HUMAN GENOME-DERIVED POLYNUCLEOTIDE AND METHOD FOR PRODUCING POLYPEPTIDE OF INTEREST USING SAME

Abstract

Provided are a human genome-derived polynucleotide having a chromatic regulator factor function, a recombinant vector and a recombinant cell, each comprising same, and a method for producing a polypeptide of interest using same.

Claims

1.-12. (canceled)

13. An expression vector for producing a polypeptide of interest, comprising (1) at least one polynucleotide selected from the group consisting of: a) a polynucleotide comprising 100 or more consecutive nucleotides within a nucleotide sequence selected from SEQ ID NOS: 1 to 16; b) a polynucleotide comprising 200 to 5,000 consecutive nucleotides, inclusive of the polypeptide a), on human chromosome X; and c) a polynucleotide comprising a nucleotide sequence complementary to the polynucleotide a) or b), and (2) a gene coding for the polypeptide of interest.

14. The expression vector of claim 13, wherein the polynucleotide comprises a nucleotide sequence selected from SEQ ID NOS: 1 to 16, a nucleotide sequence complementary thereto, or both of the nucleotide sequence and the complementary sequence.

15. The expression vector of claim 13, further comprising αpromoter and a transcription terminator.

16. The expression vector of claim 15, wherein the polynucleotide is located at a 5′ terminal side of the promoter, at a 3′ terminal side of the transcription terminator, or at both of the terminal sides.

17. The expression vector of claim 16, wherein the polynucleotide is located in opposite directions at both of the terminal sides.

18. The expression vector of claim 16, comprising one to five of the polynucleotides on the 5′ terminal side of the promoter, on the 3′ terminal side of the transcription terminator, or on both of the terminal sides, wherein the one to five of the polynucleotides are the same or at least one of them is different from the remainder.

19. The expression vector of claim 13, wherein the polypeptide of interest is at least one selected from the group consisting of an antibody, an enzyme, a receptor protein, a transporter protein, a microbiocidal or endotoxin-binding polypeptide, a structural protein, a toxin, an antibiotic, a hormone, a growth factor, and a vaccine.

20. A recombinant cell for producing a polypeptide of interest, obtained by introducing the recombinant vector of claim 13 into a host cell.

21. The recombinant cell of claim 20, wherein the polynucleotide comprises a nucleotide sequence selected from SEQ ID NOS: 1 to 16, a nucleotide sequence complementary thereto, or both of the nucleotide sequence and the complementary sequence.

22. The recombinant cell of claim 20, wherein the host cell is a eukaryotic animal cell.

23. A method for producing a polypeptide of interest, the method comprising the steps of: expressing a gene coding for the polypeptide of interest in the recombinant cell of claim 20; and recovering the expressed polypeptide of interest.

24. A method for producing a polypeptide of interest in a host cell, the method comprising a step of introducing the expression vector of claim 13 into the host cell.

Description

DESCRIPTION OF DRAWINGS

[0099] FIG. 1 is a schematic diagram of a recombinant vector for expressing a target gene.

[0100] FIG. 2 is a schematic view showing cleavage maps of recombinant vectors for assaying CCE efficacy and cloning processes therefor (in FIG. 2, the target gene accounts for a gene coding for a polypeptide of interest, the first CCE for a CCE inserted into the 5′ terminal side of the target gene, and the second CCE for a CCE inserted into the 3′ terminal side of the target gene).

[0101] FIG. 3 is a graph showing relative antibody expression levels (%) in recombinant cells anchoring CCE sequences to the control (100%).

[0102] FIG. 4 is a schematic diagram of core CCE #7 and core CCE #8 according to an embodiment.

[0103] FIG. 5a is a plot of absolute values of antibody titers after introduction of expression vectors (w/o CCE, CCE14, core CCE #7, and core CCE #8).

[0104] FIG. 5b is a plot of absolute values of Qp after introduction of expression vectors (w/o CCE, CCE14, core CCE #7, and core CCE #8).

[0105] FIG. 6a is a plot of antibody titers after introduction of expression vectors (w/o CCE, CCE14, core CCE #7, and core CCE #8) relative to the reference expression vector (CCE14).

[0106] FIG. 6b is a plot of Qp after introduction of expression vectors (w/o CCE, CCE14, core CCE #7, and core CCE #8) relative to the reference expression vector (CCE14).

[0107] FIGS. 7a and 7b are plots of antibody titer equivalent values of core CCE #7 (FIG. 7a; p value=0.001) and core CCE #8 (FIG. 7b; p value=0.000) relative to CCE14, as measured by an equivalence test.

MODE FOR INVENTION

[0108] Hereafter, the present invention will be described in detail in the following Examples.

[0109] The following examples are intended merely to illustrate the invention and are not construed to restrict the invention.

EXAMPLE 1: Discovery of Chromatin Control Element (CCE) in Human

Genomic DNA

[0110] In reporter gene (GFP gene)-overexpressing human cells (Host: 293F, Invitrogen, R790-07), the insertion position of the reporter gene was detected, and a search was made for sequences that could affect the overexpression in the vicinity of the reporter gene. Sequences located around the 5′ and 3′ termini of the reporter gene were examined.

[0111] In brief, GFP-overexpressing clones per batch were selected as follows.

[0112] [Production of Retrovirus Using GP2-293 Cell Line]

[0113] VC003-1 DNA (pRetroQ-AcGFP1-C1 vector; Clontech, #632506) and VSV-G DNA (VSV-G vector; Clontech, #631530) were transfected into the GP2-293 cell line (Clontech, #631458) with the aid of Lipofectamine® 2000. Following transfection, the cells were cultured at 37° C. for 28 hours in a 5% CO.sub.2 atmosphere with 80% humidity. The cell culture thus obtained was allowed to pass through a 0.45-μm cellulose filter to filter the viruses, and the filtrate was stored at 4° C. and used within 3 days. The filtered culture was used within 3 days while being stored at 4° C.

[0114] [Viral Transduction]

[0115] 293F cells (Invitrogen, R790-07) were treated with the virus filtrate obtained above at 37° C. for 24 hours in a 5% CO.sub.2 atmosphere with 80% humidity. Then, the medium was replaced by DMEM+10% FBS in which the cells were cultured.

[0116] [Acquisition of Monoclone]

[0117] The transduced 293F cells were subjected to geneticin selection, followed by FACS sorting to select a pool of high-expression cells.

[0118] The selected pool of high-expression cells was maintained in DMEM/F12 medium (Life technology, A11320-033) supplemented with 10% FBS (Life technology, 16000-044) and geneticin (Life technology, 10131-027). In order to obtain monoclones from the pool, the cells were counted with a cell counter (Roche Innovatis AG, Cedex standard plus M20-C), and then, limiting dilution was performed. As a result, a total of 120 monoclones were acquired.

[0119] [Examination of High Expression Level of GFP]

[0120] With respect to the acquired 120 clones, living cells were stained with DAPI (Invitrogen, Hoechst 33348) and screened by an Incell analyzer (GE Healthcare, Incell analyzer 6000). As a result, the top 24 GFP-expressing clones were selected in the order of the highest GFP expression level per unit cell.

[0121] [Confirmation of High Expression Level of GFP]

[0122] For the selected 24 clones, the top 6 clones per batch were selected by measuring the GFP expression level again using FACS (BD, FACS canto II) according to the method provided by BD.

[0123] [Examination of Single Copy Insertion Clone]

[0124] In order to examine whether the high expression of GFP of the selected top 6 clones is due to single copy insertion, a genome copy number assay was performed using real-time PCR. Selection was made of clones with a genome copy number of less than 1.05 as measured by real-time PCR.

[0125] [Search for Flanking Region]

[0126] Tail-PCR (Thermal Asymmetric Interlaced PCR) was performed using the KOD-Plus-Neo PCR kit (Cat #KOD-401, TOYOBO, Japan) to find the flanking region around the GFP in the selected clones. A band portion comprising the flanking region was purified from the agarose gel, using a gel extraction kit (QIAGEN, Cat. 28706). The purified amplicon thus obtained was cloned into pJet, using the pJet1.2 kit (Cat #K1231, Thermo Scientific, USA). The obtained sequence was analyzed by NCBI BLAST to obtain a gDNA sequence about 100 kb long. The gDNA sequence was analyzed through the MAR finder program. As a result, 14 chromatin control element (CCE) sequences were discovered (SEQ ID NOS: 1 to 14; hereinafter referred to as CCE1 to CCE14).

EXAMPLE 2: Construction of Recombinant Vector Carrying Human Chromatin Control Element (CCE) and Antigen Gene and Assay for Antibody Expression Level

[0127] The 14 CCE sequences obtained in Example 1 were each inserted in the forward direction into the 3′ terminal side of the antigen gene and/or in the reverse direction into the 5′ terminal side of the antigen gene within the vector to construct expression vectors comprising the human genome-derived chromatin control element (CCE) sequences, and the vectors were assayed for expression performance. When two CCE sequences were inserted into one vector, the CCE inserted into the 5′ terminal side of the antibody gene was named first CCE while the CCE inserted into the 3′ terminal side of the antibody gene was named second CCE.

[0128] The 14 CCE sequences obtained in Example 1 were discovered on the 5′ or 3′ terminal side of the position where the reporter gene (GFP) was inserted.

[0129] [Construction of Vector for Expressing Polypeptide of Interest]

[0130] The omalizumab antibody (Accession number DB00043) was used as a target polypeptide. The omalizumab antibody was approved by the FDA in June 2003 and has been commercially available under the trademark Xolair™ since then, and its antibody sequence is already disclosed in U.S. Pat. No. 6,329,509 or Drug Bank (https://www.drugbank.ca/drugs/DB00043).

[0131] First, a promoter and an omalizumab antibody gene were treated with Mlul (NEB, R0198L), Pvul (NEB, R0150L), Ascl (NEB, R0558L), and Pvul (NEB, R0150L) and then cloned into pcDNA™3.3 vector (Life technology) to construct a vector for expressing the target polypeptide.

[0132] [Acquisition of CCE Insert and Construction of Vector Carrying CCE]

[0133] After synthesis of primers (SEQ ID NOS: 17 to 48) for nucleotide sequences of a certain region (±2 kb) around the CCEs obtained in Example 1, vectors carrying the CCEs and omalizumab were constructed using the polymerase KOD FX Neo (TOYOBO Bio-Technology, Cat.No KFX-201) according to the protocol provided by the manufacturer, with the CHO-K1 gDNA serving as a template. The CCE inserts were ligated to the vectors, using the ligation kit (Roche, Rapid ligation kit, cat. No. 11635379001). For ligation, the vector and the insert were digested with Mull. In this regard, the CCE insert that existed on the 5′ terminal side of the position at which the reporter gene (GFP) was inserted upon the discovery of CCE on the genomic DNA was inserted in the forward direction into the 5′ terminal side of the antibody gene and in the reverse direction into the 3′ terminal side of the antibody gene (see (C) in FIG. 2). The CCE insert that existed on the 3′ terminal side of the position at which GFP was inserted in the forward direction into the 3′ terminal side of the antibody gene and in the reverse direction into the 5′ terminal side of the antibody gene (see (D) in FIG. 2). For example, the CCE insert (CCE14) was inserted in the forward direction into the 3′ terminal side of the omalizumab antibody and in the reverse direction into the 5′ terminal side of the omalizumab antibody.

[0134] A vector comprising each of CCE1 to CCE13 obtained in Example 1 was constructed in the same manner as the method for preparing a vector comprising CCE14 described above.

[0135] [Comparison of Antibody Expression Level Among CCEs]

[0136] The human-derived CCE sequence-inserted expression vectors obtained above were introduced into the genome of CHO-K1 cells (ATCC, CRL-9618), with the aid of Lipofectamine®2000 transfection reagent (Cat #11668019, Life Technologies, USA). Following transfection with the CCE vectors, puromycin selection was performed on CHO-K1 cells. The cells were counted using the CEDEX STD cell counter (ROCHE INNOVATIS, USA) and then cultured at 37° C. for 72 hours. The resulting culture medium was subjected to an antibody titer experiment using the ForteBio Octet® QK384. Antibody concentrations were measured using a protein A sensor and the measurements were analyzed with the Octet® analysis program.

[0137] Antibody expression levels (%) in the recombinant cells comprising respective CCE sequences were compared to that in the control (100%, cells having no CCE sequences) and the results are given in Table 1 and depicted in FIG. 3.

TABLE-US-00001 TABLE 1 ID Fold SD Control 100 CCE1 128 22 CCE3 197 8 CCE4 180 17 CCE5 144 32 CCE7 195 25 CCE8 217 35 CCE9 249 16 CCE11 208 35 CCE12 143 4 CCE13 242 9 CCE14 254 28

[0138] As can be seen in Table 1 and FIG. 3, the CCE sequences obtained in Example 1 showed significantly higher antibody expression rates than in the control (increased by at least 20%, compared to the control).

EXAMPLE 3: Construction of Recombinant Vector Carrying Partial Human Chromatin Control Element (CCE) and Assay for Antibody Gene Expression Level

[0139] From among the human CCEs that were confirmed to have expression effects omalizumab antibody in Example 2, CCE14 (SEQ ID NO: 14) was selected, and its partial fragments (named core CCE14 #7 and core CCE14 #8, respectively) were tested. Core CCE14 #7(SEQ ID NO: 15), which is a partial fragment of CCE14, is 913-bp long starting from the 5′ terminus of CCE14 (SEQ ID NO: 14, full length 3.5 kb) (polynucleotide extending from the 1.sup.st to the 913.sup.th nucleotide on the sequence of SEQ ID NO: 14; 0.9 kb hCCE). Core CCE14 #8 (SEQ ID NO: 16) is 1405-bp long starting from the 5′ terminus of CCE14 (SEQ ID NO: 14) (polynucleotide extending from the 1.sup.st to 1450.sup.th nucleotides on the sequence of SEQ ID NO: 14; 1.4 kb hCCE) (see FIG. 4).

[0140] The omalizumab antibody was used as a target polypeptide as in Example 2. A vector for expressing the target polypeptide was constructed and transfected into CHO cells in the same manner as in Example 2, followed by assaying the cells for expression levels of omalizumab.

[0141] Briefly, for use as a vector for expressing the target polypeptide, a CCE14 vector was designed to include a hamster EF-1α promoter and CCE14 (SEQ ID NO: 14) in the reverse direction on the 5′ terminal side of the antibody gene (first CCE) and in the forward direction on the 3′ terminal side of the antibody gene (second CCE) (see (D) in FIG. 2). Likewise, an expression vector was constructed to include the core CCE14 #7 (SEQ ID NO: 15) in the reverse direction on the 5′ terminal side of the gene coding for the polypeptide of interest and in the forward direction on the 3′ terminal side of the gene coding for the polypeptide of interest (hereinafter, referred to as “core CCE14 #7 vector” or “core CCE #7 vector”). In addition, an expression vector was constructed to include core CCE14 #8 (SEQ ID NO: 16) in the reverse direction on the 5′ terminal side of the gene coding for the polypeptide of interest and in the forward direction on the 3′ terminal side of the gene coding for the polypeptide of interest (hereinafter, referred to as “core CCE14 #8 vector” or “core CCE #8 vector”).

[0142] The vectors constructed above were transinfected by electroporation (Amaxa™ 4D-Nucleofector™ Protocol, SF kit) into CHO cells. On day 4 after transfection, the cells were measured for viable cell density (VCD) and cell viability using the CEDEX STD cell counter (ROCHE INNOVATIS, USA). Also, the cells were assayed for antibody titer and absolute and relative values of productivity (Qp) using ForteBio Octet® QK384, and the measurements were compared and analyzed.

[0143] The data thus obtained are summarized in Tables 2 and 3 and depicted in FIGS. 5a to 7b (n=5):

TABLE-US-00002 TABLE 2 Analysis of Antibody Titer Ratio (result obtained upon introduction of CCE14 set to be 1) core core w/o CCE CCE14 CCE14 #7 CCE14 #8 Average of 0.68 1.00 1.05 1.18 1.sup.st antibody titer ratio SD 0.12 0.09 0.04 0.03 Average of 0.76 1.00 0.92 1.03 2.sup.nd antibody titer ratio SD 0.04 0.08 0.04 0.07 Average of 0.67 1.00 0.95 0.93 3.sup.rd antibody titer ratio SD 0.03 0.03 0.06 0.14 Average of 0.62 1.00 0.78 0.89 4.sup.th antibody titer ratio SD 0.04 0.12 0.07 0.04 Total Average 0.68 1.00 0.93 1.00 Total SD 0.10 0.11 0.11 0.11

TABLE-US-00003 TABLE 3 Analysis of Qp (Productivity) Ratio (result obtained upon introduction of CCE14 set to be 1) core core w/o CCE CCE14 CCE14 #7 CCE14 #8 Average of 1.sup.st Qp ratio 0.68 1.00 0.97 1.00 SD 0.06 0.09 0.06 0.04 Average of 2.sup.nd Qp ratio 0.77 1.00 0.90 1.07 SD 0.09 0.07 0.03 0.06 Average of 3.sup.rd Qp ratio 0.71 1.00 0.94 1.11 SD 0.06 0.04 0.06 0.07 Average of 4.sup.th Qp ratio 0.54 1.00 0.69 0.77 SD 0.05 0.16 0.05 0.05 Total Average 0.68 1.00 0.88 0.99 Total SD 0.12 0.15 0.18 0.18

[0144] FIGS. 5a and 5b show absolute values of antibody titers and Qp measured after introduction of the expression vectors, respectively. FIGS. 6a and 6b show relative values of antibody titers and Qp measured after introduction of the expression vectors (relative to the values measured after introduction of CCE14 vector), respectively. FIGS. 7a and 7b show titers of the core CCE14 #7 vector and the core CCE14 #8 vector relative to that of CCE14 vector, as measured by an equivalence test.

[0145] As can be understood from the data, the CCE14 #7 vector including the 0.9-kb CCE fragment (core CCE14 #7) and the core CCE14 #8 vector including the 1.4-kb CCE fragment (core CCE14 #8) both exhibited similar average titer values (absolute and ratio) and Qp values (absolute and ratio) to those of the CCE14 vector including the full-length CCE14 (3.5 kb). In addition, the core CCE14 #7 vector and the core CCE14 #8 vector were both found to be equivalent in antibody titer to the CCE14 vector as measured by an equivalent test. These results indicate that the CCE fragments as well as the full-length CCE can fulfill the functional role of CCEs.