Method for producing therapeutic protein

Abstract

To provide a method for producing therapeutic protein.SOLUTION: A method for producing therapeutic protein includes: a process of culturing a cholesterol-auxotrophic and glutamine-auxotrophic cell; a process of transfecting nucleic acid (i) coding protein that can restore cholesterol biosynthesis in a the cell, nucleic acid (ii) coding protein that can restore glutamine biosynthesis in the cell, and nucleic acid (iii) coding therapeutic protein into the cell; a process of culturing the cell under a condition suitable for expression of the therapeutic protein; and a process of isolating and / or purifying the therapeutic protein.SELECTED DRAWING: None

Description

Technical Field

[0001] The production of therapeutic proteins using mammalian cell expression systems is becoming increasingly important in the biotechnology industry. There are various culture systems and transfection systems, but each system has significant limitations.

Background Art

[0002] The invention described herein is, in part, based on the discovery that by using a combination of cholesterol auxotrophic cells and one or more additional selectable markers in a method for producing a therapeutic protein, unexpected problems (e.g., low productivity) that occur during production involving cholesterol auxotrophic cells can be avoided. In one embodiment, the present disclosure features a method for producing a therapeutic protein, the method comprising culturing cholesterol auxotrophic and glutamine auxotrophic cells, transfecting the cells with a nucleic acid (i) encoding a protein capable of restoring the cholesterol biosynthetic function in the cells, a nucleic acid (ii) encoding a protein capable of restoring the glutamine biosynthetic function in the cells, and a nucleic acid (iii) encoding a therapeutic protein, culturing the cells under conditions suitable for the expression of the therapeutic protein, and isolating and / or purifying the therapeutic protein.

Summary of the Invention

[0003] In one embodiment, the cells are NS0 cells.

[0004] In some embodiments, the therapeutic protein is an antibody. In some embodiments, the therapeutic protein is a fusion protein. In some embodiments, the therapeutic protein is an Fc-containing fusion protein.

[0005] In some embodiments, in the transfection step, the cells are transfected with a nucleic acid (iii) encoding an antibody light chain and an antibody heavy chain. In some embodiments, nucleic acid (i) encodes a 3-ketosteroid reductase. In some embodiments, nucleic acid (ii) encodes a glutamine synthetase.

[0006] In some embodiments, the first expression vector contains nucleic acids (i) and (iii), and the second expression vector contains nucleic acid (ii). In some embodiments, the first expression vector contains nucleic acid (i), and the second expression vector contains nucleic acids (ii) and (iii). In some embodiments, the transfection of the first expression vector is performed prior to the transfection of the second expression vector. In some embodiments, the transfection of the first expression vector is performed simultaneously with the transfection of the second expression vector.

[0007] In some embodiments, the cells are cultured in the absence of exogenously introduced cholesterol (e.g., after transfection of nucleic acid (i)). In some embodiments, the cells are cultured in the absence of exogenously introduced glutamine (e.g., after transfection of nucleic acid (ii)). In some embodiments, the cells are cultured in the presence of a glutamine synthetase inhibitor (e.g., methionine sulfoximine) (e.g., during and / or after transfection of nucleic acid (ii)). In some embodiments, the cells are cultured in the presence of a 3-ketosteroid reductase inhibitor (e.g., during and / or after transfection of nucleic acid (i)).

[0008] In some embodiments, the cells are cultured in the absence of exogenously introduced cholesterol and in the absence of exogenously introduced glutamine. In some embodiments, the cells are cultured in the absence of exogenously introduced cholesterol, in the absence of exogenously introduced glutamine and in the presence of a glutamine synthetase inhibitor (e.g., methionine sulfoximine). In some embodiments, the cells are cultured in the absence of exogenously introduced cholesterol (e.g., after transfection of nucleic acid (i)), and the cells are then cultured in the absence of exogenously introduced glutamine (e.g., after transfection of nucleic acid (ii)).

[0009] In one aspect, the present disclosure features a method for producing a therapeutic protein, the method comprising culturing cholesterol auxotrophic cells, transfecting the cells with a nucleic acid (i) encoding a protein capable of restoring the cholesterol biosynthetic function in the cells, a nucleic acid (ii) encoding a protein expressing one or more selectable markers, and a nucleic acid (iii) encoding a therapeutic protein, culturing the cells under conditions suitable for the expression of the therapeutic protein, and isolating and / or purifying the therapeutic protein.

[0010] In one embodiment, the cells are NS0 cells.

[0011] In some embodiments, the therapeutic protein is an antibody. In some embodiments, the therapeutic protein is a fusion protein. In some embodiments, the therapeutic protein is an Fc-containing fusion protein.

[0012] In some embodiments, in the transfection step, the cells are transfected with a nucleic acid (iii) encoding an antibody light chain and an antibody heavy chain.

[0013] In some embodiments, the nucleic acid (i) encodes 3-ketosteroid reductase.

[0014] In some embodiments, nucleic acid (ii) encodes glutamine synthetase, dihydrofolate reductase (DHFR), or one or more antibiotic resistance genes (e.g., neomycin, blasticidin, hygromyocin, puromycin, zeocin, mycophenolic acid).

[0015] In some embodiments, the first expression vector contains nucleic acids (i) and (iii), and the second expression vector contains nucleic acid (ii). In some embodiments, the first expression vector contains nucleic acid (i), and the second expression vector contains nucleic acids (ii) and (iii). In some embodiments, transfection of the first expression vector is performed prior to transfection of the second expression vector. In some embodiments, transfection of the first expression vector is performed simultaneously with transfection of the second expression vector.

[0016] In some embodiments, the cells are cultured in the absence of exogenously introduced cholesterol (e.g., after transfection of nucleic acid (i)). In some embodiments, the cells are cultured in the presence of a 3-ketosteroid reductase inhibitor (e.g., during and / or after transfection of nucleic acid (i)). In some embodiments, the cells are cultured in the absence of exogenously introduced cholesterol.

[0017] In one aspect, the present disclosure features a method for producing a therapeutic protein, the method comprising culturing cholesterol auxotrophic cells that do not express functional dihydrofolate reductase (DHFR), transfecting the cells with a nucleic acid (i) that encodes a protein capable of restoring the cholesterol biosynthetic function in the cells, a nucleic acid (ii) that encodes DHFR, and a nucleic acid (iii) that encodes the therapeutic protein, culturing the cells under conditions suitable for expression of the therapeutic protein, and isolating and / or purifying the therapeutic protein.

[0018] In one embodiment, the cells are NS0 cells.

[0019] In some embodiments, the therapeutic protein is an antibody. In some embodiments, the therapeutic protein is a fusion protein. In some embodiments, the therapeutic protein is an Fc-containing fusion protein.

[0020] In some embodiments, in the transfection step, the cells are transfected with a nucleic acid (iii) encoding an antibody light chain and an antibody heavy chain. In some embodiments, the nucleic acid (i) encodes 3-ketosteroid reductase.

[0021] In some embodiments, the first expression vector contains the nucleic acids (i) and (iii), and the second expression vector contains the nucleic acid (ii). In some embodiments, the first expression vector contains the nucleic acid (i), and the second expression vector contains the nucleic acids (ii) and (iii). In some embodiments, the transfection of the first expression vector is performed prior to the transfection of the second expression vector. In some embodiments, the transfection of the first expression vector is performed simultaneously with the transfection of the second expression vector.

[0022] In some embodiments, the cells are cultured in the absence of exogenously introduced cholesterol (e.g., after transfection of the nucleic acid (i)). In some embodiments, the cells are cultured in the presence of a DHFR inhibitor (e.g., methionine sulfoximine (MSX)) (e.g., during and / or after transfection of the nucleic acid (ii)). In some embodiments, the cells are cultured in the presence of a 3-ketosteroid reductase inhibitor (e.g., during and / or after transfection of the nucleic acid (i)).

[0023] In some embodiments, the cells are cultured in the absence of exogenously introduced cholesterol. In some embodiments, the cells are cultured in the absence of exogenously introduced cholesterol and in the presence of a DHFR inhibitor (e.g., methionine sulfoximine (MSX)).

[0024] In one aspect, the present disclosure features a method for producing a therapeutic protein, the method comprising culturing cholesterol auxotrophic cells sensitive to neomycin, and transfecting the cells with: (i) a nucleic acid encoding a protein capable of restoring the cholesterol biosynthesis function in the cells, (ii) a nucleic acid encoding a neomycin resistance gene, and (iii) a nucleic acid encoding a therapeutic protein, culturing the cells under conditions suitable for expression of the therapeutic protein, and isolating and / or purifying the therapeutic protein.

[0025] In one embodiment, the cells are NS0 cells.

[0026] In some embodiments, the therapeutic protein is an antibody. In some embodiments, the therapeutic protein is a fusion protein. In some embodiments, the therapeutic protein is an Fc-containing fusion protein.

[0027] In some embodiments, in the transfection step, the cells are transfected with a nucleic acid (iii) encoding an antibody light chain and an antibody heavy chain. In some embodiments, the nucleic acid (i) encodes 3-ketosteroid reductase. In some embodiments, the nucleic acid (ii) encodes a neomycin resistance gene from Tn5 encoding aminoglycoside 3'-phosphotransferase (APH3'II).

[0028] In some embodiments, the first expression vector contains nucleic acids (i) and (iii), and the second expression vector contains nucleic acid (ii). In some embodiments, the first expression vector contains nucleic acid (i), and the second expression vector contains nucleic acids (ii) and (iii). In some embodiments, transfection of the first expression vector is performed prior to transfection of the second expression vector. In some embodiments, transfection of the first expression vector is performed simultaneously with transfection of the second expression vector.

[0029] In some embodiments, both the first and second expression vectors contain HC and LC, one vector contains KSR, and the other contains GS or another antibiotic resistance gene. In some embodiments, there are three different vectors (e.g., in the case of triple selection), all three vectors contain HC and LC, the first vector contains KSR, the second vector contains GS, and the third vector contains an arbitrary antibiotic resistance gene, such as NEO.

[0030] In some embodiments, the cells are cultured in the absence of exogenously introduced cholesterol (e.g., after transfection of nucleic acid (i)). In some embodiments, the cells are cultured in the presence of an aminoglycoside antibiotic (e.g., G418) (e.g., during and / or after transfection of nucleic acid (ii)). In some embodiments, the cells are cultured in the presence of a 3-ketosteroid reductase inhibitor (e.g., during and / or after transfection of nucleic acid (i)).

[0031] In some embodiments, the cells are cultured in the absence of exogenously introduced cholesterol. In some embodiments, the cells are cultured in the absence of exogenously introduced cholesterol and in the presence of an aminoglycoside antibiotic (e.g., G418).

[0032] In one aspect, the present disclosure features a method for producing a therapeutic protein, the method comprising culturing cholesterol auxotrophic cells sensitive to blasticidin, transfecting the cells with a nucleic acid (i) encoding a protein capable of restoring the cholesterol biosynthesis function in the cells, a nucleic acid (ii) encoding a blasticidin resistance gene, and a nucleic acid (iii) encoding a therapeutic protein, culturing the cells under conditions suitable for the expression of the therapeutic protein, and isolating and / or purifying the therapeutic protein.

[0033] In one embodiment, the cells are NS0 cells.

[0034] In some embodiments, the therapeutic protein is an antibody. In some embodiments, the therapeutic protein is a fusion protein. In some embodiments, the therapeutic protein is an Fc-containing fusion protein.

[0035] In some embodiments, in the transfection step, the cell is transfected with a nucleic acid (iii) encoding an antibody light chain and an antibody heavy chain. In some embodiments, the nucleic acid (i) encodes a 3-ketosteroid reductase. In some embodiments, the nucleic acid (ii) encodes a blasticidin resistance gene (encoding blasticidin-S deaminase) from Bacillus cereus.

[0036] In some embodiments, the first expression vector contains the nucleic acids (i) and (iii), and the second expression vector contains the nucleic acid (ii). In some embodiments, the first expression vector contains the nucleic acid (i), and the second expression vector contains the nucleic acids (ii) and (iii). In some embodiments, the transfection of the first expression vector is performed prior to the transfection of the second expression vector. In some embodiments, the transfection of the first expression vector is performed simultaneously with the transfection of the second expression vector.

[0037] In some embodiments, the cells are cultured in the absence of exogenously introduced cholesterol (e.g., after transfection of the nucleic acid (i)). In some embodiments, the cells are cultured in the presence of a peptidyl nucleoside antibiotic (e.g., blasticidin) (e.g., during and / or after transfection of the nucleic acid (ii)). In some embodiments, the cells are cultured in the presence of a 3-ketosteroid reductase inhibitor (e.g., during and / or after transfection of the nucleic acid (i)).

[0038] In some embodiments, the cells are cultured in the absence of exogenously introduced cholesterol. In some embodiments, the cells are cultured in the absence of exogenously introduced cholesterol and in the presence of peptidyl nucleoside antibiotics, aminoglycoside antibiotics (e.g., blasticidin).

[0039] In one aspect, the present disclosure features a method for producing a therapeutic protein, the method comprising culturing cholesterol auxotrophic cells sensitive to hygromycin B, transfecting the cells with a nucleic acid (i) encoding a protein capable of restoring cholesterol biosynthesis function in the cells, a nucleic acid (ii) encoding a hygromycin B resistance gene, and a nucleic acid (iii) encoding a therapeutic protein, culturing the cells under conditions suitable for the expression of the therapeutic protein, and isolating and / or purifying the therapeutic protein.

[0040] In one embodiment, the cells are NS0 cells.

[0041] In some embodiments, the therapeutic protein is an antibody. In some embodiments, the therapeutic protein is a fusion protein. In some embodiments, the therapeutic protein is an Fc-containing fusion protein.

[0042] In some embodiments, in the transfection step, the cells are transfected with a nucleic acid (iii) encoding an antibody light chain and an antibody heavy chain. In some embodiments, the nucleic acid (i) encodes 3-ketosteroid reductase. In some embodiments, the nucleic acid (ii) encodes hygromycin B phosphotransferase.

[0043] In some embodiments, the first expression vector contains nucleic acids (i) and (iii), and the second expression vector contains nucleic acid (ii). In some embodiments, the first expression vector contains nucleic acid (i), and the second expression vector contains nucleic acids (ii) and (iii). In some embodiments, transfection of the first expression vector is performed prior to transfection of the second expression vector. In some embodiments, transfection of the first expression vector is performed simultaneously with transfection of the second expression vector.

[0044] In some embodiments, the cells are cultured in the absence of exogenously introduced cholesterol (e.g., after transfection of nucleic acid (i)). In some embodiments, the cells are cultured in the presence of an aminoglycoside antibiotic (e.g., hygromycin B) (e.g., during and / or after transfection of nucleic acid (ii)). In some embodiments, the cells are cultured in the presence of a 3-ketosteroid reductase inhibitor (e.g., during and / or after transfection of nucleic acid (i)).

[0045] In some embodiments, the cells are cultured in the absence of exogenously introduced cholesterol. In some embodiments, the cells are cultured in the absence of exogenously introduced cholesterol and in the presence of a peptidyl nucleoside antibiotic and an aminoglycoside antibiotic (e.g., hygromycin B).

[0046] In one aspect, the present disclosure features a method for producing a therapeutic protein, the method comprising culturing cholesterol auxotrophic cells sensitive to puromycin, transfecting the cells with a nucleic acid (i) encoding a protein capable of restoring cholesterol biosynthetic function in the cells, a nucleic acid (ii) encoding a puromycin resistance gene, and a nucleic acid (iii) encoding a therapeutic protein, culturing the cells under conditions suitable for expression of the therapeutic protein, and isolating and / or purifying the therapeutic protein.

[0047] In one embodiment, the cell is an NS0 cell.

[0048] In some embodiments, the therapeutic protein is an antibody. In some embodiments, the therapeutic protein is a fusion protein. In some embodiments, the therapeutic protein is an Fc-containing fusion protein.

[0049] In some embodiments, in the transfection step, the cell is transfected with a nucleic acid (iii) encoding an antibody light chain and an antibody heavy chain. In some embodiments, the nucleic acid (i) encodes 3-ketosteroid reductase. In some embodiments, the nucleic acid (ii) encodes puromycin N-acetyl-transferase.

[0050] In some embodiments, the first expression vector contains the nucleic acids (i) and (iii), and the second expression vector contains the nucleic acid (ii). In some embodiments, the first expression vector contains the nucleic acid (i), and the second expression vector contains the nucleic acids (ii) and (iii). In some embodiments, the transfection of the first expression vector is performed prior to the transfection of the second expression vector. In some embodiments, the transfection of the first expression vector is performed simultaneously with the transfection of the second expression vector.

[0051] In some embodiments, the cell is cultured in the absence of exogenously introduced cholesterol (e.g., after transfection with the nucleic acid (i)). In some embodiments, the cell is cultured in the presence of an aminonucleoside antibiotic (e.g., puromycin) (e.g., during and / or after transfection with the nucleic acid (ii)). In some embodiments, the cell is cultured in the presence of a 3-ketosteroid reductase inhibitor (e.g., during and / or after transfection with the nucleic acid (i)).

[0052] In some embodiments, the cells are cultured in the absence of exogenously introduced cholesterol. In some embodiments, the cells are cultured in the absence of exogenously introduced cholesterol and in the presence of peptidyl nucleoside antibiotics, amino nucleoside antibiotics (e.g., puromycin).

[0053] In one aspect, the present disclosure features a method for producing a therapeutic protein, the method comprising culturing cholesterol auxotrophic cells sensitive to zeocin, transfecting the cells with a nucleic acid (i) encoding a protein capable of restoring cholesterol biosynthesis function in the cells, a nucleic acid (ii) encoding a zeocin resistance gene, and a nucleic acid (iii) encoding a therapeutic protein, culturing the cells under conditions suitable for expression of the therapeutic protein, and isolating and / or purifying the therapeutic protein.

[0054] In one embodiment, the cells are NS0 cells.

[0055] In some embodiments, the therapeutic protein is an antibody. In some embodiments, the therapeutic protein is a fusion protein. In some embodiments, the therapeutic protein is an Fc-containing fusion protein.

[0056] In some embodiments, in the transfection step, the cells are transfected with a nucleic acid (iii) encoding an antibody light chain and an antibody heavy chain. In some embodiments, the nucleic acid (i) encodes 3-ketosteroid reductase. In some embodiments, the nucleic acid (ii) encodes a Sh ble gene product.

[0057] In some embodiments, the first expression vector contains nucleic acids (i) and (iii), and the second expression vector contains nucleic acid (ii). In some embodiments, the first expression vector contains nucleic acid (i), and the second expression vector contains nucleic acids (ii) and (iii). In some embodiments, the transfection of the first expression vector is performed prior to the transfection of the second expression vector. In some embodiments, the transfection of the first expression vector is performed simultaneously with the transfection of the second expression vector.

[0058] In some embodiments, the cells are cultured in the absence of exogenously introduced cholesterol (e.g., after transfection of nucleic acid (i)). In some embodiments, the cells are cultured in the presence of a copper-chelating glycopeptide antibiotic (e.g., zeocin) (e.g., during and / or after transfection of nucleic acid (ii)). In some embodiments, the cells are cultured in the presence of a 3-ketosteroid reductase inhibitor (e.g., during and / or after transfection of nucleic acid (i)). In some embodiments, the cells are cultured in the absence of exogenously introduced cholesterol. In some embodiments, the cells are cultured in the absence of exogenously introduced cholesterol and in the presence of a copper-chelating glycopeptide antibiotic (e.g., zeocin).

[0059] In one aspect, the present disclosure features a method for producing a therapeutic protein, the method comprising culturing cholesterol auxotrophic cells sensitive to mycophenolic acid (MPA), transfecting the cells with a nucleic acid (i) encoding a protein capable of restoring cholesterol biosynthesis function in the cells, a nucleic acid (ii) encoding an MPA resistance gene, and a nucleic acid (iii) encoding a therapeutic protein, culturing the cells under conditions suitable for the expression of the therapeutic protein, and isolating and / or purifying the therapeutic protein.

[0060] In one embodiment, the cells are NS0 cells.

[0061] In some embodiments, the therapeutic protein is an antibody. In some embodiments, the therapeutic protein is a fusion protein. In some embodiments, the therapeutic protein is an Fc-containing fusion protein.

[0062] In some embodiments, in the transfection step, the cell is transfected with a nucleic acid (iii) encoding an antibody light chain and an antibody heavy chain. In some embodiments, the nucleic acid (i) encodes 3-ketosteroid reductase. In some embodiments, the nucleic acid (ii) encodes the xanthine-guanine phosphoribosyltransferase (Ecogpt) gene.

[0063] In some embodiments, the first expression vector contains the nucleic acids (i) and (iii), and the second expression vector contains the nucleic acid (ii). In some embodiments, the first expression vector contains the nucleic acid (i), and the second expression vector contains the nucleic acids (ii) and (iii). In some embodiments, the transfection of the first expression vector is performed prior to the transfection of the second expression vector. In some embodiments, the transfection of the first expression vector is performed simultaneously with the transfection of the second expression vector.

[0064] In some embodiments, the cells are cultured in the absence of exogenously introduced cholesterol (e.g., after transfection of the nucleic acid (i)). In some embodiments, the cells are cultured in the presence of MPA (e.g., during and / or after transfection of the nucleic acid (ii)). In some embodiments, the cells are cultured in the presence of a 3-ketosteroid reductase inhibitor (e.g., during and / or after transfection of the nucleic acid (i)).

[0065] In some embodiments, the cells are cultured in the absence of exogenously introduced cholesterol. In some embodiments, the cells are cultured in the absence of exogenously introduced cholesterol and in the presence of MPA.

[0066] In one aspect, the present disclosure features a method for producing a therapeutic protein, the method comprising culturing cholesterol auxotrophic cells sensitive to mycophenolic acid (MPA), transfecting the cells with a nucleic acid (i) encoding a protein capable of restoring the cholesterol biosynthesis function in the cells, a nucleic acid (ii) encoding an MPA resistance gene, and a nucleic acid (iii) encoding a therapeutic protein, culturing the cells under conditions suitable for the expression of the therapeutic protein, and isolating and / or purifying the therapeutic protein.

[0067] In one embodiment, the cells are NS0 cells.

[0068] In some embodiments, the therapeutic protein is an antibody. In some embodiments, the therapeutic protein is a fusion protein. In some embodiments, the therapeutic protein is an Fc-containing fusion protein.

[0069] In some embodiments, in the transfection step, the cells are transfected with a nucleic acid (iii) encoding an antibody light chain and an antibody heavy chain. In some embodiments, the nucleic acid (i) encodes 3-ketosteroid reductase. In some embodiments, the nucleic acid (ii) encodes the xanthine-guanine phosphoribosyltransferase (Ecogpt) gene.

[0070] In some embodiments, the first expression vector contains nucleic acids (i) and (iii), and the second expression vector contains nucleic acid (ii). In some embodiments, the first expression vector contains nucleic acid (i), and the second expression vector contains nucleic acids (ii) and (iii). In some embodiments, the transfection of the first expression vector is performed prior to the transfection of the second expression vector. In some embodiments, the transfection of the first expression vector is performed simultaneously with the transfection of the second expression vector.

[0071] In some embodiments, the cells are cultured in the absence of exogenously introduced cholesterol (e.g., after transfection of nucleic acid (i)). In some embodiments, the cells are cultured in the presence of MPA (e.g., during and / or after transfection of nucleic acid (ii)). In some embodiments, the cells are cultured in the presence of a 3-ketosteroid reductase inhibitor (e.g., during and / or after transfection of nucleic acid (i)).

[0072] In some embodiments, the cells are cultured in the absence of exogenously introduced cholesterol. In some embodiments, the cells are cultured in the absence of exogenously introduced cholesterol and in the presence of MPA.

[0073] In one aspect, the present disclosure features a method for producing a therapeutic protein, the method comprising culturing cholesterol auxotrophic and glutamine auxotrophic cells, transfecting the cells with a nucleic acid (i) (e.g., KSR) encoding a protein capable of restoring cholesterol biosynthesis function in the cells, a nucleic acid (ii) (e.g., glutathione synthetase) encoding a protein capable of restoring glutamine biosynthesis function in the cells, a nucleic acid (iii) (e.g., neomycin resistance gene) encoding a protein capable of conferring resistance to G418, and a nucleic acid (iv) (e.g., an antibody, e.g., a heavy chain and a light chain) encoding a therapeutic protein, culturing the cells under conditions suitable for expression of the therapeutic protein, and isolating and / or purifying the therapeutic protein.

[0074] In some embodiments, nucleic acid (iii) is transfected first, and G418 is added to the cell culture medium during and / or after transfection. In some embodiments, nucleic acid (iii) is transfected first, G418 is added to the cell culture medium during and / or after transfection, nucleic acid (ii) is transfected second, and cholesterol is absent from the cell culture medium during and / or after transfection. In some embodiments, nucleic acid (iii) is transfected first, G418 is added to the cell culture medium during and / or after transfection, nucleic acid (ii) is transfected second, cholesterol (e.g., exogenously added cholesterol) is absent from the cell culture medium during and / or after transfection, nucleic acid (i) is transfected third, and glutamine (e.g., exogenously added glutamine) is absent from the cell culture medium during and / or after transfection (optionally, methionine sulfoximine (MSX) is added to the cell culture medium).

[0075] In some embodiments, nucleic acids (ii) and (i) are transfected simultaneously, G418 is added to the cell culture medium, and the cell culture medium does not contain cholesterol (e.g., exogenously added cholesterol). In some embodiments, nucleic acids (i), (ii), (iii), and (iv) are transfected simultaneously, G418 is added to the cell culture medium, the cell culture medium does not contain cholesterol (e.g., exogenously added cholesterol), and also does not contain glutamine (e.g., exogenously added glutamine) (optionally, methionine sulfoximine (MSX) is added to the cell culture medium). In some embodiments, nucleic acid (iv) is incorporated into the same nucleic acid sequence (e.g., vector) as each of (i), (ii), and (iii). In some embodiments, nucleic acid (iv) is incorporated into the same nucleic acid sequence (e.g., vector) as each of (i) and (ii), (i) and (iii), or (ii) and (iii). In some embodiments, nucleic acid (iv) encodes the heavy and light chains of a therapeutic antibody. In some embodiments, the light chain is incorporated into the same nucleic acid sequence (e.g., vector) as each of (i) and (ii), (i) and (iii), (ii) and (iii), or (i), (ii), and (iii). In some embodiments, the heavy chain is incorporated into the same nucleic acid sequence (e.g., vector) as each of (i) and (ii), (i) and (iii), (ii) and (iii), or (i), (ii), and (iii). In some embodiments, the light and heavy chains are incorporated into the same nucleic acid sequence (e.g., vector) as each of (i) and (ii), (i) and (iii), (ii) and (iii), or (i), (ii), and (iii).

[0076] In some embodiments, any one of nucleic acids (i), (ii), (iii), and (iv) is incorporated into one nucleic acid (e.g., one vector). For example, in some embodiments, nucleic acids (i) and (ii), (i) and (iii), (i) and (iv), (i), (ii) and (iii), (i), (ii), (iii) and (iv), (ii) and (iii), or (ii) and (iv), (iii) and (iv) are incorporated into one nucleic acid. In some embodiments, nucleic acid (iv) contains two separate nucleic acids, one encoding the heavy chain of a therapeutic antibody and one encoding the light chain. In some embodiments, the heavy chain is incorporated onto another nucleic acid, such as (i), (ii), or (iii). In some embodiments, the light chain is incorporated onto another nucleic acid, such as (i), (ii), or (iii).

[0077] In one embodiment, the cell is an NS0 cell.

[0078] In some embodiments, the therapeutic protein is an antibody. In some embodiments, the therapeutic protein is a fusion protein. In some embodiments, the therapeutic protein is an Fc-containing fusion protein.

[0079] In some embodiments, in the transfection step, the cell is transfected with nucleic acid (iv) encoding the antibody light chain and the antibody heavy chain. In some embodiments, nucleic acid (i) encodes 3-ketosteroid reductase. In some embodiments, nucleic acid (ii) encodes glutamine synthetase. In some embodiments, nucleic acid (iii) encodes a neomycin resistance gene.

[0080] In some embodiments, the first expression vector contains nucleic acids (i) and (iv), and the second expression vector contains nucleic acid (ii). In some embodiments, the first expression vector contains nucleic acid (i), and the second expression vector contains nucleic acids (ii) and (iv). In some embodiments, transfection of the first expression vector is performed prior to transfection of the second expression vector. In some embodiments, transfection of the first expression vector is performed simultaneously with transfection of the second expression vector.

[0081] In some embodiments, the cells are cultured in the absence of exogenously introduced cholesterol (e.g., after transfection of nucleic acid (i)). In some embodiments, the cells are cultured in the absence of exogenously introduced glutamine (e.g., after transfection of nucleic acid (ii)). In some embodiments, the cells are cultured in the presence of a glutamine synthetase inhibitor (e.g., methionine sulfoximine) (e.g., during and / or after transfection of nucleic acid (ii)). In some embodiments, the cells are cultured in the presence of a 3-ketosteroid reductase inhibitor (e.g., during and / or after transfection of nucleic acid (i)).

[0082] In some embodiments, the cells are cultured in the absence of exogenously introduced cholesterol and in the absence of exogenously introduced glutamine. In some embodiments, the cells are cultured in the absence of exogenously introduced cholesterol, in the absence of exogenously introduced glutamine, and in the presence of a glutamine synthetase inhibitor (e.g., methionine sulfoximine). In some embodiments, the cells are cultured in the absence of exogenously introduced cholesterol (e.g., after transfection of nucleic acid (i)), and the cells are cultured in the absence of exogenously introduced glutamine (e.g., after transfection of nucleic acid (ii)).

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0084] In certain recombinant protein production systems, such as the NS0 host cell line, cholesterol auxotrophy can be exploited by incorporating the 3-ketosteroid reductase (3-KSR) gene into a vector encoding a therapeutic protein, and recombinant cells can be selected by removing cholesterol from the culture medium (U.S. Patent Application No. 2010 / 0028940). However, the inventors of the present disclosure unexpectedly discovered that in this system, amplification is difficult in this cell line, and thus productivity (e.g., protein product / cell / day) and protein titer may be low (see, for example, FIG. 8). In other NS0 host cell lines, glutamine auxotrophy can be exploited by incorporating the glutamine synthetase (GS) gene into a vector encoding a therapeutic protein, and transfected cells can be selected by removing glutamine from the culture medium. Additionally, the addition of a GS inhibitor to the culture medium can promote selection for multiple copies of the GS gene and thus integration of the gene encoding the protein of interest (Barnes et al. Cytotechnology. 2000. Advances in animal cell recombinant protein production: GS-NS0 expression system Feb;32(2):109-23). Each system has different constraints. In this specification, an alternative method for manufacturing a therapeutic product in, for example, the NS0 host cell line will be described.

[0085] As used herein, a "glutamine auxotrophic" cell is defined as a cell that does not synthesize glutamine or does not synthesize sufficient glutamine to enable survival and proliferation in a glutamine-free cell culture medium.

[0086] As used herein, a "cholesterol auxotrophic" cell is defined as a cell that does not synthesize cholesterol or does not synthesize sufficient cholesterol to enable survival and proliferation in a cholesterol-free cell culture medium.

[0087] As used herein, "restoring glutamine biosynthesis function" means increasing the glutamine biosynthesis level in a cell to at least a level that enables the survival and proliferation of the cell in a glutamine-free cell culture medium (from the level in glutamine auxotrophic cells).

[0088] As used herein, "restoring cholesterol biosynthesis function" means increasing the cholesterol biosynthesis level in a cell to at least a level that enables the survival and proliferation of the cell in a cholesterol-free cell culture medium (from the level in cholesterol auxotrophic cells).

[0089] As used herein, "survival and proliferation" refers to the ability of a cell or cell culture to maintain a cell survival rate higher than 60% during logarithmic growth when measured by the trypan blue dye exclusion method.

[0090] 3-ketosteroid reductase (KSR) The enzyme 3-ketosteroid reductase is encoded by the HSD17β7 gene and functions as 3-ketosteroid reductase in cholesterol biosynthesis (Marijanovic et al., Mol Endocrinol. 2003 Sep;17(9):1715-25). For the NCBI reference amino acid (NP_057455) and mRNA sequence (NM_016371.3), see Figure 2 (SEQ ID NO: 1) and Figure 3 (SEQ ID NO: 2), respectively.

[0091] Glutamine synthetase (GS) The enzyme glutamine synthetase encoded by the glutamine synthetase gene catalyzes the synthesis of glutamine from glutamate and ammonia (Eisenberg et al., Biochimica et Biophysica Acta, 2000, Vol 1477, 122-145). Several alternatively spliced transcript variants have been found for this gene. Refer to FIG. 4 (SEQ ID NO: 3) and FIG. 5 (SEQ ID NO: 4) for NCBI reference amino acid NP_002056.2 and mRNA sequence NM_002065.6, respectively.

[0092] Dihydrofolate reductase (DHFR) The enzyme DHFR is encoded by the DHFR gene and converts dihydrofolate to tetrahydrofolate, a methyl group shuttle required for de novo purine synthesis, thymidylate, and certain amino acids. Several alternatively spliced transcript variants have been found for this gene. Refer to FIG. 6 (SEQ ID NO: 5) and FIG. 7 (SEQ ID NO: 6) for NCBI reference amino acid (NP_000782.1) and mRNA sequence (NM_000791.3), respectively.

[0093] Antibiotic resistance gene selection marker Antibiotic resistance genes are positive selection markers commonly used in mammalian cell culture (see, e.g., Antibody Expression and Production, Editor Mohamed Al-Rubeai, Springer Netherlands, Springer Science Business Media B.V., ISBN 978-94-007-1256-0; Cell Line Development, Mohamed Al-Rubeai August 11, 2009 Springer Science&Business Media). Examples of antibiotic resistance genes include, but are not limited to, neomycin resistance gene, blasticidin, hygromycin, puromycin, zeocin, and genes conferring resistance to mycophenolic acid. Antibiotic resistance genes and corresponding inhibitors suitable for use in cell selection will be known to those skilled in the art.

[0094]

Table 1

[0095] Production of Vectors, Host Cells, and Therapeutic Proteins The therapeutic proteins of the present invention can be produced from host cells. A host cell is a medium containing the cellular components necessary to express the polypeptides and constructs described herein from their corresponding nucleic acids, such as organelles. The nucleic acid can be included in a nucleic acid vector that can be introduced into the host cell by conventional techniques known in the art (e.g., transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, infection, etc.). The selection of the nucleic acid vector depends in part on the host cell to be used. Generally, preferred host cells are derived from prokaryotes (e.g., bacteria) or eukaryotes (e.g., mammals).

[0096] Construction of Nucleic Acid Vectors and Host Cells The nucleic acid sequence encoding the amino acid sequence of the therapeutic protein of the present invention can be prepared by various methods known in the art. These methods include, but are not limited to, oligonucleotide-mediated (or site-directed) mutagenesis and PCR mutagenesis. The nucleic acid molecule encoding the therapeutic protein of the present invention can be obtained using standard techniques, such as gene synthesis. Alternatively, a nucleic acid molecule encoding a wild-type therapeutic protein can be mutated to contain specific amino acid substituents using standard techniques in the art, such as QuikChange™ mutagenesis. The nucleic acid molecule can be synthesized using a nucleotide synthesizer or PCR techniques.

[0097] The nucleic acid sequence encoding the therapeutic protein of the present invention can be replicated in a prokaryotic or eukaryotic host cell and inserted into a vector capable of expressing the nucleic acid molecule. Many vectors are available in the art and can be used for the purposes of the present invention. Each vector can contain various components that can be regulated and optimized to be compatible with a specific host cell. For example, the vector components can include, but are not limited to, an origin of replication, a selectable marker gene, a promoter, a ribosome binding site, a signal sequence, a nucleic acid sequence encoding the target protein, and a transcription termination sequence.

[0098] In one example, the vectors of the present invention include vectors containing a weak promoter for a selectable gene (e.g., 3-ketosteroid reductase and glutamine synthetase) and a strong promoter for a gene encoding a target protein (e.g., an antibody, e.g., the heavy and light chains of an antibody). The vector can be linearized or supercoiled and exhibits improved transfection efficiency. In some embodiments, codon optimization of the vector is used to minimize the use of rare codons in the coding sequence and improve protein production yields. Examples of vectors of the present invention are described in Wurm (2004) Nature Biotechnology, Vol 22; Issue 11:1393-1398).

[0099] In some embodiments, mammalian cells are used as host cells for the present invention. The glutamine auxotrophic and cholesterol auxotrophic phenotypes can be induced by genetically engineering non-glutamine auxotrophic and non-cholesterol auxotrophic cells, for example, mutations or deletions in genes required for endogenous glutamine biosynthesis, such as glutamine synthetase. General methods of genetic engineering are well known to those skilled in the art, such as site-directed mutagenesis, Zn finger nucleases, shRNA, transposons, see, for example, Cytotechnology. 2007 Apr;53(1-3):65-73. Mouse myeloma cells designated NS0 are known glutamine auxotrophic and cholesterol auxotrophic cells (see, for example, Barnes et al. Cytotechnology. 2000. Advances in animal cell recombinant protein production: GS-NS0 expression system Feb;32(2):109-23 and U.S. Patent Application No. 2010 / 0028940).

[0100] Additional examples of mammalian cell types that can be genetically engineered and used as host cells include, but are not limited to, human embryonic kidney (HEK) (e.g., HEK293, HEK293F), Chinese hamster ovary (CHO), HeLa, COS, PC3, Vero, MC3T3, NS0, VERY, BHK, MDCK, W138, BT483, Hs578T, HTB2, BT20, T47D), CRL7030, and HsS78Bst cells. In other embodiments, Escherichia coli (E. coli) cells are used as host cells for the present invention. Examples of E. coli strains include, but are not limited to, E. coli 294 (ATCC® 31446), E. coli λ1776 (ATCC® 31537), E. coli BL21(DE3) (ATCC® BAA-1025), and E. coli RV308 (ATCC® 31608). Various host cells have characteristic and specific mechanisms regarding post-translational processing and modification of protein products. By selecting an appropriate cell line or host system, the therapeutic protein to be expressed can be accurately modified and processed. The above expression vectors can be introduced into appropriate host cells using techniques conventional in the art, such as transformation, transfection, electroporation, calcium phosphate precipitation, and direct microinjection. Once the vector has been introduced into the host cell for protein production, the host cell is cultured in a conventional nutrient medium appropriately modified for induction of the promoter, selection of transformants, or amplification of the gene encoding the desired sequence.Methods for expressing therapeutic proteins are known in the art. See, for example, Paulina Balbas, Argelia Lorence (eds.) Recombinant Gene Expression: Reviews and Protocols (Methods in Molecular Biology), Humana Press; 2nd ed. 2004 (July 20, 2004) and Vladimir Voynov and Justin A. Caravella (eds.) Therapeutic Proteins: Methods and Protocols (Methods in Molecular Biology) Humana Press; 2nd ed. 2012 (June 28, 2012).

[0101] Production, recovery, and purification of proteins The host cells used for the production of the therapeutic proteins of the present invention are known in the art and can be grown in a medium suitable for culturing the selected host cells. Examples of media suitable for mammalian host cells include basal medium (MEM), Dulbecco's modified Eagle's medium (DMEM), Expi293™ Expression Medium, fetal bovine serum (FBS)-supplemented DMEM, and RPMI-1640. Examples of media suitable for bacterial host cells include Luria broth (LB) supplemented with necessary agents such as selective agents, for example, ampicillin. The host cells are cultured at an appropriate temperature, for example, about 20 to about 39°C, for example, 25 to about 37°C, preferably 37°C, and CO2 level, for example, 5 to 10% (preferably 8%). The pH of the medium is usually about 6.8 to 7.4, for example, 7.0, and mainly depends on the host organism. If an inducible promoter is used in the expression vector of the present invention, the expression of the protein is induced under conditions suitable for the activation of this promoter. Conventional cell culture conditions for producing therapeutic proteins are known in the art. See, for example, Butler, Cell Culture and Upstream Processing, Taylor & Francis; 1st edition (May 25, 2007).

[0102] In the recovery of proteins, typically, host cells are disrupted by means such as osmotic shock, sonication, or lysis. Once the cells are disrupted, cell debris can be removed by centrifugation or filtration. The protein can be further purified. The antibodies of the present invention can be purified by any protein purification method known in the art, for example, protein A affinity method, other chromatography (e.g., ion exchange, affinity, and size exclusion column chromatography), centrifugation, differential solubility method, or other standard techniques for purifying proteins. (See Process Scale Purification of Antibodies, Uwe Gottschalk (ed.) John Wiley & Sons, Inc., 2009). In some instances, a therapeutic protein can be conjugated to a marker sequence, such as a peptide, to facilitate purification. An example of a marker amino acid sequence is the hexa-histidine peptide (His-tag), which binds with micromolar affinity to a nickel-functionalized agarose affinity column. Other peptide tags useful for purification include, but are not limited to, the hemagglutinin "HA" tag, which corresponds to an epitope derived from the influenza hemagglutinin protein.

[0103] Pharmaceutical Compositions and Formulations The present invention features pharmaceutical compositions comprising one or more therapeutic proteins described herein. In addition to a therapeutically effective amount of a therapeutic protein, the pharmaceutical compositions of the present invention can contain one or more pharmaceutically acceptable carriers or excipients, which can be formulated by methods known to those skilled in the art.

[0104] Acceptable carriers and excipients in the pharmaceutical composition are non-toxic to the recipient at the dosages and concentrations employed. Acceptable carriers and excipients may include buffers, antioxidants, preservatives, polymers, amino acids, and carbohydrates. The pharmaceutical composition of the present invention can be administered in the form of a parenterally injectable formulation. The pharmaceutical composition for injection (i.e., intravenous injection) can be formulated using a sterile solution or any pharmaceutically acceptable liquid as a vehicle. Pharmaceutically acceptable vehicles include, but are not limited to, sterile water, physiological saline, and cell culture media (e.g., Dulbecco's Modified Eagle Medium (DMEM), α-Modified Eagle Medium (α-MEM), F-12 medium). The formulation methods are known in the art, see, for example, Banga (ed.) Therapeutic Peptides and Proteins: Formulation, Processing and Delivery Systems (2nd ed.) Taylor & Francis Group, CRC Press (2006).

[0105] The pharmaceutical composition can be formed in unit dosage form if necessary. The amount of the active ingredient, e.g., one or more therapeutic proteins of the present invention included in the pharmaceutical preparation, is such that it results in an appropriate dosage within the specified range (e.g., a dosage within the range of 0.01 - 500 mg / kg (body weight)).

[0106] Therapeutic protein Therapeutic proteins that can be produced by the methods described herein include all recombinant therapeutic proteins or biosimilars thereof targeted, including, but not limited to, antibodies (e.g., monoclonal antibodies, bispecific antibodies, multispecific antibodies), fusion proteins (e.g., Fc fusions), anticoagulants, blood factors, bone morphogenetic proteins, recombinant protein scaffolds, enzymes, growth factors, hormones, hormone releasing factors, interferons, interleukins, and thrombolytics. Therapeutic proteins include both glycosylated (e.g., proteins having at least one oligosaccharide chain) and non-glycosylated proteins.

[0107] Examples of monoclonal antibodies include, but are not limited to, adalimumab, infliximab, palivizumab, cetuximab, natalizumab, eculizumab, ustekinumab, golimumab, ofatumab, canakinumab, belimumab, alirocumab, mepolizumab, necitumumab, nivolumab, dinutuximab, secukinumab, evolocumab, blinatumomab, pembrolizumab, ramucirumab, vedolizumab, siltuximab, obinutuzumab, trastuzumab, lirilumab, pertuzumab, brentuximab, ipilimumab, denosumab, tocilizumab, ofatumab, canakinumab, certolizumab, catumaxomab, ranibizumab, panitumumab, bevacizumab, cetuximab, efalizumab, omalizumab, tositumomab, ibritumomab, alemtuzumab, gemtuzumab, basiliximab, daclizumab, rituximab, and abciximab.

[0108] Examples of fusion proteins include, but are not limited to, alefacept, etanercept, abatacept, belatacept, aflibercept, ziv-aflibercept, rilonacept, romiplostim, apocept, trebananib, brolucizumab, and dulaglutide.

[0109] Other embodiments The present invention will be further described by the following examples, which should not be construed as limiting. The contents of all references, patents, and published patent applications cited throughout this application are incorporated herein by reference.

Examples

[0110] Example 1: Production of Therapeutic Antibodies Using Double Auxotrophic Cells In the following examples, a method for producing a therapeutic protein, such as an antibody, is described, which method includes expression in cholesterol auxotrophic and glutamine auxotrophic cells of the therapeutic protein of interest. Desirably, this method enables amplification of the gene of interest and increase in copy number, and production in the absence of cholesterol (which is highly insoluble in aqueous solution and extremely difficult to handle). An exemplary method is outlined further in FIG. 1.

[0111] Transfect glutamine auxotrophic and cholesterol auxotrophic cells, such as NS0 cells, with a first expression vector containing a nucleic acid encoding 3-ketosteroid reductase (3-KSR). During and after this first transfection, cells are maintained in cholesterol-free medium to select cells expressing the first expression vector, and a 3-KSR-expressing glutamine auxotrophic cell culture is obtained. These cells are simultaneously or subsequently transfected with a second vector containing nucleic acids encoding glutamine synthetase (GS) and the protein of interest, such as the heavy and light chains of a therapeutic antibody. During and after this second transfection, cells are maintained in glutamine-free medium to select cells expressing the second vector. To further select cells that have incorporated multiple copies of the second vector, the cell culture medium during and / or after the second transfection is supplemented with a glutamine synthetase inhibitor, such as methionine sulfoximine (MSX). The final 3-KSR-GS antibody-producing cells can be maintained in cholesterol-free medium to avoid production problems related to cholesterol and also exhibit high productivity due to MSX selection.

[0112] Example 2: Cell viability assay In the following examples, a trypan blue assay for determining cell viability as a measure of cells showing sufficient survival and proliferation is described.

[0113] Prepare a 0.4% solution of trypan blue in buffered isotonic saline at pH 7.2 - 7.3 (i.e., phosphate buffered saline). Add 0.1 mL of the trypan blue stock solution to 1 mL of the cells. Load a hemocytometer and observe immediately under a low magnification microscope. Count the number of cells stained blue and the total number of cells. Calculate the cell viability as the number of live cells divided by the total number of cells within the grid on the hemocytometer. Consider cells that take up trypan blue as non-living cells. The cell viability should be at least 90% for a healthy logarithmic phase culture.

[0114] [Equation 1] Percentage of live cells (%) = [1.00 - (number of blue cells ÷ total number of cells)] × 100

[0115] The cell density of the cell line suspension can be determined using a hemocytometer. To calculate the number of live cells per mL of the culture, use the following formula and correct for the dilution factor: number of live cells × 10E4 × 1.1 = cells / mL (culture)

[0116] Example 3: Production of therapeutic antibodies using a triple selection method In the following example, a method for producing a therapeutic protein, such as an antibody, is described. This method involves the expression of the therapeutic protein of interest in cholesterol and glutamine auxotrophic cells, along with an additional third selection mechanism, such as a neomycin resistance gene (e.g., using G418 for selection). Desirably, this method allows for the amplification and increased copy number of the gene of interest and production in the absence of cholesterol (which is highly insoluble in aqueous solutions and extremely difficult to handle), while also overcoming unexpected manufacturing challenges associated with, for example, the 3-KSR cell culture selection system, including low productivity.

[0117] NS0 cell selection was first optimized using both single neomycin selection (Figure 9) and single GS selection (Figure 10). For neomycin optimization, cells were transfected with a nucleic acid vector encoding the neomycin resistance gene in the standard manner. Next, for selection, neomycin was added to the cell culture medium (at 100 μg / ml or 200 μg / ml). Cell viability was measured over the number of days described from harvest. For GS-based selection, cells were transfected with a nucleic acid vector encoding GS and maintained in glutamine-free cell culture medium. Cell viability was measured over the number of days described from harvest. By optimizing the 3-KSR-based triple selection method using neomycin, 3-KSR, and GS selection, the unexpected low productivity associated with 3-KSR selection was overcome (Figure 8). Cells were transfected with one or more nucleic acid vectors encoding 3-KSR, GS, and the neomycin resistance gene in the standard manner. For selection, cells were maintained in the absence of glutamine and cholesterol and in the presence of neomycin. As shown in Figure 11, the triple selection maintained sufficient cell viability after harvest. 〔Sequence Listing〕 SEQUENCE LISTING <110> MOMENTA PHARMACEUTICALS, INC. BIOFACTURA, INC. <120> METHODS OF MANUFACTURING THERAPEUTIC PROTEINS <130> M2050-7090WO <140> PCT / US2016 / 029472 <141> 2016-04-27 <150> 62 / 153,178 <151> 2015-04-27 <160> 7 <170> PatentIn version 3.5 <210> 1 <211> 341 <212> PRT <213> Homo sapiens <400> 1 Met Arg Lys Val Val Leu Ile Thr Gly Ala Ser Ser Gly Ile Gly Leu 1 5 10 15 Ala Leu Cys Lys Arg Leu Leu Ala Glu Asp Asp Glu Leu His Leu Cys 20 25 30 Leu Ala Cys Arg Asn Met Ser Lys Ala Glu Ala Val Cys Ala Ala Leu 35 40 45 Leu Ala Ser His Pro Thr Ala Glu Val Thr Ile Val Gln Val Asp Val 50 55 60 Ser Asn Leu Gln Ser Val Phe Arg Ala Ser Lys Glu Leu Lys Gln Arg 65 70 75 80 Phe Gln Arg Leu Asp Cys Ile Tyr Leu Asn Ala Gly Ile Met Pro Asn 85 90 95 Pro Gln Leu Asn Ile Lys Ala Leu Phe Phe Gly Leu Phe Ser Arg Lys 100 105 110 Val Ile His Met Phe Ser Thr Ala Glu Gly Leu Leu Thr Gln Gly Asp 115 120 125 Lys Ile Thr Ala Asp Gly Leu Gln Glu Val Phe Glu Thr Asn Val Phe 130 135 140 Gly His Phe Ile Leu Ile Arg Glu Leu Glu Pro Leu Leu Cys His Ser 145 150 155 160 Asp Asn Pro Ser Gln Leu Ile Trp Thr Ser Ser Arg Ser Ala Arg Lys 165 170 175 Ser Asn Phe Ser Leu Glu Asp Phe Gln His Ser Lys Gly Lys Glu Pro 180 185 190 Tyr Ser Ser Ser Lys Tyr Ala Thr Asp Leu Leu Ser Val Ala Leu Asn 195 200 205 Arg Asn Phe Asn Gln Gln Gly Leu Tyr Ser Asn Val Ala Cys Pro Gly 210 215 220 Thr Ala Leu Thr Asn Leu Thr Tyr Gly Ile Leu Pro Pro Phe Ile Trp 225 230 235 240 Thr Leu Leu Met Pro Ala Ile Leu Leu Leu Arg Phe Phe Ala Asn Ala 245 250 255 Phe Thr Leu Thr Pro Tyr Asn Gly Thr Glu Ala Leu Val Trp Leu Phe 260 265 270 His Gln Lys Pro Glu Ser Leu Asn Pro Leu Ile Lys Tyr Leu Ser Ala 275 280 285 Thr Thr Gly Phe Gly Arg Asn Tyr Ile Met Thr Gln Lys Met Asp Leu 290 295 300 Asp Glu Asp Thr Ala Glu Lys Phe Tyr Gln Lys Leu Leu Glu Leu Glu 305 310 315 320 Lys His Ile Arg Val Thr Ile Gln Lys Thr Asp Asn Gln Ala Arg Leu 325 330 335 Ser Gly Ser Cys Leu 340 <210> 2 <211> 1537 <212> DNA <213> Homo sapiens <400> 2 gtactctgat tggtgacggg tgaggcggcc cgaaatcgta ggacttccga aagcagcggc 60 ggcgtttgct tcactgcttg gaagtgtgag tgcgcgaaga tgcgaaaggt ggttttgatc 120 accggggcta gcagtggcat tggcctggcc ctctgcaagc ggctgctggc ggaagatgat 180 gagcttcatc tgtgtttggc gtgcaggaac atgagcaagg cagaagctgt ctgtgctgct 240 ctgctggcct ctcaccccac tgctgaggtc accattgtcc aggtggatgt cagcaacctg 300 cagtcggtct tccgggcctc caaggaactt aagcaaaggt ttcagagatt agactgtata 360 tatctaaatg ctgggatcat gcctaatcca caactaaata tcaaagcact tttctttggc 420 ctcttttcaa gaaaagtgat tcatatgttc tccacagctg aaggcctgct gacccagggt 480 gataagatca ctgctgatgg acttcaggag gtgtttgaga ccaatgtctt tggccatttt 540 atcctgattc gggaactgga gcctctcctc tgtcacagtg acaatccatc tcagctcatc 600 tggacatcat ctcgcagtgc aaggaaatct aatttcagcc tcgaggactt ccagcacagc 660 aaaggcaagg aaccctacag ctcttccaaa tatgccactg accttttgag tgtggctttg 720 aacaggaact tcaaccagca gggtctctat tccaatgtgg cctgtccagg tacagcattg 780 accaatttga catatggaat tctgcctccg tttatatgga cgctgttgat gccggcaata 840 ttgctacttc gcttttttgc aaatgcattc actttgacac catataatgg aacagaagct 900 ctggtatggc ttttccacca aaagcctgaa tctctcaatc ctctgatcaa atatctgagt 960 gccaccactg gctttggaag aaattacatt atgacccaga agatggacct agatgaagac 1020 actgctgaaa aattttatca aaagttactg gaactggaaa agcacattag ggtcactatt 1080 caaaaaacag ataatcaggc caggctcagt ggctcatgcc tataattcca gcactttggg 1140 aggccaaggc agaaggatca cttgagacca ggagttcaag accagcctga gaaacatagt 1200 gagcccttgt ctctacaaaa agaaataaaa ataatagctg ggtgtggtgg catgcgcatg 1260 tagtcccagc tactcagaag gatgaggtgg gaggatctct tgaggctggg aggcagaggt 1320 tgcagtgagc tgagattgtg ccactgcact ccagcctggg tgacagcgag accctgtctc 1380 aaaatatgta tatatttaat atatatataa aaccagagct gacaatgaca ctctggaaca 1440 ttgcatacct tctgtacatt ctggggtaca tggatttcta ctgagttgga taatatgcat 1500 ttgtaataaa ctatgaacta tgaaaaaaaa aaaaaaa 1537 <210> 3 <211> 373 <212> PRT <213> Homo sapiens <400> 3 Met Thr Thr Ser Ala Ser Ser His Leu Asn Lys Gly Ile Lys Gln Val 1 5 10 15 Tyr Met Ser Leu Pro Gln Gly Glu Lys Val Gln Ala Met Tyr Ile Trp 20 25 30 Ile Asp Gly Thr Gly Glu Gly Leu Arg Cys Lys Thr Arg Thr Leu Asp 35 40 45 Ser Glu Pro Lys Cys Val Glu Glu Leu Pro Glu Trp Asn Phe Asp Gly 50 55 60 Ser Ser Thr Leu Gln Ser Glu Gly Ser Asn Ser Asp Met Tyr Leu Val 65 70 75 80 Pro Ala Ala Met Phe Arg Asp Pro Phe Arg Lys Asp Pro Asn Lys Leu 85 90 95 Val Leu Cys Glu Val Phe Lys Tyr Asn Arg Arg Pro Ala Glu Thr Asn 100 105 110 Leu Arg His Thr Cys Lys Arg Ile Met Asp Met Val Ser Asn Gln His 115 120 125 Pro Trp Phe Gly Met Glu Gln Glu Tyr Thr Leu Met Gly Thr Asp Gly 130 135 140 His Pro Phe Gly Trp Pro Ser Asn Gly Phe Pro Gly Pro Gln Gly Pro 145 150 155 160 Tyr Tyr Cys Gly Val Gly Ala Asp Arg Ala Tyr Gly Arg Asp Ile Val 165 170 175 Glu Ala His Tyr Arg Ala Cys Leu Tyr Ala Gly Val Lys Ile Ala Gly 180 185 190 Thr Asn Ala Glu Val Met Pro Ala Gln Trp Glu Phe Gln Ile Gly Pro 195 200 205 Cys Glu Gly Ile Ser Met Gly Asp His Leu Trp Val Ala Arg Phe Ile 210 215 220 Leu His Arg Val Cys Glu Asp Phe Gly Val Ile Ala Thr Phe Asp Pro 225 230 235 240 Lys Pro Ile Pro Gly Asn Trp Asn Gly Ala Gly Cys His Thr Asn Phe 245 250 255 Ser Thr Lys Ala Met Arg Glu Glu Asn Gly Leu Lys Tyr Ile Glu Glu 260 265 270 Ala Ile Glu Lys Leu Ser Lys Arg His Gln Tyr His Ile Arg Ala Tyr 275 280 285 Asp Pro Lys Gly Gly Leu Asp Asn Ala Arg Arg Leu Thr Gly Phe His 290 295 300 Glu Thr Ser Asn Ile Asn Asp Phe Ser Ala Gly Val Ala Asn Arg Ser 305 310 315 320 Ala Ser Ile Arg Ile Pro Arg Thr Val Gly Gln Glu Lys Lys Gly Tyr 325 330 335 Phe Glu Asp Arg Arg Pro Ser Ala Asn Cys Asp Pro Phe Ser Val Thr 340 345 350 Glu Ala Leu Ile Arg Thr Cys Leu Leu Asn Glu Thr Gly Asp Glu Pro 355 360 365 Phe Gln Tyr Lys Asn 370 <210> 4 <211> 8337 <212> DNA <213> Homo sapiens <400> 4 gtaaaactat tccccgtgaa ggcggcaggg cagaggtcca gggcgggctt tgctgggagc 60 ctcgggaccc cgggttgggg gccgtggggc ggcacctggc gagctggcgg gtgggcggcg 120 agccgaggct tcccggcctg gcggcaactc gcccctctgc cctcagccct cccggctccg 180 ctcccttccc ccacgccgcc ctgcccctcc cccacgcccc tttctctttc tttctttctt 240 tcccagttcg cttgccccca ccccagcggc gcccgccggg ctcctcgccc aatggccgcg 300 gggcccggga ccgcatcagc tgatcggccc gggctcctgg ccgctgggag ccaatcaggg 360 caccgggggc ggccccgggc cgcggataaa gggtgcgggg ctgctggcgg ctctgcagag 420 tcgagagtgg gagaagagcg gagcgtgtga gcagtactgc ggcctcctct cctctcctaa 480 cctcgctctc gcggcctagc tttacccgcc cgcctgctcg gcgaccagcg gggatcctcc 540 cccagccgca agtccacgaa gaaagcaacg aatgaaaatt atgaagacaa cgagaagtca 600 gactcctccg ggtcgcgctc cagctgcttc ggcttcgtcg cctactctgt gaactccggg 660 gagagatctc gagtcaagat taagacctta acccaccaac ctgcctgttc ggacaccccc 720 cgggccggcc gctgtctgtc cccttctcca tcgccctctc ccagaaagct ccggtgcttg 780 gaccagctag agtctgagaa agaggagagg cgcgaacgcc actccaaaaa gagaagggtt 840 aaagagggca accctaacga tacgcttgac tttctgtggc tgggaacacc ttccaccatg 900 accacctcag caagttccca cttaaataaa ggcatcaagc aggtgtacat gtccctgcct 960 cagggtgaga aagtccaggc catgtatatc tggatcgatg gtactggaga aggactgcgc 1020 tgcaagaccc ggaccctgga cagtgagccc aagtgtgtgg aagagttgcc tgagtggaat 1080 ttcgatggct ctagtacttt acagtctgag ggttccaaca gtgacatgta tctcgtgcct 1140 gctgccatgt ttcgggaccc cttccgtaag gaccctaaca agctggtgtt atgtgaagtt 1200 ttcaagtaca atcgaaggcc tgcagagacc aatttgaggc acacctgtaa acggataatg 1260 gacatggtga gcaaccagca cccctggttt ggcatggagc aggagtatac cctcatgggg 1320 acagatgggc acccctttgg ttggccttcc aacggcttcc cagggcccca gggtccatat 1380 tactgtggtg tgggagcaga cagagcctat ggcagggaca tcgtggaggc ccattaccgg 1440 gcctgcttgt atgctggagt caagattgcg gggactaatg ccgaggtcat gcctgcccag 1500 tgggaatttc agattggacc ttgtgaagga atcagcatgg gagatcatct ctgggtggcc 1560 cgtttcatct tgcatcgtgt gtgtgaagac tttggagtga tagcaacctt tgatcctaag 1620 cccattcctg ggaactggaa tggtgcaggc tgccatacca acttcagcac caaggccatg 1680 cgggaggaga atggtctgaa gtacatcgag gaggccattg agaaactaag caagcggcac 1740 cagtaccaca tccgtgccta tgatcccaag ggaggcctgg acaatgcccg acgtctaact 1800 ggattccatg aaacctccaa catcaacgac ttttctgctg gtgtagccaa tcgtagcgcc 1860 agcatacgca ttccccggac tgttggccag gagaagaagg gttactttga agatcgtcgc 1920 ccctctgcca actgcgaccc cttttcggtg acagaagccc tcatccgcac gtgtcttctc 1980 aatgaaaccg gcgatgagcc cttccagtac aaaaattaag tggactagac ctccagctgt 2040 tgagcccctc ctagttcttc atcccactcc aactcttccc cctctcccag ttgtcccgat 2100 tgtaactcaa agggtggaat atcaaggtcg tttttttcat tccatgtgcc cagttaatct 2160 tgctttcttt gtttggctgg gatagagggg tcaagttatt aatttcttca cacctaccct 2220 cctttttttc cctatcactg aagcttttta gtgcattagt ggggaggagg gtggggagac 2280 ataaccactg cttccattta atggggtgca cctgtccaat aggcgtagct atccggacag 2340 agcacgtttg cagaaggggg tctcttcttc caggtagctg aaaggggaag acctgacgta 2400 ctctggttag gttaggactt gccctcgtgg tggaaacttt tcttaaaaag ttataaccaa 2460 cttttctatt aaaagtggga attaggagag aaggtagggg ttgggaatca gagagaatgg 2520 ctttggtctc ttgcttgtgg gactagcctg gcttgggact aaatgccctg ctctgaacac 2580 gaagcttagt ataaactgat ggatatccct accttgaaag aagaaaaggt tcttactgct 2640 tggtccttga tttatcacac aaagcagaat agtattttta tatttaaatg taaagacaaa 2700 aaactatatg tatggttttg tggattatgt gtgttttgct aaaggaaaaa accatccagg 2760 tcacggggca ccaaatttga gacaaatagt cggattagaa ataaagcatc tcattttgag 2820 tagagagcaa gggaagtggt tcttagatgg tgatctggga ttaggccctc aagacccttt 2880 tgggtttctg ccctgcccac cctctggaga aggtgggcac tggattagtt aacagacaac 2940 acgttactag cagtcacttg atctccgtgg ctttggttta aaagacacac ttgtccacat 3000 aggtttagag ataagagttg gctggtcaac ttgagcatgt tactgacaga gggggtattg 3060 gggttatttt ctggtaggaa tagcatgtca ctaaagcagg ccttttgata ttaaattttt 3120 taaaaagcaa aattatagaa gtttagattt taatcaaatt tgtagggttt ctaggtaatt 3180 tttacagaat tgcttgtttg cttcaactgt ctcctacctc tgctcttgga ggagatgggg 3240 acagggctgg agtcaaaaca cttgtaattt tgtatcttga tgtctttgtt aagactgctg 3300 aagaattatt ttttttcttt tataataagg aataaacccc acctttattc cttcatttca 3360 tctaccattt tctggttctt gtgttggctg tggcaggcca gctgtggttt tcttttgcca 3420 tgacaacttc taattgccat gtacagtatg ttcaaagtca aataactcct cattgtaaac 3480 aaactgtgta actgcccaaa gcagcactta taaatcagcc taacataaga tctctctgat 3540 gtgtttgtga ttctttcaaa tccctatgtg ccattatatt tctttatttc ctaaaacagg 3600 caaaataagc tcaagtttat gtactctgag tttttaaaac actggagtga tgttgctgac 3660 cagccgtttc ctgtacctct ctaagttggg tatttgggac ttaagggatt aagtttttca 3720 cctagactta gttacacaca atcttggcat ttcctagcct agaggtttgt agcagggtac 3780 aagccccact cctccccctt cctttgctcc cctgagtttg gttttggctt accataacat 3840 tgttttgacc attcctagcc taatacaata gcctaacata atgtaagatt aactggcttt 3900 acgatttcta ttctctgctc tcagtgataa gaaacaaata ttagctaccc tgctaccctg 3960 gttgaagcct tccaaggctg gctatgccct aggcatgggc tcatccttgg gtgtatcttg 4020 ccttgcagga agaccagtgg accgattgtg attctcaaaa gctctgtgtt gtcacctgtg 4080 cccttgcccc ttgctcttat cttggtccgt gtatctggga gttcttccac cttatcttgg 4140 ccaattccta ccttcgttca ttcctcatga ggttgggtaa aagctccctc cggctcccat 4200 gatgctgtgc atatacctag caaaaagcaa ttattggaca cattggagtg caatattatt 4260 aatagcatta atactactaa taatgtgggc aatagtgatt gtttttaaaa ggcagtatac 4320 tcttaccagt gcgaggtagc tggggcctgt gatagttttt agagataagt tcttcaggca 4380 actgtgtatt ttacactagt caagtaatcc tagatatccg tggtttttct taagaaagtt 4440 ggctcgtaat atgatttaat attcaaagta gagtcatcta cctattagct tgctggcgtg 4500 gtcctagttt atgcctgttt cagcatgatt gttgagtacc ctgtttcatc cttagcattt 4560 tcttgatttt gttgttaaat gatgtatacc cttatttcca ttgaatctgt gcttccaccc 4620 ccccaactga agttgtcttc cctttgcttg gccaccctta cagcctcttg gatggtgtat 4680 cctacagtgt aagcactaaa ctgaagaggc agtgacctga gcactttgga ttttgttcat 4740 tgtaatcaat tccatgacaa aatgattgca tgagaaggaa ttttaaattc ataggatcag 4800 aatttaggtg aaaacaacca gcatatttgt ttcttcaccc tctcacctag aattagcttt 4860 gacctacagg tcacagtgca atccccttgt atttctaagg tgttttttat agttcatttg 4920 cagacaatgg gttatgtgat aacttttatc agtgatagat taaacagaat aatgaccaag 4980 ctttcaacct taaggagtca ggccagtatt tacaaaagga ggtctccatg aactccttaa 5040 atatgagttc ccctaatatc atcttgccag gtactaaata acaactgata gcacaagcta 5100 tagggaattt gaaagaattc catggatggg tgttgtctag ggccttttgt tgtttttgag 5160 acggggtctg actctcaccc aggctggagt atagtgtggc gcaatcttgg ctcactgcaa 5220 cttctgcctc ccagattcaa gcgattctcc tgcctcagcc tcccaagtag ctgagactac 5280 aggtgtgcac caccatgctc agctaatttt tgtattttta gtacagatgg ggtttcacca 5340 tgttggccag gctggtcttg aactcctgat ctcccaaagt gaggtcttga actggtcttg 5400 aactcctcca cctcccaaag tgctgggatt acaggcgtga gccactgcac ccggcctagg 5460 gccatgtaaa aagccagatc tgtgctgctg tctgtgtaga agggtagaca agtggatgag 5520 aagttcctga actattcttg gcccttttac cactaagtga aagtaacttg ctgccccaaa 5580 gaaagatgtc tcatcattcg acaggacttt ctagttgaac ttcatgaaag caagagatcc 5640 tgtttttctt gctcaccact gtatcttgag acctgttgta gtgcctgcaa tacttattta 5700 ataagttatt tttaagtatc agttttgtga gctttaactc tatgaggtct ttgttgtttg 5760 actgtatttt aactctggcc atgacagcaa gacaaagttc catttttatt gagcttaaaa 5820 agaatcaagg ccaggtgaag tggcttacgc ctgtgatccc aacactttgt gaggctgcag 5880 caggaggatc tcttgagccc aggagtttga gaccgttcta ggcaatgtag tgaggtccag 5940 actccacaaa ataatttttt tttaaattgc acgcctgtag tctcagctat caggaggctg 6000 agatgggagg atgacttgag cccaggaaat tgaagctgca gtgaattgtg attgcaccac 6060 tgcactccag cctgggtgac agatcaagac cttgcctaaa caaaacaaaa caaacaaaac 6120 cccaaaaaac aaattgaaaa tgttgattct ttttactaca aacattatgg cagcactaaa 6180 aacttcgtgg gagtgtactg tggaaaatag tgtacttaat taattctcat tgtaatcagg 6240 ctaccaagag ccttgtgttg ctttaagagt tataactgcc aggcacagtg gctcatgcct 6300 ataatcccag caccttgaga ggccgaggca ggtggatcac ctgagatcgg gagtttgaga 6360 ccagccgggc caatatggtg aaacaagctg tgtctctact aaatacaaaa aattagccgg 6420 gcgtggtggc acatgcctgt aatcccagct gcttgggaga ctgagacagg agaattgctt 6480 gaacctggaa ggcggaggtt gcagtgagct gagattgcaa cattgtactc cagcctgggc 6540 aacaagaggg aaactccatc tcaaaaaaaa aaaaaaagtt gtaactgagg ctgggcatgg 6600 tggctcatac ctgtaatccc agcactttga aaagccgagg caggtagatc acttgagctc 6660 agaagttcga gactagcctg ggcaacatga caaaacccca tctctacaaa aaatacgaaa 6720 aattagctgg gcgtggtggc atgcacctgt agtcctagct acctgggagg ctgaggtggg 6780 aagattactt gaagctgcag tgagccatgg ttgtgccact gccctccagt ctgggcaaca 6840 aagtgagacc ctgtctcaaa aaaacaaaaa aaaattataa ctgatgtaaa ctggcagttt 6900 aggctgggtg tggtggctca agcctgtaat cctagcactt tgggaggcca aggcaggtgg 6960 atcacctgag ttcaggagtt cgagaccagc gtggccaaca tggtgaaacc ttgtctctat 7020 taaaaatacc aaaattagca agatgtggtg gtgggtgcct ataattccag ctactcagga 7080 ggctgaggca ggaggatcgc tggagccagg gaggcagagg ttacagtaag caaagatcac 7140 tccacttcac tccagcctgg gcaaaagagt gagacatatc aaaaaataaa caaataaata 7200 aataaataag tggcagttca tcatttaact ccaaagactt tgcgtacatt tctactgaaa 7260 acaatctgag ctgattagaa ccctgccatt ttatagcctt tagctcgatc tccgaccgtt 7320 catttaaaaa aattctactt caggccgggc atggtggctc aagcctgtaa tcccatcact 7380 gtaggaggcc aaagtgggca gatcacttaa ggtcaggagt ttgagaccag cctggccacc 7440 atggtgaaac cccatctcta ctaaaaatac aaaaattagc cgggcttggt ggtgagcacc 7500 tgtaatccca ccctgccgag tggcaggctg aggcaggaga atcgcttgag cccaagagcc 7560 ggaggttgca gtgagccaag cttgcaccat tgcactccag cctaggcaac agagtgtgac 7620 tccatctcaa gaaaaaaaaa attctatttc attttacaat atgcagatat atgtccatac 7680 acatgcataa tataaatgta taccatattt gtgagaatat gcatatatgt acacattaga 7740 tacacaatac aagcacaata catatgtctt ttgcccaaga tacagcattt tgtaaaggag 7800 acaggaattt agtaatatat gttccagaaa cagtacacaa gagaattcgc cgagatgaga 7860 aagttgtcac taggaatggg gagtggtaag atgtagaagg tataattgtt cttaaagttc 7920 tactgccaac tctttccaat taattaccca ctctgccatg ctttatggac aggaggttgt 7980 cggacactgt caattaataa atatttgagc atgatacact gcttggagct cctctaatat 8040 aggagagtga tatcctagtg catgttacag agggagtgtc cacacagttc ctattgtcat 8100 ttgatgagtt acttttcagg ggccttgtac ctgagcaagt tgtcctcttt ttgatggatt 8160 tcagattgag ttacctgcat tgtcttgaga ttgcagcgtg tttcctccac tgtacggcgt 8220 agtcagcaga tctattagtt aaactccagt gggccctcag tcactaaatc tatcctctgt 8280 gttgaaggct ttctgcattt gcctttcaat aaaggtttag aataactcct taaaaaa 8337 <210> 5 <211> 187 <212> PRT <213> Homo sapiens <400> 5 Met Val Gly Ser Leu Asn Cys Ile Val Ala Val Ser Gln Asn Met Gly 1 5 10 15 Ile Gly Lys Asn Gly Asp Leu Pro Trp Pro Pro Leu Arg Asn Glu Phe 20 25 30 Arg Tyr Phe Gln Arg Met Thr Thr Thr Ser Ser Val Glu Gly Lys Gln 35 40 45 Asn Leu Val Ile Met Gly Lys Lys Thr Trp Phe Ser Ile Pro Glu Lys 50 55 60 Asn Arg Pro Leu Lys Gly Arg Ile Asn Leu Val Leu Ser Arg Glu Leu 65 70 75 80 Lys Glu Pro Pro Gln Gly Ala His Phe Leu Ser Arg Ser Leu Asp Asp 85 90 95 Ala Leu Lys Leu Thr Glu Gln Pro Glu Leu Ala Asn Lys Val Asp Met 100 105 110 Val Trp Ile Val Gly Gly Ser Ser Val Tyr Lys Glu Ala Met Asn His 115 120 125 Pro Gly His Leu Lys Leu Phe Val Thr Arg Ile Met Gln Asp Phe Glu 130 135 140 Ser Asp Thr Phe Phe Pro Glu Ile Asp Leu Glu Lys Tyr Lys Leu Leu 145 150 155 160 Pro Glu Tyr Pro Gly Val Leu Ser Asp Val Gln Glu Glu Lys Gly Ile 165 170 175 Lys Tyr Lys Phe Glu Val Tyr Glu Lys Asn Asp 180 185 <210> 6 <211> 3932 <212> DNA <213> Homo sapiens <400> 6 tcccagacag aacctactat gtgcggcggc agctggggcg ggaaggcggg agctgggggc 60 gctgggggcg ctgcggccgc tgcggccgct gcagccgctg cagcgccagg gtccacctgg 120 tcggctgcac ctgtggagga ggaggtggat ttcaggcttc ccgtagactg gaagaatcgg 180 ctcaaaaccg cttgcctcgc aggggctgag ctggaggcag cgaggccgcc cgacgcaggc 240 ttccggcgag acatggcagg gcaaggatgg cagcccggcg gcagggcctg gcgaggagcg 300 cgagcccgcg gccgcagttc ccaggcgtct gcgggcgcga gcacgccgcg accctgcgtg 360 cgccggggcg ggggggcggg gcctcgcctg cacaaatggg gacgaggggg gcggggcggc 420 cacaatttcg cgccaaactt gaccgcgcgt tctgctgtaa cgagcgggct cggaggtcct 480 cccgctgctg tcatggttgg ttcgctaaac tgcatcgtcg ctgtgtccca gaacatgggc 540 atcggcaaga acggggacct gccctggcca ccgctcagga atgaattcag atatttccag 600 agaatgacca caacctcttc agtagaaggt aaacagaatc tggtgattat gggtaagaag 660 acctggttct ccattcctga gaagaatcga cctttaaagg gtagaattaa tttagttctc 720 agcagagaac tcaaggaacc tccacaagga gctcattttc tttccagaag tctagatgat 780 gccttaaaac ttactgaaca accagaatta gcaaataaag tagacatggt ctggatagtt 840 ggtggcagtt ctgtttataa ggaagccatg aatcacccag gccatcttaa actatttgtg 900 acaaggatca tgcaagactt tgaaagtgac acgttttttc cagaaattga tttggagaaa 960 tataaacttc tgccagaata cccaggtgtt ctctctgatg tccaggagga gaaaggcatt 1020 aagtacaaat ttgaagtata tgagaagaat gattaatatg aaggtgtttt ctagtttaag 1080 ttgttccccc tccctctgaa aaaagtatgt atttttacat tagaaaaggt tttttgttga 1140 ctttagatct ataattattt ctaagcaact agtttttatt ccccactact cttgtctcta 1200 tcagatacca tttatgagac attcttgcta taactaagtg cttctccaag accccaactg 1260 agtccccagc acctgctaca gtgagctgcc attccacacc catcacatgt ggcactcttg 1320 ccagtccttg acattgtcgg gcttttcaca tgttggtaat atttattaaa gatgaagatc 1380 cacataccct tcaactgagc agtttcacta gtggaaatac caaaagcttc ctacgtgtat 1440 atccagaggt ttgtagataa atgttgccac cttgtttgta acagtgaaaa attgaaaaca 1500 acctggaagt ccagtgatgg gaaaatgagt atgtttctgt cttagattgg ggaacccaaa 1560 gcagattgca agactgaaat ttcagtgaaa gcagtgtatt tgctaggtca taccagaaat 1620 catcaattga ggtacggaga aactgaactg agaaggtaag aaaagcaatt taaagtcagc 1680 gagcaggttc tcattgataa caagctccat actgctgaga tacagggaaa tggagggggg 1740 aaagctggag tattgatccc gcccccctcc ttggttgtca gctccctgtc ctgtgtgtgg 1800 gcggaacata gtccagctgc tctatagcaa gtctcaggtg tttgcagtaa gaagctgctg 1860 gcatgcacgg gaacagtgaa tgccaaacac ttaaagcaat tcgatgttta agtatgtaag 1920 ttcttttttt tttagacagc gtttcgctct tgttgcccag gctagcatgc aatggtgtga 1980 cctcggctta ctgcaacctc cgccttccca gattcaagcg attctcctgc ctcaggctcc 2040 caagtagcta ggaccaggtg cgcgccacca cgcccggcta atttttgtat tttgtatttt 2100 tagtagagat ggggtttcac catgttggtc aggctagtct cgaactcgtg accgcaagcg 2160 attcacccac ctcagcctcc caaagtgctg ggattaccgg cttgagccac cacacccggc 2220 acatcttcat tctttttatg tagtaaaaag tataaggcca cacatggttt atttgaagta 2280 ttttataatt taaaaaaata cagaagcagg aaaaccaatt ataagttcaa gtgagggatg 2340 atggttgctt gaaccaaagg gttgcatgta gtaagaaatt gtgatttaag atatatttta 2400 aagttataag tagcaggata ttctgatgga gtttgacttt ggttttgggc ccagggagtt 2460 tcagatgcct ttgagaaatg aatgaagtag agagaaaata aaagaaaaac cagccaggca 2520 cagtggctca cacctgtaat cccagcgctt tgggaggcta aggcaggcag atcacttgag 2580 accagcttgg gcaacatggc aaagccccat ctctacaaaa aacacaaaaa ttagctgggc 2640 attgtggcgc acacctgtat tcccatctag tcaggaagct gagatggaag aattaattga 2700 gcccacgagt tcaaggctgc agtgagtcgt gattgtgcca ctgcactcca gccggggtga 2760 cagaagagac cttgtctcga aaaggaatct gaaaacaatg gaaccatgcc ttcataattc 2820 tagaaagtta ttttcaactg ataaatctat attcacccaa ataatcaagg gtgaaggtaa 2880 aataatacat ttttagacaa gcaaagactc aggggttacc tccatgtgcc ctttttaggg 2940 aagctgttgg agaaaatact ccagcaaaat gaaggagtac acaaaccaga gaatgacatg 3000 aatccagcaa ataggatcca acacaggcaa tattccagct atggagctag ctttaaaaag 3060 gaacagtaaa aatattaatc ggttagctgg gtggaatggc ccatgcctgt agtcccagct 3120 actcaggagg ctcagcagca ggacgacttg agcccaagag ttccagacca gcctggccac 3180 cttagtgaga tcccttctct taaaaataat aacttattgc cagatttggg gcatttggaa 3240 agaagttcat tgaagataaa gcaaaagtaa aaaaaaaaaa aaaaaaaaca aggggaaagg 3300 gttggttagg caatcattct agggcagaaa gaagtacagg ataggaagag cataatacac 3360 tgtttttctc aacaaggagc agtatgtaca cagtcataat gatgtgactg cttagcccct 3420 aaatatggta actactctgg gacaatatgg gaggaaaagt gaagattgtg atggtgtaag 3480 agctaaatcc tcatctgtca tatccagaaa tcactatata atatataata atgaaatgac 3540 taagttatgt gaggaaaaaa acagaagaca ttgctaaaag agttaaaagt cattgctctg 3600 gagaattagg agggatgggg caggggactg ttaggatgca ttataaactg aaaagccttt 3660 ttaaaatttt atgtattaat atatgcattc acttgaaaaa ctaaaaaaaa acaataattt 3720 ggaaaaaccc atgaaggtaa ctaacggaag gaaaaactaa gagaatgaaa agtatttgcc 3780 tctggaaaga acaactggca ggactgttgt tttcattgta agacttttgg agccatttaa 3840 ttgtacttaa ccattttcat ctatttcttt aataagaaca attccatctt aataaagagt 3900 tacacttgtt aataagtaaa aaaaaaaaaa aa 3932 <210> 7 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic 6xHis tag <400> 7 His His His His His His 1 5

Claims

1. A method for producing a therapeutic protein, comprising: culturing cholesterol auxotrophic and glutamine auxotrophic cells; transfecting the cells with a nucleic acid (i) encoding a protein capable of restoring cholesterol biosynthesis in the cells, a nucleic acid (ii) encoding a protein capable of restoring glutamine biosynthesis in the cells, and a nucleic acid (iii) encoding a therapeutic protein; culturing the cells under conditions suitable for the expression of the therapeutic protein; isolating and / or purifying the therapeutic protein. A method characterized by including the above steps.

2. The method according to claim 1, wherein the therapeutic protein is an antibody.

3. The method according to claim 1, wherein in the transfection step, the cells are transfected with a nucleic acid (iii) encoding an antibody light chain and an antibody heavy chain.

4. The method according to claim 1, wherein the nucleic acid (i) encodes 3-ketosteroid reductase.

5. The method according to claim 1, wherein the nucleic acid (ii) encodes glutamine synthetase.

6. The method according to claim 1, wherein the first expression vector contains the nucleic acids (i) and (iii), and the second expression vector contains the nucleic acid (ii).

7. The method according to claim 1, wherein the first expression vector contains the nucleic acid (i), and the second expression vector contains the nucleic acids (ii) and (iii).

8. The method according to claim 7, wherein the transfection of the first expression vector is performed prior to the transfection of the second expression vector.

9. The method according to claim 7, wherein the transfection of the first expression vector is performed simultaneously with the transfection of the second expression vector.

10. The method according to any one of claims 1 to 9, wherein the cells are cultured in the absence of exogenously introduced cholesterol, for example, after transfection with the nucleic acid (i).

11. The method according to any one of claims 1 to 10, wherein the cells are cultured in the absence of exogenously introduced glutamine, for example, after transfection with the nucleic acid (ii).

12. The method according to claim 1, wherein the cells are cultured in the presence of a glutamine synthetase inhibitor, such as methionine sulfoximine, for example, during and / or after transfection of the nucleic acid (ii).

13. The method according to claim 1, wherein the cells are cultured in the presence of a 3-ketosteroid reductase inhibitor, for example, during and / or after transfection of the nucleic acid (i).

14. The method according to any one of claims 1 to 13, wherein the cells are NS0 cells.

15. The method according to any one of claims 1 to 14, wherein the protein is selected from the group consisting of adalimumab, infliximab, palivizumab, cetuximab, natalizumab, eculizumab, ustekinumab, golimumab, ofatumumab, canakinumab, belimumab, alirocumab, mepolizumab, necitumumab, nivolumab, dinutuximab, secukinumab, evolocumab, blinatumomab, pembrolizumab, ramucirumab, vedolizumab, siltuximab, obinutuzumab, trastuzumab, labetuzumab, pertuzumab, brentuximab, ipilimumab, denosumab, tocilizumab, ofatumumab, canakinumab, certolizumab, catumaxomab, ranibizumab, panitumumab, bevacizumab, cetuximab, efalizumab, omalizumab, tositumomab, ibritumomab, alemtuzumab, gemtuzumab, basiliximab, daclizumab, rituximab, abciximab, alefacept, etanercept, abatacept, belatacept, aflibercept, ziv-aflibercept, rilonacept, romiplostim, apoticept, trebananib, briquilimab, and dulaglutide.

16. A method for producing a therapeutic protein, comprising: culturing cholesterol auxotrophic cells; transfecting the cells with a nucleic acid (i) encoding a protein capable of restoring cholesterol biosynthesis in the cells, a nucleic acid (ii) encoding a protein expressing one or more selectable markers, and a nucleic acid (iii) encoding a therapeutic protein; culturing the cells under conditions suitable for the expression of the therapeutic protein; isolating and / or purifying the therapeutic protein; and characterized by comprising the above steps.

17. The method according to claim 16, wherein the therapeutic protein is an antibody.

18. The method according to claim 16, wherein in the transfection step, the cell is transfected with a nucleic acid (iii) encoding an antibody light chain and an antibody heavy chain.

19. The method according to claim 16, wherein the nucleic acid (i) encodes 3-ketosteroid reductase.

20. The method according to claim 16, wherein the nucleic acid (ii) encodes glutamine synthetase, dihydrofolate reductase (DHFR) or one or more antibiotic resistance genes, for example, neomycin, blasticidin, hygromycin, puromycin, zeocin, mycophenolic acid.

21. The method according to claim 16, wherein the first expression vector contains nucleic acids (i) and (iii), and the second expression vector contains nucleic acid (ii).

22. The method according to claim 16, wherein the first expression vector contains nucleic acid (i), and the second expression vector contains nucleic acids (ii) and (iii).

23. The method according to claim 22, wherein the transfection of the first expression vector is performed prior to the transfection of the second expression vector.

24. The method according to claim 22, wherein the transfection of the first expression vector is performed simultaneously with the transfection of the second expression vector.

25. The method according to claim 16, wherein the cell is cultured in the absence of exogenously introduced cholesterol, for example, after transfection of the nucleic acid (i).

26. The method according to claim 16, wherein the cell is cultured in the absence of exogenously introduced glutamine, for example, after transfection of the nucleic acid (ii).

27. The method according to claim 16, wherein the cell is cultured in the presence of a 3-ketosteroid reductase inhibitor, for example, during and / or after transfection of the nucleic acid (i).

28. The method according to claim 16, wherein the cell is an NS0 cell.

29. The method according to claim 16, wherein the protein is selected from the group consisting of adalimumab, infliximab, palivizumab, cetuximab, natalizumab, eculizumab, ustekinumab, golimumab, ofatumumab, canakinumab, belimumab, alirocumab, mepolizumab, nesitumumab, nivolumab, dinutuximab, secukinumab, evolocumab, blinatumomab, pembrolizumab, ramucirumab, vedolizumab, siltuximab, obinutuzumab, trastuzumab, lirilumab, pertuzumab, brentuximab, ipilimumab, denosumab, tocilizumab, ofatumumab, canakinumab, certolizumab, catumaxomab, ranibizumab, panitumumab, bevacizumab, cetuximab, efalizumab, omalizumab, tositumomab, ibritumomab, alemtuzumab, gemtuzumab, basiliximab, daclizumab, rituximab, abciximab, alefacept, etanercept, abatacept, belatacept, aflibercept, ziv-aflibercept, rilonacept, romiplostim, aposcept, trebananib, brisibimod, and dulaglutide.

30. A method for producing a therapeutic protein, comprising: culturing cholesterol auxotrophic cells; transfecting the cells with a nucleic acid (i) encoding a protein capable of restoring cholesterol biosynthesis in the cells, a nucleic acid (ii) encoding a protein expressing a first selection marker, a nucleic acid (iii) encoding a protein expressing a second selection marker, and a nucleic acid (iv) encoding a therapeutic protein; culturing the cells under conditions suitable for the expression of the therapeutic protein; and isolating and / or purifying the therapeutic protein. A method characterized by comprising the above steps.

31. The method according to claim 30, wherein the therapeutic protein is an antibody.

32. The method according to claim 30, wherein in the transfection step, the cells are transfected with a nucleic acid (iv) encoding an antibody light chain and an antibody heavy chain.

33. The method according to claim 30, wherein the nucleic acid (i) encodes 3-ketosteroid reductase.

34. ​ The method according to claim 30, wherein the nucleic acid (ii) encodes glutamine synthetase, dihydrofolate reductase (DHFR) or one or more antibiotic resistance genes, such as neomycin, blasticidin, hygromycin, puromycin, zeocin, mycophenolic acid.

35. The method according to claim 30, wherein the nucleic acid (iii) encodes glutamine synthetase, dihydrofolate reductase (DHFR) or one or more antibiotic resistance genes, such as neomycin, blasticidin, hygromycin, puromycin, zeocin, mycophenolic acid.

36. The method according to claim 30, wherein the cells are cultured in the absence of exogenously introduced cholesterol, for example, after transfection of the nucleic acid (i).

37. The method according to claim 30, wherein the cells are cultured in the absence of exogenously introduced glutamine, for example, after transfection of the nucleic acid (ii).

38. The method according to claim 30, wherein the cells are cultured in the presence of a 3-ketosteroid reductase inhibitor, for example, during and / or after transfection of the nucleic acid (i).

39. The method according to claim 30, wherein the cells are NS0 cells.

40. The method according to claim 30, wherein the protein is selected from the group consisting of adalimumab, infliximab, palivizumab, cetuximab, natalizumab, eculizumab, ustekinumab, golimumab, ofatumumab, canakinumab, belimumab, alirocumab, mepolizumab, nesitumumab, nivolumab, dinutuximab, secukinumab, evolocumab, blinatumomab, pembrolizumab, ramucirumab, vedolizumab, siltuximab, obinutuzumab, trastuzumab, lirilumab, pertuzumab, brentuximab, ipilimumab, denosumab, tocilizumab, ofatumumab, canakinumab, certolizumab, catumaxomab, ranibizumab, panitumumab, bevacizumab, cetuximab, efalizumab, omalizumab, tositumomab, ibritumomab, alemtuzumab, gemtuzumab, basiliximab, daclizumab, rituximab, abciximab, alefacept, etanercept, abatacept, belatacept, aflibercept, ziv-aflibercept, rilonacept, romiplostim, apoptosis, trebananib, briquilimode, and dulaglutide.

41. The method according to claim 30, wherein nucleic acid (ii) encodes glutamine synthetase and nucleic acid (iii) encodes one or more antibiotic resistance genes, such as neomycin, blasticidin, hygromycin, puromycin, zeocin, mycophenolic acid.

42. The method according to claim 30, wherein nucleic acid (ii) encodes glutamine synthetase and nucleic acid (iii) encodes a neomycin antibiotic resistance gene.

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