CN107217042B

CN107217042B - Genetic engineering cell line for producing afucosylated protein and establishing method thereof

Info

Publication number: CN107217042B
Application number: CN201710641279.5A
Authority: CN
Inventors: 魏化伟; 黄亚杰
Original assignee: Jiangsu East Against Biological Medicine Technology Co Ltd
Current assignee: Jiangsu East Against Biological Medicine Technology Co Ltd
Priority date: 2017-07-31
Filing date: 2017-07-31
Publication date: 2020-03-06
Anticipated expiration: 2037-07-31
Also published as: CN107217042A

Abstract

The invention discloses a genetic engineering cell line for producing afucosylated protein and an establishing method thereof. According to the invention, the Slc35c1 gene and/or Fut8 gene in a host cell is knocked out by using a CRISPR/Cas9 technology, so that a stable genetically engineered cell line with the Slc35c1 gene silencing and/or the Fut8 gene silencing is obtained. The genetic engineering cell line can produce protein with fucosylated completely removed, and the protein is stable after passage for 30 generations, so that the two problems of incomplete fucosylated removal and unstable passage in the prior art are solved. The afucosylated antibody produced by the method of the present invention exhibits enhanced ADCC activity to enhance the clinical therapeutic effect of the antibody.

Description

Genetic engineering cell line for producing afucosylated protein and establishing method thereof

Technical Field

The invention belongs to the technical field of bioengineering, and relates to a genetic engineering cell line capable of producing afucosylated protein and an establishment method thereof.

Technical Field

Since the first antibody drug was approved in 1986, the antibody drugs have been developed vigorously for over 30 years, and because the antibody drugs have the obvious advantages of high targeting property, specificity and the like, the treatment field is gradually expanded from the traditional treatment fields of cancers and autoimmune diseases to the treatment fields of anti-infection, metabolic diseases and cardiovascular diseases, and the like, and the antibody drugs have remarkable curative effects, and create great economic benefits while improving the life quality of patients. In addition to blocking target-mediated signal transduction and thereby inducing apoptosis by tumor-directed antibodies, more therapeutic antibodies exert therapeutic effects through the following effector functions: cell-dependent cytotoxicity (ADCC), cell-dependent phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), and the like.

Cell-dependent cellular cytotoxicity (ADCC) refers to an action of natural killer cells (NK), macrophages, neutrophils, or the like expressing an antibody Fc receptor (e.g., fcyriiia) to directly kill target cells by binding Fc fragments of IgG antibodies bound to the surface of the target cells via the Fc receptor, and activating and releasing cytotoxic substances such as perforin and granzyme. ADCC is one of the key mechanisms of the clinical curative effect of a therapeutic antibody, and is crucial to the development of the curative effect of the antibody, which is also supported by clinical research. The binding condition of the antibody and an Fc receptor (such as Fc gamma RIIIa) directly influences the ADCC effect function of the antibody, and the binding capacity of the antibody and the Fc receptor (such as Fc gamma RIIIa) can be enhanced by changing the protein structure of the Fc segment of the antibody or modifying the oligosaccharide chain of the Fc segment, so that the ADCC effect is enhanced, and the clinical treatment effect of the antibody is enhanced. Enhancing the effector function of therapeutic antibodies not only allows for better therapeutic results, but also allows for a reduction in the amount of antibody required for therapy, thereby reducing the cost of therapy.

In recent years, it has been found that therapeutic antibodies show better therapeutic efficacy in animals or clinically after removal or reduction of fucosylation. Mainly because the removal or reduction of fucosylation of antibodies to improve and Fc gamma RIIIa affinity, can be at lower concentrations through Fc gamma RIIIa high affinity and show stronger ADCC effects, removal or reduction of fucosylation into the next generation of therapeutic antibody efficacy of effective method. Therefore, the development of engineered cell lines capable of producing afucosylated antibodies is a problem to be solved by those skilled in the art.

However, most of the monoclonal antibodies are prepared by Chinese Hamster Ovary (CHO) cells, the core fucosylation level of the antibodies is high, and how to remove the core fucose in the Fc region of the antibodies to improve the ADCC effect is one of the main hotspots of the research on the antibodies at present. Wherein Gazyva (Obinutuzumab) approved for marketing in 2013 is Gazyva (Obinutuzumab)Approved glycoengineered monoclonal antibodies whose fucose knockdown is based on the Roche GlycoMab^TMThe technology platform promotes the addition of N-sugar terminal bisection galactoside by highly expressing β -1,4-N acetylglucosamine-transferase III (GnTIII enzyme), further inhibits the modification of antibody core fucose, and further enhances the ADCC activity of the antibody, but the glycosylation modification technology has certain defects that the technology leads the glycoform on the antibody to be more than 30 by introducing the bisection galactoside, has more and more complicated types, increases the heterogeneity of the glycoform and the quality control difficulty of the product, and has low fucose removal efficiency and about 30 percent of the glycoform contains core fucose.

According to the method, fucosylation of the protein can be completely removed by simultaneously knocking out two genes Slc35c1 and Fut8, and the fucosylation protein can not be detected after 30 generations, so that two problems of incomplete fucosylation removal and unstable generation are solved.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention provides a method for completely removing protein fucosylation, which aims to remove the protein fucosylation produced by cells by knocking out Slc35c1 gene and/or Fut8 gene. According to the above method, the present invention also provides a genetically engineered cell line useful for producing an afucosylated protein, and an afucosylated protein can be prepared using the genetically engineered cell line. In a specific embodiment of the invention, afucosylated monoclonal antibodies against CD20 can be prepared using the genetically engineered cell line. The afucosylated monoclonal antibody against CD20 has an enhanced ADCC effect compared to the fucosylated monoclonal antibody against CD 20.

According to one aspect of the invention, the invention provides a genetically engineered cell line for the production of afucosylated proteins, said genetically engineered cell line being a cell line with a Slc35c1 gene silencing and/or a Fut8 gene silencing.

Further, the genetically engineered cell line is resistant to at least one of the following lectins: lentil lectin, pea lectin, broad bean lectin, and pericarpium Citri Tangerinae Auricularia lectin.

Further, the protein is a protein containing an Fc region.

Further, the genetically engineered cell line contains a nucleotide sequence encoding a protein comprising an Fc region. The nucleotide sequence encoding the Fc region-containing protein may be present independently of the genome of the genetically engineered cell line or may be integrated into the genome of the genetically engineered cell line.

Further, the genetically engineered cell line contains a Cas9 gene sequence. The Cas9 gene sequence may exist independently of the genome of the genetically engineered cell line or may be integrated into the genome of the genetically engineered cell line.

Cas9 of the present invention includes, but is not limited to, SpCas9, Cas9-nickase (Cas9n), dCas9-FokI, and the like.

In a specific embodiment of the invention, the invention employs SpCas 9.

Furthermore, the genetically engineered cell line contains a gene sequence of sgRNA directed to the Slc35c1 gene and/or a gene sequence of sgRNA directed to the Fut8 gene. The sgRNA gene sequence may be present independently of the genome of the genetically engineered cell line or may be integrated into the genome of the genetically engineered cell line.

According to another aspect of the present invention, there is provided a method for establishing the above-mentioned genetically engineered cell line, comprising the steps of: knocking out the Slc35c1 gene and/or the Fut8 gene in the host cell to obtain a cell line with a Slc35c1 gene silencing and/or a Fut8 gene silencing.

Further, the establishing method comprises the following steps: knocking out the Slc35c1 gene and/or the Fut8 gene in a host cell by using a CRISPR/Cas9 technology to obtain a cell line with the Slc35c1 gene silencing and/or the Fut8 gene silencing.

Still further, the establishing method comprises the following steps:

for the knockout of the Slc35c1 gene alone,

(1) designing a sgRNA sequence aiming at an Slc35c1 gene in a host cell by using a CRISPR/Cas9 technology, and simultaneously introducing the gene sequence and a Cas9 gene sequence into the host cell;

(2) screening resistance pressure to obtain a genetic engineering cell line for producing the afucosylated protein;

for the single knockout of the Fut8 gene,

(1) designing a sgRNA sequence aiming at a Fut8 gene in a host cell by using a CRISPR/Cas9 technology, and simultaneously introducing the gene sequence and a Cas9 gene sequence into the host cell;

for the simultaneous knockout of the Slc35c1 gene and the Fut8 gene,

(1) designing sgRNA sequences aiming at Slc35c1 gene and Fut8 gene in host cells respectively by using CRISPR/Cas9 technology, and introducing the gene sequences and Cas9 gene sequences into the host cells simultaneously;

(2) and (3) obtaining a stable gene engineering cell line for producing the afucosylated protein by resistance pressure screening.

In the above establishment method, the step of introducing the sgRNA gene sequence and the Cas9 gene sequence into the host cell at the same time may include: and respectively connecting the sgRNA gene sequence and the Cas9 gene sequence to vectors to construct knockout vectors, and simultaneously transfecting the two knockout vectors into host cells.

Alternatively, the step of introducing the sgRNA gene sequence into the host cell simultaneously with the Cas9 gene sequence may comprise: the sgRNA gene sequence and the Cas9 gene sequence are jointly connected to a vector to construct a knockout vector, and the constructed knockout vector is transfected into a host cell.

In a specific embodiment of the invention, the step of introducing the sgRNA gene sequence into the host cell simultaneously with the Cas9 gene sequence comprises: the sgRNA gene sequence and the Cas9 gene sequence are jointly connected to a vector to construct a knockout vector, and the constructed knockout vector is transfected into a host cell.

According to yet another aspect of the invention, the invention provides an afucosylated protein. The protein is produced by the genetically engineered cell line described above.

According to a further aspect of the present invention, there is provided a method for producing the afucosylated protein as described above, characterized in that said production method comprises the steps of:

(1) establishing the genetically engineered cell line according to the establishing method;

(2) introducing the coding sequence of the protein into the genetic engineering cell line obtained in the step (1) to obtain the afucosylated protein.

According to a further aspect of the invention, there is provided the use of a genetically engineered cell line as hereinbefore described for the preparation of an afucosylated protein.

The protein of the present invention may be a recombinant protein or an endogenous protein. Recombinant proteins are foreign proteins formed by constructing a nucleic acid sequence encoding the protein into an expression vector and introducing it into a host cell for expression.

In a specific embodiment, the protein of the invention is a recombinant protein.

In a specific embodiment, the protein of the invention is an Fc-containing protein.

Fc-containing proteins of the invention include antibodies, bispecific antibodies, immunoadhesins and other binding proteins comprising at least a functional portion of the immunoglobulin CH2 and CH3 regions. "functional portion" refers to the regions of CH2 and CH3 that are capable of binding Fc receptors (e.g., fcyr or FcRN), and/or that are capable of participating in complement activation. The CH2 and CH3 regions are not functional if the CH2 and CH3 regions contain deletions, substitutions, and/or insertions or other modifications that render them incapable of binding any Fc receptor and incapable of activating complement.

Fc-containing proteins of the invention also include antibody derivatives comprising an antibody or an Fc domain or Fc region of an antibody, modified by covalent binding to a heterologous molecule, examples of antibody derivatives include binding domain-Ig fusions, wherein the binding domain may be, for example, the extracellular domain of a ligand, receptor, peptide, non-naturally occurring peptide, etc. exemplary fusions of immunoglobulins or Fc region include etanercept, which is a fusion protein of sTNFRII with the Fc region (U.S. Pat. No. 5,605,690), alexan, which is a fusion protein of LFA-3 with the Fc region expressed on antigen presenting cells (U.S. Pat. No. 5,914,111), cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) with the Fc region (J.exp.Med.181: 9(1995), interleukin 15 with the Fc region (IL-Pro), such as interleukin-CD-Pro-CD-Ser-No. 5, No. 5, No. 5, No. 5, No. 5, No. 5, No. 5.

Any kind of afucosylated antibodies or antibody derivatives can be prepared using the genetically engineered cell lines of the invention as described above. Examples of afucosylated antibodies or antibody derivatives prepared using the previously described genetically engineered cell lines of the invention include, but are not limited to: anti-HER 2 mab: trastuzumab (Trastuzumab, U.S. patent No 5821337), Pertuzumab (Pertuzumab, U.S. patent No 7862817); anti-EGFR monoclonal antibody: cetuximab (Cetuximab, U.S. patent No 6217866); anti-CD 20 mab: rituximab (Rituximab, world patent WO9411026), Obinutuzumab (Chinese patent publication No.: CN1902231B), Ocrelizumab (Chinese patent publication No.: CN 101151278); anti-CD 19 mab: chimeric antibody IgG1 with light heavy chain CDRs identical to HD37 hybridoma (pezzutto (1997), j.immunol.138, 2793-9); anti-CLD 18a2 antibody: IMAB362 (Chinese patent grant publication No. CN 101687929).

In a specific embodiment of the invention, the afucosylated protein prepared with the previously described genetically engineered cell lines of the invention is an afucosylated antibody against CD 20.

The heavy chain protein sequence of the anti-CD 20 antibody is shown in SEQ ID NO.1, and the DNA sequence of the coding heavy chain protein is shown in SEQ ID NO. 29; the light chain protein sequence is shown as SEQ ID NO.2, and the DNA sequence of the coding light chain protein is shown as SEQ ID NO. 30.

Antibodies prepared by using the genetically engineered cells of the invention include, but are not limited to, murine antibodies, humanized antibodies, chimeric antibodies, fully human antibodies. Preferably, the antibody produced by the genetically engineered cell of the invention is an IgG-type monoclonal antibody.

The antibody prepared by the gene engineering cell can be different subtypes of IgG, including IgG1, IgG2, IgG3 and IgG 4.

Antibodies and antibody derivatives prepared according to the methods of the invention can be used in a variety of therapeutic or non-therapeutic applications. For example, the antibodies can be used as therapeutic antibodies. Antibody derivatives (e.g., receptor-Fc fusions) are useful as therapeutic molecules. The antibody or antibody derivative may be conjugated to other molecules. The antibody is conjugated to a suitable drug (e.g., an antibody drug conjugate) or other active agent. The antibodies and antibody derivatives may also be used for non-therapeutic purposes, such as diagnostic assays, prognostic assays, release assays, and the like.

Antibodies and antibody derivatives prepared according to the methods of the invention can be formulated as therapeutic or non-therapeutic products. The antibodies and derivatives can be formulated into pharmaceutical compositions comprising a therapeutically or prophylactically effective amount of the antibody or derivative and one or more pharmaceutically compatible (acceptable) ingredients. For example, pharmaceutical or non-pharmaceutical compositions typically comprise one or more pharmaceutical carriers (e.g., sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like). Water is the more common carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions may also be employed as liquid carriers, particularly for injectable solutions. Suitable excipients include, for example, amino acids, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk (chalk), silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The compositions may also contain minor amounts of wetting or emulsifying agents or pH buffering agents, if desired. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. Martin, Remington's pharmaceutical Sciences describes examples of suitable drug carriers. Such compositions typically contain a therapeutically effective amount of the protein, usually in purified form, and an appropriate amount of carrier to provide a form suitable for administration to a patient. The formulation corresponds to the mode of administration.

Compositions for intravenous administration are typically solutions in sterile isotonic aqueous buffer. If desired, the drug may also contain a solubilizing agent and a local anesthetic, such as lidocaine, to relieve pain at the site of injection. Typically, the ingredients are provided separately or mixed together in unit dosage form, e.g., as a lyophilized powder or as a water-free concentrate in a sealed container such as an ampoule or sachet indicating the active ingredient content. When the drug is administered by infusion, the drug can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. When the medicament is administered by injection, an ampoule of sterile water for injection or saline may be provided to mix the ingredients prior to administration.

The above-mentioned genetically engineered cell lines constructed according to the present invention may be transient transfection/transduction-derived cell lines or may be stable transfection/transduction-derived cell lines.

In a specific embodiment, the genetically engineered cell line of the invention is a stable cell line.

The term "stable cell line" as used herein refers to a cell line whose nature is stably inherited, and generally refers to a cell line produced by stable transfection/transduction, i.e., the nucleotide sequence introduced into the cell is stably inherited by the genome of the cell.

The term "afucosylated" herein refers to a glycosylation modification of a protein that does not contain fucosylation.

The term "core fucose" herein means: in the core pentasaccharide of the N-saccharide, fucose is linked to the asparagine-linked GlcNAC.

The term "ADCC" as used herein refers to a cell-mediated cytotoxic reaction.

The term "enhancing ADCC" as used herein refers to any measurable increase in cell lysis, e.g., an increase in cell lysis of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, or 325%, in the presence of effector cells, when contacted with an afucosylated antibody, as compared to cell killing of the same cells contacted with the fucosylated antibody.

The term "vector" as used herein refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operatively linked. These vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Expression vectors commonly used in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" may be used interchangeably, as plasmids are the most commonly used form of vector. However, the invention also includes other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

The term "host cell" herein includes any cell suitable for expression of a recombinant nucleic acid sequence. Cells include eukaryotic cells (unicellular or multicellular), yeast cells (e.g., Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, Pichia methanolica, and the like), plant cells, insect cells (e.g., SF-9, SF-21, baculovirus-infected insect cells, Trichoplusia ni, and the like), non-human animal cells, human cells, or cell fusions, such as hybridomas or quadromas (quadromas).

The host cell may be a human, monkey, ape, hamster, rat or mouse cell. In some embodiments, the cell is a eukaryotic cell and is selected from the group consisting of: CHO (e.g., CHOK1, DXB-11CHO, Veggie-CHO), COS (e.g., COS-7), Syrian hamster, rat melanoma, mouse melanoma (e.g., SP2/0, NS0), retinal cells, Vero, CV1, kidney (e.g., HEK293, 293EBNA, MSR293, MDCK, HaK, BHK, BHK21), HeLa, HepG2, WI38, MRC5, Colo205, HB8065, HL-60, Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3, L cells, C127 cells, MMT 060562, Sertoli cells, BRL 3A cells, 1080 HT cells, human melanoma cells, tumor cells, human lymphoma cells (e.g., Namalwa cells) and cell lines derived from the foregoing cells.

In a particular embodiment of the invention, the host cell is selected from CHO.

The term "antibody" herein means: (1) immunoglobulin polypeptides and immunologically active portions of immunoglobulin polypeptides, i.e., polypeptides of the immunoglobulin family or portions thereof, comprise an antigen binding site for an immunospecific binding domain specific antigen (e.g., CD20) and an Fc domain comprising complex N-glycoside-linked sugar chains, or (b) conservatively substituted derivatives of such immunoglobulin polypeptides or fragments of immunospecific binding antigens (e.g., CD 20). Antibodies are generally described in, for example, Harlow & Lane, antibodies: a Laboratory Manual (Cold Spring Harbor Laboratory Press, 1988).

The term "bispecific antibody" includes antibodies that are capable of selectively binding two or more epitopes. Bispecific antibodies typically comprise 2 different heavy chains, each of which specifically binds to a different epitope-either a different epitope on two different molecules (e.g., a different epitope on two different antigens) or a different epitope on the same molecule (e.g., a different epitope on the same antigen). If the bispecific antibody is capable of selectively binding two different epitopes (a first epitope and a second epitope), the affinity of the first heavy chain for the first epitope is typically at least 1 to 2 or 3 or 4 or more orders of magnitude lower than the affinity of the first heavy chain for the second epitope, or vice versa. The epitopes specifically bound by the bispecific antibody may be on the same or different targets (e.g., on the same or different proteins). Bispecific antibodies can be made, for example, by combining heavy chains that recognize different epitopes on the same antigen. For example, nucleic acid sequences encoding heavy chain variable sequences that recognize different epitopes on the same antigen can be fused to nucleic acid sequences encoding the same or different heavy chain constant regions, and such sequences can be expressed in cells expressing immunoglobulin light chains. A typical bispecific antibody has 2 heavy chains, each with 3 heavy chain CDRs, followed (from N-terminus to C-terminus) by a CH1 domain, a hinge region, a CH2 domain, and a CH3 domain; there is also an immunoglobulin light chain which does not confer epitope binding specificity but may bind to each heavy chain, or may bind to each heavy chain and may bind to one or more epitopes bound by the heavy chain epitope binding region, or may bind to each heavy chain and is capable of binding 1 or 2 heavy chains to 1 or 2 epitopes.

The term "monoclonal antibody" refers to an antibody derived from a single cell clone, including any eukaryotic or prokaryotic cell clone or phage clone.

The term "Fc region" denotes the constant region of an antibody, e.g., the CH 1-hinge-CH 2-CH3 domain, optionally with the CH4 domain, or conservatively substituted derivatives of such Fc region.

The term "Fc domain" denotes a constant region domain of an antibody, e.g., a CH1, hinge, CH2, CH3, or CH4 domain, or conservatively substituted derivatives of such Fc domains.

The invention has the beneficial effects that:

the invention utilizes CRISPR/Cas9 technology to construct a gene engineering cell line with Slc35c1 gene silencing and/or Fut8 gene silencing, the gene engineering cell line can prepare afucosylated antibody, and the property of the cell line can be inherited stably for a long time.

Drawings

FIG. 1 is a schematic diagram of the structure and insertion site of plasmid pX 330;

FIG. 2 shows N-glycoform analysis of EM201 antibody expressed by different cell lines;

FIG. 3 shows a graph of the N-glycoform analysis of the passage stability of different cell lines;

figure 4 shows a graph of the binding activity of an anti-CD 20 antibody to Fc γ RIIIa;

figure 5 shows ADCC effector function of anti-CD 20 antibodies against Daudi cells;

figure 6 shows a graph of the effect of anti-CD 20 antibody treatment on Daudi cell nude mouse subcutaneous graft tumor volume.

Detailed Description

Embodiments of the present invention will be described in detail with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples do not show the specific techniques or conditions, and the techniques or conditions are described in the literature in the art (for example, refer to molecular cloning, a laboratory Manual, third edition, scientific Press, written by J. SammBruker et al, Huang Petang et al) or according to the product instructions. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Example 1 construction of Slc35c1 Gene knockout vector

The coding region targeting the mRNA sequence of the Chinese hamster Slc35c1 gene (NM-001246808.1, shown in SEQ ID NO. 3) 4 gRNAs (Table 1) were designed and synthesized by Nanjing Kingsler Biotech, Inc., and after gradient annealing of two paired single strands (F and R), ligated with BbsI restriction enzyme digested pX 330-U6-Chimerac-BB-CBh-hSpCas 9 (purchased from Addgene, Cathasmid 42230; Multiplex Genome Engineering Using CRISPR/Cassys. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hau PD, Wu X, Jiang W, Marraffini LA, Zhang F. science, Jan 3.10.1126/science.1143 Pub23287718) vector. A schematic of the carrier structure and insertion site is shown in FIG. 1.

After the sequencing verification, the correct clone is confirmed to be obtained, Top-10 competent cells are transfected, a single clone is selected and cultured in a shake flask, and the plasmid used for transfecting Chinese hamster cell CHO is obtained by an endotoxin-free plasmid macroextraction kit (Tiangen Biochemical technology Beijing GmbH).

TABLE 1 gRNA design targeting Slc35c1 Gene

Example 2: construction of FUT8 Gene knockout vector

The coding region targeting the mRNA sequence of the Chinese hamster FUT8 gene (XM _003501735.2, shown in SEQ ID NO. 14) 5 segments of gRNAs (Table 2) were designed, synthesized by Nanjing Kingsler Biotech, Inc., and after gradient annealing of two paired single strands (F and R), ligated with BbsI restriction enzyme digested pX330-U6-Chimeric _ BB-CBh-hSpCas9 (purchased from Addgene, Cathasmid 42230; Multiplex Genome Engineering Using/Cassys. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, PD, Wu X, Jiang W, Marraffini LA, ZhangF. science.2013Jan 3.10.1126/science.1141233 Pub23287718) vector.

Table 2 gRNA design targeting FUT8 gene

Example 3 construction of CHO cells knockout of Slc35c1 Gene

Inoculating 30ml of 5X 10 density cell culture shake flask⁵The CHO-K1(ATCC) cells having a cell viability of 98% or more, centrifuged, and diluted to 1X 10 with an electrotransfer buffer⁷cell/ml density, adding 40 μ g of constructed pX330 plasmid targeting Slc35c1 gene into an electric shock cup, adding 0.7ml of cell suspension, finally adding electric transfer buffer solution to 0.8ml, mixing gently, performing electric shock once under the conditions of 300V and 20ms, placing the electric shock cup in an ice box for 5min, adding culture medium after transfection, placing at 37 ℃ and 5% CO₂And culturing on a cell culture shaking table with the rotating speed of 130 rpm. After 2 days, adding lentil lectin LCA (Lensculinaris agglutinin) to 200 mu g/ml, and screening stable resistant clones; adding drugs for 5 days, centrifugally collecting cells, and carrying out negative sorting on the co-transfected cells by using biotin-LCA markers in combination with anti-biotin microBeads and MACs LD columns by utilizing the specific affinity of lentil lectin LCA to protein fucosyl; after sorting, the cells were cultured for 24hr in 300. mu.g/ml lentil lectin LCA, and the cells were seeded into a 96-well plate by limiting dilution to perform cell monoclonality, cultured in 300. mu.g/ml lentil lectin LCA by cloning, and then further seeded into 12-well or 6-well plates until transferred to culture flasks, to obtain monoclonal cell strains. After extracting genomic DNA from the obtained monoclonal cell strain, amplifying a Slc35c1 gene fragment by PCR to detect the mutation condition of the gene, wherein the sequence of a PCR forward primer is as follows: 5'-ATGTGACAATTGAAGGCTGTACC-3' (SEQ ID NO. 12); the reverse primer sequence is as follows: 5'-AGAAGGAAGTGGTCTGTTTGAG-3' (SEQ ID NO: 5'-AGAAGGAAGTGGTCTGTTTGAG-3')ID NO. 13). The PCR product was purified and recovered by agarose gel electrophoresis, cloned into a T-Vector pGM-Simple-T Fast Vector (Tiangen Biochemical technology Beijing Co., Ltd.), the cloned Vector was used for sequencing, and monoclonal cell lines in which the reading frames of the Slc35c1 genes all had frameshift mutations were selected and designated CHO-K1-S^-/-。

Example 4 construction of Fut8 Gene knockout CHO cells

Inoculating 30ml of 5X 10 density cell culture shake flask⁵The CHO-K1(ATCC) cells having a cell viability of 98% or more, centrifuged, and diluted to 1X 10 with an electrotransfer buffer⁷cell/ml density, adding 40 μ g of constructed pX330 plasmid targeting Fut8 gene into an electric shock cup, adding 0.7ml of cell suspension, adding electric transfer buffer solution to 0.8ml, mixing, performing electric shock once under the conditions of 300V and 20ms, placing the electric shock cup in an ice box for 5min in ice bath, adding culture medium after transfection, placing at 37 ℃ and 5% CO₂And culturing on a cell culture shaking table with the rotating speed of 130 rpm. After 2 days, adding lentil agglutinin LCA (lens culiaris agglutinin) to 200 mu g/ml, and screening stable resistant clones; adding drugs for 5 days, centrifugally collecting cells, and carrying out negative sorting on the co-transfected cells by using biotin-LCA markers in combination with anti-biotin microBeads and MACs LD columns by utilizing the specific affinity of lentil lectin LCA to protein fucosyl; after sorting, the cells were cultured for 24hr in 300. mu.g/ml lentil lectin LCA, and the cells were seeded into a 96-well plate by limiting dilution to perform cell monoclonality, cultured in 300. mu.g/ml lentil lectin LCA by cloning, and then further seeded into 12-well or 6-well plates until transferred to culture flasks, to obtain monoclonal cell strains.

After extracting genomic DNA from the obtained monoclonal cell strain, PCR amplifying a Fut8 gene fragment to detect the mutation condition of the gene, wherein the PCR forward primer sequence is as follows: 5'-GATTCCAGGTTCCCATATATTC-3' (SEQ ID NO.25), the reverse primer sequence is: 5'-TGATGACTGCTAGTGATGCTAC-3' (SEQ ID NO.26), or a forward primer sequence: 5'-TGTCTGAAGCATCATGTGTTG-3' (SEQ ID NO.27), the reverse primer sequence being: 5'-ACAGTATTTCATCAAATCCTTG-3' (SEQ ID NO. 28). The PCR product is purified and recovered by agarose gel electrophoresis, and then cloned to a T vector pGM-Simple-T Fast Vector (Tiangen Biochemical technology Beijing, Ltd.) cloned Vector is used for sequencing, and monoclonal cell strains with frame shift mutation of Fut8 gene reading frames are selected and recorded as CHO-K1-F^-/-。

Example 5 construction of CHO cells double-knocked-out of Slc35c1 and Fut8 genes

The constructed stable strain CHO-K1-S of which the Slc35c1 gene is knocked out^-/-The Fut8 gene is knocked out according to the embodiment 4, and the screened clone strains are the CHO cells with Slc35c1 and Fut8 genes double-knocked out and are marked as CHO-K1-SF^-/-。

Example 6 expression and purification of anti-CD 20 antibody

Adopting pcDNA3.1 as a skeleton vector, cutting off a Neomycin selection label, replacing the Neomycin selection label with a GS (glutamine synthetase) expression cassette, and using the GS expression cassette as a screening marker; wherein the GS cDNA can be obtained from a cell line CHO expressing GS by RT-PCR, and the modified vector is named as a GS screening vector. DNA sequences of an EM201 heavy chain and an EM201 light chain are respectively synthesized (the DNA sequence of the coding heavy chain is shown as SEQ ID NO.29, and the DNA sequence of the coding light chain is shown as SEQ ID NO. 30), after a CMV promoter is inserted between the light chain and the heavy chain, a HindIII enzyme cutting site is introduced into the 5 'end through the conventional PCR, a NotI enzyme cutting site is introduced into the 3' end, and the DNA sequences are inserted between HindIII and NotI of a GS screening vector to construct and obtain a eukaryotic expression vector pEM 201.

Inoculating 30ml of 5X 10 density cell culture shake flask⁵CHO-K1, CHO-K1-S^-/-、CHO-K1-F^-/-And CHO-K1-SF^-/-The cell viability rate is more than 98%, and the cell viability rate is 1X 10⁷cells/ml density was inoculated in the electroporation buffer, 40. mu.g of pEM201 plasmid was added to the cuvette, 0.7ml of cell suspension was added, and finally the electroporation buffer was added to 0.8ml, gently mixed without air bubbles. Shocking once at 300V for 20ms, placing the shocking cup in ice box, ice-cooling for 5min, diluting to 6ml culture medium, and adding CO at 37 deg.C₂The incubator is recovered for 24h, and 75 μ M MSX is added to the seed culture medium for subclone screening by limiting dilution. The monoclonal cells were separated into 96-well plates by limiting dilution at 37 ℃ with 5% CO₂Culturing in an incubator, performing ELISA verification when the cells are amplified to a certain number, and selecting positive clones and sequentially amplifying the positive clones to a 24-pore plate, a 6-pore plate and a T25 square bottle. And culturing and screening for 6-8 weeks to obtain the monoclonal cell strain capable of efficiently expressing the anti-CD 20 antibody. The anti-CD 20 antibody is named as EM201, and the protein sequences of the heavy chain and the light chain are shown in SEQ ID NO.1 and SEQ ID NO. 2.

The monoclonal cell strain is subjected to multi-step amplification culture in a culture medium, and the inoculation density is 0.5 multiplied by 10⁶cells/ml, adding MSX to 75. mu.M for 2 weeks, centrifuging to collect cells, culturing in 125ml shake flask, placing at 37 deg.C and 5% CO₂The culture was carried out in suspension on a shaker at 130rpm for 12 days, in which glucose was supplemented at a final concentration of 4g/L on

days

3, 6 and 9, and after the completion of the culture, the supernatant was harvested and purified. The EM201 antibody was isolated and purified by AKTA (GE). The eluate, which was passed through a Protein A affinity column (MabSelect Sure) at pH ranging from 3.4 to 3.6 (monitored at 280 nm), was first collected, adjusted to pH 8.0, applied to an anion exchange chromatography column (Q-Sepharose FF), monitored at 280nm, and the sample was collected. Adjusting the pH value of the collected solution to 5.5, loading the solution to a cation exchange chromatography column (Poros) to collect a sample, and performing ultrafiltration concentration to obtain the anti-CD 20 antibody.

Example 7 glycoform analysis of anti-CD 20 antibody (EM201)

Collecting 200 μ g anti-CD 20 antibody, desalting with ultrafiltration tube, adding 10 μ L G7 enzyme digestion buffer solution, 1 μ LPNGase F, and water to 100 μ L, and keeping the temperature at 37 deg.C for 10 hr. Adding the enzyme-digested sample into 300 mu L of ethanol with the temperature of-20 ℃, uniformly mixing, standing for 20min, centrifuging at 12000rpm for 3min, taking supernatant, and concentrating and drying in vacuum to obtain the N-sugar dry powder. Mixing DMSO and acetic acid uniformly according to a ratio of 350 mu L to 150 mu L, dissolving 5mg of 2-AB and 6mg of SodiumCyanobiohydride in 100 mu L of mixed solution of DMSO and acetic acid, adding 10 mu L of mixed solution into vacuum-concentrated N-sugar dry powder, deriving for 3 hours at 65 ℃, adding 200 mu L of mixed solution of 80% acetonitrile and water, centrifuging for 2mins, taking supernatant, and performing liquid phase analysis: a chromatographic column: water Acquity UPLC BEH amide1.7 μm; column temperature: 40 ℃; excitation wavelength: λ ex 330 nm; λ em ═ 420 nm; sample introduction amount: 10 mu L of the solution; the liquid phase gradient is shown in table 3.

TABLE 3 gradient elution schedule

Time (min)	Flow A% (ammonium formate)	Mobile phase B% (acetonitrile)
			0	20	80
20	20	80
			25	25	75
75	40	60
			80	65	35
85	65	35
			86	20	80
96	20	80

The anti-CD 20 antibody EM201 is composed of CHO-K1 and CHO-K1-F^-/-、CHO-K1-S^-/-And CHO-K1-SF^-/-The glycoform of the cell expression is shown in FIG. 2, and it can be seen from FIG. 2 and Table 4 below that, compared with EM201 expressed by CHO-K1 cell line, CHO-K1-F^-/-、CHO-K1-S^-/-The content of antibody fucose expressed by the cells is obviously reduced, and only 0.6 percent and 1.8 percent of glycoforms contain fucose; and CHO-K1-SF^-/-No glycoforms containing fucose were detected by the cell-expressed antibodies, indicating that the double knockout removed fucose more completely than knockout of the Slc35c1 and Fut8 genes alone.

TABLE 4 proportion of N-glycoforms of EM201 antibody expressed by different cell lines

Legend N-acetylglucosamine ■, fucose:

(ii) a Mannose and galactose: ●

Example 8 passage stability of fucose knockout Stable strains

In order to meet the requirements of the industrialized production scale of protein drugs on passage and cell multiplication level, passage stability research is carried out on the constructed stable cell strain. The passage from the cell bank to 30 generations covers the requirements of the planned industrialized production scale on the passage and the cell multiplication level, and 15 generations are added on the basis of the passage to ensure the coverage of the stability research on the requirements of the future production scale. The cells were passed every 2-4 days for 30 passages, and the passage stability of the cells was analyzed by examining the doubling time, doubling level, maximum cell density, productivity, specific antibody production rate of the cells at different stages, and evaluating the expression amount and quality of the antibody. The cell strain has stable passageThe qualitative result shows that the cell density, the cell doubling time, the survival rate, the antibody specific generation rate, the antibody expression quantity and the antibody quality are basically stable when the cell is passaged to P30, and the cell is in accordance with the requirements of engineering cell strains. However, CHO-K1-S^-/-And CHO-K1-F^-/-The proportion of fucose forms increased significantly with increasing passage number of the cell lines (fig. 3 and table 5), to 1.7% and 4.7%, respectively, probably due to the activation of other glycosylated bypasses; and the double gene knockout CHO-K1-FS^-/-The cell line was passaged to 30 passages, and no fucose-containing glycoform was detected (FIG. 4 and Table 5). Shows that compared with the single knockout of Slc35c1 and Fut8 genes, the cell line with double knockout has CHO-K1-FS^-/-The generation stability is better, the core fucose knockout is more thorough, and the cell strain which is preferably used as the fucose thorough knockout cell strain is preferably applied and developed.

TABLE 5N-glycoform ratio of the subculture stabilities of different cell lines

Legend N-acetylglucosamine ■, fucose:

(ii) a Mannose and galactose: ●

Example 9 analysis of the binding Activity of anti-CD 20 antibodies with Fc γ RIIIa

Anti-His(R&Company D) the antibody was diluted to 1. mu.g/mL with a carbonate buffer solution of pH9.6, and added to an microplate and incubated overnight at 4 ℃. After washing and blocking (5% skim milk in phosphate buffer), 1. mu.g/ml of Fc. gamma. RIIIa (158V) (R) was added&Company D), incubated at 37 ℃ for 1 h. And (3) preparing a sample to be tested: EM201 (CHO-K1-FS)^-/-Expression), EM201(CHO-K1 expression), Gazyva (Genentech Co.) and anti-human kappa (R)&Company D) were mixed at a ratio of 1:2, incubated at 37 ℃ for 1 hour, and then the antibody was diluted to 50. mu.g/ml and further subjected to 3-fold gradient dilution to obtain 10 concentration points. The diluted antibody and anti-human kappa mixture (100. mu.l) was added to the microplate and incubated at 37 ℃ for 1 hour. After washing the plate, HRP-conjugated goatF (ab') 2anti-huma was addedn IgG F(ab′)2(R&Company D) was diluted at a ratio of 1:5000, added to an ELISA plate in an amount of 100. mu.l/well, and incubated at 37 ℃ for 1 hour. After washing the plate, 100. mu.l/well of TMB was added and the plate was developed at room temperature for 30min, 100. mu.l/well of 2mol/L H₂SO₄And (3) terminating the color reaction, measuring the light absorption value at the position of 450nm wavelength on an enzyme labeling instrument, and fitting a four-parameter equation curve by taking the sample concentration as the abscissa and the average value of the light absorption as the ordinate.

EM201(CHO-K1-FS^-/-Expressed) anti-CD 20 antibody in glycoform without core fucose; EM201(CHO-K1 expression) is a glycoform of core fucose (>84% ratio) of anti-CD 20 antibody; gazyva is an anti-CD 20 antibody mainly composed of a glycoform (about 50% ratio) partially containing core fucose. EM201 (CHO-K1-FS)^-/-Expression), EM201(CHO-K1 expression) and Gazyva (Genentech) binding Activity to Fc γ RIIIa (158V) the results of the experiments are shown in FIG. 4, which indicate that EM201 (CHO-K1-FS)^-/-Expressed) had about 2-fold greater binding activity to Fc γ RIIIa (158V) than Gazyva, and was significantly stronger (about 6-fold) than the binding activity of EM201(CHO-K1 expressed), indicating that the afucosylated anti-CD 20 antibody had a higher affinity for Fc γ RIIIa.

Example 10 ADCC Effect of anti-CD 20 antibodies on human B-cell lymphoma Daudi cells

Human Burkitt's lymphoma cell line Daudi cells were purchased from the American Type Culture Collection (ATCC) as target cells in ADCC assays; NK-92MI-CD16a, which highly expresses Fc gamma RIIa on cell membranes, is used as an effector cell (Huabo biomedical technology Co., Ltd.). 96-well plates with 5X 10 additions per well⁴NK-92MI-CD16a and 1X 10⁴40. mu.l of Daudi cells; EM201 (CHO-K1-FS)^-/-Expression), EM201(CHO-K1 expression), Gazyva (Genentech Co.) were added to 96-well plates to a final sample concentration of 1X 10-1 to 1X 10-8. mu.g/ml (10-fold gradient, total of 8 concentration points), 37 ℃ and 5% CO₂Culturing in a cell culture box for 4 h. And (3) reading an OD value at 492nm of the enzyme labeling instrument after the lactate dehydrogenase LDH test kit is developed, calculating the target cell lysis rate, and performing four-parameter equation fitting analysis by taking the sample concentration as an abscissa and the target cell lysis rate as an ordinate.

EM201(CHO-K1-FS^-/-The results of ADCC activity experiments on EM201(CHO-K1 expression) and Gazyva (Genentech) are shown in FIG. 5, and the results show that EM201 (CHO-K1-FS)^-/-Expressed) ADCC activity was about 4-fold that of Gazyva, more significantly stronger than that of EM201(CHO-K1 expressed) (about 20-fold), indicating that the afucosylated anti-CD 20 antibody has higher ADCC potency than the fucosylated antibody.

Example 11 therapeutic Effect of anti-CD 20 antibody on human B-cell lymphoma Daudi nude mouse subcutaneous transplantation tumor

To evaluate the in vivo anti-tumor activity of anti-CD 20 antibody, a human tumor transplantation model with BALB/cA-nude mice (6-7 weeks female, purchased from Shanghai Ling Biotech, Inc.) as hosts was used. Nude mice were inoculated subcutaneously with human B-cell lymphoma Daudi cells and animals were randomly grouped after tumors grew to 130-180mm 3. Blank control (PBS) and three anti-CD 20 antibodies: EM201 (CHO-K1-FS)^-/-Expression), EM201(CHO-K1 expression) and Gazyva (Genentech) were administered separately, with a three anti-CD 20 antibody dosing regimen of 3mg/kg 2 times per week for a total of 4 times. Tumor volumes were measured 2-3 times a week, mice weighed, and data recorded.

The results show that: as shown in fig. 6, tumor volume was significantly increased in the placebo mice; three groups of antibodies against CD20 were able to significantly inhibit tumor growth compared to the control group; EM201 (CHO-K1-FS)^-/-Expressed) anti-CD 20 antibody in glycoform without core fucose; antibody EM201 (CHO-K1-FS) with lower fucose content than EM201(CHO-K1 expression) antibody with predominant glycoform of core fucose^-/-Expression) and Gazyva (Genentech company), and the tumor-bearing mice can well tolerate the anti-CD 20 antibody medicament for knocking out fucose and have no symptoms of weight loss and the like.

The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.

SEQUENCE LISTING

<110> eastern Biocide science and technology Co., Ltd of Jiangsu

<120> a genetic engineering cell line for producing afucosylated protein and its establishment method

<160>30

<170>PatentIn version 3.5

<210>1

<211>449

<212>PRT

<213> human source

<400>1

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Tyr Ser

20 25 30

Trp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210>2

<211>219

<212>PRT

<213> human source

<400>2

Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser

20 25 30

Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Val Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala Gln Asn

85 90 95

Leu Glu Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210>3

<211>1098

<212>DNA

<213> mouse source

<400>3

atgaacaggg cgcctctgaa gcggtccagg atcctgcgca tggcgctgac tggaggctcc 60

actgcctctg aggaggcaga tgaggacagc aggaacaagc cgtttctgct gcgggcgctg 120

cagatcgcgc tggtcgtctc tctctactgg gtcacctcca tctccatggt attcctcaac 180

aagtacctgc tggacagccc ctccctgcag ctggataccc ctatcttcgt cactttctac 240

caatgcctgg tgacctctct gctgtgcaag ggcctcagca ctctggccac ctgctgccct 300

ggcaccgttg acttccccac cctgaacctg gaccttaagg tggcccgcag cgtgctgcca 360

ctgtcggtag tcttcattgg catgataagt ttcaataacc tctgcctcaa gtacgtaggg 420

gtggccttct acaacgtggg gcgctcgctc accaccgtgt tcaatgtgct tctgtcctac 480

ctgctgctca aacagaccac ttccttctat gccctgctca catgtggcat catcattggt 540

ggtttctggc tgggtataga ccaagaggga gctgagggca ccctgtccct cataggcacc 600

atcttcgggg tgctggccag cctctgcgtc tccctcaatg ccatctatac caagaaggtg 660

ctcccagcag tggacaacag catctggcgc ctaaccttct ataacaatgt caatgcctgt 720

gtgctcttct tgcccctgat ggttctgctg ggtgagctcc gtgccctcct tgactttgct 780

catctgtaca gtgcccactt ctggctcatg atgacgctgg gtggcctctt cggctttgcc 840

attggctatg tgacaggact gcagatcaaa ttcaccagtc ccctgaccca caatgtatca 900

ggcacagcca aggcctgtgc gcagacagtg ctggccgtgc tctactatga agagactaag 960

agcttcctgt ggtggacaag caacctgatg gtgctgggtg gctcctcagc ctatacctgg 1020

gtcaggggct gggagatgca gaagacccaa gaggacccca gctccaaaga gggtgagaag 1080

agtgctatca gggtgtga 1098

<210>4

<211>28

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<222>(26)..(26)

<223>n is a, c, g, or t

<400>4

caccggcggt ccaggatcct gcgcangg 28

<210>5

<211>25

<212>DNA

<213> Artificial sequence

<400>5

aaactgcgca ggatcctgga ccgcc 25

<210>6

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<213> Artificial sequence

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<212>DNA

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<212>DNA

<213> Artificial sequence

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<210>9

<211>25

<212>DNA

<213> Artificial sequence

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aaacgcgcga tctgcagcgc ccgcc 25

<210>10

<211>28

<212>DNA

<213> Artificial sequence

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<221>misc_feature

<222>(26)..(26)

<223>n is a, c, g, or t

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caccgaacct ctgcctcaag tacgtngg 28

<210>11

<211>25

<212>DNA

<213> Artificial sequence

<400>11

aaacacgtac ttgaggcaga ggttc 25

<210>12

<211>23

<212>DNA

<213> Artificial sequence

<400>12

atgtgacaat tgaaggctgt acc 23

<210>13

<211>22

<212>DNA

<213> Artificial sequence

<400>13

agaaggaagt ggtctgtttg ag 22

<210>14

<211>2823

<212>DNA

<213> mouse source

<400>14

ttcaggttgc tgctctggct taggccatct atgaccctgg tggtgttttc attcactata 60

agtccttccc atctttatta actgagcaag ttcagctagt aattttagag accgaggttc 120

aagcaataac acctatctct gcaataccgt gtggctttct tcaatgtctt acatcctaag 180

gaaaggaagc atgtagagcc caggaagcac aggacaagaa agctgcctcc ttgtatcacc 240

aggaagatct ttttgtaaga gtcatcacag taaccagaga gactaatttt gtctgaagca 300

tcatgtgttg aaacaacaga aacttatttt cctgtgtggc taactagaac cagagtacaa 360

tgtttccaat tctttgagct ccgagaagac agaagggagt tgaaactctg aaaatgcggg 420

catggactgg ttcctggcgt tggattatgc tcattctttt tgcctggggg accttattgt 480

tttatatagg tggtcatttg gttcgagata atgaccaccc tgaccattct agcagagaac 540

tctccaagat tcttgcaaag ctggagcgct taaaacaaca aaatgaagac ttgaggagaa 600

tggctgagtc tctccgaata ccagaaggcc ctattgatca ggggacagct acaggaagag 660

tccgtgtttt agaagaacag cttgttaagg ccaaagaaca gattgaaaat tacaagaaac 720

aagctaggaa tgatctggga aaggatcatg aaatcttaag gaggaggatt gaaaatggag 780

ctaaagagct ctggtttttt ctacaaagtg aattgaagaa attaaagaaa ttagaaggaa 840

acgaactcca aagacatgca gatgaaattc ttttggattt aggacatcat gaaaggtcta 900

tcatgacaga tctatactac ctcagtcaaa cagatggagc aggtgagtgg cgggaaaaag 960

aagccaaaga tctgacagag ctggtccagc ggagaataac atatctgcag aatcccaagg 1020

actgcagcaa agccagaaag ctggtatgta atatcaacaa aggctgtggc tatggatgtc 1080

aactccatca tgtggtttac tgcttcatga ttgcttatgg cacccagcga acactcatct 1140

tggaatctca gaattggcgc tatgctactg gaggatggga gactgtgttt agacctgtaa 1200

gtgagacatg cacagacagg tctggcctct ccactggaca ctggtcaggt gaagtgaagg 1260

acaaaaatgt tcaagtggtc gagctcccca ttgtagacag cctccatcct cgtcctcctt 1320

acttaccctt ggctgtacca gaagaccttg cagatcgact cctgagagtc catggtgatc 1380

ctgcagtgtg gtgggtatcc cagtttgtca aatacttgat ccgtccacaa ccttggctgg 1440

aaagggaaat agaagaaacc accaagaagc ttggcttcaa acatccagtt attggagtcc 1500

atgtcagacg cactgacaaa gtgggaacag aagcagcctt ccatcccatt gaggaataca 1560

tggtacacgt tgaagaacat tttcagcttc tcgaacgcag aatgaaagtg gataaaaaaa 1620

gagtgtatct ggccactgat gacccttctt tgttaaagga ggcaaagaca aagtactcca 1680

attatgaatt tattagtgat aactctattt cttggtcagc tggactacac aaccgataca 1740

cagaaaattc acttcggggc gtgatcctgg atatacactt tctctcccag gctgacttcc 1800

ttgtgtgtac tttttcatcc caggtctgta gggttgctta tgaaatcatg caaacactgc 1860

atcctgatgc ctctgcaaac ttccattctt tagatgacat ctactatttt ggaggccaaa 1920

atgcccacaa ccagattgca gtttatcctc accaacctcg aactaaagag gaaatcccca 1980

tggaacctgg agatatcatt ggtgtggctg gaaaccattg gaatggttac tctaaaggtg 2040

tcaacagaaa actaggaaaa acaggcctgt acccttccta caaagtccga gagaagatag 2100

aaacagtcaa ataccctaca tatcctgaag ctgaaaaata gagatggagt gtaagagatt2160

aacaacagaa tttagttcag accatctcag ccaagcagaa gacccagact aacatatggt 2220

tcattgacag acatgctccg caccaagagc aagtgggaac cctcagatgc tgcactggtg 2280

gaacgcctct ttgtgaaggg ctgctgtgcc ctcaagccca tgcacagtaa aataatgtac 2340

tcacacataa catacaaatg gattattttc tactttgccc tttaaatatt ctgtccccat 2400

gaaacaaaca ctgccacatt atgtaattta agtgacacag acgttttgtg tgagacttca 2460

aacatggtgc ctatatctga gagacctctg tgatttactg agaagatgag aacagctccc 2520

ttctgtgggg aagttggttc ttagtcagtg gtggactggc cactgaattc actgcaatca 2580

acagattcag aatgagaatg gatgtttttc ctttatatgg ttgtctggat tttttttaaa 2640

gtaatttcat cagttcagtt catccacctc attaataaat gaaggaatat accaataaaa 2700

tcaaatgaaa tattcactgt ccattaggaa gttttataaa acaatgccat gaacaaaaaa 2760

ttctttagta ctcaatgttt ctggacattc tctttgataa caaaaataaa ttttaaaaag 2820

gaa 2823

<210>15

<211>28

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<222>(26)..(26)

<223>n is a, c, g, or t

<400>15

caccggtcag acgcactgac aaagtngg 28

<210>16

<211>25

<212>DNA

<213> Artificial sequence

<400>16

aaacactttg tcagtgcgtc tgacc 25

<210>17

<211>28

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<222>(26)..(26)

<223>n is a, c, g, or t

<400>17

caccgggata aaaaaagagt gtatcngg 28

<210>18

<211>25

<212>DNA

<213> Artificial sequence

<400>18

aaacgataca ctcttttttt atccc 25

<210>19

<211>28

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<222>(26)..(26)

<223>n is a, c, g, or t

<400>19

caccggtgta ccatgtattc ctcaangg 28

<210>20

<211>25

<212>DNA

<213> Artificial sequence

<400>20

aaacttgagg aatacatggt acacc 25

<210>21

<211>28

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<222>(26)..(26)

<223>n is a, c, g, or t

<400>21

caccggggac cttattgttt tatatngg 28

<210>22

<211>25

<212>DNA

<213> Artificial sequence

<400>22

aaacatataa aacaataagg tcccc 25

<210>23

<211>28

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<222>(26)..(26)

<223>n is a, c, g, or t

<400>23

caccggtttt atataggtgg tcattngg 28

<210>24

<211>25

<212>DNA

<213> Artificial sequence

<400>24

aaacaatgac cacctatata aaacc 25

<210>25

<211>22

<212>DNA

<213> Artificial sequence

<400>25

gattccaggt tcccatatat tc 22

<210>26

<211>22

<212>DNA

<213> Artificial sequence

<400>26

tgatgactgc tagtgatgct ac 22

<210>27

<211>21

<212>DNA

<213> Artificial sequence

<400>27

tgtctgaagc atcatgtgtt g 21

<210>28

<211>22

<212>DNA

<213> Artificial sequence

<400>28

acagtatttc atcaaatcct tg 22

<210>29

<211>1347

<212>DNA

<213> Artificial sequence

<400>29

caggtgcagc tggtgcagtc cggcgccgag gtgaagaagc ccggctcctc cgtgaaggtg 60

tcctgcaagg cctccggcta cgccttctcc tactcctgga tcaactgggt gaggcaggcc 120

cccggccagg gcctggagtg gatgggcagg atcttccccg gcgacggcga caccgactac 180

aacggcaagt tcaagggcag ggtgaccatc accgccgaca agtccacctc caccgcctac 240

atggagctgt cctccctgag gtccgaggac accgccgtgt actactgcgc caggaacgtg 300

ttcgacggct actggctggt gtactggggc cagggcaccc tggtgaccgt gtcctccgcc 360

tccaccaagg gcccctccgt gttccccctg gccccctcct ccaagtccac ctccggcggc 420

accgccgccc tgggctgcct ggtgaaggac tacttccccg agcccgtgac cgtgtcctgg 480

aactccggcg ccctgacctc cggcgtgcac accttccccg ccgtgctgca gtcctccggc 540

ctgtactccc tgtcctccgt ggtgaccgtg ccctcctcct ccctgggcac ccagacctac 600

atctgcaacg tgaaccacaa gccctccaac accaaggtgg acaagaaggt ggagcccaag 660

tcctgcgaca agacccacac ctgccccccc tgccccgccc ccgagctgct gggcggcccc 720

tccgtgttcc tgttcccccc caagcccaag gacaccctga tgatctccag gacccccgag 780

gtgacctgcg tggtggtgga cgtgtcccac gaggaccccg aggtgaagtt caactggtac 840

gtggacggcg tggaggtgca caacgccaag accaagccca gggaggagca gtacaactcc 900

acctacaggg tggtgtccgt gctgaccgtg ctgcaccagg actggctgaa cggcaaggag 960

tacaagtgca aggtgtccaa caaggccctg cccgccccca tcgagaagac catctccaag 1020

gccaagggcc agcccaggga gccccaggtg tacaccctgc ccccctccag ggacgagctg 1080

accaagaacc aggtgtccct gacctgcctg gtgaagggct tctacccctc cgacatcgcc 1140

gtggagtggg agtccaacgg ccagcccgag aacaactaca agaccacccc ccccgtgctg 1200

gactccgacg gctccttctt cctgtactcc aagctgaccg tggacaagtc caggtggcag 1260

cagggcaacg tgttctcctg ctccgtgatg cacgaggccc tgcacaacca ctacacccag 1320

aagtccctgt ccctgtcccc cggcaag 1347

<210>30

<211>657

<212>DNA

<213> Artificial sequence

<400>30

gacatcgtga tgacccagac ccccctgtcc ctgcccgtga cccccggcga gcccgcctcc 60

atctcctgca ggtcctccaa gtccctgctg cactccaacg gcatcaccta cctgtactgg 120

tacctgcaga agcccggcca gtccccccag ctgctgatct accagatgtc caacctggtg 180

tccggcgtgc ccgacaggtt ctccggctcc ggctccggca ccgacttcac cctgaagatc 240

tccagggtgg aggccgagga cgtgggcgtg tactactgcg cccagaacct ggagctgccc 300

tacaccttcg gcggcggcac caaggtggag atcaagagga ccgtggccgc cccctccgtg 360

ttcatcttcc ccccctccga cgagcagctg aagtccggca ccgcctccgt ggtgtgcctg 420

ctgaacaact tctaccccag ggaggccaag gtgcagtgga aggtggacaa cgccctgcag 480

tccggcaact cccaggagtc cgtgaccgag caggactcca aggactccac ctactccctg 540

tcctccaccc tgaccctgtc caaggccgac tacgagaagc acaaggtgta cgcctgcgag 600

gtgacccacc agggcctgtc ctcccccgtg accaagtcct tcaacagggg cgagtgc 657

Claims

1. A genetically engineered cell line for production of an afucosylated protein, said genetically engineered cell line being a Slc35c1 gene-silenced and Fut8 gene-silenced cell line, said cell line being selected from the group consisting of: human, monkey, ape, hamster, rat, mouse cells.

2. The genetically engineered cell line of claim 1, wherein the protein is a protein comprising an Fc region.

3. The genetically engineered cell line of claim 2, comprising a nucleotide sequence encoding a protein comprising an Fc region.

4. The genetically engineered cell line of claim 1 containing a Cas9 gene sequence.

5. The genetically engineered cell line of claim 1, comprising a gene sequence for a sgRNA of the Slc35c1 gene and a gene sequence for a sgRNA of the Fut8 gene.

6. The method for establishing a genetically engineered cell line according to any one of claims 1 to 5, wherein the establishing method comprises the steps of: knocking out the genes of Slc35c1 and Fut8 in the cells to obtain a cell line with the genes of Slc35c1 and Fut8 silenced.

7. The method according to claim 6, wherein the establishing method comprises the steps of: knocking out the Slc35c1 gene and the Fut8 gene in the cell by using a CRISPR/Cas9 technology to obtain a cell line with the Slc35c1 gene silencing and the Fut8 gene silencing.

8. A method for producing an afucosylated protein, said method comprising using the genetically engineered cell line of any one of claims 1 to 5.

9. The method according to claim 8, characterized in that it comprises the steps of:

(1) obtaining a genetically engineered cell line with both Slc35c1 gene silencing and Fut8 gene silencing by the establishment method of claim 6 or 7;

(2) introducing the coding sequence of the protein into the genetic engineering cell line obtained in the step (1).

10. Use of the genetically engineered cell line of any one of claims 1 to 5 for the preparation of an afucosylated protein.