WO2005116060A1

WO2005116060A1 - Polypeptide increasing the monoclonal antibody production, nucleic acid encoding same and use thereof

Info

Publication number: WO2005116060A1
Application number: PCT/KR2005/001541
Authority: WO
Inventors: Jin-Soo Kim; Ryuk Jun Kwon; Wongi Seol
Original assignee: Toolgen, Inc.
Priority date: 2004-05-25
Filing date: 2005-05-25
Publication date: 2005-12-08
Also published as: KR20070030203A; KR100857125B1

Abstract

Disclosed are a polypeptide capable of increasing the monoclonal antibody production in a eukaryotic cell, a nucleic acid encoding same and a eukaryotic cell transformed with said nucleic acid. The polypeptide and the nucleic acid can be used to increase the production of a monoclonal antibody useful as a medicament for treating various diseases, in a eukaryotic cell.

Description

POLYPEPTIDE INCREASING THE MONOCLONAL ANTIBODY PRODUCTION, NUCLEIC ACID ENCODING SAME AND USE THEREOF

Field of the Invention

The present invention relates to a polypeptide increasing the monoclonal antibody production in a eukaryotic cell, a nucleic acid encoding same and a eukaryotic cell transformed with the polypeptide or nucleic acid.

Background of the Invention

Since the first production of the human growth hormone by DNA recombination technology in early 1980's, numerous recombinant proteins have been developed and used for treating various diseases. Further, herceptin, a drug for breast cancer approved by FDA (USA) in 1998, has not only established that monoclonal antibody can be effective as a drug, but it also expanded the conventional concept for drug. Thereafter, drugs using monoclonal antibody have been developed for treating cancers and other diseases. Such protein drugs, specifically, monoclonal antibody drugs have diversified with the progress in the field of biology. Except for a few, e.g. growth hormone, these drugs have to be produced in an animal cell to keep treatment titer at certain level because the protein must go through post-translational modification such as proteolytic cleavage,_" phosphorylation and glycosylation on specific amino acid to keep in vivo activity which is impossible or inefficient in bacteria or yeast. With the increase in the number of clinical tests of monoclonal antibodies allowed as useful drugs, demand for mass culture facilities for the protein drugs production has risen explosively (see Dove A, Nature Biotech. (2001) 19:117-120). The solution for the problem is the expansion of culture facilities and increase of yield rate using present facilities (see Fussenegger M, et al, Trends Biotechnol. (1999) 17:35-42). There have been two approaches taken to increase yield rate, one study being related increasing unit production in one cell (Lim L.H. et al., Biochem. Biophys. Res. Commun. (2004) 316:991-996) while the other study being related to optimizing the condition of a growth medium to make more cells grow in identical volume (Lee G. M. et al., J. Biotechnol. (1999) 69:85-93):" The followings exemplify the former approach: a reconstructing vector for the production of target proteins so that it has optimal and maximal productivity (Ted, H. J. et ah, Nature Biotech. (2003) 21:553-558); developing methods for the fast screening of a clone having high productivity after gene amplification (Charles, G. et al, Biotechnol.&Bioeng. (2002) 80:670-676); and controlling proliferation of a clone having high productivity or preventing its apoptosis (Lai, D. et al, Biotechnol.&Bioeng. (2004) 85:20-28). These attempts aim at increasing gene expression of target protein. Cells being used for producing recombinant protein are a CHO cell from Chinese Hamster Ovary, a NS-O as a hybridoma cell of a mouse and a Per.C6 as a human cell, and a CHO cell is the most popular for the production of proteins^' for medical treatment. The production of a recombinant protein in a CHO cell is maximized by gene amplification using dihydrofolate reductase (DHFR) and methotrexate (MTX) (Kim, S. J., et al, Biotechnol.&Bioeng. (1998) 58:73-84). Aforementioned attempts are about increasing the number of mRNA of genes by amplifying the number of genes in a cell. However, protein production will not be enhanced as much unless the capacity of the intracellular systems working at post-transcriptional process such as translation, post- translational modification and secretion are increased as much as mRNA. Actually, the increase in protein production reaches its plateau once the number of genes is increased more than some point (Schroder, M. et al, Biotechnol.&Bioeng. (1997) 53:547-559). Therefore, if this bottleneck problem is resolved by increasing the number of proteins involved in the critical step of post-transcriptional process, notable increase in the protein production can be achieved. The present invention is to correlate transcriptional regulatory technique of a gene with increase of the intracellular protein production using an artificial transcription factor made of zinc finger. There are many reports showing that increase or decrease in the specific protein expression is caused by the use of a zinc finger (Zhang L, et al, J Biol Chem. (2000) 275:33850-33860; Liu PQ, et al, JBiol Chem. (2001) 276:11323-11334; Beerli RR, et al, Proc Natl Acad Sci USA. (2000) 97:1495-1500; Beerli RR, et al, JBiol Chem. (2000) 275:32617- 32627; and Bae K.-H. et al, Nature Biotech. (2003) 21:275-280). However, these examples were intended to regulate the transcription of a gene using a zinc finger binding to a promoter of specific gene. The present inventors have endeavored to develop, a method for increasing the production of a specific protein by regulating an unspecified gene using a library of artificial transcription factors having a random array of zinc finger domains (ZFDs).

Summary of the Invention

Accordingly, it is an object of the present invention to provide a polypeptide increasing the monoclonal antibody production in a eukaryotic cell and a nucleic acid encoding same. It is another object of the present invention to provide a eukaryotic cell transformed with the polypeptide or nucleic acid. It is further object of the present invention to provide a method for increasing the monoclonal antibody production in the eukaryotic cell. In accordance with one aspect of the present invention, there is provided a polypeptide capable of increasing the amount of a monoclonal antibody produced by a eukaryotic cell relative to a cell that does not include the polypeptide, wherein the polypeptide comprises a DNA binding domain that includes a plurality of zinc finger domains and a nucleic acid encoding same. In accordance with another aspect of the present invention, there is provided a eukaryotic cell containing a gene encoding a monoclonal antibody and the nucleic acid comprising a sequence encoding the polypeptide. In accordance with further aspect of the present invention, there is provided a method for increasing the monoclonal antibody production in a eukaryotic cell comprising the step of introducing the polypeptide or the nucleic acid into the cell.

Brief Description of the Drawings

The above and other objects and features of the present invention will become apparent from the following description of the invention taken in conjunction with the following accompanying drawings, which respectively show:

Fig. 1: a schematic diagram of the procedure of preparing pLNCXl, pLNCX2 and pLNCX2-(ZFD)₄-Kid library; Fig. 2: a schematic diagram of screening a zinc finger protein capable of increasing monoclonal antibody production in a mammal, including the procedures of virus production and enzyme-linked immunosorbent assay (ELISA); Fig. 3: a graph showing increase of the monoclonal antibody production in AKA cell by LK33-Kid, LK35-Kid, LK50-KM and LK52-Kid, as compared with that in the control cells (LNCX2-Kid and LNCX2); Fig. 4: a graph showing increase of the monoclonal antibody production in AKA cell by LK52-Kid and its mutant (mLK52-Kid) according to the culture period, as compared with that in the control cell (LNCX2-Kid); Fig. 5: a sequence of a plasmid p3 including one ZFD; Fig. 6: a sequence of a pLNCX2-Kid; Fig. 7: a sequence of pLNCX2-(ZFD)₄-Kid, wherein (Apal/Claϊ) represents a deleted restriction enzyme region, and FI to F4 represent a zinc finger domain, respectively. Fig. 8: a schematic diagram of the procedure of mutant insertion to LK52 in pLNCX2-LK52-Kid, wherein "* vertical line" represents a position of a mutation; and Fig. 9: a graph of quantitative analysis by RT-PCR of mRNA of a monoclonal antibody in AKA cell having Zinc Finger Proteins (ZFPs). Detailed Description of the Invention

A polypeptide of the present invention, which is capable of increasing the amount of a monoclonal antibody produced by a eukaryotic cell, preferably, a mammalian cell, relative to a cell that does not include the polypeptide, comprises a DNA binding domain that includes a plurality of zinc finger domains (ZFDs). ZFDs are small polypeptide domains of approximately 30 amino acid residues in which there are four amino acids, either cysteine or histidine, appropriately spaced such that they can coordinate a zinc ion (For reviews, see, e.g., Klug and Rhodes, (1987) Trends Biochem. Sci.12:464-469(1987); Evans and Hollenberg, (1988) Cell 52:1-3; Payre and Vincent, (1988) FEBS Lett. 234:245-250; Miller et al, (1985) EMBO J. 4:1609-1614; Berg, (1988) Proc. Natl. Acad. Sci. U.S.A. 85:99-102; Rosenfeld and Margalit, (1993) J. Biomol Struct. Dyn. 11 :557-570). Hence, zinc finger domains can be categorized according to the identity of the residues that coordinate the zinc ion, e.g., as the Cys₂-His₂ class, the Cys₂-Cys₂ class, the Cys₂-CysHis class, and so forth. The zinc coordinating residues of Cys₂-His₂ zinc fingers are typically spaced as follows: N^-X_a-X-C-X^-C-Xs-X_a-Xs-ψ-Xi-H-Xs-s-H-COOH, where ψ (psi) is a hydrophobic residue (Wolfe et al, (1999) Annu. Rev. Biophys. Biomol. Struct. 3:183-212), wherein "X" represents any amino acid, wherein X_a is phenylalanine or tyrosine, the subscript indicates the number of amino acids, and a subscript with two hyphenated numbers indicates a typical range of intervening amino acids. Typically, the intervening amino acids fold to form an anti-parallel β-sheet that packs against an α-helix, although the anti-parallel β-sheets can be short, non-ideal, or non-existent. The fold positions the zinc- coordinating side chains so they are in a tetrahedral conformation appropriate for coordinating the zinc ion. The base contacting residues are at the N- terminus of the finger and in the preceding loop region. For convenience, the primary DNA contacting residues of a zinc finger domain are numbered: -1, 2, 3, and 6 based on the following example:

-1 1 2 3 4 5 6 X_a-X-C-X₂_₅-C-X₃-X_a-X-C-X-S-N-X_b-X-R-H-X₃_₅-H (SEQ ID NO: 1), where X_a is phenylalanine or tyrosine, and X_b is a hydrophobic residue. As noted in the example above, the DNA contacting residues are Cys (C), Ser (S), Asn (N), and Arg (R). The above motif can be abbreviated CSNR. As used herein, such abbreviation refers to a particular polypeptide sequence. Where two sequences have the same motif, a number may be used to indicate the sequence. In certain contexts where made explicitly apparent, the four letter abbreviation refers to the motif in general. The term "zinc finger protein" refers to any protein that includes a zinc finger domain. A zinc finger protein typically includes a tandem array of at least two zinc finger domains. The zinc finger domain (or "ZFD") is one of the most common eukaryotic DNA-binding motifs, found in species from yeast to higher plants and to humans. By one estimate, there are at least several thousand zinc finger domains in the human genome alone, possibly at least 4,500. Zinc fmger domains can be isolated from zinc finger proteins. Non-limiting examples of zinc finger proteins include CF2-II, Kruppel, WT1, basonuclin, BCL-6/LAZ-3, erythroid Kruppel-like transcription factor, Spl, Sp2, Sp3, Sp4, transcriptional repressor YY1, EGRl/Krox24, EGR2/Krox20, EGR3/Pilot, EGR4/AT133, Evi- 1, GLI1, GLI2, GLI3, HIV-EP1/ZNF40, HIV-EP2, KR1, ZfX, ZfY, and ZNF7. Computational methods described below can be used to identify all zinc finger domains encoded in a sequenced genome or in a nucleic acid database.

Any such zinc finger domain can be utilized. In addition, artificial zinc finger domains have been designed, e.g., using computational methods (e.g., Dahiyat and Mayo, (1997) Science 278:82-7). It is also noteworthy that at least some zinc finger domains bind to ligands other than DNA, e.g., RNA or protein. Thus, a chimera of zinc finger domains or of a zinc finger domain and another type of domain can be used to recognize a variety of targets compounds, not just DNA. U.S. Patent Application Serial No. 60/374,355, titled "Zinc Finger

Domain Libraries," and filed April 22, 2002 describes exemplary zinc finger domains, which can be used to construct an artificial zinc finger protein. See also the Table 1. Preferably, the DNA binding domain comprises at least 2 ZFDs selected from the group consisting of the following amino acid sequences: CX₍₂.₅₎CXXXBXPvXXHJXTHX_(3-5)H (SEQ ID NO: 2); CX_(2-5)CXXXBXQXXHJXRHX_(3-5)H (SEQ ID NO: 3); CX_(2-5)CXXXBXQXXNJXKHX_(3-5)H (SEQ ID NO: 4); CX_(2-5)CXXXBXQXXSJXRHX₍₃_₅₎H (SEQ ID NO: 5); CX₍₂-₅₎CXXXBXQXXNJXRHX_(3-5)H (SEQ ID NO: 6); CX₍₂.₅₎CXXXBXQXXNJXIHX_(3-5)H (SEQ ID NO: 7); CX_(2-5)CXXXBXRXXKJXRHX_(3-5)H (SEQ ID NO: 8); CX_(2-5)CXXXBXQXXHJXVHX_(3-5)H (SEQ ID NO: 9); and CX_(2-5)CXXXBXQXXHJXQHX_(3-5)H (SEQ ID NO: 10), where B is phenylalanine or tyrosine; J is a hydrophobic amino acid; and

X is any amino acid. More preferably, the polypeptide of the present invention further comprises a domain selected from the group consisting of a transcription activation domain, a transcription repression domain, a protein transduction domain (PTD) and a combination thereof bound to the ZFD-including DNA binding domain. In the present invention, each zinc finger domain is named after its DNA contacting amino acid residues, i.e., amino acid residues at positions -1, 2, 3 and 6 along the alpha helix structure. For example, when the amino acid residues at positions -1, 2, 3 and 6 along the alpha helix structure are arginine (R), aspartic acid (D), histidine (H) and threonine (T), the zinc finger domain is represented as RDHT. The term "DNA contacting residue" as used herein refers to the four amino acid positions of zinc finger domains that structurally correspond to the positions of amino acids arginine 73, aspartic acid 75, glutamic acid 76, and arginine 79 of finger 3 of zinc finger protein zif268 (Kim and Pabo, J. Biol. Chem., (1997)

272(47): 29795-800). Said positions are also referred as positions -1, 2, 3 and 6, respectively. Among these four DNA contacting residues, the amino acid residues at positions -1, 3 and 6 play a key role for DNA recognition and the amino acid residue at position 2 plays an auxiliary role. The polypeptide of the present invention may comprises at least 2, preferably 2 to 6 zinc finger domains selected from the group consisting of the zinc finger domains having the amino acid sequence of SEQ ID Nos. 2 to 10, and each zinc finger domain can be a wild type, non-wild type or a combination thereof. Additionally, the zinc fmger domains may include various conventional linkers e.g. a peptide linker between the domains. The utility and design of linkers are well known in the art. A particularly useful linker is a peptide linker that is encoded by a nucleic acid. Thus, one can construct a synthetic gene that encodes a first DNA binding domain, the peptide linker, and a second DNA binding domain. This design can be repeated in order to construct large, synthetic, multi-domain DNA binding proteins. PCT WO 99/45132 and Kim and Pabo ((1998) Proc. Natl. Acad. Sci. USA 95:2812-7) describe the design of peptide linkers suitable for j oining zinc finger domains . Additional peptide linkers are available that form random coil, α-helical or β-pleated tertiary structures. Polypeptides that form suitable flexible linkers are well known in the art (see, e.g., Robinson and Sauer (1998) Proc Natl Acad Sci U S A. 95:5929-34). Flexible linkers typically include glycine, because this amino acid, which lacks a side chain, is unique in its rotational freedom. Serine or threonine can be interspersed in the linker to increase hydrophilicity. In additional, amino acids capable of interacting with the phosphate backbone of DNA can be utilized in order to increase binding affinity. Judicious use of such amino acids allows for balancing increases in affinity with loss of sequence specificity. If a rigid extension is desirable as a linker, α-helical linkers, such as the helical linker described in Pantoliano et al. (1991) Biochem. 30:10117-10125, can be used. Linkers can also be designed by computer modeling (see, e.g., U.S. Patent. No. 4,946,778). Software for molecular modeling is commercially available (e.g., from Molecular Simulations, Inc., San Diego, CA). The linker is optionally optimized, e.g., to reduce antigenicity and/or to increase stability, using standard mutagenesis techniques and appropriate biophysical tests as practiced in the art of protein engineering, and functional assays as described herein. For implementations utilizing zinc finger domains, the peptide that occurs naturally between zinc fingers can be used as a linker to join fingers together. A typical such naturally occurring linker is: Thr-Gly-(Glu/Gln)-

(Lys/Arg)-Pro-(Tyr/Phe) (SEQ ID NO: 11) (Agata et al, (1998) Gene 213:55-

64). More preferably, the DNA binding domain of the inventive polypeptide includes, in the order of N-terminal to C-terminal, the first, second, third and fourth zinc finger domains, wherein (1) the DNA contacting residues at positions -1, 3 and 6 of the first zinc finger domain are R, H and T, respectively; those of the second zinc finger domain are Q, H and R, respectively; those of the third zinc finger domain are Q, N and K, respectively; and those of the fourth zinc finger domain are Q, S and R, respectively; (2) the DNA contacting residues at positions -1, 3 and 6 of the first zinc finger domain are R, H and T, respectively; those of the second zinc finger domain are Q, N and R, respectively; those of the third zinc finger domain are Q, H and R, respectively; and those of the fourth zinc fmger domain are Q, N and I, respectively; (3) the DNA contacting residues at positions -1, 3 and 6 of the first zinc finger domain are Q, H and R, respectively; those of the second zinc finger domain are R, K and R, respectively; those of the third zinc finger domain are R, H and T, respectively; and those of the fourth zinc finger domain are Q, H and R, respectively; or (4) the DNA contacting residues at positions -1, 3 and 6 of the first zinc finger domain are R, H and T, respectively; those of the second zinc finger domain are Q, H and V, respectively; those of the third zinc finger domain are Q, H and Q, respectively; and those of the fourth zinc finger domain are Q, S and R, respectively. The most preferable polypeptides of the present invention have the amino acid sequence of any one of SEQ ID NOs: 12 to 15, and are designated as LK33, LK35, LK50 and LK52, respectively. The polypeptide of the present invention may further comprise a domain selected form the group consisting of a transcription activation domain, a transcription repression domain, a protein transduction domain (PTD) and a combination thereof to induce the increase in the monoclonal antibody production in a eukaryotic cell. Exemplary transcription activation domains include a Gal4 activation domain of yeast (Laughon, A. and Gesteland, R. F. Mol. Cell. Biol, (1984) 4: 260- 267; Amino acid No. 768-878 of NCBI accession number AAA45766), a VP16 domain of herpes simplex virus (Pellett, P. E. et al, Proc. Natl. Acad. Sci. USA (1985) 82:5870-5874; Amino acid No. 402-479 of NCBI accession number AAA45766) and a p65 of mammalian cell (Nolan G. P. et al, Cell (1991) 64:961- 969; Amino acid No. 275-535 of NCBI accession number NP_068810), but not limited thereto. Examples of the eukaryotic transcription repression domains include repression domains from Kid, UME6, ORANGE, groucho, and WRPW (Dawson et al., Mol. Cell Biol. (1995) 15: 6923-6931). Specifically, it can be a "KRAB" domain of Kid protein of rat (Witzgall R. et al, Proc. Natl. Acad. Sci. USA (1994) 91: 4514-4518) and a "KRAB" domain (amino acid No. 2-97 of Koxl) of human Koxl protein (NCBI protein database AAH24182; GI: 18848329). In one embodiment of the present invention, a Kid transcription repression domain was used, whose amino acid sequence and nucleic acid sequence encoding same are represented by SEQ ID NOs: 69 and 68, respectively. The transcription activation or repression domains can be fused to the DNA binding domain to form a fusion protein. Meanwhile, protein transduction domains result in uptake of the transduction domain and attached polypeptide into a cell. Representative PTDs useful in the present invention include a part of HIV TAT protein (e.g., amino acid region 47-57 of human HIV-1 virus Tat protein), a part of VP22 protein (e.g., HSV VP22 C-terminal 34 amino acid residues; Elliott and O'Hare Cell (1997) 88: 223- 234 and U.S. Patent No. 6,184,038) or a part of an Antennapedia homeodomain (Derossi et al, J. Bio. Chem. 269: 10444-10450). Typically a PTD is linked to a zinc finger protein by producing the DNA binding domain of the zinc finger protein and the PTD as a single polypeptide chain, but other methods of for physically associating a PTD can be used. For example, the PTD can be associated by a non-covalent interaction (e.g., using biotin-avidin, coiled-coils, etc.) More typically, a PTD can be linked to a zinc fmger protein, for example, using a flexible linker. Flexible linkers can include one or more glycine residues to allow for free rotation. For example, the PTD can be spaced from a DNA binding domain of the transcription factor by at least 10, 20, or 50 amino acids. A PTD can be located N- or C-terminal relative to a DNA binding domain. The inventive polypeptides are useful to screen genes associated with enhancing the production of monoclonal antibody or heterogeneous protein in a eukaryotic cell, preferably, in a mammalian cell, and they can up- or down- regulate the expression of the genes depending on the kinds of transcription regulation domains bound to the ZFD. In addition, the present invention provides a transformed eukaryotic cell, preferably, a mammalian cell, which_cornprises the inventive polypeptide directly introduced into the cell or produced from a nucleic acid encoding same introduced into the cell. Non-limiting examples of the cells useful in the present invention include a CHO cell, a NS-O cell, a Per.C6 cell, a HEK293 cell and a HEK293T cell, wherein a CHO cell is preferred. By using the transformed cell of the present invention, yield of the monoclonal antibody can be increased significantly under the same culture condition, as compared with the existing methods such as that using DHFR gene. In one preferred embodiment of the present invention, the inventive polypeptide is selected from a library of ZFP-Kid artificial transcription factors prepared by fusing a transcription repression factor of mammalian cell, Kid, to be used as a zinc finger protein. There are two representative methods available for obtaining a zinc finger domain as a building unit of the library. One is about mutating a DNA contacting residue of existing zinc finger (Rebar E. J. & Pabo, C. O., Science (1994) 263: 671-673), and the other is about isolating a zinc finger domain from the genome of an organism (Bae K. -H. et al, Nature Biotechnol, (2003) 21: 275-280). Specific target base sequences of the zinc finger can be determined by phage display or the yeast one-hybrid method. In one embodiment of the present invention, in order to transform a CHO cell with the zinc finger library in a high efficiency, a Kid domain and a (ZFD)₄ domain, which was obtained from existing zinc finger library, p3-(ZFD)₄ (WO 03/048345 and Park K. -S. et al, Nature Biotech, (2003) 21: 1208-1214), are inserted into pLNCX2 vector, which was prepared by removing 2 EcoRI sites from a pLNCX retrovirus vector (Clontech, USA), to obtain a pLNCX2-(ZFD)₄- Kid library (Fig. 1). A procedure of screening a zinc finger protein increasing the monoclonal antibody production from the library is shown in Fig. 2. Each DNA of the library is introduced together with pGag-Pol (Takara, Japan) and pVpack-VSV-G (Stratagene, USA) to a HEK 293T cell for transformation, and the supernatant of the culture medium including virus particles packed with different ZFP artificial transcription factors are collected. The supernatant is then added to the AKA cell, which was prepared by transforming a CHO cell with a gene of humanized antibody against TAG72 and amplifying in 20 nM MTX, to insert other genes of a ZFP-Kid artificial transcription factor into genome of the AKA cell in each well. The increase of the monoclonal antibody production induced by a ZFP- Kid gene is compared with the antibody production induced by an AKA cell having ZFP-free pLNCX-Kid. Quantity of the monoclonal antibody is analyzed with ELISA using an antibody against human immunoglobulin, and ZFPs increasing the monoclonal antibody production in an AKA cell, i.e., ZFP LK33- Kid, LK35-KM, LK50-Kid and LK52-Kid, are identified (Fig. 3 and Table 2). ZFP LK52-Kid increases the monoclonal antibody production about 3 to 9 times than that of the Kid control group, depending on the culture period (Fig. 4). Further, when one amino acid at each of two zinc finger domains of LK52-Kid is mutated, no increase in the monoclonal antibody production is observed (Fig. 4). From these results, it is assumed that the inventive polypeptides control the transcription of intracellular genes, thereby controlling the expression of genes causing increase of the monoclonal antibody production. In addition, the present invention provides a method of identifying the gene causing the increase of the monoclonal antibody production using the inventive polypeptide or nucleic acid encoding same. For example, a gene inducing a significant increase in the monoclonal antibody production can be identified as follows. A mammalian cell producing monoclonal antibody, e.g., a CHO cell transformed with a monoclonal antibody gene ("AKA cell"), is transformed with a zinc finger polypeptide comprising at least 2, preferably 2 to 6 zinc finger domains and a transcription repression domain connected thereto, or a nucleic acid encoding the zinc finger polypeptide. RNA is extracted from the transformed AKA cell, and cDNA probe is prepared by the reverse transcription. The probe is then subjected to reaction with a DNA chip having microarray of CHO cell genes to select a gene of which expression is repressed by ZFP, thereby causing the increase of the monoclonal antibody production. In the present invention, the microarray can be conducted in accordance with a conventional method (Schena M. et al, Science (1995) 270:467-470). The inventive screening method using a ZFP library can also be applied to other animal cells (e.g., CHO, NS-O, Per.C6, HeLa, HEK293 and 293T cell) which is industrially used to produce a heterogeneous useful protein, for improving their useful characters (e.g., increase of protein production and prevention of apoptosis).

The following Examples are intended to further illustrate the present invention without limiting its scope.

Example 1: Construction of pLNCX-(ZFD)₄-Kid library

(Step 1) Preparation of a zinc finger protein expression library, p3-(ZFD)₄

A zinc finger library was prepared in accordance with the method of Bae, K. -H. et al. (Nature Biotechnol, (2003) 21:275-280) using zinc fmger domains of Table 1 which were separated from the human genome by PCR method or cloning and their specific target base sequences were determined by yeast one- hybrid method (Korean Patent Laid-open Publication No.10-2001-0084880; WO 01/60970; Bae, K. -H. et al, supra), respectively. Specifically, a HA-tag (SEQ ID NO: 16) and a nuclear localization signal (NLS, SEQ ID NO: 17) were inserted into the multiple cloning site of plasmid pCDNA3 (Invitrogen, USA), and the resulting vector was designated as plasmid p3 (Bae, K. -H. et al, supra).

Table 1

Any one of DNA sequences encoding respective zinc finger domain listed in Table 1 was cloned into EcoRllNotl restriction site of plasmid p3 to make plasmid p3-ZFD (see Fig. 5). Equal amounts of each plasmid p3 including any one of the 25 domains listed in Table 1 were collected to make a domain pool. A part of the pool was digested with Xmal and Notl restriction enzymes to obtain DΝA inserts of each zinc finger domain. The other part of the pool was digested with Agel and Notl restriction enzymes to obtain long DΝA fragments corresponding to vectors containing one zinc finger domain.- The DΝA inserts were cloned into the long DΝA fragments to obtain a vector library expressing zinc finger proteins including two zinc finger domains. A part of the library was digested with Agel and Notl restriction enzymes to obtain DΝA inserts including two zinc finger domains, and the DΝA inserts were cloned into AgeVNotl restriction sites of the library vectors including two zinc finger domains to obtain a library of vectors p3-(ZFD)₄ expressing zinc finger proteins including four zinc finger domains. A DΝA encoding a zinc finger protein including four zinc finger domains can be separated from a vector of the library by digesting with EcoRI and Notl restriction enzymes.

(Step 2) Preparation of pLΝCX2-(ZFD)₄-Kid library

A vector expressing a zinc finger protein fused with a transcription repression domain was prepared as follows (see Fig. 1). First of all, in order to remove the EcoRI restriction site at nucleotide No. 1475 of retrovirus vector pLNCX (Clontech), site-directed mutagenesis was carried out using a mutagenesis kit (Stratagene, USA) and a pair of forward oligomer LNCX1-F (SEQ ID NO: 70, 5^' -

TAGGCGCCGGAATTtCGATCTGATCAAGA-3^' ; hereinafter, a small letter represents a mutated base) and reverse oligomer LNCX1-R (SEQ ID NO: 71, 5' -TCTTGATCAGATCGaAATTCCGGCGCCTA-3^' ), thereby obtaining plasmid pLNCXl . The plasmid pLNCXl was digested with EcoRI, and then treated with T4 polymerase to make blunt ends, which were then self-ligated to remove two EcoRI restriction sites therefrom to produce plasmid pLNCX2. A transcription repression domain was prepared as follows. To amplify Kid domain of a rat, PCR was carried out using plasmid pTet-tTS (Clontech) being used as a template and a pair of forward oligomer Kid-F (SEQ ID NO: 72, 5' -GGGCGGCCGCTAAATTCGTGTCAGTGACA-3' ) and reverse oligomer Kid-Rl (SEQ ID NO: 73, 5' -

CCGCTCGAGTTACCAGGGATCCTCTCC-3' ). The resulting product was digested with Notl and Xhol restriction enzymes and inserted into NotllXhol- digested plasmid pLFD-p65 (Bae, K. -H. et al, Nature Biotechnol, supra) to obtain a plasmid pLFD-Kid. The plasmid pLFD-Kid was sequentially treated with Apal and T4 polymerase to have blunt ends, and then treated with Hindlll. to obtain a Kid fragment. The plasmid pLΝCX2 was sequentially treated with Cla and T4 polymerase to have blunt ends, and then digested with Hindlll. The Kid fragment obtained above was inserted thereto to produce plasmid pLNCX2-Kid (see Fig. 6). In Fig.6, (Apal/Claϊ) represents that the two restriction enzyme sites were removed by the above procedure. The p3-(ZFD)₄ library prepared in (Step 1) was digested with EcoRI and Notl to obtain (ZFD)₄ DΝA fragments, and the fragments were inserted into a plasmid pLΝCX2-Kid digested with EcoRI and Notl to obtain a plasmid pLΝCX2-(ZFD)₄-Kid (see Fig. 7). In Fig.7, each of FI, F2, F3 and F4 represents one zinc finger domain, and (Apal/Clal) has the same meaning as defined in Fig. 6. In order to prepare a library, E. coli cells were transformed with pLNCX2-(ZFD)₄-Kid vectors, and library plasmids were isolated from more than 5,000 colonies of E. coli using an Accuprep plasmid extraction kit (Bioneer, Korea).

Example 2: Screening of ZFP increasing the production of monoclonal antibody

(Step 1) Production of ZFP-containing virus in 293T cell

6 X 10⁴ of 293T cells (ATCC CRL-11268) were put into each well of a 96- well culture plate provided with DMEM (JBI, Korea) containing heat-treated 10 % FBS (JBI, Korea), and cultured for a day at 37 °C in a CO₂ incubator. Transformation was conducted as follows. First of all, 100 ng of plasmid ρLNCX2-(ZFD)₄-Kid prepared in Example 1 or ZFP-free pLNCX2-Kid control group vector, 50 ng of pGag-Pol virus vector (Takara, Japan) and 50 ng of plasmid pVpack-VSV-G (Stratagene, USA) were mixed with 25 μl of Opti-MEM (Invitrogen, USA) (Mixture A). Aside from this, 0.5 μl of Lipofectamine 2000 (Invitrogen) was mixed with 25 μl of Opti-MEM (Mixture B). Mixture B was placed at room temperature for 5 minutes, and mixed with mixture A. The resulting mixture was placed at room temperature for 20 minutes (Mixture C). The medium was removed from each well of the above culture plate, and mixture C was added thereto to transform 293T cells. After 10-hour culture, the medium was replaced with DMEM containing 10 mM sodium butyrate and 10 % FBS (JBI, Korea) to increase the virus production. Further, after 12-hour culture, the medium was replaced with DMEM including 10 % FBS (JBI, Korea). The cells were cultured in a CO₂ incubator for 24 hours and a supernatant including virus was collected from the culture.

(Step 2) Transduction of AKA cell using ZFP-containing virus

In order to insert and screen a zinc finger library, an AKA cell (obtained from Dr. Hyo-Jeong Hong of Korea Research Institute of Bioscience and Biotechnology) was used. AKA cell was prepared by transforming a CHO cell with a gene of humanized antibody against TAG72 glycoprotein (Korea Patent Laid-open Publication No. 2000-0005885) and amplified using 20 nM MTX, in accordance with a conventional method (Kim, S. J., et al, Biotechnol&Bioeng. (1998) 58: 73-84). One day before collecting the supernatant in (Step 1), 1.5 X 10⁴ of AKA cells were put into each well of a 96-well culture plate containing alpha-MEM (JBI, Korea) containing dialyzed 10 % FBS (JBI, Korea), and cultured for a day at 37 °C in a CO₂ incubator. 25 μl of the supernatant of (Step 1) was mixed with 25 μl of alpha-MEM containing 10 % dialyzed FBS and 0.05 μl of polybrene (80 μg/ml), and the resulting mixture was added to each well having cultured AKA cells to transform the AKA cells. After 24 hours, the medium was replaced with fresh alpha-MEM and the cells were further incubated for 24 hours. 50 μl of the supernatant was taken from the transformed AKA cell culture and frozen.

(Step 3) ELISA for screening of ZFP increasing the monoclonal antibody production

50 μl of 0.05 M sodium carbonate (pH 9.6) containing 0.5 μg of anti- mouse IgG (Sigma) was put into each well of a 96-well immuno plate (Nunc, Denmark) and the plate was stored in a 37 °C incubator for 2 hours. Then the plate was washed three times with distilled water, and 50 μl of diluted supernatant of (Step 2), which was 100 times diluted with PBS (0.01 M phosphate buffer, 0.0027 M potassium chloride, 0.137 M sodium chloride, pH 7.4) containing 0.25 % BSA and 0.05 % Tween-20, was added to each well of the plate. After 2 hours at room temperature, the plate was washed three times with distilled water, and 50 μl of diluted anti-human IgG-peroxide conjugate (Sigma), which was 10,000 times diluted with PBS containing 0.25 % BSA, and 0.05 % Tween-20, was added to each well of the plate. After 1 hour, the plate was washed three times with distilled water. 70 μl of TMB solution (Sigma) was added to each well of the plate, and the reaction was carried out for 10 minutes. The absorbance of the wells was measured at 655 nm by powerwave 340 (Bio-TEK Instruments, INC, USA). As a result, most of the wells showed similar absorbance to that of ZFP-free pLNCX2-Kid control group vector. However, four pLNCX2-(ZFD)₄-Kid plasmids showed higher absorbance than that of the control group and they were subjected to further experiment.

Example 3: Change of the monoclonal antibody production by four ZFPs in AKA cell

The antibody productions of four ZFP's were shown to be higher than that of the control group with pLNCX2-Kid inserted therein. For confirmation, Example 2 was repeated, except that length of time the reaction mixture was placed at room temperature has changed. Lipofectamine 2000 was mixed with Opti-MEM and placed at room temperature for 20 minutes. The mixture was mixed with Opti-MEM including plasmids pLNCX2-ZFP-Kid, pGag-Pol and pVpack-VSV-G and then was placed at room temperature for 40 minutes. 293T cells cultured in a 96-well plate were transformed with the mixture, each plasmid of pLNCX2 and pLNCX2-Kid being used as control plasmid. AKA cells were transformed with virus in accordance with the procedure of (Step 2) of Example 2, and the medium was replaced with new medium after 24 hours and cultured for 24 hours. Each supernatant including antibody was collected and then diluted 250 and 500 times. Next, they were subjected to ELISA. As can be seen in Fig. 3, four ZFP-Kids, i.e., LK33-Kid, LK35-Kid, LK50-Kid and LK52-Kid showed about 2 to 6 times increase in the production of antibody in AKA cells. Zinc finger domains and target sequences of these ZFPs are listed in Table 2.

Table 2

Example 4: Change in the monoclonal antibody production by LK52 in AKA cell in accordance with length of time

(Step 1) Production of ZFP-containing virus in 293T cell

5 X 105³ 293T cells (ATCC CRL-11268) were put into each well of a 24- well culture plate provided with DMEM (JBI, Korea) containing heat-treated 10 % FBS (JBI, Korea), and cultured for 1 day at 37 °C in a CO₂ incubator. 400 ng of plasmid pLNCX2-LK52-Kid, 200 ng of pGag-Pol virus vector (Takara, Japan) and 200 ng of plasmid pVpack-VSV-G (Stratagene, USA) were mixed with 50 μl of Opti-MEM (Invitrogen, USA) (Mixture A). Aside from this, 2 μl of Lipofectamine 2000 (Invitrogen) was mixed with 50 μl of Opti- MEM (Mixture B). Mixture B was placed at room temperature for 20 minutes before being mixed with mixture A. The resulting mixture was placed at room temperature for 40 minutes (Mixture C). The medium was removed from each well of the above culture plate, and mixture C was added thereto to transform 293T cells. After 10-hour culture, the medium were replaced with DMEM containing 10 mM sodium butyrate and 10 % FBS (JBI, Korea) to increase the production of virus. Further, after 12 hour culture, the medium was replaced with fresh DMEM containing 10 % FBS (JBI, Korea). The cells were cultured in a CO₂ incubator for 24 hours and a supernatant of transformed 293T cells including virus was collected from the culture.

(Step 2) Transduction of AKA cell using ZFP-containing virus One day before collecting the supernatant in (Step 1), 3 X 10⁴ AKA cells were put into each well of a 24-well culture plate containing alpha-MEM (JBI, Korea) containing dialyzed 10 % FBS (JBI, Korea), and cultured for a day at 37 °C in a CO₂ incubator. 125 μl of the supernatant of (Step 1) was mixed with 125 μl of alpha-MEM containing 10 % dialyzed FBS and 0.25 μl of polybrene (80 μg/ml), and the resulting mixture was added to each well having cultured AKA cells to transform the AKA cells. After 24 hours, the medium was replaced with new alpha-MEM containing 10 % FBS and the cells were further incubated for 24, 48, 72, 96 and 120 hours (i.e., 3, 4, 5, 6 and 7 days shown in Fig. 4). 200 μl of each supernatant after transformation, 200 μl of each supernatant before transformation (1 day shown in Fig. 4), and 200 μl of each supernatant after transformation but before replacement of medium (2 day shown in Fig. 4) were taken from the transformed AKA cell cultures and frozen. (Step 3) ELSIA for screening of ZFP increasing the monoclonal antibody production

The stored supernatants of (Step 2) were subjected to ELISAin accordance with the same procedure of (Step 3) of Example 2. As shown in Fig. 4, with the increase in culture time, the monoclonal antibody production of LK52-Kid increased to as high as 37 μg/ml, 9 times higher when compared with the control group.

Example 5: Activity of mLK52-Kid transduced with mutant ZFP

To examine whether the ZFP is a direct cause of the increase in the monoclonal antibody production, both arginine residues at DNA contacting residue -1 of the first finger RDHT and arginine residues at DNA contacting residue 6 of the fourth finger QSSR1 of LK52-Kid were mutated to alanines to change the DNA binding site of LK52. The mutagenesis was conducted as shown in Fig. 8. First of all, PCRs were carried out by using plasmid pLNCX2-LK52- Kid as a template and a respective oligomer pairs of φ NLS-F (SEQ ID NO: 78, 5' -CCTCCAAAAAAGAAGAGAAAGGTA-3^' ) and mutRDHT-R (SEQ ID NO: 79, 5' -CAGGTGGTCGGAggcGGAGAACTTTCG-3^' ), (2) mutRDHT-F (SEQ ID NO: 80, 5^' -

CGAAAGTTCTCCgccTCCGACCACCTG-3^' ) and QTHQ-R (SEQ ID NO: 81, 5' -CCTAAAGGACTTTCCGCAATCGTGACACTC-3' ), © QTHQ-F (SEQ ID NO: 82, 5' -GAGTGTCACGATTGCGGAAAGTCCTTTAGG-3^' ) and mutQSSRl-R (SEQ ID NO: 83, 5^' -

GTGTCCGCTGGTGGgcAATGAGGCTGGAAC-3' ) and © mutQSSRl-F (SEQ ID NO: 84, 5^' -GTTCCAGCCTCATTgcCCACCAGCGGACAC-3^' ) and Kid-R2 (SEQ ID NO: 85, 5^' -CCGCTCGAGCCAGGGGTCCTCTCC- 3' ) to obtain the first products. Further, the 2^nd PCRs were carried out by employing the first products of φ and © as templates and an oligomer pair of NLS-F (SEQ ID NO: 78) and QTHQ-R (SEQ ID NO: 81) (©); and by employing the first products of © and © as templates and an oligomer pair of QTHQ-F (SEQ ID NO: 82) and Kid-R2 (SEQ ID NO: 85) (©), respectively. Finally, a 3^rd PCR was carried out by employing the products of © and © as templates and an oligomer pair of NLS-F (SEQ ID NO: 78) and Kid-R2 (SEQ ID NO: 85) to obtain a DNA product having desired mutation. The final PCR product was digested with EcoRI and Notl to obtain mutated mLK52 DΝA fragment. The DΝA fragment was cloned into the EcoRΪ/Notl restriction site of plasmid pLΝCX2-LK52-Kid, wherein wild type LK52 gene was removed. E. coli DH5 α cells were transformed with the resulting plasmid. Plasmids were isolated from colonies thus obtained and sequenced to obtain a plasmid pLNCX2-mLK52-Kid including a mutated mLK52 gene. AKA cells were transformed with this mutated pLNCX2-mLK52-Kid using virus as described in Example 4, and increase in the monoclonal antibody production by the ZFP was examined. As shown in Fig. 4, an AKA cell including mLK52-Kid showed similar monoclonal antibody production to that of a ZFP-free control cell expressing Kid only. Considering that AKA cell transformed with LK52-Kid showed about 9 times increase of the production^ this result demonstrate that specific DNA binding ability of LK52 is responsible for the increase of the monoclonal antibody production.

Example 6: Change of the monoclonal antibody production by LK52 in various cells In order to examine whether LK33-Kid, LK35-Kid, LK50-Kid and

LK52-Kid also induce an increase in the monoclonal antibody production in other monoclonal antibody-producing cells than AKA cell, the ZFP-Kids were introduced into SH2-0.32 and ISU-ABC cells (obtained from Professor Gyun Min LEE of Korea Advanced Institute of Science and Technology), which are CHO cells producing monoclonal antibody, and the amount of produced monoclonal antibody was examined. SH2-0.32 cell was prepared by transforming a CHO cell with a gene for a monoclonal antibody against the surface antigen of Hepatitis B virus and amplifying the gene to increase the monoclonal antibody production (Kim, N. S. et al, Biotech&Bioeng. (2001) 71:184-193). ISU-ABC cell was prepared by transforming CHO cell with a gene for a monoclonal antibody against the glycoprotein receptor of human blood platelet and amplifying the gene to increase the monoclonal antibody production. The amplification of a specific gene in CHO cell was carried out in accordance with conventional methods such as gene amplification using DHFR (dihydrofolate reductase) gene and MTX (Kim, S. J. et al, Biotechnol&Bioeng. (1998) 58: 73-84). The examination was conducted in accordance with the same procedure as described in Example 2, except that the medium was replaced with 100 μl of new one 1 day after the transformation with virus; 30 μl of the supernatant was collected after 1 day; the residual supernatant was collected after 3 days; and the concentration of antibody secreted into the medium was determined by using the supernatants. The average and standard deviation were calculated using 3-4 wells per each plasmid sample, and the ELISA was conducted as follows. 100 μl of 0.05 M sodium carbonate (pH 9.6) including 0.2 μg of anti- mouse IgG (Sigma) was put into each well of a 96-well immuno plate (Nunc, Denmark) and placed in a 37 °C incubator for 2 hours. The plate was washed three times with PBST (PBS including 0.05 % Tween-20). 300 μl/well of 2 % BSA was added to the wells and the plate was placed at 37 ^°C for 2 hours to block the non-specific reactions, and washed three times with PBST. 100 μl of diluted supernatant, which was prepared by diluting 1,000 times the supernatants obtained above with PBS containing 0.25 % BSA and 0.05 % Tween-20, was added to each well of the plate. After 2 hours at room temperature, the plate was washed three times with PBST, and 100 μl of diluted anti-human IgG-HRP conjugate (Sigma), which was 2,000 times diluted with PBS containing 0.25 % BSA and 0.05 % Tween-20, was added to each well of the plate. After 1 hour, the plate was washed three times with distilled water. 100 μl of substrate mixture (mixture of 10 ml of PC buffer [2.55 g of C₆H₈O₇ ^• H₂O and 3.65 g of Na₂HPO₄ in 500 ml of distilled water, pH 5.0], 12 μl of O-phenylenediamine and 4 μl of H₂O₂) was added to each well of the plate, and left there for reaction for 2-3 minutes. The reaction was terminated by adding 50 μl of 1 M H₂SO₄ to each well. The result was detected by ELISA reader (Bio-rad, model-680) at 490 nm, and shown in Tables 3 and 4.

Table 3: Monoclonal antibody production in SH2-0.32 cell

Table 4: Monoclonal antibody production in ISU-ABC cell

As can be seen in Tables 3 and 4, LK33-Kid, LK35-Kid, LK50-Kid and LK52-Kid increased the monoclonal antibody productions after 3 days in SH2- 0.32 and ISU-ABC cells by 2.5 to 4.1 folds and by 1.3 to 1.8 folds, respectively, and LK52-Kid showed the best result. This result indicates that ZFP screened in an AKA cell worked similarly in CHO cells producing other kind of antibody. Accordingly, it is expected that ZFP screened in one cell may have similar effect in other homogeneous cells having a similar function.

Example 7: Real time PCR In order to examine the mechanisms of LK33-Kid, LK35-Kid, LK50- Kid and LK52-Kid increasing the monoclonal antibody production in an AKA cell, mRNAs of monoclonal antibody of AKA cells expressing the 4 ZFPs were quantified by real time PCR. Viruses having LK33-Kid, LK35-Kid, LK50-Kid and LK52-Kid were produced and transformed into AKA cells by the method described in Example 3. The plasmid pLNCX2-Kid was used as a control. The media was replaced after 24 hours and then the cells were collected after 2 days. RNA was extracted from the cells using Trizol (Invitrogen). Reverse transcription reaction was conducted using 1 μg of the extracted RNA and Superscript II kit (Invitrogen). 8 μl of the product was mixed with 10 μl of 2X SYBR (Qiagen), and the mixture was subjected to real time PCR (Rotor-Gene 2000, Corbett research, Australia) using an oligomer pair of 1 μl of AKA-F (SEQ ID NO: 86, 5' -GATGGGCCCTTGGTGCTGGCT-3^' ) and 1 μl of AKA-R (SEQ ID NO: 87, 5^' -GACGAATTCACTCTAACCATGGAA-3^' ), or a pair of 1 ≠ of GAPDH-F (SEQ ID NO: 88, 5^' - CCGAGTATGTTGTGGAATCTACTG-3^' ) and 1 μi of GAPDH-R (SEQ ID NO: 89, 5^' -GACAATCTTGAGGGAGTTGTCATA-3^' ). The concentration of each oligomer was 10 pmol/ £. As can be seen in Fig. 9, all ZFPs, i.e., LK33-Kid, LK35-Kid, LK50-Kid and LK52-Kid, increased the amount of mRNA of monoclonal antibody 3-6 time than that of GAPDH mRNA. In addition, the ZFPs may also act on the genes of proteins related to the translation or secretion of a monoclonal antibody.

While the invention has been described with respect to the above specific embodiments, it should be recognized that various modifications and changes may be made and also fall within the scope of the invention as defined by the claims that follow.

Claims

What is claimed is:

1. A polypeptide capable of increasing the amount of a monoclonal antibody produced by a eukaryotic cell relative to a cell that does not include the polypeptide, wherein the polypeptide comprises a DNA binding domain that includes a plurality of zinc finger domains.

2. The polypeptide of claim 1, wherein the DNA binding domain comprises at least 2 zinc finger domains selected from the group consisting of the following amino acid sequences: CX₍₂-₅₎CXXXBXRXXHJXTHX₍₃_₅₎H (SEQ ID NO: 2); CX₍₂-₅₎CXXXBXQXXHJXRHX_(3-5)H (SEQ ID NO: 3); CX_(2-5)CXXXBXQXXNJXKHX_(3-5)H (SEQ ID NO: 4); CX_(2-5)CXXXBXQXXSJXRHX₍₃-₅₎H (SEQ ID NO: 5); CX_(2-5)CXXXBXQXXNJXRHX_(3-5)H (SEQ ID NO: 6); CX_(2-5)CXXXBXQXXNJXIHX_(3-5)H (SEQ ID NO: 7); CX_(2-5)CXXXBXRXXKJXRHX_(3-5)H (SEQ ID NO: 8); CX₍₂.₅₎CXXXBXQXXHJXVHX_(3-5)H (SEQ ID NO: 9); and CX_(2-5)CXXXBXQXXHJXQHX_(3-5)H (SEQ ID NO: 10); where B is phenylalanine or tyrosine; J is a hydrophobic amino acid; and

X is any amino acid.

3. The polypeptide of claim 1, wherein the DNA binding domain includes, in N-terminal to C-terminal order, the first, second, third and fourth zinc finger domains, wherein (1) the DNA contacting residues at positions -1, 3 and 6 of the first zinc finger domain are R, H and T, respectively; those of the second zinc fmger domain are Q, H and R, respectively; those of the third zinc finger domain are Q, N and K, respectively; and those of the fourth zinc finger domain are Q, S and R, respectively; (2) the DNA contacting residues at positions -1, 3 and 6 of the first zinc finger domain are R, H and T, respectively; those of the second zinc finger domain are Q, N and R, respectively; those of the third zinc finger domain are Q, H and R, respectively; and those of the fourth zinc finger domain are Q, N and I, respectively; (3) the DNA contacting residues at positions -1, 3 and 6 of the first zinc finger domain are Q, H and R, respectively; those of the second zinc fmger domain are R, K and R, respectively; those of the third zinc finger domain are R, H and T, respectively; and those of the fourth zinc finger domain are Q, H and R, respectively; or (4) the DNA contacting residues at positions -1, 3 and 6 of the first zinc finger domain are R, H and T, respectively; those of the second zinc finger domain are Q, H and V, respectively; those of the third zinc finger domain are Q, H and Q, respectively; and those of the fourth zinc finger domain are Q, S and R, respectively.

4. The polypeptide of claim 3, wherein the DNA binding domain has the amino acid sequence of any one of SEQ ID NOs: 12 to 15.

5. The polypeptide of claim 1, which further comprising a domain selected form the group consisting of a transcription activation domain, a transcription repression domain, a protein transduction domain and a combination thereof connected to the DNA binding domain.

6. The polypeptide of claim 5, wherein the transcription activation domain is selected from the group consisting of a Gal4 activation domain of yeast, a VP16 domain of herpes simplex virus and a p65 domain of a mammalian cell.

7. The polypeptide of claim 5, wherein the transcription repression domain is Kid or KOX repression domain.

8. The polypeptide of claim 5, wherein the protein transduction domain is a part of a TAT protein, a VP22 protein or an Antennapedia homeodomain.

9. The polypeptide of claim 1, wherein the eukaryotic cell is a mammalian cell.

10. The polypeptide of claim 9, wherein the mammalian cell is selected from the group consisting of a CHO (Chinese Hamster Ovary) cell, a HEK293T cell, a HEK293 cell, a PerC6 cell and a NS-O cell.

11. A nucleic acid that comprises a sequence encoding the polypeptide of claim 1.

12. The nucleic acid of claim 11, wherein the polypeptide comprises a

DNA binding domain that includes at least 2 zinc finger domains selected from the group consisting of the following amino acid sequences: CX₍₂.₅₎CXXXBXRXXHJXTHX_(3-5)H (SEQ ID NO: 2); CX_(2-5)CXXXBXQXXHJXRHX_(3-5)H (SEQ ID NO: 3); CX_(2-5)CXXXBXQXXNJXKHX_(3-5)H (SEQ ID NO: 4); CX_(2-5)CXXXBXQXXSJXRHX_(3-5)H (SEQ ID NO: 5); CX₍₂.₅₎CXXXBXQXXNJXRHX_(3-5)H (SEQ ID NO: 6); CX_(2-5)CXXXBXQXXNJXIHX_(3-5)H (SEQ ID NO: 7); CX_(2-5)CXXXBXRXXKJXRHX_(3-5)H (SEQ ID NO: 8); CX_(2-5)CXXXBXQXXHJXVHX_(3-5)H (SEQ ID NO: 9); and CX_(2-5)CXXXBXQXXHJXQHX_(3-5)H (SEQ ID NO: 10); where B is phenylalanine or tyrosine; J is a hydrophobic amino acid; and X is any amino acid.

13. The nucleic acid of claim 11, wherein the polypeptide comprises a

DNA binding domain that includes, in N-terminal to C-terminal order, the first, second, third and fourth zinc finger domains, wherein (1) the DNA contacting residues at positions -1, 3 and 6 of the first zinc finger domain are R, H and T, respectively; those of the second zinc finger domain are Q, H and R, respectively; those of the third zinc finger domain are Q, N and K, respectively; and those of the fourth zinc finger domain are Q, S and R, respectively; (2) the DNA contacting residues at positions -1, 3 and 6 of the first zinc finger domain are R, H and T, respectively; those of the second zinc finger domain are Q, N and R, respectively; those of the third zinc finger domain are Q, H and R, respectively; and those of the fourth zinc fmger domain are Q, N and I, respectively; (3) the DNA contacting residues at positions -1, 3 and 6 of the first zinc finger domain are Q, H and R, respectively; those of the second zinc finger domain are R, K and R, respectively; those of the third zinc finger domain are R, H and T, respectively; and those of the fourth zinc finger domain are Q, H and R, respectively; or (4) the DNA contacting residues at positions -1, 3 and 6 of the first zinc finger domain are R, H and T, respectively; those of the second zinc fmger domain are Q, H and V, respectively; those of the third zinc finger domain are Q, H and Q, respectively; and those of the fourth zinc finger domain are Q, S and R, respectively.

14. The nucleic acid of claim 11, wherein the polypeptide further comprises a transcription activation domain, a transcription repression domain, or a protein transduction domain connected to the DNA binding domain.

15. A eukaryotic cell containing a gene encoding a monoclonal antibody and the nucleic acid that comprises a sequence encoding the polypeptide of claim 1.

16. The eukaryotic cell of claim 15, which is a mammalian cell.

17. The eukaryotic cell of claim 15, which is selected from the group consisting of a CHO cell, a HEK293T cell, a HEK293 cell, a PerC6 cell and a NS-O cell.

18. The eukaryotic cell of claim 15, wherein the polypeptide comprises a

DNA binding domain that includes at least 2 zinc finger domains selected from the group consisting of the following amino acid sequences: CX_(2-5)CXXXBXRXXHJXTHX₍₃-₅₎H (SEQ ID NO: 2); CX, _2-5)CXXXBXQXXHJXRHX_(3-5)H (SEQ ID NO: 3); CX_(2-5)CXXXBXQXXNJXKHX_(3-5)H (SEQ ID NO: 4); CX_(2-5)CXXXBXQXXSJXRHX₍₃-₅₎H (SEQ ID NO: 5); CX_(2-5)CXXXBXQXXNJXRHX_(3-5)H (SEQ ID NO: 6); CX_(2-5)CXXXBXQXXNJXIHX_(3-5)H (SEQ ID NO: 7); CX_(2-5)CXXXBXRXXKJXRHX_(3-5)H (SEQ ID NO: 8); CX_(2-5)CXXXBXQXXHJXVHX_(3-5)H (SEQ ID NO: 9); and CX_(2-5)CXXXBXQXXHJXQHX_(3-5)H (SEQ ID NO: 10); where B is phenylalanine or tyrosine; J is a hydrophobic amino acid; and X is any amino acid.

19. The eukaryotic cell of claim 15, wherein the polypeptide comprises a DNA binding domain that includes, in N-terminal to C-terminal order, the first, second, third and fourth zinc finger domains, wherein (1) the DNA contacting residues at positions -1, 3 and 6 of the first zinc fmger domain are R, H and T, respectively; those of the second zinc fmger domain are Q, H and R, respectively; those of the third zinc finger domain are Q, N and K, respectively; and those of the fourth zinc finger domain are Q, S and R, respectively; (2) the DNA contacting residues at positions -1, 3 and 6 of the first zinc finger domain are R, H and T, respectively; those of the second zinc finger domain are Q, N and R, respectively; those of the third zinc finger domain are Q, H and R, respectively; and those of the fourth zinc finger domain are Q, N and I, respectively; (3) the DNA contacting residues at positions -1, 3 and 6 of the first zinc finger domain are Q, H and R, respectively; those of the second zinc finger domain are R, K and R, respectively; those of the third zinc finger domain are R, H and T, respectively; and those of the fourth zinc finger domain are Q, H and R, respectively; or (4) the DNA contacting residues at positions -1, 3 and 6 of the first zinc finger domain are R, H and T, respectively; those of the second zinc finger domain are Q, H and V, respectively; those of the third zinc finger domain are Q, H and Q, respectively; and those of the fourth zinc finger domain are Q, S and R, respectively.

20. The eukaryotic cell of claim 15, wherein the polypeptide further comprises a transcription activation domain, a transcription repression domain, or a protein transduction domain connected to the DNA binding domain.

21. A method for increasing the monoclonal antibody production in a eukaryotic cell, which comprises the step of introducing the polypeptide of claim 1 or the nucleic acid of claim 11 into the cell.