WO2014126254A1

WO2014126254A1 - Membrane-binding protein expression vector

Info

Publication number: WO2014126254A1
Application number: PCT/JP2014/053730
Authority: WO
Inventors: 恵黒川; 恵奈山口; 梨沙小川; 正義塚原; 洋子林
Original assignee: 協和発酵キリン株式会社
Priority date: 2013-02-18
Filing date: 2014-02-18
Publication date: 2014-08-21

Abstract

A desired-protein expression vector which can be used for producing a protein composition with high efficiency and in a large quantity and for producing a protein-containing medicine in a safe manner and a method for producing a desired protein using the vector have been demanded. According to the present invention, it becomes possible to provide: a desired-protein expression vector which can express both a fusion protein (a membrane-binding protein or a fragment of the membrane binding-protein) of a desired protein and a transmembrane domain and the desired protein at the same time; a cell having the vector introduced thereinto; a method for screening for a cell having the vector introduced thereinto; a method for expressing both the desired protein and the membrane-binding protein at the same time by introducing the vector into a host cell; a cell capable of producing the desired protein, which can produce the desired protein at a high level and can be obtained by screening for a cell, into which the vector has been introduced to express the membrane-binding protein, by employing the expression amount of the membrane-binding protein as a measure; and a method for producing the desired protein using the cell.

Description

Membrane-bound protein expression vector

The present invention provides a target protein expression vector, target protein, or target protein capable of simultaneously expressing a target protein and a target protein fused with a membrane-binding domain or a fragment of the target protein using one gene. The present invention relates to a method for screening a cell that expresses a fragment, a cell that highly produces a target protein, a method for producing a cell that highly produces a target protein, and a method for producing the target protein using the cell.

In the production of protein drugs such as cytokines, growth factors, hormones, and antibodies using mammalian cells as hosts, it is important to select and screen high-producing cells simply and rapidly for shortening the drug development period. However, conventional selection of target protein producing cells has the following three problems.
(1) Disagreement between cells with drug resistance phenotype and cells expressing the target protein Currently, cells producing the target protein have host cells with the drug resistance marker gene incorporated in the same expression vector as the target protein or in another expression vector. As a result, the cells are indirectly screened by selecting cells having a drug-resistant phenotype (Non-patent Document 1). Since the drug resistance marker and the target protein are expressed in different and different gene expression cassettes, the expression of the drug resistance gene does not necessarily match the expression of the target protein. That is, a cell exhibiting a drug resistance trait does not always express the target protein at a high level. Therefore, select a cell population based on drug resistance traits, and measure the amount of the target protein directly using the enzyme-linked immuno-sorbent assay (ELISA) method for cells that highly express the target protein. Thus, the protein producing cells must be rescreened (Non-patent Document 2). Since cell selection is performed in a process with two or more steps, screening time and labor are required.
(2) Effect of drug resistance marker on protein production When cell selection is performed based on a drug resistance phenotype, the expressed drug resistance marker protein is an impurity protein that is unnecessary in the production of the target protein. That is, when the target protein is used as a pharmaceutical, it is necessary to remove this drug resistance marker protein in the purification process. Furthermore, it has been reported that high expression of a drug resistance marker protein used for cell selection changes the metabolism of the host cell and conversely reduces the expression of the target protein (Non-patent Document 3). In addition, fluorescent proteins are generally used as marker proteins for cell selection, but are known to have cytotoxicity, and improvements to fluorescent proteins with low toxicity are being promoted (Patent Document 1).
(3) Single-cell cloning Cells that produce recombinant DNA technology-applied pharmaceuticals such as protein drugs must be derived from single clones (ICH-Q5D Guidelines, Pharmaceutical Examination No. 873 dated July 14, 2000) ). Since the cells selected for drug resistance are not necessarily single clones, they must be newly cloned by a method such as limiting dilution after drug selection.

As a means for solving the above-mentioned problem (3), there is a cell selection method using a fluorescence-activated cell sorter (SFACS) by a flow cytometry method (flow cytometry). The cell sorter can select cells in the state of individual clones. Examples of cell selection marker genes that can be used in the cell sorter include green fluorescent protein and red fluorescent protein (Non-patent Document 4). However, as with the drug resistance gene, if the fluorescent protein and the target protein are different and different gene cassettes, the expression of the fluorescent protein does not necessarily match the expression of the target protein. That is, as described in (1), cells that highly express the fluorescent protein do not necessarily express the target protein at a high level. As means for solving this problem, a method is known in which a target protein and a fluorescent protein are fused and expressed in the same gene cassette (Patent Document 2). However, in the present protein expression method, since the target protein and the fluorescent protein are fused, the activity and quality of the target protein may be affected. Particularly in the case of pharmaceuticals, it is not a practical protein production method because of concerns such as antigenicity.

Separately, the use of an internal ribosome entry site (IRES) sequence is known as a method for expressing two genes in one gene expression cassette (Non-patent Document 3, Patent Document 5). In the protein expression method using this IRES, it has been clarified that the expression of the target protein increases in proportion to the expression of the drug resistance marker or the fluorescent protein. In these protein expression methods, the expression level of the marker protein correlates with the expression level of the target protein, and it is possible to obtain production cells expressing the target protein. An unwanted marker protein must be expressed.

In addition, a method has been reported in which the target protein is expressed on the cell membrane of the production cell and the expression level of the target protein is directly detected using a cell sorter (Patent Document 4). According to this method, a protein expression vector in which a polypeptide for cell membrane binding is arranged immediately downstream of the stop codon of the target protein is introduced into a host cell, and the protein is translated by treating the cell with an aminoglycoside antibiotic. The target protein expressed on the cell membrane can be expressed by reducing the recognition of the termination codon by the translation initiation factor complex (read-through). Is detected with a cell sorter, and it is reported that cells expressing the target protein can be selected. However, although this method requires a short period of time, it is necessary to treat the target protein-producing cells with the drug. The drug may adversely affect the producing cells, and an unnecessary drug may be mixed into the target protein. As a result of other proteins other than the target protein being translated by read-through, there is a demerit such as the possibility that an unexpected protein may be produced. Absent.

As described above, as a result of expressing a part of the target protein on the cell membrane, a method of selecting a high-expressing cell using a cell sorter with the protein expression level on the cell membrane as an index and performing single cloning is known. However, the risk of inefficiency and contamination with impurities, such as the expression of unnecessary proteins and the use of unnecessary drugs for producing the target protein, has not been solved.
On the other hand, by introducing a genomic sequence encoding an antibody gene into a host cell and shortening the intron located between the C-terminal antibody constant region and the membrane-type IgG sequence, alternative splicing of the mRNA encoding the antibody gene can be achieved. A method for producing a library of antibody-expressing cells in which expression of membrane-type antibodies is increased (Patent Document 5) is known.

In addition, using antibody genes present in the human genome, using a splicing donor sequence at the 5 'end of the stop codon of the antibody gene, expressing a membrane-type antibody bound with a natural transmembrane domain, antibody expression It is known to produce cells (Patent Document 6). However, these methods do not disclose the production of protein-producing cells in which the rate of alternative splicing is controlled. Furthermore, as antibody-producing cells such as pharmaceuticals, membrane-type antibodies are mixed as impurities in the final pharmaceutical, which causes a problem in the pharmaceutical manufacturing process.

Special table 2004-536571 Special table 2003-525605 U.S. Patent No. 6,632,637 International Publication No. 2005/073375 Pamphlet Japanese Patent No.4553584 International Publication 2007/131774 Pamphlet

There is a need for a target protein expression vector for efficiently and mass-producing a protein composition and safely manufacturing a protein drug, a cell into which the expression vector is introduced, and a method for producing the target protein using the cell .

The present invention relates to the following (1) to (13).
(1) A target protein expression vector comprising a DNA sequence encoding a target protein including a splicing donor sequence, a polypyrimidine sequence, a splicing acceptor sequence, and a DNA sequence encoding a transmembrane region in this order.
(2) DNA sequence encoding target protein including splicing donor sequence, first polyadenylation signal sequence, polypyrimidine sequence, splicing acceptor sequence, DNA sequence encoding transmembrane region and second polyadenylation signal sequence An expression vector for the protein of interest, in order.
(3) A cell into which the expression vector according to any one of (1) or (2) has been introduced.
(4) The cell according to (3), wherein the cell is a cell selected from (a) and (b).
(A) A cell in which messenger RNA (mRNA) encoding a protein in which a target protein or a fragment of the target protein is fused with a transmembrane region is expressed in 0.1% to 5% of the total mRNA of the target protein.
(B) A cell in which at least 1 × 10 ³ molecules / cell of a protein in which the target protein or a fragment of the target protein and the transmembrane region are fused is expressed.

(5) A screening method for cells that produce a target protein in a high amount, comprising detecting the target protein or a fragment of the target protein expressed on the cell membrane of the cell according to (3) or (4).
(6) A method for producing the cell according to (3) or (4).
(7) A method for producing a target protein, comprising the steps of culturing the cell according to (3) or (4), accumulating the target protein in a culture solution, and purifying the target protein from the cell culture solution.
(8) Simultaneous expression of both the target protein and the target protein or a protein in which the target protein fragment and the transmembrane region are fused, including the step of introducing the target protein expression vector described in (1) into a host cell How to make.

(9) A target protein-expressing cell selected from (a) and (b), into which a protein expression vector containing a DNA sequence encoding the target protein has been introduced and which expresses a secreted target protein.
(a) A cell in which messenger RNA (mRNA) encoding a protein in which a target protein or a fragment of the target protein is fused with a transmembrane region is expressed in 0.1% to 5% of the total mRNA of the target protein.
(b) A cell expressing 1 × 10 ³ molecules / cell or more of a protein in which the target protein or a fragment of the target protein and the transmembrane region are fused.
(10) The cell according to (9), wherein the cell is a cell selected from (a) and (b).
(a) A cell in which mRNA encoding a target protein or a protein in which a fragment of the target protein is fused with a transmembrane region is expressed in 0.1% to 2% of the total mRNA of the target protein.
(b) A cell in which 1 × 10 ³ to 1 × 10 ⁷ molecules / cell of a protein in which the target protein or a fragment of the target protein and the transmembrane region are fused is expressed.

(11) A screening method for cells that produce a target protein in a high amount, comprising detecting the target protein or a fragment of the target protein expressed on the cell membrane of the cell according to (9) or (10).
(12) A method for producing the cell according to (9) or (10).
(13) A method for producing a target protein comprising the steps of culturing the cell according to (9) or (10), accumulating the target protein in a culture solution, and purifying the target protein from the cell culture solution.

According to the present invention, an expression vector for a target protein that can simultaneously express a target protein and a protein in which the target protein or a fragment of the target protein and a transmembrane region are fused using one gene, It is possible to provide a method for screening cells to be expressed, a cell that produces a target protein at a high level, a method for producing a cell that produces a target protein at a high level, and a method for producing the target protein using the cell.

FIG. 2 shows a schematic diagram of a human antibody expression vector pINC_OX40L / CD98H vector containing DNA fragments encoding the anti-human OX40 antibody L chain and the anti-human CD98 / LAT1 antibody H chain. In the figure, CMV is the CMV promoter, Poly A is the BGH polyadenylation site, Hc is the anti-human CD98 / LAT1 complex antibody heavy chain, Lc is the anti-human OX40 antibody light chain, and SV is the SV40 polyadenylation site. CHX-r represents a cycloheximide resistance gene, respectively. (a) DNA fragment encoding anti-human CD98 / LAT1 antibody H chain, including anti-human OX40 antibody L chain, splicing donor (SD) sequence, first bovine growth factor (BGH) polyadenylation signal sequence, polypyrimidine sequence Of the secreted human antibody and membrane-bound human antibody expression vector pINC_TMSP_AC27, including DNA sequences encoding (poly-Pyr), splicing acceptor (SA) sequence, platelet 、 derived growth factor receptor (PDGFR) transmembrane region (PDGFRtm) A schematic diagram is shown. In the figure, CMV is the CMV promoter, Poly A is the BGH polyadenylation site, Hc is the anti-human CD98 / LAT1 complex antibody heavy chain, Lc is the anti-human OX40 antibody light chain, and SV is the SV40 polyadenylation site. CHX-r represents a cycloheximide resistance gene, and TM represents PDGFRtm. (b) Among the secreted human antibody and membrane-bound human antibody expression vector pINC_TMSP_AC27, the downstream of the platelet derived growth factor receptor membrane region (PDGFRtm) from the DNA fragment (Hc) encoding the H chain of the CD98 / LAT1 antibody. A schematic diagram of the region up to the 2BGH polyadenylation signal sequence is shown. In the figure, Hc is the anti-human CD98 / LAT1 complex antibody heavy chain, SD is the splicing donor sequence, polyA is the BGH polyadenylation signal sequence, and T / C25 is the 25-base polypyrimidine sequence consisting of thymine and cytosine. , SA represents a splicing acceptor sequence, and TM represents PDGFRtm.

Including anti-human OX40 antibody L chain, DNA fragment encoding anti-human CD98 / LAT1 antibody H chain containing SD sequence, 1st BGH-polyA sequence, poly-Pyr sequence, SA sequence, including DNA sequence encoding PDGFRtm, A schematic diagram of a secreted human antibody and a membrane-bound human antibody expressing Tol2 transposon vector AC27 / TnPMug is shown. In the figure, CMV is the CMV promoter, Poly A is the BGH polyadenylation site, Hc is the anti-human CD98 / LAT1 complex antibody heavy chain, Lc is the anti-human OX40 antibody light chain, and SV is the SV40 polyadenylation site. CHX-r represents a cycloheximide resistance gene, TM represents PDGFRtm, Tol2-L represents a left-end Tol2 transposon, and Tol2-R represents a right-end Tol2 transposon. The correlation between the antibody concentration of the secretory antibody (mg / L) and the fluorescence intensity when stained with a PE-conjugated anti-human IgG mouse antibody when CHO-K1 cells transformed with the pINC_TMSP_AC27 vector are cultured is shown. The vertical axis represents the fluorescence intensity (FI), and the horizontal axis represents the antibody concentration (mg / L) of the culture supernatant. (A) The gate diagram developed by forward scatter (FS) and side scatter (SS) in flow cytometer (FCM) analysis is shown. (B) Fluorescence intensity due to binding of PE-conjugated anti-human mouse antibody is shown on the horizontal axis, and the number of counts on the vertical axis. The horizontal line represents the 0.1% fraction of the total count. Secreted / membrane-bound human antibody-expressing Tol2 transposon vector AC10 / TnPMug (a), AC17 / TnPMug (b) or AC27 / TnPMug (c) secreted antibody when cultured with CHO-K1 cells Antibody concentration (mg / L) is shown on the vertical axis, and FITC fluorescence intensity due to the binding of the FITC-conjugated recombinant protein A / G is shown on the horizontal axis. (A) analysis of all H chain mRNA of membrane-bound human antibody in CHO-K1 cells into which secreted / membrane-bound human antibody-expressing Tol2 transposon vector AC10 / TnPMug, AC17 / TnPMug or AC27 / TnPMug vector was introduced, and (b) Real-time PCR schematic diagram of secreted human antibody and membrane-bound human antibody H chain mRNA analysis is shown. In the figure, Fw1 represents a probe-1 Fw primer, Rv1 represents a probe-1 Rf primer, Fw3 represents a probe 3 Fw primer, and Rv3 represents a probe 3 Rv primer. The analysis result of the antibody H chain mRNA expression level of the CHO-K1 cell into which the secreted / membrane-bound human antibody-expressing Tol2 transposon vector AC10 / TnPMug, AC17 / TnPMug, or AC27 / TnPMug vector was introduced is shown. (A) shows the mRNA expression level of the membrane-bound human antibody H chain in each cell when the expression in the cell into which the AC10 / TnPMug vector is introduced is 1. (B) shows the total antibody H chain including the secretory human antibody H chain mRNA and the membrane-bound human antibody H chain mRNA in each cell when the expression in the cell into which the AC10 / TnPMug vector is introduced is 1. Shows the mRNA expression level. (C) shows the ratio (%) of the mRNA expression level of the membrane-bound human antibody H chain in the mRNA expression level of all antibody H chains. A schematic diagram of a membrane-bound Fab fragment encoded by a membrane-bound Fab fragment expression vector is shown. In the figure, VH is the heavy chain variable region, CH1-3 is the heavy chain constant region, SD is the region corresponding to the splicing donor sequence, SA is the region corresponding to the splicing acceptor sequence, and PDGFRtm is the platelet derived growth factor Each of the receptor (PDGFR) transmembrane regions is shown.

In the present invention, a protein in which a target protein and a transmembrane region are fused is defined as a membrane-bound protein. A protein in which a fragment of a target protein and a transmembrane region are fused is defined as a fragment of a membrane-bound protein.
In the present invention, the DNA sequence consists of adenine, thymine, guanine and cytosine bases and is abbreviated as A, T, G and C, respectively. The RNA sequence consists of adenine, uracil, guanine and cytosine bases and is abbreviated as A, U, G and C, respectively.

In the present invention, the expression vector means a DNA in which exogenous DNA is incorporated and can increase in the introduced host cell, and may be simply described as a vector. The vector includes a nucleic acid sequence having a gene expression control region necessary for expressing foreign DNA and a sequence encoding a target protein. Examples of the gene expression control region include enhancers, promoters, and terminators.

In the present invention, splicing refers to a splicing donor sequence (abbreviated as SD), a branch site (branch site), and a splicing acceptor sequence (splicing acceptor sequence) present in a messenger 存在 RNA (mRNA) precursor (pre-mRNA). , Abbreviated as SA) refers to a reaction in which an intron is excluded from an exon by a cleavage-binding reaction by a spliceosome reaction that is a complex of RNA and protein.

Examples of the expression vector of the present invention include an expression vector containing a DNA sequence encoding a target protein and a DNA sequence encoding a transmembrane region, which are cis-actuated on one gene. By integrating the vector into a host cell, a protein (membrane-bound protein or membrane-bound protein fragment) in which the target protein or a fragment of the target protein and the transmembrane region are fused by a splicing reaction of messenger RNA (mRNA) Both of the secretory target proteins can be expressed simultaneously.

Specific examples of the expression vector of the present invention include an expression vector comprising a DNA sequence encoding a target protein including a splicing donor sequence, a polypyrimidine sequence, a splicing acceptor sequence, and a DNA sequence encoding a transmembrane region in this order. .
More specifically, such an expression vector of the present invention, more specifically, a DNA encoding a target protein containing a splicing donor sequence present at the 5 ′ end of a stop codon (TAG, TAA or TGA) of the DNA sequence encoding the protein. An expression vector comprising a DNA sequence encoding a sequence, a polypyrimidine sequence, a splicing acceptor sequence and a transmembrane region.

The splicing donor sequence may be a naturally occurring sequence or an artificially inserted sequence. When an artificial splicing donor sequence is inserted into an expression vector, artificial splicing is performed by designing the codon of the amino acid sequence encoded by the inserted splicing donor sequence and the codon of the target protein to match. Donor sequences can be inserted into expression vectors.

The expression vector of the present invention includes a DNA sequence encoding a protein containing a splicing donor sequence, a first polyadenylation signal sequence, a polypyrimidine sequence, a splicing acceptor sequence, and a DNA sequence encoding a transmembrane region in this order. Also included are expression vectors.
Further, the expression vector of the present invention includes a DNA sequence encoding a protein containing a splicing donor sequence, a first polyadenylation signal sequence, a polypyrimidine sequence, a splicing acceptor sequence, a DNA sequence encoding a transmembrane region, and a second sequence. An expression vector containing a polyadenylation signal sequence in order is mentioned.

The expression vector of the present invention includes a DNA sequence encoding a polyadenylation signal sequence, a polypyrimidine sequence, a splicing acceptor sequence and a transmembrane region on the 3 ′ end side of a DNA sequence encoding a protein containing a splicing donor sequence. And an expression vector containing.
As the expression vector, when animal cells are used as a host, any of those capable of functioning in animal cells can be used. For example, pcDNAI, pCDM8 (manufactured by Funakoshi), pAGE107 [JP-A-3-22979]. ; Cytotechnology, 3, 133 (1990)], pAS3-3 (JP-A-2-27075), pCDM8 [Nature, 329, 840 (1987)], pcDNAI / Amp (Invitrogen), pcDNA3.1 (Invitrogen) ), PREP4 (manufactured by Invitrogen), pAGE103 [J. Biochemistry, 101, 1307 (1987)], pAGE210, pME18SFL3, pKANTEX93 (WO97 / 10354), and transposon vectors (WO2010 / 143698).

Any promoter can be used as long as it can function in mammalian cells. For example, cytomegalovirus (CMV) IE (immediate early) gene promoter, SV40 early promoter, retrovirus promoter, metallothionein promoter, heat shock promoter, SRα promoter, moloney murine leukemia virus promoter And enhancers. In addition, an IE gene enhancer of human CMV may be used together with a promoter.

When the expression vector of the present invention is introduced into a cell, it generates a mature mRNA encoding a membrane-bound protein or the membrane-bound protein fragment by a splicing reaction simultaneously with the generation of a mature mRNA encoding the protein. Can do.
In the present invention, the splicing donor sequence may be any of a sequence that exists in nature and is known to function as a splicing donor sequence, and an artificially created splicing donor sequence, such as M (A / G / T) GGT (A / G) (A / T) (A / G) (A / T) -containing sequence (M is A or C), preferably GTA (A / T) GT-containing sequence, Examples include sequences containing GTATGT and sequences containing M (A / G) GGT (A / G) A (A / G) T (M is A or C) (US Pat. No. 6,642,028).

More specifically, the splicing donor sequence includes a sequence containing CAGGTAAGT (SEQ ID NO: 1), a sequence containing GACGTAAGT (SEQ ID NO: 2) (Lucas et al., Nucleic Acids Research, 24, 1774-1779, 1996.), CGGGTAAAT And a sequence containing (SEQ ID NO: 3).
Further, since the splicing donor sequence may function even if it is a slightly different sequence from the above consensus sequence, the homology with the above consensus sequence is 80% or more, 90% or more, 95% or more, and A splicing donor sequence that causes a splicing reaction can also be used in the present invention.

The immunoglobulin constant region includes a sequence containing a naturally occurring splicing donor sequence, and the immunoglobulin constant region can also be applied as a splicing donor.
In the present invention, examples of the splicing acceptor sequence include a sequence containing N (C / T) AGG (N may be any nucleotide). Specific examples of the splicing acceptor sequence include a sequence containing CAGAA (SEQ ID NO: 4) and a sequence containing GCAGG (SEQ ID NO: 5).

In addition, since the splicing acceptor sequence may function even if the sequence is slightly different from the above consensus sequence, the homology with the above consensus sequence is 80% or more, 90% or more, 95% or more, A splicing acceptor sequence that causes a splicing reaction can also be used in the present invention.
In the present invention, a branch site exists between the splicing donor sequence and the splicing acceptor sequence.

Examples of the branch site include a sequence containing (C / T) N (C / T) T (A / G) A (C / T) (N may be any nucleotide), and 3 'of the splicing donor sequence. Exists on the side. Specific examples include sequences including CATTAACT, TCCTAAT, and the like.
In addition, a polypyrimidine sequence (hereinafter sometimes abbreviated as Poly-Pyr) is a sequence in which bases consisting of C and / or T, which are pyrimidine bases, are continuous between the splicing donor sequence and the branch site. To do.

The number of consecutive pyrimidine bases in the polypyrimidine sequence in the present invention may be 8 bases or more, but preferably 8-30 bases, 9-30 bases, 10-30 bases, 11-30 bases, 12 Examples include lengths of -30 bases, 13-30 bases, 15-30 bases, more preferably 15-29 bases, 15-28 bases, 15-27 bases, 15-26 bases and 15-25 bases.
Specific examples of the Poly-Pyr sequence include the nucleotide sequences represented by SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8.

In the present invention, the polypyrimidine sequence is present between the splicing donor sequence and the splicing acceptor sequence and between the polyadenylation sequence and the splicing acceptor sequence. When the expression vector of the present invention includes a first polyadenylation signal sequence and a second polyadenylation signal sequence, the polypyrimidine sequence is present between the first polyadenylation signal sequence and the splicing acceptor sequence.

In a cell into which a vector capable of simultaneously expressing both the target protein and the membrane-bound protein of the present invention is inserted, the membrane-bound type produced by splicing after the mRNA encoding the target protein transcribed from the introduced vector Both the protein and the mRNA encoding the membrane-bound protein fragment are simultaneously expressed, and both the secreted protein and the membrane-bound protein or the membrane-bound protein fragment are expressed.

When the target protein is a secreted protein, the expression level ratio between the secreted protein (sP) and the membrane-bound protein or the membrane-bound protein fragment (mP) may be in the range of sP / mP = 1000-50. preferable. By adjusting the length of the poly-Pyr sequence used in the present invention, the sP / mP ratio can be controlled. Specifically, when the poly-Pyr sequence is shortened, the splicing reaction efficiency decreases, and when the poly-Pyr sequence is lengthened, the splicing reaction efficiency increases.

In the present invention, an amino acid sequence derived from any protein can be used as the transmembrane region as long as the protein penetrates the cell membrane or is anchored to the cell membrane. The transmembrane region used in the present invention may be any of 1 to 12 transmembrane membrane proteins or GPI anchor proteins (European Patent Publication No. 1716233), such as a cell membrane ligand (or precursor ligand), Examples include a transmembrane region derived from a membrane protein selected from a cell membrane receptor, a T cell receptor, an adhesion molecule, a major histocompatibility complex (MHC), a membrane immunoglobulin and a GPI anchor protein.

Cell membrane ligands include epidermal growth factor (EGF) ligand family, EGF, transforming growth factor-α (TGF-α), amphiregulin, betacellulin, epiregulin, heparin-binding epidermal growth factor-like growth factor (HB-EGF ), Transmembrane regions such as NTAK, vascular endothelial growth factor (VEGF) and heregulin (neuregulin), FAS ligand, TRAIL ligand.

Cell membrane receptors include EGF receptor (EGFR), insulin-like growth factor receptor I (IGF-IR), hepatocyte growth factor receptor (HGFR, cMet), platelet derived growth factor receptor (PDGFR), vascular endothelial growth Transmembrane regions such as VEGFR), amino acid transporters, tetraspanin-type proteins, G-protein coupled receptors (GPCR), and Cluster of differentiation (CD) antigens.

Examples of MHC include MHC class I and class II transmembrane regions.
Membrane immunoglobulins include transmembrane regions of membrane-bound IgM, IgD, IgG, IgE and IgA antibodies (Peterson et al., PNAS, 83, 8883-8887, 1986, Rebecca et al., 32, 277-285, 1995, Tsurushita and Korn et al., Mol. Cell Biol., 1987, 7, 2602-2605).

Specific examples of the transmembrane region of the present invention include the PDGFR transmembrane region. Examples of the DNA encoding the transmembrane region include a DNA comprising the base sequence represented by SEQ ID NO: 12.
In the present invention, the polyadenylation signal (hereinafter abbreviated as polyA) may be any polyadenylation signal as long as it contains an AATAAA consensus sequence and can be polyadenylated, and specifically, siman virus 40 (SV40) late polyadenylation signal (Scheck et al., Mol. Cell Biol., 12, 5386-5393, 1992.), HIV-1 poly A (Klasens et al., 26, 1870-1876, 1998.), βglobin poly A (Gil et al., Cell, 49, 399-406, 1987.), HSV TK poly A (Cole et al., Mol.Cell Biol., 5, 2104-2113, 1985.), polyoma virus poly A (Batt et al., 15, 4783-4790, 1995.) and bovine growth hormone poly A (Gimmi et al., Necleic Acids Res., 17, 6983-6998, 1989.) βgalactosidase poly A, αglobin poly A, human growth hormone poly A (JP 2002) -233374).

In the present invention, as the first polyadenylation signal or the second polyadenylation signal, the same sequence may be used, or two different types of poly A signals may be used.
The DNA encoding the target protein in the present invention is a DNA sequence encoding the amino acid sequence of the full-length protein or a partial fragment of the protein, and starting from the start codon (ATG) to the stop codon (TAA, TAG or TGA) Say. A full-length protein or a partial fragment of the protein may be a functional protein or a simple structural protein.

In the present invention, a DNA encoding a protein containing a splicing donor sequence is a DNA sequence encoding a target protein containing a splicing donor sequence that exists in nature in the DNA sequence encoding the amino acid sequence of the target protein. Examples of the DNA sequence encoding the target protein include a splicing donor sequence artificially inserted in the DNA sequence encoding the amino acid sequence, and any of them may be used.

Specifically, as a DNA sequence encoding a target protein including a splicing donor, a DNA sequence having a splicing donor sequence at the 5 ′ end side of the stop codon of the full length or partial fragment of the target protein to be expressed, and a purpose to be expressed Examples thereof include a DNA sequence in which a splicing donor sequence is artificially inserted at the 5 ′ end side of the stop codon of the full-length protein or a partial fragment.

When inserting a splicing donor sequence, the codon of the DNA sequence into which the SD sequence is inserted matches the codon encoding the amino acid sequence of the target protein, and the codon of the DNA sequence generated as a result of the splicing reaction is a membrane. By designing the splicing donor sequence so as to match the codon of the amino acid sequence of the bound protein, a vector that simultaneously expresses the target protein and the membrane-bound protein or a fragment of the membrane-bound protein can be prepared.

For example, when an immunoglobulin fragment is expressed as a membrane-bound protein fragment, it can be carried out by introducing a splicing donor sequence into the antibody constant region. The antibody constant region may be any of a CH1 domain, a hinge domain, a CH2 domain, and a CH3 domain, and preferred examples include insertion of a splicing donor sequence into the CH2 domain. When using an antibody expression vector in which a splicing donor sequence is inserted into the CH2 domain, a secretory antibody and a Fab fragment spliced with the CH2 domain and fused to the transmembrane region are expressed on the cell membrane of the cell into which the vector has been introduced. be able to.

Specifically, the codon encoding the 51st glycine of the IgG2 antibody CH2 domain was replaced from GGC to GGT, and the codon encoding the 6th glycine of the IgG2 antibody CH2 domain was replaced from GGG to GGT SD sequence in which the codon encoding the 86th glycine of the CH2 domain of IgG1 antibody is replaced from GGC to GGT, and the codon encoding the 48th tyrosine of the CH2 domain of IgG1 antibody is changed from TAC to TAT. Examples include substituted SD sequences.

The splicing donor sequence may be naturally occurring in an immunoglobulin constant region or an artificial sequence, depending on the target protein to be expressed and the membrane-bound protein or the membrane-bound protein fragment. In addition, it may be designed by inserting it at any position as long as it is at the 5 ′ end side of the stop codon in the target protein.
The protein in the present invention refers to a protein produced using the above-described expression vector.
In the present invention, the protein composition refers to a composition containing protein molecules produced and purified by the protein production method of the present invention. In the present invention, a protein composition includes a protein composition that includes two or more different variations of post-translational modifications.

Protein post-translational modifications include SS-linked cross-linking of amino acid side chains, glycosylation (N-linked sugar chains, O-linked sugar chains), phosphorylation, sulfation, methylation, myristoylation, and peptide chain specificity For example.
The protein composition in the present invention is a composition containing any degree of post-translational modification protein as long as it is a modified protein variation within a range acceptable as a protein pharmaceutical obtained by the protein production method of the present invention. Also good.

In the present invention, the target protein may be any protein as long as it can be expressed as a membrane-bound protein and protein using the expression vector of the present invention. Examples include human serum proteins, albumin binding proteins, peptide hormones, growth factors, cytokines, blood clotting factors, fibrinolytic proteins, antibodies, membrane proteins, and partial fragments of various proteins. Specifically, human intravenous immunoglobulin (IVIG), erythropoietin (EPO), albumin, growth hormone (GH), follicle stimulating hormone (FSH), hepatocyte growth factor (HGF), insulin, insulin-like growth factor- I (IGF-I), interferon (IFN), Fas ligand, blood coagulation factor (II, VII, VIII, IX, X), prothrombin, fibrinogen, protein C, protein S, antithrombin III (ATIII), tissue plasminol Examples include a gene activator (tPA), a monoclonal antibody, an oligoclonal antibody, and a polyclonal antibody.

Examples of the monoclonal antibody in the present invention include monoclonal antibodies and gene recombinant antibodies produced from hybridomas. Examples of the recombinant antibody include a chimeric antibody, a humanized antibody [also referred to as a complementarity determining region (CDR) transplanted antibody], a human antibody, and the like.
The monoclonal antibody in the present invention is an antibody secreted by antibody-producing cells of a single clone, recognizes only one epitope (also called an antigenic determinant), and has a uniform amino acid sequence (primary structure) constituting the monoclonal antibody. It is. Oligoclonal antibodies and polyclonal antibodies are antibody mixtures containing two or more monoclonal antibodies.

Antibody molecules are also called immunoglobulins (hereinafter referred to as Ig), and human antibodies are classified into IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, and IgM isotypes according to the difference in molecular structure. Is done. IgG1, IgG2, IgG3, and IgG4 having relatively high amino acid sequence homology are collectively referred to as IgG.
Antibody molecules are composed of polypeptides called heavy chains (hereinafter referred to as H chains) and light chains (hereinafter referred to as L chains). In addition, the H chain is the H chain variable region (also referred to as VH) and the H chain constant region (also referred to as CH) from the N terminal side, and the L chain is also expressed as the L chain variable region (VL) from the N terminal side. ) And L chain constant region (also referred to as CL). As for CH, α, δ, ε, γ, and μ chains are known for each subclass. CH is further composed of each domain of the CH1 domain, hinge domain, CH2 domain, and CH3 domain from the N-terminal side. A domain refers to a functional structural unit constituting each polypeptide of an antibody molecule. The CH2 domain and the CH3 domain are collectively referred to as Fc region or simply Fc. As for CL, Cλ chain and Cκ chain are known.

In addition, examples of the fusion protein in the present invention include those obtained by fusing different protein fragments with an appropriate linker or the like. Specifically, an Fc fusion protein (also referred to as an immunoadhesin) in which an antibody Fc region is fused with another protein, an Fc fusion protein in which multiple Fc regions are fused, a glutathioneathS-transferase (GST) fusion protein, FLAG fusion protein, Histidine tag fusion protein, green fluorescence protein (GFP) fusion protein and the like are also included in the protein of the present invention. Also included is an Fc region (Fc variant) containing an amino acid residue modification that has been modified to enhance or lack the effector activity of the antibody, stabilize the antibody, and control the blood half-life. It can be used for an antibody expressed by the expression vector of the invention.

Furthermore, examples of the protein in the present invention include bispecific antibodies and multivalent antibodies (WO1998 / 050431, WO2001 / 7734, WO2002 / 002773, WO2009 / 131239).
The protein fragment in the present invention may be either a functional fragment or a structural fragment of a protein, particularly an antibody fragment. Examples of antibody fragments include Fab, Fab ′, F (ab ′) ₂ , single chain Fv (scFv), diabody, dsFv, peptides containing multiple CDRs (particularly peptides containing 6 CDRs of VH and VL), etc. Can be mentioned.

Examples of the cell of the present invention include a cell into which the above-described expression vector is introduced, and specifically includes a cell into which the expression vector of the present invention has been introduced. The cell can simultaneously express both a membrane-bound protein or the membrane-bound protein fragment and a target protein.
The cell of the present invention is a cell into which the expression vector of the present invention has been introduced, and an mRNA encoding a target protein transcribed from the vector and an mRNA encoding a membrane-bound protein generated by splicing after transcription. Among these two types of mRNAs, 0.1% or more of the cells express mRNA that encodes a membrane-bound protein. The mRNA expression ratio (%) of the membrane-bound protein to the total mRNA of the protein of interest (mRNA that encodes the membrane-bound protein or the membrane-bound protein fragment + mRNA that encodes the protein of interest) is 0.1-5%, 0.1 The range of ˜4%, 0.1 to 3%, and 0.1 to 2% is preferable, but the range of 0.5 to 2% is preferable, more preferably 1 to 2%, 1.1 to 2%, 1.2 to 2%, and 1.3 to 2%. 1.4 to 2% range.

A cell into which the protein expression vector of the present invention has been introduced can simultaneously express both the target protein and a membrane-bound protein or a fragment of the membrane-bound protein, and the membrane-bound protein is expressed on the cell membrane. Yes.
In the present invention, when the target protein is a secretory protein, the expression level ratio between the secreted protein (sP) and the membrane-bound protein or the membrane-bound protein fragment (mP) is sP / mP = 1000-50. A range is preferable. Alternatively, the expression level ratio of mRNA encoding secreted protein (sP-mRNA) and mRNA encoding membrane-bound protein or membrane-bound protein fragment (mP-mRNA) is sP-mRNA / mP-mRNA = 1000 A range of ˜50 is preferred.

In the present invention, the expression level of the membrane-bound protein or the fragment of the membrane-bound protein is a conjugate (chemical substance, antibody, protein ligand, etc.) that specifically binds to the membrane-bound protein or the fragment of the membrane-bound protein. The expression level may be any level as long as it can be detected by binding analysis using a cell, and can be measured by methods such as Cell binding ELISA, flow cytometer (FCM), FMAT8100HTS system (Applied Biosystems).

Specific examples of the cell of the present invention include a cell expressing 1 × 10 ³ molecules / cell or more of a membrane-bound protein or a fragment of the membrane-bound protein on the cell membrane. As the expression level of the membrane-bound protein or the fragment of the membrane-bound protein on the cell membrane, cells expressing 1 × 10 ³ to 1 × 10 ⁷ molecules / cell are preferable. The expression level of the mRNA encoding the membrane-bound protein or the fragment of the membrane-bound protein is preferably expressed by about 0.1% to 2% of the total protein mRNA (sum of the expression levels of sP-mRNA and mP-mRNA). Cell.

As the cell into which the expression vector of the present invention is introduced, any cell generally used for recombinant protein production, such as animal cells, plant cells, and microorganisms, can be used.
Examples of cells into which the expression vector of the present invention is introduced include Chinese hamster ovary tissue-derived CHO cells, human cell PER.C6, human leukemia cell Namalwa cell, monkey cell COS, rat myeloma cell line YB2 / 3HL.P2.G11.16Ag. 20 cells, mouse myeloma cell line NS0 cell, mouse myeloma cell line SP2 / 0-Ag14 cell, Syrian hamster kidney tissue-derived BHK cell, human Burkitt lymphoma-derived Namalva cell, human retinoblastoma-derived PER.C6 cell, human embryonic Examples include kidney tissue-derived HEK293 cells, human myeloid leukemia-derived NM-F9 cells, embryonic stem cells, and fertilized egg cells. Preferably, a host cell for producing a recombinant glycoprotein pharmaceutical, an embryonic stem cell or a fertilized egg cell used for producing a transgenic non-human animal producing the recombinant glycoprotein pharmaceutical, and a recombinant sugar Examples include plant cells used to produce transgenic plants that produce protein drugs.

Preferred examples of the parent cell line include the following cells.
NS0 cell parent cell lines include NS0 cells described in literature such as BIO / TECHNOLOGY, 10, 169 (1992), Biotechnol. Bioeng., 73, 261, (2001). In addition, NS0 cell line (RCB0213) registered in RIKEN Cell Bank, or sub-strains obtained by acclimating these strains to various serum-free media are also included.

Examples of parental cells of SP2 / 0-Ag14 cells include J. Immunol., 126, 317, (1981), Nature, 276, 269, (1978), Human Antibodies and Hybridomas, 3, 129, (1992), etc. SP2 / 0-Ag14 cells described in the above. In addition, SP2 / 0-Ag14 cells (ATCC CRL-1581) registered in ATCC or sub-strains (ATCC CRL-1581.1) in which these strains are conditioned to various serum-free media are also included.

As a parent cell of Chinese hamster ovary tissue-derived CHO cells, JournalJof Experimental Medicine, 108, 945 (1958), Proc. Natl. Acad. Sci. USA, 60, 1275 (1968), Genetics, 55, 513 (1968) , Chromosoma, 41, 129 (1973), Methods in Cell Science, 18, 115 (1996), Radiation Research, 148, 260 (1997), Proc. Natl. Acad. Sci. USA, 77, 4216Pro (1980), Proc CHO cells described in literature such as “Natl.” Acad. “Sci.” 60, “1275” (1968), Cell, “6”, “121” (1975), Molecular “Cell” Genetics, “Appendix I, II” (p883-900), and the like. In addition, CHO-K1 strain (ATCC CL CCL-61), DUXB11 strain (ATCC CRL-9096), Pro-5 strain (ATCC CRL-1781) registered in ATCC, and commercially available CHO-S strain (Life technologies) (Seizo Cat. No. 11619) or sub-strains obtained by acclimating these strains to various serum-free media.

The parent cell of the rat myeloma cell line YB2 / 3HL.P2.G11.16Ag.20 cell includes a cell line established from Y3 / Ag1.2.3 cell (ATCC CRL-1631). Specific examples thereof include YB2 / 3HL.P2.G11.16Ag.20 cells described in J. CellBiol., 93, 576 (1982), Methods Enzymol. 73B, 1 (1981) and the like. Can be mentioned. In addition, YB2 / 3HL.P2.G11.16Ag.20 cells (ATCC CRL-1662) registered in ATCC or sub-strains obtained by acclimating these strains to various serum-free media are also included.

In order to express antibodies with high ADCC activity, the amount of fucose that binds α1,6 to N-acetylglucosamine G (GlcNAc) present at the reducing end of the N-glycoside-linked sugar chain that binds to the antibody Fc region is reduced or eliminated. Cells can also be used. The cell is a cell in which at least one enzyme associated with a series of fucose metabolic reactions is reduced or deficient. As an enzyme related to fucose metabolic reaction, protein such as an enzyme involved in synthesis of intracellular sugar nucleotide GDP-fucose or N-acetylglucosamine at the reducing terminal of N-glycoside-linked complex sugar chain is located at position 1 of fucose at α-position. Examples include proteins such as enzymes involved in modification of the sugar chain to be bound, proteins involved in transport of intracellular sugar nucleotide GDP-fucose to the Golgi apparatus, and the like. Examples of cells in which these activities are reduced or deleted include CHO cells in which the α1,6-fucose transferase (FUT8) gene is reduced or deleted (WO2005 / 035586, WO02 / 31140), lentil lectin LCA, pea lectin PSA , Cells that have acquired resistance to lectins such as broad bean lectin VFA or yellow chawantake lectin AAL (WO02 / 31140), Lec13 [Somatic Cell and Molecular genetics, with reduced GMD-mannose-4,6-dehydratase (GMD) activity, 12, 55 (1986)], cells with reduced GMD activity, cells with reduced or deficient GDP-fucose transporter (WO2003 / 85102) and the like.

Mammalian cells used in the present invention are suspended in a culture solution without adhering to a cell culture support in a serum-free medium not containing fetal calf serum (hereinafter referred to as FCS). Cells that can survive and proliferate are preferred, and mammalian cells that can float and survive and proliferate in a protein-free medium without protein are more preferred.
The tissue culture incubator for confirming mammalian cells that can survive in a serum-free medium may be any incubator as long as it is a flask, petri dish or the like that is coated with an adhesion culture. Specifically, for example, by culturing a cell using a commercially available tissue culture flask (manufactured by Greiner) and an adhesion culture flask (manufactured by Sumitomo Bakelite Co., Ltd.), the property that the cells do not adhere to the adhesive culture flask By confirming, it is possible to confirm floating mammalian cells.

The suspension mammalian cell used in the present invention may be a cell originally adapted to suspension culture having suspension properties, or a suspension in which adhesive mammalian cells are adapted to suspension culture conditions. Any mammalian cell may be used.
The “floating mammalian cell obtained by acclimatizing an adherent mammalian cell to a floating culture condition” is, for example, according to the method described in Mol. Biotechnol., 2000, 15 (3), 249-257, etc. It can be produced by establishing cells that exhibit the same proliferation and viability as before suspension culture acclimation or better than those before acclimation to suspension culture (J. Biotechnol., 2007, 130 (3), 282-290).

As a method for introducing an expression vector into cells, any method can be used as long as it introduces DNA into animal cells. For example, electroporation [Cytotechnology, 3, 133 (1990)], calcium phosphate method (specialized) Kaihei 2-227075), a method using calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972), Gene, 17, 107 (1982), Molecular & General Genetics, 168, 111 (1979)] Or the lipofection method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)].

Examples of the method for expressing the target protein of the present invention include a method in which the expression vector of the present invention is introduced into a host cell and both the target protein and the membrane-bound protein or a fragment of the membrane-bound protein are expressed simultaneously.
The method of expressing the protein of the present invention can be used to select cells expressing a membrane-bound protein without expressing a drug selection marker gene or an unnecessary fusion protein and without selecting a drug. it can. As a result of selecting a cell that highly expresses a membrane-bound protein or a fragment of the membrane-bound protein, a cell that highly expresses the target protein can be obtained. Furthermore, by adjusting the position of the splicing donor sequence of the present invention and the likelihood (frequency) of the splicing reaction, a desired plurality of target proteins such as a full length (or partial fragment) of the target protein and a membrane-bound protein can be simultaneously obtained. It can also be expressed.

Examples of membrane-bound target protein fragments include antibody Fab fragments when the target protein is an antibody. Fab fragments can be expressed by artificial insertion of splicing donor sequences anywhere in the antibody constant region. The Fab fragment can be expressed particularly by inserting an artificial splicing donor sequence in the CH2 domain.

Examples of the method for producing the target protein of the present invention include a method of introducing the expression vector of the present invention into a host cell and producing the target protein using the cell.
Examples of the method for producing a target protein of the present invention include a method for producing a target protein including the following steps (a) to (e).
(a) Introducing into a host cell a protein expression vector comprising a DNA sequence encoding a protein containing a splicing donor sequence, a polyadenylation signal sequence, a polypyrimidine sequence, a splicing acceptor sequence and a DNA sequence encoding a transmembrane region. Process.
(b) A step of expressing a target protein outside the protein producing cell and expressing a membrane-bound protein or a fragment of the membrane-bound protein on the cell membrane of the protein-producing cell.
(c) A step of detecting a membrane-bound protein or a fragment of the membrane-bound protein and selecting a protein-producing cell.
(d) A step of culturing the selected protein-producing cells in large quantities.
(e) A step of purifying the target protein from the culture supernatant.

In addition, as a screening method for cells that produce the target protein of the present invention at a high level, the expression vector of the present invention is introduced into a host cell, and a cell that highly expresses a membrane-bound protein or a fragment of the membrane-bound protein is obtained. A screening method is included. As a result of selecting cells that highly express a membrane-bound protein or a fragment of the membrane-bound protein by this screening method, cells that produce the target protein at a high level can be obtained.

Animal cell culture media include RPMI1640 medium (Invitrogen), GIT medium (Nippon Pharmaceutical), EX-CELL301 medium (JRH), EX-CELL302 medium (JRH), IMDM medium (Invitrogen) ), Hybridoma-SFM medium (Invitrogen), CD-CHO medium (Invitrogen), EX-CELL 325-PF medium (SAFC Biosciences) and SFM4CHO medium (HyClone) it can. It can also be obtained by blending and preparing necessary sugars, amino acids, etc. in animal cell culture media.

As the culture conditions, for example, static culture can be performed in a 5% CO ₂ atmosphere at a culture temperature of 37 ° C. It can also be cultured by methods such as Wave bioreactor (GE Healthcare Bioscience), swirl stirrer, bioreactor culture [Cytotechnology, (2006), 52: 199-207] it can.
Cells expressing the membrane-bound protein of the present invention or a fragment of the membrane-bound protein are detected using a compound or protein that specifically binds to the expressed membrane-bound protein or the fragment of the membrane-bound protein. can do. Any of them can be used as long as it binds to a membrane-bound protein or a fragment of the membrane-bound protein. For example, a fluorescent protein (FITC, TRITC, etc.) can be used as a ligand protein, a receptor protein, a polyclonal antibody, a monoclonal antibody and an antigen protein. PE, such as Cy-5), an enzyme (peroxidase, alkaline phosphatase), biotin, with a radioactive substance ^{^{^{(3 H, 14 C, 32}}} P, 35 S, 67 Ga, 99 Tc) detector conjugated etc. In addition, cells expressing a membrane-bound protein or a fragment of the membrane-bound protein can be detected.

When the target protein to be expressed or the fragment of the target protein is a human IgG antibody or the antibody fragment, an anti-human IgG antibody, protein A / G or an antigen specifically recognized by the human IgG antibody is conjugated with an appropriate detection substance The membrane-bound human IgG antibody can be detected using the resulting product. In addition, when the target protein to be expressed is a TNFR-Fc fusion protein, a membrane-bound TNFR is prepared using a TNF-α, anti-Fc antibody or protein A / G conjugated with an appropriate detection substance. -Fc fusion protein can be detected.

In addition, the present invention includes a method for analyzing the protein modification ability and modification characteristics of a cell producing the target protein. That is, a cell into which a secretory / membrane-bound protein expression vector has been introduced expresses a membrane-bound protein or the membrane-bound protein fragment on the cell membrane and produces a secreted protein. By analyzing the degree of protein modification of the expressed membrane-bound protein or the membrane-bound protein fragment, the protein modification ability and modification characteristics of the cell can be identified.

Proteins are modified as a result of post-translational modifications such as sugar chains, amino groups, carboxyl groups, phosphoric acid, and sulfuric acid bound to the protein. Specifically, the protein modification level can be directly analyzed using a substance that specifically binds to a modified residue of the membrane-bound protein or the membrane-bound protein fragment, and the protein on the cell membrane can be analyzed with an enzyme or the like. It can also be analyzed indirectly by analyzing the free protein by releasing it by digesting with.

When the protein modification is a sugar chain modification, it is specific to the sugar chain structure, such as a protein (lectin) that specifically binds to each sugar chain binding structure, an anti-sugar chain antibody, a ligand protein, a receptor protein, a low molecular weight compound, etc. Any one may be used as long as it is coupled.
A method for analyzing the sugar chain-modifying ability and sugar chain-modifying properties of protein-producing cells by the above-described method is also included in the present invention. By this method, a target protein-producing cell capable of producing a specific sugar chain structure of the target protein can be screened and obtained.

As a detection method, measurement using a method described in monoclonal antibody-Principles-and-practice, Third-edition, Academic Press (1996), monoclonal antibody experiment manual (Kodansha Scientific 1987), and specifically enzyme Examples include -linked immunosorbent assay (ELISA), fluorescence-activated cell sorter (FACS) analysis using flow cytometer (FCM) method, and fluorescence staining using FMAT8100HTS system (Applied Biosystems). Further, by sorting cells that highly express a membrane-bound protein using FACS or the like, that is, cells having high fluorescence intensity, a high protein-producing cell can be obtained more directly.

Examples of the method for measuring secretory protein in the present invention include the above-mentioned ELISA method, sandwich ELISA method, Biacore system using surface plasmon resonance (SPR) method, measurement method using high performance liquid chromatography (HPLC), and the like. It is done.
In the present invention, the target protein can be purified using affinity chromatography, ion exchange chromatography, ultrafiltration, gel filtration, or the like. For example, antibodies are purified from the culture supernatant of transformants using protein A-columns [Monoclonal Antibodies-Principles and practice, Third edition, Academic Press (1996), Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory (1988) )]. Also, methods used in protein purification such as gel filtration, ion exchange chromatography and ultrafiltration can be combined.

Examples of the present invention will be described below, but the present invention is not limited thereto.

Preparation of vector capable of simultaneously expressing secretory antibody and membrane-bound antibody Vectors capable of simultaneously expressing secretory antibody and membrane-bound antibody (hereinafter abbreviated as secretory / membrane-bound antibody expression vectors) pINC_TMSP_AC27, pINC_TMSP_AC17 and pINC_TMSP_AC10 It was produced as follows.
(1) Preparation of expression vector pINC_OX40L / CD98H containing DNA encoding anti-OX40 antibody L chain and anti-CD98 / LAT1 antibody H chain CMV promoter, cycloheximide resistance gene and anti-M2 antibody expression vector described in WO2010 / 143698 An antibody expression vector (pINC vector) was prepared by reversing the gene fragment containing the SV40 early polyadenylation site.
Next, a gene fragment containing the anti-OX40 antibody light chain (L chain) linked under the control of CMV enhancer / promoter (Special Table No. 2009-518005) (SEQ ID NO: 9), and linked under the control of CMV enhancer / promoter A gene fragment containing the anti-CD98 / LAT1 complex antibody heavy chain (H chain) (JP 2009-189376 A) (SEQ ID NO: 10) was prepared. A DNA sequence containing a splicing donor sequence GGGTAAAT and a stop codon (TGA) was ligated to the 3 ′ end of the DNA sequence encoding the H chain.

The prepared gene fragment was inserted into an appropriate position of the pINC vector to prepare an expression vector pINC_OX40L / CD98H containing DNA encoding the anti-OX40 antibody L chain and the anti-CD98 / LAT1 complex antibody H chain (FIG. 1). (Hereinafter, in this example, an antibody comprising the L chain of an anti-OX40 antibody and the H chain of an anti-CD98 / LAT1 antibody is abbreviated as a human IgG antibody).
(2) Preparation of secretory / membrane-bound human IgG antibody expression vectors pINC_TMSP_AC27, pINC_TMSP_AC17 and pINC_TMSP_AC10 The vector pINC_TMSP_AC27 (Fig. 2) capable of simultaneously expressing secreted human and membrane-bound human antibodies is the bovine growth hormone (BGH) polya Denylation signal sequence (hereinafter abbreviated as BGH-PolyA) (SEQ ID NO: 11), 25-base polypyrimidine sequence (hereinafter abbreviated as T / C sequence 25) consisting of thymine and cytosine (SEQ ID NO: 8), splicing DNA sequence encoding the acceptor sequence (hereinafter abbreviated as SA sequence) (SEQ ID NO: 5) and the platelet-derived growth factor receptor (PDGFR) transmembrane region (SEQ ID NO: 13) (hereinafter abbreviated as PDGFR transmembrane; PDGFRtm) ) (SEQ ID NO: 12) ligated DNA fragments were inserted into the 3 ′ end of the H chain stop codon and prepared as follows.

Using the pINC_OX40L / CD98H expression vector as a template, polymerase chain reaction (PCR) was performed using synthetic DNA primers TM-F1 (SEQ ID NO: 14) and TM-R1 (SEQ ID NO: 15) to amplify DNA fragments. Next, after the PCR reaction using the amplified DNA fragment as a template and the synthetic DNA primers TM-F1 and TM-R2 (SEQ ID NO: 16), the synthetic DNA primers TM-F1 and TM-R3 (SEQ ID NO: 17) were used. A DNA fragment in which the DNA sequence encoding the transmembrane region (SEQ ID NO: 12) was bound to the 3 ′ end of the human antibody H chain was prepared. Next, the obtained DNA fragment and the pINC_OX40L / CD98H vector were treated with restriction enzymes BgIII and EcoRI, respectively, and the DNA sequence encoding the PDGFR transmembrane region was found at the 3 ′ end of the DNA sequence encoding the human antibody H chain. A membrane-bound human IgG antibody expression vector pINC_TM vector containing the inserted DNA fragment was prepared.

Next, using the pINC_TM vector as a template, PCR reaction was performed using the synthetic DNA primers AC27-F (SEQ ID NO: 18) and TMSP-R (SEQ ID NO: 19), T / C sequence 25 (SEQ ID NO: 8), SA sequence (SEQ ID NO: 5), a DNA fragment (SEQ ID NO: 11) containing PDGFRtm and BGH-PolyA sequences was prepared. The first BGH-PolyA sequence, the DNA encoding the human antibody H chain including the SD sequence, by inserting the prepared DNA fragment into the DNA encoding the human antibody H chain of the pINC_OX40L / CD98H expression vector treated with the restriction enzyme EcoRV , A secreted / membrane-bound human antibody expression vector pINC_TMSP_AC27 containing a poly-Pyr sequence (T / C sequence 25), an SA sequence, a PDGFRtm sequence, and a second BGH-PolyA sequence in order was prepared (FIG. 2).

Similarly, the poly-Pyr sequence T / C25 (SEQ ID NO: 8) on the 3 ′ end side of the first BGH-PolyA sequence is replaced with a 15-base poly-Pyr sequence T / C15 (SEQ ID NO: 7) consisting of T and C. Alternatively, secreted / membrane-bound human IgG antibody expression vectors pINC_TMSP_AC17 and pINC_TMSP_AC10 substituted with 8-base poly-Pyr sequence T / C8 (SEQ ID NO: 6), respectively, were prepared.

Expression of human IgG antibody in secretory / membrane-bound human IgG antibody expression vector-introduced cells (1) Preparation of secreted / membrane-bound human IgG antibody expression vector pINC_TMSP_AC27-introduced cells Secreted / membrane prepared in Example 1 (2) The conjugated human IgG antibody expression vector is a Chinese hamster ovary cell line CHO-K1 (American Type Culture Collection, Cat. No. CCL) using a gene introduction reagent LipofectAmine (registered trademark) 2000 (Life Technologies) according to the attached manual. -61).

Specifically, CHO-K1 cells that became 70-80% confluent in a 100 mm culture plate were washed with serum-free medium and then α-minimum essential medium medium (hereinafter referred to as MEM medium) (Life Technologies) Was added. Next, 24 μg of secreted / membrane-bound human IgG antibody expression vector pINC_TMSP_AC27 and 60 μL of LipofectAmine (registered trademark) 2000 solution were mixed and added to the cells, followed by static culture in a 37 ° C., 5% CO ₂ incubator for 6 hours Was done. After culturing, the medium was replaced with MEM medium supplemented with 10% fetal bovine serum (hereinafter abbreviated as FCS) (SAFC Biosciences), and diluted appropriately so that the cells did not exceed 90% confluence.

After culturing for 7 days, the cells were detached by trypsin treatment to collect CHO-K1 cells, and 1 × 10 ⁶ cells / mL of cells using phosphate buffer saline (hereinafter abbreviated as PBS) (Life Technologies). A suspension was prepared. 50 μL of mouse Anti-Human IgG (Fc) -PE (hereinafter referred to as “anti-human IgG mouse antibody”) (BECKMAN, Cat. No. 736007) was added to the cell suspension and reacted at room temperature for 1 hour. After the reaction, the cell suspension was centrifuged, and the cells were washed with PBS to remove excess anti-human IgG mouse antibody. Next, after centrifugation, 10 mL of MEM medium was added to the cells and suspended, and the cell suspension diluted stepwise to 1 cell / well was dispensed into a 96-well plate.

The fluorescence intensity of the cells contained in each well was measured using a cell image analysis system (Olympus CELAVIEW RS100). As a result, wells containing multiple cells were visually excluded, and wells where fluorescence was not detected were also excluded. Wells in which fluorescence was detected at 1 cell / well were selected. The cells were cultured until the cells in the selected wells were approximately 90% confluent, and the antibody concentration in the secreted culture medium was measured. The antibody concentration in the culture was measured using HPLC (Waters).

As a result, the anti-human IgG mouse antibody bound to the pINC_TMSP_AC27 expression vector-introduced cell, and the fluorescently stained CHO-K1 cell expressed the IgG antibody in the culture medium. Moreover, the fluorescence intensity of CHO-K1 cells and the antibody concentration in the culture supernatant showed a positive correlation from the results of multiple cell clones (FIG. 4).
Therefore, the cells into which the expression vector pINC_TMSP_AC27 containing the DNA sequence encoding the human antibody H chain containing the SD sequence, the first BGH-PolyA sequence, the poly-Pyr sequence, the SA sequence, the PDGFRtm and the second BGH-PolyA sequence are introduced on the cell membrane It was revealed that a membrane-bound human IgG antibody can be expressed at the same time and a secretory IgG antibody can be expressed simultaneously in the culture supernatant. It was also revealed that the amount of membrane-bound human IgG antibody expressed on the cell membrane increased with a positive correlation with the amount of secreted human IgG antibody.
(2) Examination of polypyrimidine sequence in secretory / membrane-bound human IgG antibody expression vector Secretion / membrane-bound human IgG antibody expression vector pINC_TMSP_AC10 with different lengths of the poly-Pyr sequence prepared in Example 1 (2) PINC_TMSP_AC17 and pINC_TMSP_AC27 were introduced into CHO-K1 cells in the same manner as in Example 2 (1), and fluorescence staining of the cells and measurement of the antibody concentration in the culture supernatant were performed. Table 1 shows the relative values of AC17 and AC10-introduced cells when the amount of secretory antibody in the AC27 culture supernatant and the intensity of fluorescence staining with anti-human IgG mouse antibody are taken as 100.

As a result, the anti-human IgG mouse antibody also bound to CHO-K1 cells into which any of the expression vectors pINC_TMSP_AC10, pINC_TMSP_AC17 and pINC_TMSP_AC27 was introduced. Further, IgG antibody was detected in the culture supernatant.
Therefore, it was revealed that an expression vector containing a polypyrimidine sequence having a length of 8 bases or more can express both a secretory IgG antibody and a membrane-bound IgG antibody at the same time.

In addition, in cells transfected with pINC_TMSP_AC10 whose poly-Pyr sequence is 8 bases, the fluorescence intensity of the membrane-bound IgG antibody expressed on the cell membrane is higher than that of cells introduced with pINC_TMSP_AC27 whose poly-Pyr sequence is 25 bases. It was low.
Therefore, splicing efficiency from mRNA encoding secretory human IgG antibody to mRNA encoding membrane-bound IgG antibody is reduced in cells introduced with pINC_TMSP_AC10, which has a shorter poly-Pyr sequence than pINC_TMSP_AC27. It was revealed.

Production of secretory / membrane-bound anti-human antibody expression vector using Tol2 transposon The pINC_TMSP_AC27 expression vector produced in Example 1 (2) was treated with restriction enzymes SalI and NotI, and human IgG antibody L chain , Including a human H chain containing an SD sequence linked under CMV-derived enhancer / promoter control, a DNA fragment containing the first BGH-polyA, SA sequence and PDGFRtm, and a cycloheximide resistance gene linked under CMV-derived enhancer / promoter control The gene fragment was excised. After the restriction enzyme treatment, PCR was performed using the purified gene fragment as a template and the synthetic DNA primers AC27-TnPMug FW (SEQ ID NO: 20) and AC27-TnPMug RV P2 (SEQ ID NO: 21), and restriction enzyme sites SalI and MluI A gene fragment to which was added was prepared.

Tol2 transposon vector excluding the antibody L chain, CMV promoter, DNA encoding cassette encoding H chain and cycloheximide resistance gene expression cassette contained in the anti-M2 antibody expressing Tol2 transposon vector described in WO2010 / 143698 (hereinafter referred to as TnPMug vector and the abbreviated) was treated with the restriction enzymes SalI and MluI, the gene fragments generated by ^{PCR in-Fusion TM Advantage PCR Cloning} Kit (Clontech , Inc., according to catalog number. six hundred and thirty-nine thousand six hundred and nineteen ) with the attached manual, restrictions TnPMug vector A secreted / membrane-bound human IgG antibody-expressing Tol2 transposon vector AC27 / TnPMug inserted between the enzyme sites SalI and MluI and introduced in a Tol2 transposase-dependent manner was prepared (FIG. 3). In the same manner, using the pINC_TMSP_AC17 vector or the pINC_TMSP_AC10 vector prepared in Example 1 (2), an expression vector AC17 containing a T / C15 sequence consisting of 15 bases and a T / C8 sequence consisting of 8 bases, respectively. / TnPMug and AC10 / TnPMug were prepared.

Preparation of cells that simultaneously produce secreted / membrane-bound human IgG antibodies using transposon vectors (1) Preparation of CHO cell transformants using Tol2 transposon CHO-K1 cells (American Type Culture Collection) adapted to floating cells The vector was introduced into Cat. No. CCL-61) by electroporation using Gene Pulser Xcell (registered trademark) (BIO-RAD) as follows.

Centrifugation of 4.0 × 10 ⁶ floating CHO-K1 cells at 1000 rpm for 5 minutes to remove the supernatant, and then add ice-cooled 400 μL PBS (Life Technologies) to prepare a cell suspension did. To the cell suspension was added secreted / membrane-bound human IgG antibody-expressing Tol2 transposon vector AC27 / TnPMug 10 μg, Tol2 transposase expression plasmid (WO2010 / 143698) 20 μg, and placed in an electroporation cuvette. Electroporation was performed with a capacitance of 500 μF. After electroporation, the cell suspension is suspended by adding 20 mL of CD opti CHO medium (Life Technologies) containing 200 mM L-Glutamine (hereinafter referred to as cell culture medium) to a 6-well plate. After dispensing at 3 mL / well, the cells were statically cultured for 24 hours in a 37 ° C., 5% CO ₂ incubator. After the culture, stirring culture was performed in a 37 ° C., 5% CO ₂ incubator for 10 days.
(2) Selection of anti-human antibody-expressing cells using a cell sorter (a) Selection of antibody-producing cells using PE-labeled mouse anti-human IgG antibody CHO-K1 introduced with a secreted / membrane-bound human antibody-expressing Tol2 transposon vector The cell expresses a membrane-bound human antibody on the cell membrane and produces a secreted human antibody. Therefore, it is possible to select cells that highly express secreted anti-human antibody by staining antibody-producing cells with PE-labeled mouse anti-human IgG antibody and selecting cells with strong fluorescence intensity. Or we examined as follows.

After culturing, CHO-K1 cells introduced with AC27 / TnPMug were centrifuged at 1000 培養 rpm for 5 minutes, and the supernatant was removed. Then, 10µL (1µg) PE-labeled anti-human IgG mouse antibody (mouse Anti-Human IgG (Fc ) -PE, Cat. No.736007, manufactured by BECKMAN), and the mixture was allowed to react for 30 to 60 minutes under light shielding at ice temperature. After the reaction, after washing with a cell culture medium, the cells were collected by centrifugation at 1000 rpm for 5 minutes, and washed twice to remove non-specifically bound PE-labeled anti-human IgG mouse antibody. . After washing, the cells were stirred with 1 mL of cell culture medium and filtered through a tube with a cell strainer.

Using a cell sorter FACSAria (BD Pharmigen), cells with high fluorescence intensity were sorted from cells stained with PE-labeled mouse anti-human antibody.
Cell sorting is developed with forward scatter and side scatter, and cell populations exhibiting the desired laser scatter are gated to eliminate cells and cell debris that exhibit abnormal laser scatter. (Figure 5 (a)). Next, in the cells in the gate, a histogram with the fluorescence intensity of PE as the horizontal axis was developed, the top 0.1% cells with the strongest PE fluorescence intensity were sorted, and seeded in a 384-well plate at 1 cell / well. (FIG. 5 (b)). After visually confirming that it was 1 cell / 1 well, it was statically cultured for 2 weeks in a 37 ° C., 5% CO ₂ incubator.

(B) Selection of antibody-producing cells using Recombinant protein A / G-FITC In the same manner as in (a) above, CHO-K1 cells into which a secreted / membrane-bound human antibody-expressing Tol2 transposon vector was introduced were recombinant We examined whether it is possible to select cells that highly express secreted human antibodies by staining with protein A / G-FITC (BioVision, Cat. No. 6504-1). .

As a result, as with the PE-labeled anti-human IgG mouse antibody, it was possible to stain cells and sort cells using recombinant protein A / G-FITC.
From the results of (a) and (b) above, in the cells into which the secretory / membrane-bound human antibody expression vector of the present invention has been introduced, the membrane-bound human antibody is expressed on the cell membrane, and the membrane-bound human antibody is expressed. It was revealed that antibody-producing cells that highly express membrane-bound human antibodies can be screened using anti-human IgG antibodies, recombinant protein A and recombinant protein G that bind to human IgG antibodies. In addition, the membrane-bound antibody retains the binding epitope of the anti-human IgG antibody and the recombinant protein A that binds to the Fc region of the antibody, and a plurality of detection bodies that detect the membrane-bound antibody produce the antibody of the present invention. It was also revealed that it can be applied to cell screening.
(3) Membrane-bound human antibody expression level on the cell membrane of antibody-producing cells Secretion / membrane-bound human antibody expression Tol2 transposon vector AC27 / TnPMug having 25, 15 or 8 bases contained in the polypyrimidine sequence, The amount of membrane-bound human antibody expressed on the cell membrane in CHO-K1 cells into which AC17 / TnPMug or AC10 / TnPMug was introduced was examined.

Each vector was introduced into CHO-K1 cells in the same manner as in Example 4 (1), and cells expressing membrane-bound human antibodies were measured by the same method as in Example 4 (2) (b). . The ratio of the number of cells in which the fluorescence intensity of Recombinant protein A / G-FITC binding per certain number of cells was in the range of 1.0 × 10 ³ to 1.0 × 10 ⁵ was calculated.

As a result, compared with the negative control without the expression vector, the recombinant / protein-binding human antibody-expressing Tol2 transposon vector AC27 / TnPMug, AC17 / TnPMug, or AC10 / TnPMug was significantly recombinant protein A Fluorescence intensity increased due to / G-FITC binding. Therefore, as in Example 2, it was revealed that the membrane-bound human antibody was expressed in the secretory / membrane-bound human antibody-expressing Tol2 transposon vector-introduced cells.

In addition, in the cells into which AC17 / TnPMug or AC27 / TnPMug vectors have been introduced, the number of cells having a fluorescence intensity of 1 × 10 ³ to 1 × 10 ⁵ is about 2.4 times or 2.1 times that of cells into which AC10 / TnPMug vectors have been introduced. Doubled. Therefore, a secretory / membrane-bound human antibody expression vector containing a polypyrimidine sequence of 8 bases or more is capable of expressing a membrane-bound human antibody, and is a secreted / membrane-bound antibody comprising a polypyrimidine sequence of 15 bases or more. It has been clarified that the human antibody expression vector can highly efficiently express a membrane-bound human antibody.

Expression and production of secreted human and membrane-bound human antibodies by antibody-producing cells (1) Secreted / membrane-bound human antibody expressing Tol2 transposon vectors AC27 / TnPMug, AC17 / TnPMug, and AC10 / TnPMug-introduced cells Expression of antibody and membrane-bound human antibody In the same manner as in Example 4 (1), CHO-K1 cells into which each AC27 / TnPMug, AC17 / TnPMug, and AC10 / TnPMug vectors were introduced were prepared. Using a cell sorter FACSAria (BD Pharmigen), set the gate at around 2.0 × 10 ³ , 3.0 × 10 ³ and 4.0 × 10 ^{3 for the} fluorescence intensity due to the binding of recombinant protein A / G-FITC, and perform single cell sorting Carried out.

Sorted cells were seeded in a 384-well plate containing 50 μL of cell culture medium, and statically cultured at 37 ° C. in a 5% CO ₂ incubator for 2 weeks. After culturing, the cells that formed colonies were transferred to a 96-well plate in which a cell culture medium was dispensed at 150 μL / well, followed by stationary culture at 37 ° C. in a 5% CO ₂ incubator for 1 week. After culturing, the culture supernatant was collected, and the expression level of the secretory antibody was quantified by the sandwich method (LENCE ^TM, PerkinElmer) using FRET (fluorescence resonance energy transfer) (FIGS. 6 (a), (b) and (c)).

As a result, in CHO-K1 cells into which any expression vector was introduced, an increase in fluorescence intensity due to the binding of recombinant protein A / G-FITC was positively correlated with an increase in the concentration of secreted human antibody (Fig. 6). (a), (b) and (c)). Therefore, it has been clarified that the production of secreted human antibodies increases as the expression level of membrane-bound human antibodies increases. It was also clarified that cells expressing highly secreted antibodies can be screened by selecting antibody-expressing cells using conjugates such as fluorescently labeled antibodies that bind to membrane-bound antibodies and protein A. It was.
(2) Secretory / membrane-bound human antibody-expressing Tol2 transposon vectors AC27 / TnPMug, AC17 / TnPMug, and AC10 / TnPMug Introducing cells into cells In Example 4 (2) (a), AC27 / TnPMug, AC17 / TnPMug As a result of single cell sorting of 0.1% of the cells with the highest fluorescence intensity of AC10 / TnPMug-introduced cells, 8 clones in AC27 / TnPMug-introduced cells, 16 in AC17 / TnPMug-introduced cells, and 6 in AC10 / TnPMug-introduced cells Obtained a single clone. The average antibody production amount of each cell was AC10 / TnPMug <AC17 / TnPMug≈AC27 / TnPMug.

Furthermore, the CHO-K1 cell line expressing membrane-bound human antibodies is cultured, the expression level of membrane-bound human antibodies is examined for each clone, and the most antibody is expressed among the clones into which each expression vector has been introduced. Selected clones. The production amount of the human antibody of the antibody-expressing cell line into which each finally selected expression vector was introduced was examined.
Using a production medium with CD opti CHO (Life Technologies) plus L-Glutamine (Life Technologies), seed 1 × 10 ⁶ cells / 3 mL in a 6-well plate (Corning) at 37 ° C. Then, stirring culture was performed in a 5% CO ₂ incubator for 7 days. The IgG antibody concentration was measured by HPLC (Waters) (Table 3).

As a result, CHO-K1 cells into which the secreted / membrane-bound human antibody-expressing Tol2 transposon vectors AC10 / TnPMug, AC17 / TnPMug, and AC27 / TnPMug were respectively introduced were 0.1 μg / L in the culture supernatant after 7 days of culture. , 0.5 g / L and 0.4 g / L secretory human IgG antibodies were produced. In addition, cells transfected with the AC17 / TnPMug or AC27 / TnPMug vector produced

antibodies

5 or 4 times higher than cells transfected with the AC10 / TnPMug vector.

Therefore, it is clear that antibody-producing cells can be cleaned from cells into which secreted / membrane-bound antibody expression vectors have been introduced using the expression level of membrane-bound antibody as an indicator, and antibody production can be performed using the selected cells. became. In addition, it was revealed that the antibody-producing cells selected based on the high expression level of the membrane-bound antibody produced a high amount of secretory antibody.
Furthermore, from the result of Example 4 (3) and the result of this example, an expression vector containing an 8-base poly-Pyr sequence was introduced into a cell into which an expression vector containing a 15-base poly-Pyr sequence was introduced. As a result of efficient splicing, the cell-bound antibody is highly expressed, and the cells that are screened using the high expression of the membrane-bound antibody as a marker show high expression of the secretory antibody. It became clear that Thus, it has also been clarified that by introducing an expression vector containing a poly-Pyr sequence of 15 bases or more into a host cell, a high antibody-producing cell can be produced more efficiently and the antibody can be produced.

Analysis of mRNA expression in secretory / membrane-bound human antibody-producing cells To analyze the expression of secretory human IgG antibody and membrane-bound human antibody mRNA in cells into which secreted / membrane-bound human antibody expression vectors have been introduced. The mRNA expression of CHO-K1 cells selected in Example 5 (2) using membrane-bound human IgG antibody expression as an index was analyzed by real-time PCR.
Total RNA was extracted from cultured cells containing the secreted / membrane-bound human antibody expression vector using the RNeasy (registered trademark) Mini Kit (250) (Cat. No. 74106, QIAGEN) according to the attached instructions. Extracted. Using the extracted total RNA as a template, probe 3 Fw primer (SEQ ID NO: 22) and probe 3 Rv primer (SEQ ID NO: 23) for amplifying partial fragments of membrane-bound human antibody H chain mRNA, membrane-bound human antibody and PCR of each mRNA fragment was performed using probe 1 Fw primer (SEQ ID NO: 24) and probe 1 Rv primer (SEQ ID NO: 25) for amplifying partial fragments of H chain mRNA of both secretory human antibodies (Fig. 7). The amplified fragment was detected using probe 3 (SEQ ID NO: 26) and probe 1 (SEQ ID NO: 27).

Real time-PCR was performed using 7900HT Fast Real-Time PCR System (Applied Biosystems), and data analysis was performed using SDS software version 2.3 (Applied Biosystems) to quantify mRNA.
As a result, assuming that the mRNA expression level of the membrane-bound antibody H chain of AC10 / TnPMug vector-introduced cells containing poly-Pyr sequences containing 8 T / C sequences is 1, poly-Pyr containing 15 T / C sequences. AC27 / TnPMug vector-introduced cell membrane-bound antibody H chain mRNA expression level is 23 fold and membrane-bound antibody H in AC27 / TnPMug vector-introduced cell with 25 T / C poly-Pyr sequences The mRNA expression level of the strand was 27 times (FIG. 8 (a)).

On the other hand, the total antibody H chain mRNA amount including secreted human antibody H chain and membrane-bound human antibody H chain is AC17 / TnPMug when the mRNA expression level of all antibody H chains in AC10 / TnPMug vector-introduced cells is 1. The H chain mRNA expression level of the vector-introduced cells was doubled, and the H chain mRNA expression level of the AC27 / TnPMug vector-introduced cells was 1.4 times (FIG. 8 (b)). Moreover, the membrane-bound human antibody H chain mRNA expression level in the total antibody H chain mRNA expression level in each vector-introduced cell was 0.1%, 1.4% or 1.6%, respectively (FIG. 8 (c)).

Based on the above results, in cells into which a secretory / membrane-bound human antibody expression vector has been introduced, the pre-mRNA encoding the antibody H chain is present in the downstream region and the splicing donor sequence at the 3 ′ end of the antibody H chain. Splicing occurs in the splicing acceptor sequence and expresses both mRNA encoding the membrane-bound human antibody H chain in which the antibody H chain and the PDGFR transmembrane region are fused and mRNA encoding the secreted human antibody H chain. (Figs. 8 (a) and (b)). The sequences of secretory antibody mRNA and membrane-bound mRNA expressed in the cells are shown in SEQ ID NO: 28 and SEQ ID NO: 30, and the amino acid sequences encoded by the respective mRNAs are shown in SEQ ID NOs: 29 and 31.

Also, cells into which the secretory / membrane-bound human antibody expression vector has been introduced may express membrane-bound human antibody H chain mRNA at a rate of 0.1% or more of the total human antibody H chain mRNA expression level. For example, it was revealed that antibody-producing cells can be obtained by screening with the expression level of membrane-bound antibody (FIG. 8 (c)).
In particular, in cells transfected with AC17 / TnPMug and AC27 / TnPMug vectors containing poly-Pyr sequences of 15 bases or more, the mRNA expression level of membrane-bound human antibody H chain is higher than cells transfected with AC10 / TnMug vector (Figs. 8 (a) and (c)), in cells into which an expression vector containing a poly-Pyr sequence of 15 bases or more has been introduced, an efficient splicing reaction is performed and mRNA expression of membrane-bound human antibodies is increased. It was revealed that

Furthermore, in the cells into which AC17 / TnPMug and AC27 / TnPMug vectors were introduced, not only the expression level of the membrane-bound human antibody H chain mRNA was increased compared to the cells into which AC10 / TnMug vector was introduced (FIG. 8 (a)). ), Because the mRNA expression level of all human antibody H chains including secreted type increased (Fig. 8 (b)), from the cells into which AC17 / TnPMug or AC27 / TnPMug vectors were introduced, membrane-bound antibody was used as an index. By screening antibody-expressing cells, it became clear that high antibody-producing cell lines can be obtained.

Therefore, by screening antibody-producing cells that express 1% or more of the membrane-bound antibody H chain mRNA expression level relative to the total antibody H chain mRNA expression level, cells that produce a secretory antibody at a higher level It was revealed that the expression level ratio of secretory antibody H chain mRNA and membrane-bound antibody H chain mRNA can be controlled by adjusting the length of the poly-Pyr sequence.

Production of Fab fragment splicing vector (1) Production of secretory / membrane-bound antibody expression vector 4D5TM_Hc Encoding the heavy chain amino acid sequence of anti-HER2 humanized antibody trastuzumab (Herceptin (registered trademark)) (US Pat. No. 5,821,337) The DNA sequence to be inserted was inserted into a Tol2 transposon vector, and a secreted / membrane-bound antibody expression vector 4D5TM_Hc containing the polypyrimidine sequence T / C25 was prepared in the same manner as in Example 3. On the other hand, a DNA sequence encoding a light chain amino acid sequence (US Pat. No. 5,821,337) was inserted, and a Herceptin light chain expression Tol2 transposon vector 4D5_Lc was prepared in the same manner as in Example 3. In addition, a heavy chain expression Tol2 transposon vector 4D5 into which no splicing donor or acceptor sequence was inserted was prepared.
(2) Preparation of membrane-bound Fab fragment expression vector Since the splicing donor (SD) sequence GGGTAAAT is present at the 3 'end of the CH3 domain of the DNA sequence encoding the antibody H chain, the SD sequence was modified. In addition, among the DNA sequences encoding the CH2 domain amino acid sequence, an SD consensus sequence was introduced by replacing the DNA sequence so that the amino acid residue of each codon was not changed. The H chain in which the SD sequence is introduced into the CH2 domain is spliced in the CH2 domain, and a membrane-bound Fab protein in which the Fab fragment is fused with the transmembrane domain protein can be expressed on the cell membrane.

Each Fab fragment expression construct Fab, Fab1, Fab2, and Fab3 produced SD sequences by introducing mutations into genes encoding the following amino acids (FIG. 9).
The codon encoding the 51st glycine of the CH2 domain of Fab: IgG1 antibody was replaced from GGC to GGT.
The codon encoding the 6th glycine of the CH2 domain of Fab1: IgG1 antibody was replaced from GGG to GGT.
The codon encoding the 86th glycine of the CH2 domain of Fab2: IgG1 antibody was replaced from GGC to GGT.
The codon encoding the 48th tyrosine of the CH2 domain of Fab3: IgG1 antibody was replaced from TAC to TAT.

Membrane-bound Fab fragment expression vector 4D5TM_Fab was prepared by PCR using QuikChange II Site-Directed Mutagenesis Kit (Invitrogen) using 4D5TM_Hc as a template and primers having DNA sequences described in SEQ ID NOs: 32-35 (Table 1). Four).
Further, membrane-bound Fab fragment expression vectors 4D5TM_Fab1, 4D5TM_Fab2 and 4D5TM_Fab3 are 4D5TM_Fab as a template, DNA sequence primers set forth in SEQ ID NOs: 36-39, DNA set forth in SEQ ID NOs: 36, 37, 40 and 41, respectively. The primers were prepared in the same manner as described above using the primers for the sequences and the DNA sequences described in SEQ ID NOs: 42 and 43 (Table 4).

Production of antibody-producing cells and protein production by the cells (1) Production of antibody-producing cells In the same manner as in Example 4 (1), Herceptin secretory / membrane-bound heavy chain expression vector 4D5TM_Hc and membrane-bound Fab fragment expression Vectors 4D5TM_Fab_Hc, 4D5TM_Fab1_Hc, 4D5TM_Fab2_Hc and 4D5TM_Fab3_Hc were introduced into CHO-K1 cells together with Herceptin light chain expression vector 4D5 Lc, and the cells were cultured. Expression of membrane-bound IgG antibody or Fab fragment was confirmed using protein A / G-FITC as in Example 4 (2) (b), and cells expressing membrane-bound IgG antibody or Fab fragment Selected.

Therefore, it was confirmed that IgG antibody or Fab fragment to which protein A / G-FITC binds is expressed on the cell membrane.
(2) Antibody production by batch culture In the same manner as in Example 5 (2), the antibody-producing cells obtained in Example 8 (1) above were cultured, and the amount of antibody produced in the culture supernatant was measured (Table Five).

As a result, antibody production was confirmed in cells into which both the herceptin secretory / membrane-bound heavy chain expression vector and the membrane-bound Fab fragment expression vector were introduced. Thus, it was revealed that any cell expressing IgG antibody and Fab fragment as a membrane-bound protein can produce secretory IgG antibody (Table 5).

(3) Antibody production by Fed-batch culture The antibody-producing cells obtained in Example 8 (1) above were subjected to Fed-batch culture, and the amount of antibody production was examined. The production medium was prepared by adding L-Glutamine (Life Technologies) and soybean hydrolyzate (SAFC) to CD opti CHO (Life Technologies).
The Feed medium was prepared by mixing CHO CD EFFICIENTFEED A (Invitrogen) and CHO CD EFFICIENTFEED B (Invitrogen) in equal amounts and adding Glucose (Wako) and Glutamine (Wako).

As a specific culture method, cells were seeded in a production medium prepared in an Erlenmeyer flask (CORNING) to a concentration of 0.3 × 10 ⁶ cells / mL, and in an incubator at 37 ° C. and 5% CO ₂ . Stirring culture was performed for 14 days. Glucose and Glutamine were appropriately added by measuring ingredients in the culture solution every few days and adding Feed medium.
As a result, cells into which both the herceptin secretory / membrane-bound heavy chain expression vector and the membrane-bound Fab fragment expression vector were introduced produced antibodies under Fed-batch culture. Therefore, it was clarified that any cell expressing IgG antibody and Fab fragment as a membrane-bound protein can produce secretory IgG antibody (Table 6).

In addition, cells expressing membrane-bound IgG, membrane-bound Fab, or membrane-bound Fab1 all produce antibodies higher than cells expressing membrane-bound Fab2 or membrane-bound Fab3. It became clear.
Therefore, cells expressing an antibody gene having a splicing donor sequence inserted into the CH2 domain of the antibody constant region can be selected by the expression of the spliced membrane-bound Fab, and the selected cells Was confirmed to produce antibodies.

Binding activity of membrane-bound antibody to protein A and protein G In the same manner as in Example 4 (1), Herceptin heavy chain expression vector 4D5, secreted / membrane-bound heavy chain expression vector 4D5TM, and membrane-bound Fab Fragment expression vectors 4D5TM_Fab, 4D5TM_Fab1, 4D5TM_Fab2 and 4D5TM_Fab3 were introduced into CHO-K1 cells together with Herceptin's light chain expression vector 4D5 Lc to produce a transfectant, and cycloheximide (Cat. No. C4859, SIGMA) The drug selection was carried out.

Five days after gene transfer, CD opti CHO (Life Technologies) is added with L-Glutamine (Life Technologies) and cultured in a medium containing 3 mg / mL cycloheximide to select only cells that exhibit drug resistance. did.
Cycloheximide-resistant CHO cells are stained with FITC-Protein A (Cat. No. 101011, Invitrogen) or Protein G, Alexa Fluor 488 conjugate (Cat. No. P11065, Invitrogen), and using FACSCalibur (BD Pharmigen) The fluorescence intensity on the cell surface was measured. Gates with fluorescence intensities of 10 ² to 10 ⁴ were set to show the cell population (%) that reacted with each label (Table 7).

As a result, CHO-K1 introduced with secretory / membrane-bound Herceptin expression vector 4D5TM and membrane-bound Fab fragment expression vector compared to CHO-K1 into which host cell CHO-K1 and secreted Herceptin expression vector 4D5 were introduced. Both bound to both Protein A and Protein G.
Moreover, since CHO-K1 introduced with 4D5TM bound most strongly to both Protein A and Protein G, it was confirmed that the membrane-bound IgG antibody was expressed efficiently and predominantly.

On the other hand, CHO-K1 introduced with membrane-bound Fab fragment expression vectors 4D5TM-Fab, 4D5TM-Fab1, 4D5TM-Fab2, and 4D5TM-Fab3 all have a binding activity against Protein A compared to CHO-K1 introduced with 4D5TM. 4D5TM-Fab and 4D5TM-Fab1 decreased to about 1/2, and 4D5TM-Fab2 and 4D5TM-Fab3 decreased to about 1/4.
As for Protein G, the membrane-bound Fab fragment expression vector 4D5TM-Fab or 4D5TM-Fab1 was slightly reduced in binding amount compared to CHO-K1 into which 4D5TM was introduced, but 4D5TM-Fab2 or 4D5TM-Fab3 Protein G binding activity decreased to about 1/2.

Based on the above results, cells introduced with 4D5TM expressing membrane-bound IgG and cells transfected with 4D5TM-Fab and 4D5TM-Fab1 expressing membrane-bound Fab fragments efficiently expressed IgG or Fab fragments. However, it was revealed that the expressed Fab fragment has a reduced binding activity to Protein A. In addition, cells introduced with 4D5TM-Fab2 or 4D5TM-Fab3 have a protein-A binding activity as well as a protein-G binding activity reduced, so that compared with cells introduced with 4D5TM-Fab and 4D5TM-Fab1, membrane-bound type The possibility that the expression level of the Fab fragment was low was revealed.

Membrane-bound IgG may be contaminated with secretory IgG antibody in the manufacturing process of antibody drugs, but it is difficult to separate even with Protein A column purification. Therefore, by using a cell that expresses a membrane-bound Fab fragment that does not bind to Protein A, contamination in the production process of the antibody drug can be prevented.

According to the present invention, an expression vector for a target protein capable of simultaneously expressing a target protein and a protein in which the target protein or the target protein fragment and the transmembrane region are fused using a single gene is expressed. A method for screening cells to be produced, a cell for producing a target protein at a high level, a method for producing a cell for producing a target protein at a high level, and a method for producing the target protein using the cell.

SEQ ID NO: 1: Splicing donor sequence SEQ ID NO: 2: Splicing donor sequence SEQ ID NO: 3: Splicing donor sequence SEQ ID NO: 4: Splicing acceptor sequence SEQ ID NO: 5: Splicing acceptor sequence SEQ ID NO: 6: Polypyrimidine sequence T / C8
SEQ ID NO: 7: Polypyrimidine sequence T / C15
SEQ ID NO: 8: Polypyrimidine sequence T / C25
SEQ ID NO: 9: OX40 L chain SEQ ID NO: 10: CD98 / LAT1 H chain SEQ ID NO: 11: BGH polyadenylation signal sequence SEQ ID NO: 12: PDGFR transmembrane region SEQ ID NO: 13: Synthetic construct SEQ ID NO: 14: TM-F1 primer SEQ ID NO: 15: TM-R1 primer SEQ ID NO: 16: TM-R2 primer SEQ ID NO: 17: TM-R3 primer SEQ ID NO: 18: AC27-F primer SEQ ID NO: 19: TMSP-R primer SEQ ID NO: 20: AC27-TnPMug FW primer SEQ ID NO: 21 : AC27-TnPMug Rv P2 primer SEQ ID NO: 22: Probe 3 Fw primer SEQ ID NO: 23: Probe 3 Rv primer SEQ ID NO: 24: Probe 1 Fw primer SEQ ID NO: 25: Probe 1 Rv primer SEQ ID NO: 26: Probe 3
SEQ ID NO: 27: Probe 1
SEQ ID NO: 28: Secretory human IgG antibody H chain mRNA
SEQ ID NO: 29: Synthetic construct SEQ ID NO: 30: Membrane-bound human IgG antibody H chain mRNA
SEQ ID NO: 31: Synthetic construct SEQ ID NO: 32: Hinge primer Fw
SEQ ID NO: 33: Hinge primer Rv
SEQ ID NO: 34: 4D5 mutant Fv
SEQ ID NO: 35: 4D5 mutation Rv
SEQ ID NO: 36: Fab donor Fw
SEQ ID NO: 37: Fab donor Rv
SEQ ID NO: 38: Fab1 donor Fw
SEQ ID NO: 39: Fab1 donor Rv
SEQ ID NO: 40: Fab2 donor Fw
SEQ ID NO: 41: Fab2 donor Rv
SEQ ID NO: 42: Fab3 donor Fw
SEQ ID NO: 43: Fab3 donor Rv

Claims

An expression vector for a target protein comprising a DNA sequence encoding a target protein including a splicing donor sequence, a polypyrimidine sequence, a splicing acceptor sequence, and a DNA sequence encoding a transmembrane region in this order.
Including DNA sequence encoding target protein including splicing donor sequence, first polyadenylation signal sequence, polypyrimidine sequence, splicing acceptor sequence, DNA sequence encoding transmembrane region and second polyadenylation signal sequence An expression vector for the target protein.
A cell into which the expression vector according to any one of claims 1 and 2 has been introduced.
4. The cell according to claim 3, wherein the cell is a cell selected from (a) and (b).
(A) A cell in which messenger RNA (mRNA) encoding a protein in which a target protein or a fragment of the target protein is fused with a transmembrane region is expressed in 0.1% to 5% of the total mRNA of the target protein.
(B) A cell in which at least 1 × 10 3 molecules / cell of a protein in which the target protein or a fragment of the target protein and the transmembrane region are fused is expressed.
5. A screening method for cells that produce a target protein in a high amount, comprising detecting the target protein or a fragment of the target protein expressed on the cell membrane of the cell according to claim 3 or 4.
A method for producing the cell according to claim 3 or 4.
5. A method for producing a target protein, comprising the steps of culturing the cell according to claim 3 or 4, accumulating the target protein in a culture solution, and purifying the target protein from the cell culture solution.
A method of simultaneously expressing both a target protein and a target protein or a protein in which a fragment of the target protein and a transmembrane region are fused, comprising the step of introducing the target protein expression vector according to claim 1 into a host cell.
A target protein-expressing cell selected from (a) and (b), wherein a protein expression vector containing a DNA sequence encoding the target protein is introduced and a secreted target protein is expressed.
(a) A cell in which messenger RNA (mRNA) encoding a protein in which a target protein or a fragment of the target protein is fused with a transmembrane region is expressed in 0.1% to 5% of the total mRNA of the target protein.
(B) A cell in which at least 1 × 10 3 molecules / cell of a protein in which the target protein or a fragment of the target protein and the transmembrane region are fused is expressed.
10. The cell according to claim 9, wherein the cell is a cell selected from (a) and (b).
(a) A cell in which mRNA encoding a target protein or a protein in which a fragment of the target protein is fused with a transmembrane region is expressed in 0.1% to 2% of the total mRNA of the target protein.
(b) A cell in which 1 × 10 3 to 1 × 10 7 molecules / cell of a protein in which the target protein or a fragment of the target protein and the transmembrane region are fused is expressed.
11. A screening method for cells that produce a target protein in a high amount, comprising detecting the target protein or a fragment of the target protein expressed on the cell membrane of the cell according to claim 9 or 10.
A method for producing the cell according to claim 9 or 10.
11. A method for producing a target protein comprising culturing the cell according to claim 9 or 10, accumulating the target protein in a culture medium, and purifying the target protein from the cell culture medium.