WO2025140609A1 - Method for constructing cell which produces defucosylated protein and use of cell - Google Patents

Abstract

Provided is a method for constructing a cell which produces defucosylated protein using a zinc finger nuclease which contains a zinc finger protein capable of binding to the FUT8 gene in a targeted manner and a DNA cleavage domain or cleavage half-domain. Also provided are a polynucleotide which encodes the zinc finger protein and the zinc finger nuclease, and a cell containing the polynucleotide and the zinc finger nuclease.

Description

A method for constructing a cell for producing defucosylated protein and its application Technical Field

The present disclosure relates to the fields of molecular biology and biopharmaceuticals; in particular, to cells for producing defucosylated proteins constructed by gene editing using zinc finger protein nuclease technology and applications thereof.

Background Art

In recent years, with the successful preparation and widespread application of genetically engineered antibodies and the significant improvement in antibody production levels, the research and development of antibody drugs has entered a new stage of rapid development. According to literature reports, as of December 2019, there were 79 therapeutic antibodies on the market, mainly used for tumors, autoimmune diseases, metabolic and infectious diseases (Lu RM et al. (2020) J Biomed Sci. 27(1): 1-30). The top ten best-selling anti-cancer drugs in the world in 2022 include four antibody drugs, with a total sales of nearly US$42.492 billion.

Antibodies activate the immune system to destroy attacking cells through two mechanisms: complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC). ADCC is an immune response produced primarily by natural killer (NK) cells against antibody-coated targets. In ADCC, NK cells recognize the constant (Fc) region of antibodies primarily through interaction with NK cell FcγRIII receptors. NK cells then deposit perforins and granzymes on the surface of target cells, inducing cell lysis and apoptosis, respectively.

Most mammalian immunoglobulins are fucosylated, including those produced by Chinese hamster ovary cells (CHO cells). α-1,6-fucosyltransferase (FUT8) catalyzes the transfer of fucose residues from guanosine diphosphate-fucose (GDP-Fuc) to the end of the innermost acetylglucosamine (GlcNAc) of the N-glycan core to form fucose attached to the antibody Fc core region. Studies have shown that when fucose is removed from the sugar chain structure at the Asn297 position of the CH2 domain of the IgG antibody Fc fragment, it can enhance the binding between NK cell FcγRIIIa and the IgG antibody Fc fragment, thereby enhancing the ADCC effect of the antibody (Shinkawa T et al. (2003) J Biol Chem. 278 (5): 3466-73).

At present, there are three common methods for industrial production of defucosylated antibodies: (1) adding fucosylation inhibitors such as 2-fluorofucose (2FF) to the culture medium (Rillahan CD et al. (2012) Nat Chem Biol. 8(7): 661-8); (2) stably overexpressing bacterial RMD protein (GDP-6-deoxy-D-lyso-4-hexulose reductase) in engineered cells (von Horsten HH et al. (2010) Glycobiology. 20(12): 1607-18) to block the fucosylation modification of the Fc region by interfering with the formation of substrate GDP-fucose; 3) Interfering with key genes in the fucose production pathway, such as introducing expression of β(1,4)-N-acetylglucosamine transferase III (GnTIII) (Popp O et al. (2018) MAbs. 10(2): 290-303), knocking out GDP-fucose transporter (GFT) (Omasa T et al. (2008) J Biosci Bioeng. 106(2): 168-73), or knocking out α-1,6-fucosyltransferase (FUT8) (Yamane-Ohnuki N et al. (2004) Biotechnol Bioeng. 87(5): 614-22), etc.

There is still a need in the art for new methods to construct defucosylated protein-producing cells.

Summary of the invention

To meet the above needs, after long-term and in-depth exploration and research, the inventors have developed a new method for constructing defucosylated protein production cells. This method uses zinc finger nuclease to inactivate the FUT8 gene in cells, and the resulting cells produce defucosylated proteins (e.g., antibodies) with strong effector functions, which has broad application prospects in industry.

In one aspect, the present invention provides a zinc finger protein capable of targeting and binding to the FUT8 gene, which comprises the zinc finger recognition region amino acid sequence of zinc finger F1 to F5 or to F6 shown in the same row in the following table in order from N to C terminus:

In another aspect, the present invention provides a zinc finger nuclease (ZFN) against the FUT8 gene, which is a fusion protein and comprises the ZFP described herein and at least one DNA cleavage domain or cleavage half-domain.

Yet another aspect herein provides a polynucleotide encoding a ZFP or ZFN as described herein.

Another aspect herein provides a vector comprising the polynucleotide as described above.

Another aspect herein provides an isolated cell (eg, a host cell) comprising a ZFP, ZFN, polynucleotide or vector described herein.

Another aspect herein provides a cell line in which the FUT8 gene is inactivated or partially inactivated, comprising a ZFP, ZFN, polynucleotide or vector described herein.

Another aspect herein provides a method of producing a FUT8-deficient cell, the method comprising:

(a) introducing a polynucleotide encoding one or more ZFNs into a cell, wherein the ZFN comprises: (i) a ZFP as described herein, which is capable of binding to a target site in a FUT8 gene in the cell; and (ii) a DNA cleavage domain or cleavage half-domain;

(b) expressing the ZFN in the cell, so that the ZFN binds to the target site and cleaves the FUT8 gene.

Another aspect herein provides a method of generating a FUT8-deficient cell line, the method comprising:

(a) inactivating the endogenous FUT8 gene in the cell by the method for producing FUT8-deficient cells as described herein; and

(b) culturing the cells under conditions suitable for generating a FUT8-deficient cell line.

Another aspect of the present invention provides a method for producing a target recombinant protein in a host cell, the method comprising:

(a) providing a FUT8-deficient cell as described herein,

(b) introducing an expression vector comprising a nucleic acid encoding the target recombinant protein into the cell, thereby producing the target recombinant protein.

Another aspect of the present invention provides a method for producing a target recombinant protein in a host cell, the method comprising:

(a) providing a cell comprising an endogenous FUT8 gene;

(b) inactivating the endogenous FUT8 gene in the cell by the method for producing a FUT8-deficient cell as described herein; and

(c) introducing an expression vector comprising a nucleic acid encoding the target recombinant protein into the cell, thereby producing the target recombinant protein.

Another aspect herein provides the use of the ZFPs, ZFNs, polynucleotides, vectors described herein for generating FUT8-deficient cells.

Another aspect herein provides use of a ZFP, ZFN, polynucleotide, vector, cell or cell line described herein for producing a defucosylated Fc-containing recombinant protein.

Another aspect provided herein is the use of a ZFP, ZFN, polynucleotide, vector, cell or cell line described herein for producing an antibody (eg, a monoclonal antibody) capable of eliciting an enhanced ADCC effect.

Another aspect herein provides a FUT8-deficient cell line prepared according to the method for generating a FUT8-deficient cell line described herein.

On the other hand, the present invention provides a FUT8-deficient cell line, which comprises a nucleotide sequence as shown in one or more of SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58 and SEQ ID NO: 59 at the FUT8 locus.

Another aspect of the present invention provides a FUT8-deficient cell line comprising an altered FUT8 locus, wherein the altered FUT8 locus can be detected by the following primers:

Primer pair (i):

Forward primer: AGCCTGAAGTACATAGCCGA (SEQ ID NO: 46); and

Reverse primer: TGCCACTGCTTCTATATACTGATTC (SEQ ID NO: 47);

and/or

Primer pair (ii):

Forward primer: GACGCACTGACAAAGTGGGA (SEQ ID NO: 48); and

Reverse primer: GGTCTGTTCCATCCCCAGAATG (SEQ ID NO: 49);

and/or

Primer pair (iii):

Forward primer: CTGTTGATTCCAGGTTCCCA (SEQ ID NO: 50); and

Reverse primer: TGTTACTTAAGCCCCAGGC (SEQ ID NO: 51).

Another aspect of the present invention provides a method for cultivating a FUT8-deficient cell line, comprising:

(a) providing a FUT8-deficient cell line according to any one of claims 15 to 17; and

(b) culturing or expanding cells of said cell line.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be further described below in conjunction with the accompanying drawings, wherein these drawings are only for illustrating the embodiments of the present invention rather than for limiting the scope of the present invention.

FIG. 1 shows the binding site, cleavage site, and PCR primer sites of zinc finger nuclease targeting FUT8 exon 3 and exon 10 and their vicinity according to one of the exemplary embodiments herein.

Figure 2A shows a schematic diagram of the expression plasmid of the zinc finger protein nuclease according to one of the exemplary embodiments of the present invention. In which the zinc finger protein and the restriction endonuclease FokI cleavage half domain are fused and cloned in the plasmid. The fusion protein is under the control of the CMV promoter. Figure 2B shows a schematic diagram of the specific structure of the zinc finger protein nuclease according to one of the exemplary embodiments of the present invention, in which each zinc finger binding domain is sequentially inserted between the framework sequence of the natural zinc finger protein and connected to the FokI cleavage half domain at the C-terminus.

Figure 3 shows the T7E1 digestion experiment to verify whether the ZFN plasmid can effectively remove the target gene sequence. Lane 1: S2-L and S4-R transfection, the editing efficiency calculated according to the band brightness measured by ImageJ is 4.7%; Lane 2: L3 and R3 transfection, the editing efficiency is 6.2%; NC: negative control group, that is, the cell genomic DNA without knockout of the FUT8 gene; PC: positive control group, that is, the cell genome containing 50% of the knockout FUT8 gene.

Figure 4 shows the results of flow cytometric analysis of cell pools stained with green fluorescent marker lentil agglutinin (LCA-FITC). (A) Quantitative analysis of different sub-cell populations, where the circled cell population with lower green fluorescence is the FUT8 ^-/- cell population, which accounts for 27.44% of the cell pool. (B) Peak shifts of different sub-cell populations.

Figure 5 shows the flow cytometry analysis results of clone 154-F8ZFN-09-08 and negative control after LCA-FITC staining. Solid line: 154-F8ZFN-09-08 cells; dotted line: negative control without knockout of FUT8 gene.

FIG6 shows the NGS validation results, demonstrating that a pair of alleles of clones 154-F8ZFN-09-08 and 154-F8ZFN-10-02 were knocked out.

Figure 7 shows the application of FUT8 ^-/- cell lines (i.e., 154-F8ZFN-09-08 and 154-F8ZFN-10-02) in the screening of monoclonal antibody expression. NC: Negative control in which the FUT8 gene was not knocked out. (A) Changes in viability during FB culture after cell pool recovery; (B) Changes in viable cell density VCD during FB culture of cell pool; (C) Protein expression level titer on day 14 during fed-batch culture; (D) The harvested supernatant was purified by one-step ProA and then analyzed by size exclusion chromatography SEC-HPLC; (E) One-step purified samples were subjected to SDS Caliper NR and SDS Caliper R; (F) Glycoform detection results of one-step purified samples.

Figure 8 shows the performance evaluation results of the FUT8-/- cell line (154-F8ZFN-09-08) in antibody production. (A) Cell viability of the 154-F8ZFN-09-08 group during FB culture; (B) Changes in viable cell density (VCD); (C) Protein expression titer; (D) SEC-HPLC and SDS Caliper NR analysis results; (E) Glycoform analysis results.

Figure 9 shows the results of gene editing efficiency detection. Lane 1: 31-L1/31-R1 knockout sample, editing efficiency is 9.4%; Lane 2: 32-L1/32-R1 knockout sample, editing efficiency is 10.9%; NC: wild-type CHO-K1.

Figure 10 shows the results of SCP monoclonal sorting. The unstained sample is CHO K1; Fut8 ^-/- clone C6 is the monoclonal clone selected after LCA-FITC staining after sorting monoclonal clones; Fut8 wt is CHO K1 stained with LCA-FITC.

Figure 11 shows the results of SCP monoclonal sorting. The unstained sample is CHO K1-N079; Fut8 ^-/- clone F2 is a monoclonal selected after LCA-FITC staining after sorting; Fut8 wt is CHO K1 stained with LCA-FITC.

FIG12 shows the NGS validation results, demonstrating that a pair of alleles in clones C6 and F2 were knocked out.

FIG. 13 shows that the ADCC effect of adalimumab produced by host cells after knocking out the Fut8 gene is significantly enhanced compared with the wild-type control.

DETAILED DESCRIPTION

The meaning of scientific and technological terms in this application is consistent with the general understanding of those skilled in the art, unless otherwise specified. In this application, "one" or its combination with various quantifiers includes both singular and plural meanings, unless otherwise specified. In this application, for the same parameter or variable, when multiple numerical values, numerical ranges, or combinations thereof are given for description, it is equivalent to specifically revealing these numerical values, range ends, and numerical ranges formed by any combination thereof. In this application, any numerical value, whether or not it carries a modifier such as "about", covers an approximate range that can be understood by those skilled in the art, such as plus or minus 10%, 5%, etc. In this article, each "implementation method" refers to and covers the implementation methods of various methods and systems of this application equally. In this application, one or more technical features in any implementation method can be freely combined with one or more technical features in any one or more other implementation methods, and the implementation methods obtained thereby also belong to the content disclosed in this application.

In mammalian cells, Fut8 attaches core fucose to oligosaccharides present on the Fc region of antibodies, which is generally considered to be important for the effector function of antibody-dependent cellular cytotoxicity. Three-dimensional analysis of the structure of human Fut8 revealed that three α2/α6 fucosyltransferase motifs form the catalytic core of the enzyme. See, Ihara et al. (2007) Glycobiology 17:455-66. In this region, many single residue point mutations to alanine result in complete inactivation of the enzyme. See, Ihara et al. (2007) Glycobiology 17:455-66; Takahashi et al. (2000) Glycobiology 10:503-10. It has been shown that cells in which the expression of FUT8 is reduced or eliminated (e.g., knockout cell lines or using siRNA) can produce non-fucosylated antibodies with enhanced effector function. See, for example, Kanada et al. (2007) Biotechnol. 130(3): 300-310; Kanada et al. (2007) Glycobiology 18: 104-118; Mori et al. (2004) Biotechnol. Bioeng. 88: 901-908.

The present invention provides a method for constructing a defucosylated protein production cell, which inactivates the FUT8 gene in the cell by using a zinc finger nuclease targeting the FUT8 gene. The zinc finger protein nuclease (ZFN) comprises a zinc finger protein (ZFP) and a nuclease cleavage domain or a cleavage partner domain. The inventors surprisingly found that the FUT8 gene in the cell can be highly effectively knocked out using the zinc finger nuclease specially designed as described herein, and the resulting cell produces a defucosylated protein (e.g., antibody) with stronger effector function.

A. Zinc finger proteins

In one aspect, the present invention provides a zinc finger protein (ZFP) capable of targeting and binding to the FUT8 gene.

As used herein, the term "zinc finger protein" or "ZFP" refers to a protein that binds DNA in a sequence-specific manner via one or more zinc fingers (or zinc finger recognition regions). The zinc fingers are amino acid sequence regions within the protein whose structure is stabilized by the coordination of zinc ions.

The ZFP described herein is a non-naturally occurring ZFP. The ZFP may have 1, 2, 3, 4, 5, 6 or more zinc fingers, each of which has a zinc finger recognition region capable of binding to a target site in the FUT8 gene. In some embodiments, the ZFP comprises five or six zinc fingers.

Herein, the term "target site" refers to a nucleotide sequence of a portion of the DNA of a target gene (eg, FUT8 gene) to which the ZFP or ZFN described herein is to bind.

In some embodiments, the target site may include exon 3 of the FUT8 gene (the relevant sequence is shown in SEQ ID NO: 1). In some embodiments, the target site may include exon 10 of the FUT8 gene and its vicinity (the relevant sequence is shown in SEQ ID NO: 2).

Herein, the zinc fingers in ZFP are named in the form of "F+sequential numbers from N to C". For example, for a certain ZFP, in the order from N to C, the first zinc finger is named "F1", the second zinc finger is named "F2", and so on.

In some embodiments, one or more zinc fingers of the ZFP have an engineered zinc finger recognition region. In some embodiments, the ZFP has the recognition region amino acid sequence of the zinc finger (F1 to F5 or to F6) shown in the same row in Table 1:

Table 1. Zinc finger proteins targeting FUT8 gene

In Table 1, each row describes a single ZFP. The name and DNA target sequence (in 5'-3' order) of each ZFP are shown in the first column, and the amino acid sequence of the recognition region of the zinc finger (F1 to F5 or to F6) in each ZFP is shown in columns 2 to 6 or 7.

In some embodiments, the multiple zinc fingers of the ZFP can be connected by a suitable linker sequence. In some embodiments, the linker sequence can include a linker of 1, 2, 3, 4, 5 or more amino acids in length.

Further, ZFP pairs are also provided herein, which include left ZFP and right ZFP. The target site of the left ZFP in the ZFP pair is located on the relative strand of the target gene DNA sequence at a close position (e.g., 5 to 10 nucleotide base pairs apart). For example, the left ZFP in the ZFP pair can bind to a target site on a strand (e.g., template strand or coding strand) of the target gene DNA sequence, and the right ZFP can bind to a target site located near the left ZFP target site (e.g., 5 to 20 nucleotide base pairs apart) on the opposite strand (e.g., coding strand or template strand) of the target gene DNA sequence. In some embodiments, the target site of the left ZFP and the target site of the right ZFP are separated by 5 to 20 nucleotide base pairs, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotide base pairs.

In some embodiments, the left ZFP can have the following recognition region amino acid sequence (ZFP 32-L1):

F1: QLATLNR (SEQ ID NO: 13);

F2: TRWNLRA (SEQ ID NO: 14);

F3: SRRDLRR (SEQ ID NO: 15);

F4: WRRRLLS(SEQ ID NO: 16);

F5:RSDYLTN(SEQ ID NO:17), and

F6: FHSNLLA (SEQ ID NO: 18).

In some embodiments, the left ZFP can have the following recognition region amino acid sequence (ZFP L3):

F1:SKWNLRS(SEQ ID NO:24);

F2: AQSNLLS(SEQ ID NO: 25);

F3: LRHQLRR (SEQ ID NO: 26);

F4: RSDYLTN (SEQ ID NO: 17);

F5: RSDYLTN (SEQ ID NO: 17).

In some embodiments, the left ZFP can have the following recognition region amino acid sequence (ZFP S2-L):

F1: RADNLTE(SEQ ID NO: 30);

F2: TSGNLTE (SEQ ID NO: 31);

F3: TSGHLVR (SEQ ID NO: 32);

F4: RKDNLKN (SEQ ID NO: 33); and

F5: RKDNLKN (SEQ ID NO: 33).

In some embodiments, the left ZFP can have the following recognition region amino acid sequence (ZFP 31-L1):

F1: DRSNLLS(SEQ ID NO: 39);

F2: NQSNLLR (SEQ ID NO: 40);

F3: FHSNLLA (SEQ ID NO: 18);

F4: QLSTLNY(SEQ ID NO: 41); and

F5: QSGNLSR (SEQ ID NO: 42).

In some embodiments, the right ZFP can have the following recognition region amino acid sequence (ZFP 32-R1):

F1: RKSHLTM (SEQ ID NO: 19);

F2: FHSGLLA (SEQ ID NO: 20);

F3: WRRRLLS(SEQ ID NO: 16);

F4: RKYVLLR (SEQ ID NO: 21);

F5: RKDYLVL (SEQ ID NO: 22); and

F6: QQAGLIN (SEQ ID NO: 23).

In some embodiments, the right ZFP can have the following recognition region amino acid sequence (ZFP R3):

RSDYLTN (SEQ ID NO: 17);

QKITLVR (SEQ ID NO: 27);

RSDYLTN (SEQ ID NO: 17);

QLATLNR (SEQ ID NO: 13);

SRFNLTR (SEQ ID NO: 28); and

TKYILTN (SEQ ID NO: 29).

In some embodiments, the right ZFP can have the following recognition region amino acid sequence (ZFP S4-R):

RSDNLSV (SEQ ID NO: 34);

SPADLTR (SEQ ID NO: 35);

RSDHLSQ (SEQ ID NO: 36);

QSGDLRR (SEQ ID NO: 37);

RSDNLVR (SEQ ID NO: 38); and

RKDNLKN (SEQ ID NO: 33).

In some embodiments, the right ZFP can have the following recognition region amino acid sequence (ZFP 31-R1):

FHSGLLA (SEQ ID NO: 20);

HPSTLSK (SEQ ID NO: 43);

ARWTLDC (SEQ ID NO: 44);

HPSTLSK (SEQ ID NO: 43);

HPSTLSK (SEQ ID NO: 43); and

RKFTLTN (SEQ ID NO: 45).

In some embodiments, the ZFP pair may consist of ZFP 32-L1 and ZFP 32-R1. In some embodiments, the ZFP pair may consist of ZFP L3 and ZFP R3. In some embodiments, the ZFP pair may consist of ZFP S2-L and S4-R. In some embodiments, the ZFP pair may consist of ZFP 31-L1 and ZFP 31-R1.

B. Zinc Finger Nuclease

In another aspect, the present invention provides a zinc finger nuclease (ZFN) against the FUT8 gene, which is a fusion protein and comprises the ZFP described herein and at least one DNA cleavage domain or cleavage half-domain.

As used herein, the term "fusion protein" refers to a protein molecule in which two or more subunit molecules (eg, a ZFP and a DNA cleavage domain or cleavage half-domain as described herein) are linked to each other (preferably covalently linked).

As used herein, the term "cleavage" refers to the breakage of the covalent backbone of a DNA molecule. Cutting can be initiated by various methods, including but not limited to enzymatic or chemical hydrolysis of phosphodiester bonds. In some embodiments, cutting can be single-stranded cutting or double-stranded cutting. In some embodiments, double-stranded cutting can be caused by two different single-stranded cutting events. Cutting of DNA can result in the generation of flat ends or staggered ends. In certain embodiments, the ZFN is used to target double-stranded DNA cutting.

As used herein, the term "cleavage half-domain" refers to a polypeptide sequence that can be conjugated with another polypeptide (the same or different) to form a complex having cleavage activity (preferably double-chain cleavage activity). In some embodiments, two cleavage half-domains typically function as a dimer (in pairs) to exert cleavage activity.

In some embodiments, the ZFP and at least one DNA cleavage domain or cleavage half-domain are operably linked.

As used herein, the term "operably linked" may refer to a component being linked to another component in a functional manner. For example, in the case of a fusion protein in which a ZFP is fused to a cleavage domain, if the ZFP in the fusion protein can bind to its target site and/or its binding site, and the cleavage domain can cleave DNA near the target site, then the ZFP and the cleavage domain are in an operably linked relationship.

The zinc finger nuclease described in this article can be used as a tool for targeted cutting and inactivation of a target gene (such as the FUT8 gene), which can achieve the purpose of targeted knockout of the target gene by introducing a double-stranded DNA break (DSB) at the target site of the target gene and utilizing non-homologous end joining (NHEJ)-mediated DSB repair.

Further, the present invention also provides a ZFN pair, which includes a left ZFN and a right ZFN. The left ZFN and the right ZFN are each fused with the ZFP described herein and at least one DNA cleavage domain or cleavage half-domain. For example, ZFN 32-L1 is a fusion protein comprising ZFP 32-L1 and at least one DNA cleavage domain or cleavage half-domain.

The target site of the left ZFN in the ZFN pair and the target site of the right ZFN are located on opposite strands at a close position (e.g., 5 to 10 nucleotide base pairs apart) on the target gene DNA sequence. For example, the left ZFN in the ZFN pair can bind to a target site on one strand (e.g., template strand or coding strand) of the target gene DNA sequence, while the right ZFN can bind to a target site on the opposite strand (e.g., coding strand or template strand) of the target gene DNA sequence that is located near the left ZFN target site (e.g., 5 to 20 nucleotide base pairs apart). In some embodiments, the target site of the left ZFN and the target site of the right ZFN are separated by 5 to 20 nucleotide base pairs, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotide base pairs.

In some embodiments, the ZFN pair may consist of ZFN 32-L1 and ZFN 32-R1. In some embodiments, the ZFN pair may consist of ZFN L3 and ZFN R3. In some embodiments, the ZFN pair may consist of ZFN S2-L and S4-R. In some embodiments, the ZFN pair may consist of ZFN 31-L1 and ZFN 31-R1.

The cleavage domain portion of the fusion protein disclosed herein can be obtained from any endonuclease or exonuclease. Exemplary endonucleases from which the cleavage domain can be derived include, but are not limited to, restriction endonucleases and homing endonucleases. See, e.g., New England Biolabs 2002-2003 Catalog, Beverly, Massachusetts; and Belfort et al. (1997) Nucleic Acids Res. 25: 3379-3388. Other enzymes capable of cleaving DNA are known (e.g., S1 nuclease; mung bean nuclease; pancreatic DNase I; micrococcal nuclease; yeast HO endonuclease; see also Linn et al. (eds.) Nucleases (Nucleases), Cold Spring Harbor Press, 1993). One or more of these enzymes (or functional fragments thereof) can be used as a source of cleavage domains and cleavage half-domains.

Similarly, the cleavage half-domains may be derived from any nuclease or fragment thereof whose cleavage activity requires dimerization as mentioned above. In general, if a fusion protein comprises a cleavage half-domain, two fusion proteins are required to perform cleavage. Optionally, a single protein comprising two cleavage half-domains may be used. The two cleavage half-domains may be derived from the same endonuclease (or a functional fragment thereof), or each cleavage half-domain may be derived from a different endonuclease (or a functional fragment thereof). In addition, the target sites of the two fusion proteins are arranged so that the binding of the two fusion proteins to their respective target sites places the cleavage half-domains in a spatial orientation relative to each other that allows the cleavage half-domains to form a functional cleavage domain (e.g., by dimerization). Thus, in certain embodiments, the target sites may be separated by 5-20 or more nucleotide base pairs. In general, the cleavage site is located between the target sites.

Restriction endonucleases (restriction enzymes) are present in many species and can sequence-specifically bind to DNA (at a recognition site) and cleave the DNA at or near the binding site. Certain restriction enzymes (e.g., Type IIS) cleave DNA at a site distal to the recognition site and have separable binding domains and cleavage domains. Exemplary Type IIS restriction endonucleases are described in International Publication WO 07/014275, the entirety of which is incorporated herein by reference. Thus, in one embodiment, the fusion protein comprises the zinc finger protein and a cleavage domain (or cleavage half-domain) from at least one Type IIS restriction enzyme.

A cleavage domain or cleavage half-domain can be any portion of a protein that retains cleavage activity or retains the ability to multimerize (e.g., dimerize) to form a functional cleavage domain.

In some embodiments, the type IIS restriction enzyme is FokI. The enzyme is active as a dimer. Accordingly, in some embodiments, a portion of the FokI enzyme used in the fusion protein is considered to be a cleavage half-domain. Thus, for targeted double-stranded cleavage and/or targeted replacement of cellular sequences using zinc finger protein-FokI fusions, two fusion proteins, each comprising a FokI cleavage half-domain, can be used to reconstitute a catalytically active cleavage domain. Optionally, a single polypeptide molecule containing a zinc finger protein and two FokI cleavage half-domains can also be used. In some embodiments, the cleavage half-domain is a FokI cleavage half-domain. In some embodiments, the cleavage half-domain is a wild-type FokI cleavage half-domain. In other embodiments, the cleavage half-domain is a modified FokI cleavage half-domain.

In some exemplary embodiments, the cleavage half-domain is derived from Sharkey FokI nuclease obtained by amino acid mutation based on wild-type FokI, and its exemplary amino acid sequence is shown in SEQ ID NO: 3.

The engineered cleavage half-domains described herein can be prepared using any suitable method, for example, by site-directed mutagenesis.

Other restriction enzymes comprising separable binding and cleavage domains are also contemplated herein. See, e.g., Roberts et al. (2003) Nucleic Acids Res. 31: 418-420.

In some embodiments, the cleavage domain may comprise one or more engineered cleavage half-domains (also referred to as dimerization domain mutants) that minimize or prevent homodimerization, such as described in U.S. Patent Publication Nos. 20050064474; 20060188987 and 20080131962 (the disclosures of all of which are incorporated herein by reference in their entireties). Amino acid residues located at positions 446, 447, 479, 483, 484, 486, 487, 490, 491, 496, 498, 499, 500, 531, 534, 537, and 538 of FokI are all targets for influencing dimerization of the FokI cleavage half-domain.

In some embodiments, exemplary engineered cleavage half-domains of FokI that form obligate heterodimers include pairs wherein a first cleavage half-domain includes mutations in amino acid residues at positions 490 and 538 of FokI and a second cleavage half-domain includes mutations in amino acid residues 486 and 499.

In one embodiment, the mutation at 490 replaces Glu (E) with Lys (K); the mutation at 538 replaces Iso (I) with Lys (K); the mutation at 486 replaces Gln (Q) with Glu (E); and the mutation at 499 replaces Iso (I) with Lys (K). Specifically, the engineered cleavage half-domains described herein are prepared by mutating positions 490 (E→K) and 538 (I→K) in one cleavage half-domain to generate an engineered cleavage half-domain designated "E490K:I538K" and by mutating positions 486 (Q→E) and 499 (I→L) in the other cleavage half-domain to generate an engineered cleavage half-domain designated "Q486E:I499L". The engineered cleavage half-domains described herein are obligate heterodimer mutants in which aberrant cleavage is minimized or eliminated when one or more pairs of nucleases containing these cleavage half-domains are used for cleavage. See, for example, US Patent Publication No. 20080131962, the disclosure of which is incorporated by reference in its entirety for all purposes.

C. Delivery

The ZFNs described herein can be delivered to cells to inactivate the FUT8 gene in the cells. The delivery of the ZFNs to cells can be achieved by introducing a polynucleotide encoding the ZFN into the cells, wherein the polynucleotide is transcribed in the cells and the transcript is translated to produce the ZFN fusion protein. The expression of proteins in cells can also involve trans-splicing, polypeptide cleavage, and polypeptide ligation.

Another aspect of the present invention provides a polynucleotide encoding a ZFP or ZFN as described herein. In some embodiments, the polynucleotide may be mRNA.

The ZFNs described herein can be delivered to target cells by a variety of suitable methods.

In some embodiments, a vector (or vector system) can be used to deliver the ZFN to a target cell.

Another aspect of the present invention provides a vector comprising a polynucleotide as described above. Available vectors include, but are not limited to, one or more of the following: plasmid vectors, retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, poxvirus vectors, herpesvirus vectors, and the like.

In some embodiments, the vector may contain a polynucleotide encoding one or more ZFNs. In some embodiments, the polynucleotide encoding one or more ZFNs may be located on one (same) or multiple (different) vectors. In embodiments using multiple vectors, each vector may contain a polynucleotide encoding one or more ZFNs. In embodiments using multiple vectors, each vector may be delivered to the target cell simultaneously or sequentially.

In some embodiments, the ZFN can be delivered to the target cell using a non-vector delivery method. Available non-vector delivery methods include, but are not limited to, electroporation, lipofection, microinjection, gene guns, liposomes, immunoliposomes, polycations or lipids: nucleic acid conjugates, naked DNA, and agents that enhance DNA uptake. The polynucleotide encoding the ZFN can also be delivered using, for example, the Sonitron 2000 system (Rich-Mar).

Another aspect herein provides an isolated cell (eg, a host cell) comprising a ZFP, ZFN, polynucleotide or vector described herein.

Suitable cells include, but are not limited to, eukaryotic and prokaryotic cells and/or cell lines. Non-limiting examples of such cells or cell lines include: COS, CHO (e.g., CHO-S, CHO-K1, CHO-DG44, CHO-DUXB11, CHO-DUKX, CHOK1SV), VERO, MDCK, WI38, V79, B14AF28-G3, BHK, HaK, NS0, SP2/0-Ag14, HeLa, HEK293 (e.g., HEK293-F, HEK293-H, HEK293-T) and perC6 cells and insect cells such as fall armyworm (Sf) or fungal cells such as saccharomyces cerevisiae, pichia pastoris and fission yeast. In some embodiments, the cell is a mammalian cell. In a specific embodiment, the cell is a Chinese hamster ovary cell (CHO cell). Progeny, variants and derivatives of these cell lines can also be used.

Another aspect herein provides a cell line in which the FUT8 gene is inactivated or partially inactivated, comprising a ZFP, ZFN, polynucleotide or vector described herein.

Also provided herein is a cell line in which the FUT8 gene is inactivated or partially inactivated, which is generated by using the ZFP, ZFN, polynucleotide and/or vector described herein.

The present invention also provides a FUT8-deficient cell line, which comprises a nucleotide sequence as shown in one or more of SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58 and SEQ ID NO: 59 at the FUT8 locus.

In some embodiments, the FUT8-deficient cell line comprises the nucleotide sequences set forth in SEQ ID NO: 52 and SEQ ID NO: 53 at the FUT8 locus. In some embodiments, the FUT8-deficient cell line comprises the nucleotide sequences set forth in SEQ ID NO: 54 and SEQ ID NO: 55 at the FUT8 locus. In some embodiments, the FUT8-deficient cell line comprises the nucleotide sequences set forth in SEQ ID NO: 56 and SEQ ID NO: 57 at the FUT8 locus. In some embodiments, the FUT8-deficient cell line comprises the nucleotide sequences set forth in SEQ ID NO: 58 and SEQ ID NO: 59 at the FUT8 locus.

Also provided herein is a FUT8-deficient cell line comprising an altered FUT8 locus, wherein the altered FUT8 locus can be detected by the following primer pair:

Primer pair (i):

Forward primer: AGCCTGAAGTACATAGCCGA (SEQ ID NO: 46); and

Reverse primer: TGCCACTGCTTCTATATACTGATTC (SEQ ID NO: 47);

and/or

Primer pair (ii):

Forward primer: GACGCACTGACAAAGTGGGA (SEQ ID NO: 48); and

Reverse primer: GGTCTGTTCCATCCCCAGAATG (SEQ ID NO: 49);

and/or

Primer pair (iii):

Forward primer: CTGTTGATTCCAGGTTCCCA (SEQ ID NO: 50); and

Reverse primer: TGTTACTTAAGCCCCAGGC (SEQ ID NO: 51).

In some embodiments, the detection uses an equivalent cell line with normal (non-defective) FUT8 as a control cell line. In some embodiments, the detection shows a difference in results between the FUT8-deficient cell line and the control cell line (e.g., a difference in molecular size and band brightness measured by electrophoresis, etc.).

D. Application

The ZFNs described herein can be used to inactivate the FUT8 gene in a cell. In some embodiments, inactivation comprises partial or complete inhibition of expression of the FUT8 gene in a cell. Inactivation of the FUT8 gene can be achieved, for example, by a single cleavage event, by cleavage followed by non-homologous end joining, by cleavage at two sites followed by joining to delete the sequence between the two cleavage sites, by targeted recombination of missense or nonsense codons to the coding region, by targeted recombination of an unrelated sequence (i.e., a "stuffer" sequence) to a gene or its regulatory region to disrupt a gene or regulatory region, or by targeted recombination of a splice acceptor sequence to an intron to cause mis-splicing of the transcript.

The ZFN-mediated inactivation (knockout or inhibition) described herein has a variety of applications.

Another aspect herein provides a method of producing a FUT8-deficient cell, the method comprising:

(a) introducing a polynucleotide encoding one or more ZFNs into a cell, wherein the ZFN comprises: (i) a ZFP as described herein, which is capable of binding to a target site in a FUT8 gene in the cell; and (ii) a DNA cleavage domain or cleavage half-domain;

(b) expressing the ZFN in the cell, so that the ZFN binds to the target site and cleaves the FUT8 gene.

In some embodiments, the one or more ZFNs include a ZFN pair described herein.

In some embodiments, the method may further include introducing an additional nuclease having a target site in the FUT8 gene into the cell. Examples of the additional nuclease may include, for example, homing endonucleases and meganucleases.

Another aspect herein provides a method of generating a FUT8-deficient cell line, the method comprising:

(a) inactivating the endogenous FUT8 gene in a cell by the method for producing a FUT8-deficient cell as described herein; and

(b) culturing the cells under conditions suitable for generating a FUT8-deficient cell line.

Exemplary homing endonucleases include I-Scel, I-Ceul, PI-PspI, PI-Sce, I-SceIV, I-Csml, I-Panl, I-Scel, I-Ppol, I-Scelll, I-Crel, I-Tevl, I-Tevll, and I-TevIII. Their recognition sequences are known. See also U.S. Pat. No. 5,420,032; U.S. Pat. No. 6,833,252; Belfort et al. (1997) Nucleic Acids Res. 25:3379-3388; Dujon et al. (1989) Gene 82:115-118; Perler et al. (1994) Nucleic Acids Res. 22, 1125-1127; Jasin (1996) Trends Genet. 12:224-228; Gimble et al. (1996) J Mol. Biol. 263:163-180; Argast et al. (1998) J. Mol. Biol. 280:345-353 and the New England Biolabs catalog.

Although the cleavage specificity of most homing endonucleases is not absolute with respect to their recognition sites, these sites are of sufficient length to enable a single cleavage event per mammalian-sized genome to be obtained by expressing the homing endonuclease in cells containing a single copy of its recognition site. It has also been reported that the specificity of homing endonucleases and meganucleases can be engineered to bind to non-natural target sites. See, e.g., Chevalier et al. (2002) Molec. Cell 10:895-905; Epinat et al. (2003) Nucleic Acids Res. 31:2952-2962; Ashworth et al. (2006) Nature 441:656-659; Paques et al. (2007) Current Gene Therapy 7:49-66.

Also provided herein are FUT8-deficient cell lines produced by the methods of generating FUT8-deficient cell lines described herein.

Another aspect of the present invention provides a method for cultivating a FUT8-deficient cell line, comprising:

(a) providing a FUT8-deficient cell line obtained as described herein; and

(b) culturing or expanding cells of said cell line.

Another aspect of the present invention provides a method for producing a target recombinant protein in a host cell, the method comprising:

(a) providing a FUT8-deficient cell as described herein,

(b) introducing an expression vector comprising a nucleic acid encoding the target recombinant protein into the cell, thereby producing the target recombinant protein.

Another aspect of the present invention provides a method for producing a target recombinant protein in a host cell, the method comprising:

(a) providing a cell comprising an endogenous FUT8 gene;

(b) inactivating the endogenous FUT8 gene in the cell by the method for producing a FUT8-deficient cell as described herein; and

(c) introducing an expression vector comprising a nucleic acid encoding the target recombinant protein into the cell, thereby producing the target recombinant protein.

In some embodiments, the step of inactivating the endogenous FUT8 gene in the cell as described herein and the step of introducing an expression vector comprising a nucleic acid encoding the target recombinant protein into the cell may be performed in reverse order or simultaneously.

Suitable cells include, but are not limited to, eukaryotic and prokaryotic cells and/or cell lines. Non-limiting examples of such cells or cell lines include: COS, CHO (e.g., CHO-S, CHO-K1, CHO-DG44, CHO-DUXB11, CHO-DUKX, CHOK1SV), VERO, MDCK, WI38, V79, B14AF28-G3, BHK, HaK, NS0, SP2/0-Ag14, HeLa, HEK293 (e.g., HEK293-F, HEK293-H, HEK293-T) and perC6 cells and insect cells such as fall armyworm (Sf) or fungal cells such as saccharomyces cerevisiae, pichia pastoris and fission yeast. In some embodiments, the cell is a mammalian cell. In a specific embodiment, the cell is a Chinese hamster ovary cell (CHO cell). Progeny, variants and derivatives of these cell lines may also be used. In some embodiments, the cell is a mammalian cell. In a specific embodiment, the cell is a Chinese hamster ovary cell (CHO cell).

In some embodiments, the target recombinant protein includes an Fc-containing recombinant protein. In some embodiments, the target recombinant protein is a defucosylated Fc-containing recombinant protein.

Another aspect herein provides the use of the ZFPs, ZFNs, polynucleotides, vectors described herein for generating FUT8-deficient cells.

Another aspect herein provides use of a ZFP, ZFN, polynucleotide, vector, cell or cell line described herein for producing a defucosylated Fc-containing recombinant protein.

Another aspect provided herein is the use of a ZFP, ZFN, polynucleotide, vector, cell or cell line described herein for producing an antibody (eg, a monoclonal antibody) capable of eliciting an enhanced ADCC effect.

In some embodiments, the Fc-containing recombinant protein can be an antibody (eg, monoclonal antibody, polyclonal antibody) or an Fc fusion protein. The defucosylated Fc-containing recombinant protein provided by the method described herein exhibits stronger effector function, particularly in the induction of ADCC.

Example

The technical scheme of the present invention will be described in more detail below in conjunction with specific embodiments. The following embodiments are only examples and do not constitute any limitation or restriction on the technical scheme of the present invention. The specific materials, steps, conditions, values or numerical ranges and other technical parameters in the following embodiments are only examples and are not exhaustive or limiting.

In addition to the specific methods, equipment, and materials used in the embodiments, based on the understanding of the prior art by those skilled in the art and the description of the present invention, any methods, equipment, and materials of the prior art that are similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention may also be used to implement the present invention.

The equipment and reagents used in the examples are listed in Table 2 and Table 3, respectively.

Table 2: Experimental equipment list

Table 3: List of experimental reagents

Example 1 Application of FUT8 ^-/- cell line in developing stable expression of adalimumab monoclonal antibody molecules

1 Expression vector construction

1.1 As shown in Figure 2A, ZFP proteins L3/R3 and S2-L/S4-R are each grouped and constructed on expression vectors, and transcription is initiated by CMV as a promoter. ZFP protein is connected to the downstream FokI cleavage half domain as a DNA binding domain to form a complete protein, which is expressed through the same ORF. The expression vector does not pass the resistance screening after transfection into CHO cells, so no eukaryotic cell screening marker is inserted in the plasmid construction. Figure 2B shows an exemplary sequence structure, in which 5-6 zinc finger recognition regions are sequentially inserted into the natural zinc finger protein framework sequence (such as ZIF268 or SP1C) and connected to the FokI cleavage half domain at the C-terminus. The full-length sequences of S2-L-FokI and S4-R-FokI used in this example are shown as SEQ ID NO: 60 and SEQ ID NO: 61. The other ZFP proteins used are constructed in a similar manner to construct the corresponding zinc finger recognition regions into the same framework sequence and connected to FokI.

1.2 The HC sequence of Adalimumab (SEQ ID NO: 4) was constructed into the expression vector pWX039-HC-Z-105 and connected to the Zeocin resistance gene through EMCV IRES. The LC sequence of Adalimumab (SEQ ID NO: 5) was constructed into the expression vector pWX040-LC-B-105 and placed upstream of the Blasticidin resistance gene. The LC and resistance gene elements belong to two independent reading frames. The Blasticidin resistance gene is driven by the SV40 promoter.

2. Cell transfection and cell pool screening

2.1 Take 1E7 CHO-K1 host cell suspension, take 30μg plasmid and mix it evenly in 250μL DPBS solution, mix the plasmid solution and cell suspension thoroughly and add them to the electroporation cup. Place the electroporation cup in the card slot of the Biorad electroporator and perform electroporation according to the corresponding program of the host cell. After transfection, aspirate the cell suspension from the electroporation cup and transfer it to a T25 cell culture bottle with 7.5mL preheated pressure-free culture medium, and place it in a carbon dioxide incubator at 36.5℃, 5% _CO2 , and 85% humidity for static culture.

2.2 After 48 hours of transfection, the T25 culture flask was transferred to a carbon dioxide incubator at 36.5℃. After 24 hours of static culture, the cell pool was sampled and counted to extract 1E6 cells for genomic DNA extraction and quantified using a micro-UV spectrophotometer. 100ng~200ng genomic DNA was selected and high-fidelity DNA polymerase was used to select appropriate primers (primer sequences: primer 2F: CTGTTGATTCCAGGTTCCCA (SEQ ID NO 50); primer R: TGTTACTTAAGCCCCAGGC (SEQ ID NO 51)) to PCR amplify the target gene sequence, and then the system was re-denatured and annealed. After annealing, 1μL T7E1 enzyme (10U/μL) was added to the system and incubated at 37℃ for 15 minutes. After enzyme digestion, 2% agarose gel electrophoresis was performed to detect the enzyme digestion bands and editing efficiency.

3. Clone Screening

The cell pool with a viability of more than 90% was sorted by limiting dilution method for monoclonal selection. After the cells were cultured in 96-well plates for about 2 weeks, the clones were transferred to 24-well plates and cultured for about 2-3 days before being expanded to shake tubes for culture. At the same time, the sequence of FUT8 gene in the cell line was checked by NGS method. The cells were subcultured every 2-4 days in shake tubes under the culture conditions of 36.5°C, 6% CO ₂ , 85% humidity, and 225 rpm.

4. Application of target cell lines in expressing monoclonal antibody molecules

4.1 After the growth of the target cell strain to be screened (monoclonal 154-F8ZFN-09-08 and monoclonal 154-F8ZFN-10-02 were selected in this example), the target cell strain and the cell strain without knockout of FUT8 gene (negative control group) were used as host cells, and 1E7 cells and 20ug of adalimumab monoclonal antibody HC and LC plasmids were taken. The cells and plasmid solution were mixed evenly and transferred to an electroporation cup, and the Biorad electroporator was used to select appropriate electroporation parameters according to the corresponding cell type for electroporation. After electroporation, all cells were transferred to a shaking tube filled with preheated culture medium and placed in a shaking incubator for culture. The culture conditions were 36.5°C, 6% CO ₂ , 85% humidity, and a rotation speed of 225rpm.

4.2 24 hours after transfection, add screening stress medium and continue culturing, subculturing every 2-4 days.

4.3 After 3-4 weeks of continuous subculturing, when the viability of the cell pool recovers to more than 95%, it is inoculated into the production medium at a cell density of 4E5/mL for fed-batch culture. Samples are taken on the 3rd, 5th, 7th, 9th and 11th days of culture to detect the density and viability of live cells, and feed and sugar are supplemented. After 14 days of culture, the supernatant is harvested by centrifugation, and the titer level of the monoclonal antibody in the supernatant is detected by the ProA-HPLC method. In addition, the harvested supernatant samples are further purified by proA and then subjected to SEC-HPLC high polymer content detection, SDS Caliper and N-glycan-LC glycoform analysis. In this embodiment, the negative control group is a host cell in which the FUT8 gene has not been knocked out and the monoclonal antibody vector is transfected.

5 Experimental results

The results of the enzyme digestion experiment successfully verified that the ZFN plasmid can effectively remove the target gene sequence (Figure 3). The cell pool was analyzed by flow cytometry (Figure 4), and a certain proportion of FUT8 ^-/- cell populations were found. Through the clone screening process, clones 154-F8ZFN-09-08 and 154-F8ZFN-10-02 were successfully verified to have a pair of alleles knocked out (Figures 5 and 6). As can be seen from Figures 7A and 7B, the live cell density and peak VCD of 154-F8ZFN-09-08 and 154-F8ZFN-10-02 during FB culture were comparable to those of the control group; Figure 7C showed that there was no significant difference in the monoclonal antibody expression titer of the 154-F8ZFN-09-08 group and the control group. And the results of SEC-HPLC and Caliper R/NR showed that there was no significant difference in the high polymer level of the one-step purified samples of the cell pool of the 154-F8ZFN-09-08 and 154-F8ZFN-10-02 groups and the control group (Figure 7D, 7E). Finally, the glycoform results (Figure 7F) showed that the fucose level of the 154-F8ZFN-09-08 and 154-F8ZFN-10-02 groups was much lower than that of the negative control group in which the FUT8 gene was not knocked out, and dropped to a level close to zero.

Example 2 Application of FUT8 ^-/- cell line in developing stable expression of fusion protein molecule Dulaglutide

1 Expression vector construction

Dulaglutide is a glucagon-like peptide 1 (GLP-1) Fc fusion protein.

The sequence of dulaglutide (SEQ ID NO 6) was constructed into the expression vectors pWX039-Pr-Z-126A3 and pWX040-Pr-B-126A3. In the pWX039-Pr-Z-126A3 vector, the sequence of dulaglutide was connected by EMCV IRES and Zeocin resistance gene. In pWX040-Pr-B-126A3, the sequence of dulaglutide was placed upstream of the blasticidin resistance gene, and the target gene and resistance gene elements belonged to two independent reading frames. The expression of blasticidin resistance gene was driven by SV40 promoter.

2 Cell transfection and fed-batch culture

2.1 The 154-F8ZFN-09-08 selected in Example 1 was used as a host cell, 1E7 cells and 20 μg of the expression vector were mixed evenly and then transferred to an electroporation cup, and electroporated using a Biorad electroporator with appropriate electroporation parameters selected according to the corresponding cell type. After electroporation, all cells were transferred to a shaking tube filled with preheated culture medium and cultured in a shaking incubator at 36.5°C, 6% CO ₂ , 85% humidity, and a rotation speed of 225 rpm.

2.2 24 hours after transfection, add an equal volume of screening stress medium and continue culturing, subculturing every 2-4 days.

2.3 After 3-4 weeks of continuous subculturing, when the viability of the cell pool recovers to more than 95%, it is inoculated into the production culture medium at a cell density of 4E5/mL for fed-batch culture. Samples are taken on the 3rd, 5th, 7th, 9th and 11th days of culture to detect the density and viability of live cells, and feed and sugar are supplemented. After 14 days of culture, the supernatant is harvested by centrifugation, and the titer level of the monoclonal antibody in the supernatant is detected by the ProA-HPLC method. In addition, the harvested supernatant samples are further purified by proA and then subjected to SEC HPLC high polymer content detection, SDS CaliperNR purity detection and N-glycan glycotype analysis. In this embodiment, the positive control group is a host cell in which the FUT8 gene is knocked out and transfected with a fusion protein vector.

3 Experimental results

Figure 8A shows that the live cell density and viability data of the 154-F8ZFN-09-08 group during FB culture are within the acceptable range; Figures 8C, D, and E show that the protein expression titer, SEC-HPLC main peak, and SDS CaliperNR main peak of the 154-F8ZFN-09-08 group are not much different from those of the control group. Finally, the glycoform results (Figure 8E) show that the fucose level of the 154-F8ZFN-09-08 group is much lower than that of the negative control group in which the FUT8 gene has not been knocked out, and has dropped to a level close to zero.

Example 3 ZFN editing at exon 3

1. Expression vector construction

1.1 As shown in FIG. 2A , similarly to Example 1, the ZFP proteins 31-L1/31-R1 and 32-L1/32-R1 were each grouped and constructed on expression vectors.

2. Cell transfection and cell population (library) screening

2.2 1×10 ⁷ CHO-K1 cells were mixed evenly with 32-L1/32-R1 and 31-L1/31-R1 plasmids and transfected in a Bio-rad electroporator using program WE. After transfection, the cells were recovered in 5 mL CD CHO medium and cultured in a static incubator at 30°C.

2.3 On the second day after transfection, cells were collected, genomic DNA was prepared, and primers Fut8-EX3-F1 (SEQ ID NO: 46) and Fut8-EX3-R1 (SEQ ID NO: 47) were used to amplify the targeted region of the FUT8 gene; finally, the gene editing efficiency was detected using the T7E1 kit. As shown in lane 2 of Figure 9, according to the calculation of the band brightness by ImageJ, 32-L1/32-R1 modified the FUT8 exon 3 region, resulting in 10.9% DNA mutation; as shown in lane 1, 31-L1/31-R1 modified the FUT8 exon 3 region, resulting in 9.4% DNA mutation.

3 Monoclonal screening

On the 34th day after transfection, SCP monoclonal sorting was performed. All grown monoclones were stained with LCA-FITC, and finally two FUT8 ^-/- phenotype monoclones C6 and F2 were obtained (Figures 10 and 11). Through sequence verification, the sequencing results of these two clones are shown in Figure 12, and both alleles were knocked out.

Example 4 The ADCC activity of the antibody expressed by the FUT8 ^−/− clone knocked out by ZFN was significantly improved

1. Protein Expression

The FUT8 ^-/- double knockout clone (154-F8ZFN-09-08) obtained according to the method described herein was used as a host cell, and wild-type CHO cells without mutation were used as a control. Adalimumab was expressed and purified according to the method described above.

2. Target Cell Labeling

HT1080 cells expressing TNFα were first labeled with DELFIA BATDA (Cat# C136-100, Perkin Elmer, China) at 37°C in a humidified incubator with 5% _CO2 for 30-40 minutes. After incubation, HT1080 cells were centrifuged and washed with assay medium and then resuspended in assay medium (RPMI 1640 + 10% HI-FBS).

3. Co-incubation of target cells and effector cells

Peripheral blood mononuclear cells (PBMC) were resuspended in assay medium. 1×10 ⁶ PBMCs were thoroughly mixed with 1×10 ⁴ HT1080 cells expressing TNFα, and 0 to 50 ng/mL anti-TNFα therapeutic antibody samples were added. The samples were co-cultured in a 96-well plate at 200 μL/well in a 37°C, 5% CO ₂ incubator for 1 to 2 hours.

4. Supernatant reaction

After centrifugation of the 96-well plate at 500 × g for 3–5 min, 20 μL of supernatant was removed from each well and transferred to the corresponding well in a 96-well white microplate (3912, Corning, USA). Subsequently, 200 μL of DELFIA Europium solution (Cat# C135-100, Perkin Elmer, China) was added to each well, and the plate was incubated for 20–30 min.

5. Data processing

Data was collected on a microplate reader M5e (Molecular Devices, California, USA). Relative fluorescence units (RFU) were plotted against antibody concentration to generate a 4-parameter logistic response curve. When all system and sample suitability were met, the relative potency of the sample was calculated by the EC50 ratio. As shown in Figure 13, the ADCC activity of adalimumab produced by the FUT8 ^-/- double knockout host was significantly higher than that of the same protein produced by the wild-type CHO host in the control group.

Claims (18)

A zinc finger protein capable of targeting and binding to the FUT8 gene, comprising the amino acid sequences of the zinc finger recognition regions of zinc fingers F1 to F5 or to F6 shown in the same row in the following table in order from N to C terminus:
A zinc finger nuclease comprising the zinc finger protein according to claim 1 and at least one DNA cleavage domain or cleavage half-domain. A polynucleotide encoding the zinc finger protein according to claim 1. A polynucleotide encoding the zinc finger nuclease according to claim 2. A vector comprising the polynucleotide according to claim 3 or 4. An isolated cell comprising the zinc finger protein according to claim 1, the zinc finger nuclease according to claim 2, the polynucleotide according to claim 3 or 4, or the vector according to claim 5. A method for generating a FUT8-deficient cell, the method comprising: (a) introducing a polynucleotide encoding one or more zinc finger nucleases according to claim 2 into a cell; (b) expressing the zinc finger nuclease in the cell, so that the zinc finger nuclease binds to the target site and cleaves the FUT8 gene. The method according to claim 7, wherein, in step (a), a zinc finger nuclease pair comprising a left zinc finger nuclease and a right zinc finger nuclease is introduced into the cell, wherein the zinc finger nuclease pair comprises: Zinc finger nuclease pair (a): Left zinc finger nuclease having the following zinc finger recognition region amino acid sequence: F1: QLATLNR (SEQ ID NO: 13); F2: TRWNLRA (SEQ ID NO: 14); F3: SRRDLRR (SEQ ID NO: 15); F4: WRRRLLS(SEQ ID NO: 16); F5:RSDYLTN (SEQ ID NO:17); and F6: FHSNLLA (SEQ ID NO: 18); and A right zinc finger nuclease having the following zinc finger recognition region amino acid sequence: F1: RKSHLTM (SEQ ID NO: 19); F2: FHSGLLA (SEQ ID NO: 20); F3: WRRRLLS(SEQ ID NO: 16); F4: RKYVLLR (SEQ ID NO: 21); F5: RKDYLVL (SEQ ID NO: 22); and F6: QQAGLIN(SEQ ID NO: 23); and/or Zinc finger nuclease pair (b): Left zinc finger nuclease having the following zinc finger recognition region amino acid sequence: F1:SKWNLRS(SEQ ID NO:24); F2: AQSNLLS(SEQ ID NO: 25); F3: LRHQLRR (SEQ ID NO: 26); F4:RSDYLTN (SEQ ID NO:17); and F5: RSDYLTN (SEQ ID NO: 17); and A right zinc finger nuclease having the following zinc finger recognition region amino acid sequence: RSDYLTN (SEQ ID NO: 17); QKITLVR (SEQ ID NO: 27); RSDYLTN (SEQ ID NO: 17); QLATLNR (SEQ ID NO: 13); SRFNLTR (SEQ ID NO: 28); and TKYILTN (SEQ ID NO: 29); and/or Zinc finger nuclease pair (c): Left zinc finger nuclease having the following zinc finger recognition region amino acid sequence: F1: RADNLTE(SEQ ID NO: 30); F2: TSGNLTE (SEQ ID NO: 31); F3: TSGHLVR (SEQ ID NO: 32); F4: RKDNLKN (SEQ ID NO: 33); and F5: RKDNLKN (SEQ ID NO: 33); and A right zinc finger nuclease having the following zinc finger recognition region amino acid sequence: RSDNLSV (SEQ ID NO: 34); SPADLTR (SEQ ID NO: 35); RSDHLSQ (SEQ ID NO: 36); QSGDLRR (SEQ ID NO: 37); RSDNLVR (SEQ ID NO: 38); and RKDNLKN (SEQ ID NO: 33); and/or Zinc finger nuclease pair (d): Left zinc finger nuclease having the following zinc finger recognition region amino acid sequence: F1: DRSNLLS(SEQ ID NO: 39); F2: NQSNLLR (SEQ ID NO: 40); F3: FHSNLLA (SEQ ID NO: 18); F4: QLSTLNY(SEQ ID NO: 41); and F5: QSGNLSR (SEQ ID NO: 42); or A right zinc finger nuclease having the following zinc finger recognition region amino acid sequence: FHSGLLA (SEQ ID NO: 20); HPSTLSK (SEQ ID NO: 43); ARWTLDC (SEQ ID NO: 44); HPSTLSK (SEQ ID NO: 43); HPSTLSK (SEQ ID NO: 43); and RKFTLTN (SEQ ID NO: 45). A method for generating a FUT8-deficient cell line, the method comprising: (a) inactivating the FUT8 gene in a cell by the method according to claim 7; and (b) culturing the cells under conditions suitable for generating a FUT8-deficient cell line; Specifically, the cell is a mammalian cell; more specifically, the cell is a CHO cell. A method for producing a target recombinant protein in a host cell, the method comprising: (a) providing the cell according to claim 6, wherein the cell is a FUT8-deficient cell, (b) introducing an expression vector comprising a nucleic acid encoding the target recombinant protein into the cell, thereby producing the target recombinant protein; Specifically, the cell is a mammalian cell; more specifically, the cell is a CHO cell. A method for producing a target recombinant protein in a host cell, the method comprising: (a) providing a cell comprising an endogenous FUT8 gene; (b) inactivating the endogenous FUT8 gene in the cell by the method according to claim 7; and (c) introducing an expression vector comprising a nucleic acid encoding the target recombinant protein into the cell, thereby producing the target recombinant protein; More specifically, step (b) is performed before, simultaneously with or after step (c). Use of the zinc finger protein according to claim 1, the zinc finger nuclease according to claim 2, the polynucleotide according to claim 3 or 4, or the vector according to claim 5 for producing FUT8-deficient cells. Use of the zinc finger protein according to claim 1, the zinc finger nuclease according to claim 2, the polynucleotide according to claim 3 or 4, the vector according to claim 5 or the cell according to claim 6 for producing a defucosylated Fc-containing recombinant protein. Use of the zinc finger protein according to claim 1, the zinc finger nuclease according to claim 2, the polynucleotide according to claim 3 or 4, the vector according to claim 5 or the cell according to claim 6 for producing an antibody (such as a monoclonal antibody) that can induce an enhanced ADCC effect. A FUT8-deficient cell line prepared according to the method of claim 9. A FUT8-deficient cell line, which comprises a nucleotide sequence as shown in one or more of SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58 and SEQ ID NO: 59 at the FUT8 locus. A FUT8-deficient cell line comprising an altered FUT8 locus, wherein the altered FUT8 locus can be detected by the following primer pair: Primer pair (i): Forward primer: AGCCTGAAGTACATAGCCGA (SEQ ID NO: 46); and Reverse primer: TGCCACTGCTTCTATATACTGATTC (SEQ ID NO: 47); and/or Primer pair (ii): Forward primer: GACGCACTGACAAAGTGGGA (SEQ ID NO: 48); and Reverse primer: GGTCTGTTCCATCCCCAGAATG (SEQ ID NO: 49); and/or Primer pair (iii): Forward primer: CTGTTGATTCCAGGTTCCCA (SEQ ID NO: 50); and Reverse primer: TGTTACTTAAGCCCCAGGC (SEQ ID NO: 51). A method for cultivating a FUT8-deficient cell line, comprising: (a) providing a FUT8-deficient cell line according to any one of claims 15 to 17; and (b) culturing or expanding cells of said cell line.