Genetically modified vertebrate cell

Abstract

Provided is a genetically modified vertebrate cell for use in producing a recombinant polypeptide, said genetically modified vertebrate cell being modified such that the functional expression of an mRNA decapping activator gene is reduced or eliminated.

Description

Genetically engineered vertebrate cells

[0001] The present invention relates to methods for producing recombinant polypeptides in vertebrate cells, genetically engineered vertebrate cells and methods for their production, and methods for increasing the productivity of recombinant polypeptides by vertebrate cells.

[0002] Many biopharmaceuticals are produced using vertebrate cells, such as mammalian cells. Biopharmaceuticals include antibodies, antibody fragments, ADCs (antibody-drug conjugates), nanobodies, Fc fusion proteins, growth factors, hormones, cytokines, enzymes, and other therapeutic polypeptides. Polypeptides, which are active ingredients in biopharmaceuticals, can be produced by expressing them as recombinant polypeptides in mammalian cells. Considering the cost of producing recombinant polypeptides, improved production efficiency in production methods is needed. Methods using genetically engineered cell lines for the expression of recombinant polypeptides have been reported (Patent Documents 2 to 5). Furthermore, the use of EDC3 in constructing a cell-free protein synthesis system using yeast, etc. has been reported (Patent Document 1). Other studies on the involvement of EDC3 in polypeptide expression have also been reported (Non-Patent Documents 1 to 4).

[0003] International Patent Application Publication No. WO2020 / 135623A1 Special Publication No. 2017-514484 Special Publication No. 2021-530225 Special Publication No. 2022-552323 Special Publication No. 2023-542228

[0004] Robert W. Walters et al., G3, Vol. 4, April 2014, pp.613-622, doi: 10.1534 / g3.114.010470; Mark Larance et al., Molecular & Cellular Proteomics 9.4, pp.682-694; Trach Nissan et al., Mol Cell. 2010 September 10, 39(5): pp.773-783; Darach Miller et al., PLOS Genetics, May 21, 2018, https: / / doi.org / 10.1371 / journal.pgen.1007406.

[0005] There is a need for a method for improving the production efficiency of recombinant polypeptides by cell culture, particularly a method for improving the productivity of recombinant polypeptide production in vertebrate cells, and for vertebrate cells that can be used in said method.

[0006] The inventors have discovered that efficient production of recombinant polypeptides is possible by using vertebrate cells genetically engineered to reduce or eliminate functional expression of an mRNA decapping activator gene.

[0007] [1-1] A vertebrate cell for use in producing a recombinant polypeptide, wherein the vertebrate cell is a genetically engineered vertebrate cell modified to reduce or eliminate functional expression of an mRNA decapping activator gene. [1-2] The vertebrate cell according to [1-1], wherein functional expression of the mRNA decapping activator gene is reduced or eliminated by gene knockout, gene modification, gene deletion, gene silencing, or a combination thereof. [1-3] The vertebrate cell according to [1-1] or [1-2], wherein functional expression of the mRNA decapping activator gene is eliminated by heterozygous knockout or homozygous knockout.

[0008] [1-4] The vertebrate cell according to any one of [1-1] to [1-3], wherein the vertebrate cell comprises at least one heterologous polynucleotide encoding a recombinant polypeptide. [1-5] The vertebrate cell according to any one of [1-1] to [1-4], wherein the vertebrate cell secretes a recombinant polypeptide. [1-6] The vertebrate cell according to any one of [1-1] to [1-5], wherein the genome of the vertebrate cell has been modified to reduce or eliminate functional expression of an mRNA decapping activator.

[0009] [1-7] The vertebrate cell according to any of [1-1] to [1-6], wherein the vertebrate cell contains at least one modification in at least one copy or all copies of the mRNA decapping activator gene. [1-8] The vertebrate cell according to [1-7], wherein the at least one modification is a modification that causes a frameshift in an exon of one or both alleles of the mRNA decapping activator gene. [1-9] The vertebrate cell according to any of [1-1] to [1-8], wherein the amino acid sequence of the endogenous mRNA decapping activator after the modification has at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NOs: 1 to 4 as mRNA decapping activator reference proteins.

[0010] [1-10] The vertebrate cell according to any one of [1-1] to [1-9], wherein the mRNA decapping activator is PAT1, SCD6, EDC3, or DHH1. [1-11] The vertebrate cell according to any one of [1-1] to [1-10], wherein the mRNA decapping activator is EDC3. [1-12] The vertebrate cell according to any one of [1-1] to [1-11], wherein the vertebrate cell is a mammalian cell.

[0011] [1-13] The vertebrate cell according to any one of [1-1] to [1-12], wherein the vertebrate cell is a human cell. [1-14] The vertebrate cell according to any one of [1-1] to [1-12], wherein the vertebrate cell is a rodent cell. [1-15] The vertebrate cell according to any one of [1-1] to [1-12] and [1-14], wherein the vertebrate cell is a hamster cell.

[0012] [1-16] The vertebrate cell according to any one of [1-1] to [1-12], [1-14] and [1-15], wherein the vertebrate cell is a CHO cell. [1-17] The vertebrate cell according to any one of [1-1] to [1-16], wherein the vertebrate cell is a mammalian cell provided as a cell clone or a cell line. [1-18] The vertebrate cell according to any one of [1-1] to [1-17], wherein the recombinant polypeptide is used for therapy or diagnosis.

[0013] [1-19] The vertebrate cell according to any one of [1-1] to [1-18], wherein the recombinant polypeptide is selected from the group consisting of a glycoprotein, an antibody, an IgG antibody, an IgG1 antibody, a peptide, an Fc fusion protein, a Fab fragment, a protein complex, a peptidase, a signal peptide, a nanobody, a growth factor, a hormone, a cytokine, a blood factor, and an enzyme. [1-20] The vertebrate cell according to any one of [1-1] to [1-19], wherein the recombinant polypeptide is an antibody, an IgG antibody, an IgG1 antibody, or a bispecific antibody. [1-21] The vertebrate cell according to any one of [1-1] to [1-20], wherein the vertebrate cell comprises at least one heterologous polynucleotide encoding a selectable marker or a reporter polypeptide.

[0014] [1-22] The vertebrate cell according to any of [1-1] to [1-21], wherein at least one heterologous polynucleotide encoding the recombinant polypeptide is contained in an expression cassette. [1-23] The vertebrate cell according to any of [1-1] to [1-22], wherein productivity of the recombinant polypeptide by a vertebrate cell in which functional expression of an mRNA decapping activator gene has been reduced or eliminated has been increased by at least 10% compared to a vertebrate cell having the same genotype except for the modification. [1-24] The vertebrate cell according to [1-23], wherein the increase in productivity of the recombinant polypeptide is an increase of at least 10% in the amount of recombinant polypeptide produced by a vertebrate cell in which functional expression of an mRNA decapping activator gene has been reduced or eliminated, compared to the amount of recombinant polypeptide produced by a vertebrate cell having the same genotype except for the modification, cultured under the same conditions.

[0015] [1-25] The vertebrate cell according to [1-23] or [1-24], wherein the increase in productivity of the recombinant polypeptide is an increase of at least 10% in the titer of the recombinant polypeptide in a culture medium of vertebrate cells in which functional expression of an mRNA decapping activator gene has been reduced or eliminated, compared to the titer of the recombinant polypeptide in a culture medium of vertebrate cells having the same genotype except for the modification, cultured under the same conditions. [1-26] The vertebrate cell according to any of [1-1] to [1-25], which does not express a detectable level of an mRNA decapping activator.

[0016] [1-27] The vertebrate cell according to any one of [1-1] to [1-12] and [1-14] to [1-26], wherein the vertebrate cell is a CHO cell and contains at least one modification in at least one copy or all copies of the mRNA decapping activator gene, the at least one modification causing a frameshift in an exon of one or both alleles of the mRNA decapping activator gene, the recombinant polypeptide is an IgG antibody, and the mRNA decapping activator is EDC3.

[0017] [1-28] A genetically engineered vertebrate cell that has been modified to reduce or eliminate functional expression of an mRNA decapping activator gene, for use in producing a recombinant polypeptide, wherein the vertebrate cell is a CHO cell and contains at least one modification in at least one copy or all copies of the mRNA decapping activator gene, the at least one modification causing a frameshift in an exon of one or both alleles of the mRNA decapping activator gene, the recombinant polypeptide is an IgG antibody, and the mRNA decapping activator is EDC3. [1-29] The vertebrate cell of [1-23] or [1-24], wherein the increase in productivity of the recombinant polypeptide is at least a 10% increase in the antibody secretion rate of the recombinant polypeptide in a culture medium of vertebrate cells in which functional expression of the mRNA decapping activator gene has been reduced or eliminated, compared to the antibody secretion rate of the recombinant polypeptide in a culture medium of vertebrate cells having the same genotype except for the modification, cultured under the same conditions.

[0018] [2-1] A method for increasing recombinant polypeptide productivity in vertebrate cells, comprising preparing recombinant vertebrate cells modified to reduce or eliminate functional expression of an mRNA decapping activator gene in the vertebrate cells, wherein the productivity of the recombinant polypeptide is increased when cultured under the same conditions as vertebrate cells having the same genotype except for the modification. [2-2] The method according to [2-1], wherein the functional expression of the mRNA decapping activator gene is reduced or eliminated by gene knockout, gene modification, gene deletion, gene silencing, or a combination thereof. [2-3] The method according to [2-1] or [2-2], wherein the functional expression of the mRNA decapping activator gene is eliminated by heterozygous knockout or homozygous knockout.

[0019] [2-4] The method according to any one of [2-1] to [2-3], which comprises introducing at least one heterologous polynucleotide encoding a recombinant polypeptide into the vertebrate cell. [2-5] The method according to any one of [2-1] to [2-4], wherein the genome of the vertebrate cell is modified to reduce or eliminate functional expression of an mRNA decapping activator gene. [2-6] The method according to any one of [2-1] to [2-5], wherein the vertebrate cell contains at least one modification in at least one copy or all copies of the mRNA decapping activator gene.

[0020] [2-7] The method of [2-6], wherein the at least one modification is a modification that causes a frameshift in an exon of one or both alleles of the mRNA decapping activator gene. [2-8] The method of any of [2-1] to [2-7], wherein the amino acid sequence of the endogenous mRNA decapping activator after the modification has at least 90% identity to the amino acid sequences shown in SEQ ID NOs: 1 to 4 as mRNA decapping activator reference proteins. [2-9] The method of any of [2-1] to [2-8], wherein the mRNA decapping activator is PAT1, SCD6, EDC3, or DHH1.

[0021] [2-10] The method according to any one of [2-1] to [2-9], wherein the mRNA decapping activator is EDC3. [2-11] The method according to any one of [2-1] to [2-10], wherein the vertebrate cell is a mammalian cell. [2-12] The method according to any one of [2-1] to [2-11], wherein the vertebrate cell is a human cell.

[0022] [2-13] The method according to any one of [2-1] to [2-11], wherein the vertebrate cell is a rodent cell. [2-14] The method according to any one of [2-1] to [2-11] and [2-13], wherein the vertebrate cell is a hamster cell. [2-15] The method according to any one of [2-1] to [2-11], [2-13] and [2-14], wherein the vertebrate cell is a CHO cell.

[0023] [2-16] The method according to any one of [2-1] to [2-15], wherein the vertebrate cell is a mammalian cell provided as a cell clone or a cell line. [2-17] The method according to any one of [2-1] to [2-16], wherein the recombinant polypeptide is a polypeptide used in therapy or diagnosis. [2-18] The method according to any one of [2-1] to [2-17], wherein the recombinant polypeptide is selected from the group consisting of a glycoprotein, an antibody, an IgG antibody, an IgG1 antibody, a peptide, an Fc fusion protein, a Fab fragment, a protein complex, a peptidase, a signal peptide, a nanobody, a growth factor, a hormone, a cytokine, a blood factor, and an enzyme.

[0024] [2-19] The method according to any one of [2-1] to [2-18], wherein the recombinant polypeptide is an antibody, an IgG antibody, an IgG1 antibody, or a bispecific antibody. [2-20] The method according to any one of [2-1] to [2-19], further comprising introducing into the vertebrate cell at least one heterologous polynucleotide encoding a selectable marker or a reporter polypeptide. [2-21] The method according to any one of [2-1] to [2-20], wherein the at least one heterologous polynucleotide encoding the peptide of interest is contained in an expression cassette.

[0025] [2-22] The method according to any of [2-1] to [2-21], wherein the productivity of a recombinant polypeptide by a vertebrate cell in which functional expression of an mRNA decapping activator gene has been reduced or eliminated is increased by at least 10% compared to a vertebrate cell having the same genotype except for the modification. [2-23] The method according to [2-22], wherein the increase in recombinant polypeptide productivity is an increase of at least 10% in the amount of recombinant polypeptide produced by a vertebrate cell in which functional expression of an mRNA decapping activator gene has been reduced or eliminated, compared to the amount of recombinant polypeptide produced by a vertebrate cell having the same genotype except for the modification, cultured under the same conditions.

[0026] [2-24] The method of [2-22] or [2-23], wherein the increase in productivity of the recombinant polypeptide is an increase of at least 10% in the titer of the recombinant polypeptide in a culture medium of vertebrate cells in which functional expression of the mRNA decapping activator gene has been reduced or eliminated, compared to the titer of the recombinant polypeptide in a culture medium of vertebrate cells having the same genotype except for the modification, cultured under the same conditions. [2-25] The method of any of [2-1] to [2-24], wherein the vertebrate cells do not express a detectable level of the mRNA decapping activator. [2-26] The method of any of [2-1] to [2-25], wherein the reduction or elimination of functional expression of the mRNA decapping activator gene is mediated by a gene targeting system selected from the group consisting of CRISPR / Cas9, CRISPR / Cpf1, zinc finger nuclease, TALEN, or meganuclease.

[0027] [2-27] The method of any of [2-1] to [2-26], wherein the elimination of functional expression of the mRNA decapping activator gene is carried out by gene knockout before or after introduction of a polynucleotide encoding a recombinant polypeptide. [2-28] The method of any of [2-1] to [2-11] or [2-13] to [2-27], wherein the vertebrate cell is a CHO cell and contains at least one modification in at least one copy or all copies of the mRNA decapping activator gene, the at least one modification causing a frameshift in an exon of one or both alleles of the mRNA decapping activator gene, the recombinant polypeptide is an IgG antibody, and the mRNA decapping activator is EDC3.

[0028] [2-29] A method for increasing the productivity of a recombinant polypeptide in a vertebrate cell, comprising preparing a recombinant vertebrate cell that has been modified to reduce or eliminate functional expression of an mRNA decapping activator gene in the vertebrate cell, wherein the productivity of the recombinant polypeptide is increased when cultured under the same conditions compared to a vertebrate cell having the same genotype except for the modification, cultured under the same conditions; wherein the vertebrate cell is a CHO cell and contains at least one modification in at least one copy or all copies of the mRNA decapping activator gene, the at least one modification causing a frameshift in an exon of one or both alleles of the mRNA decapping activator gene; the recombinant polypeptide is an IgG antibody; and the mRNA decapping activator is EDC3. [2-30] The method according to [2-22] or [2-23], wherein the increase in productivity of the recombinant polypeptide is at least a 10% increase in the antibody secretion rate of the recombinant polypeptide in a culture medium of vertebrate cells in which functional expression of the mRNA decapping activator gene has been reduced or eliminated, compared to the antibody secretion rate of the recombinant polypeptide in a culture medium of vertebrate cells having the same genotype except for the modification, cultured under the same conditions.

[0029] [3-1] A method for producing the vertebrate cell according to any one of [1-1] to [1-29], comprising: preparing a recombinant vertebrate cell modified so that functional expression of the mRNA decapping activator gene is reduced or eliminated in the vertebrate cell; and introducing a polynucleotide encoding a recombinant polypeptide into the vertebrate cell, wherein the recombinant polypeptide is expressed in the vertebrate cell. [3-2] The method according to [3-1], wherein the reduction or elimination of functional expression of the mRNA decapping activator gene is mediated by a gene targeting system selected from the group consisting of CRISPR / Cas9, CRISPR / Cpf1, zinc finger nuclease, TALEN, or meganuclease. [3-3] The method according to [3-1] or [3-2], wherein gene knockout is performed before or after introduction of the polynucleotide encoding the recombinant polypeptide.

[0030] [3-4] A method for producing a vertebrate cell, wherein the recombinant polypeptide is expressed in the vertebrate cell, comprising: preparing a recombinant vertebrate cell that has been modified so that functional expression of the mRNA decapping activator gene is reduced or eliminated in the vertebrate cell; and introducing a polynucleotide encoding a recombinant polypeptide into the vertebrate cell, wherein the vertebrate cell is a genetically engineered vertebrate cell that has been modified so that functional expression of the mRNA decapping activator gene is reduced or eliminated, for use in producing a recombinant polypeptide; the vertebrate cell is a CHO cell, and contains at least one modification in at least one copy or all copies of the mRNA decapping activator gene, wherein the at least one modification causes a frameshift in an exon of one or both alleles of the mRNA decapping activator gene; the recombinant polypeptide is an IgG antibody; and the mRNA decapping activator is EDC3.

[0031] [4-1] A method for producing a recombinant polypeptide, comprising: (a) culturing a vertebrate cell according to any one of [1-1] to [1-29] under conditions that allow expression and secretion of the recombinant polypeptide into a cell culture medium; and (b) isolating the recombinant polypeptide from the cell culture medium.

[0032] [4-2] The method according to [4-1], further comprising (c) processing the isolated recombinant polypeptide. [4-3] A method for producing a recombinant polypeptide, comprising: (a) culturing vertebrate cells under conditions that allow expression and secretion of the recombinant polypeptide into a cell culture medium, and (b) isolating the recombinant polypeptide from the cell culture medium, and optionally comprising processing the isolated recombinant polypeptide, wherein the vertebrate cells are genetically engineered vertebrate cells that have been modified to reduce or eliminate functional expression of an mRNA decapping activator gene for use in producing a recombinant polypeptide, wherein the vertebrate cells are CHO cells and contain at least one modification in at least one copy or all copies of the mRNA decapping activator gene, the at least one modification causing a frameshift in an exon of one or both alleles of the mRNA decapping activator gene, the recombinant polypeptide is an IgG antibody, and the mRNA decapping activator is EDC3.

[0033] [5-1] A method for increasing the titer of an antibody obtained in a medium by CHO cell culture, comprising preparing recombinant CHO cells modified to reduce or eliminate functional expression of an mRNA decapping activator gene, wherein the antibody titer in the CHO cell medium is increased compared to when CHO cells having the same genotype except for the modification are cultured under the same conditions. [5-2] The method according to [5-1], wherein the functional expression of the mRNA decapping activator gene is reduced or eliminated by gene knockout, gene modification, gene deletion, gene silencing, or a combination thereof. [5-3] The method according to [5-1] or [5-2], wherein the functional expression of the mRNA decapping activator gene is eliminated by heterozygous knockout or homozygous knockout.

[0034] [5-4] The method of any of [5-1] to [5-3], which comprises introducing at least one heterologous polynucleotide encoding an antibody into the CHO cells. [5-5] The method of any of [5-1] to [5-4], wherein the genome of the CHO cells is modified to reduce or eliminate functional expression of an mRNA decapping activator gene. [5-6] The method of any of [5-1] to [5-5], wherein the CHO cells contain at least one modification in at least one copy or all copies of the mRNA decapping activator gene.

[0035] [5-7] The method according to [5-6], wherein the at least one modification is a modification that causes a frameshift in an exon of one or both alleles of the mRNA decapping activator gene. [5-8] The method according to any one of [5-1] to [5-7], wherein the amino acid sequence of the endogenous mRNA decapping activator after the modification has at least 90% identity to the amino acid sequences shown in SEQ ID NOs: 1 to 4 as mRNA decapping activator reference proteins. [5-9] The method according to any one of [5-1] to [5-8], wherein the mRNA decapping activator is PAT1, SCD6, EDC3, or DHH1.

[0036] [5-10] The method according to any one of [5-1] to [5-9], wherein the mRNA decapping activator is EDC3.

[0037] [5-11] The method according to any one of [5-1] to [5-10], wherein the CHO cells are provided as a cell clone or a cell line. [5-12] The method according to any one of [5-1] to [5-11], wherein the antibody is an antibody used in therapy or diagnosis.

[0038] [5-13] The method according to any one of [5-1] to [5-12], wherein the antibody is an IgG antibody, an IgG1 antibody, or a bispecific antibody. [5-14] The method according to any one of [5-1] to [5-13], further comprising introducing into the CHO cells at least one heterologous polynucleotide encoding a selection marker or a reporter polypeptide. [5-15] The method according to any one of [5-1] to [5-14], wherein the at least one heterologous polynucleotide encoding the peptide of interest is contained in an expression cassette.

[0039] [5-16] The method of any of [5-1] to [5-15], wherein the antibody titer in the culture medium of the CHO cells is increased by at least 10% compared to when CHO cells having the same genotype except for the modification are cultured under the same conditions. [5-17] The method of any of [5-1] to [5-16], wherein the CHO cells do not express a detectable level of the mRNA decapping activator. [5-18] The method of any of [5-1] to [5-17], wherein the reduction or elimination of functional expression of the mRNA decapping activator gene is mediated by a gene targeting system selected from the group consisting of CRISPR / Cas9, CRISPR / Cpf1, zinc finger nuclease, TALEN, or meganuclease.

[0040] [5-19] The method of any of [5-1] to [5-18], wherein the functional expression of the mRNA decapping activator gene is reduced or eliminated by gene knockout before or after introduction of a polynucleotide encoding an antibody. [5-20] The method of any of [5-1] to [5-19], wherein the CHO cells contain at least one modification in at least one copy or all copies of the mRNA decapping activator gene, the at least one modification causing a frameshift in an exon of one or both alleles of the mRNA decapping activator gene, the antibody is an IgG antibody, and the mRNA decapping activator is EDC3.

[0041] [5-21] A method for increasing the titer of an antibody obtained in a medium by culturing CHO cells, comprising preparing CHO cells modified to reduce or eliminate functional expression of an mRNA decapping activator gene in the CHO cells, wherein the antibody titer in the CHO cell medium is increased compared to when CHO cells having the same genotype except for the modification are cultured under the same conditions, wherein the CHO cells contain at least one modification in at least one copy or all copies of the mRNA decapping activator gene, and the at least one modification is a modification that causes a frameshift in an exon of one or both alleles of the mRNA decapping activator gene, the antibody is an IgG antibody, and the mRNA decapping activator is EDC3.

[0042] [6-1] A genetically engineered vertebrate cell that has been modified to reduce or eliminate functional expression of an mRNA decapping activator gene. [6-2] The vertebrate cell according to [6-1], in which functional expression of the mRNA decapping activator gene is reduced or eliminated by gene knockout, gene modification, gene deletion, gene silencing, or a combination thereof. [6-3] The vertebrate cell according to [6-1] or [6-2], in which functional expression of the mRNA decapping activator gene is eliminated by heterozygous knockout or homozygous knockout.

[0043] [6-4] A vertebrate cell described in any of [6-1] to [6-3], wherein the genome of the vertebrate cell has been modified to reduce or eliminate functional expression of an mRNA decapping activator.

[0044] [6-5] The vertebrate cell according to any of [6-1] to [6-4], wherein the vertebrate cell contains at least one modification in at least one copy or all copies of the mRNA decapping activator gene. [6-6] The vertebrate cell according to [6-5], wherein the at least one modification is a modification that causes a frameshift in an exon of one or both alleles of the mRNA decapping activator gene. [6-7] The vertebrate cell according to any of [6-1] to [6-6], wherein the amino acid sequence of the endogenous mRNA decapping activator after the modification has at least 90% sequence identity to the amino acid sequence shown in SEQ ID NOs: 1 to 4 as mRNA decapping activator reference proteins.

[0045] [6-8] The vertebrate cell according to any one of [6-1] to [6-7], wherein the mRNA decapping activator is PAT1, SCD6, EDC3, or DHH1. [6-9] The vertebrate cell according to any one of [6-1] to [6-8], wherein the mRNA decapping activator is EDC3. [6-10] The vertebrate cell according to any one of [6-1] to [6-9], wherein the vertebrate cell is a mammalian cell.

[0046] [6-11] The vertebrate cell according to any one of [6-1] to [6-10], wherein the vertebrate cell is a human cell. [6-12] The vertebrate cell according to any one of [6-1] to [6-10], wherein the vertebrate cell is a rodent cell. [6-13] The vertebrate cell according to any one of [6-1] to [6-10] and [6-12], wherein the vertebrate cell is a hamster cell.

[0047] [6-14] The vertebrate cell according to any one of [6-1] to [6-10], [6-12] and [6-13], wherein the vertebrate cell is a CHO cell. [6-15] The vertebrate cell according to any one of [6-1] to [6-14], wherein the vertebrate cell is a mammalian cell provided as a cell clone or cell line. [6-16] The vertebrate cell according to any one of [6-1] to [6-10], [6-12] to [6-15], which does not express a detectable level of an mRNA decapping activator.

[0048] [6-17] The vertebrate cell according to any of [6-1] to [6-16], wherein the vertebrate cell is a CHO cell and contains at least one modification in at least one copy or all copies of the mRNA decapping activator gene, the at least one modification causing a frameshift in an exon of one or both alleles of the mRNA decapping activator gene, and the mRNA decapping activator is EDC3.

[0049] [6-18] A genetically engineered vertebrate cell that has been modified to reduce or eliminate functional expression of an mRNA decapping activator gene, wherein the vertebrate cell is a CHO cell and contains at least one modification in at least one copy or all copies of the mRNA decapping activator gene, the at least one modification causing a frameshift in an exon of one or both alleles of the mRNA decapping activator gene, and the mRNA decapping activator is EDC3.

[0050] [7-1] A method for increasing the secretion rate of an antibody obtained into a medium by CHO cell culture, comprising preparing recombinant CHO cells modified to reduce or eliminate functional expression of an mRNA decapping activator gene, wherein the secretion rate of the antibody into the CHO cell medium is increased compared to when CHO cells having the same genotype except for the modification are cultured under the same conditions. [7-2] The method according to [7-1], wherein the functional expression of the mRNA decapping activator gene is reduced or eliminated by gene knockout, gene modification, gene deletion, gene silencing, or a combination thereof. [7-3] The method according to [7-1] or [7-2], wherein the functional expression of the mRNA decapping activator gene is eliminated by heterozygous knockout or homozygous knockout.

[0051] [7-4] The method of any of [7-1] to [7-3], which comprises introducing at least one heterologous polynucleotide encoding an antibody into the CHO cells. [7-5] The method of any of [7-1] to [7-4], wherein the genome of the CHO cells is modified to reduce or eliminate functional expression of an mRNA decapping activator gene. [7-6] The method of any of [7-1] to [7-5], wherein the CHO cells contain at least one modification in at least one copy or all copies of the mRNA decapping activator gene.

[0052] [7-7] The method of [7-6], wherein the at least one modification is a modification that causes a frameshift in an exon of one or both alleles of the mRNA decapping activator gene. [7-8] The method of any of [7-1] to [7-7], wherein the amino acid sequence of the endogenous mRNA decapping activator after the modification has at least 90% identity to the amino acid sequences shown in SEQ ID NOs: 1 to 4 as mRNA decapping activator reference proteins. [7-9] The method of any of [7-1] to [7-8], wherein the mRNA decapping activator is PAT1, SCD6, EDC3, or DHH1.

[0053] [7-10] The method according to any one of [7-1] to [7-9], wherein the mRNA decapping activator is EDC3.

[0054] [7-11] The method according to any one of [7-1] to [7-10], wherein the CHO cells are provided as a cell clone or a cell line. [7-12] The method according to any one of [7-1] to [7-11], wherein the antibody is an antibody used in therapy or diagnosis.

[0055] [7-13] The method according to any one of [7-1] to [7-12], wherein the antibody is an IgG antibody, an IgG1 antibody, or a bispecific antibody. [7-14] The method according to any one of [7-1] to [7-13], further comprising introducing into the CHO cells at least one heterologous polynucleotide encoding a selection marker or a reporter polypeptide. [7-15] The method according to any one of [7-1] to [7-14], wherein the at least one heterologous polynucleotide encoding the peptide of interest is contained in an expression cassette.

[0056] [7-16] The method of any of [7-1] to [7-15], wherein the antibody secretion rate in the culture medium of the CHO cells is increased by at least 10% compared to when CHO cells having the same genotype except for the modification are cultured under the same conditions. [7-17] The method of any of [7-1] to [7-16], wherein the CHO cells do not express a detectable level of mRNA decapping activator. [7-18] The method of any of [7-1] to [7-17], wherein the reduction or elimination of functional expression of the mRNA decapping activator gene is mediated by a gene targeting system selected from the group consisting of CRISPR / Cas9, CRISPR / Cpf1, zinc finger nuclease, TALEN, or meganuclease.

[0057] [7-19] The method of any of [7-1] to [7-18], wherein the functional expression of the mRNA decapping activator gene is reduced or eliminated by gene knockout before or after introduction of a polynucleotide encoding an antibody. [7-20] The method of any of [7-1] to [7-19], wherein the CHO cells contain at least one modification in at least one copy or all copies of the mRNA decapping activator gene, the at least one modification causing a frameshift in an exon of one or both alleles of the mRNA decapping activator gene, the antibody is an IgG antibody, and the mRNA decapping activator is EDC3.

[0058] [7-21] A method for increasing the secretion rate of an antibody obtained in a medium by CHO cell culture, comprising preparing CHO cells modified to reduce or eliminate functional expression of an mRNA decapping activator gene in the CHO cells, wherein the secretion rate of the antibody in the medium of the CHO cells is increased compared to when CHO cells having the same genotype except for the modification are cultured under the same conditions, wherein the CHO cells contain at least one modification in at least one copy or all copies of the mRNA decapping activator gene, the at least one modification causing a frameshift in an exon of one or both alleles of the mRNA decapping activator gene, the antibody is an IgG antibody, and the mRNA decapping activator is EDC3.

[0059] Figure 1 is a diagram of a plasmid in which the heavy and light chains of SOF10 are combined to incorporate a hygromycin resistance gene and a DHFR gene, and Figure 2 is a diagram of a plasmid in which the heavy and light chains of RAY121 are combined to incorporate a hygromycin resistance gene and a DHFR gene.

[0060] DETAILED DESCRIPTION OF THE INVENTION I. General Definitions

[0061] As used herein, the term "about" refers to a range of ±20% of the preceding numerical value. In certain embodiments, the term "about" refers to a range of ±10% of the preceding numerical value. In certain embodiments, the term "about" refers to a range of ±5% of the preceding numerical value.

[0062] As used herein, the term "genetically engineered vertebrate cell" refers to a vertebrate cell containing a heterologous polynucleotide capable of expressing a polypeptide. Such genetically engineered vertebrate cells are cells into which one or more heterologous nucleic acid(s) have been introduced, including the progeny of such cells. Thus, the term "vertebrate cell containing at least one heterologous polynucleotide encoding a recombinant polypeptide" refers to a cell containing a heterologous polynucleotide that has been integrated into the vertebrate cell and is capable of expressing the recombinant polypeptide. In one aspect of the invention, the heterologous polynucleotide is introduced into the genome of the vertebrate cell, and in certain embodiments, the vertebrate cell containing the heterologous polynucleotide is a cell that contains a heterologous polynucleotide sequence integrated at a single site within a locus in the genome of the cell.

[0063] As used herein, the term "genetically engineered" refers to a state in which, after genetic modification, the organism has been engineered to be able to express, for example, a recombinant polypeptide of interest and to be used for the production of the recombinant polypeptide of interest on any scale.

[0064] Both "genetically engineered vertebrate cells" and "vertebrate cells containing at least one heterologous polynucleotide encoding a recombinant polypeptide" are "transformed cells." This term includes the primary transformed cell and its progeny, regardless of the number of transfers. The progeny may not be completely identical to the parent cell in nucleic acid content, for example, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are within the scope of the term.

[0065] In one embodiment of the present invention, recombinant polypeptides can be expressed in vertebrate cells. As used herein, the term "polypeptide" refers to a molecule comprising a polymer of amino acids linked by peptide bond(s). Polypeptides include polypeptides of any length, including proteins (e.g., having more than 50 amino acids) and peptides (e.g., 2-49 amino acids). Polypeptides include proteins and / or peptides of any activity, function, or size, and may include, for example, antibodies, enzymes (e.g., kinases, phosphatases), receptors, transporters, bacterial and / or endotoxin-binding proteins, structural polypeptides, membrane-bound polypeptides, glycopolypeptides, globular proteins, immune polypeptides, toxins, antibiotics, hormones, growth factors, blood factors, vaccines, viral glycopolypeptides, and the like. Recombinant polypeptides expressed in one embodiment of the present invention can also be subunits or domains of polypeptides, such as heavy or light chains of antibodies, or functional fragments or derivatives thereof. Depending on the context, the term "recombinant polypeptide" can refer to such individual subunits or domains or to the final protein composed of the respective subunits or domains.

[0066] As used herein, "recombinant polypeptide" refers to a polypeptide that is artificially engineered relative to the vertebrate cell that expresses it, regardless of whether the polypeptide is a naturally occurring polypeptide derived from a different cell / organism or an artificial polypeptide. Recombinant polypeptides are not derived from a specific cell, and the respective encoding nucleic acid is introduced into the cell by a DNA delivery method, such as transfection, electroporation, or transformation. Recombinant polypeptides are encoded by heterologous polynucleotides contained in vertebrate cells. Host vertebrate cells can contain two or more heterologous polynucleotides encoding recombinant polypeptides. Host vertebrate cells express or contain the recombinant polypeptide. Recombinant polypeptides are secreted by vertebrate cells into cell culture medium and can be recovered therefrom, e.g., isolated and purified.

[0067] In one aspect of the invention, the recombinant polypeptide is an immunoglobulin molecule, such as an antibody. In a particular aspect, the antibody is a multispecific antibody, e.g., a bispecific antibody. In one aspect of the invention, the recombinant polypeptide is selected from the group of multispecific antibodies and antibody-multimeric fusion polypeptides. In one aspect of the invention, the recombinant polypeptide may be a protein that is difficult to express, or a protein with a complex and / or non-native structure, e.g., a next-generation biological protein, e.g., a bispecific antibody, a fusion protein, or a glycosylated protein.

[0068] As used herein, expressions such as "heterologous polynucleotide" or "heterologous nucleic acid" refer specifically to a polynucleotide sequence introduced into a vertebrate cell by the use of recombinant techniques such as transfection. "Polynucleotide" refers specifically to a polymer of nucleotides, usually linked from one deoxyribose or ribose to another, and refers to DNA as well as RNA, depending on the context. The term "polynucleotide" does not imply any size limitation.

[0069] As used herein, an "isolated" polypeptide or antibody refers to a polypeptide or antibody molecule that has been separated from components of the natural environment in which it occurs.

[0070] As used herein, the term "integration site" refers to a nucleic acid sequence in a cell's genome where a heterologous polynucleotide is inserted into the cell's genome. In one embodiment of the present invention, the integration site is between two adjacent nucleotides in the cell's genome. In one embodiment of the present invention, the integration site comprises a stretch of nucleotide sequence. In one embodiment of the present invention, the integration site is located within a specific locus in the genome of a vertebrate cell. In one embodiment of the present invention, the integration site is within an endogenous gene of a vertebrate cell.

[0071] As used herein, the terms "vector" and "plasmid" can be used interchangeably and refer to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors as autonomously replicating nucleic acid structures and vectors that integrate into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0072] As used herein, the term "selection marker" refers to a gene that allows cells carrying the gene to be specifically selected, either positively or negatively, in the presence of a corresponding selection agent. For example, but not limited to, a selection marker may allow host cells transformed with the selection marker gene to be positively selected in the presence of the respective selection agent (under selective culture conditions). Untransformed host cells would not be able to grow or survive under selective culture conditions. A selection marker may be positive, negative, or bifunctional. A positive selection marker may allow the selection of cells carrying the marker, while a negative selection marker may allow the selective elimination of cells carrying the marker. A selection marker may confer resistance to a drug in a host cell or complement a metabolic or catabolic defect. In prokaryotic cells, genes that confer resistance to ampicillin, tetracycline, kanamycin, or chloramphenicol, among others, may be used. Resistance genes useful as selectable markers in eukaryotic cells include, but are not limited to, genes for aminoglycoside phosphotransferases (APH) (e.g., hygromycin phosphotransferase (HYG), neomycin, and G418APH), dihydrofolate reductase (DHFR), thymidine kinase (TK), glutamine synthetase (GS), asparagine synthetase, tryptophan synthetase (indole), histidinol dehydrogenase (histidinol D)), and genes encoding resistance to puromycin, blasticidin, bleomycin, phleomycin, chloramphenicol, zeocin, and mycophenolic acid. Additional marker genes are described in WO 92 / 08796 and WO 94 / 28143.

[0073] Beyond facilitating selection in the presence of a corresponding selection agent, a selectable marker may alternatively be a molecule not normally present in cells, such as green fluorescent protein (GFP), enhanced GFP (eGFP), synthetic GFP, yellow fluorescent protein (YFP), enhanced YFP (eYFP), cyan fluorescent protein (CFP), mPlum, mCherry, tdTomato, mStrawberry, J-red, DsRed monomer, mOrange, mKO, mCitrine, Venus, YPet, Emerald, CyPet, mCFPm, Cerulean, and T-Sapphire. Cells expressing such molecules can be distinguished from cells that do not harbor the gene based, for example, on the detection or absence, respectively, of fluorescence emitted by the encoded polypeptide.

[0074] As used herein, the term "endogenous" refers to a nucleotide sequence derived from a cell or a product based on that nucleotide sequence. The product may be an mRNA decapping activator.

[0075] II. Genetically Engineered Vertebrate Cells In one aspect of the present invention, the vertebrate cell is preferably a mammalian cell, e.g., selected from the group consisting of rodent cells, human cells, and monkey cells. Preferred vertebrate cells are rodent cells, such as cells derived from hamsters or mice. The rodent cell may be a cell line selected from the group consisting of Chinese hamster cell lines (e.g., Chinese hamster ovary (CHO) cell lines), BHK cell lines, NS0 cell lines, C127 cell lines, mouse 3T3 fibroblast cell lines, and SP2 / 0 cell lines. CHO cells, such as CHO-K1-derived CHO cells, are particularly preferred. The mRNA decapping activator is expressed in human cells. Thus, in one aspect of the present invention, the vertebrate cell is derived from a human cell, e.g., selected from the group consisting of HEK293 cells, MCF-7 cells, PerC6 cells, CAP cells, hematopoietic cells, and HeLa cells. Another alternative is a monkey cell, which may be selected from the group consisting of, for example, COS cells, COS-1, COS-7 cells and Vero cells. According to one embodiment, the vertebrate cell is provided as a cell clone, a cell line or a cell culture.

[0076] As used herein, the term "host cell" refers to a cell into which a heterologous polynucleotide has been introduced and which is used to express a recombinant polypeptide. In one aspect of the present invention, a "vertebrate cell" can be used as the "host cell," and examples of the "vertebrate cell" include mammalian cells that are commonly used in the production of bioproducts using recombinant DNA technology.

[0077] In one embodiment of the present invention, the vertebrate cell is a mammalian cell, for example, a Chinese hamster ovary (CHO) cell (e.g., CHOK1, CHODG44, etc.), a human embryonic kidney (HEK) cell, a lymphoid cell (e.g., YO, NS0, Sp20 cell), or a human amniotic cell (e.g., CAP-T, etc.). In a preferred embodiment, the mammalian cell is a CHO cell.

[0078] In one embodiment of the invention, the cell is a stem cell. In one aspect, the cell is a differentiated form of any of the cells described herein. In one aspect, the cell is derived from any primary cell in culture.

[0079] As used herein, the terms "cell," "cell line," and "cell culture" are used to refer to all cell progeny. For example, CHO cells are the cellular progeny of Chinese hamster ovary cells and may be removed from the original primary cellular parent by any number of generations, including transformed progeny. Transformants and transformed cells include the primary subject cell and cultures derived therefrom, regardless of the number of introductions. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included.

[0080] In one embodiment of the present invention, the components of the culture medium used may be any of those typically used in cell (preferably animal cell) culture media, including amino acids, vitamins, lipid factors, energy sources, osmotic pressure regulators, iron sources, and pH buffers. In addition to the above components, trace metal elements, surfactants, growth cofactors, and nucleotides may also be added.

[0081] Specific examples include L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-cystine, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-ornithine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine, and preferably L-alanine, L-arginine, and L-aspartic acid. Amino acids such as paragine, L-aspartic acid, L-cystine, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine; i-inositol, biotin, folic acid, lipoic acid, nicotinamide, nicotinic acid, p-aminobenzoic acid, calcium pantothenate, and pyridoxine hydrochloride. lipid factors such as choline chloride, choline tartrate, linoleic acid, oleic acid, cholesterol, etc., preferably choline chloride; energy sources such as glucose, galactose, mannose, fructose, etc., preferably glucose; osmotic regulators such as sodium chloride, potassium chloride, potassium nitrate, etc., preferably sodium chloride; iron sources such as iron EDTA, iron citrate, ferrous chloride, ferric chloride, ferrous sulfate, ferric sulfate, ferric nitrate, etc., preferably ferric chloride, iron EDTA, and iron citrate; and pH buffer solutions such as sodium bicarbonate, calcium chloride, sodium dihydrogen phosphate, HEPES, MOPS, etc., preferably sodium bicarbonate.

[0082] In addition to the above components, trace metal elements such as copper sulfate, manganese sulfate, zinc sulfate, magnesium sulfate, nickel chloride, tin chloride, magnesium chloride, and sodium silicate, preferably copper sulfate, zinc sulfate, and magnesium sulfate; surfactants such as Tween 80 and Pluronic® F68; growth cofactors such as recombinant insulin, recombinant IGF, recombinant EGF, recombinant FGF, recombinant PDGF, recombinant TGF-α, ethanolamine hydrochloride, sodium selenite, retinoic acid, and putrescine hydrochloride, preferably sodium selenite, ethanolamine hydrochloride, recombinant IGF, and putrescine hydrochloride; and nucleosides such as deoxyadenosine, deoxycytidine, deoxyguanosine, adenosine, cytidine, guanosine, and uridine may also be added. In a preferred embodiment of the present invention, antibiotics such as streptomycin, penicillin G potassium, and gentamicin, and pH indicators such as phenol red may also be added.

[0083] The contents of other components in the medium are suitable within the ranges of 0.05-1500 mg / mL for amino acids, 0.001-10 mg / mL for vitamins, 0-200 mg / mL for lipid factors, 1-20 g / mL for energy sources, 0.1-10,000 mg / mL for osmotic pressure regulators, 0.1-500 mg / mL for iron sources, 1-10,000 mg / mL for pH buffers, 0.00001-200 mg / mL for trace metal elements, 0-5,000 mg / mL for surfactants, 0.05-10,000 μg / mL for growth cofactors, and 0.001-50 mg / mL for nucleosides, and can be determined appropriately depending on the type of cells being cultured and the type of desired protein. The pH of the medium varies depending on the cells being cultured, but is generally between 6.8 and 7.6, with a pH of 7.0-7.4 being most common.

[0084] In one embodiment of the present invention, the culture method is not particularly limited and can be used to culture various vertebrate cells. For example, it is possible to culture COS cells or CHO cells into which a gene encoding a desired protein has been inserted by genetic engineering, or fusion cells such as mouse-human, mouse-mouse, or mouse-rat hybridomas that produce antibodies. The method of the present invention can also be used to culture vertebrate cells to obtain native proteins produced by the vertebrate cells, and can be used to culture BHK cells, HeLa cells, and other cells in addition to the above-mentioned cells.

[0085] In one embodiment of the present invention, a vector can be introduced into a host cell by, for example, the calcium phosphate method, the DEAE-dextran method, a method using the cationic liposome DOTAP (manufactured by Boehringer Mannheim), electroporation, lipofection, or the like.

[0086] In one embodiment of the present invention, protein production in vertebrate cells involves both simple culturing and special manipulations, and these manipulations or conditions may be appropriately determined depending on the vertebrate cells being cultured. For example, CHO cells transformed with a vector containing a gene encoding a mouse-human chimeric antibody by genetic engineering can produce an antibody in the medium under the conditions described below. The desired protein can be obtained by isolating and purifying the antibody according to standard methods (see, for example, "Introduction to Antibody Engineering," Chijin Shokan, pp. 102-104; "Affinity Chromatography Principles & Methods," Amersham Pharmacia Biotech, pp. 56-60, etc.).

[0087] In one embodiment of the present invention, culture conditions may be appropriately determined depending on the type of cells used. For example, CHO cells may typically be cultured for 1 to 14 days in an atmosphere with a CO2 concentration in the gas phase of 0 to 40%, preferably 2 to 10%, at 30 to 39°C, preferably about 37°C. Various culture devices for animal cell culture, such as a fermenter-type tank culture device, an airlift-type culture device, a culture flask-type culture device, a spinner flask-type culture device, a microcarrier-type culture device, a fluidized bed-type culture device, a hollow fiber-type culture device, a roller bottle-type culture device, and a packed bed-type culture device, may be used.

[0088] In one embodiment of the present invention, proteins secreted into the medium from cultured animal cells can be recovered from the culture medium by conventional methods. Alternatively, proteins can be recovered from cell lysates of host cells by conventional methods. Specifically, the desired protein can be recovered by removing cells and cell debris from the cell culture medium or cell lysate by centrifugation or other methods, followed by application of common protein isolation and purification techniques. Examples of techniques that can be used include salting out (e.g., ammonium sulfate fractionation), alcohol precipitation (e.g., ethanol precipitation), PEG, electrophoresis, ion exchange chromatography, ultracentrifugation, gel filtration, hydrophobic chromatography, and affinity chromatography. When the desired protein is an antibody, protein A chromatography is preferably used, but is not limited to this. Furthermore, various affinity-based separation or fractionation methods can be used to separate antibodies into immunoglobulin classes or to separate them based on their antigen binding ability.

[0089] Regulation of mRNA translation and degradation is mediated by proteins that inhibit translation, promote decapping, and act to assemble cytoplasmic mRNA granules called processing bodies (P-bodies). Known mRNA decapping proteins include Pat1, Scd6, Edc3, and Dhh1.

[0090] In one embodiment of the present invention, the genetically engineered vertebrate cells are modified to reduce or eliminate the functional expression of the mRNA decapping activator gene compared to unmodified vertebrate cells that endogenously express the mRNA decapping activator. In one embodiment of the present invention, when the functional expression of the mRNA decapping activator gene is reduced or eliminated, the protein expression level is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, preferably 100%, compared to unmodified vertebrate cells that endogenously express the mRNA decapping activator. The protein expression level can be measured by known methods, for example, by Western blotting. In order to provide a manufacturing cell line with stable, uniform, and therefore predictable characteristics, it is preferable to modify the genome of the vertebrate cells to reduce or eliminate the functional expression of the mRNA decapping activator gene. Genetically engineered vertebrate cells can be transfected with an expression vector comprising a polynucleotide encoding a recombinant polypeptide to provide a host cell of the present disclosure that contains a heterologous polynucleotide encoding a recombinant polypeptide and secretes the recombinant polypeptide into cell culture medium.

[0091] In one embodiment of the present invention, the functional expression of an mRNA decapping activator gene in a cell is eliminated by "gene knockout." Gene knockout is a genetic technique in which a gene is disabled by disrupting its function. For example, a nucleic acid can be inserted into the coding sequence, thereby disrupting gene function. Furthermore, the mRNA decapping activator gene or a portion thereof can be deleted, thereby preventing the individually modified cell from expressing a protein or functional protein. Another option is to introduce one or more knockout mutations into the coding sequence. Alternatively, in one embodiment of the present invention, one or more stop codons can be introduced into the coding sequence. Thus, in one embodiment of the present invention, the mRNA decapping activator gene contains one or more mutations that reduce or eliminate gene expression. In one embodiment of the present invention, the one or more mutations are frameshift or stop codon mutations. Methods for achieving gene knockout, which suppress or eliminate the functional expression of a target gene, are also well known to those skilled in the art and therefore do not require any detailed description herein.

[0092] In one aspect of the present invention, the mRNA decapping activator gene is functionally knocked out by genetic engineering.For example, genome editing, such as genome editing using recombinant nucleases (GENE).This is a type of genetic engineering in which DNA is inserted, replaced, or removed from the genome using recombinant nucleases or "molecular scissors".Nucleases create specific double-strand breaks (DSBs) at desired positions in the genome, and utilize the endogenous mechanisms of cells to repair the breaks induced by the natural processes of homologous recombination (HR) and non-homologous end joining (NHEJ).At least four families of recombinant nucleases that can be used for this purpose are known: zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), nucleases that recognize clustered regularly interspaced short palindromic repeats (CRISPRs), and homing endonucleases reengineered by recombinant meganucleases. In one aspect of the present invention, the reduction, elimination or knockout of endogenous genes is mediated by a nuclease-assisted gene targeting system. The nuclease-assisted gene targeting system is selected from the group consisting of CRISPR / Cas9, CRISPR / Cpf1, zinc finger nuclease, TALEN and meganuclease. In one aspect of the present invention, the knockout of endogenous genes is carried out i) before the introduction of heterologous nucleic acid encoding recombinant polypeptide, or ii) after the introduction of heterologous nucleic acid encoding recombinant polypeptide.

[0093] In one embodiment of the invention, the gene knockout is a heterozygous knockout or a homozygous knockout.

[0094] In one aspect of the present invention, "gene deletion" refers to removing at least a portion of a DNA sequence from or adjacent to a gene. In some aspects, the sequence subjected to gene deletion comprises an exon sequence of the gene. In some aspects, the sequence subjected to gene deletion comprises a promoter sequence of the gene. In some aspects, the sequence subjected to gene deletion comprises a flanking sequence of the gene.

[0095] In one embodiment of the present invention, the mRNA decapping activator has the amino acid sequence shown in SEQ ID NO: 1 to 4. In one embodiment of the present invention, the mRNA decapping activator can be PAT1, SCD6, EDC3, or DHH1. PAT1, SCD6, EDC3, and DHH1 bind to mRNA decapping enzymes.

[0096] III. Antibodies In one embodiment of the present invention, recombinant polypeptides are produced by genetically engineered vertebrate cells. In one aspect, the recombinant polypeptide is an antibody.

[0097] Herein, the amino acid positions of all heavy and light chain constant regions and domains are numbered according to the Kabat numbering system described in Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), and are referred to herein as "Kabat numbering." Specifically, the Kabat numbering system described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. The Kabat numbering system of the National Institutes of Health, Bethesda, MD (1991) (see pages 647-660) is used for the light chain constant domains CL of kappa and lambda isotypes, and the Kabat EU index numbering system (see pages 661-723) is used for the heavy chain constant domains (CH1, hinge, CH2, and CH3, which is further clarified herein by referring to "Kabat EU index numbering" in this case).

[0098] The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, full length antibodies, monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody-antibody fragment-fusions and combinations thereof.

[0099] As used herein, the term "natural antibody" refers to naturally occurring immunoglobulin molecules with a variety of structures. For example, natural IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons composed of two identical disulfide-bonded light chains and two identical heavy chains. From the N- to C-terminus, each heavy chain has a heavy chain variable region (VH) followed by three heavy chain constant domains (CH1, CH2, and CH3), thereby positioning a hinge region between the first heavy chain constant domain and the second heavy chain constant domain. Similarly, from the N- to C-terminus, each light chain has a light chain variable region (VL) followed by a light chain constant domain (CL). Based on the amino acid sequence of its constant domain, the light chain of an antibody may be assigned to one of two types, called kappa (κ) or lambda (λ).

[0100] As used herein, the term "full-length antibody" refers to an antibody having a structure substantially similar to that of a naturally occurring antibody. A full-length antibody comprises two full-length antibody light chains, each comprising, from N- to C-terminus, a light chain variable region and a light chain constant domain, and two full-length antibody heavy chains, each comprising, from N- to C-terminus, a heavy chain variable region, a first heavy chain constant domain, a hinge region, a second heavy chain constant domain, and a third heavy chain constant domain. In contrast to naturally occurring antibodies, full-length antibodies may comprise additional immunoglobulin domains, such as one or more additional scFvs, or heavy or light chain Fab fragments, or scFabs conjugated to one or more ends of different chains of the full-length antibody, but only one fragment at each end. These conjugates are also encompassed by the term full-length antibody.

[0101] As used herein, the term "antibody combining site" refers to a pair of heavy-chain and light-chain variable domains. To ensure proper binding to an antigen, these variable domains are cognate variable domains, i.e., belong together. An antibody combining site comprises at least three HVRs (e.g., in the case of a VHH) or three to six HVRs (e.g., in the case of a naturally occurring, i.e., conventional antibody having a VH / VL pair). Generally, the amino acid residues of an antibody involved in antigen binding form the combining site. These residues are usually contained in a pair of antibody heavy-chain variable domains and corresponding antibody light-chain variable domains. An antibody antigen-binding site comprises amino acid residues from "hypervariable regions" or "HVRs." "Framework" or "FR" regions are the regions of the variable domain other than the hypervariable region residues as defined herein. Thus, the light and heavy chain variable domains of an antibody comprise, from N- to C-terminus, the regions FR1, HVR1, FR2, HVR2, FR3, HVR3, and FR4. In particular, the HVR3 region of the heavy chain variable domain is the region that contributes most to antigen binding and defines the binding specificity of the antibody. A "functional binding site" is capable of specifically binding to its target. The term "specifically binds" refers to the binding of a binding site to its target in an in vitro assay, and in a specific embodiment, in a binding assay. Such a binding assay can be any assay as long as a binding event can be detected. For example, a binding assay in which an antibody is bound to a surface and the binding of an antigen to the antibody is measured by surface plasmon resonance (SPR). Alternatively, a bridging ELISA can be used.

[0102] The term "hypervariable region" or "HVR" as used herein refers to each region of an antibody variable domain that comprises stretches of amino acid residues that are hypervariable sequences ("complementarity determining regions" or "CDRs") and / or form structurally defined loops ("hypervariable loops") and / or contain residues that contact antigen ("antigen contacts"). Typically, antibodies contain six HVRs: three in the heavy chain variable domain (VH) (H1, H2, H3) and three in the light chain variable domain (VL) (L1, L2, L3).

[0103] Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra.

[0104] As used herein, the "class" of an antibody refers to the type of constant domain or constant region, preferably the Fc region, of the heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into "subclasses" (isotypes), for example, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different immunoglobulin classes are called α, δ, ε, γ, and μ, respectively.

[0105] As used herein, the term "heavy chain constant region" refers to the region of an immunoglobulin heavy chain comprising the constant domains, i.e., the CH1 domain, hinge region, CH2 domain, and CH3 domain. In a specific aspect, a human IgG constant region extends from Ala118 to the carboxyl terminus of the heavy chain (numbering according to the Kabat EU index). However, the C-terminal lysine (Lys447) of the constant region may or may not be present (numbering according to the Kabat EU index). The term "constant region" refers to a dimer comprising two heavy chain constant regions that can be covalently linked to each other via hinge region cysteine ​​residues that form interchain disulfide bonds.

[0106] As used herein, the term "heavy chain Fc region" refers to the C-terminal region of an immunoglobulin heavy chain, including at least a portion of the hinge region, the CH2 domain, and the CH3 domain. In a specific embodiment, the human IgG heavy chain Fc region extends from Asp221 or Cys226 or Pro230 to the carboxyl terminus of the heavy chain (numbering according to the Kabat EU index). Thus, although the Fc region is smaller than the constant region, the C-terminal portion is identical to it. However, the C-terminal lysine (Lys447) of the heavy chain Fc region may or may not be present (numbering according to the Kabat EU index). The term "Fc region" refers to a dimer comprising two heavy chain Fc regions that can be covalently bound to each other via hinge region cysteine ​​residues that form interchain disulfide bonds.

[0107] The constant region of an antibody, more precisely the Fc region (and similarly the constant region), is directly involved in complement activation, C1q binding, C3 activation, and Fc receptor binding. The effect of an antibody on the complement system depends on the specific conditions, but binding to C1q is caused by a defined binding site in the Fc region. Such binding sites are known in the prior art and are described, for example, in Lukas, T. J. et al., J. Immunol. 127 (1981) 2555-2560; Brunhouse, R. and Cebra, J. J., Mol. Immunol. 16 (1979) 907-917; Burton, D. R. et al., Nature 288 (1980) 338-344; Thommesen, J. E. et al., Mol. Immunol. 37 (2000) 995-1004, Idusogie, E. E. et al., J. Immunol. 164 (2000) 4178-4184, Hezareh, M. et al., J. Virol. 75 (2001) 12161-12168, Morgan, A. et al., Immunology 86 (1995) 319-324, and EP 0 307 434. Such binding sites are, for example, L234, L235, D270, N297, E318, K320, K322, P331 and P329 (numbering according to the EU index of Kabat). Antibodies of the subclasses IgG1, IgG2, and IgG3 typically exhibit complement activation, C1q binding, and C3 activation, whereas IgG4 does not activate the complement system, does not bind C1q, and does not activate C3. The term "Fc region of an antibody" is well known to those skilled in the art and is defined based on papain cleavage of an antibody.

[0108] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain comprising at least a portion of the constant region. This term includes native-sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl terminus of the heavy chain, with the exception that the C-terminal lysine (Lys447) or glycine-lysine (Gly446-Lys447) residue at the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is in accordance with Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. According to the EU numbering system (also called the EU index) as set out in the Public Health Service, National Institutes of Health, Bethesda, MD 1991.

[0109] Fc Region Variants In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) containing an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0110] In certain embodiments, antibody variants that retain some, but not all, effector functions are also contemplated by the present invention, which make the antibody a desirable candidate for applications where its in vivo half-life is important but certain effector functions (such as complement and ADCC) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / lack of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to confirm that an antibody lacks FcγR binding (and thus likely lacks ADCC activity) while retaining FcRn binding ability. NK cells, the primary cells for mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. Expression of FcRs on hematopoietic cells is discussed in detail in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991) at page 464, Table 3. Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are those described in U.S. Patent No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); U.S. Pat. No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays may be used (see, e.g., ACT1™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA); and CytoTox96® non-radioactive cytotoxicity assays (Promega, Madison, WI)).Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that described in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays may also be performed to confirm that the antibody is unable to bind C1q and thereby lacks CDC activity. See, e.g., C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. CDC measurements may also be performed to assess complement activation (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M.S. et al., Blood 101:1045-1052 (2003); and Cragg, M.S. and M.J. Glennie, Blood 103:2738-2743 (2004)). Furthermore, FcRn binding and in vivo clearance / half-life determinations may also be performed using methods known in the art (see, e.g., Petkova, S.B. et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0111] Antibodies with reduced effector function include those with one or more substitutions at Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). Such Fc variants include Fc variants with substitutions at two or more amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc variant with substitutions of residues 265 and 297 to alanine (U.S. Patent No. 7,332,581).

[0112] Certain antibody variants with increased or decreased binding to FcRs have been described (see U.S. Pat. No. 6,737,056; WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001)).

[0113] In certain embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC (e.g., substitutions at positions 298, 333, and / or 334 (EU numbering) of the Fc region).

[0114] In some embodiments, modifications are made in the Fc region that result in altered (i.e., either increased or decreased) C1q binding and / or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0115] Antibodies with increased half-lives and increased binding to the neonatal Fc receptor (FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) are described in U.S. Patent Application Publication No. 2005 / 0014934 A1 (Hinton et al.). These antibodies comprise an Fc region with one or more substitutions therein that increase binding of the Fc region to FcRn. Such Fc variants include those with substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434 (e.g., substitution of Fc region residue 434 (U.S. Patent No. 7,371,826)).

[0116] For other examples of Fc region variants, see also Duncan & Winter, Nature 322:738-40 (1988); US Pat. No. 5,648,260; US Pat. No. 5,624,821; and WO 94 / 29351.

[0117] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies. The individual antibodies comprising the population are identical or bind to the same epitope, with the exception of possible variant antibodies that contain, for example, naturally occurring mutations or that arise during production of the monoclonal antibody preparation; such variants are generally present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention may be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci.

[0118] The term "valency," as used herein in this application, refers to the presence of a particular number of binding sites within an antibody. Thus, the terms "bivalent," "tetravalent," and "hexavalent" refer to the presence of two, four, and six binding sites, respectively, within an antibody.

[0119] As used herein, the term "monospecific antibody" refers to an antibody that has a single binding specificity, i.e., that specifically binds to one antigen. Monospecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2), or combinations thereof (e.g., a full-length antibody with an additional scFv or Fab fragment). Monospecific antibodies need not be monovalent; that is, they may contain more than one binding site that specifically binds to one antigen. For example, natural antibodies are monospecific but bivalent.

[0120] As used herein, the term "multispecific antibody" refers to an antibody that has binding specificities for at least two different epitopes on the same antigen or two different antigens. Multispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies), or combinations thereof (e.g., a full-length antibody with an additional scFv or Fab fragment). Multispecific antibodies are at least bivalent, i.e., contain two antigen-binding sites. Furthermore, multispecific antibodies are at least bispecific. Thus, bivalent bispecific antibodies are the simplest form of multispecific antibodies. Engineered antibodies with two, three, or more (e.g., four) functional antigen-binding sites have been reported (see, e.g., US 2002 / 0004587).

[0121] In one aspect of the present invention, the antibody is a multispecific antibody, e.g., at least a bispecific antibody. A multispecific antibody is a monoclonal antibody that has binding specificities for at least two different antigens or epitopes. In certain aspects, one of the binding specificities is for a first antigen and the other is for a different second antigen. In certain aspects, a multispecific antibody can bind to two different epitopes of the same antigen. Multispecific antibodies can also be used to localize cytotoxic agents to cells expressing the antigen.

[0122] Multispecific antibodies can be prepared as full-length antibodies or antibody-antibody fragment-fusions.

[0123] Techniques for producing multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein, C. and Cuello, A.C., Nature 305 (1983) 537-540, WO 93 / 08829, and Traunecker, A. et al., EMBO J. 10 (1991) 3655-3659), and "knob-in-hole" engineering (see, e.g., U.S. Pat. No. 5,731,168). Multispecific antibodies can also be produced by modifying electrostatic steering effects to create antibody Fc heterodimeric molecules (WO 2009 / 089004); cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan, M., et al., Science 229 (1985) 81-83); producing bispecific antibodies using leucine zippers (see, e.g., Kostelny, S.A., et al., J. Immunol. 2009, 10, 141-143). bispecific antibody fragments can be generated by using conventional light chain technology to circumvent light chain mispairing problems (see, e.g., WO 98 / 50431); by using "diabody" technology to generate bispecific antibody fragments (see, e.g., Holliger, P., et al., Proc. Natl. Acad. Sci. USA 90 (1993) 6444-6448); and by preparing trispecific antibodies as described, for example, in Tutt, A., et al., J. Immunol. 147 (1991) 60-69.

[0124] Also included herein are engineered antibodies with three or more antigen-binding sites, including, for example, "Octopus antibodies," or DVD-binding proteins (see, e.g., WO 2001 / 77342 and WO 2008 / 024715). Other examples of multispecific antibodies with three or more antigen-binding sites can be found in WO 2010 / 115589, WO 2010 / 112193, WO 2010 / 136172, WO 2010 / 145792, and WO 2013 / 026831. Bispecific antibodies or antigen-binding fragments thereof also include "dual acting Fabs" or "DAFs" (see, e.g., U.S. Patent Application Publication Nos. 2008 / 0069820 and WO 2015 / 095539).

[0125] Multispecific antibodies can also be provided in an asymmetric manner with domain crossover in one or more binding arms of the same antigen specificity, i.e., by exchanging VH / VL domains (see, e.g., WO 2009 / 080252 and WO 2015 / 150447), CH1 / CL domains (see, e.g., WO 2009 / 080253), or complete Fab arms (see, WO 2009 / 080251, WO 2016 / 016299; see also Schaefer et al., Proc. Natl. Acad. Sci. USA 108 (2011) 1187-1191, and Klein et al., MAbs 8 (2016) 1010-1020). In one aspect, the multispecific antibody comprises cross-Fab fragments. The term "cross-Fab fragment" or "xFab fragment" or "crossover Fab fragment" refers to a Fab fragment in which either the variable or constant regions of the heavy and light chains have been exchanged. A cross-Fab fragment comprises a polypeptide chain composed of a light chain variable region (VL) and a heavy chain constant region 1 (CH1), and a polypeptide chain composed of a heavy chain variable region (VH) and a light chain constant region (CL). Asymmetric Fab arms can also be engineered by introducing charged or uncharged amino acid mutations at the domain interface to direct correct Fab pairing. See, e.g., WO 2016 / 172485.

[0126] In one aspect of the invention, the antibody or fragment may also be a multispecific antibody as described in WO2009 / 080254, WO2010 / 112193, WO2010 / 115589, WO2010 / 136172, WO2010 / 145792 or WO2010 / 145793.

[0127] In one aspect of the present invention, the antibody or fragment thereof may also be a multispecific antibody as disclosed in WO2012 / 163520.

[0128] A variety of additional molecular formats of multispecific antibodies are known in the art and are included herein (see, e.g., Spiess et al., Mol. Immunol. 67 (2015) 95-106).

[0129] Bispecific antibodies are generally antibody molecules that specifically bind to two different, non-overlapping epitopes on the same antigen or to two epitopes on different antigens.

[0130] "Knob-into-hole" dimerization modules and their use in antibody engineering are described in Carter P.; Ridgway J. B. B.; Presta L. G.: Immunotechnology, Volume 2, Number 1, February 1996, pp. 73-73(1).

[0131] The CH3 domain of an antibody heavy chain can be modified using the "knob-into-hole" technique. This technique is described in detail with some examples in, for example, WO 96 / 027011; Ridgway, J. B., et al., Protein Eng. 9 (1996) 617-621; and Merchant, A. M., et al., Nat. Biotechnol. 16 (1998) 677-681. In this method, the interaction surfaces of two CH3 domains are modified to increase heterodimerization of these two CH3 domains, thereby increasing heterodimerization of polypeptides containing them. Each of the two CH3 domains (of the two heavy chains) can be a "knob," and the other a "hole." The introduction of disulfide bridges further stabilizes the heterodimer (Merchant, AM et al., Nature Biotech. 16 (1998) 677-681; Atwell, S. et al., J. Mol. Biol. 270 (1997) 26-35) and increases the yield.

[0132] The mutation T366W in the CH3 domain (of an antibody heavy chain) is designated as a "knob mutation" or "mutation knob," and the mutations T366S, L368A, and Y407V in the CH3 domain (of an antibody heavy chain) are designated as "hole mutations" or "mutation hole" (numbering according to the EU index of Kabat). Additional interchain disulfide bridges between CH3 domains (Merchant, A.M. et al., Nature Biotech. 16 (1998) 677-681) can also be used, for example, by introducing a S354C mutation in the CH3 domain of a heavy chain bearing a "knob mutation" (designated "knob-cys-mutation" or "mutation knob-cys") and a Y349C mutation in the CH3 domain of a heavy chain bearing a "hole mutation" (designated "hole-cys-mutation" or "mutation hole-cys") (numbering according to the EU index of Kabat).

[0133] The term "domain crossover" as used herein refers to deviations in domain sequence from that of a native antibody in that in a pair of antibody heavy chain VH-CH1 fragment and its corresponding cognate antibody light chain, i.e., antibody Fab (Fragment-Antigen Binding), at least one heavy chain domain is replaced by its corresponding light chain domain, and vice versa. There are three general types of domain crossovers: (i) crossovers of CH1 and CL domains, where the domain crossover in the light chain results in a VL-CH1 domain sequence and the domain crossover in the heavy chain fragment results in a VH-CL domain sequence (or a full-length antibody heavy chain having a VH-CL-hinge-CH2-CH3 domain sequence); (ii) domain crossovers of VH and VL domains, where the domain crossover in the light chain results in a VH-CL domain sequence and the domain crossover in the heavy chain fragment results in a VL-CH1 domain sequence; and (iii) domain crossovers of an intact light chain (VL-CL) and an intact VH-CH1 heavy chain fragment ("Fab crossover"), where the domain crossover results in a light chain with a VH-CH1 domain sequence and the domain crossover results in a heavy chain fragment with a VL-CL domain sequence (all domain sequences listed above are in the N-terminal to C-terminal direction).

[0134] As used herein, the term "replaced by one another" with respect to corresponding heavy and light chain domains refers to the domain crossover described above. Thus, when the CH1 and CL domains are "replaced by one another," the term refers to the domain crossover referred to under item (i) and the resulting heavy and light chain domain sequences. Thus, when the VH and VL are "replaced by one another," the term refers to the domain crossover referred to under item (ii), and when the CH1 and CL domains are "replaced by one another" and the VH and VL domains are "replaced by one another," the term refers to the domain crossover referred to under item (iii). Bispecific antibodies comprising domain crossovers are described, for example, in WO 2009 / 080251, WO 2009 / 080252, WO 2009 / 080253, WO 2009 / 080254, and Schaefer, W., et al., Proc. This is reported in Natl. Acad. Sci. USA 108 (2011) 11187-11192. Such antibodies are generally called CrossMab.

[0135] In one aspect, a multispecific antibody also comprises at least one Fab fragment comprising either the domain crossover of the CH1 and CL domains described in the above item (i), the domain crossover of the VH and VL domains described in the above item (ii), or the domain crossover of the VH-CH1 and VL-VL domains described in the above item (iii). In the case of a multispecific antibody with domain crossover, Fabs that specifically bind to the same antigen are constructed to have the same domain sequence. Therefore, when more than one Fab with domain crossover is included in a multispecific antibody, the Fabs specifically bind to the same antigen.

[0136] As used herein, a "humanized" antibody refers to an antibody that comprises amino acid residues from non-human HVRs and amino acid residues from human FRs. In one aspect of the invention, a humanized antibody comprises substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody refers to an antibody that has undergone humanization.

[0137] The term "recombinant antibody," as used herein, refers to all antibodies (chimeric, humanized, and human) that are prepared, expressed, produced, or isolated by recombinant means, such as recombinant cells. This includes antibodies isolated from recombinant cells, such as NS0, HEK, BHK, amniotic cells, CHO cells, etc.

[0138] As used herein, the term "antibody fragment" refers to a molecule other than an intact antibody that contains a portion of the intact antibody that binds to the antigen to which the intact antibody binds, i.e., it is a functional fragment. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, bispecific Fab, diabodies, linear antibodies, and single-chain antibody molecules (e.g., scFv or scFab).

[0139] IV. Methods for Producing Recombinant Polypeptides One aspect of the present invention provides methods for producing a recombinant polypeptide, comprising culturing a host cell comprising nucleic acid encoding the recombinant polypeptide under conditions suitable for expression of the recombinant polypeptide, and optionally recovering the recombinant polypeptide from the host cell (or host cell culture).

[0140] For the production of recombinant polypeptides, such as recombinant antibodies, for example, nucleic acids encoding the recombinant polypeptides described above are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be readily isolated and sequenced using standard procedures.

[0141] Typically, for large-scale production of a recombinant polypeptide, such as a therapeutic antibody, cells that stably express and secrete the polypeptide are required. These cells are called "recombinant cells" or "recombinant production cells," and the methods used to generate such cells are called "cell line development." In the first step of the cell line development method, suitable host cells, such as CHO cells, are transfected with a nucleic acid sequence suitable for expression of the recombinant polypeptide. In the second step, cells that stably express the recombinant polypeptide are selected based on the co-expression of a selectable marker that has been co-transfected with the nucleic acid encoding the recombinant polypeptide.

[0142] A nucleic acid that encodes a polypeptide, i.e., a coding sequence, is called a structural gene. Such a structural gene is purely coding information. Therefore, its expression requires additional regulatory elements. Therefore, structural genes are usually incorporated into so-called expression cassettes. The minimum regulatory elements required for an expression cassette to be functional in mammalian cells are a promoter functional in mammalian cells located upstream, i.e., 5', of the structural gene, and a polyadenylation signal sequence functional in mammalian cells located downstream, i.e., 3', of the structural gene. The promoter sequence, structural gene sequence, and polyadenylation signal sequence are arranged in an operably linked form.

[0143] When the recombinant polypeptide is a heteromultimeric polypeptide composed of different (monomeric) polypeptides, such as an antibody or a complex antibody format, not only a single expression cassette is required, but multiple expression cassettes containing different structural genes are required, i.e., at least one expression cassette for each of the different (monomeric) polypeptides of the heteromultimeric polypeptide. For example, a full-length antibody is a heteromultimeric polypeptide containing two copies of a light chain and two copies of a heavy chain. Thus, a full-length antibody is composed of two different polypeptides. Therefore, two expression cassettes are required for the expression of a full-length antibody: one for the light chain and one for the heavy chain. For example, if the full-length antibody is a bispecific antibody, i.e., if the antibody contains two different binding sites that specifically bind to two different antigens, the two light chains and the two heavy chains are also different from each other. Therefore, such a bispecific full-length antibody is composed of four different polypeptides, and therefore requires four expression cassettes.

[0144] The expression cassette for the recombinant polypeptide is then incorporated into one or more so-called "expression vectors." An "expression vector" is a nucleic acid that provides all the elements required to amplify the vector in bacterial cells and to express the contained structural gene in mammalian cells. Typically, an expression vector comprises a prokaryotic plasmid propagation unit, which, for example in the case of E. coli, contains an origin of replication and a prokaryotic or eukaryotic selection marker, as well as an expression cassette required for expression of the structural gene of interest. An "expression vector" is a delivery vehicle for introducing an expression cassette into mammalian cells.

[0145] Cell line development (CLD) for generating recombinant cells expressing recombinant polypeptides, such as multispecific antibodies, uses either random integration (RI) or targeted integration (TI) of nucleic acids containing the respective expression cassettes necessary for the expression and production of the recombinant polypeptide of interest. In one aspect of the present invention, the genes introduced into the host cells can also be transient transfections that are present in the cells for a limited period of time.

[0146] Using RI, multiple vectors or fragments thereof are typically integrated into the genome of a cell at the same or different loci.

[0147] Using TI, typically a single copy of a transgene containing different expression cassettes is integrated into a defined "hot spot" in the host cell's genome.

[0148] In one aspect of the present invention, efficient production of a recombinant polypeptide using a genetically engineered vertebrate cell obtained by the present invention means, for example, production that is more efficient than production using a non-genetically engineered vertebrate cell, where efficient production means, for example, improved productivity in the production of the recombinant polypeptide using a vertebrate cell.

[0149] In one aspect of the present invention, the productivity of a recombinant polypeptide by a vertebrate cell is expressed by an index such as titer, antibody secretion rate per cell, etc. Here, productivity refers to the efficiency of recombinant polypeptide production by vertebrate cell culture, and varies depending on the expression level of the recombinant polypeptide in each cell, the growth rate of the cells in culture, etc.

[0150] In one aspect of the present invention, increased productivity refers to an increase in the amount of polypeptide produced compared to a control. Here, the amount of polypeptide produced can be measured by, for example, weighing the polypeptide isolated after purification or by HPLC. In one aspect of the present invention, the amount of polypeptide produced, for example, the amount of antibody produced, can be calculated by measuring the titer or the antibody secretion rate per cell. Specifically, the antibody titer in the medium when non-genetically modified vertebrate cells are cultured under the same conditions as the control can be measured, and the increase in productivity can be calculated from the increase in antibody production.

[0151] In one aspect of the present invention, the recombinant polypeptide of interest produced is isolated from the cell culture medium and optionally further processed by methods known in the art. For example, the polypeptide can be collected from the nutrient medium by conventional procedures, including, but not limited to, centrifugation, filtration, ultrafiltration, extraction, or precipitation. Further processing steps, such as purification steps, can be carried out by various procedures known in the art, including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, isoelectric focusing, protein A or protein G chromatography, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), or extraction. Furthermore, the isolated, purified polypeptide of interest can be further processed, for example, modified and / or formulated into a composition, e.g., a pharmaceutical composition.

[0152] [Example 1] Preparation of knockout cells [Culturing of CHO cells] A vial containing CHO cells (DXB11-derived) was thawed in a water bath at 37°C, and the contents were transferred to a centrifuge tube containing cell culture medium. The cells were then collected by centrifugation and seeded in a flask containing cell culture medium for shake culture. The cell culture after shake culture was subcultured in the cell culture medium, and the cell culture was maintained and subcultured on a 3-day / 4-day schedule. At this time, the cell culture was diluted to 2 x 10 cells in 20 mL of maintenance medium. ５ Culture was initiated at a seeding density of 1000 cells / mL. [Transfection of Cas9-guide RNA complex] 1) Cas9 protein and 2) a mixture of annealed guide RNA and tracrRNA specific to the EDC3 gene were mixed to prepare a Cas9-guide RNA complex. The prepared Cas9-guide RNA complex was transfected into the cells cultured above by electroporation, and then cultured at 37°C and 5% CO2 gas for 4 days with a shaking speed of 2.5 cm and 100 rpm.

[0153] [Single-cell cloning of transfectant pools and cell expansion] Single-cell cloning was performed from the transfectant pool using CellCelector™ Nanowell Plate H100-100 330K Microplate 24-well 100 μm nanowells uncoated (Sartorius). Cells were seeded into the nanowells, and photographs of the cells were taken at the time of seeding, and on days 1, 2, and 3. From the images, cells that were single cells at the time of seeding and showed proliferation by day 3 were selected and expanded to a 384-well plate on day 4 after seeding. Culture was continued for nine days, and on day 13, when the cells had sufficiently proliferated, the entire volume was expanded to a 96-well plate.

[0154] [Establishment of knockout cells] After 7 days of culture in a 96-well plate, the cells were uniformly subcultured at 1 / 10 of their original size in a 96-well plate, and genome extraction was performed from each of the remaining cells. Using the extracted genome, the genomic sequence near the designed guide RNA was amplified by PCR. The obtained DNA was used as a library for next-generation sequencing (NGS), and sequencing was performed using NextSeq™ 2000 (Illumina Inc.). The obtained sequence data was mapped to a reference sequence to confirm whether frameshifting due to genome editing had occurred. Only cells in which frameshifting was confirmed were picked to obtain knockout cells.

[0155] [Example 2] Preparation of expression vectors For IgG1 antibodies (antibody A: SOF10, antibody B: RAY121), antibody gene expression units were prepared by attaching a CAG promoter upstream of each of the heavy chain (H chain) and light chain (L chain) genes of each antibody and further attaching a rabbit beta globin (rBG) polyA signal sequence downstream thereof.

[0156] The constructed antibody gene expression units were ligated in the order of L, L, H, L, H, L or L, L, H, L, L, H, and further ligated to a vector plasmid for introduction into CHO cells incorporating a DHFR gene to prepare a plasmid with an H:L ratio of 2:4, consisting of two copies of the antibody gene H chain and four copies of the L chain (Figures 1 and 2).

[0157] [Example 2] Production of antibody-producing cells and antibodies The plasmid prepared in Example 1 was introduced by electroporation into the knockout cells prepared by the method described above and into unmodified CHO cell DXB11-derived cells, respectively, to integrate two copies of the plasmid into the same specific position in the CHO cell genome. The cells were then cultured in the presence of 400 mg / mL hygromycin B to obtain a cell pool into which the expression plasmid had been introduced, and the culture was evaluated.

[0158] Using the obtained plasmid-transfected cell pools, antibody titers were compared in fed-batch culture using ambr15. Culture was carried out under conditions of an initial culture volume of 12 mL, a culture temperature of 37°C, and an agitation speed of 800 rpm. The antibody concentration in the medium on day 14 after the start of culture was measured as the titer. The titer was determined by measuring the binding of antibodies present in the culture supernatant to a Protein A sensor using a biomolecular interaction analysis system (Octet Systems, Sartorius).

[0159] Culture was performed using a cell pool producing antibody A (samples 1 to 8) and a cell pool producing antibody B (samples 9 to 11). Different clonal strains of EDC3 knockout cells were used for samples 1 to 7, 9, and 10, and wild-type CHO cells were used for samples 8 and 11, and the antibody titer and secretion rate per cell for each sample were confirmed. As a result, when EDC3 knockout cells for antibody A and antibody B were used, the antibody titer and secretion rate per cell were higher than when wild-type cells were used.

[0160]

[0161] Here, the antibody secretion rate (ng / cells / day) in Table 1 is the value obtained by dividing the titer by the integral viable cell density (IVCD). Here, IVCD represents the sum of the viable cell densities during the culture period. It was calculated from the viable cell densities at each time point (days 0, 3, 5, 7, 10, 12, and 14). The formula for calculating IVCD is shown below. The average of the viable cell densities (VCD) at adjacent time points was calculated assuming that the period continued (t: period (days)).

[0162]

Claims

1. A genetically engineered vertebrate cell for use in the production of a recombinant polypeptide, wherein the vertebrate cell has been modified to reduce or eliminate functional expression of an mRNA decapping activator gene.

2. The vertebrate cell of claim 1, in which functional expression of the mRNA decapping activator gene is reduced or eliminated by gene knockout, gene modification, gene deletion, gene silencing, or a combination thereof.

3. A vertebrate cell according to claim 1 or 2, wherein said vertebrate cell contains at least one heterologous polynucleotide encoding a recombinant polypeptide.

4. A vertebrate cell according to any one of claims 1 to 3, wherein the genome of said vertebrate cell has been modified to reduce or eliminate expression of an mRNA decapping activator.

5. The vertebrate cell of any one of claims 1 to 4, wherein the vertebrate cell comprises at least one modification in at least one copy or all copies of the mRNA decapping activator gene.

6. A vertebrate cell described in any one of claims 1 to 5, wherein the amino acid sequence of the modified endogenous mRNA decapping activator has at least 90% sequence identity to the amino acid sequence shown in SEQ ID NOs: 1 to 4 as the mRNA decapping activator reference protein.

7. An isolated vertebrate cell according to any one of claims 1 to 6, wherein the mRNA decapping activator is PAT1, SCD6, EDC3, or DHH1.

8. A vertebrate cell according to any one of claims 1 to 7, wherein the mRNA decapping activator is EDC3.

9. The vertebrate cell according to any one of claims 1 to 8, wherein the vertebrate cell is a mammalian cell.

10. The vertebrate cell according to any one of claims 1 to 9, wherein the vertebrate cell is a CHO cell.

11. The vertebrate cell of any one of claims 1 to 10, wherein the recombinant polypeptide is an antibody, an IgG antibody, an IgG1 antibody, or a bispecific antibody.

12. A vertebrate cell according to any one of claims 1 to 11, wherein the productivity of the vertebrate cell in which functional expression of the mRNA decapping activator gene has been reduced or eliminated is increased by at least 10% compared to a vertebrate cell having the same genotype except for the modification.

13. A method for increasing recombinant polypeptide productivity in vertebrate cells, comprising preparing recombinant vertebrate cells that have been modified to reduce or eliminate functional expression of an mRNA decapping activator gene in the vertebrate cells, wherein the productivity of the recombinant polypeptide is increased when cultured under the same conditions compared to vertebrate cells having the same genotype except for the modification.

14. A method for producing a vertebrate cell according to any one of claims 1 to 12, comprising: preparing a recombinant vertebrate cell modified so that functional expression of the mRNA decapping activator gene is reduced or eliminated in the vertebrate cell; and introducing a polynucleotide encoding a recombinant polypeptide into the vertebrate cell, wherein the recombinant polypeptide is expressed in the vertebrate cell.

15. A method for producing a recombinant polypeptide, comprising: (a) culturing a vertebrate cell according to any one of claims 1 to 12 under conditions that allow expression and secretion of the recombinant polypeptide into a cell culture medium; and (b) isolating the recombinant polypeptide from the cell culture medium.

16. The method of claim 15, further comprising (c) processing the isolated recombinant polypeptide.

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