharides derived from BAT4306F each had one less fucose residue than the N-polysaccharide derived from 4306. The left image shows the 4306 antibody molecule produced from parent cells, and the right image shows the BAT4306F antibody molecule. [Figure 10] FIG. 10 shows that BAT4306F has a lower fucose content than GAZYVA (obinutuzumab), resulting in a lower degree of heterogeneity in the glycans and a better uniformity of the product. [Figure 11] Figure 11 compares the ADCC activity of anti-CD20 antibodies such as wild-type BAT4306, glycosylated BAT4306F, obinutuzumab, and rituximab when Raji was used as target cells and PBMC was used as effector cells. [Figure 12]FIG. 12 is a graph comparing the ability of three types of antibodies, glycosylated BAT4306F, obinutuzumab, and rituximab, to eliminate B cells in whole blood in vitro at concentrations of 50, 25, and 10 ng / mL. [Figure 13] 13 is a diagram comparing the glycan spectra of the anti-CD20 antibodies BAT4306F and BAT4406F, the anti-EGFR antibody BAT0206F, and the anti-Trop2 antibody BAT0808, all produced in CHO-BAT-KF cells. The genome-edited CHO host cells of the present invention were deposited with the China Center for Typical Culture Collections (CCTCC), with deposit number CTCCC NO: C2017127, date of deposit 2017.8.10, depository address: Wuhan University, Wuhan, China, classification and name: Chinese hamster ovary cell CHO-BAT-KF fut8(- / -). DETAILED DESCRIPTION OF THE INVENTION

[0043] The technical configuration of the present invention will be further explained below based on specific examples. The scope of the invention is not limited to these. Any non-essential modifications or adjustments made by the present invention are also included within the scope of the invention sought to be protected.

[0044] In the present invention, expressions such as "level" or "content" of the sugar moiety of an antibody are used in the same sense. Each of these terms refers to the mass ratio of a certain sugar moiety to the total sugar moieties of an antibody.

[0045] For example, in the present invention, "amino acids" refer to those that are encoded directly or as precursors from nucleic acids. A single amino acid can be synthesized from three nucleotides (so-called Codons, or triplet bases, are encoded by nucleic acids. , encoded by at least one codon. The same amino acid is coded by different codons. This is called "codon degeneracy." The term "amino acid" as used in this application means Alanine (three-letter code: ala, single letter code: alanine) is a naturally occurring carboxy α-amino acid. Mother code: A), arginine (arg, R), asparagine (asn, N), asparagine Phosphate (asp, D), cysteine ​​(cys, C), glutamine (gln, Q), glutamine Phosphoric acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine leucine (leu, L), lysine (lys, K), methionine (me t, M), phenylalanine (phe, F), proline (pro, P), serine (ser , S), threonine (thr, T), tryptothia (trp, W), tryptophan ( tyr (Y) and valine (val (V)).

[0046] In the present invention, the interchangeable terms "polynucleotide" or "nucleic acid" or "nucleic acid sequence" are used. It is a combination of the nucleotides (also called bases) a, c, g, and t (or in RNA). and u) a polymer molecule, such as DNA, RNA or a modified form thereof. The polynucleotide molecule may be a naturally occurring polynucleotide molecule or may be synthetic. a polynucleotide molecule that is identical to one or more naturally occurring polynucleotide molecules The definition may be a combination of one or more synthetic polynucleotide molecules. In this case, one or more of the nucleotides are changed (e.g., by mutagenesis) or deleted. The term "nucleic acid" also encompasses naturally occurring polynucleotide molecules with deletions or additions. It may be alone or in combination with other nucleic acids, such as expression cassettes, plasmids or host cell staining. Nucleic acids can be nucleic acid sequences composed of single nucleotides. For example, the amino acid sequence of a polypeptide can be characterized by Procedures and methods for transforming the corresponding nucleic acid sequences are well known to those skilled in the art. Thus, a nucleic acid is characterized by a nucleic acid sequence composed of corresponding single nucleotides. and may be characterized by the amino acid sequence of the polypeptide encoded thereby. It can also be done as follows.

[0047] The term "polynucleotide" or "nucleic acid" or "nucleic acid sequence" refers to a sequence present in a nucleic acid molecule. The percentage of modified nucleotides in the total number of nucleotides in the sequence is, 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 is 100%, including modified nucleotides.

[0048] In the present invention, a "polypeptide" is a molecule consisting of amino acids linked by peptide bonds. It is a polymer that can be natural or synthetic. A polypeptide having two or more amino acid residues is called a "peptide." A molecule made up of peptides, or a single polypeptide with more than 100 amino acid residues. Molecules that contain peptides are called "proteins." Polypeptides include, for example, glycosyltransferases. The non-amino acid components may be carboxylates, metal ions, or non-amino acid components. The components can be added to cells that express the polypeptide, with variations in cell type. Here, a polypeptide can be defined as a polypeptide having a structure of its amino acid backbone or a structure thereof. For example, glycosyl addition is usually not specified. However, the term "polypeptide" includes those that are present in amino acid molecules. The percentage of modified amino acids in the total number of amino acids is, for example, at least Approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100 %, including modified amino acids.

[0049] In the present invention, the term "host cell" refers to a mononuclear culture of a microorganism or a eukaryotic cell or cell line. means a recombinant vector or other transferred polynucleotide. Recipients of progenitor cells or progeny of transfected progenitor cells. In some embodiments, the host cells are non-lymphocytes, and the host cells may be In some embodiments, the host cell is, for example, a NS These include human COS cells, monkey COS cells, and Chinese hamster ovary (CHO) cells. In one embodiment, the host cell is selected from the group consisting of Chinese hamster ovary (CHO) cells. In some embodiments, the host cell is a CHO-K1, CHO- S, DUXB11, CHO-1E5, CHO3F, CHO / DG44, CHO-BAT In some embodiments, the host is selected from the group consisting of CHO-2.6 cells. The cells produce antibodies that exhibit a unique glycosylation spectrum. The CHO host cells with edited fut8, e.g., CHO-BAT-KF fut8(- / -), were cultured The cells can be grown in a medium, a device that allows for the growth of cultures (including fermentation vessels). The host cells may grow as a monolayer or may be attached to a surface. Alternatively, the host cells may be in suspension. The cells may 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 do not retain their specific ubiquitination even after many passages in culture. For example, edited CHO host cells can maintain a unique glycan spectrum. Even after about 20, 30, 40, or 50 generations, the specific unique glycan spectrum is maintained. In some embodiments, the modified CHO host cells are maintained after at least about 60 generations. In another embodiment, the modified The CHO host cells may have at least about 100, 150, 200, 500, or 1000 or more Generation after generation, it also maintains its specific and unique glycan spectrum.

[0050] The glycosylation pattern of the host cell may include N- or O-glycosylation of any protein moiety. Cosylation may occur at the nitrogen position of the asparagine amide, or is one or more hydroxyl groups at the hydroxyl oxygen position of hydroxylysine, serine or threonine. The glycosylation pattern is characterized by the presence of at least two or more sugar molecules. The molecule or sugar (e.g., monosaccharide, disaccharide, polysaccharide, or oligosaccharide) level is altered. For example, the sugar molecule may be a trisaccharide, tetrasaccharide, pentasaccharide, hexasaccharide, heptasaccharide, octasaccharide, nonasaccharide, or a derivative thereof, e.g. Deoxy sugars (e.g., deoxyhexasaccharides), N- or O-substituted derivatives such as sialic acid The sugar molecule may be galactose (Gal), bromine (Br), or a sugar having an amino group. Glucose (Glc), mannose (Man), N-acetylneuraminic acid (NeuAc), Fucose (Fuc), N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GalNAc), These may include glycerol, ... Sugar molecules may be linked to other sugar molecules by α or β linkages, but are not limited to these.

[0051] The "antibody" of the present invention includes any type of antibody, such as a recombinant antibody, a humanized antibody, or a chimeric antibody. Antibodies include antibodies, single-chain antibodies, fusion antibodies, monoclonal antibodies, polyclonal antibodies, etc. The antibody may also be a fragment. The antibody may also be a drug, toxin or therapeutic agent. The bispecific antibody may be bound to a radioisotope for use in the treatment of cancer. Fusion proteins can also be produced, including heterologous antibodies that bind to more than one antigen. Therefore, antibodies can be classified as naked antibodies, conjugated antibodies, or or antibody fragments, which may be monospecific or multispecific. stomach.

[0052] In an alternative embodiment, the antibody or antibody fragment is, but is not limited to, an anti-HER2 antibody or an anti-HER2 antibody. 2, anti-CD20, anti-EGF, anti-VEGF, anti-PDGF, anti-EpCam, anti-CD3, anti-CD 4, anti-CD19, anti-CD30, anti-CD33, anti-CD40, anti-CD51, anti-CD55, anti-C D80, anti-CD95, anti-CCR2, anti-CCR3, anti-CCR4, anti-CCR5, anti-folate, anti-C Examples include XCR4, anti-EGFR, and Trop2 antibodies. The antibody may be humanized or of fully human origin. In the pharmaceutical composition of the present invention, an antibody having a required degree of purity and a selectable physiologically acceptable Carriers, excipients, or stabilizers (Remington's Pharmaceuticals al Sciences 16th edition,Osol,A.Ed.(1980 )) to prepare a pharmaceutical preparation of the antibody of the present invention for storage as a lyophilized formulation or aqueous solution. Acceptable carriers, excipients, or stabilizers may be used in the amount and concentration of the formulation to obtain a product formulation. non-toxic to phosphates, e.g., phosphites, citrates and other organic acids buffers, ascorbic acid and methionine antioxidants (e.g., stearyl dimethyl ether benzyl ammonium chloride, hexamethyl ammonium chloride, benzalkonium chloride, Benzethonium chloride, phenol, butanol or benzyl alcohol, alkylparaben methylparaben or propylparaben, catechol, resorcinol, cyclohexyl Preservatives of low molecular weight (approximately 10 residues) polypeptides of less than 1000 bases, such as serum albumin, gelatin, or immunoglobulin Proteins, e.g., hydrophilic polymers of polyethylene pyrrolidone, e.g., glycine, glutamine amino acids of arginine, asparagine, histidine, arginine or lysine, glucose, mannose Monosaccharides, disaccharides and other carbohydrates consisting of sugars or dextrins, e.g., EDTA Chelating agents, such as sugars such as sucrose, mannitol, trehalose or sorbitol, e.g. salt-forming counterions, such as sodium, metal complexes (e.g., Zinc-protein complexes), and / or For example, Tween, Pluronics TM or polyethylene glycol (macroglycol Examples of nonionic surfactants include methyl methyl acrylate and methyl methacrylate.

[0053] The antibodies, pharmaceutical compositions, and pharmaceutical formulations of the present invention may be administered by any suitable means. It is also possible to administer the drug by parenteral, subcutaneous, intraperitoneal, intrapulmonary or intranasal routes, but if necessary Therefore, local immunosuppressive therapy can be administered intralesionally. Oral irrigation means include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration methods. The antibody is preferably perfused in a pulsed manner (especially with a gradient of the antibody dose of the present invention). Depending on the length or duration of administration, injection is preferred. It is most preferably administered intravenously or subcutaneously. Activation) is a response mediated by cells, among which effector cells expressing FcRs Cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) bind to target cells. The primary cells that mediate ADCC recognize the bound antibody and subsequently cleave the target cells. These include NK cells, monocytes, and macrophages. NK cells typically express primarily FcγRI. The present invention relates to the expression of FcγRI, FcγRII, and FcγRIII in human monocytes. So that the parent CHO cell line produces a CHO cell line with a unique glycan spectrum. The edited CHO cell line was then used to generate antibodies produced by the parent CHO cells. It is possible to produce antibodies with higher ADCC activity than the ADCC activity of

[0054] Example 1 Screening of parent cells suitable for serum-free suspension culture CHO-K1 cells cultured in DMEM / F12 medium containing 10% FBS showed a confluence of cells. When the fluence reached 80-90%, the cells were washed with PBS, digested with trypsin, and then diluted with 5% FB. The culture was terminated in DMEM / F12 medium containing 5% FBS, counted, and centrifuged. Resuspend the cells in DMEM / F12 medium containing 1 x 10 5 Inoculate cells at a density of 100 cells / ml. When the cells reached 80%-90% confluence, they were washed with PBS and trypsinized. The cells were digested with ethanol, and the culture was terminated with DMEM / F12 containing 2% FBS. The cells were resuspended in DMEM / F12 medium containing 2% FBS and diluted to 1 × 10 5 Cells / The cells were seeded at a density of 1000 ml. When the cells reached 80-90% confluence, The cells were digested with trypsin and cultured in DMEM / F12 medium containing 1% FBS. The culture was terminated in medium and subcultured for 3-4 generations. The glutamine solution was mixed at a ratio of 1 (V / V) to a final concentration of 6 mM. The content was adjusted to 1%. The obtained low serum-adapted CHO-K1 cells were cultured at 3 × 10 5 Inoculate cells / mL into a T25 bottle and incubate at 37°C in a 5% CO2 incubator. When the cells reached 80-90% confluence, they were digested with trypsin and then resuspended in 1% FBS. A mixture of DMEM / F12 containing α- and β-CD CHO medium (volume ratio 1:2) The culture was terminated at 200°C, counted, centrifuged, and 3 x 10 5 T25 bottle at density of cells / mL The cells were inoculated into the DMEM / F medium and cultured at 37°C in a 5% CO2 incubator. Gradually reduce the ratio of 12 to (1:8) and when the cell viability is above 90%, The components from DMEM / F12 in the cell culture medium at the time can be completely eliminated, and the chemical composition CHO-K1 cells suitable for culturing in a defined CD CHO medium containing 1% serum were established. CHO-K1 cells cultured in a chemically defined CD CHO medium containing 1% FBS When the cells reached 80-90% confluence, they were washed with PBS, digested with trypsin, and then incubated for 1 hour. The culture was terminated with CD CHO medium containing 0.5% FBS, counted, and centrifuged. Resuspend the cells in CD CHO medium containing 10% FBS and dilute to 1 x 10 5 T at a density of 10 cells / ml Inoculate 25 bottles, and when the cell proliferation rate reaches 80%-90%, wash with PBS and The cells were digested with psoriasis, cultured in CD CHO medium containing 0.25% FBS, and counted. The cells were resuspended in CD CHO medium containing 0.25% FBS and diluted to 1 × 10 5 The cells were seeded at a density of 100 cells / ml. The serum concentration in the serum-free CD stage began to decrease. The cells were diluted to a limit and seeded into 30 96-well plates at a cell density of 1 cell / well. After two weeks, the cells were observed under a microscope to identify the cells that produced the monoclonal antibody. Clones with large cell areas were transferred to 24-well plates, and after one cycle, they were observed under a microscope. The cell clones with relatively high growth density and uniform cell size were labeled and cultured in 6 well plates. After one week, the cells were observed under a microscope and found to be completely suspended and aggregated into clumps. The cells were transferred to a 100 mL conical bottle, where the density was high and the clones were labeled. The culture volume was 10 mL. Cell density and proliferation activity were recorded for each cell line. After adaptation, CHO-K1 cells suitable for serum-free culture were newly named CHO-BAT. .

[0055] Example 2 Construction of FUT8 TALEN Recombinant Plasmid

[0056] The complete genome sequence of Chinese hamster ovary cancer cell line CHO-K1 (NW.003613 By analyzing the Fut8 genome sequence (Gene ID: 100751648 ) and its cDNA (shown in Table 1, SEQ ID NO. 8) sequence was obtained. The genome consists of 9 exons and 11 introns. The amino acids encoded by exon 1 (SEQ ID NO. 7) O.9, the underlined amino acid sequence), so it is the target of Talen The binding sequences were designed using the left and right wings of exon 1 of the Fut8 gene. Fut8 TALENs based on LEN design guiding principles and gene editing mechanism of action Proteins L130P (SEQ ID NO. 10), R184P (SEQ ID NO. 11) has been designed. L130-FokI (SEQ ID NO. 14) and the fusion protein The fusion protein recognizes the base L130PTN (SEQ ID NO. 3) in the left wing of ATPase 1. The nucleic acid sequence L130-FokIN corresponding to protein L130-FokI is set forth in SEQ ID NO: The length is 19 bp. The fusion protein R184P-FokI (SEQ ID NO. 16) was formed. The protein recognizes base R184PTN (SEQ ID NO. 4) in the right wing of exon 1. The nucleic acid sequence R184P-FokIN corresponding to the fusion protein R184P-FokI is , as shown in SEQ ID NO. 17. Its length is 17 bp. TALEN protein A plasmid vector encoding exon 1 of the left wing L130PTN and right wing R184PTN (See Figure 2) was described in Tomas Cermak et al. (2011) The L130-FokIN and R184P-FokIN were constructed as follows. Add enzyme digestion sites for NcoI and XbaI, and synthesize the two fragment sequences. The resulting mixture was cloned into the pCS2-peas-T vector using NcoI and XbaI (Fig. 2 Between the left and right wing binding sequences, there is a 19 bp long spacer (Space , SEQ ID NO. 5). Fut8 TALEN plasmids: L1 The DNA sequencing results of 30N and R184N are shown in Table 1, SEQ ID NO. 12 The L130N and R184N nucleic acid sequences are shown in SEQ ID NO. 13 and are translated into amino acids. The corresponding amino acid sequences L130P and R184P are shown in Table 1, SEQ ID NO. .10, as shown in SEQ ID NO.11. Table 1 Sequence Listing

[0057] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11]

[0058] Example 3 Analysis of the functional efficacy of FUT8 TALEN proteins

[0059] CEL-I enzyme recognizes mismatched bases in double-stranded DNA and separates the double-stranded DNA. It is a nuclease that can cleave at mismatched positions. 8When edited by TALEN, the region containing the target sequence amplified from the parent genome and a region containing the targeting sequence amplified from the genome of the transformed cell. After denaturing and annealing, the annealed double-stranded DNA contains no base mismatches. The CEL-I enzyme then cuts the annealed double-stranded DNA, The expression revealed both bands in the agarose gel electrophoresis results. 5 CHO- BAT cells were collected and seeded in a 6-well plate the day before transfection. The plasmids L130N and R184N were The cells are transiently transfected using the methods provided in the reagent description. Three days after transfection, cells were harvested by centrifugation and genomes were extracted using a genome extraction kit. This was used as a template and primer L130 for (SEQ ID NO. 1) was used. PCR was performed using Primer L130 rev (SEQ ID NO. 2). PCR amplification of the targeting sequence region contained in the cells was performed in the same manner. 20 μl of each CR product was mixed and heated to 94°C, then allowed to cool to room temperature. 0.5 μl of CEL-I enzyme was added to 200 ng of annealed DNA, and the resulting mixture was stirred for 42 The PCR reaction product was subjected to agarose gel electrophoresis. The results of the agarose electrophoresis analysis are shown in FIG.

[0060] The results showed that the gene-edited PCR products were 500 bp and 750 bp longer than the wild type. The wild type showed a single band at 750 bp, which was the expected result. This result was consistent with the previous results, demonstrating that the Talen protein pair is functional.

[0061] Example 4 Effect of FUT8 TALEN protein on the fucose content of antibodies

[0062] The designed FUT8 TALEN protein regulates the host genome. The aim of this study was to determine whether the sugar chains of the antibodies affected the fucose content of the L The 130N and R184N plasmids were used to stably express the established anti-CD20 antibody. The cell lines were transiently transfected using Lipofectamine 2000 ( I referred to the method provided in the Invitrogen reagent description. In cell culture dish inner, 1 x 10 6 L130 cells, respectively 24 μL of liposomes containing 4 μg of N and R184N plasmid DNA was added. Two days after transfection, 10% (v / v) FBS (GBICO) and 400 μg / m The medium was replaced with DMEM / F12 containing LLCA (Vector). After one week, the cells Most of the cells became round and were suspended in the medium, while some of the cells adhered and grew normally. LCA-resistant cells were digested with 0.25% (v / v) trypsin and centrifuged. The cells were resuspended in DMEM / F12 medium containing 100% (v / v) FBS. After two weeks of culture, monoclonal cells were selected. The cells grown in the 24-well plate were analyzed by FACS. Cells that were analyzed and showed negative binding to FITC-labeled LCA (Figure 4) were scaled up. The antibodies were cultured and produced. The oligosaccharide content of the antibodies produced from the two types of cells was Measurements were performed by Biodonor, and the results are shown in Figure 5.

[0063] As a result, transient transfection of L130 and R184 plasmids into antibody-producing cells was performed. The transfection reduced the fucose content of the antibody.

[0064] Example 5 Establishment of genome-modified host cells

[0065] Genome-tuned CHO-K1 for the production of fucose-free proteins and antibodies To establish the cells, the L130 and R184 plasmids were transiently transfected into the CHO-K1 cell line. The screening of anti-LCA monoclonal cells was carried out as described in Example 3. The genomes of the candidate cell clones were extracted, and the primers L130 for (Table 1, SEQ ID NO. 1) and primer L130 rev( The candidate cell clones were subjected to PCR reaction using the sequences shown in Table 1 and SEQ ID NO. 2. CEL-1 base mismatches were detected in the PCR amplification products of the target sequence region. If the candidate clones were heterozygous, they were analyzed by agarose electrophoresis after enzymatic digestion with CEL-1. In electrophoresis, both bands are present, and if the gene is homozygous, it is not digested with CEL-1. Since the PCR fragment cannot be separated, it shows one band in agarose gel electrophoresis. The vector was directly cloned into a T vector (pGEM-T Easy Vector), The sequencing results and the frequency of the corresponding region in the parent cells were compared. The pair ratio of the fragment sequences is shown in Figure 6. The homozygous clones were selected and designated CHO-2G8 and CHO-1D6.

[0066] Example 6 Evaluation of host cell growth characteristics

[0067] The host cell clone CHO-2G8, in which the fut8 gene was knocked out, was selected. The final concentration of 30 mL of CD containing 6 mM Gln was CHO AGT TM Each of the three CHO-BAC strains was taken and cultured at a cell density of 300,000 / mL. T-KF and one strain of CHO-BAT were inoculated into a small 125 mL roller bottle. 0.5 mL of cells were taken on days 0, 3, 6, and 7, and counted to determine the cell density and The changes in growth characteristics of cells after Fut8 gene knockout were measured by measuring cell viability. The cell growth density is shown in Figure 7, and the cell growth proliferation ability is shown in Figure 8. As shown in Fig. 1, CHO-BAT-KF, in which the fut8 gene was knocked out, There was no significant difference in growth density and activity compared to CHO-BAT cells in which the gene was not knocked out. stomach.

[0068] Example 7 Analysis of the glycosylation spectrum of antibodies produced from host cells

[0069] Glycosylation of antibodies produced from the genome-modified CHO-2G8 cell line described in this invention To confirm that the mutant N-glycosylation is present, protein A affinity chromatography was performed. BAT4306F was produced from CHO-2G8 cells in the culture medium, and BAT4306F was produced from CHO-K1 cells. The 4306 produced from the 4306 was purified and quantified by UV280. The monoclonal antibody (1 mg) was incubated with PNGaseF overnight at 37°C to separate the antibody into N-terminal fragments. The released N-glycans were purified by 30K Amicon ultrafiltration. The flow-through was lyophilized and resuspended in 200 μl deionized water. MALDI-TOF MS mass spectrometer was used to detect the N-glycosides from both of the antibody molecules mentioned above. The BAT430 produced from CHO-2G8 was analyzed by MALDI-TOF MS. The oligosaccharides of the 6F antibody were present in a single peak and were essentially the same population. The population belonged to the oligosaccharides of antibody 4306 produced by the parent host cells. This spectrum is different from that of the previous one (Figure 9).

[0070] As a result, the three peaks of N-polysaccharide of 4306 were G0F, G1F, and G2F, respectively. The time when the N-polysaccharide peak of BAT4306F appeared and the molecular weight indicated that the N-polysaccharide peak was 3. The three peaks were estimated to be G0, G1, and G2, respectively, i.e., N-polysaccharides from 4306. There is one less fucose per N-polysaccharide from BAT4306F than from BAT4306F.

[0071] At the same time, the heterogeneity of the glycans of the commercially available Gazyva was compared to that of BAT4306F. The results of analyzing the degree of heterogeneity of the N-polysaccharide of BAT4306F are shown in Figure 10. The heterologous glycans produced from CHO-BAT-KF cells were The sugar chain types of the antibodies were analyzed. The results are shown in Table 4.

[0072] Example 8 Analysis of ADCC activity of antibodies produced from host cells

[0073] Modification of antibodies with N-polysaccharides of the present invention may enhance the biological function (e.g., ADCC activity) of the antibody. To determine whether CD20 enhances the immune response, a purified antibody targeted to CD20 was used. The in vitro ADCC activity of these cells was measured (LDH method, Promega). The antibody was isolated from the medium by a protein A affinity column and produced by CHO-2G8 cells. The BAT4306F antibody was purified and quantified using UV280. The 4306 antibody was expressed in wild-type CHO cells and purified in a similar manner. To achieve this, Wil2-s cells were cultured in RPMI-1640 medium containing 10% FBS. The cells are in good condition (4-7 days). Take the cells in the logarithmic growth phase and centrifuge them at 1000 rpm for 10 minutes. The supernatant was discarded. Solution A (phenol red-free RPMI-164 containing 10% FBS) The cells were centrifuged as above, washed twice, counted, and then diluted with solution A. 3 x 10 cells 5 Adjust the concentration to 100 cells / mL and add 100 μl to each well of a U-96 well cell culture plate. The final antibody concentrations were 1.2, 0.24, 0.048, and 0. 0096, 0.00192, 0.000384, 0.0000768, 0.000015 The solution was adjusted to 36 μg / mL. The solution was placed in a 37°C, 5% CO2 incubator and incubated for 30 min. The effector cells, PBMC, were collected and cultured in solution B (serum-free phenol- The cells were centrifuged and washed twice in the same manner as above. Count and culture 3 x 10 cells in solution B. 5 Adjust to 50 μg / ml, mix well, and add 50 μg per well. The cells were then added to the U-96 well cell culture plate at 1 μL. The cells were then incubated at 37°C in 5% CO The cells were placed in a centrifuge tube and incubated for 3 hours. When 45 minutes remained in the 3-hour incubation period, the target cells were first Add 20 μl of lysis solution to the large release well and incubate at 37°C in a 5% CO2 incubator for 45 min. The culture was continued. The U-96 well cell culture plate was placed in a centrifuge and centrifuged at 250 g for 4 min. 50 μl / well of the supernatant was transferred to another flat-bottom 96-well plate. Add 50 μl of the prepared colorimetric substrate solution to each well, shake gently to mix well, and let stand at room temperature for 30 min. The reaction was averted from light. 50 μl of stop solution was added to each well, and the plate was gently shaken to mix well. The results were read at OD490 using a thermometer reader.

[0074] The results showed that the 4306 produced in serum-free medium was significantly higher than that produced in unmodified CHO cells. BAT4306F, which has N-polysaccharide produced from CHO-2G8 cell clone, was R The ADCC activity against aji cells and Wil2-s cells is significantly improved (Fig. 11).

[0075] Example 9 Analysis of the affinity of antibodies produced from host cells to CD20

[0076] The N-glycosylation-modified antibodies produced from the cells described in this invention bind to CD20-positive cells. To see if this affected the ability of the cells to bind to the Klervi Even -Referring to the method of Desrumeaux et al. (2012), B was calculated using the FASC method. AT4306F, 4306, and the control rituximab target CD20 on different cell surfaces Briefly, Wil2-s cells in the logarithmic growth phase were harvested and incubated for 800 r The cells were centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. The cells were washed once with PBS, and the density was calculated. and resuspend in 1.5 mL centrifuge tubes to give 500,000 cells per tube. The mixture was centrifuged at 1200 rpm for 5 minutes, and the supernatant was discarded. 30, 3.33, 1.11, 0.37, 0.1, 0.04, 0.014, 0.0046 Add 200µl of antibody to the cells in order, resuspend the cells and mix well. At the same time, the same volume of PBS was added as a negative control. The mixture was centrifuged at 1200 rpm for 5 minutes, the supernatant was discarded, and the mixture was washed once with PBS. The cells were resuspended in 100 μl of PBS and then diluted with 2 μl of FITC-sheep anti-human Ig. The secondary antibody G1 Fab was added, and the mixture was left at 4°C for 30 minutes in the dark. Centrifuge for 5 min, discard the supernatant, and wash once with PBS. Detected. Result calculation formula: Kd = [Ab] * {Fmax / (F-Fback) - 1}. The results are shown in the table below.

[0077] Table 2. Statistics of IC50 and Kd values ​​from antibody cell binding experiments [Table 2] As a result, the N-glycosylation of the antibody affects its affinity to bind to CD20. do not have.

[0078] Example 10 Ability of BAT4306F to eliminate B cells in whole blood of different NHL patients In Vitro Assessment of Force

[0079] The mechanism of action of anti-CD20 antibodies in B lymphoma patients is being investigated directly by ADCC and CDC. However, there are various mechanisms for the induction of B cell apoptosis, including the specific type of anti-CD20 The effect of antibodies is not to enhance a single mechanism of action, but rather to develop a mechanism that will eventually This is reflected by the ability to eliminate B cells in the patient. To determine whether the modification enhances its ability to eliminate B cells, In vitro BAT4306F depletes B cells in whole blood from different NHL patients The biofunction of the heparin sodium anticoagulation tube was evaluated. Approximately 3 mL of blood was taken from a patient newly diagnosed with NHL. The blood was left to stand at room temperature and stored. 90 μL of each sample was taken and placed in a new FACS tube. The final antibody concentrations in the test samples in each tube were 10 nM, 1 nM, and 0 Add 0.1 nM, 0.01 nM, and 0.001 nM of each sample to each tube. 10 μL of BAT4306F antibody diluted solution at different concentrations was added. The plate was then placed in a 37°C incubator for 3 After leaving the tubes for 4 hours, 50 μL of blood was taken from each tube and analyzed using BD TruCount In addition to the tubes, BD's B cell counting antibody mixture (anti-CD45 (lymphocyte population)) was added to the blood. ), anti-CD3 (T cells), or anti-CD19 (B cells) was added. The cells were incubated at room temperature in the dark for 15 min. After leaving the cells to stand, BD FACS lysis solution was added, and the cells were placed on the instrument and measured (BD C6). The results are shown in Figure 12.

[0080] As a result, the BAT4306F antibody was found to eliminate B cells at the three concentration levels tested. The ability to do this is stronger than that of the antibody rituximab, which has not had any N-glycosylation modifications.

[0081] Example 11 Enhancement of the affinity of BAT4306F for FcγRIIIa molecule

[0082] The unique glycan spectrum produced by genome-edited CHO-BAT-KF cells To verify that the recombinant antibody having the above-mentioned nucleotide sequence has enhanced affinity for FcγRIIIA, The affinity of BAT4306F, commercially available GAZYVA, and rituximab for FcγRIIIA The sensor was placed in PBS and allowed to soak for 10 minutes. 2.5 μg of labeled FcγRIIIa 158V and FcγRIIIa 158F in AB / mL. Loading: Biotin-labeled FcγRIIIa 158V Loading into dilution solution for 10 min (until the signal reaches approximately 1.3 nM), 3.6 Testing affinity of .3 with FcγRIIIa 158V: Test drug BAT4306F, Obinutuzumab was diluted to 500 nM in AB. Rituximab was diluted to 3000 nM in AB. The solution was then diluted in seven 2-fold gradients using the same buffer. B, FcγRIIa V158, regeneration buffer, pharmaceutical diluent, and neutralization buffer, in that order. The following steps were performed with the SA sensor: eline: Baseline detected in AB. 150s, Association: Concentration gradient Drug diluent samples of the agent and blank (AB) were allowed to bind for 90 s. Dissociation: Dissociated in AB for 120 seconds. Regeneration: Regenerated in NaOH at pH 10.5 for 5 seconds. Neutralization: Neutralized with AB for 5 seconds. Regeneration and neutralization were repeated three times. After collecting the data, The data was analyzed using the instrument's data analysis software, Acquisition 8.2. The signal collected at Baseline is used as the baseline, and the reference signal (sample The group was then subtracted from the data obtained (double subtraction of the pull blank and the sensor blank). was analyzed and fitted. Table 3. Statistical results of the affinity of BAT4306F for FcγRIIIa 158F

[0083] [Table 3] As a result, the three antibodies tested were found to be soluble in the ubiquitin-derived CHO-BAT-KF cells. Recombinant antibodies with unique glycosylation spectrum have the strongest affinity for FcγRIIIA. stomach.

[0084] Example 12

[0085] Glycans of antibodies expressed from other antibody sequences in the above CHO-BAT-KF host cells To verify that the spectra are stable and consistent, CHO-BAT-KF cells were Several other antibodies were expressed, one of which is shown in SEQ ID NO. 22. BAT4406F has both light chains shown in SEQ ID NO. 23 and both heavy chains shown in SEQ ID NO. 24. One of the antibodies has both light chains as shown in SEQ ID NO. 24 and SEQ The anti-EGFR antibody BAT0206F has both heavy chains and is shown in ID NO. 25. One of the antibodies has both light chains as shown in SEQ ID NO. 26 and SEQ ID NO. Anti-Trop2 antibody BAT0808, which has both heavy chains, is shown in Figure 0.27. Product Description (LudgerTag TM PROC(procainamide)Glyca After denaturing and reducing the sample, glycosidase was used to label the The glycans of the sample were removed from the glycosylation site. After labeling with the inconjugate, the eluate was isolated on a HILIC column and treated with mobile phase A at pH 4.5, 100 mM ammonium formate, with acetonitrile as mobile phase B. The elution gradient was 28% A to 38% A over 0 to 36 minutes. The resolution of the G1 and G1' sugar chain types in the system-suitable solution was 1.0 or greater. The results are shown in Figure 13 and Table 4. The sugar chain types of the four antibodies were The method and cells of the present invention have general applicability. This method can be applied not only to the preparation of anti-CD20 antibodies but also to the preparation of antibodies with other action sites. Homogeneous targeting antibodies can be produced that have enhanced DCC activity.

[0086] Table 4. Percentage of glycan types (%) in four antibodies prepared in CHO-BAT-KF cells [Table 4]

Claims

1. The amino acid sequences shown in SEQ ID NO. 10 and SEQ ID NO. 11, or has at least one sequence identical to that shown in SEQ ID NO. 10 and SEQ ID NO.

11. 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 less A pair of polypeptides having amino acid sequences that are at least 99% identical.

2. A pair of polynucleotides encoding the pair of polypeptides of claim 1, Preferably, the sequences of the pair of polynucleotides are SEQ ID NO. 1 2 and the sequence shown in SEQ ID NO. 13, or SEQ ID NO. 12 and the sequence shown in SEQ ID NO. 13, and at least 90%, at least 91%, At least 92%, at least 93%, at least 94%, at least 95%, at least also have 96%, at least 97%, at least 98% or at least 99% identity A pair of polynucleotides that are sequences.

3. The pair of polypeptides according to claim 1 each comprise a natural or artificial Fok I DNA. A pair of fusion proteins fused to a cleavage domain, preferably The amino acid sequences of the proteins are SEQ ID NO. 14 and SEQ ID NO. 16, or the sequence shown in SEQ ID NO. 14 and SEQ ID NO. 1 6 and at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% 99%, at least 98%, or at least 99% identity. Protein.

4. A pair of polynucleotides encoding the pair of fusion proteins of claim 3, respectively. and preferably, the polynucleotide sequences are SEQ ID NO. 15 and SEQ ID NO. The sequence shown in SEQ ID NO. 17, or SEQ ID NO. 15 and SEQ ID NO. The sequence shown in EQ ID NO. 17 has at least 90%, at least 91%, or at least at least 92%, at least 93%, at least 94%, at least 95%, at least 9 6%, at least 97%, at least 98% or at least 99% identity A pair of polynucleotides,

5. At least one polynucleotide in the pair of polynucleotides according to claim 2 or 4 A vector comprising a vector, preferably the vector is a plasmid.

6. A host cell transfected with the vector of claim 5, preferably Preferably, the host cells are CHO cells, and more preferably, the CHO cells are CHO-K 1, and more preferably, the CHO-K1 is suitable for serum-free culture; Preferably, the host cell is for expressing an antibody, more preferably, the host cell is for the expression of antibodies with a unique glycan spectrum, preferably wild-type glycans The level of at least one sugar moiety in the unique glycan spectrum is compared with the spectrum. The rule has been modified, Preferably, the sugar moiety is glucose, fucose, galactose, mannose, high mannosaccharide. selected from the group consisting of glucose, glucosamine, G0, and acetoglucosamine; Preferably, the carbohydrate moiety of the antibody satisfies one or more of the following conditions: a. the antibody has a reduced level of fucose, preferably the level of fucose is 5% or less, more preferably the level of fucose is 0, b. The antibody has reduced levels of mannose, preferably the mannose content The rate is less than 5% c. The antibody has reduced levels of high mannose, preferably The content is 5% or less, d. The antibody has reduced levels of galactose, preferably levels of 30% or less, more preferably levels of galactose of 5% or less; e. The G0 content of the antibody is increased, preferably the G0 content is 60% or more, more preferably Preferably, the G0 level is 80% or more, Preferably, the G0 content is 60% or more, does not contain fucose, and more preferably, 0 content is 60% or more, no fucose is contained, and mannose or / and high mannose is contained The content of is 5% or less, Preferably, the antibody has a glycan spectrum shown in Figure 10; Preferably, the antibody is an anti-EGFR or CD20 or Trop2 antibody, more preferably Alternatively, the antibody is a humanized or fully human anti-CD20 antibody, Preferably, the antibody is BAT1206F, more preferably the antibody BAT1 206F contains both light chains shown in SEQ ID NO. 18 and SEQ ID NO.

19. and Preferably, the antibody is BAT4406F, more preferably BAT440 The 6F antibody contains both light chains shown in SEQ ID NO. 22 and SEQ ID NO.

23. and Preferably, the antibody is BAT4306F, more preferably, the antibody is SEQ Both light chains shown in SEQ ID NO. 20 and heavy chains shown in SEQ ID NO. 21 Both books are available. Preferably, the antibody is BAT0206F, more preferably the antibody BAT0 206F contains both light chains shown in SEQ ID NO. 24 and SEQ ID NO. 25 and Preferably, the antibody is BAT0808, more preferably the antibody BAT08 08 contains both light chains shown in SEQ ID NO. 26 and SEQ ID NO.

27. A host cell having both heavy chains that are encoded by the antibody.

7. A kit comprising at least one polypeptide of the pair of polypeptides of claim 1. and preferably at least one of the pair of polynucleotides according to claim 2 The polynucleotide preferably comprises a pair of fusion proteins according to claim 3. At least one fusion protein, preferably a pair of polynucleotides according to claim 4. The nucleic acid sequence of the present invention comprises at least one polynucleotide among the nucleotides, preferably as defined in claim 5. A kit comprising the vector described above and preferably a host cell according to claim 6.

8. A pair of polypeptides / a pair of polynucleotides / a pair according to any one of claims 1 to 5. Paired fusion proteins / paired polynucleotides / vectors for Fut8 gene expression in CHO cells Use for editing.

9. A pair of polypeptides / a pair of polynucleotides / a pair according to any one of claims 1 to 6. Use of paired fusion proteins / paired polynucleotides / vectors / host cells for the preparation of antibodies or antibodies prepared using them, Preferably, the antibody has a unique glycosylation spectrum, more preferably, a wild-type glycosylation spectrum. Compared to the chain spectrum, the unique glycan spectrum contains the spectrum of at least one sugar moiety. The bell has been modified Preferably, the sugar moiety is glucose, fucose, galactose, mannose, high mannosaccharide. selected from the group consisting of glucose, glucosamine, G0, and acetoglucosamine; Preferably, the carbohydrate moiety of the antibody satisfies one or more of the following conditions: a. the antibody has a reduced level of fucose, preferably the level of fucose is 5% or less, more preferably, the antibody has a level of fucose of 0; b. The antibody has reduced levels of mannose, preferably the mannose content The rate is less than 5% c. The antibody has reduced levels of high mannose, preferably The content is 5% or less, d. The antibody has reduced levels of galactose, preferably levels of 30% or less, more preferably levels of galactose of 5% or less; e. The G0 content of the antibody is increased, preferably the G0 content is 60% or more, more preferably Preferably, the G0 level is 80% or more, Preferably, the antibody has a G0 content of 60% or more and does not contain fucose, and more preferably The G0 content is 60% or more, does not contain fucose, and is mannose or / and high mannose. The content of North is less than 5%. Preferably, the antibody has a glycan spectrum shown in Figure 10; Preferably, the antibody binds to EGFR or CD20 or Trop2, more preferably Preferably, the antibody binds to CD20, and more preferably, the antibody is humanized or fully human. an anti-CD20 antibody of the present invention; Preferably, the antibody is BAT1206F, more preferably BAT120 The 6F antibody contains both light chains shown in SEQ ID NO. 18 and SEQ ID NO.

19. and Preferably, the antibody is BAT4406F, more preferably BAT440 The 6F antibody contains both light chains shown in SEQ ID NO. 22 and SEQ ID NO.

23. and Preferably, the antibody is BAT4306F, more preferably BAT430 The 6F antibody contains both light chains shown in SEQ ID NO. 20 and SEQ ID NO.

21. and Preferably, the antibody is BAT0206F, more preferably the antibody BAT0 206F contains both light chains shown in SEQ ID NO. 24 and SEQ ID NO. 25 and Preferably, the antibody is BAT0808, more preferably the antibody BAT08 08 contains both the light chain shown in SEQ ID NO. 26 and the light chain shown in SEQ ID NO.

27. and a heavy chain that is to be used for preparing an antibody having both the heavy chain and the polypeptide chain, or an antibody prepared using the same.

10. 1. A method for editing the Fut8 gene in CHO, comprising: A pair of fusion proteins according to claim 3 or a pair of polynucleotides according to claim 4. or the vector according to claim 5 is introduced into CHO cells and the cells are cultured. CHO Fut8 gene knockout CHO cells are obtained.

8. Methods for editing genes.

11. A method for producing an antibody or an antibody produced using the method, (1) A pair of fusion proteins according to claim 3 or a pair of polynucleotides according to claim 4. or a CHO cell transfected with the vector of claim 5. The α-1-6 fragment in the CHO genome was identified by agglutinin pressure screening and limiting dilution. The CHO cell line CH obtaining O-BAT-KF, (2) A eukaryotic expression vector plasmid containing an antibody gene expression cassette was transfected into CHO-BAT- The antibody tag was electroporated into KF and defucosed by pressure screening. Protein-producing process A method for producing an antibody, or an antibody produced using the same, comprising:

12. In step (1), the vector according to claim 5 is transfected into wild-type CHO cells. and more preferably, transfecting the plasmid of claim 5 into wild-type CHO cells.

12. The method for producing an antibody according to claim 11, or an antibody produced by the method. and Preferably, the antibody has a unique glycosylation spectrum, more preferably, a wild-type glycosylation spectrum. Compared to the chain spectrum, the unique glycan spectrum contains the spectrum of at least one sugar moiety. The bell has been modified, Preferably, the sugar moiety is glucose, fucose, galactose, mannose, high mannosaccharide. selected from the group consisting of glucose, glucosamine, G0, and acetoglucosamine; Preferably, the carbohydrate moiety of the antibody satisfies one or more of the following conditions: a. the antibody has a reduced level of fucose, preferably the level of fucose is 5% or less, more preferably, the antibody has a level of fucose of 0; b. The antibody has reduced levels of mannose, preferably the mannose content The rate is less than 5% c. The antibody has reduced levels of high mannose, preferably The content is 5% or less, d. The antibody has reduced levels of galactose, preferably levels of 30% or less, more preferably levels of galactose of 5% or less; e. The G0 content of the antibody is increased, preferably the G0 content is 60% or more, more preferably Preferably, the G0 level is 80% or more, Preferably, the antibody has a G0 content of 60% or more and does not contain fucose, and more preferably The G0 content is 60% or more, does not contain fucose, and is mannose or / and high mannose. The content of North is less than 5%. Preferably, the antibody has a glycan spectrum shown in Figure 10; Preferably, the antibody binds to EGFR or CD20 or Trop2, more preferably Preferably, the antibody binds to CD20, and more preferably, the antibody is humanized or fully human. an anti-CD20 antibody of the present invention; Preferably, the antibody is BAT1206F, more preferably BAT120 The 6F antibody contains both light chains shown in SEQ ID NO. 18 and SEQ ID NO.

19. and Preferably, the antibody is BAT4406F, more preferably BAT440 The 6F antibody contains both light chains shown in SEQ ID NO. 22 and SEQ ID NO.

23. and Preferably, the antibody is BAT4306F, more preferably BAT430 The 6F antibody contains both light chains shown in SEQ ID NO. 20 and SEQ ID NO.

21. and Preferably, the antibody is BAT0206F, more preferably the antibody BAT0 206F contains both light chains shown in SEQ ID NO. 24 and SEQ ID NO. 25 and Preferably, the antibody is BAT0808, more preferably the antibody BAT08 08 contains both light chains shown in SEQ ID NO. 26 and SEQ ID NO.

27. and Preferably, the CHO cells are CHO-K1, and more preferably, the CHO-K 12. The method for producing an antibody according to claim 11, wherein the antibody is suitable for serum-free culture. Antibodies produced using the antibody.

13. The antibody is a BAT4306F antibody, and G of the BAT4306F antibody 0 content is 60% or more, does not contain fucose, More preferably, the G0 content of the BAT4306F antibody is 60% or more and the antibody contains fucose. and the mannose or / and high mannose content is 5% or less, More preferably, the antibody has a glycan spectrum shown in BAT4306F in FIG. Has, Preferably, the BAT4306F antibody has a light chain as shown in SEQ ID NO.

20. Both have the heavy chain shown in SEQ ID NO. 21, Preferably, the antibody is prepared by the method of claim 11.

14. Deposit number: CCTCC NO: C2017127, August 10, 2017, China Cells deposited at a culture depository center, Preferably, the Fut8 gene of the cell has the sequence shown in SEQ ID NO.

28. Having, cells.

15. 15. The cell of claim 14, wherein the cell expresses an antibody; Preferably, the host cell expresses an antibody, Preferably, the host cell expresses an antibody with a unique glycosylation spectrum, and more preferably Preferably, the unique glycan spectrum has at least one of the following characteristics compared to the wild-type glycan spectrum: The level of one sugar moiety has also been altered, Preferably, the sugar moiety is glucose, fucose, galactose, mannose, high mannosaccharide. selected from the group consisting of glucose, glucosamine, G0, and acetoglucosamine; Preferably, the carbohydrate moiety of the antibody satisfies one or more of the following conditions: a. The antibody has a reduced level of fucose, preferably the level of fucose is 5% or less, more preferably the level of fucose is 0, b. The antibody has reduced levels of mannose, preferably the mannose content The prevalence is less than 5%. c. The antibody has reduced levels of high mannose, preferably The content of is 5% or less, d. The antibody has reduced levels of galactose, preferably the level of galactose in the antibody is 30% or less, more preferably the level of galactose in the antibody is 5% or less and e. The G0 content of the antibody is increased, preferably, the G0 content is 60% or more, Preferably, the level of G0 is 80% or more, Preferably, the antibody has a G0 content of 60% or more and does not contain fucose, and more preferably The G0 content is 60% or more, does not contain fucose, and is mannose or / and high mannose. The content of North is less than 5%. Preferably, the antibody according to claim 14 has a glycosylation spectrum as shown in Figure 10. cells.

16. 15. The use of the cell according to claim 14 in the field of antibodies or antibodies prepared therefrom, Preferably, the antibody has a unique glycosylation spectrum, preferably a wild-type glycosylation spectrum. The unique glycan spectrum contains at least one sugar moiety at a level different from the original spectrum. has been modified, Preferably, the sugar moiety is glucose, fucose, galactose, mannose, high mannosaccharide. selected from the group consisting of glucose, glucosamine, G0, and acetoglucosamine; Preferably, the carbohydrate moiety of the antibody satisfies one or more of the following conditions: a. The antibody has a reduced level of fucose, preferably the level of fucose is 5% or less, more preferably the antibody has a level of fucose of 0, b. The antibody has reduced levels of mannose, preferably the mannose content The prevalence is less than 5%. c. The antibody has reduced levels of high mannose, preferably The content of is 5% or less, d. The antibody has reduced levels of galactose, preferably the level of galactose is 30% or less, more preferably the level of galactose is 5% or less, e. The G0 content of the antibody is increased, preferably, the G0 content is 60% or more, Preferably, the level of G0 is 80% or more, Preferably, the antibody has a G0 content of 60% or more and does not contain fucose, and more preferably The G0 content is 60% or more, does not contain fucose, and is mannose or / and high mannose. The content of North is less than 5%. Preferably, the antibody has the glycan spectrum shown in Figure 10. It is an antibody used or prepared using it.

17. A pharmaceutical composition comprising the antibody of any one of claims 9, 11, 13 or 16, Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

18. Administering an effective amount of the antibody of claim 9, 11, 13 or 16 to a subject in need thereof. A method for preventing / treating a disease, comprising:

19. The sequence shown in SEQ ID NO. 28, or the sequence shown in 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 or at least 98%, at least 99%, at least 99.5%, or at least 99.8% identical A nucleic acid sequence comprising a sequence having a specific activity, or a CHO cell comprising said sequence.

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