Targeted integration of nucleic acids

Abstract

The disclosed subject matter relates to targeted integration (TI) host cells suitable for expressing recombinant proteins, wherein those TI host cells have been supertransfected such that exogenous nucleic acid is randomly integrated (RI) into their genome, and to methods of producing and using said supertransfected TI host cells.

Description

[0001] Cross-references to related patent applications

[0002] This application claims priority to U.S. Provisional Patent Application Serial No. 62 / 784,019 filed on December 21, 2018, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] The disclosed subject matter relates to targeted integration (TI) host cells suitable for expressing recombinant proteins, wherein those TI host cells have been supertransfected such that exogenous nucleic acid is randomly integrated (RI) into their genome, and to methods of producing and using said supertransfected TI host cells.

[0004] Sequence Listing

[0005] This specification refers to the sequence listing (.txt file named "00B2060866SEQ.txt" submitted in electronic form on December 19, 2019). The 00B2060866SEQ.txt file was generated on December 10, 2019 and is 1,251,200 bytes in size. The entire contents of the sequence listing are hereby incorporated by reference. Background Art

[0006] Due to the rapid development of cell biology and immunology, there is an increasing demand for the development of new therapeutic recombinant proteins for various diseases, including cancer, cardiovascular disease, and metabolic diseases. These candidate biopharmaceuticals are usually produced by commercial cell lines that can express the target protein. For example, Chinese hamster ovary (CHO) cells have been widely used to produce monoclonal antibodies.

[0007] The conventional strategy for developing commercial cell lines involves random integration of nucleotide sequences encoding target polypeptides, and then selecting and isolating cell lines that produce target polypeptides. However, this method has several disadvantages. First, such integration is not only a rare event, but also in view of the randomness of nucleotide sequence integration, these rare events may lead to a variety of gene expression and cell growth phenotypes. Such changes are called "position effect changes", which are at least partially derived from the complex gene regulatory network present in the eukaryotic cell genome and the accessibility of certain genomic sites for integration and gene expression. Secondly, random integration strategies are generally unable to provide control over the number of gene copies integrated in the host cell genome. In fact, gene amplification methods are generally used to obtain high-yield cells. However, such gene amplification may lead to unwanted cell phenotypes, such as unstable cell growth and / or product expression. Third, due to the inherent heterogeneity of the integrated loci in the random integration process, the process of screening thousands of clones after transfection to isolate the cell line of the target polypeptide showing the desired expression level is time-consuming and laborious. Even after isolating such cell lines, the stable expression of the target polypeptide cannot be guaranteed, and further screening may be required to obtain a stable commercial cell line. Finally, polypeptides produced by randomly integrated cell lines exhibited a high degree of sequence variation, which may be due in part to the mutagenicity of the selection agents used to select for polypeptides of interest that express high levels. Summary of the invention

[0008] The subject matter of the present disclosure relates, at least in part, to targeted integration (TI) host cells suitable for expressing recombinant proteins, wherein the TI host cells have been supertransfected such that exogenous nucleic acid encoding is randomly integrated (RI) into their genome, and methods of producing and using the supertransfected TI host cells. The subject matter of the present disclosure not only provides host cell TI sites with high yields, but also provides the following novel methods, which introduce multiple target sequences into a single TI locus in a host cell by recombinase-mediated cassette exchange (RMCE), and as outlined herein, by subjecting the cells to supertransfection such that exogenous nucleic acid encoding is randomly integrated (RI) into the TI host cell genome to achieve increased expression of the target sequence.

[0009] In certain embodiments, the supertransfected TI host cell initially (before undergoing RI) or subsequently (after undergoing RI) will contain an exogenous nucleotide sequence integrated at an integration site within a locus in the genome of the host cell, wherein the locus is at least about 90% homologous to a sequence selected from SEQ ID No. 1 to SEQ ID No. 7. In certain embodiments, the nucleotide sequence immediately adjacent to the 5' end of the integrated exogenous nucleotide sequence is selected from the group consisting of sequences that are at least about 90% homologous to nucleotides 41190-45269 of NW_006874047.1, nucleotides 63590-207911 of NW_006884592.1, nucleotides 253831-491909 of NW_006881296.1, nucleotides 69303-79768 of NW_003616412.1, nucleotides 293481-315265 of NW_003615063.1, nucleotides 2650443-2662054 of NW_006882936.1, and nucleotides 82214-97705 of NW_003615411.1. In certain embodiments, the nucleotide sequence immediately adjacent to the 3' end of the integrated exogenous nucleotide sequence is selected from the group consisting of sequences that are at least about 90% homologous to nucleotides 45270-45490 of NW_006874047.1, nucleotides 207912-792374 of NW_006884592.1, nucleotides 491910-667813 of NW_006881296.1, nucleotides 79769-100059 of NW_003616412.1, nucleotides 315266-362442 of NW_003615063.1, nucleotides 2662055-2701768 of NW_006882936.1, or nucleotides 97706-105117 of NW_003615411.1. In certain embodiments, the integrated exogenous nucleotide sequence is operably linked to a nucleotide sequence selected from the group consisting of a sequence that is at least about 90% homologous to a sequence of SEQ ID No. 1 to SEQ ID No. 7.

[0010] In certain embodiments, the supertransfected TI host cells initially (before undergoing RI) or subsequently (after undergoing RI) will contain an exogenous nucleotide sequence integrated at an integration site within an endogenous gene, the endogenous gene being selected from the group consisting of LOC107977062 (SEQ ID NO.1), LOC100768845 (SEQ ID NO.2), ITPR2 (SEQ ID NO.3), ERE67000.1 (SEQ ID NO.4), UBAP2 (SEQ ID NO.5), MTMR2 (SEQ ID NO.6), XP_003512331.2 (SEQ ID NO.7), and sequences at least about 90% homologous thereto. In certain embodiments, the exogenous nucleotide sequence is integrated at an integration site operably linked to an endogenous gene selected from the group consisting of LOC107977062 (SEQ ID NO.1), LOC100768845 (SEQ ID NO.2), ITPR2 (SEQ ID NO.3), ERE67000.1 (SEQ ID NO.4), UBAP2 (SEQ ID NO.5), MTMR2 (SEQ ID NO.6), XP_003512331.2 (SEQ ID NO.7), and sequences at least about 90% homologous thereto. In certain embodiments, the integrated exogenous nucleotide is flanked by a nucleotide sequence selected from the group consisting of: a sequence at least about 90% homologous to a sequence of SEQ ID No.1 to SEQ ID No.7. In certain embodiments, the exogenous nucleotide sequence is integrated at an integration site immediately adjacent to all or a portion of a sequence selected from the group consisting of: a sequence at least about 90% homologous to a sequence of SEQ ID No.1 to SEQ ID No.7.

[0011] In certain embodiments, the TI host cell is a mammalian host cell. In certain embodiments, the TI host cell is a hamster host cell, a human host cell, a rat host cell, or a mouse host cell. In certain embodiments, the TI host cell is a Chinese hamster ovary (CHO) host cell, a CHO K1 host cell, a CHO K1SV host cell, a DG44 host cell, a DUKXB-11 host cell, a CHOK1S host cell, or a CHO K1M host cell.

[0012] In certain embodiments, the host cell comprises an exogenous nucleotide sequence, wherein the integration of the sequence is targeted, and wherein the exogenous nucleotide sequence comprises one or more recombination recognition sequences (RRS). In certain embodiments, the exogenous nucleotide sequence comprises at least two RRS. RRS can be recognized by a recombinase (e.g., Cre recombinase, FLP recombinase, Bxb1 integrase or φC31 integrase). RRS can be selected from the group consisting of the following items: LoxP sequence, LoxP L3 sequence, LoxP 2L sequence, LoxFas sequence, Lox511 sequence, Lox2272 sequence, Lox2372 sequence, Lox5171 sequence, Loxm2 sequence, Lox71 sequence, Lox66 sequence, FRT sequence, Bxb1 attP sequence, Bxb1 attB sequence, φC31 attP sequence and φC31 attB sequence.

[0013] In certain embodiments, the exogenous nucleotide sequence integrated by targeted integration includes the first RRS and the second RRS and at least one selection marker between the first RRS and the second RRS. In certain embodiments, the exogenous nucleotide sequence integrated by targeted integration further includes the 3rd RRS, wherein the 3rd RRS is between the first RRS and the second RRS, and the 3rd RRS is different from the first RRS or the second RRS. In certain embodiments, the exogenous nucleotide sequence integrated by targeted integration includes the first RRS and the second RRS and the first selection marker between the first RRS and the second RRS. In certain embodiments, the exogenous nucleotide sequence integrated by targeted integration further includes the second selection marker, and the first selection marker is different from the second selection marker. In certain embodiments, the exogenous nucleotide sequence integrated by targeted integration may further include the third selection marker and internal ribosome entry site (IRES), wherein IRES is operably connected to the third selection marker. The third selection marker may be different from the first selection marker or the second selection marker. The selectable marker can be selected from the group consisting of aminoglycoside phosphotransferase (APH) (e.g., hygromycin phosphotransferase (HYG), neomycin and G418 APH), 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. The selection marker can also be selected from the group consisting of: green fluorescent protein (GFP) marker, enhanced GFP (eGFP) marker, synthetic GFP marker, yellow fluorescent protein (YFP) marker, enhanced YFP (eYFP) marker, cyan fluorescent protein (CFP) marker, mPlum marker, mCherry marker, tdTomato marker, mStrawberry marker, J-red marker, DsRed-monomer marker, mOrange marker, mKO marker, mCitrine marker, Venus marker, YPet marker, Emerald6 marker, CyPet marker, mCFPm marker, Cerulean marker and T-Sapphire marker. In some embodiments, the selection marker is selected from the group consisting of: green fluorescent protein (GFP) marker, enhanced GFP (eGFP) marker, synthetic GFP marker.

[0014] In certain embodiments, the exogenous nucleotide sequence integrated by targeted integration comprises at least one selection marker and at least one exogenous sequence of interest (SOI). In certain embodiments, the exogenous nucleotide sequence integrated by targeted integration comprises a first RRS and a second RRS and at least one selection marker and at least one exogenous SOI located between the first RRS and the second RRS. In certain embodiments, the exogenous nucleotide sequence integrated by targeted integration comprises a first RRS, a second RRS and a third RRS, at least one selection marker and at least one exogenous SOI located between the first RRS and the third RRS, and at least one exogenous SOI located between the third RRS and the second RRS. The target sequence can encode any target polypeptide, including but not limited to the examples listed herein. SOIs may be the same or different, for example, SOIs may include coding sequences for two chains of an antibody. In the case where two or more different polypeptides are expressed, the target sequence may include two polypeptides of various ratios, for example, when eight coding sequences encode two different polypeptides, the ratio includes but is not limited to 1:7, 2:6, etc. The SOI may encode a single chain antibody, an antibody light chain, an antibody heavy chain, a single chain Fv fragment (scFv), or an Fc fusion protein.

[0015] The subject matter of the present disclosure also provides a method for targeted integration of exogenous nucleic acids in a host cell to promote the expression of a polypeptide of interest, wherein the targeted integration is introduced before, after, or simultaneously with a randomly integrated exogenous nucleic acid encoding the same or different polypeptide of interest. In certain embodiments, such methods include targeted integration of exogenous nucleic acids in a host cell by recombinase-mediated recombination. In certain embodiments, such methods relate to a cell comprising an exogenous nucleotide sequence integrated into a site within an endogenous gene, the endogenous gene being selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, XP_003512331.2, and a sequence at least 90% homologous thereto. In certain embodiments, such methods relate to a cell comprising an exogenous nucleotide sequence integrated into a locus of the genome of the host cell, wherein the locus comprises a nucleotide sequence at least about 90% homologous to a sequence selected from SEQ ID No. 1 to SEQ ID No. 7.

[0016] In certain embodiments, the present disclosure provides a method for preparing a TI host cell expressing a polypeptide of interest, wherein targeted integration of a sequence encoding the polypeptide of interest is introduced before, after, or simultaneously with randomly integrated exogenous nucleic acids encoding the same or different polypeptide of interest, and wherein the method further comprises: a) providing a TI host cell comprising an exogenous nucleotide sequence integrated at a site within a locus of the genome of the TI host cell, wherein the locus is at least about 90% homologous to a sequence selected from SEQ ID No. 1 to SEQ ID No. 7, wherein the exogenous nucleotide sequence comprises two RRSs flanked by at least one first selection marker; b) introducing a vector into the cell provided in a), wherein the vector comprises two RRSs that match the two RRSs on the integrated exogenous nucleotide sequence and are flanked by at least one exogenous SOI and at least one second selection marker; c) introducing a recombinase, wherein the recombinase recognizes the RRS; and d) selecting TI cells expressing the second selection marker, thereby isolating TI host cells expressing the polypeptide of interest.

[0017] In certain embodiments, the present disclosure provides a method for preparing a TI host cell expressing a first polypeptide of interest and a second polypeptide of interest (wherein the first polypeptide and the second polypeptide may be the same or different), wherein the targeted integration of a sequence encoding the polypeptide of interest is introduced before, after or simultaneously with the randomly integrated exogenous nucleic acid encoding the same or different polypeptide of interest, and wherein the method further comprises: a) providing a TI host cell, the TI host cell comprising an exogenous nucleotide sequence integrated into a locus of the genome of the host cell, wherein the locus is at least about 90% homologous to a sequence selected from SEQ ID No. 1 to SEQ ID No. 7, wherein the exogenous nucleotide sequence comprises a first RRS and a second RRS flanked by at least one first selection marker and a third RRS located between the first RRS and the second RRS, wherein the first RRS, the second RRS and the third RRS have heterologous specificity, that is, the first RRS, the second RRS and the third RRS are not matching RRSs, and therefore any two of the first RRS, the second RRS and the third RRS will not be suitable for recombinase-mediated recombination; b) introducing a first vector into the cell provided in a), the first vector comprising two RRSs, and the two RRSs are integrated c) introducing into the cells provided in a) a second vector, the second vector comprising two RRSs, which match the second RRS and the third RRS on the integrated exogenous nucleotide sequence and are flanked by at least one second exogenous SOI; d) introducing one or more recombinases, wherein the one or more recombinases recognize the RRSs; and e) selecting TI cells expressing the second selection marker, thereby isolating TI host cells expressing the first polypeptide of interest and the second polypeptide of interest.

[0018] In certain embodiments, the present disclosure provides a method for expressing a polypeptide of interest, wherein targeted integration of a sequence encoding the polypeptide of interest is introduced before, after, or simultaneously with randomly integrated exogenous nucleic acid encoding the same or different polypeptide of interest, and wherein the method further comprises: a) providing a host cell comprising at least one exogenous SOI flanked by two RRSs and at least one selection marker integrated into a locus in the genome of the host cell, wherein the locus is at least about 90% homologous to a sequence selected from SEQ ID No. 1 to SEQ ID No. 7; and b) culturing the cells in a) under conditions suitable for expression of the SOI, and recovering the polypeptide of interest therefrom.

[0019] In certain embodiments, the present disclosure provides a method for expressing a polypeptide of interest, wherein targeted integration of a sequence encoding the polypeptide of interest is introduced before, after, or simultaneously with randomly integrated exogenous nucleic acids encoding the same or different polypeptide of interest, wherein the method further comprises: a) providing a host cell comprising at least two exogenous SOIs and at least one selection marker integrated into a locus of the genome of the host cell, wherein the locus is at least about 90% homologous to a sequence selected from SEQ ID No. 1 to SEQ ID No. 7, wherein the at least one exogenous SOI and one selection marker are flanked by a first RRS and a third RRS, and the at least one exogenous SOI is flanked by a second RRS and a third RRS; and b) culturing the cells in a) under conditions suitable for expression of the SOI, and recovering the polypeptide of interest therefrom.

[0020] The subject matter of the present disclosure also provides compositions and methods for producing a polypeptide of interest by using a TI host cell, wherein the targeted integration of a sequence encoding a polypeptide of interest is introduced before, after, or simultaneously with a randomly integrated exogenous nucleic acid encoding the same or different polypeptide of interest. Such compositions and methods include, but are not limited to, polynucleotides and vectors that facilitate integration of exogenous nucleotide sequences in TI host cells, as well as TI host cells and compositions comprising exogenous nucleotide sequences, and methods of use thereof. In certain embodiments, the vector may comprise a nucleotide sequence that is at least 50% homologous to a sequence selected from SEQ ID No. 1 to SEQ ID No. 7 flanked by a DNA cassette, wherein the DNA cassette comprises at least one selection marker flanked by two RRSs and at least one exogenous SOI. The vector may be selected from the group consisting of an adenoviral vector, an adeno-associated viral vector, a lentiviral vector, a retroviral vector, an integrating phage vector, a non-viral vector, a transposon and / or a transposase vector, an integrase substrate, and a plasmid.

[0021] In certain embodiments, the methods of the present disclosure include targeted integration of an exogenous nucleic acid encoding a polypeptide of interest into a host cell by homologous recombination, homology-mediated repair (HDR) and / or non-homologous end joining (NHEJ). In certain embodiments, such methods involve integrating an exogenous nucleic acid encoding a polypeptide of interest into a cell at a site within an endogenous gene, the endogenous gene being selected from the group consisting of LOC107977062 (SEQ ID NO.1), LOC100768845 (SEQ ID NO.2), ITPR2 (SEQ ID NO.3), ERE67000.1 (SEQ ID NO.4), UBAP2 (SEQ ID NO.5), MTMR2 (SEQ ID NO.6), XP_003512331.2 (SEQ ID NO.7), and a sequence at least about 90% homologous thereto. In certain embodiments, such methods involve integrating an exogenous nucleic acid encoding a polypeptide of interest into a cell at a site within a locus in the genome of the host cell, wherein the locus comprises a nucleotide sequence that is at least about 90% homologous to a sequence selected from SEQ ID No. 1 to SEQ ID No. 7.

[0022] In certain embodiments, the present disclosure provides a method for preparing a TI host cell expressing a polypeptide of interest, wherein the targeted integration of a sequence encoding the polypeptide of interest is introduced before, after, or simultaneously with the randomly integrated exogenous nucleic acid encoding the same or different polypeptide of interest, and wherein the method further comprises: a) providing a host cell comprising a locus of the genome of the host cell, wherein the locus is at least about 90% homologous to a sequence selected from SEQ ID No. 1 to SEQ ID No. 7; b) introducing a vector into the host cell, wherein the vector comprises a nucleotide sequence at least 50% homologous to a sequence selected from SEQ ID No. 1 to SEQ ID No. 7 flanked by a DNA cassette, wherein the DNA cassette comprises at least one selection marker flanked by two RRSs and at least one exogenous SOI; and c) selecting for the selection marker to isolate a TI host cell comprising the SOI integrated within the locus of the genome and expressing the polypeptide of interest. In certain embodiments, such methods involve using an exogenous nuclease to promote targeted integration. The exogenous nuclease can be selected from the group consisting of zinc finger nucleases (ZFNs), ZFN dimers, transcription activator-like effector nucleases (TALENs), TAL effector domain fusion proteins, RNA-guided DNA endonucleases, engineered large-range nucleases, and clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) endonucleases.

[0023] In certain embodiments, the present disclosure provides a method for preparing a TI host cell that expresses a polypeptide of interest, wherein targeted integration of a sequence encoding the polypeptide of interest is introduced before, after, or simultaneously with randomly integrated exogenous nucleic acids encoding the same or different polypeptide of interest, and wherein the method further comprises: a) providing a TI host cell comprising at least one exogenous nucleotide sequence integrated into a site within one or more loci of the genome of the TI host cell, wherein the one or more loci are at least about 90% homologous to a sequence selected from SEQ ID No. 1 to SEQ ID No. 7, wherein the exogenous nucleotide sequence comprises one or more RRSs; b) introducing into the cell provided in a) a vector comprising one or more RRSs that match the one or more RRSs on the integrated exogenous nucleotide sequence and are flanked by at least one exogenous SOI that is operably linked to a regulatable promoter; c) introducing a recombinase or a nucleic acid encoding a recombinase, wherein the recombinase recognizes the RRS; and d) selecting a TI cell that expresses the exogenous SOI in the presence of an inducer, thereby isolating a TI host cell that expresses the polypeptide of interest.

[0024] In certain embodiments, the present disclosure provides a method for expressing a polypeptide of interest, wherein targeted integration of a sequence encoding the polypeptide of interest is introduced before, after, or simultaneously with randomly integrated exogenous nucleic acid encoding the same or different polypeptide of interest, and wherein the method further comprises: a) providing a host cell comprising at least one exogenous SOI integrated into a locus in the genome of the host cell, flanked by two RRSs and operably linked to a regulatable promoter, wherein the locus is at least about 90% homologous to a sequence selected from SEQ ID No. 1 to SEQ ID No. 7; and b) culturing the cell under conditions suitable for expression of the SOI, and recovering the polypeptide of interest therefrom.

[0025] In certain embodiments, the present disclosure provides a method for preparing a TI host cell expressing a first polypeptide of interest and a second polypeptide of interest (wherein the first polypeptide and the second polypeptide may be the same or different), wherein the targeted integration of a sequence encoding the polypeptide of interest is introduced before, after or simultaneously with the randomly integrated exogenous nucleic acid encoding the same or different polypeptide of interest, and wherein the method further comprises: a) providing a TI host cell, the TI host cell comprising an exogenous nucleotide sequence integrated into a locus of the genome of the host cell, wherein the locus is at least about 90% homologous to a sequence selected from SEQ ID No. 1 to SEQ ID No. 7, wherein the exogenous nucleotide sequence comprises a first RRS and a second RRS and a third RRS located between the first RRS and the second RRS, wherein the first RRS, the second RRS and the third RRS have heterologous specificity, that is, the first RRS, the second RRS and the third RRS are not matched RRSs, and therefore any two of the first RRS, the second RRS and the third RRS will not be suitable for recombinase-mediated recombination; b) introducing a first vector into the cell provided in a), the first vector comprising two RRSs, which match the first RRS and the third RRS on the integrated exogenous nucleotide sequence and and flanked by at least one first exogenous SOI operably linked to a regulatable promoter; c) introducing into the cells provided in a) a second vector comprising two RRSs, which match the second RRS and the third RRS on the integrated exogenous nucleotide sequence and flanked by at least one second exogenous SOI operably linked to a regulatable promoter; d) introducing one or more recombinases or one or more nucleic acids encoding one or more recombinases, wherein the one or more recombinases recognize the RRSs; and e) selecting TI cells that express the exogenous SOI in the presence of an inducer, thereby isolating TI host cells that express the first polypeptide of interest and the second polypeptide of interest.

[0026] In certain embodiments, the present disclosure provides a host cell capable of expressing a polypeptide of interest, the host cell comprising: a) a targeted integration exogenous nucleic acid sequence of interest (SOI) encoding a first polypeptide of interest flanked by two recombination recognition sequences (RRS) and a first selection marker, wherein the targeted integration exogenous SOI is integrated into a targeted locus of the genome of the host cell; and b) a randomly integrated exogenous nucleic acid SOI encoding a second polypeptide of interest and a second selection marker, wherein the randomly integrated SOI is integrated at least once into the genome of the host cell. In certain embodiments, wherein the first polypeptide of interest and the second polypeptide of interest may be the same. In certain embodiments, the first selection marker and the second selection marker may be the same. In certain embodiments, the host cell may comprise one to ten randomly integrated exogenous nucleic acid SOIs. In certain embodiments, the targeted locus may be at least about 90% homologous to a sequence selected from SEQ ID No.1 to SEQ ID No.7. In certain embodiments, the host cell of the present disclosure may further comprise a second targeted integration exogenous nucleic acid SOI encoding a second polypeptide of interest integrated into the targeted locus of the genome of the host cell and a second selection marker, wherein the first targeted integration exogenous nucleic acid SOI and the first selection marker may be flanked by a first RRS and a third RRS, and the second targeted exogenous SOI and the second selection marker may be flanked by a second RRS and a third RRS. In certain embodiments, the polypeptide of interest may be selected from the group consisting of: a single-chain antibody, an antibody light chain, an antibody heavy chain, a single-chain Fv fragment (scFv), and an Fc fusion protein. In certain embodiments, the host cell may be a mammalian host cell. In certain embodiments, the host cell may be a hamster host cell, a human host cell, a rat host cell, or a mouse host cell. In certain embodiments, the host cell may be a CHO host cell, a CHO K1 host cell, a CHO K1SV host cell, a DG44 host cell, a DUKXB-11 host cell, a CHOK1S host cell, or a CHO K1M host cell. In certain embodiments, the targeted integration of SOI and the selection marker can be promoted by an exogenous nuclease. In certain embodiments, the exogenous nuclease can be selected from the group consisting of zinc finger nucleases (ZFNs), ZFN dimers, transcription activator-like effector nucleases (TALENs), TAL effector domain fusion proteins, RNA-guided DNA endonucleases, engineered meganucleases, and clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) endonucleases.

[0027] The subject matter of the present disclosure also provides a method for expressing a polypeptide of interest. In certain embodiments, the present disclosure provides a method for expressing a polypeptide of interest, the method comprising: a) providing a host cell comprising an exogenous nucleotide sequence integrated at a targeted locus of the genome of the host cell, wherein the exogenous nucleotide sequence comprises two RRS flanking a first selection marker; b) introducing a nucleic acid into the cell provided in (a), the nucleic acid comprising two RRSs, which match the two RRSs of the integrated exogenous nucleotide sequence and flank the first exogenous SOI encoding the first polypeptide of interest and the second selection marker; c) introducing a recombinase or a nucleic acid encoding a recombinase, wherein the recombinase recognizes the RRS; d) selecting a cell expressing the second selection marker; e) introducing a second exogenous SOI encoding the second polypeptide of interest and a third selection marker into the genome of the host cell by random integration; f) selecting a cell expressing the third selection marker; and g) culturing the host cell under conditions sufficient to express the first polypeptide of interest and the second polypeptide of interest. In certain embodiments, such methods may further include recovering the first polypeptide of interest and the second polypeptide of interest from the host cell culture. In certain embodiments, the first polypeptide of interest and the second polypeptide of interest may be the same. In certain embodiments, the targeted locus may be at least about 90% homologous to a sequence selected from SEQ ID No. 1 to SEQ ID No. 7. In certain embodiments, the first polypeptide of interest and the second polypeptide of interest may be selected from the group consisting of: a single-chain antibody, an antibody light chain, an antibody heavy chain, a single-chain Fv fragment (scFv), and an Fc fusion protein. In certain embodiments, the host cell may be a mammalian host cell. In certain embodiments, the host cell may be a hamster host cell, a human host cell, a rat host cell, or a mouse host cell. In certain embodiments, the host cell may be a CHO host cell, a CHO K1 host cell, a CHO K1SV host cell, a DG44 host cell, a DUKXB-11 host cell, a CHOK1S host cell, or a CHO K1M host cell. In certain embodiments, the targeted integration of any of the SOIs may be promoted by an exogenous nuclease. In certain embodiments, the exogenous nuclease may be selected from the group consisting of zinc finger nucleases (ZFNs), ZFN dimers, transcription activator-like effector nucleases (TALENs), TAL effector domain fusion proteins, RNA-guided DNA endonucleases, engineered meganucleases, and clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) endonucleases. In certain embodiments, the expression of SOI may be controlled by a regulatable promoter. In certain embodiments, the regulatable promoter may be selected from the group consisting of SV40 and CMV promoters. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1Figure 2 is a flow chart showing the steps of genome-wide screening for identifying CHO TI loci that allow stable and high-level expression of antibodies.

[0029] Figures 2A and 2B show two dual selection schemes for generating targeted antibody expression populations. The TI host generated by transposase is shown in Figure 2A, and the TI host generated by random recombination is shown in Figure 2B.

[0030] Figure 3 Shown are the productivity of the first few clones generated from two specific TI hosts.

[0031] Figure 4 shows a dual plasmid RMCE strategy involving two independent RMCEs performed simultaneously using three RRS sites. One vector (front) contains RRS1, the first SOI and the promoter (P) followed by the start codon (ATG) and RRS3. Another vector (rear) contains RRS3 fused to the coding sequence of the marker without the start codon (ATG) as well as SOI 2 and RRS2. Only when both plasmids are correctly targeted, the complete expression cassette of the selection marker is assembled, thereby making the cell resistant to selection.

[0032] Figure 5 Results are shown for the expression of antibodies D, E, F, G, and J using either single or dual vector RMCE. FACS, genomic PCR, and gene copy analysis were evaluated to confirm that both plasmid cassettes were correctly targeted to the TI site. In dual plasmid RMCE, more total HC and LC copies were targeted to the TI site compared to single plasmid RMCE.

[0033] Figure 6 Titers of six molecules are shown.

[0034] Figure 7 Results are shown for complex mAb formats expressed using dual vector RMCE. Production of two bispecific molecules (each requiring four different chains (two HCs and two LCs)) and % bispecific molecule formation were determined. In both cases, cell lines above 1.5 g / L were developed with bispecific content above 80%.

[0035] Figure 8 Titers at day 14 are shown for the four bispecific molecules.

[0036] Fig.9A and Fig. 9B Shown are the generation of cell lines expressing mAb-I or mAb-II in the RTI system. Fig.9A shows histological results of cell line development, and Fig. 9B A schematic diagram of the RTI cell line development (CLD) process is shown.

[0037] Fig.10 A to Fig.10 C shows the titer and specific productivity results for cell lines expressing mAb-I or mAb-II. Fig.10 A shows the production end titer, Fig.10 B shows the production side IVCC, and Fig.10 C shows the production side productivity.

[0038] Fig.11 A and Fig.11 B shows the expression levels of HC and LC mRNA of cell lines expressing mAb-I and mAb-II. Fig.11 A shows the expression level of HC mRNA, and Fig.11 B shows the expression level of HL mRNA.

[0039] Fig.12 A to Fig.12 D shows the intracellular levels of HC, HL and BiP molecules in cell lines expressing mAb-I or mAb-II. Fig.12 A Schematic diagram showing the effects of cycloheximide and Dox on the RTI system. Fig.12 B shows the intracellular levels of HC, LC and BiP as well as the levels of HC and LC in the supernatants of cell lines expressing mAb-A or mAb-B. Fig.12 C shows the intracellular levels of HC, LC and BiP in cell lines expressing mAb-A or mAb-B after overnight CHX treatment. Fig.12 C shows the intracellular levels of HC, LC and BiP in cell lines expressing mAb-A or mAb-B after removal of doxycycline from the culture medium.

[0040] Fig.13 A to Fig.13 D shows the correlation between BiP intracellular accumulation and mAb-I LC expression. Fig.13 A shows the production end titer, Fig.13 B shows the accumulation of HC, LC and BiP. Fig.13 C shows a schematic diagram of the strand exchange experiment. Fig.13 D shows the intracellular levels of HC, LC and BiP of RTI libraries expressing mAb-I, mAb-II or strand-swapped versions of these molecules.

[0041] Fig.14 A to Fig.14 C shows the contribution of mAb-A HC and HL to the library expression of this molecule. Fig.14 A shows the production end titer, Fig.13 B shows the production side IVCC, and Fig.14 C shows the production side productivity.

[0042] Fig.15 A to Fig.15 B shows that the CDR regions of LC and HC may lead to poor expression. Fig.15 A shows a schematic diagram of mAb-I and mAb-II LC subunits. Fig.15 B shows a schematic diagram of the HC region of mAb-I and mAb-II.

[0043] Fig.16 A to Fig.16 C shows binding of HC using two point mutations in the HC constant region A The result of expression. Fig.16 A shows production end titers for various chain swaps between mAb-I and mAb-II HC and LC subunits. Fig.16 B shows the intracellular levels of HC, LC and BiP of various RTI pools. Fig.16 C shows a schematic diagram of the RTI system used as a diagnostic tool for low protein expression.

[0044] Fig.17 Production using two different cell culture methods is shown.

[0045] Fig.18 A and Fig.18 D shows the titer and specific productivity results of the RMCE pool of three mAbs. Fig.18 A shows the titers at day 14 comparing the RMCE pools of three mAbs in HL and HLL configurations. Fig.18 B shows the specific productivity (Qp) at day 14 comparing the RMCE pools of three mAbs in HL and HLL configurations. Fig.18 C is shown by Fig.18 Titers of clones generated from library A on day 14. Fig.18 D is shown by Fig.18 The clones generated from the library of A were at Qp on day 14. Fig.18 C and 18D, six clones of each configuration were tested.

[0046] Fig.19 A to Fig.19 F shows the comparison of titer, Qp and growth (expressed as the integrated viable cell concentration (IVCC)), mRNA expression and protein expression of the RMCE pool of mAb Y of three different plasmid configurations. Fig.19 A. Fig.19 B and Fig.19C shows the titer, Qp and growth (expressed as the integral of viable cell concentration (IVCC)) on day 14, respectively. Each data bar represents data from a single library. Seed series mRNA expression and intracellular antibody protein expression from the library in 19A. The mRNA expression of heavy and light chains was normalized to the HLL-HL configuration; each data bar is the average of four technical replicates (error bars are standard deviations). The mRNA expression of heavy and light chains was normalized to the HLL-HL configuration. The intracellular heavy and light chain protein expression of all libraries was quantitatively analyzed by Western blotting and normalized to the HLL-HL configuration, as shown in Figure 1. Fig.19 E; each data bar is the average of three technical replicates (error bars are standard deviations). Fig.19 Representative Western blots of the data shown in E Fig.19 As shown in F.

[0047] Fig. 20 A to Fig. 20 F shows the titer, Qp and growth (expressed by the integral of viable cell concentration (IVCC)) of monoclones expressing mAb Y or mAb III generated from RMCE pools with three different configurations. Fig. 20 A shows the titer of monoclones expressing mAb Y on day 14. Fig. 20 B shows the Qp of the monoclone expressing mAb Y at day 14. Fig. 20 C shows IVCC at day 14 of clones expressing mAb Y. Fig. 20 D shows the titer of monoclones expressing mAb III on day 14. Fig. 20 E shows the Qp of the monoclone expressing mAb III at day 14. Fig. 20 F shows IVCC at day 14 of clones expressing mAb III.

[0048] Fig.21 A to Fig.21 D shows the expression level, titer and Qp of RMCE libraries of various configurations. Fig.21 A shows a schematic diagram of transfection of one heavy chain and one light chain plasmid to evaluate the effect of plasmid position on chain expression. Antibody heavy chain (abbreviated as H) and light chain (abbreviated as L) are expressed on the same plasmid or separated between the front and back plasmids. L3, Loxfas and 2L sites are marked, as is the pac puromycin resistance gene in the back plasmid. Fig.23 B shows the Fig.21 Seed series mRNA expression of heavy and light chains of the library with the DNA configuration outlined in A. Fig.21 C shows the titer of RMCE libraries with various configurations on day 14. Fig.21 D shows the Qp at day 14 with various configurations. Fig.21E shows the heavy and light chain DNA copy numbers of the library.

[0049] Fig. 22 A to Fig. 22 D shows the effect of HC:LC ratio on the percentage of high molecular weight species (HMWS) of mAbs regardless of TI site. Fig. 22 A shows the titer of antibody S on host 7 cells at day 14. Fig. 22 B shows the percentage of high molecular weight species (HMWS) of antibody S on host 7 cells. Fig. 22 C shows the titer of antibody S on host 4 cells at day 14. Fig. 22 D shows the percentage of high molecular weight species (HMWS) of antibody S on host 4 cells.

[0050] Fig.23 A and Fig.23 B shows the effect of HC:LC ratio on productivity and high molecular weight species (HMWS) of Antibody IV. Fig.23 A shows the titer of antibody IV on host 4 cells at day 14. Fig.23 D shows the percentage of high molecular weight species (HMWS) of antibody IV on host 4 cells.

[0051] Fig.24 A and Fig.24 B shows the effect of HC:LC ratio on the productivity and high molecular weight species (HMWS) of antibody VI in dual-site TI host cells. Fig.24 A shows the titer of antibody VI on day 14 on dual-site TI host cells. Fig.24 B shows the percentage of high molecular weight species (HMWS) of antibody VI on dual site TI host cells.

[0052] Fig.25 Two clones are shown with titers of 8.4 g / L and 8.7 g / L, respectively, and containing 7.4% and 5.6% aggregates, respectively.

[0053] Fig.26 The titers of antibody Z on day 14 are shown in different tunable targeted integration cell lines.

[0054] Fig. 27 The specific productivity of antibody Z in different tunable targeted integration cell lines is shown.

[0055] Fig.28 A to Fig.28 D shows titer, Qp and growth (expressed as the integrated viable cell concentration (IVCC)) for mAb Z on supertransfected and mock-transfected clones. Fig.28A shows the titers of supertransfected and mock transfected clones at day 14 generated by mAb Z expressing HLL-HL TI clone. Fig.28 B shows Qp at day 14. Fig.28 C shows IVCC on day 14. Fig.28 D shows the heavy and light chain DNA copy numbers of four supertransfected clones and four mock-transfected clones generated from HLL-HLHL and HLL-HL parental clones.

[0056] Fig.29 A to Fig.29 C shows the titer of tunable supertransfection of each clone of antibody Z. Fig.29 A shows the titer of regulatable expression of antibody Z on days 10 and 13. Fig.29 B shows a comparison of the titers of the selected clones from two different production methods. Fig.29 C shows the titer of antibody Z on tunable supertransfected cells. DETAILED DESCRIPTION

[0057] In certain embodiments, the host cells, gene constructs (e.g., vectors), compositions and methods described herein can be used to develop and / or use targeted integration (TI) host cells. In certain embodiments, such TI host cells comprise exogenous nucleotide sequences integrated into a specific gene or a specific locus of the genome of the host cell.

[0058] For the sake of clarity and not limitation, the detailed description is divided into the following subsections:

[0059] 1. Definition

[0060] 2. Integration site

[0061] 3. Exogenous nucleotide sequences

[0062] 4. Host Cells

[0063] 5. Targeted Integration

[0064] 6. Preparation and Use of TI Host Cells

[0065] 7. Products

[0066] 8. Exemplary Non-Limiting Embodiments

[0067] 1. Definition

[0068] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those of ordinary skill in the art. In the event of ambiguity, this patent specification (including definitions) shall prevail. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used to practice or test the currently disclosed subject matter. All publications, patent applications, patents and other references mentioned herein are incorporated herein by reference in their entirety. The materials, methods and examples disclosed herein are illustrative only and are not intended to be limiting.

[0069] As used herein, the terms "comprises," "including," "having," "having," "may," "containing," and variations thereof are intended to be open-ended transitional phrases, terms, or words that do not exclude the possibility of additional actions or structures. Unless expressly provided otherwise, the singular forms "a," "an," and "the" include plural referents. The present disclosure also contemplates other embodiments that "comprise," "consist of," and "consist essentially of," whether or not expressly set forth, the embodiments or elements presented herein.

[0070] For enumerating the numerical ranges herein, each intermediate number with the same degree of precision therebetween is expressly contemplated. For example, for the range of 6-9, in addition to 6 and 9, the numbers 7 and 8 are also contemplated; and for the range of 6.0-7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are expressly contemplated.

[0071] As used herein, the term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, according to practice in the art, "about" can mean 3 or more than 3 standard deviations. Alternatively, "about" can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within a certain order of magnitude of a certain value, preferably within 5 times, and more preferably within 2 times.

[0072] As used herein, the term "selectable marker" can be a gene that allows specific selection or exclusion of cells carrying the gene in the presence of a corresponding selection agent. For example, but not as a limitation, a selectable marker can allow positive selection of host cells transfected with the selectable marker gene in the presence of the gene; untransformed host cells will not be able to grow or survive under the selection conditions. Selectable markers can be positive markers, negative markers, or bifunctional markers. Positive selection markers can allow selection of cells carrying the marker, while negative selection markers can allow selective elimination of cells carrying the marker. Selectable markers can confer resistance to drugs, or compensate for metabolic or catabolic defects in host cells. In prokaryotic cells, genes that confer resistance to ampicillin, tetracycline, kanamycin, or chloramphenicol can be used. Resistance genes can be used as selection markers in eukaryotic cells, including but not limited to genes for aminoglycoside phosphotransferase (APH) (e.g., hygromycin phosphotransferase (HYG), neomycin and G418 APH), 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. Other marker genes are described in WO 92 / 08796 and WO 94 / 28143.

[0073] In addition to facilitating selection in the presence of a corresponding selection agent, a selectable marker may alternatively provide a gene encoding a molecule not normally present in the cell, 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 carrying such genes can be distinguished from cells that do not carry the gene, for example, by detecting fluorescence emitted by the encoded polypeptide.

[0074] As used herein, the term "operably connected" refers to the juxtaposition of two or more components, wherein the relationship of these components allows them to function in an expected manner. For example, if a promoter and / or enhancer are used to regulate the transcription of a coding sequence, the promoter and / or enhancer are operably connected to the coding sequence. In certain embodiments, the DNA sequence of "operably connected" is connected and adjacent on a single chromosome. In certain embodiments, for example, when it is necessary to connect two protein coding regions (such as secretory leaders and polypeptides), these sequences are connected, adjacent, and in the same reading frame. In certain embodiments, the promoter that can be operably connected is located upstream of the coding sequence and can be adjacent to the coding sequence. In certain embodiments, for example, with respect to the enhancer sequence that regulates the expression of the coding sequence, the two components are operably connected, but not adjacent. If the enhancer increases the transcription of the coding sequence, the enhancer is operably connected to the coding sequence. The enhancer that can be operably connected can be located upstream, internal, or downstream of the coding sequence, and can be located at a position quite far away from the promoter of the coding sequence. The operable connection can be completed by recombinant methods known in the art, for example, using PCR methods and / or by connecting at convenient restriction sites. If no convenient restriction sites exist, synthetic oligonucleotide adaptors or linkers may be used in accordance with conventional practice.An internal ribosome entry site (IRES) is operably linked to an open reading frame (ORF) if it allows initiation of translation of the ORF at an internal location independent of the 5' end.

[0075] As used herein, the term "expression" refers to transcription and / or translation. In certain embodiments, the level of transcription of the desired product can be determined based on the amount of the corresponding mRNA present. For example, the mRNA transcribed from the target sequence can be quantified by PCR or Northern blot hybridization. In certain embodiments, the protein encoded by the target sequence can be quantified by a variety of methods, for example, by ELISA, by measuring the biological activity of the protein, or by using antibodies that recognize and bind to the protein, using assays unrelated to this activity, such as Western blotting or radioimmunoassay.

[0076] The term "antibody" herein is used in the broadest sense and includes various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), half antibodies, and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0077] As used herein, the term "antibody fragment" refers to a molecule other than an intact antibody that includes a portion of an intact antibody and binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. For a review of certain antibody fragments, see Holliger and Hudson, Nature Biotechnology 23:1126-1136 (2005).

[0078] As used herein, the term "variable region" or "variable domain" refers to the domain of an antibody heavy chain or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of a native antibody (V H and V L ) generally have a similar structure, wherein each domain comprises four conserved framework regions (FR) and three hypervariable regions (HVR). (See, e.g., Kindt et al., Kuby Immunology, 6th edition, WH Freeman and Co., p. 91 (2007)). A single V H or V L The domain may be sufficient to confer antigen binding specificity. In addition, antibodies that bind to a specific antigen may use V domains from antibodies that bind to that antigen, respectively. H or V L domains to separate and screen for complementary V L or V H Libraries of domains. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0079] As used herein, the term "heavy chain" refers to an immunoglobulin heavy chain.

[0080] As used herein, the term "light chain" refers to an immunoglobulin light chain.

[0081] The "class" of an antibody refers to the type of constant domain or constant region possessed by the heavy chain of the antibody. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of them can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0082] The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous antibody population, that is, except for possible variant antibodies (e.g., containing naturally occurring mutations or produced during the production of monoclonal antibody preparations, such variants are usually present in small amounts), each antibody comprising the population is identical and / or binds to the same epitope. Contrary to the polyclonal antibody preparations that typically include different antibodies for different determinants (epitopes), each monoclonal antibody in the monoclonal antibody preparation is directed to a single determinant on the antigen. Therefore, the modifier "monoclonal" represents that the feature of an antibody is obtained from a substantially homogeneous antibody population, and should not be interpreted as requiring antibody to be produced by any ad hoc method. For example, the monoclonal antibody according to the present invention can be prepared 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 human immunoglobulin loci, such methods and other exemplary methods for preparing monoclonal antibodies are described herein.

[0083] "Multispecific antibodies" are monoclonal antibodies that have binding specificity for at least two different sites (i.e., different epitopes on different antigens or different epitopes on the same antigen). In certain aspects, multispecific antibodies have three or more binding specificities. Multispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0084] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region as defined herein.

[0085] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody and binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv and scFab); single domain antibodies (dAb); and multispecific antibodies formed from antibody fragments. For a review of certain antibody fragments, see Holliger and Hudson, Nature Biotechnology 23:1126-1136 (2005).

[0086] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0087] A "human antibody" is an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell, or derived from a non-human antibody using a human antibody library or other human antibody encoding sequences. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen binding residues.

[0088] "Humanized" antibody refers to such chimeric antibody, which comprises amino acid residues from non-human CDR and amino acid residues from human FR. In some aspects, humanized antibody will substantially comprise at least one, usually two variable domains, wherein all or substantially all CDRs correspond to the CDRs of non-human antibodies, and all or substantially all FRs correspond to the FRs of human antibodies. Humanized antibody optionally can comprise at least a portion of the antibody constant region derived from human antibodies. "Humanized form" antibody, such as non-human antibody, refers to humanized antibody. As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, that is, except for possible variant antibodies (e.g., containing naturally occurring mutations or produced in the production process of monoclonal antibody preparations, such variants are generally present in small amounts), each antibody comprising the population is identical and / or combines the same epitope. Contrary to the polyclonal antibody preparations that generally include different antibodies for different determinants (epitopes), each monoclonal antibody in the monoclonal antibody preparation is directed to a single determinant on the antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.

[0089] The term "therapeutic antibody" refers to an antibody used to treat a disease. Therapeutic antibodies may have various mechanisms of action. Therapeutic antibodies may bind to and neutralize the normal function of a target associated with an antigen. For example, a monoclonal antibody that blocks the activity of a protein required for cancer cell survival causes cell death. Another therapeutic monoclonal antibody may bind to and activate the normal function of a target associated with an antigen. For example, a monoclonal antibody may bind to a protein on a cell and trigger a cell apoptosis signal. Another monoclonal antibody may bind to a target antigen that is expressed only on diseased tissue; conjugating a toxic payload (effective agent), such as a chemotherapeutic agent or a radioactive agent, to a monoclonal antibody may form a toxic payload that is specifically delivered to diseased tissue, reducing damage to healthy tissue. The "biologically functional fragment" of a therapeutic antibody will exhibit at least one (if not part or all) of the biological function attributed to the complete antibody, which includes at least specific binding to the target antigen.

[0090] The term "diagnostic antibody" refers to an antibody used as a disease diagnostic agent. The diagnostic antibody can bind to a target antigen that is specifically associated with a particular disease or exhibits increased expression therein. The diagnostic antibody can be used, for example, to detect a target in a biological sample from a patient, or for diagnostic imaging of a disease site such as a tumor in a patient. A "biologically functional fragment" of a diagnostic antibody will exhibit at least one (if not part or all) of the biological function attributed to the intact antibody, including at least specific binding to the target antigen.

[0091] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived from the primary transformed cell, regardless of the number of passages. Progeny may not be identical to the nucleic acid content of the parent cell, but may contain mutations. Mutant progeny as screened or selected for the same function or biological activity as in the original transformed cell are included herein.

[0092] The term "nucleic acid molecule" or "polynucleotide" includes any compound and / or substance comprising a nucleotide polymer. Each nucleotide is composed of a base, particularly a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e., deoxyribose or ribose) and a phosphate group. Typically, nucleic acid molecules are described by base sequences, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule. Base sequences are typically expressed as from 5' to 3'. In this article, the term nucleic acid molecule encompasses synthetic forms of deoxyribonucleic acid (DNA) (including, for example, complementary DNA (cDNA) and genomic DNA), ribonucleic acid (RNA) (particularly messenger RNA (mRNA)), DNA or RNA, and mixed polymers comprising two or more of these molecules. Nucleic acid molecules can be linear or cyclic. In addition, the term nucleic acid molecule includes sense strands and antisense strands, as well as single-stranded and double-stranded forms. In addition, nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases with derivatized sugar or phosphate backbone bonds or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules suitable as carriers for direct expression in vitro and / or in vivo (e.g., in a host or patient) of the antibodies of the present invention. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors may be unmodified or modified. For example, mRNA may be chemically modified to enhance the stability of the RNA vector and / or the expression of the coding molecule so that mRNA may be injected into a subject to produce antibodies in vivo (see, e.g., Stadler et al., Nature Medicine 2017, published online on June 12, 2017, doi: 10.1038 / nm.4356 or EP 2 101 823B1).

[0093] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0094] As used herein, the term "vector" refers to a nucleic acid molecule capable of carrying another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures, as well as vectors that integrate into the genome of a host cell into which it has been introduced. In certain embodiments, a vector directs the expression of a nucleic acid to which it is operably linked. Such vectors are referred to herein as "expression vectors."

[0095] As used herein, the term "homologous sequence" refers to a sequence that shares significant sequence similarity, as determined by sequence alignment. For example, two sequences can be 50%, 60%, 70%, 80%, 90%, 95%, 99% or 99.9% homologous. Alignment is performed by algorithms and computer programs (including but not limited to BLAST, FASTA and HMME), which can compare sequences and calculate the statistical significance of matching based on factors such as sequence length, sequence identity and similarity, and the presence and length of sequence mismatches and gaps. Homologous sequences can refer to both DNA and protein sequences.

[0096] As used herein, the term "flanking" refers to a first nucleotide sequence located at the 5' end or 3' end or both ends of a second base sequence. The flanking nucleotide sequence may be adjacent to or at a certain distance from the second nucleotide sequence. The length of the flanking nucleotide sequence is not specifically limited. For example, the flanking sequence may have a few base pairs or may have several thousand base pairs. In certain embodiments, the length of the flanking nucleotide sequence may be about at least 15 base pairs, at least 20 base pairs, at least 30 base pairs, at least 40 base pairs, at least 50 base pairs, at least 75 base pairs, at least 100 base pairs, at least 150 base pairs, at least 200 base pairs, at least 300 base pairs, at least 400 base pairs, at least 500 base pairs, at least 1,000 base pairs, at least 1,500 base pairs, at least 2,000 base pairs, at least 3,000 base pairs, at least 4,000 base pairs, at least 5,000 base pairs, at least 6,000 base pairs, at least 7,000 base pairs, at least 8,000 base pairs, at least 9,000 base pairs, or at least 10,000 base pairs.

[0097] As used herein, the term "exogenous" refers to a nucleotide sequence that is not derived from a host cell, but is introduced into a host cell by conventional DNA delivery methods (e.g., by transfection, electroporation, transformation methods). The term "endogenous" refers to a nucleotide sequence derived from a host cell. An "exogenous" nucleotide sequence may have an "endogenous" counterpart with the same base composition, wherein the "exogenous" sequence is introduced into a host cell by, for example, recombinant DNA technology.

[0098] 2. Integration site

[0099] The presently disclosed subject matter provides host cells suitable for targeted integration of exogenous nucleotide sequences.In certain embodiments, the host cell comprises an exogenous nucleotide sequence integrated at an integration site on the genome of the host cell (ie, a TI host cell).

[0100] An "integration site" comprises a nucleic acid sequence within the host cell genome into which an exogenous nucleotide sequence is inserted. In certain embodiments, the integration site is between two adjacent nucleotides on the host cell genome. In certain embodiments, the integration site comprises a stretch of nucleotides into which an exogenous nucleotide sequence may be inserted between any of the nucleotides. In certain embodiments, the integration site is located within a locus of the genome of a specific TI host cell. In certain embodiments, the integration site is within an endogenous gene of the TI host cell.

[0101] In certain embodiments, the exogenous nucleotide sequence is integrated at a site within a specific locus of the genome of the TI host cell. In certain embodiments, the locus into which the exogenous nucleotide sequence is integrated is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or at least about 99.9% homologous to a sequence selected from SEQ ID No. 1 to SEQ ID No. 7.

[0102] In certain embodiments, the exogenous nucleotide sequence is integrated at a site within a specific locus of the genome of the TI host cell. In certain embodiments, the locus into which the exogenous nucleotide sequence is integrated is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or at least about 99.9% homologous to the following sequences selected from Contig NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1.

[0103] In certain embodiments, the exogenous nucleotide sequence is integrated at an integration site located at a position selected from the following nucleotides, which are the nucleotides numbered as follows in SEQ ID No. 1: 1-1,000 bp; 1,000-2,000 bp; 2,000-3,000 bp; 3,000-4,000 bp; and 4,000-4,301 bp. In certain embodiments, the exogenous nucleotide sequence is integrated at an integration site located at a position selected from the following nucleotides, which are the nucleotides numbered as follows in SEQ ID No. 2: 1-100,000 bp; 100,000-200,000 bp; 200,000-300,000 bp; 300,000-400,000 bp; 400,000-500,000 bp; 500,000-600,000 bp; 600,000-700,000 bp; and 700,000-728785 bp. In certain embodiments, the exogenous nucleotide sequence is integrated at an integration site located at a position selected from the following nucleotides, which are the nucleotides numbered as follows in SEQ ID No. 3: 1-100,000 bp; 100,000-200,000 bp; 200,000-300,000 bp; 300,000-400,000 bp; and 400,000-413,983 bp. In certain embodiments, the exogenous nucleotide sequence is integrated at an integration site located at a position selected from the following nucleotides, which are the nucleotides numbered as follows in SEQ ID No. 4: 1-10,000 bp; 10,000-20,000 bp; 20,000-30,000 bp; and 30,000-30,757 bp. In certain embodiments, the exogenous nucleotide sequence is integrated at an integration site located at a position selected from the following nucleotides, which are the nucleotides numbered as follows in SEQ ID No. 5: 1-10,000 bp; 10,000-20,000 bp; 20,000-30,000 bp; 30,000-40,000 bp; 40,000-50,000 bp; 50,000-60,000 bp; and 60,000-68,962 bp. In certain embodiments, the exogenous nucleotide sequence is integrated at an integration site located at a position selected from the following nucleotides, which are the nucleotides numbered as follows in SEQ ID No. 6: 1-10,000 bp; 10,000-20,000 bp; 20,000-30,000 bp; 30,000-40,000 bp; 40,000-50,000 bp; and 50,000-51,326 bp.In certain embodiments, the exogenous nucleotide sequence is integrated at an integration site located at a position selected from the following nucleotides, which are the nucleotides numbered as follows in SEQ ID No. 7: 1-10,000 bp; 10,000-20,000 bp; and 20,000-22,904 bp.

[0104] In certain embodiments, the nucleotide sequence immediately adjacent to the 5' end of the integrated exogenous sequence is selected from the group consisting of nucleotides 41190-45269 of NW_006874047.1, nucleotides 63590-207911 of NW_006884592.1, nucleotides 253831-491909 of NW_006881296.1, nucleotides 69303-79768 of NW_003616412.1, nucleotides 293481-315265 of NW_003615063.1, nucleotides 2650443-2662054 of NW_006882936.1, or nucleotides 82214-97705 of NW_003615411.1, and sequences at least 50% homologous thereto. In certain embodiments, the nucleotide sequence immediately 5' to the integrated exogenous sequence is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or at least about 99.9% homologous to nucleotides 41190-45269 of NW_006874047.1, nucleotides 63590-207911 of NW_006884592.1, and nucleotides 63610-206911 of NW_006874047.1. _006881296.1, nucleotides 253831-491909, nucleotides 69303-79768 of NW_003616412.1, nucleotides 293481-315265 of NW_003615063.1, nucleotides 2650443-2662054 of NW_006882936.1, or nucleotides 82214-97705 of NW_003615411.1.

[0105] In certain embodiments, the nucleotide sequence immediately adjacent to the 3' end of the integrated exogenous sequence is selected from the group consisting of nucleotides 45270-45490 of NW_006874047.1, nucleotides 207912-792374 of NW_006884592.1, nucleotides 491910-667813 of NW_006881296.1, nucleotides 79769-100059 of NW_003616412.1, nucleotides 315266-362442 of NW_003615063.1, nucleotides 2662055-2701768 of NW_006882936.1, or nucleotides 97706-105117 of NW_003615411.1, and sequences at least 50% homologous thereto. In certain embodiments, the nucleotide sequence immediately 3' to the integrated exogenous sequence is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or at least about 99.9% homologous to nucleotides 45270-45490 of NW_006874047.1, nucleotides 207912-792374 of NW_006884592.1, and nucleotides 207920-207167 of NW_006874047.1. _006881296.1, nucleotides 491910-667813, nucleotides 79769-100059 of NW_003616412.1, nucleotides 315266-362442 of NW_003615063.1, nucleotides 2662055-2701768 of NW_006882936.1, or nucleotides 97706-105117 of NW_003615411.1.

[0106] In certain embodiments, the integrated exogenous nucleotide sequence is operably linked to a nucleotide sequence selected from the group consisting of SEQ ID. No. 1 to SEQ ID No. 7 and sequences at least 50% homologous thereto. In certain embodiments, the nucleotide sequence operably linked to the exogenous nucleotide sequence is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or at least about 99.9% homologous to a sequence selected from SEQ ID No. 1 to SEQ ID No. 7. In certain embodiments, the integrated exogenous nucleotide sequence comprises at least one SOI. In certain embodiments, the operably linked nucleotide sequence increases the expression level of the SOI compared to the randomly integrated SOI. In certain embodiments, the expression level of the integrated exogenous SOI is about 20%, 30%, 40%, 50%, 100%, 2 times, 3 times, 5 times, or 10 times higher than the randomly integrated SOI.

[0107] In certain embodiments, the integrated exogenous sequence is flanked at the 5' end by a nucleotide sequence selected from the group consisting of nucleotides 41190-45269 of NW_006874047.1, nucleotides 63590-207911 of NW_006884592.1, nucleotides 253831-491909 of NW_006881296.1, nucleotides 69303-79768 of NW_003616412.1, nucleotides 293481-315265 of NW_003615063.1, nucleotides 2650443-2662054 of NW_006882936.1, and nucleotides 82214-97705 of NW_003615411.1, and at least 50% identical The present invention relates to a sequence of a nucleic acid source, and is flanked at the 3' end by a nucleotide sequence selected from the group consisting of nucleotides 45270-45490 of NW_006874047.1, nucleotides 207912-792374 of NW_006884592.1, nucleotides 491910-667813 of NW_006881296.1, nucleotides 79769-100059 of NW_003616412.1, nucleotides 315266-362442 of NW_003615063.1, nucleotides 2662055-2701768 of NW_006882936.1 and nucleotides 97706-105117 of NW_003615411.1, and sequences at least 50% homologous thereto.In certain embodiments, the nucleotide sequence flanking the 5' end of the integrated exogenous nucleotide sequence is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or at least about 99.9% homologous to nucleotides 41190-45269 of NW_006874047.1, nucleotides 63590-207911 of NW_006884592.1, nucleotides 253831-491909 of NW_006881296.1, or nucleotides 41616412 of NW_006874047.1. 69303-79768 of NW_003615063.1, nucleotides 293481-315265 of NW_006882936.1, nucleotides 2650443-2662054 of NW_003615411.1, and the nucleotide sequence flanking the 3' end of the integrated exogenous nucleotide sequence is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or at least about 99.9% homologous to: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: Nucleotides 45270-45490 of ID No. NW_006874047.1, nucleotides 207912-792374 of NW_006884592.1, nucleotides 491910-667813 of NW_006881296.1, nucleotides 79769-100059 of NW_003616412.1, nucleotides 315266-362442 of NW_003615063.1, nucleotides 2662055-2701768 of NW_006882936.1 and nucleotides 97706-105117 of NW_003615411.1.

[0108] In certain embodiments, the integrated exogenous nucleotide is integrated in a locus immediately adjacent to all or a portion of a sequence selected from the group consisting of a sequence at least about 90% homologous to a sequence of SEQ ID No. 1 to SEQ ID No. 7.

[0109] In certain embodiments, the integrated exogenous nucleotide sequence is adjacent to a nucleotide sequence selected from the group consisting of SEQ ID. No. 1 to SEQ ID No. 7 and sequences at least 50% homologous thereto. In certain embodiments, the integrated exogenous nucleotide sequence is within about 100 bp, about 200 bp, about 500 bp, about 1 kb of a sequence selected from the group consisting of SEQ ID. No. 1 to SEQ ID No. 7 and sequences at least 50% homologous thereto. In certain embodiments, the nucleotide sequence adjacent to the exogenous nucleotide sequence is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or at least about 99.9% homologous to a sequence selected from SEQ ID No. 1 to SEQ ID No. 7.

[0110] In certain embodiments, the exogenous nucleotide sequence is integrated at an integration site adjacent to a position selected from the following nucleotides, which are the nucleotides numbered as follows in SEQ ID No. 1: 1-1,000 bp; 1,000-2,000 bp; 2,000-3,000 bp; 3,000-4,000 bp; and 4,000-4,301 bp. In certain embodiments, the exogenous nucleotide sequence is integrated at an integration site adjacent to a position selected from the following nucleotides, which are the nucleotides numbered as follows in SEQ ID No. 2: 1-100,000 bp; 100,000-200,000 bp; 200,000-300,000 bp; 300,000-400,000 bp; 400,000-500,000 bp; 500,000-600,000 bp; 600,000-700,000 bp; and 700,000-728785 bp. In certain embodiments, the exogenous nucleotide sequence is integrated at an integration site adjacent to a position selected from the following nucleotides, which are nucleotides numbered as follows in SEQ ID No. 3: 1-100,000 bp; 100,000-200,000 bp; 200,000-300,000 bp; 300,000-400,000 bp; and 400,000-413,983 bp. In certain embodiments, the exogenous nucleotide sequence is integrated at an integration site adjacent to a position selected from the following nucleotides, which are nucleotides numbered as follows in SEQ ID No. 4: 1-10,000 bp; 10,000-20,000 bp; 20,000-30,000 bp; and 30,000-30,757 bp. In certain embodiments, the exogenous nucleotide sequence is integrated at an integration site adjacent to a position selected from the following nucleotides, which are the nucleotides numbered as follows in SEQ ID No. 5: 1-10,000 bp; 10,000-20,000 bp; 20,000-30,000 bp; 30,000-40,000 bp; 40,000-50,000 bp; 50,000-60,000 bp; and 60,000-68,962 bp. In certain embodiments, the exogenous nucleotide sequence is integrated at an integration site adjacent to a position selected from the following nucleotides, which are the nucleotides numbered as follows in SEQ ID No. 6: 1-10,000 bp; 10,000-20,000 bp; 20,000-30,000 bp; 30,000-40,000 bp; 40,000-50,000 bp; and 50,000-51,326 bp.In certain embodiments, the exogenous nucleotide sequence is integrated at an integration site adjacent to a position selected from the following nucleotides, which are the nucleotides numbered as follows in SEQ ID No. 7: 1-10,000 bp; 10,000-20,000 bp; and 20,000-22,904 bp.

[0111] In certain embodiments, the locus comprising the integration site of the exogenous nucleotide sequence does not encode an open reading frame (ORF). In certain embodiments, the locus comprising the integration site of the exogenous nucleotide sequence includes cis-acting elements, such as promoters and enhancers. In certain embodiments, the locus comprising the integration site of the exogenous nucleotide sequence does not contain any cis-acting elements, such as promoters and enhancers that enhance gene expression.

[0112] In certain embodiments, the exogenous nucleotide sequence is integrated at an integration site within an endogenous gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2. The endogenous LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2 genes include wild type and all homologous sequences of the LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2 genes. In certain embodiments, homologous sequences of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2 genes may be at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or at least about 99.9% homologous to the wild-type LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2 genes. In certain embodiments, the LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2 genes are wild-type mammalian LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2 genes. In certain embodiments, the LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2 genes are wild-type human LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2 genes. In certain embodiments, the LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2 genes are wild-type hamster LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2 genes.

[0113] In certain embodiments, the integration site is operably linked to an endogenous gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, XP_003512331.2, and sequences at least about 90% homologous thereto. In certain embodiments, the integration site is flanked by an endogenous gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, XP_003512331.2, and sequences at least about 90% homologous thereto.

[0114] Table 1 provides exemplary TI host cell integration sites:

[0115] Table 1 - TI host cell integration sites

[0116]

[0117] In certain embodiments, the nucleotide sequence of the integration site and / or the lateral joint of this integration site can be identified by experiment.In certain embodiments, the nucleotide sequence of the integration site and / or the lateral joint of this integration site can be identified by a full genome screening method to isolate a host cell, the target polypeptide encoded by one or more SOIs integrated in one or more exogenous nucleotide sequences expressed by this host cell at the desired level, wherein the exogenous sequence itself is integrated in one or more loci in the genome of the host cell.In certain embodiments, the nucleotide sequence of the integration site and / or the lateral joint of this integration site can be identified by a full genome screening method after the box integration event based on transposase.In certain embodiments, the nucleotide sequence of the integration site and / or the lateral joint of this integration site can be identified by a strong random integration screening.In certain embodiments, the nucleotide sequence of the integration site and / or the lateral joint of this integration site can be determined by conventional sequencing methods such as target locus amplification (TLA) and subsequent new generation sequencing (NGS) and full genome NGS.In certain embodiments, the position of the integration site on the chromosome can be determined by conventional cell biology methods such as fluorescence in situ hybridization (FISH) analysis.

[0118] In certain embodiments, the TI host cell comprises: a first exogenous nucleotide sequence integrated into a first integration site within a specific first locus in the genome of the TI host cell; and a second exogenous nucleotide sequence integrated into a second integration site within a specific second locus in the genome. In certain embodiments, the TI host cell comprises multiple exogenous nucleotide sequences integrated into multiple integration sites in the genome of the TI host cell.

[0119] In certain embodiments, the TI host cell of the present disclosure comprises at least two different exogenous nucleotide sequences, for example, an exogenous nucleotide sequence comprising at least one RRS. In certain embodiments, when one or more SOIs are introduced, two or more exogenous nucleotide sequences can be targeted. In certain embodiments, the SOIs are the same. In certain embodiments, the SOIs are different. In certain embodiments, a parent TI host cell comprising a first exogenous nucleotide sequence may comprise a second exogenous nucleotide sequence, the integration site of which is different from the integration site of the first exogenous nucleotide sequence.

[0120] In certain embodiments, the integration site is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or at least about 99.9% homologous to a sequence selected from SEQ ID No. 1 to SEQ ID No. 7. In certain embodiments, the integration site may be the same chromosome. In certain embodiments, the integration sites are 1-1,000 nucleotides, 1,000-100,000 nucleotides, 100,000-1,000,000 nucleotides or more away from each other within the same chromosome. In certain embodiments, the integration sites are on different chromosomes. In certain embodiments, a TI host cell comprising an exogenous nucleotide sequence at one integration site can be used to insert at least two, at least three, at least four, at least five, at least six, at least seven, at least eight or more exogenous nucleotide sequences at the same or different integration sites.

[0121] In certain embodiments, the feasibility of recombinase-mediated cassette exchange (RMCE) at least two integration sites of each site can be evaluated separately. In certain embodiments, the feasibility of RMCE at at least two integration sites can be evaluated simultaneously. The feasibility of RMCE at multiple sites can be evaluated by methods known in the art (e.g., measuring polypeptide titer or polypeptide-specific production). In certain embodiments, the evaluation can be performed by methods known in the art (e.g., by evaluating the titer and / or specific productivity of a culture of TI host cells expressing one or more SOIs). Exemplary culture strategies include, but are not limited to, batch-fed shake flask culture and bioreactor batch-fed culture. The titer and specific productivity of TI host cells expressing the polypeptide of interest can be evaluated by methods known in the art (e.g., but not limited to ELISA, FACS, fluorescent microwell analysis technology (FMAT), protein A affinity chromatography, Western blot analysis).

[0122] 3. Exogenous nucleotide sequences

[0123] An exogenous nucleotide sequence is a nucleotide sequence that is not derived from a host cell but can be introduced into a host cell by conventional DNA delivery methods (e.g., by transfection, electroporation, transformation methods). In certain embodiments, the exogenous nucleotide sequence is a sequence of interest (SOI), such as a nucleotide sequence encoding a polypeptide of interest. However, in certain embodiments, the exogenous nucleotide sequence employed in the context of the present disclosure comprises elements that facilitate the introduction of additional nucleic acid sequences (e.g., SOI), such as one or more recombination recognition sequences (RRS) and one or more selection markers. In certain embodiments, the exogenous nucleotide sequence that facilitates the introduction of additional nucleic acid sequences is referred to herein as a "landing pad." Thus, in certain embodiments, a TI host cell may comprise: (1) an exogenous nucleotide sequence comprising one or more SOIs, such as an SOI targeted for integration into a specific locus of the host cell genome mediated by an exogenous site-specific nuclease (e.g., CRISPR / Cas9); (2) an exogenous nucleotide sequence comprising one or more landing pads; or (3) an exogenous nucleotide sequence comprising one or more landing pads into which one or more SOIs have been integrated.

[0124] In certain embodiments, the TI host cell comprises at least one exogenous nucleotide sequence integrated into one or more integration sites in the genome of the TI host cell. In certain embodiments, the exogenous nucleotide sequence is integrated into one or more integration sites within a locus of the genome of a particular TI host cell. For example, but not by way of limitation, at least one exogenous nucleic acid sequence may be integrated into one or more loci that have at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or at least about 99.9% homology to a sequence selected from SEQ ID No. 1 to SEQ ID No. 7.

[0125] 3.1 Landing Pad

[0126] In certain embodiments, the exogenous nucleotide sequence of integration comprises one or more recombination recognition sequences (RRS), wherein the RRS can be recognized by a recombinase. In certain embodiments, the exogenous nucleotide sequence of integration comprises at least two RRS. In certain embodiments, the exogenous nucleotide sequence of integration comprises two RRS, and the two RRS are identical. In certain embodiments, the exogenous nucleotide sequence of integration comprises two RRS, and the two RRS have heterospecificity, i.e., they are not recognized by the same recombinase. In certain embodiments, the exogenous nucleotide sequence of integration comprises three RRS, wherein the third RRS is located between the first RRS and the second RRS. In certain embodiments, the first RRS is identical to the second RRS, and the third RRS is different from the first RRS or the second RRS. In certain embodiments, all three RRS have heterospecificity. In certain embodiments, the exogenous nucleotide sequence of integration comprises four, five, six, seven or eight RRS. In certain embodiments, the exogenous nucleotide sequence of integration comprises multiple RRS. In certain embodiments, the multiple (two or more) RRS are identical. In certain embodiments, the two or more RRS have heterospecificity. In certain embodiments, each RRS can be recognized by different recombinases. In certain embodiments, a subset of all RRSs are homospecific, i.e., recognized by the same recombinase, and a subset of all RRSs are heterospecific, i.e., not recognized by the same recombinase. In certain embodiments, one or more RRSs may be selected from the group consisting of LoxP sequence, LoxP L3 sequence, LoxP 2L sequence, LoxFas sequence, Lox511 sequence, Lox2272 sequence, Lox2372 sequence, Lox5171 sequence, Loxm2 sequence, Lox71 sequence, Lox66 sequence, FRT sequence, Bxb1 attP sequence, Bxb1 attB sequence, φC31 attP sequence, and φC31 attB sequence.

[0127] In certain embodiments, the exogenous nucleotide sequence of integration comprises at least one selection marker. In certain embodiments, the exogenous nucleotide sequence of integration comprises an RRS and at least one selection marker. In certain embodiments, the exogenous nucleotide sequence of integration comprises the first RRS and the second RRS and at least one selection marker. In certain embodiments, the selection marker is located between the first RRS and the second RRS. In certain embodiments, two RRS flank at least one selection marker, i.e., the first RRS is located at the 5' end upstream of the selection marker and the second RRS is located at the 3' end downstream of the selection marker. In certain embodiments, the first RRS is adjacent to the 5' end of the selection marker, and the second RRS is adjacent to the 3' end of the selection marker.

[0128] In certain embodiments, the selection marker is located between the first RRS and the second RRS, and the two flanking RRS are identical. In certain embodiments, both RRSs flanking the selection marker are LoxP sequences. In certain embodiments, both RRSs flanking the selection marker are FRT sequences. In certain embodiments, the selection marker is located between the first RRS and the second RRS, and the two flanking RRS have heterologous specificity. In certain embodiments, the first flanking RRS is a LoxP L3 sequence, and the second flanking RRS is a LoxP 2L sequence. In certain embodiments, the LoxP L3 sequence is located at the 5' end of the selection marker, and the LoxP 2L sequence is located at the 3' end of the selection marker. In certain embodiments, the first flanking RRS is a wild-type FRT sequence, and the second flanking RRS is a mutant FRT sequence. In certain embodiments, the first flanking RRS is a Bxb1 attP sequence, and the second flanking RRS is a Bxb1 attB sequence. In some embodiments, the first flanking RRS is a φC31 attP sequence and the second flanking RRS is a φC31 attB sequence. In some embodiments, the two RRSs are positioned in the same orientation. In some embodiments, the two RRSs are both in a forward or reverse orientation. In some embodiments, the two RRSs are positioned in opposite orientations.

[0129] In certain embodiments, the selection marker may be an aminoglycoside phosphotransferase (APH) (e.g., hygromycin phosphotransferase (HYG), neomycin and G418 APH), 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 or mycophenolic acid. In certain embodiments, the selection marker may be a GFP, eGFP, synthetic GFP, YFP, eYFP, CFP, mPlum, mCherry, tdTomato, mStrawberry, J-red, DsRed monomer, mOrange, mKO, mCitrine, Venus, YPet, Emerald, CyPet, mCFPm, Cerulean or T-Sapphire marker. In certain embodiments, the selection marker may be a fusion construct comprising at least two selection markers. In certain embodiments, a gene encoding a selection marker or a fragment of a selection marker may be fused to a gene encoding a different selection marker or a fragment thereof.

[0130] In certain embodiments, the integrated exogenous nucleotide sequence comprises two selection markers flanked by two RRS, wherein the first selection marker is different from the second selection marker. In certain embodiments, both selection markers are selected from the group consisting of: glutamine synthetase selection marker, thymidine kinase selection marker, HYG selection marker and puromycin resistance selection marker. In certain embodiments, the integrated exogenous nucleotide sequence comprises a thymidine kinase selection marker and a HYG selection marker. In certain embodiments, the first selection marker is selected from the group consisting of aminoglycoside phosphotransferase (APH) (e.g., hygromycin phosphotransferase (HYG), neomycin and G418 APH), 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 or mycophenolic acid, and the second selection marker is selected from the group consisting of GFP, eGFP, synthetic GFP, YFP, eYFP, CFP, mPlum, mCherry, tdTomato, mStrawberry, J-red, DsRed monomer, mOrange, mKO, mCitrine, Venus, YPet, Emerald, CyPet, mCFPm, Cerulean and T-Sapphire markers. In certain embodiments, the first selection marker is a glutamine synthetase selection marker, and the second selection marker is a GFP marker. In certain embodiments, the two RRSs flanking the two selection markers are identical. In certain embodiments, the two RRSs flanking the two selection markers are different.

[0131] In certain embodiments, the selection marker is operably linked to a promoter sequence. In certain embodiments, the selection marker is operably linked to an SV40 promoter. In certain embodiments, the selection marker is operably linked to a cytomegalovirus (CMV) promoter.

[0132] In certain embodiments, the exogenous nucleotide sequence of integration includes at least one selection marker and IRES, wherein the IRES is operably connected to the selection marker. In certain embodiments, the selection marker operably connected to IRES is selected from the group consisting of the following items: GFP, eGFP, synthetic GFP, YFP, eYFP, CFP, mPlum, mCherry, tdTomato, mStrawberry, J-red, DsRed monomer, mOrange, mKO, mCitrine, Venus, YPet, Emerald, CyPet, mCFPm, Cerulean and T-Sapphire markers. In certain embodiments, the selection marker operably connected to IRES is a GFP marker. In certain embodiments, the exogenous nucleotide sequence of integration includes an IRES and two selection markers flanked by two RRS, wherein the IRES is operably connected to the second selection marker. In certain embodiments, the exogenous nucleotide sequence of integration includes an IRES and three selection markers flanked by two RRS, wherein the IRES is operably connected to the third selection marker. In certain embodiments, the exogenous nucleotide sequence of integration comprises an IRES and three kinds of selection markers flanked by two RRS, wherein the IRES is operably connected to the third selection marker. In certain embodiments, the third selection marker is different from the first selection marker or the second selection marker. In certain embodiments, the exogenous nucleotide sequence of integration comprises the first selection marker operably connected to a promoter and the second selection marker operably connected to IRES. In certain embodiments, the exogenous nucleotide sequence of integration comprises a glutamine synthetase selection marker operably connected to an SV40 promoter and a GFP selection marker operably connected to an IRES. In certain embodiments, the exogenous nucleotide sequence of integration comprises a thymidine kinase selection marker operably connected to a CMV promoter and a HYG selection marker and a GFP selection marker operably connected to an IRES.

[0133] In certain embodiments, the exogenous nucleotide sequence of integration comprises three RRS. In certain embodiments, the third RRS is located between the first RRS and the second RRS. In certain embodiments, all three RRS are identical. In certain embodiments, the first RRS is identical to the second RRS, and the third RRS is different from the first RRS or the second RRS. In certain embodiments, all three RRS have heterologous specificity.

[0134] 3.2 Sequence of Interest (SOI)

[0135] In certain embodiments, the integrated exogenous nucleotide sequence comprises at least one exogenous SOI. In certain embodiments, the integrated exogenous nucleotide sequence comprises at least one selection marker and at least one exogenous SOI. In certain embodiments, the integrated exogenous nucleotide sequence comprises at least one selection marker, at least one exogenous SOI and at least one RRS. In certain embodiments, the integrated exogenous nucleotide sequence comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight or more SOIs. In certain embodiments, the SOIs are the same. In certain embodiments, the SOIs are different.

[0136] In certain embodiments, SOI encodes a single-chain antibody or a fragment thereof. In certain embodiments, SOI encodes an antibody heavy chain sequence or a fragment thereof. In certain embodiments, SOI encodes an antibody light chain sequence or a fragment thereof. In certain embodiments, the integrated exogenous nucleotide sequence comprises an SOI encoding an antibody heavy chain sequence or a fragment thereof and an SOI encoding an antibody light chain sequence or a fragment thereof. In certain embodiments, the integrated exogenous nucleotide sequence comprises an SOI encoding a first antibody heavy chain sequence or a fragment thereof, an SOI encoding a second antibody heavy chain sequence or a fragment thereof, and an SOI encoding an antibody light chain sequence or a fragment thereof. In certain embodiments, the integrated exogenous nucleotide sequence comprises an SOI encoding a first antibody heavy chain sequence or a fragment thereof, an SOI encoding a second antibody heavy chain sequence or a fragment thereof, an SOI encoding a first antibody light chain sequence or a fragment thereof, and a second SOI encoding an antibody light chain sequence or a fragment thereof. In certain embodiments, the number of SOIs encoding heavy and light chain sequences can be selected to achieve the desired expression level of heavy and light chain polypeptides, for example, to achieve the desired bispecific antibody yield. In certain embodiments, separate SOI encoding heavy and light chain sequences can be integrated, for example, in a single exogenous nucleic acid sequence at an integration site, in multiple exogenous nucleic acid sequences at a single integration site, or in multiple exogenous nucleic acid sequences at different integration sites within the TI host cell.

[0137] In certain embodiments, the integrated exogenous nucleotide sequence comprises at least one selection marker, at least one exogenous SOI and one RRS. In certain embodiments, the RRS is adjacent to at least one selection marker or at least one exogenous SOI. In certain embodiments, the integrated exogenous nucleotide sequence comprises at least one selection marker, at least one exogenous SOI and two RRS. In certain embodiments, the integrated exogenous nucleotide sequence comprises at least one selection marker and at least one exogenous SOI located between the first RRS and the second RRS. In certain embodiments, the two RRSs flanking the selection marker and the exogenous SOI are the same. In certain embodiments, the two RRSs flanking the selection marker and the exogenous SOI are different. In certain embodiments, the first flanking RRS is a LoxP L3 sequence, and the second flanking RRS is a LoxP 2L sequence. In certain embodiments, the L3 LoxP sequence is located at the 5' end of the selection marker and the exogenous SOI, and the LoxP 2L sequence is located at the 3' end of the selection marker and the exogenous SOI.

[0138] In certain embodiments, the integrated exogenous nucleotide sequence comprises three RRS and two exogenous SOIs, and the third RRS is located between the first RRS and the second RRS. In certain embodiments, the first SOI is located between the first RRS and the third RRS, and the second SOI is located between the third RRS and the second RRS. In certain embodiments, the first SOI is different from the second SOI. In certain embodiments, the first RRS is the same as the second RRS, and the third RRS is different from the first RRS or the second RRS. In certain embodiments, all three RRS have heterologous specificity. In certain embodiments, the first RRS is a LoxP L3 site, the second RRS is a LoxP 2L site, and the third RRS is a LoxFas site. In certain embodiments, the integrated exogenous nucleotide sequence comprises three RRS, an exogenous SOI, and a selection marker. In certain embodiments, SOI is located between the first RRS and the third RRS, and the selection marker is located between the third RRS and the second RRS. In certain embodiments, the integrated exogenous nucleotide sequence comprises three RRS, two exogenous SOIs, and a selection marker. In certain embodiments, the first SOI and the selection marker are located between the first RRS and the third RRS, and the second SOI is located between the third RRS and the second RRS.

[0139] In certain embodiments, the exogenous SOI encodes a polypeptide of interest. Such polypeptide of interest may be selected from the group consisting of, but not limited to, antibodies, enzymes, cytokines, growth factors, hormones, viral proteins, bacterial proteins, vaccine proteins, or proteins with therapeutic functions. In certain embodiments, the exogenous SOI encodes an antibody or an antigen-binding fragment thereof. In certain embodiments, the exogenous SOI encodes a single-chain antibody, an antibody light chain, an antibody heavy chain, a single-chain Fv fragment (scFv), or an Fc fusion protein. In certain embodiments, the exogenous SOI (or SOI) encodes a standard antibody. In certain embodiments, the exogenous SOI (or SOI) encodes a half antibody, such as, but not limited to, antibody B, Q, T, and mAb I of the present disclosure. In certain embodiments, the exogenous SOI (or SOI) encodes a composite antibody. In certain embodiments, the composite antibody may be a bispecific antibody, such as, but not limited to, bispecific molecule A, bispecific molecule B, bispecific molecule C, or bispecific molecule D of the present disclosure. In certain embodiments, the exogenous SOI may be operably linked to at least one cis-acting element, such as a promoter or enhancer. In certain embodiments, the exogenous SOI is operably linked to a CMV promoter.

[0140] In certain embodiments, the integrated exogenous nucleotide sequence comprises two RRS and at least two exogenous SOIs located between the two RRS. In certain embodiments, the SOI encoding one heavy chain and one light chain of an antibody is located between the two RRS. In certain embodiments, the SOI encoding one heavy chain and two light chains of an antibody is located between the two RRS. In certain embodiments, SOIs encoding different combinations of copies of the heavy and light chains of an antibody are located between the two RRS.

[0141] In certain embodiments, the integrated exogenous nucleotide sequence comprises three RRSs and at least two exogenous SOIs, and the third RRS is located between the first RRS and the second RRS. In certain embodiments, at least one SOI is located between the first RRS and the third RRS, and at least one SOI is located between the third RRS and the second RRS. In certain embodiments, the first RRS is identical to the second RRS, and the third RRS is different from the first RRS or the second RRS. In certain embodiments, all three RRSs have heterologous specificity. In certain embodiments, the SOI encoding one heavy chain and one light chain of the first antibody is located between the first RRS and the third RRS, and the SOI encoding one heavy chain and one light chain of the second antibody is located between the third RRS and the second RRS. In certain embodiments, the SOI encoding one heavy chain and two light chains of the first antibody is located between the first RRS and the third RRS, and the SOI encoding one heavy chain and one light chain of the second antibody is located between the third RRS and the second RRS. In certain embodiments, the SOI encoding one heavy chain and three light chains of the first antibody is located between the first RRS and the third RRS, and the SOI encoding one light chain of the first antibody and one heavy chain and one light chain of the second antibody is located between the third RRS and the second RRS. In certain embodiments, the SOI encoding one heavy chain and one light chain of the first antibody is located between the first RRS and the third RRS, and the SOI encoding two light chains of the first antibody and one heavy chain and one light chain of the second antibody is located between the third RRS and the second RRS. In certain embodiments, SOIs encoding different combinations of copies of heavy and light chains of multiple antibodies are located between the first RRS and the third RRS and between the third RRS and the second RRS.

[0142] In certain embodiments, the number of SOIs is selected to increase the expression titer and / or specific productivity of the host cell expressing the SOI. For example, but not by way of limitation, combining two, three, four, five, six, seven, eight or more SOIs may result in higher titers and / or specific productivity.

[0143] In the context of antibody expression, including additional heavy chain or light chain encoding SOIs can achieve higher titers and / or specific productivity. For example, but not by way of limitation, when the copy number is increased from one heavy chain and one light chain (HL) to one heavy chain and two light chain encoding sequences (HLL), higher titers and / or specific productivity can be achieved. Similarly, as outlined in the examples below, when HLL (three SOIs) is increased to HLL-HL (five SOIs) or HLL-HLL (six), higher titers and / or specific productivity can be provided. In addition, when the copy number is increased to HLL-HL (five SOIs) or HLL-HLHL (seven SOIs), higher titers and / or specific productivity can be provided. Further options for heavy and light chain SOI copy numbers include, but are not limited to: HHL; HHL-H; HLL-H; HHL-HH; HHL-HL; HHL-LL; HLL-HH; HLL-HL; HLL-LL; HHL-HHL; HHL-HHH; HHL-HLL; HHK-LLL; HLL-HHL; HLL-HHH; HLL-LLL; HHL-HHHL; HHL-HHHH; HHL-HHLL; HHL-HLLL; HHL-LLLL; HLL-HHHL; HHL-HHHH; HHL-HHLL; HHL-HLLL; HHL-LLLL; HLL-HHHL; HLL-HHHH; HLL-HLLL; and HLL-LLLL. In certain embodiments, inclusion of additional copies occurs at a single genomic locus, while in certain embodiments, SOI copies may be integrated at two or more loci, for example, multiple copies may be integrated at a single locus and one or more copies integrated at one or more additional loci.

[0144] In certain embodiments, the position of the SOI (e.g., whether one SOI is located at the 3' or 5' end relative to another SOI) is selected to improve the titer and / or specific productivity of the host cell expressing the SOI. For example, but not by way of limitation, in the context of antibody production, the integration position of the heavy and light chain SOIs can result in higher titers and / or specific productivity. Fig.23 As outlined in AD and the Examples shown below, the relative positions of the heavy and light chain SOIs can affect titer and specific productivity despite unchanged SOI copy number.

[0145] 4. Host Cells

[0146] The disclosed subject matter provides a host cell suitable for targeted integration of a nucleotide sequence and expression of a polypeptide of interest. In certain embodiments, the host cell comprises an endogenous gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, XP_003512331.2, and sequences at least 50% homologous thereto, or a locus comprising the genome of the host cell, wherein the locus comprises a nucleotide sequence selected from the group consisting of SEQ ID No. 1 to SEQ ID No. 7, and sequences at least 50% homologous thereto.

[0147] In certain embodiments, the host cell is a eukaryotic host cell. In certain embodiments, the host cell is a mammalian host cell. In certain embodiments, the host cell is a hamster host cell, a human host cell, a rat host cell or a mouse host cell. In certain embodiments, the host cell is a Chinese hamster ovary (CHO) host cell, a CHO K1 host cell, a CHO K1SV host cell, a DG44 host cell, a DUKXB-11 host cell, a CHOK1S host cell or a CHO K1M host cell.

[0148] In certain embodiments, the host cell is selected from the group consisting of: monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293 cells, as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells, as described, e.g., in Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); Buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor cells (MMT 060562); TRI cells (as described, e.g., in Mather et al., Annals NY Acad. Sci. 383: 44-68 (1982)); MRC 5 cells; FS4 cells; Y0 cells; NS0 cells; Sp2 / 0 cells; and cell.

[0149] In certain embodiments, the host cell is a cell line. In certain embodiments, the host cell is a cell line that has been cultured for a certain number of generations. In certain embodiments, the host cell is a primary cell.

[0150] In certain embodiments, the expression of the polypeptide of interest is stable if the expression level of the polypeptide of interest is maintained at a certain level, increased or decreased by less than 20% in 10, 20, 30, 50, 100, 200 or 300 generations. In certain embodiments, the expression of the polypeptide of interest is stable if the culture can be maintained without any selection. In certain embodiments, the expression level of the polypeptide of interest is high if the polypeptide product of the gene of interest reaches about 1 g / L, about 2 g / L, about 3 g / L, about 4 g / L, about 5 g / L, about 10 g / L, about 12 g / L, about 14 g / L or about 16 g / L.

[0151] The disclosed subject matter also relates to methods of producing a polypeptide of interest. In certain embodiments, such methods include: a) providing a host cell comprising at least one exogenous SOI integrated into a locus of the genome of the host cell and flanked by two RRSs and at least one selection marker, wherein the locus is at least about 90% homologous to a sequence selected from SEQ ID No. 1 to SEQ ID No. 7; and b) culturing the cell in a) under conditions suitable for expression of the SOI, and recovering the polypeptide of interest therefrom. In certain embodiments, such methods include: a) providing a host cell comprising at least two exogenous SOIs integrated into a locus of the genome of the host cell and at least one selection marker, wherein the locus is at least about 90% homologous to a sequence selected from SEQ ID No. 1 to SEQ ID No. 7, wherein at least one exogenous SOI and one selection marker are flanked by a first RRS and a third RRS and at least one exogenous SOI is flanked by a second RRS and a third RRS; and b) culturing the cell in a) under conditions suitable for expression of the SOI, and recovering the polypeptide of interest therefrom. In certain embodiments, such methods comprise: a) providing a host cell comprising at least one exogenous SOI flanked by two RRSs integrated into an endogenous gene and at least one selection marker, wherein the endogenous gene is selected from the group consisting of: LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, XP_003512331.2, and a sequence at least about 90% homologous thereto; and b) culturing the cells in a) under conditions suitable for expressing the SOI, and recovering the polypeptide of interest therefrom. In certain embodiments, such methods comprise: a) providing a host cell comprising at least two exogenous SOIs integrated into an endogenous gene and at least one selection marker, wherein the endogenous gene is selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, XP_003512331.2, and a sequence at least about 90% homologous thereto, wherein at least one exogenous SOI and one selection marker are flanked by a first RRS and a third RRS and at least one exogenous SOI is flanked by a second RRS and a third RRS; and b) culturing the cells in a) under conditions suitable for expression of the SOI, and recovering the polypeptide of interest therefrom.

[0152] In certain embodiments, the target polypeptide is produced and secreted into a cell culture medium. In certain embodiments, the target polypeptide is expressed and retained in a host cell. In certain embodiments, the target polypeptide is expressed, inserted into and retained in a host cell membrane.

[0153] Conventional cell biology methods can be used to introduce the target exogenous nucleotide or vector into the host cell, including but not limited to transfection, transduction, electroporation or injection. In certain embodiments, the target exogenous nucleotide or vector is introduced into the host cell using a chemical-based transfection method, including a lipid-based transfection method, a calcium phosphate-based transfection method, a cationic polymer-based transfection method, or a nanoparticle-based transfection method. In certain embodiments, the target exogenous nucleotide or vector is introduced into the host cell using viral-mediated transduction, including but not limited to transduction mediated by lentivirus, retrovirus, adenovirus or adeno-associated virus. In certain embodiments, the target exogenous nucleotide or vector is introduced into the host cell by gene gun-mediated injection. In certain embodiments, both DNA and RNA molecules are introduced into the host cell using the methods described herein.

[0154] 5. Targeted Integration

[0155] Targeted integration allows the exogenous nucleotide sequence to be integrated into one or more predetermined sites of the host cell genome. In certain embodiments, targeted integration is mediated by a recombinase that recognizes one or more RRS. In certain embodiments, targeted integration is mediated by homologous recombination. In certain embodiments, targeted integration is mediated by exogenous site-specific nucleases and subsequent HDR and / or NHEJ.

[0156] In certain embodiments, targeted integration can be combined with random integration. In certain embodiments, targeted integration can be followed by random integration. In certain embodiments, random integration can be mediated by any method or system known in the art. In certain embodiments, random integration is mediated by MaxCyte Electroporation system mediated.

[0157] 5.1. Targeted Integration via Recombinase-Mediated Recombination

[0158] A "recombination recognition sequence" (RRS) is a nucleotide sequence that is recognized by a recombinase and is necessary and sufficient for a recombinase-mediated recombination event. An RRS can be used to define the position in a nucleotide sequence where a recombination event will occur.

[0159] In certain embodiments, the RRS is selected from the group consisting of a LoxP sequence, a LoxP L3 sequence, a LoxP 2L sequence, a LoxFas sequence, a Lox511 sequence, a Lox2272 sequence, a Lox2372 sequence, a Lox5171 sequence, a Loxm2 sequence, a Lox71 sequence, a Lox66 sequence, a FRT sequence, a Bxb1 attP sequence, a Bxb1 attB sequence, a φC31 attP sequence, and a φC31 attB sequence.

[0160] In certain embodiments, the RRS is recognized by the Cre recombinase. In certain embodiments, the RRS is recognized by the FLP recombinase. In certain embodiments, the RRS is recognized by the Bxb1 integrase. In certain embodiments, the RRS is recognized by the φC31 integrase.

[0161] In certain embodiments, when the RRS is a LoxP site, the host cell requires Cre recombinase to perform recombination. In certain embodiments, when the RRS is a FRT site, the host cell requires FLP recombinase to perform recombination. In certain embodiments, when the RRS is a Bxb1 attP or Bxb1 attB site, the host cell requires Bxb1 integrase to perform recombination. In certain embodiments, when the RRS is a φC31 attP or φC31attB site, the host cell requires φC31 integrase to perform recombination. The recombinase can be introduced into the host cell using an expression vector comprising the coding sequence of the enzyme.

[0162] The Cre-LoxP site-specific recombination system has been widely used in many biological experimental systems. Cre is a 38-kDa site-specific DNA recombinase that recognizes a 34bp LoxP sequence. Cre is derived from bacteriophage P1 and belongs to the tyrosine family site-specific recombinase. Cre recombinase can mediate intramolecular and intermolecular recombination between LoxP sequences. The LoxP sequence consists of an 8bp non-palindromic core region flanked by two 13bp inverted repeat sequences. The Cre recombinase binds to the 13bp repeat sequence, thereby mediating recombination within the 8bp core region. Cre-LoxP-mediated recombination occurs with high efficiency and does not require any other host factors. If two LoxP sequences are placed in the same nucleotide sequence in the same orientation, Cre-mediated recombination will excise the DNA sequence between the two LoxP sequences to form a covalent closed loop. If two LoxP sequences are placed in the same nucleotide sequence in opposite orientations, Cre-mediated recombination will reverse the orientation of the DNA sequence between the two sequences. LoxP sequences can also be placed on different chromosomes to promote recombination between different chromosomes. If the two LoxP sequences are on two different DNA molecules and if one DNA molecule is a circular molecule, Cre-mediated recombination will result in the integration of the circular DNA sequence.

[0163] In certain embodiments, the LoxP sequence is a wild-type LoxP sequence. In certain embodiments, the LoxP sequence is a mutant LoxP sequence. Mutant LoxP sequences have been developed to improve Cre-mediated integration or replacement. In certain embodiments, the mutant LoxP sequence is selected from the group consisting of the following items: LoxP L3 sequence, LoxP 2L sequence, LoxFas sequence, Lox511 sequence, Lox2272 sequence, Lox2372 sequence, Lox5171 sequence, Loxm2 sequence, Lox71 sequence and Lox66 sequence. For example, the Lox71 sequence has 5bp mutations in the left 13bp repeat sequence. The Lox66 sequence has 5bp mutations in the right 13bp repeat sequence. Both wild-type and mutant LoxP sequences can mediate Cre-dependent recombination.

[0164] The FLP-FRT site-specific recombination system is similar to the Cre-Lox system. It relates to a flip enzyme (FLP) recombinase, which is derived from a 2 μm plasmid of Saccharomyces cerevisiae. FLP also belongs to the tyrosine family site-specific recombinase. The FRT sequence is a 34 bp sequence, consisting of two 13 bp palindromic sequences each flanked by an 8 bp spacer. FLP binds to the 13 bp palindromic sequence and mediates DNA breakage, exchange and connection within the 8 bp spacer. Similar to the Cre recombinase, the position and orientation of the two FRT sequences determine the FLP-mediated recombination results. In certain embodiments, the FRT sequence is a wild-type FRT sequence. In certain embodiments, the FRT sequence is a mutant FRT sequence. Both wild-type and mutant FRT sequences can mediate recombination that depends on FLP. In certain embodiments, the FRT sequence is fused to a reactive receptor domain sequence, such as, but not limited to, a tamoxifen reactive receptor domain sequence.

[0165] Bxb1 and φC31 belong to the family of serine recombinases. They are both derived from bacteriophages and are used by these phages to establish lysogeny to promote site-specific integration of the phage genome into the bacterial genome. These integrases catalyze site-specific recombination events between short (40-60 bp) DNA substrates called attP and attB sequences, which are originally attachment sites on the phage DNA and bacterial DNA, respectively. After recombination, two new sequences are formed, called attL and attR sequences, each containing half of the sequence from attP and attB. Recombination can also occur between attL and attR sequences, excising the integrated phage from the bacterial DNA. Both integrases can catalyze recombination without the aid of any additional host factors. In the absence of any auxiliary factors, these integrases mediate unidirectional recombination between attP and attB with an efficiency of more than 80%. Due to the short DNA sequences that can be recognized by these integrases and the unidirectional recombination, these recombination systems have been developed as a complement to the widely used Cre-LoxP and FRT-FLP systems for genetic engineering purposes.

[0166] The terms "matching RRS" and "homologous RRS" indicate that recombination has occurred between two RRSs. In certain embodiments, the two matching RRSs are identical. In certain embodiments, both RRSs are wild-type LoxP sequences. In certain embodiments, both RRSs are mutant LoxP sequences. In certain embodiments, both RRSs are wild-type FRT sequences. In certain embodiments, both RRSs are mutant FRT sequences. In certain embodiments, the two RRSs are different sequences but can be recognized by the same recombinase. In certain embodiments, the first matching RRS is a Bxb1 attP sequence, and the second matching RRS is a Bxb1 attB sequence. In certain embodiments, the first matching RRS is a φC31 attB sequence, and the second matching RRS is a φC31 attB sequence.

[0167] In certain embodiments, the integrated exogenous nucleotide sequence comprises two RRSs, and the vector comprises two RRSs, and the two RRSs match the two RRSs on the integrated exogenous nucleotide sequence, that is, the first RRS on the integrated exogenous nucleotide sequence matches the first RRS on the vector, and the second RRS on the integrated exogenous nucleotide sequence matches the second RRS on the vector. In certain embodiments, the first RRS on the integrated exogenous nucleotide sequence and the first RRS on the vector are the same as the second RRS on the integrated exogenous nucleotide sequence and the second RRS on the vector. A non-limiting example of such a "single vector RMCE" strategy is shown in Figure 2A. In certain embodiments, the first RRS on the integrated exogenous nucleotide sequence and the first RRS on the vector are different from the second RRS on the integrated exogenous nucleotide sequence and the second RRS on the vector. In certain embodiments, the first RRS on the integrated exogenous nucleotide sequence and the first RRS on the vector are both LoxP L3 sequences, and the second RRS on the integrated exogenous nucleotide sequence and the second RRS on the vector are both LoxP 2L sequences.

[0168] In certain embodiments, a "dual vector RMCE" strategy is employed. For example, but not by way of limitation, the integrated exogenous nucleotide sequence may comprise three RRSs, for example, having the following arrangement, wherein the third RRS ("RRS3") is present between the first RRS ("RRS1") and the second RRS ("RRS2"), and the first vector comprises two RRSs that match the first RRS and the third RRS on the integrated exogenous nucleotide sequence, and the second vector comprises two RRSs that match the third RRS and the second RRS on the integrated exogenous nucleotide sequence. An example of a dual vector RMCE strategy is shown in FIG4. In such examples, RRS1, RRS2, and RRS3 have heterologous specificity, for example, they do not cross-react with each other. In some embodiments, one vector (front) comprises RRS1, the first SOI and a promoter, followed by a start codon and RRS3 (in this order). Another vector (rear) comprises RRS3 fused to a coding sequence of a marker without a start codon (ATG), as well as SOI 2 and RRS2 (in this order). Additional nucleotides may be inserted between the RRS3 site and the selection marker sequence to ensure that the fusion protein undergoes frame translation. In some embodiments, the first SOI encodes an antibody. In some embodiments, the antibody is a single chain antibody, an antibody light chain, an antibody heavy chain, a single chain Fv fragment (scFv) or an Fc fusion protein. In some embodiments, the second SOI encodes an antibody. In some embodiments, the antibody is a single chain antibody, an antibody light chain, an antibody heavy chain, a single chain Fv fragment (scFv) or an Fc fusion protein. In certain embodiments, the first SOI and the second SOI are paired to encode an antibody to form a multispecific (e.g., bispecific antibody).

[0169] Such a dual-carrier RMCE strategy allows the introduction of eight or more SOIs by combining an appropriate number of SOIs between each pair of RRSs.

[0170] Both single-vector RMCE and dual-vector RMCE allow one or more donor DNA molecules to be unidirectionally integrated into a predetermined site of the host cell genome, and precisely exchange the DNA cassette present on the donor DNA with the DNA cassette on the host genome where the integration site is located. The DNA cassette is characterized by two heterospecific RRSs flanking at least one selection marker (although in some dual-vector RMCE examples, "separate selection markers" as outlined herein may be used) and / or at least one exogenous SOI. RMCE involves double recombination exchange events between two heterospecific RRSs within the target genomic locus and the donor DNA molecule, which are catalyzed by recombinases. RMCE is designed to introduce a copy of an SOI or selection marker into a predetermined locus of the host cell genome. Unlike recombination involving only one crossover event, RMCE can be implemented so that the prokaryotic vector sequence is not introduced into the host cell genome, thereby reducing and / or preventing unnecessary triggering of host immunity or defense mechanisms. The RMCE method can be repeated through multiple DNA cassettes.

[0171] In certain embodiments, targeted integration is achieved by a single crossover recombination event, wherein an exogenous nucleotide sequence is integrated into a predetermined site of the host cell genome, the exogenous nucleotide sequence comprising an RRS adjacent to at least one exogenous SOI or at least one selection marker. In certain embodiments, targeted integration is achieved by a single RMCE, wherein a DNA cassette is integrated into a predetermined site of the host cell genome, the DNA cassette comprising at least one exogenous SOI or at least one selection marker flanked by two heterospecific RRS. In certain embodiments, targeted integration is achieved by two RMCEs, wherein two different DNA cassettes are integrated into a predetermined site of the host cell genome, each of the two DNA cassettes comprising at least one exogenous SOI or at least one selection marker flanked by two heterospecific RRS. In certain embodiments, targeted integration is achieved by multiple RMCEs, wherein DNA cassettes from multiple vectors are all integrated into a predetermined site of the host cell genome, each of the DNA cassettes comprising at least one exogenous SOI or at least one selection marker flanked by two heterospecific RRS. In certain embodiments, the selection marker may be partially encoded on the first vector and partially encoded on the second vector, such that integration of two RMCEs allows expression of the selection marker. An example of such a system is shown in FIG4 .

[0172] In certain embodiments, targeted integration by recombinase-mediated recombination results in the integration of a selection marker or one or more exogenous SOIs into one or more predetermined integration sites of the host cell genome comprising sequences from a prokaryotic vector. In certain embodiments, targeted integration by recombinase-mediated recombination results in the integration of a selection marker or one or more exogenous SOIs into one or more predetermined integration sites of the host cell genome that does not contain sequences from a prokaryotic vector.

[0173] 5.2 Targeted integration via homologous recombination, HDR or NHEJ

[0174] The presently disclosed subject matter also relates to targeted integration mediated by homologous recombination or by exogenous site-specific nucleases followed by HDR or NHEJ.

[0175] Homologous recombination is the recombination between DNA molecules that share extensive sequence homology. It can be used to guide the error-free repair of double-stranded DNA breaks, and produces sequence changes in gametes during meiosis. Since homologous recombination involves the exchange of genetic information between two homologous DNA molecules, the overall arrangement of genes on the chromosome is not changed. During homologous recombination, a gap or break is formed in double-stranded DNA (dsDNA), and then a homologous dsDNA molecule is introduced by the end of a single-stranded DNA, and homologous sequence pairing, branch displacement, and Holliday junction formation occur, and the Holliday junction is finally separated.

[0176] Double-strand breaks (DSBs) are the most severe form of DNA damage, and the repair of such DNA damage is essential for maintaining the genome integrity of all organisms. There are two major DSB repair pathways. The first repair pathway is the homology-mediated repair (HDR) pathway, and homologous recombination is the most common form of HDR. Since HDR requires the presence of homologous DNA in the cell, this repair pathway is usually active in the S and G2 phases of the cell cycle, where the newly replicated sister chromatids can be used as homologous templates. HDR is also the main repair pathway for repairing collapsed replication forks during DNA replication. HDR is considered to be a relatively error-free repair pathway. The second DSB repair pathway is non-homologous end joining (NHEJ). NHEJ is a repair pathway that joins the ends of broken DNA together without the need for a homologous DNA template.

[0177] Exogenous site-specific nucleases and subsequent HDR can promote targeted integration. This is because the frequency of homologous recombination can be increased by introducing DSBs at specific target genomic sites. In certain embodiments, the exogenous nuclease can be selected from the group consisting of zinc finger nucleases (ZFNs), ZFN dimers, transcription activator-like effector nucleases (TALENs), TAL effector domain fusion proteins, RNA-guided DNA endonucleases, engineered large-range nucleases, and clustered regularly spaced short palindromic repeats (CRISPR)-related (Cas) endonucleases.

[0178] CRISPR / Cas and TALEN systems are two genome editing tools that can provide optimal construction simplicity and high efficiency. CRISPR / Cas has been identified as an immune defense mechanism for bacteria against invading phages. Cas is a nuclease that, when guided by a synthetic guide RNA (gRNA), can be associated with a specific nucleotide sequence in a cell and edit the DNA in or around the nucleotide sequence, for example, by forming one or more of a single-strand break, DSB and / or a point mutation. TALEN is an engineered site-specific nuclease consisting of a DNA binding domain of a TALE (class transcription activator effector nuclease) and a catalytic domain of a restriction endonuclease FokI. By changing the amino acids in the highly variable residue region of the monomer present in the DNA binding domain, artificial TALENs targeting various nucleotide sequences can be created. The DNA binding domain then directs the nuclease to the target sequence and forms a DSB.

[0179] Targeted integration by homologous recombination or HDR involves the presence of homologous sequences at the integration site. In certain embodiments, the homologous sequences are present on a vector. In certain embodiments, the homologous sequences are present on a polynucleotide.

[0180] In certain embodiments, the vector for targeted integration of an exogenous nucleotide sequence in a host cell comprises a nucleotide sequence homologous to an endogenous gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2, or homologous to a sequence selected from SEQ ID No. 1 to SEQ ID No. 7 flanked by at least one selection marker. In certain embodiments, the vector for targeted integration of an exogenous nucleotide sequence in a host cell comprises a nucleotide sequence homologous to an endogenous gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2, or homologous to a sequence selected from SEQ ID No. 1 to SEQ ID No. 7 flanked by at least one selection marker and at least one exogenous SOI. In certain embodiments, the vector for targeted integration of an exogenous nucleotide sequence in a host cell comprises a nucleotide sequence at least 50% homologous to a sequence selected from SEQ ID No. 1 to SEQ ID No. 7 flanked by a DNA cassette, wherein the DNA cassette comprises at least one selection marker flanked by two RRSs and at least one exogenous SOI. In certain embodiments, the vector for targeted integration of an exogenous nucleotide sequence in a host cell comprises a nucleotide sequence that is at least 50% homologous to an endogenous gene flanked by DNA selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2, wherein the DNA cassette comprises at least one selection marker flanked by two RRSs and at least one exogenous SOI. In certain embodiments, the vector nucleotide sequence is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or at least about 99.9% homologous to an endogenous gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2; or to a sequence selected from SEQ ID No. 1 to SEQ ID No. 7. In certain embodiments, the vector is selected from the group consisting of an adenoviral vector, an adeno-associated viral vector, a lentiviral vector, a retroviral vector, an integrating phage vector, a non-viral vector, a transposon and / or transposase vector, an integrase substrate, and a plasmid.

[0181] In certain embodiments, the polynucleotide for targeted integration of an exogenous nucleotide sequence in a host cell comprises a nucleotide sequence homologous to an endogenous gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2, or homologous to a sequence selected from SEQ ID No. 1 to SEQ ID No. 7 flanked by at least one selection marker. In certain embodiments, the polynucleotide for targeted integration of an exogenous nucleotide sequence in a host cell comprises a nucleotide sequence homologous to an endogenous gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2, or homologous to a sequence selected from SEQ ID No. 1 to SEQ ID No. 7 flanked by at least one selection marker and at least one exogenous SOI. In certain embodiments, the polynucleotide for targeted integration of an exogenous nucleotide sequence in a host cell comprises a nucleotide sequence at least 50% homologous to a sequence selected from SEQ ID No. 1 to SEQ ID No. 7 flanked by a DNA cassette, wherein the DNA cassette comprises at least one selection marker flanked by two RRSs and at least one exogenous SOI. In certain embodiments, the polynucleotide for targeted integration of an exogenous nucleotide sequence in a host cell comprises a nucleotide sequence that is at least 50% homologous to an endogenous gene flanked by DNA selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2, wherein the DNA cassette comprises at least one selection marker flanked by two RRSs and at least one exogenous SOI. In certain embodiments, the flanking nucleotide sequence is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99% or at least about 99.9% homologous to an endogenous gene selected from the group consisting of: LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2; or to a sequence selected from SEQ ID No.1 to SEQ ID No.7.

[0182] In certain embodiments, homologous recombination is performed in the absence of any auxiliary factors. In certain embodiments, there is a vector capable of integration to promote homologous recombination. In certain embodiments, the integration vector is selected from the group consisting of: an adeno-associated virus vector, a lentiviral vector, a retroviral vector, and an integrating phage vector.

[0183] 5.3 Adjustable targeted integration

[0184] In many cases, protein expression levels are not optimal, mainly because the encoded protein is difficult to express. The low expression level of difficult-to-express proteins may have a variety of difficult-to-identify reasons. One possibility is the toxicity of the protein expressed in the host cell. In such cases, a regulatable expression system can be used to express toxic proteins, in which the target sequence encoding the protein is under the control of an inducible promoter. In these systems, the expression of difficult-to-express proteins is promoted only when regulatory factors (e.g., small molecules, such as but not limited to tetracycline or its analogs, doxycycline (DOX)) are added to the culture medium. Regulating the expression of toxic proteins can alleviate toxic effects and enable the culture to achieve the desired cell growth before production. In certain embodiments, the regulatable targeted integration (RTI) system comprises an SOI integrated into a specific locus (e.g., an exogenous nucleic acid sequence comprising one or more RRSs) and transcribed in the presence of an regulatable promoter operably connected thereto. In certain embodiments, the RTI system can be used to determine the root cause of the low protein expression level of molecules that are difficult to express (such as but not limited to antibodies). In certain embodiments, the ability to selectively shut down the expression of SOI in the RTI system can be used to link the expression of SOI to the observed adverse effects.

[0185] In certain embodiments, to minimize the effects of transcription and cell line variability during root cause analysis of difficult-to-express molecules, a regulatable targeted integration (RTI) system can be used. For example, but not by way of limitation, expression of the SOI in a TI host can be triggered by adding a regulatory factor (e.g., doxycycline) to the culture medium. In certain embodiments, an RTI vector utilizes a promoter regulated by tetracycline to express the SOI, which is integrated into an exogenous nucleic acid sequence, such as one comprising an RRS, which itself is integrated into an integration site in the host cell genome, thereby achieving regulatable expression of the SOI.

[0186] In certain embodiments, the RTI system described in the present disclosure can be used to successfully determine the potential cause of low protein expression levels of SOI (e.g., therapeutic antibodies) compared to control cell lines. In certain embodiments, once the relative expression of SOI (e.g., therapeutic antibodies) in RTI cell lines is confirmed to be lower, protein translation inhibitor treatment (e.g., Dox and cycloheximide) can be used to assess the intracellular accumulation and secretion level of SOI.

[0187] 5.4 Adjustable system

[0188] The subject matter of the present disclosure also relates to adjustable systems for use in TI. For example, but not as a limitation, such regulation can be based on a gene switch for blocking or activating mRNA synthesis by adjustable coupling of a transcriptional repressor or activator with a constitutive or minimal promoter. In certain non-limiting embodiments, repression can be achieved by binding to a repressor protein, for example, wherein the protein spatially blocks transcription initiation, or actively represses transcription by a transcriptional silencer. In certain non-limiting embodiments, activation of a minimal promoter of a mammal or a virus-free enhancer can be achieved by adjustable coupling with an activation domain.

[0189] In certain embodiments, conditional coupling of transcriptional repressors or activators can be achieved by using allosteric proteins that bind to promoters in response to external stimuli. In certain embodiments, conditional coupling of transcriptional repressors or activators can be achieved by using intracellular receptors released from chelating proteins, thereby binding to target promoters. In certain embodiments, conditional coupling of transcriptional repressors or activators can be achieved by using chemically induced dimers.

[0190] In certain embodiments, the allosteric protein used in the TI system of the present disclosure can be a protein that regulates transcriptional activity in response to antibiotics, bacterial quorum sensing messengers, catabolites, or culture parameters (such as temperature, e.g., cold or hot). In certain embodiments, such RTI systems can be based on catabolites, e.g., bacterial inhibitors that control catabolism genes for alternative carbon sources have been transferred to mammalian cells. In certain embodiments, inhibition of target promoters can be achieved by cumate-reactive binding of inhibitor CymR. In certain embodiments, catabolite-based systems can rely on the 6-hydroxynicotine-reactive binding activation of a chimeric promoter by the prokaryotic inhibitor HdnoR fused to the herpes simplex VP16 transactivation domain.

[0191] In certain embodiments, the TI system can be a quorum sensing-based expression system derived from prokaryotes that manages intra- and inter-population communication through quorum sensing molecules. These quorum sensing molecules bind to receptors in target cells, modulate the affinity of the receptors to cognate promoters, and thereby activate specific regulator switches. In certain embodiments, the quorum sensing molecule can be N-(3-oxo-octanoyl)-homoserine lactone, in the presence of which the TraR-p65 fusion protein activates expression of a minimal promoter fused to a TraR-specific operator sequence. In certain embodiments, the quorum sensing molecule can be butyrolactone SCB1 (racemic 2-(1'-hydroxy-6-methylheptyl)-3-(hydroxymethyl)-butyrolactone) in a system based on the Streptomyces coelicolor A3(2) ScbR inhibitor, which binds to its cognate operator OScbR in the absence of SCB1. In certain embodiments, the quorum sensing molecule can be a homoserine-derived inducer used in the RTI system, in which the Pseudomonas aeruginosa quorum sensing suppressors RhlR and LasR are fused to the SV40T antigen nuclear localization sequence and the herpes simplex VP16 domain, and can activate a promoter containing a specific operator gene sequence (las box).

[0192] In certain embodiments, the inducing molecule that regulates the allosteric protein used in the RTI system of the present disclosure may be, but is not limited to, cumate, isopropyl-β-D-galactopyranoside (IPTG), macrolide, 6-hydroxynicotine, doxycycline, streptogramin, NADH, tetracycline.

[0193] In certain embodiments, the intracellular receptor used in the RTI system of the present disclosure may be a cytoplasmic or nuclear receptor. In certain embodiments, the RTI system of the present disclosure may use small molecules to release transcription factors from chelated and inhibited proteins. In certain embodiments, the RTI system of the present disclosure relies on steroid regulation, in which hormone receptors are fused to natural or artificial transcription factors, which can be released by HSP90 in the cytosol, migrate to the nucleus and activate the selected promoter. In certain embodiments, mutant receptors regulated by synthetic steroid analogs can be used to avoid crosstalk with endogenous steroid hormones. In certain embodiments, the receptor may be an estrogen receptor variant that responds to 4-hydroxytamoxifen or a progesterone receptor mutant induced by RU486. In certain embodiments, a rosiglitazone-responsive transcription switch derived from a nuclear receptor of human nuclear peroxisome proliferator-activated receptor γ (PPARγ) can be used in the RTI system of the present disclosure. In certain embodiments, a variant of a steroid response receptor may be RheoSwitch, which is based on a modified Choristoneura fumiferana ecdysone receptor and mouse retinoid X receptor (RXR) fused to a Gal4 DNA binding domain and a VP16 transactivator. In the presence of synthetic ecdysone, the RheoSwitch variant can bind and activate a minimal promoter fused to several repeats of a Gal4 response element.

[0194] In certain embodiments, the RTI systems disclosed herein may utilize chemically induced dimerization of DNA binding proteins and transcriptional activators to activate a minimal core promoter fused to a cognate operator gene. In certain embodiments, the RTI systems disclosed herein may utilize dimerization of FKBP and FRB regulated by rapamycin. In this system, FRB is fused to the p65 transactivator, and FKBP is fused to zinc finger domains for specific cognate operator gene sites, which are located upstream of the engineered minimal interleukin-12 promoter. In certain embodiments, FKBP may be mutated. In certain embodiments, the RTI systems disclosed herein may utilize bacterial gyrase B subunit (GyrB), wherein GyrB dimerizes in the presence of the antibiotic coumermycin and dissociates from neomycin.

[0195] In certain embodiments, the RTI system of the present disclosure can be used for tunable siRNA expression. In certain embodiments, the tunable siRNA expression system can be tetracycline, macrolide or closed and open QuoRex system. In certain embodiments, the RTI system can utilize the Xenopus terminal oligopyrimidine element (TOP), which blocks translation initiation by forming a hairpin structure in the 5' untranslated region.

[0196] In certain embodiments, the RTI systems described in the present disclosure can utilize gas-phase controlled expression, such as the acetaldehyde-inducible regulation (AIR) system. The AIR system can employ the Aspergillus nidulans AlcR transcription factor, which specifically activates the PAIR promoter assembled by the AlcR-specific operator gene in the presence of non-toxic concentrations of gaseous or liquid acetaldehyde.

[0197] In certain embodiments, the RTI system of the present disclosure may utilize a Tet-On or Tet-Off system. In such a system, the expression of one or more SOIs may be regulated by tetracycline or its analog doxycycline.

[0198] In certain embodiments, the RTI system of the present disclosure may utilize a PIP-on or PIP-off system. In such a system, the expression of the SOI may be regulated by, for example, pristine, tetracycline, and / or erythromycin.

[0199] 6. Preparation and Use of TI Host Cells

[0200] The subject matter of the present disclosure relates to methods for targeted integration of exogenous nucleotide sequences in host cells. In certain embodiments, these methods relate to integrating exogenous nucleotide sequences in host cells to produce host cells suitable for subsequent targeted integration of SOI. In certain embodiments, the methods include recombinase-mediated recombination. In certain embodiments, the methods relate to homologous recombination, HDR and / or NHEJ.

[0201] In certain embodiments, the subject matter of the present disclosure relates to methods for targeted integration of exogenous nucleotide sequences in host cells and combined with random integration of exogenous nucleotide sequences in host cells. In certain embodiments, these methods relate to targeted integration of exogenous nucleotide sequences in host cells to produce host cells suitable for subsequent targeted integration of SOIs, and combined with random integration of the same or different SOIs. In certain embodiments, the methods include recombinase-mediated recombination. In certain embodiments, the methods relate to homologous recombination, HDR and / or NHEJ.

[0202] 6.1 Preparation of TI host cells using recombinase-mediated recombination

[0203] In certain embodiments, the present disclosure provides methods for preparing TI host cells to express a polypeptide of interest, the methods comprising: a) providing a TI host cell comprising an exogenous nucleotide sequence integrated at a site within a locus in the genome of the host cell, wherein the locus is at least about 90% homologous to SEQ ID No. 1 to SEQ ID No. 7, wherein the exogenous nucleotide sequence comprises two RRSs flanked by at least one first selection marker; b) introducing a vector into the cell provided in a), the vector comprising two RRSs that match the two RRSs on the integrated exogenous nucleotide sequence and are flanked by at least one exogenous SOI and at least one second selection marker; c) introducing a recombinase that recognizes the RRS; and d) selecting TI cells that express the second selection marker, thereby isolating TI host cells that express the polypeptide of interest.

[0204] In certain embodiments, the present disclosure provides methods for preparing TI host cells to express a polypeptide of interest, the methods comprising: a) providing a TI host cell comprising an exogenous nucleotide sequence integrated at a site within an endogenous gene, the endogenous gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, XP_003512331.2, and sequences at least about 90% homologous thereto, wherein the exogenous nucleotide sequence comprises two RRSs flanked by at least one first selection marker; b) introducing a vector into the cells provided in a), the vector comprising two RRSs that match the two RRSs on the integrated exogenous nucleotide sequence and are flanked by at least one exogenous SOI and at least one second selection marker; c) introducing a recombinase that recognizes the RRS; and d) selecting TI cells that express the second selection marker, thereby isolating TI host cells that express the polypeptide of interest.

[0205] In certain embodiments, the present disclosure provides methods for preparing TI host cells to express a polypeptide of interest, the methods comprising: a) providing a TI host cell comprising an exogenous nucleotide sequence integrated at a site within a locus of the genome of the TI host cell, wherein the locus is at least about 90% homologous to SEQ ID No. 1 to SEQ ID No. 7, wherein the exogenous nucleotide sequence comprises a first DNA cassette comprising two heterologous specific RRSs flanked by at least one first selection marker; b) introducing into the cell provided in a) a vector comprising a second DNA cassette comprising two heterologous specific RRSs that match the two RRSs on the integrated exogenous nucleotide sequence and are flanked by at least one exogenous SOI and at least one second selection marker; c) introducing a recombinase, wherein the recombinase recognizes the RRS and performs a RMCE; and d) selecting TI cells expressing the second selection marker, thereby isolating TI host cells expressing the polypeptide of interest.

[0206] In certain embodiments, the present disclosure provides methods for preparing TI host cells for expressing a polypeptide of interest, the methods comprising: a) providing a TI host cell, the TI host cell comprising an exogenous nucleotide sequence integrated into a site within an endogenous gene, the endogenous gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, XP_003512331.2, and sequences at least about 90% homologous thereto, wherein the exogenous nucleotide sequence comprises a first DNA cassette, the first a) introducing into the cells provided in a) a vector comprising a second DNA cassette comprising two heterologous specific RRSs that match the two RRSs on the integrated exogenous nucleotide sequence and are flanked by at least one exogenous SOI and at least one second selection marker; c) introducing a recombinase that recognizes the RRSs and performs a RMCE; and d) selecting TI cells that express the second selection marker, thereby isolating TI host cells that express the polypeptide of interest.

[0207] In certain embodiments, the present disclosure provides methods for preparing a TI host cell to express a first polypeptide of interest and a second polypeptide of interest (wherein the first polypeptide and the second polypeptide may be the same or different), the methods comprising: a) providing a TI host cell, the TI host cell comprising an exogenous nucleotide sequence integrated into a locus within the genome of the host cell, wherein the locus is identical to SEQ ID No. 1 to SEQ ID No.7 is at least about 90% homologous, wherein the exogenous nucleotide sequence comprises a first RRS and a second RRS flanked by at least one first selection marker and a third RRS located between the first RRS and the second RRS, and all RRSs have heterologous specificity; b) introducing a first vector into the cells provided in a), the first vector comprising two RRSs, which match the first RRS and the third RRS on the integrated exogenous nucleotide sequence and are flanked by at least one first exogenous SOI and at least one second selection marker; c) introducing a second vector into the cells provided in a), the second vector comprising two RRSs, which match the second RRS and the third RRS on the integrated exogenous nucleotide sequence and are flanked by at least one second exogenous SOI; d) introducing one or more recombinases, wherein the one or more recombinases recognize RRS; and e) selecting TI cells expressing the second selection marker, thereby isolating TI host cells expressing the first target polypeptide and the second target polypeptide. In certain embodiments, as opposed to having the entire selection marker on the first vector, the first vector comprises a promoter sequence operably linked to the codon ATG flanked by a first SOI upstream and by an RRS downstream; and the second vector comprises a selection marker lacking the ATG transcription start codon flanked by an RRS upstream and by a second SOI downstream.

[0208] In certain embodiments, the present disclosure provides methods for preparing a TI host cell to express a first polypeptide of interest and a second polypeptide of interest (wherein the first polypeptide and the second polypeptide may be the same or different), the methods comprising: a) providing a TI host cell, the TI host cell comprising an exogenous nucleotide sequence integrated into a site within an endogenous gene, the endogenous gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, XP_003512331.2, and sequences at least about 90% homologous thereto, wherein the exogenous nucleotide sequence comprises a first RRS and a second RRS flanked by at least one first selection marker and a third RRS located between the first RRS and the second RRS S, and all RRSs are heterologous specific; b) introducing into the cells provided in a) a first vector comprising two RRSs, which match the first RRS and the third RRS on the integrated exogenous nucleotide sequence and are flanked by at least one first exogenous SOI and at least one second selection marker; c) introducing into the cells provided in a) a second vector comprising two RRSs, which match the second RRS and the third RRS on the integrated exogenous nucleotide sequence and are flanked by at least one second exogenous SOI; d) introducing one or more recombinases, wherein the one or more recombinases recognize the RRSs; and e) selecting TI cells expressing the second selection marker, thereby isolating TI host cells expressing the first polypeptide of interest and the second polypeptide of interest. In certain embodiments, in contrast to having the entire selection marker on the first vector, the first vector comprises a promoter sequence operably linked to a codon ATG flanked by the first SOI upstream and the RRS downstream; and the second vector comprises a selection marker lacking the ATG transcription start codon flanked by the RRS upstream and the second SOI downstream.

[0209] In certain embodiments, the present disclosure provides methods for preparing a TI host cell to express a first polypeptide of interest and a second polypeptide of interest (wherein the first polypeptide and the second polypeptide may be the same or different), the methods comprising: a) providing a TI host cell, the TI host cell comprising an exogenous nucleotide sequence integrated into a locus within the genome of the host cell, wherein the locus is identical to SEQ ID No. 1 to SEQ ID No.7 is at least about 90% homologous, wherein the exogenous nucleotide sequence comprises a first DNA cassette, the first DNA cassette comprising a first RRS and a second RRS flanked by at least one first selection marker and a third RRS located between the first RRS and the second RRS, and all three RRSs have heterologous specificity; b) introducing a first vector comprising a second DNA cassette into the cells provided in a), wherein the second DNA cassette comprises two heterologous specific RRSs, which match the first RRS and the third RRS of the first DNA cassette and are flanked by at least one first exogenous SOI and at least one second selection marker; c) introducing a second vector comprising a third DNA cassette into the cells provided in a), wherein the third DNA cassette comprises two heterologous specific RRSs, which match the second RRS and the third RRS of the first DNA cassette and are flanked by at least one second exogenous SOI; d) introducing one or more recombinases, wherein the one or more recombinases recognize RRS and perform two RMCEs; and e) selecting TI cells expressing the second selection marker, thereby isolating TI host cells expressing the first target polypeptide and the second target polypeptide. In certain embodiments, as opposed to having the entire selection marker on the first vector, the first vector comprises a promoter sequence operably linked to the codon ATG flanked by a first SOI upstream and by an RRS downstream; and the second vector comprises a selection marker lacking the ATG transcription start codon flanked by an RRS upstream and by a second SOI downstream.

[0210] In certain embodiments, the present disclosure provides methods for preparing a TI host cell to express a first polypeptide of interest and a second polypeptide of interest (wherein the first polypeptide and the second polypeptide may be the same or different), the methods comprising: a) providing a TI host cell, the TI host cell comprising an exogenous nucleotide sequence integrated into a site within an endogenous gene, the endogenous gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, XP_003512331.2, and sequences at least about 90% homologous thereto, wherein the exogenous nucleotide sequence comprises a first DNA cassette comprising a first RRS and a second RRS flanked by at least one first selection marker and a third RRS located between the first RRS and the second RRS, and all three RRSs have a heterologous b) introducing into the cells provided in a) a first vector comprising a second DNA cassette, wherein the second DNA cassette comprises two heterologous specific RRSs, which match the first RRS and the third RRS of the first DNA cassette and are flanked by at least one first exogenous SOI and at least one second selection marker; c) introducing into the cells provided in a) a second vector comprising a third DNA cassette, wherein the third DNA cassette comprises two heterologous specific RRSs, which match the second RRS and the third RRS of the first DNA cassette and are flanked by at least one second exogenous SOI; d) introducing one or more recombinases, wherein the one or more recombinases recognize the RRS and perform two RMCEs; and e) selecting TI cells expressing the second selection marker, thereby isolating TI host cells expressing the first polypeptide of interest and the second polypeptide of interest. In certain embodiments, as opposed to having the entire selection marker on the first vector, the first vector comprises a promoter sequence operably linked to the codon ATG flanked by a first SOI upstream and by an RRS downstream; and the second vector comprises a selection marker lacking the ATG transcription start codon flanked by an RRS upstream and by a second SOI downstream.

[0211] In certain embodiments, the present disclosure provides methods for preparing TI host cells to express a polypeptide of interest, the methods comprising: a) providing a TI host cell comprising an exogenous nucleotide sequence integrated at a site within a locus in the genome of the host cell, wherein the locus is at least about 90% homologous to a sequence selected from SEQ ID No. 1 to SEQ ID No. 7, wherein the exogenous nucleotide sequence comprises an RRS adjacent to at least one first selection marker; b) introducing a vector into the cell provided in a), the vector comprising an RRS that matches the RRS on the integrated exogenous nucleotide sequence and is flanked by at least one exogenous SOI and at least one second selection marker; c) introducing a recombinase, wherein the recombinase recognizes the RRS; and d) selecting TI cells expressing the second selection marker, thereby isolating TI host cells expressing the polypeptide of interest.

[0212] In certain embodiments, the present disclosure provides methods for preparing TI host cells to express a polypeptide of interest, the methods comprising: a) providing a TI host cell comprising an exogenous nucleotide sequence integrated at a site within an endogenous gene, the endogenous gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, XP_003512331.2, and sequences at least about 90% homologous thereto, wherein the exogenous nucleotide sequence comprises an RRS adjacent to at least one first selection marker; b) introducing a vector into the cell provided in a), the vector comprising an RRS that matches the RRS on the integrated exogenous nucleotide sequence and is flanked by at least one exogenous SOI and at least one second selection marker; c) introducing a recombinase, wherein the recombinase recognizes the RRS; and d) selecting TI cells expressing the second selection marker, thereby isolating TI host cells expressing the polypeptide of interest.

[0213] The disclosed subject matter also relates to methods of producing a polypeptide of interest, the methods comprising: a) providing a TI host cell as described herein; b) culturing the TI host cell in a) under conditions suitable for expressing the SOI, and recovering the polypeptide of interest therefrom.

[0214] In certain embodiments, the present disclosure provides methods for preparing TI host cells suitable for subsequent targeted integration, the methods comprising: a) providing a TI host cell, the TI host cell comprising an exogenous nucleotide sequence integrated at a site within a locus in the genome of the host cell, wherein the locus is at least about 90% homologous to a sequence selected from SEQ ID No. 1 to SEQ ID No. 7, wherein the exogenous nucleotide sequence comprises two RRSs flanked by at least one exogenous SOI and at least one first selection marker; b) introducing a vector into the cell provided in a), the vector comprising two RRSs that match the two RRSs on the integrated exogenous nucleotide sequence and are flanked by at least one second selection marker; c) introducing a recombinase, wherein the recombinase recognizes the RRS; and d) selecting TI cells expressing the second selection marker, thereby isolating TI host cells suitable for subsequent targeted integration.

[0215] In certain embodiments, the present disclosure provides methods for preparing TI host cells suitable for subsequent targeted integration, the methods comprising: a) providing a TI host cell comprising an exogenous nucleotide sequence integrated at a site within an endogenous gene, the endogenous gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, XP_003512331.2, and sequences at least about 90% homologous thereto, wherein the exogenous nucleotide sequence comprises two RRSs flanked by at least one exogenous SOI and at least one first selection marker; b) introducing a vector into the cells provided in a), the vector comprising two RRSs that match the two RRSs on the integrated exogenous nucleotide sequence and are flanked by at least one second selection marker; c) introducing a recombinase that recognizes the RRS; and d) selecting TI cells that express the second selection marker, thereby isolating TI host cells suitable for subsequent targeted integration.

[0216] In certain embodiments, the present disclosure provides methods for preparing TI host cells suitable for subsequent targeted integration, the methods comprising: a) providing a TI host cell, the TI host cell comprising an exogenous nucleotide sequence integrated at a site within a locus of the genome of the host cell, wherein the locus is at least about 90% homologous to a sequence selected from SEQ ID No. 1 to SEQ ID No. 7, wherein the exogenous nucleotide sequence comprises a first RRS and a second RRS flanked by at least one exogenous SOI and at least one first selection marker; b) introducing a vector into the cell provided in a), the vector comprising three RRSs, wherein the first RRS of the vector matches the first RRS on the integrated exogenous nucleotide sequence, the second RRS of the vector matches the second RRS on the integrated exogenous nucleotide sequence, and at least one second selection marker is located between the first RRS and the second RRS; c) introducing a recombinase, wherein the recombinase recognizes the first RRS and the second RRS on both the vector and the integrated exogenous nucleotide sequence; and d) selecting TI host cells expressing the second selection marker, thereby isolating TI host cells suitable for subsequent targeted integration.

[0217] In certain embodiments, the present disclosure provides methods for preparing a TI host cell suitable for subsequent targeted integration, the methods comprising: a) providing a TI host cell comprising an exogenous nucleotide sequence integrated at a site within an endogenous gene, the endogenous gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, XP_003512331.2, and sequences at least about 90% homologous thereto, wherein the exogenous nucleotide sequence comprises at least one exogenous SOI flanked by at least one first selection marker. a) introducing into the cells provided in a) a vector comprising three RRSs, wherein the first RRS of the vector matches the first RRS on the integrated exogenous nucleotide sequence, the second RRS of the vector matches the second RRS on the integrated exogenous nucleotide sequence, and at least one second selection marker is located between the first RRS and the second RRS; c) introducing a recombinase, wherein the recombinase recognizes the first RRS and the second RRS on both the vector and the integrated exogenous nucleotide sequence; and d) selecting TI host cells expressing the second selection marker, thereby isolating TI host cells suitable for subsequent targeted integration.

[0218] 6.2 Methods for targeted modification of host cells using homologous recombination, HDR or NHEJ

[0219] In certain embodiments, the present disclosure provides methods for preparing a TI host cell to express a polypeptide of interest, the methods comprising: a) providing a TI host cell, the TI host cell comprising a locus in the genome of the host cell, wherein the locus is at least about 90% homologous to SEQ ID No. 1 to SEQ ID No. 7; b) introducing a vector into the TI host cell, wherein the vector comprises a nucleotide sequence at least 50% homologous to a sequence selected from SEQ ID No. 1 to SEQ ID No. 7 flanked by a DNA cassette, wherein the DNA cassette comprises at least one selection marker and at least one exogenous SOI; and c) selecting for the selection marker to isolate a TI host cell, the TI host cell comprising the SOI integrated into the locus in the genome, and expressing the polypeptide of interest. In certain embodiments, the DNA cassette of the vector further comprises at least one selection marker flanked by two RRSs and at least one exogenous SOI.

[0220] In certain embodiments, the present disclosure provides methods for preparing a TI host cell to express a polypeptide of interest, the methods comprising: a) providing a TI host cell, the TI host cell comprising a locus in the genome of the host cell, wherein the locus is at least about 90% homologous to a sequence selected from SEQ ID No. 1 to SEQ ID No. 7; b) introducing a polynucleotide into the TI host cell, wherein the polynucleotide comprises a nucleotide sequence at least 50% homologous to a sequence selected from SEQ ID No. 1 to SEQ ID No. 7 flanked by a DNA cassette, wherein the DNA cassette comprises at least one selection marker and at least one exogenous SOI; and c) selecting for the selection marker to isolate a TI host cell, the TI host cell comprising the SOI integrated into the locus in the genome, and expressing the polypeptide of interest. In certain embodiments, the DNA cassette of the vector further comprises at least one selection marker flanked by two RRSs and at least one exogenous SOI.

[0221] In certain embodiments, homologous recombination is promoted by an integration vector. In certain embodiments, the vector is selected from the group consisting of an adenoviral vector, an adeno-associated viral vector, a lentiviral vector, a retroviral vector, an integrating phage vector, a non-viral vector, a transposon and / or a transposase vector, an integrase substrate, and a plasmid. In certain embodiments, the transposon may be a PiggyBac (PB) transposon system.

[0222] In certain embodiments, integration is promoted by exogenous nucleases. In certain embodiments, the exogenous nuclease is selected from the group consisting of zinc finger nucleases (ZFNs), ZFN dimers, transcription activator-like effector nucleases (TALENs), TAL effector domain fusion proteins, RNA-guided DNA endonucleases, engineered meganucleases, and clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) endonucleases.

[0223] In certain embodiments, the present disclosure provides methods for preparing TI host cells suitable for subsequent targeted integration, the methods comprising: a) providing a TI host cell, the TI host cell comprising a locus in the genome of the host cell, wherein the locus is at least about 90% homologous to a sequence selected from SEQ ID No.1 to SEQ ID No.7; b) introducing a vector into the TI host cell, wherein the vector comprises a nucleotide sequence at least 50% homologous to a sequence selected from SEQ ID No.1 to SEQ ID No.7 flanked by a DNA cassette, wherein the DNA cassette comprises at least one selection marker flanked by two RRSs; c) selecting for the selection marker to isolate the TI host cell suitable for subsequent targeted integration.

[0224] In certain embodiments, the present disclosure provides methods for preparing TI host cells suitable for subsequent targeted integration, the methods comprising: a) providing a TI host cell, the TI host cell comprising a locus in the genome of the host cell, wherein the locus is at least about 90% homologous to a sequence selected from SEQ ID No.1 to SEQ ID No.7; b) introducing a polynucleotide into the TI host cell, wherein the polynucleotide comprises a nucleotide sequence that is at least 50% homologous to a sequence selected from SEQ ID No.1 to SEQ ID No.7 flanked by a DNA cassette, wherein the DNA cassette comprises at least one selection marker flanked by two RRSs; c) selecting for the selection marker to isolate the TI host cell suitable for subsequent targeted integration.

[0225] In certain embodiments, the present disclosure provides methods for preparing TI host cells suitable for subsequent targeted integration, the methods comprising: a) providing a TI host cell, the TI host cell comprising a locus in the genome of the host cell, wherein the locus is at least about 90% homologous to a sequence selected from SEQ ID No.1 to SEQ ID No.7; b) introducing a vector into the host cell, wherein the vector comprises a nucleotide sequence at least 50% homologous to a sequence selected from SEQ ID No.1 to SEQ ID No.7 flanked by a DNA cassette, wherein the DNA cassette comprises three RRSs, wherein the third RRS and at least one selection marker are located between the first RRS and the second RRS; c) selecting for the selection marker to isolate the TI host cell suitable for subsequent targeted integration.

[0226] In certain embodiments, the present disclosure provides methods for preparing TI host cells suitable for subsequent targeted integration, the methods comprising: a) providing a TI host cell, the TI host cell comprising a locus in the genome of the host cell, wherein the locus is at least about 90% homologous to a sequence selected from SEQ ID No.1 to SEQ ID No.7; b) introducing a polynucleotide into the host cell, wherein the polynucleotide comprises a nucleotide sequence that is at least 50% homologous to a sequence selected from SEQ ID No.1 to SEQ ID No.7 flanked by a DNA cassette, wherein the DNA cassette comprises three RRSs, wherein the third RRS and at least one selection marker are located between the first RRS and the second RRS; and c) selecting for the selection marker to isolate the TI host cell suitable for subsequent targeted integration.

[0227] In certain embodiments, the present disclosure provides methods for preparing a TI host cell that expresses at least one polypeptide of interest, the methods comprising: a) providing a TI host cell comprising at least one exogenous nucleotide sequence integrated into a site within one or more loci in the genome of the TI host cell, wherein the one or more loci are at least about 90% homologous to a sequence selected from SEQ ID No. 1 to SEQ ID No. 7, wherein the at least one exogenous nucleotide sequence comprises two RRSs flanked by at least one first selection marker; b) introducing a vector into the cell provided in a), the vector comprising two RRSs that match the two RRSs on the integrated exogenous nucleotide sequence and are flanked by at least one exogenous SOI and at least one second selection marker; c) introducing a recombinase or a nucleic acid encoding a recombinase, wherein the recombinase recognizes the RRS; and selecting TI cells that express the second selection marker, thereby isolating TI host cells that express the at least one polypeptide of interest.

[0228] In certain embodiments, the present disclosure provides methods for preparing a TI host cell expressing at least a first polypeptide of interest and a second polypeptide of interest (wherein the first polypeptide and the second polypeptide may be the same or different), the methods comprising: a) providing a TI host cell, the TI host cell comprising at least one exogenous nucleotide sequence integrated into a site within one or more loci of the genome of the host cell, wherein the one or more loci are selected from SEQ ID No. 1 to SEQ ID No.7 is at least about 90% homologous to the sequence of No.7, wherein the exogenous nucleotide sequence comprises a first RRS and a second RRS flanked by at least one first selection marker and a third RRS located between the first RRS and the second RRS, and all RRSs have heterologous specificity; b) introducing a first vector into the cells provided in a), the first vector comprising two RRSs, the two RRSs matching the first RRS and the third RRS on at least one integrated exogenous nucleotide sequence and flanked by at least one first exogenous SOI and at least one second selection marker; c) introducing a second vector into the cells provided in a), the second vector comprising two RRSs matching the second RRS and the third RRS on at least one integrated exogenous nucleotide sequence and flanked by at least one second exogenous SOI; d) introducing one or more recombinases or one or more nucleic acids encoding one or more recombinases, wherein the one or more recombinases recognize RRS; and e) selecting TI cells expressing the second selection marker, thereby isolating TI host cells expressing at least one first target polypeptide and a second target polypeptide. In certain embodiments, as opposed to having the entire selection marker on the first vector, the first vector comprises a promoter sequence operably linked to the codon ATG flanked by a first SOI upstream and by an RRS downstream; and the second vector comprises a selection marker lacking the ATG transcription start codon flanked by an RRS upstream and by a second SOI downstream.

[0229] In certain embodiments, the present disclosure provides methods for preparing TI host cells that express a polypeptide of interest, the methods comprising: a) providing a TI host cell comprising at least one exogenous nucleotide sequence integrated into a site within one or more loci of the genome of the TI host cell, wherein the one or more loci are at least about 90% homologous to a sequence selected from SEQ ID No. 1 to SEQ ID No. 7, wherein the exogenous nucleotide sequence comprises one or more RRSs; b) introducing a vector into the cell provided in a), the vector comprising one or more RRSs that match the one or more RRSs on the integrated exogenous nucleotide sequence and flanked by at least one exogenous SOI that is operably linked to a regulatable promoter; c) introducing a recombinase or a nucleic acid encoding a recombinase, wherein the recombinase recognizes the RRS; and d) selecting a TI cell that expresses the exogenous SOI in the presence of an inducer, thereby isolating a TI host cell that expresses the polypeptide of interest.

[0230] In certain embodiments, the present disclosure provides methods for expressing a polypeptide of interest, the methods comprising: a) providing a host cell comprising at least one exogenous SOI flanked by two RRSs and a regulatable promoter integrated into a locus in the genome of the host cell, wherein the locus is at least about 90% homologous to a sequence selected from SEQ ID No. 1 to SEQ ID No. 7; and b) culturing the cell under conditions suitable for expressing the SOI, and recovering the polypeptide of interest therefrom.

[0231] In certain embodiments, the present disclosure provides methods for preparing a TI host cell expressing a first polypeptide of interest and a second polypeptide of interest (wherein the first polypeptide and the second polypeptide may be the same or different), the methods comprising: a) providing a TI host cell, the TI host cell comprising an exogenous nucleotide sequence integrated into a locus within the genome of the host cell, wherein the locus is selected from SEQ ID No. 1 to SEQ ID No.7 is at least about 90% homologous to the sequence of No.7, wherein the exogenous nucleotide sequence comprises a first RRS, a second RRS, and a third RRS located between the first RRS and the second RRS, and all RRSs have heterologous specificity; b) introducing a first vector into the cell provided in a), the first vector comprising two RRSs, the two RRSs matching the first RRS and the third RRS on the integrated exogenous nucleotide sequence and flanking at least one first exogenous SOI operably linked to a regulatable promoter; c) introducing a second vector into the cell provided in a), the second vector comprising two RRSs, the two RRSs matching the second RRS and the third RRS on the integrated exogenous nucleotide sequence and flanking at least one second SOI operably linked to a regulatable promoter; d) introducing one or more recombinases or one or more nucleic acids encoding one or more recombinases, wherein the one or more recombinases recognize RRS; and e) selecting TI cells expressing the first exogenous SOI and the second exogenous SOI in the presence of an inducer, thereby isolating TI host cells expressing the polypeptide of interest. In certain embodiments, as opposed to having the entire selection marker on the first vector, the first vector comprises a promoter sequence operably linked to the codon ATG flanked by a first SOI upstream and by an RRS downstream; and the second vector comprises a selection marker lacking the ATG transcription start codon flanked by an RRS upstream and by a second SOI downstream.

[0232] 7. Products

[0233] Host cells of the present disclosure can be used to express any target molecule (e.g., target polypeptide). In certain embodiments, host cells of the present disclosure can be used to express polypeptides (e.g., mammalian polypeptides). Non-limiting examples of such polypeptides include hormones, receptors, fusion proteins, regulatory factors, growth factors, complement system factors, enzymes, coagulation factors, anticoagulation factors, kinases, cytokines, CD proteins, interleukins, therapeutic proteins, diagnostic proteins, and antibodies. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a therapeutic antibody. In some embodiments, the antibody is a diagnostic antibody. In some embodiments, the antibody is a human antibody. In some embodiments, the antibody is a humanized antibody.

[0234] In certain embodiments, examples of polypeptides encompassed within the definitions herein include mammalian polypeptides such as, for example, renin; growth hormones, including human growth hormone and bovine growth hormone; growth hormone releasing factor; parathyroid hormone; thyroid stimulating hormone; lipoprotein; alpha-1-antitrypsin; insulin A chain; insulin B chain; proinsulin; follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon; leptin; coagulation factors such as factor VIIIC, factor IX, tissue factor, and von Willebrands factor; anticoagulant factors such as protein C; atrial natriuretic peptide; pulmonary surfactant; plasminogen activators, such as urokinase or human urinary or tissue-type plasminogen activator (t-PA); bombesin; thrombin; hematopoietic growth factors; tumor necrosis factor-α and tumor necrosis factor-β; tumor necrosis factor receptors such as death receptor 5 and CD120; TNF-related apoptosis-inducing ligand (TRAIL); B cell maturation antigen (BCMA); B lymphocyte stimulating factor (BLyS); enhancer apoptosis-inducing ligand (APRIL); enkephalinase; RANTES (regulatory T-cell activation normally expressed and secreted); human macrophage inflammatory protein (MIP-1-α); serum albumin such as human serum albumin; Muellerian inhibitory substance; relaxin A chain; relaxin B chain; prorelaxin; mouse gonadotropin-related peptide; microbial proteins such as beta-lactamase, deoxyribonuclease; IgE; cytotoxic T lymphocyte-associated antigen (CTLA), such as CTLA-4; inhibin; activin; Platelet-derived endothelial cell growth factor (PD-ECGF); vascular endothelial growth factor family proteins (e.g., VEGF-A, VEGF-B, VEGF-C, VEGF-D and P1GF); platelet-derived growth factor (PDGF) family proteins (e.g., PDGF-A, PDGF-B, PDGF-C, PDGF-D and dimers thereof); fibroblast growth factor (FGF) family such as aFGF, bFGF, FGF4 and FGF9; epidermal growth factor (EGF); hormones or growth factors receptors such as VEGF receptors (e.g., VEGFR1, VEGFR2, and VEGFR3), epidermal growth factor (EGF) receptors (e.g., ErbB1, ErbB2, ErbB3, and ErbB4 receptors), platelet-derived growth factor (PDGF) receptors (e.g., PDGFR-α and PDGFR-β), and fibroblast growth factor receptors; TIE ligands (angiogenin, ANGPT1, ANGPT2); angiogenin receptors such as TIE1 and TIE2; protein A or D; rheumatoid factor;Neurotrophic factors such as bone-derived neurotrophic factor (BDNF), neurotrophin-3, neurotrophin-4, neurotrophin-5 or neurotrophin-6 (NT-3, NT-4, NT-5 or NT-6) or nerve growth factors such as NGF-b; transforming growth factors (TGFs) such as TGF-α and TGF-β, including TGF-β1, TGF-β2, TGF-β3, TGF-β4 or TGF-β5; insulin-like growth factor-I and -II (IGF-I and IGF-II); des(1-3)-IGF-I (brain IGF-I), Insulin-like growth factor binding protein (IGFBP); CD proteins such as CD3, CD4, CD8, CD19 and CD20; erythropoietin; osteoinductive factors; immunotoxins; bone morphogenetic proteins (BMPs); chemokines such as CXCL12 and CXCR4; interferons such as interferon-α, -β and -γ; colony stimulating factors (CSFs), e.g., M-CSF, GM-CSF and G-CSF; cytokines such as interleukins (ILs), e.g., IL-1 to IL-10; midkine; superoxide dismutase; T cell receptors; surface membrane proteins; decay accelerating factors; Viral antigens such as, for example, a portion of the AIDS envelope; transporters; homing receptors; addressing proteins; regulatory proteins; integrins such as CD11a, CD11b, CD11c, CD18, ICAM, VLA-4, and VCAM; ephrins; Bv8; delta-like ligand 4 (DLL4); Del-1; BMP9; BMP10; follistatin; hepatocyte growth factor (HGF) / scatter factor (SF); Alk1; Robo4; ESM1; perlecan; EGF-like domain 7 (EGFL7); CTGF and its family members; thrombospondins such as platelet-receptor kinase inhibitors (PRE-1). 1 and thrombospondin 2; collagens such as collagen IV and collagen XVIII; neuropilins such as NRP1 and NRP2; pleiotrophin (PTN); progranulin; synuclein; Notch proteins such as Notch1 and Notch4; inducible proteins such as Sema3A, Sema3C and Sema3F; tumor-associated antigens such as CA125 (ovarian cancer antigen); immunoadhesins; any of the polypeptides listed above and fragments and / or variants of antibodies (including antibody fragments) and one or more proteins (including, for example, any of the proteins listed above) are bound. ;

[0235] In certain embodiments, the polypeptide of interest is a bispecific antibody, a trispecific antibody, or a multispecific antibody (e.g., a bispecific antibody). Various molecular forms of multispecific antibodies are known in the art and are included herein (see, e.g., Spiess et al., Mol Immunol 67 (2015) 95-106). A specific type of multispecific antibody also included herein is a bispecific antibody designed to simultaneously bind to a surface antigen on a target cell (e.g., a tumor cell) and an activation-invariant component of a T cell receptor (TCR) complex (such as CD3) for retargeting T cells to kill target cells. Other examples of bispecific antibody formats include, but are not limited to, so-called "BiTE" (bispecific T cell engager) molecules, in which two scFv molecules are fused via a flexible linker (see, e.g., WO2004 / 106381, WO2005 / 061547, WO2007 / 042261, and WO2008 / 119567; Nagorsen and Exp Cell Res 317, 1255-1260 (2011)); diabodies (Holliger et al., Prot Eng 9, 299-305 (1996)) and their derivatives, such as tandem diabodies ("TandAb"; Kipriyanov et al., J Mol Biol 293, 41-56 (1999)); "DART" (dual affinity retargeting) molecules, which are based on the diabody format but feature a C-terminal disulfide bridge for additional stabilization (Johnson et al., J Mol Biol 399, 436-449 (2010)), and so-called triomabs, which are fully hybrid mouse / rat IgG molecules (reviewed in Seimetz et al., Cancer Treat Rev 36, 458-467 (2010)). Specific T cell bispecific antibody formats encompassed herein are described in the following documents: WO 2013 / 026833; WO 2013 / 026839; WO 2016 / 020309; Bacac et al., Oncoimmunology 5(8) (2016) e1203498.

[0236] In certain embodiments, the host cells of the present disclosure can be used to express molecular chaperones, protein modifying enzymes, shRNAs, gRNAs, or other proteins or peptides, while constitutively or regulatably expressing therapeutic proteins or molecules of interest.

[0237] In some embodiments, the polypeptides expressed by the host cells of the present disclosure may bind to or interact with any protein including, but not limited to, cytokines, cytokine-related proteins, and cytokine receptors selected from the group consisting of 8MPI, 8MP2, 8MP38 (GDFIO), 8MP4, 8MP6, 8MP8, CSFI (M-CSF), CSF2 (GM-CSF), CSF3 (G-CSF), EPO, FGF1 (αFGF), FGF2 (βFGF), FGF3 (int-2), FGF4 (HST), FGF5, FGF6 (HST-2), FGF7 (KGF), FGF9 ... (HST-2), FGF7 (KGF), FGF1 (KGF), FGF1 (KGF), FGF2 (GM-CSF), CSF3 (G-CSF), EPO, FGF1 (αFGF), FGF2 (βFGF), FGF3 (int-2), FGF4 (HST), FGF5 (HST-2), FGF6 (HST-2), FGF7 (KGF), FGF1 (KGF), FGF2 (GM-CSF), CSF3 (G-CSF), EPO, FGF1 (αFGF), FGF2 (βFGF), FGF3 (int-2), FGF4 (HST), FGF5 0. FGF11, FGF12, FGF12B, FGF14, FGF16, FGF17, FGF19, FGF20, FGF21, FGF23, IGF1, IGF2, IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFN81, IFNG, IFNWI, FEL1, FEL1(EPSELON), FEL1(ZETA), IL 1A, IL 1B, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL1 0, IL 11, IL 12A, IL 12B, IL 13, IL 14, IL 15, IL 16, IL 17, IL 17B, IL 18, IL19, IL20, IL22, IL23, IL24, IL25, IL26, IL27, IL28A, IL28B, IL29, IL30, PDGFA, PDGFB, TGFA, TGFB1, TGFB2, TGFBb3, LTA (TNF-β), LTB, TNF (TNF-α), TNFSF4 (OX40 ligand), TNFSF5 (CD40 ligand), TNFSF6 (FasL), TNFSF7 (CD27 ligand), TNFSF8 (CD30 ligand), TNFSF9 (4-1BB ligand), TNFSF10 (TRAI L), TNFSF11(TRANCE), TNFSF12(APO3L), TNFSF13(April), TNFSF13B, TNFSF14(HVEM-L), TNFSF15(VEGI), TNFSF18, HGF(VEGFD), VEGF, VE GFB, VEGFC, IL1R1, IL1R2, IL1RL1, IL1RL2, IL2RA, IL2RB, IL2RG, IL3RA, IL4R, IL5RA, IL6R, IL7R, IL8RA, IL8RB, IL9R, IL10RA, IL10RB, IL11RA, IL12RB1, IL12RB2, IL13RA1, IL13RA2, IL15RA, IL17R, IL18R1, IL20RA, IL21R, IL22R, IL1HY1, IL1RAP, IL1RAPL1, IL1RAPL2, IL1RN, IL6ST, IL18BP, IL18RAP, IL22RA2, AIF1, HGF, LEP (leptin), PTN and THPO.k.

[0238]

[0239] In certain embodiments, the target molecules of the antibodies (or bispecific antibodies) produced according to the methods disclosed herein include CD proteins, such as CD3, CD4, CDS, CD16, CD19, CD20, CD21 (CR2 (complement receptor 2) or C3DR (C3d / Epstein-Barr virus receptor) or Hs.73792); CD33; CD34; CD64; CD72 (B cell differentiation antigen CD72, Lyb-2); CD79b (CD79B, CD79β, IGb (immunoglobulin-related β), B29); CD200 members of the ErbB receptor family, such as EGFR; F receptor, HER2, HER3 or HER4 receptor; cell adhesion molecules such as LFA-1, Mac1, p150.95, VLA-4, ICAM-1, VCAM, α4 / β7 integrin and αv / β3 integrin, including their α subunits or β subunits (e.g., anti-CD11a, anti-CD18 or anti-CD11b antibodies); growth factors such as VEGF-A, VEGF-C; tissue factor (TF); alpha interferon (αIFN); TNFα, interleukins such as IL-1β, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-13, IL 17AF, IL-1S, IL-13Rα1, IL13Rα2, IL-4R, IL-5R, IL-9R, IgE; blood group antigens; flk2 / flt3 receptors; obesity (OB) receptor; mpl receptor; CTLA-4; RANKL, RANK, RSV F protein, protein C, etc. In certain embodiments, the methods provided herein can be used to generate antibodies (or multispecific antibodies, such as bispecific antibodies) that specifically bind to complement protein C5 (e.g., anti-C5 agonist antibodies that specifically bind to human C5.

[0240] In certain embodiments, the methods provided herein can be used to generate antibodies (or multispecific antibodies, such as bispecific antibodies) that specifically bind to influenza B virus hemagglutinin, i.e., "fluB" (e.g., antibodies that bind in vitro and / or in vivo to a hemagglutinin of influenza B virus from the Yamagata lineage, to a hemagglutinin of influenza B virus from the Victoria lineage, or to a hemagglutinin of influenza B virus from the Yamagata lineage, the Victoria lineage, and the ancestral lineage). Further details on anti-FluB antibodies are described in WO 2015 / 148806, which is incorporated herein by reference in its entirety.

[0241] In certain embodiments, the antibodies (or bispecific antibodies) produced according to the methods provided herein bind to low-density lipoprotein receptor-related protein (LRP)-1 or LRP-8 or transferrin receptor and at least one target selected from the group consisting of: β-secretase (BACE1 or BACE2), α-secretase, γ-secretase, τ-secretase, amyloid precursor protein (APP), death receptor 6 (DR6), amyloid β, α-synuclein, parkin, huntingtin, p75 NTR, CD40 and caspase-6.

[0242] In certain embodiments, the antibody produced according to the methods provided herein is a human IgG2 antibody against CD40. In certain embodiments, the anti-CD40 antibody is RG7876.

[0243] In certain embodiments, the polypeptide produced according to the methods provided herein is a targeted immunocytokine. In certain embodiments, the targeted immunocytokine is a CEA-IL2v immunocytokine. In certain embodiments, the CEA-IL2v immunocytokine is RG7813. In certain embodiments, the targeted immunocytokine is a FAP-IL2v immunocytokine. In certain embodiments, the FAP-IL2v immunocytokine is RG7461.

[0244] In certain embodiments, the multispecific antibodies (such as bispecific antibodies) produced according to the methods provided herein bind to CEA and at least one other target molecule. In certain embodiments, the multispecific antibodies (such as bispecific antibodies) produced according to the methods provided herein bind to tumor-targeted cytokines and at least one other target molecule. In certain embodiments, the multispecific antibodies (such as bispecific antibodies) produced according to the methods provided herein fuse to IL2v (i.e., interleukin 2 variants) and bind to immune cytokines based on IL1 and at least one other target molecule. In certain embodiments, the multispecific antibodies (such as bispecific antibodies) produced according to the methods provided herein are T cell bispecific antibodies (i.e., bispecific T cell engagers or BiTEs).

[0245] In certain embodiments, the multispecific antibodies (such as bispecific antibodies) produced according to the methods provided herein bind to at least two target molecules selected from the group consisting of IL-1α and IL-1β, IL-12 and IL-1S; IL-13 and IL-9; IL-13 and IL-4; IL-13 and IL-5; IL-5 and IL-4; IL-13 and IL-1β; IL-13 and IL-25; IL-13 and TARC; IL-13 and MDC; IL-13 and MEF; IL-13 and TGF-β ... IL-4; IL-13 and IL-5; IL-5 and IL-4; IL-13 and IL-1β; IL-13 and IL-25; IL-13 and TARC; IL-13 and MDC; IL-13 and MEF; IL-13 and TGF-β; IL -13 and LHR agonist; IL-12 and TWEAK, IL-13 and CL25; IL-13 and SPRR2a; IL-13 and SPRR2b; IL-13 and ADAMS, IL-13 and PED2, IL17A and IL17F, CEA and CD3, CD3 and CD19, CD138 and CD20; CD138 and CD40; CD19 and CD20; CD20 and CD3; CD3S and CD13S; CD3S and CD20; CD3S and CD40; CD40 and CD 20; CD-S and IL-6; CD20 and BR3, TNFα and TGF-β, TNFα and IL-1β; TNFα and IL-2, TNFα and IL-3, TNFα and IL-4, TNFα and IL-5, TNFα and IL6, TNFα and IL8, TNFα and IL-9, TNFα and IL-10, TNFα and IL-11, TNFα and IL-12, TNFα and IL-13, TNFα and IL-14, TNFα and IL-15, TNFα and IL-16, TNF Fα and IL-17, TNFα and IL-18, TNFα and IL-19, TNFα and IL-20, TNFα and IL-23, TNFα and IFNα, TNFα and CD4, TNFα and VEGF, TNFα and MIF, TNFα and ICAM-1, TNFα and PGE4, TNFα and PEG2, TNFα and RANK ligand, TNFα and Te38, TNFα and BAFF, TNFα and CD22, TNFα and CTLA-4, TNFα and GP130, TNF a and IL-12p40, VEGF and angiopoietin, VEGF and HER2, VEGF-A and HER2, VEGF-A and PDGF, HER1 and HER2, VEGFA and ANG2, VEGF-A and VEGF-C, VEGF-C and VEGF-D, HER2 and DR5, VEGF and IL-8, VEGF and MET, VEGFR and MET receptor, EGFR and MET, VEGFR and EGFR, HER2 and CD64, HER2 and CD3, HER2 and CD16, HER2 and HER3;EGFR (HER1) and HER2, EGFR and HER3, EGFR and HER4, IL-14 and IL-13, IL-13 and CD40L, IL4 and CD40L, TNFR1 and IL-1R, TNFR1 and IL-6R and TNFR1 and IL-18R, EpCAM and CD3, MAPG and CD28, EGFR and CD64, CSPGs and RGM A; CTLA-4 and BTN02; IGF1 and IGF2; IGF1 / 2 and Erb2B; MAG and RGM A; NgR and RGM A; NogoA and RGM A; OMGp and RGMA; POL-1 and CTLA-4; and RGM A and RGM B. ;

[0246] In certain embodiments, the multispecific antibody (such as a bispecific antibody) is an anti-CEA / anti-CD3 bispecific antibody. In certain embodiments, the anti-CEA / anti-CD3 bispecific antibody is RG7802. Further details of anti-CEA / anti-CD3 bispecific antibodies are provided in WO 2014 / 121712, which is incorporated herein by reference in its entirety.

[0247] In certain embodiments, the multispecific antibody (such as a bispecific antibody) is an anti-VEGF / anti-angiogenin bispecific antibody. In certain embodiments, the anti-VEGF / anti-angiogenin bispecific antibody is Crossmab. In certain embodiments, the anti-VEGF / anti-angiogenin bispecific antibody is RG7716.

[0248] In certain embodiments, the multispecific antibody (such as a bispecific antibody) is an anti-Ang2 / anti-VEGF bispecific antibody. In certain embodiments, the anti-Ang2 / anti-VEGF bispecific antibody is RG7221. In certain embodiments, the anti-Ang2 / anti-VEGF bispecific antibody is CAS No. 1448221-05-3.

[0249] Host cells according to the present disclosure may express numerous other antibodies and / or other proteins, and the above list is not intended to be limiting.

[0250] The host cells of the present disclosure can be used to produce the target molecule at a production scale. "Manufacturing scale" production of therapeutic proteins or other proteins utilizes cell cultures ranging from about 400 L to about 80,000 L, depending on the protein produced and the demand. Typically, such manufacturing scale production utilizes cell cultures ranging from about 400 L to about 25,000 L. Within this range, specific cell cultures such as 4,000 L, about 6,000 L, about 8,000 L, about 10,000 L, about 12,000 L, about 14,000 L, or about 16,000 L can be utilized.

[0251] In certain embodiments, the polypeptide of interest is a bispecific antibody, a trispecific antibody, or a multispecific antibody (eg, a bispecific antibody).

[0252] The host cells of the present disclosure can be used to produce large amounts of target molecules in a shorter time than non-TI cells used in current cell culture methods. In certain embodiments, the host cells of the present disclosure can be used to improve the quality of target molecules compared to non-TI cells used in current cell culture methods. In certain embodiments, the host cells of the present disclosure can enhance the stability of seed lines by preventing chronic toxicity caused by unstable products that may cause cell stress and clone over time. In certain embodiments, the host cells of the present disclosure can be used to optimize the expression of acute toxic products.

[0253] In certain embodiments, the host cells (TI systems) of the present disclosure can be used for cell culture process optimization and / or process development.

[0254] In certain embodiments, the host cells of the present disclosure can be used to accelerate the production of the target molecule by about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, or about 10 weeks compared to non-TI cells used in conventional cell culture methods. In certain embodiments, the host cells of the present disclosure can be used to accelerate the harvest of the target molecule by about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, or about 10 weeks compared to non-TI cells used in conventional cell culture methods.

[0255] In certain embodiments, the host cells of this embodiment can be used to reduce the aggregation level of a molecule of interest compared to non-TI cells used in conventional cell culture methods.

[0256] In certain embodiments, host cells of the present disclosure can be used to achieve higher levels of expression of one or more polypeptides of interest relative to random integration host cells. For example, but not limited to, host cells of the present disclosure can achieve expression of standard antibodies and half antibodies at a titer of at least 3 g / L, 3.5 g / L, 4 g / L, 4.5 g / L, 5 g / L, 5.5 g / L, 6 g / L, 6.5 g / L, 7 g / L, 7.5 g / L, 8 g / L, 8.5 g / L, 9 g / L, 9.5 g / L, 10 g / L, 10.5 g / L, 11 g / L or more, and achieve expression of multispecific antibodies (e.g., bispecific antibodies) at a titer of at least 1.5 g / L, 2 g / L, 2.5 g / L, 3 g / L, 3.5 g / L, 4 g / L, 4.5 g / L, 5 g / L, 5.5 g / L, 6 g / L or more. In certain embodiments, host cells of the present disclosure can achieve higher bispecific content relative to random integration host cells. For example, but not limited to, host cells of the present disclosure can achieve at least 80%, 85%, 90%, 95%, 96%, 98%, 99% or more bispecific content.

[0257] In certain embodiments, the host cells of the present disclosure can be used for constitutive expression of selected subunits of a therapeutic molecule and regulated expression of other different subunits of the same therapeutic molecule. In certain embodiments, the therapeutic molecule can be a fusion protein. In certain embodiments, the host cells of the present disclosure can be used to understand the role and influence of various antibody subunits in the expression and secretion of fully assembled antibody molecules.

[0258] In certain embodiments, the host cells of the present disclosure can be used as research tools. In certain embodiments, the host cells of the present disclosure can be used as diagnostic tools to identify the root causes of low protein expression levels of problematic molecules in various cells. In certain embodiments, the host cells of the present disclosure can be used to directly link observed phenomena or cell behaviors to transgenic expression in cells. The host cells of the present disclosure can also be used to demonstrate whether the observed behavior is reversible in cells. In certain embodiments, the host cells of the present disclosure can be used to identify and alleviate problems related to transgenic transcription and expression in cells.

[0259] In certain embodiments, the host cells of the present disclosure can be used to exchange transgenic subunits of difficult-to-express molecules (such as, but not limited to, HC and LC subunits of antibodies) with transgenic subunits of common molecules in the TI system to identify one or more subunits that are problematic. In certain embodiments, amino acid sequence analysis can then be used to narrow down and focus on amino acid residues or regions that may be responsible for low protein expression levels.

[0260] 8. Exemplary Non-Limiting Embodiments

[0261] A. A targeted integration (TI) host cell, comprising: a targeted integration exogenous nucleotide sequence, which is integrated at an integration site within a specific locus in the genome of the host cell, wherein the locus is at least about 90% homologous to a sequence selected from SEQ ID No.1 to SEQ ID No.7; and a randomly integrated exogenous nucleic acid sequence, wherein the randomly integrated sequence is integrated into the genome of the host cell at least once.

[0262] A1. The TI host cell according to A, wherein the nucleotide sequence immediately adjacent to the 5' end of the integrated targeted integration exogenous nucleotide sequence is selected from the group consisting of the following sequences, which are at least about 90% homologous to the following items: nucleotides 41190-45269 of NW_006874047.1, nucleotides 63590-207911 of NW_006884592.1, .1, nucleotides 253831-491909 of NW_003616412.1, nucleotides 69303-79768 of NW_003615063.1, nucleotides 293481-315265 of NW_006882936.1, nucleotides 2650443-2662054 of NW_003615411.1.

[0263] A2. The TI host cell according to A, wherein the nucleotide sequence immediately adjacent to the 5' end of the integrated targeted integration exogenous nucleotide sequence is selected from the group consisting of the following sequences, which sequences include nucleotide 45269 from NW_006874047.1, nucleotide 207911 of NW_006884592.1, nucleotide 491909 of NW_006881296.1, nucleotide 79768 of NW_003616412.1, nucleotide 315265 of NW_003615063.1, nucleotide 315266 of NW_006882936 .1 or nucleotide 2662054 of NW_003615411.1 or at least 15 base pairs, at least 20 base pairs, at least 30 base pairs, at least 40 base pairs, at least 50 base pairs, at least 75 base pairs, at least 100 base pairs, at least 150 base pairs, at least 200 base pairs, at least 300 base pairs, at least 400 base pairs, at least 500 base pairs, at least 1,000 base pairs, at least 1,500 base pairs, at least 2,000 base pairs, at least 3,000 base pairs.

[0264] A3. The TI host cell according to A, wherein the nucleotide sequence immediately adjacent to the 3' end of the integrated targeted integration exogenous nucleotide sequence is selected from the group consisting of the following sequences, which are at least about 90% homologous to the following: nucleotides 45270-45490 of NW_006874047.1, nucleotides 207912-792374 of NW_006884592.1, .1, nucleotides 491910-667813 of NW_003616412.1, nucleotides 79769-100059 of NW_003615063.1, nucleotides 315266-362442 of NW_003615063.1, nucleotides 2662055-2701768 of NW_006882936.1, or nucleotides 97706-105117 of NW_003615411.1.

[0265] A4. The TI host cell according to A, wherein the nucleotide sequence immediately adjacent to the 3' end of the integrated targeted exogenous nucleotide sequence is selected from the group consisting of the following sequences, which sequences include nucleotide 45270 from NW_006874047.1, nucleotide 207912 from NW_006884592.1, nucleotide 491910 from NW_006881296.1, nucleotide 79769 from NW_003616412.1, nucleotide 315266 from NW_003615063.1, NW_006882936. 1 or nucleotide 97706 of NW_003615411.1.

[0266] A5. The TI host cell according to any one of A-A4, wherein the integrated targeted exogenous nucleotide sequence is operably linked to a nucleotide sequence selected from the group consisting of: a sequence that is at least about 90% homologous to the sequences of SEQ ID No. 1 to SEQ ID No. 7.

[0267] A6. A TI host cell, comprising: an exogenous nucleotide sequence for targeted integration integrated at an integration site within an endogenous gene, the endogenous gene being selected from the group consisting of: LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, XP_003512331.2, and sequences at least about 90% homologous thereto; and a randomly integrated exogenous nucleic acid sequence, wherein the randomly integrated sequence is integrated into the genome of the host cell at least once.

[0268] A7. A TI host cell, comprising: a targeted integration exogenous nucleotide sequence integrated at an integration site operably linked to an endogenous gene, the endogenous gene selected from the group consisting of: LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, XP_003512331.2 and sequences at least about 90% homologous thereto; and a randomly integrated exogenous nucleic acid sequence, wherein the randomly integrated sequence is integrated into the genome of the host cell at least once.

[0269] A8. A TI host cell, comprising: a targeted integration exogenous nucleotide sequence integrated at an integration site adjacent to all or part of the following sequence, the sequence being selected from the group consisting of sequences that are at least about 90% homologous to a sequence selected from SEQ ID No.1 to SEQ ID No.7; and a randomly integrated exogenous nucleic acid sequence, wherein the randomly integrated sequence is integrated into the genome of the host cell at least once.

[0270] A9. The TI host cell according to any one of A-A8, wherein the TI host cell is a mammalian host cell.

[0271] A10. The TI host cell according to A9, wherein the TI host cell is a hamster host cell, a human host cell, a rat host cell or a mouse host cell.

[0272] A11. The TI host cell according to A9 or A10, wherein the TI host cell is a Chinese hamster ovary (CHO) host cell, a CHO K1 host cell, a CHO K1SV host cell, a DG44 host cell, a DUKXB-11 host cell, a CHOK1S host cell or a CHO K1M host cell.

[0273] A12. The TI host cell according to any one of A-A11, wherein the exogenous nucleotide sequence targeted for integration comprises one or more recombination recognition sequences (RRS), wherein the RRS can be recognized by a recombinase.

[0274] A13. The TI host cell according to A12, wherein the exogenous nucleotide sequence targeted for integration comprises at least two RRSs.

[0275] A14. The TI host cell according to A12 or A13, wherein the recombinase is Cre recombinase or FLP recombinase.

[0276] A15. The TI host cell according to A12 or A13, wherein the recombinase is Bxb1 integrase or φC31 integrase.

[0277] A16. The TI host cell according to any one of A12-A15, wherein RRS is selected from the group consisting of: LoxP sequence, LoxP L3 sequence, LoxP 2L sequence, LoxFas sequence, Lox511 sequence, Lox2272 sequence, Lox2372 sequence, Lox5171 sequence, Loxm2 sequence, Lox71 sequence, Lox66 sequence, FRT sequence, Bxb1 attP sequence, Bxb1 attB sequence, φC31 attP sequence and φC31 attB sequence.

[0278] A17. The TI host cell according to any one of A13 to A16, wherein the exogenous nucleotide sequence targeted for integration comprises a first RRS and a second RRS and at least one selection marker located between the first RRS and the second RRS.

[0279] A18. The TI host cell according to A17, comprising a first selection marker, wherein the first selection marker is selected from the group consisting of aminoglycoside phosphotransferase (APH) (hygromycin phosphotransferase (HYG), neomycin, G418 APH), dihydrofolate reductase (DHFR), thymidine kinase (TK), glutamine synthetase (GS), asparagine synthetase, tryptophan synthetase (indole), histidinol dehydrogenase (histidinol D), blasticidin, bleomycin, phleomycin, chloramphenicol, Zeocin and mycophenolic acid.

[0280] A19. The TI host cell according to A17 or A18, further comprising a second selection marker, wherein the first selection marker and the second selection marker are different.

[0281] A20. According to the TI host cell described in A19, the second selection marker is selected from the group consisting of: aminoglycoside phosphotransferase (APH) (hygromycin phosphotransferase (HYG), neomycin, G418 APH), dihydrofolate reductase (DHFR), thymidine kinase (TK), glutamine synthetase (GS), asparagine synthetase, tryptophan synthetase (indole), histidinol dehydrogenase (histidinol D), blasticidin, bleomycin, phleomycin, chloramphenicol, Zeocin and mycophenolic acid.

[0282] A21. The TI host cell according to A19 or A20, further comprising a third selection marker and an internal ribosome entry site (IRES), wherein the IRES is operably linked to the third selection marker.

[0283] A22. The TI host cell according to A21, wherein the third selection marker is different from the first selection marker or the second selection marker.

[0284] A23. The TI host cell according to A21 or A22, wherein the third selection marker is selected from the group consisting of a green fluorescent protein (GFP) marker, an enhanced GFP (eGFP) marker, a synthetic GFP marker, a yellow fluorescent protein (YFP) marker, an enhanced YFP (eYFP) marker, a cyan fluorescent protein (CFP) marker, an mPlum marker, an mCherry marker, a tdTomato marker, an mStrawberry marker, a J-red marker, a DsRed-monomer marker, a mOrange marker, an mKO marker, an mCitrine marker, a Venus marker, a YPet marker, an Emerald6 marker, a CyPet marker, a mCFPm marker, a Cerulean marker and a T-Sapphire marker.

[0285] A24. The TI host cell according to any one of A17-A23, wherein the exogenous nucleotide sequence for targeted integration further comprises a third RRS, wherein the third RRS is located between the first RRS and the second RRS, and the third RRS has heterologous specificity relative to the first RRS or the second RRS.

[0286] A25. The TI host cell according to any one of A12-A24, wherein the exogenous nucleotide sequence targeted for integration comprises at least one selection marker and at least one exogenous sequence of interest (SOI).

[0287] A26. The TI host cell according to any one of A12-A25, wherein the exogenous nucleotide sequence targeted for integration further comprises at least one exogenous SOI.

[0288] A27. The TI host cell according to A26, wherein the exogenous SOI for targeted integration is located between the first RRS and the second RRS.

[0289] A28. The TI host cell according to A26 or A27, wherein the SOI targeted for integration encodes a single-chain antibody, an antibody light chain, an antibody heavy chain, a single-chain Fv fragment (scFv) or an Fc fusion protein.

[0290] A29. The TI host cell according to any one of A24-A28, wherein the exogenous nucleotide sequence for targeted integration further comprises at least one exogenous SOI for targeted integration located between the first RRS and the third RRS and at least one exogenous SOI for targeted integration located between the third RRS and the second RRS.

[0291] A30. The TI host cell according to A29, wherein the SOI targeted for integration encodes a single-chain antibody, an antibody light chain, an antibody heavy chain, a single-chain Fv fragment (scFv) or an Fc fusion protein.

[0292] A31. The TI host cell of A26, wherein the targeted integrated SOI encodes an antibody expressed at a higher level relative to the randomly integrated host cell.

[0293] A32. The TI host cell according to A31, wherein the antibody is a standard antibody or a half antibody, and its expression titer is at least 3g / L, 3.5g / L, 4g / L, 4.5g / L, 5g / L, 5.5g / L, 6g / L, 6.5g / L, 7g / L, 7.5g / L, 8g / L, 8.5g / L, 9g / L, 9.5g / L, 10g / L, 10.5g / L, 11g / L or higher.

[0294] A33. The TI host cell of A31, wherein the antibody is a multispecific antibody (e.g., a bispecific antibody) and its expression titer is at least 1.5 g / L, 2 g / L, 2.5 g / L, 3 g / L, 3.5 g / L, 4 g / L, 4.5 g / L, 5 g / L, 5.5 g / L, 6 g / L.

[0295] A34. The TI host cell of A31, wherein the antibody is a bispecific antibody and the bispecific content is increased relative to the random integration host cell.

[0296] A35. The TI host cell according to A1, wherein the antibody is a bispecific antibody and the bispecific content is at least 80%, 85%, 90%, 95%, 96%, 98%, 99% or more.

[0297] B. A method for preparing a TI host cell expressing a polypeptide of interest, the method comprising: providing a TI host cell, the TI host cell comprising: an exogenous nucleotide sequence for targeted integration, the exogenous nucleotide sequence for targeted integration being integrated at a site within a locus of the genome of the TI host cell, wherein the locus is at least about 90% homologous to a sequence selected from SEQ ID No. 1 to SEQ ID No. 7; and a randomly integrated exogenous nucleic acid sequence, wherein the randomly integrated sequence is integrated into the genome of the host cell at least once, wherein the exogenous nucleotide sequence for targeted integration comprises two RRSs, and the two RRSs are flanked by at least one first selection marker; introducing a vector into the cell provided in a), the vector comprising two RRSs, the two RRSs matching the two RRSs on the exogenous nucleotide sequence for targeted integration and flanked by at least one exogenous SOI and at least one second selection marker; introducing a recombinase or a nucleic acid encoding a recombinase, wherein the recombinase recognizes the RRS; and selecting TI cells expressing the second selection marker, thereby isolating TI host cells expressing the polypeptide.

[0298] B1. The method according to B, wherein the recombinase is Cre recombinase or FLP recombinase.

[0299] B2. The method according to B, wherein the recombinase is Bxb1 integrase or φC31 integrase.

[0300] B3. A method according to any one of B-B2, wherein SOI encodes a single-chain antibody, an antibody light chain, an antibody heavy chain, a single-chain Fv fragment (scFv) or an Fc fusion protein.

[0301] B4. The method according to any one of claims B-B3, wherein the TI host cell is a mammalian host cell.

[0302] B5. The method according to B4, wherein the TI host cell is a hamster host cell, a human host cell, a rat host cell or a mouse host cell.

[0303] B6. The method according to B4 or B5, wherein the TI host cell is a CHO host cell, a CHO K1 host cell, a CHO K1SV host cell, a DG44 host cell, a DUKXB-11 host cell, a CHOK1S host cell or a CHO K1M host cell.

[0304] C. A method for expressing a target polypeptide, the method comprising: providing a TI host cell, the TI host cell comprising at least one targeted integrated exogenous SOI and at least one selection marker, wherein the at least one targeted integrated exogenous SOI and the at least one selection marker are flanked by two RRSs, the two RSSs are integrated into a locus of the genome of the TI host cell, wherein the locus is at least about 90% homologous to a sequence selected from SEQ ID No.1 to SEQ ID No.7, and the TI host cell comprises a randomly integrated exogenous nucleic acid sequence, wherein the randomly integrated sequence is integrated into the genome of the host cell at least once; and culturing the cells in a) under conditions suitable for expressing the target polypeptide, and recovering the target polypeptide therefrom.

[0305] C1. The method according to C, wherein the at least one exogenous SOI targeted for integration encodes a single-chain antibody, an antibody light chain, an antibody heavy chain, a single-chain Fv fragment (scFv) or an Fc fusion protein.

[0306] D. A method for preparing a TI host cell expressing at least a first target polypeptide and a second target polypeptide, the method comprising: providing a TI host cell, the TI host cell comprising a targeted integration exogenous nucleotide sequence integrated into a locus within the genome of the host cell, wherein the locus is selected from SEQ ID No. 1 to SEQ ID No.7 is at least about 90% homologous, and the TI host cell comprises a randomly integrated exogenous nucleic acid sequence, wherein the randomly integrated sequence is integrated into the genome of the host cell at least once, wherein the exogenous nucleotide sequence for targeted integration comprises a first RRS and a second RRS flanked by at least one first selection marker and a third RRS located between the first RRS and the second RRS, and all RRSs have heterologous specificity; introducing a first vector into the cell provided in a), the first vector comprising two RRSs, the two RRSs matching the first RRS and the third RRS on the integrated targeted integration exogenous nucleotide sequence and flanked by at least one first targeted integration exogenous SOI and at least one second selection marker; introducing a second vector into the cell provided in a), the second vector comprising two RRSs, the two RRSs matching the second RRS and the third RRS on the integrated targeted integration exogenous nucleotide sequence and flanked by at least one second targeted integration exogenous SOI; introducing one or more recombinases or one or more nucleic acids encoding one or more recombinases, wherein the one or more recombinases recognize RRS; and selecting TI cells expressing the second selection marker, thereby isolating TI host cells expressing the first target polypeptide and the second target polypeptide.

[0307] D1. The method according to D, wherein the recombinase is Cre recombinase or FLP recombinase.

[0308] D2. The method according to D, wherein the recombinase is Bxb1 integrase or φC31 integrase.

[0309] D3. The method according to D, wherein: the first vector further comprises a promoter sequence, which is operably linked to the codon ATG with a first SOI on the upstream side and an RRS on the downstream side; and the second vector further comprises a selection marker, which lacks the ATG transcription start codon with an RRS on the upstream side and a second SOI on the downstream side.

[0310] D4. The method according to any one of D-D3, wherein the first SOI encodes a single-chain antibody, an antibody light chain, an antibody heavy chain, a single-chain Fv fragment (scFv) or an Fc fusion protein.

[0311] D5. The method according to any one of D-D3, wherein the second SOI encodes a single-chain antibody, an antibody light chain, an antibody heavy chain, a single-chain Fv fragment (scFv) or an Fc fusion protein.

[0312] D6. A method according to any one of C or D-D5, wherein the TI host cell is a mammalian host cell.

[0313] D7. The method according to D6, wherein the TI host cell is a hamster host cell, a human host cell, a rat host cell or a mouse host cell.

[0314] D8. The method according to D7, wherein the TI host cell is a CHO host cell, a CHO K1 host cell, a CHOK1SV host cell, a DG44 host cell, a DUKXB-11 host cell, a CHOK1S host cell or a CHO K1M host cell.

[0315] E. A method for expressing a polypeptide of interest, the method comprising: providing a host cell, the host cell comprising at least two targeted-integrated exogenous SOIs and at least one selection marker integrated into a locus of the host cell's genome, wherein the locus is at least about 90% homologous to a sequence selected from SEQ ID No. 1 to SEQ ID No. 7, and the host cell comprising a randomly integrated exogenous nucleic acid sequence, wherein the randomly integrated sequence is integrated at least once into the genome of the host cell, wherein at least one targeted-integrated exogenous SOI and one selection marker are flanked by a first RRS and a third RRS, and at least one targeted-integrated exogenous SOI is flanked by a second RRS and a third RRS; and culturing the cells in a) under conditions suitable for expressing the polypeptide of interest, and recovering the polypeptide of interest therefrom.

[0316] E1. The method according to E, wherein the first SOI encodes a single chain antibody, an antibody light chain, an antibody heavy chain, a single chain Fv fragment (scFv) or an Fc fusion protein.

[0317] E2. The method according to E or E1, wherein the second SOI encodes a single chain antibody, an antibody light chain, an antibody heavy chain, a single chain Fv fragment (scFv) or an Fc fusion protein.

[0318] E3. A method according to any one of E-E2, wherein the TI host cell is a mammalian host cell.

[0319] E4. The method according to E3, wherein the TI host cell is a hamster host cell, a human host cell, a rat host cell or a mouse host cell.

[0320] E5. The method according to E4, wherein the TI host cell is a CHO host cell, a CHO K1 host cell, a CHOK1SV host cell, a DG44 host cell, a DUKXB-11 host cell, a CHOK1S host cell or a CHO K1M host cell.

[0321] F. A method for expressing a target polypeptide, the method comprising: providing a TI host cell, the TI host cell comprising at least one targeted integrated exogenous SOI and at least one selection marker and a randomly integrated exogenous nucleic acid sequence, the randomly integrated exogenous nucleic acid sequence comprising at least one randomly integrated exogenous SOI, wherein the randomly integrated sequence is integrated into the genome of the host cell at least once, wherein the expression of the at least one randomly integrated exogenous SOI is regulatable; and culturing the cells in a) under conditions suitable for expressing the target polypeptide from the randomly integrated SOI, and recovering the target polypeptide therefrom.

[0322] F1. A method according to F, wherein the at least one randomly integrated exogenous SOI encodes a single-chain antibody, an antibody light chain, an antibody heavy chain, a single-chain Fv fragment (scFv) or an Fc fusion protein.

[0323] G. A method for preparing a TI host cell expressing a polypeptide of interest, the method comprising: providing a host cell, the host cell comprising a locus in the genome of the host cell, wherein the locus is at least about 90% homologous to a sequence selected from SEQ ID No. 1 to SEQ ID No. 7, and the host cell comprises a randomly integrated exogenous nucleic acid sequence, wherein the randomly integrated sequence is integrated at least once into the genome of the host cell; introducing a vector into the host cell, wherein the vector comprises a nucleotide sequence at least 50% homologous to a sequence selected from SEQ ID No. 1 to SEQ ID No. 7 flanked by a DNA cassette, wherein the DNA cassette comprises at least one selection marker flanked by two RRSs and at least one exogenous SOI for targeted integration; and selecting for the selection marker to isolate a TI host cell comprising the SOI for targeted integration integrated into the locus of the genome and expressing the polypeptide of interest.

[0324] G1. The method according to G, wherein the vector is selected from the group consisting of: an adenoviral vector, an adeno-associated viral vector, a lentiviral vector, a retroviral vector, an integrating phage vector, a non-viral vector, a transposon and / or transposase vector, an integrase substrate and a plasmid.

[0325] G2. The method according to G or G1, wherein the at least one SOI targeted for integration encodes a single-chain antibody, an antibody light chain, an antibody heavy chain, a single-chain Fv fragment (scFv) or an Fc fusion protein.

[0326] G3. A method according to any one of G-G2, wherein the TI host cell is a mammalian host cell.

[0327] G4. The method according to G3, wherein the TI host cell is a hamster host cell, a human host cell, a rat host cell or a mouse host cell.

[0328] G5. The method according to G3 or G4, wherein the TI host cell is a CHO host cell, a CHO K1 host cell, a CHO K1SV host cell, a DG44 host cell, a DUKXB-11 host cell, a CHOK1S host cell or a CHO K1M host cell.

[0329] G6. The method according to any one of G1-G5, wherein the integration of the targeted integration nucleic acid comprising at least one SOI and a selection marker is promoted by an exogenous nuclease.

[0330] G7. A method according to G6, wherein the exogenous nuclease is selected from the group consisting of: zinc finger nucleases (ZFNs), ZFN dimers, transcription activator-like effector nucleases (TALENs), TAL effector domain fusion proteins, RNA-guided DNA endonucleases, engineered large-range nucleases and clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) endonucleases.

[0331] G8. A method according to G, wherein the target polypeptide is a multispecific antibody.

[0332] G9. The method according to G8, wherein the multispecific antibody is a bispecific antibody.

[0333] H. A TI host cell, comprising: at least one exogenous nucleotide sequence for targeted integration, which is integrated at an integration site within one or more specific loci of the genome of the host cell, wherein the locus is at least about 90% homologous to a sequence selected from SEQ ID No.1 to SEQ ID No.7; and a randomly integrated exogenous nucleic acid sequence, wherein the randomly integrated sequence is integrated into the genome of the host cell at least once.

[0334] H1. A TI host cell, comprising: at least one targeted integration exogenous nucleotide sequence integrated into one or more integration sites within an endogenous gene, the endogenous gene being selected from the group consisting of: LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, XP_003512331.2, and sequences at least about 90% homologous thereto; and a randomly integrated exogenous nucleic acid sequence, wherein the randomly integrated sequence is integrated into the genome of the host cell at least once.

[0335] H2. The TI host cell according to any one of H-H1, wherein the at least one targeted integration exogenous nucleotide sequence comprises one or more recombination recognition sequences (RRS), wherein the RRS can be recognized by a recombinase.

[0336] H3. The TI host cell according to H2, wherein the at least one targeted integration exogenous nucleotide sequence further comprises at least one exogenous SOI.

[0337] H4. A TI host cell according to any one of H-H3, wherein the host cell comprises at least one targeted integration exogenous nucleotide sequence in a locus of the genome of a first host cell and at least one targeted integration exogenous nucleotide sequence in a locus of the genome of one or more second host cells.

[0338] H5. A TI host cell according to H4, wherein the first locus comprises a sequence that is at least 90% homologous to all or part of SEQ ID No.1, and the second locus comprises at least one sequence that is at least 90% homologous to all or part of the following sequences: SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6 or SEQID No.7.

[0339] H5.1. A TI host cell according to H4, wherein the first locus comprises a sequence that is at least 90% homologous to all or part of SEQ ID No.1, and the second locus comprises at least one sequence that is at least 90% homologous to all or part of SEQ ID No.2.

[0340] H5.2. A TI host cell according to H4, wherein the first locus comprises a sequence that is at least 90% homologous to all or part of SEQ ID No.1, and the second locus comprises at least one sequence that is at least 90% homologous to all or part of SEQ ID No.3.

[0341] H5.3. A TI host cell according to H4, wherein the first locus comprises a sequence that is at least 90% homologous to all or part of SEQ ID No.1, and the second locus comprises at least one sequence that is at least 90% homologous to all or part of SEQ ID No.4.

[0342] H5.4. A TI host cell according to H4, wherein the first locus comprises a sequence that is at least 90% homologous to all or part of SEQ ID No.1, and the second locus comprises at least one sequence that is at least 90% homologous to all or part of SEQ ID No.5.

[0343] H5.5. A TI host cell according to H4, wherein the first locus comprises a sequence that is at least 90% homologous to all or part of SEQ ID No.1, and the second locus comprises at least one sequence that is at least 90% homologous to all or part of SEQ ID No.6.

[0344] H5.6. A TI host cell according to H4, wherein the first locus comprises a sequence that is at least 90% homologous to all or part of SEQ ID No.1, and the second locus comprises at least one sequence that is at least 90% homologous to all or part of SEQ ID No.7.

[0345] H6. A TI host cell according to H4, wherein the first locus comprises a sequence that is at least 90% homologous to all or part of SEQ ID No.2, and the second locus comprises at least one sequence that is at least 90% homologous to all or part of the following sequences: SEQ ID No.1, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6 or SEQID No.7.

[0346] H6.1. A TI host cell according to H4, wherein the first locus comprises a sequence that is at least 90% homologous to all or part of SEQ ID No.2, and the second locus comprises at least one sequence that is at least 90% homologous to all or part of SEQ ID No.1.

[0347] H6.2. A TI host cell according to H4, wherein the first locus comprises a sequence that is at least 90% homologous to all or part of SEQ ID No.2, and the second locus comprises at least one sequence that is at least 90% homologous to all or part of SEQ ID No.3.

[0348] H6.3. A TI host cell according to H4, wherein the first locus comprises a sequence that is at least 90% homologous to all or part of SEQ ID No.2, and the second locus comprises at least one sequence that is at least 90% homologous to all or part of SEQ ID No.4.

[0349] H6.4. A TI host cell according to H4, wherein the first locus comprises a sequence that is at least 90% homologous to all or part of SEQ ID No.2, and the second locus comprises at least one sequence that is at least 90% homologous to all or part of SEQ ID No.5.

[0350] H6.5. A TI host cell according to H4, wherein the first locus comprises a sequence that is at least 90% homologous to all or part of SEQ ID No.2, and the second locus comprises at least one sequence that is at least 90% homologous to all or part of SEQ ID No.6.

[0351] H6.6. A TI host cell according to H4, wherein the first locus comprises a sequence that is at least 90% homologous to all or part of SEQ ID No.2, and the second locus comprises at least one sequence that is at least 90% homologous to all or part of SEQ ID No.7.

[0352] H7. A TI host cell according to H4, wherein the first locus comprises a sequence that is at least 90% homologous to all or part of SEQ ID No.3, and the second locus comprises at least one sequence that is at least 90% homologous to all or part of the following sequences: SEQ ID No.1, SEQ ID No.2, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6 or SEQID No.7.

[0353] H7.1. A TI host cell according to H4, wherein the first locus comprises a sequence that is at least 90% homologous to all or part of SEQ ID No.3, and the second locus comprises at least one sequence that is at least 90% homologous to all or part of SEQ ID No.1.

[0354] H7.2. A TI host cell according to H4, wherein the first locus comprises a sequence that is at least 90% homologous to all or part of SEQ ID No.3, and the second locus comprises at least one sequence that is at least 90% homologous to all or part of SEQ ID No.2.

[0355] H7.3. A TI host cell according to H4, wherein the first locus comprises a sequence that is at least 90% homologous to all or part of SEQ ID No.3, and the second locus comprises at least one sequence that is at least 90% homologous to all or part of SEQ ID No.4.

[0356] H7.4. A TI host cell according to H4, wherein the first locus comprises a sequence that is at least 90% homologous to all or part of SEQ ID No.3, and the second locus comprises at least one sequence that is at least 90% homologous to all or part of SEQ ID No.5.

[0357] H7.5. A TI host cell according to H4, wherein the first locus comprises a sequence that is at least 90% homologous to all or part of SEQ ID No.3, and the second locus comprises at least one sequence that is at least 90% homologous to all or part of SEQ ID No.6.

[0358] H7.6. A TI host cell according to H4, wherein the first locus comprises a sequence that is at least 90% homologous to all or part of SEQ ID No.3, and the second locus comprises at least one sequence that is at least 90% homologous to all or part of SEQ ID No.7.

[0359] H8. A TI host cell according to H4, wherein the first locus comprises a sequence that is at least 90% homologous to all or part of SEQ ID No.4, and the second locus comprises at least one sequence that is at least 90% homologous to all or part of the following sequences: SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.5, SEQ ID No.6 or SEQID No.7.

[0360] H8.1. A TI host cell according to H4, wherein the first locus comprises a sequence that is at least 90% homologous to all or part of SEQ ID No.4, and the second locus comprises at least one sequence that is at least 90% homologous to all or part of SEQ ID No.1.

[0361] H8.2. A TI host cell according to H4, wherein the first locus comprises a sequence that is at least 90% homologous to all or part of SEQ ID No.4, and the second locus comprises at least one sequence that is at least 90% homologous to all or part of SEQ ID No.2.

[0362] H8.3. A TI host cell according to H4, wherein the first locus comprises a sequence that is at least 90% homologous to all or part of SEQ ID No.4, and the second locus comprises at least one sequence that is at least 90% homologous to all or part of SEQ ID No.3.

[0363] H8.4. A TI host cell according to H4, wherein the first locus comprises a sequence that is at least 90% homologous to all or part of SEQ ID No.4, and the second locus comprises at least one sequence that is at least 90% homologous to all or part of SEQ ID No.5.

[0364] H8.5. A TI host cell according to H4, wherein the first locus comprises a sequence that is at least 90% homologous to all or part of SEQ ID No.4, and the second locus comprises at least one sequence that is at least 90% homologous to all or part of SEQ ID No.6.

[0365] H8.6. A TI host cell according to H4, wherein the first locus comprises a sequence that is at least 90% homologous to all or part of SEQ ID No.4, and the second locus comprises at least one sequence that is at least 90% homologous to all or part of SEQ ID No.7.

[0366] H9. A TI host cell according to H4, wherein the first locus comprises a sequence that is at least 90% homologous to all or part of SEQ ID No.5, and the second locus comprises at least one sequence that is at least 90% homologous to all or part of the following sequences: SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.6 or SEQID No.7.

[0367] H9.1. A TI host cell according to H4, wherein the first locus comprises a sequence that is at least 90% homologous to all or part of SEQ ID No.5, and the second locus comprises at least one sequence that is at least 90% homologous to all or part of SEQ ID No.1.

[0368] H9.2. A TI host cell according to H4, wherein the first locus comprises a sequence that is at least 90% homologous to all or part of SEQ ID No.5, and the second locus comprises at least one sequence that is at least 90% homologous to all or part of SEQ ID No.2.

[0369] H9.3. A TI host cell according to H4, wherein the first locus comprises a sequence that is at least 90% homologous to all or part of SEQ ID No.5, and the second locus comprises at least one sequence that is at least 90% homologous to all or part of SEQ ID No.3.

[0370] H9.4. A TI host cell according to H4, wherein the first locus comprises a sequence that is at least 90% homologous to all or part of SEQ ID No.5, and the second locus comprises at least one sequence that is at least 90% homologous to all or part of SEQ ID No.4.

[0371] H9.5. A TI host cell according to H4, wherein the first locus comprises a sequence that is at least 90% homologous to all or part of SEQ ID No.5, and the second locus comprises at least one sequence that is at least 90% homologous to all or part of SEQ ID No.6.

[0372] H9.6. A TI host cell according to H4, wherein the first locus comprises a sequence that is at least 90% homologous to all or part of SEQ ID No.5, and the second locus comprises at least one sequence that is at least 90% homologous to all or part of SEQ ID No.7.

[0373] H10. A TI host cell according to H4, wherein the first locus comprises a sequence that is at least 90% homologous to all or part of SEQ ID No.6, and the second locus comprises at least one sequence that is at least 90% homologous to all or part of the following sequences: SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5 or SEQ ID No.7.

[0374] H10.1. A TI host cell according to H4, wherein the first locus comprises a sequence that is at least 90% homologous to all or part of SEQ ID No.6, and the second locus comprises at least one sequence that is at least 90% homologous to all or part of SEQ ID No.1.

[0375] H10.2. A TI host cell according to H4, wherein the first locus comprises a sequence that is at least 90% homologous to all or part of SEQ ID No.6, and the second locus comprises at least one sequence that is at least 90% homologous to all or part of SEQ ID No.2.

[0376] H10.3. A TI host cell according to H4, wherein the first locus comprises a sequence that is at least 90% homologous to all or part of SEQ ID No.6, and the second locus comprises at least one sequence that is at least 90% homologous to all or part of SEQ ID No.3.

[0377] H10.4. A TI host cell according to H4, wherein the first locus comprises a sequence that is at least 90% homologous to all or part of SEQ ID No.6, and the second locus comprises at least one sequence that is at least 90% homologous to all or part of SEQ ID No.4.

[0378] H10.5. A TI host cell according to H4, wherein the first locus comprises a sequence that is at least 90% homologous to all or part of SEQ ID No.6, and the second locus comprises at least one sequence that is at least 90% homologous to all or part of SEQ ID No.5.

[0379] H10.6. A TI host cell according to H4, wherein the first locus comprises a sequence that is at least 90% homologous to all or part of SEQ ID No.6, and the second locus comprises at least one sequence that is at least 90% homologous to all or part of SEQ ID No.7.

[0380] H11. A TI host cell according to H4, wherein the first locus comprises a sequence that is at least 90% homologous to all or part of SEQ ID No.7, and the second locus comprises at least one sequence that is at least 90% homologous to all or part of the following sequences: SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5 or SEQ ID No.6.

[0381] H11.1. A TI host cell according to H4, wherein the first locus comprises a sequence that is at least 90% homologous to all or part of SEQ ID No.7, and the second locus comprises at least one sequence that is at least 90% homologous to all or part of SEQ ID No.1.

[0382] H11.2. A TI host cell according to H4, wherein the first locus comprises a sequence that is at least 90% homologous to all or part of SEQ ID No.7, and the second locus comprises at least one sequence that is at least 90% homologous to all or part of SEQ ID No.2.

[0383] H11.3. A TI host cell according to H4, wherein the first locus comprises a sequence that is at least 90% homologous to all or part of SEQ ID No.7, and the second locus comprises at least one sequence that is at least 90% homologous to all or part of SEQ ID No.3.

[0384] H11.4. A TI host cell according to H4, wherein the first locus comprises a sequence that is at least 90% homologous to all or part of SEQ ID No.7, and the second locus comprises at least one sequence that is at least 90% homologous to all or part of SEQ ID No.4.

[0385] H11.5. A TI host cell according to H4, wherein the first locus comprises a sequence that is at least 90% homologous to all or part of SEQ ID No.7, and the second locus comprises at least one sequence that is at least 90% homologous to all or part of SEQ ID No.5.

[0386] H11.6. A TI host cell according to H4, wherein the first locus comprises a sequence that is at least 90% homologous to all or part of SEQ ID No.7, and the second locus comprises at least one sequence that is at least 90% homologous to all or part of SEQ ID No.6.

[0387] H12. A TI host cell according to H4, wherein the first locus is an integration site within the LOC107977062 gene, and the second locus is an integration site within a gene selected from the group consisting of LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, XP_003512331.2, and sequences at least about 90% homologous thereto.

[0388] H13. A TI host cell according to H4, wherein the first locus is an integration site within the LOC100768845 gene, and the second locus is an integration site within a gene selected from the group consisting of LOC107977062, ITPR2, ERE67000.1, UBAP2, MTMR2, XP_003512331.2, and sequences at least about 90% homologous thereto.

[0389] H14. A TI host cell according to H4, wherein the first locus is an integration site within the ITPR2 gene, and the second locus is an integration site within a gene selected from the group consisting of LOC107977062, LOC100768845, ERE67000.1, UBAP2, MTMR2, XP_003512331.2, and sequences at least about 90% homologous thereto.

[0390] H15. A TI host cell according to H4, wherein the first locus is an integration site within the ERE67000.1 gene, and the second locus is an integration site within a gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, UBAP2, MTMR2, XP_003512331.2, and sequences at least about 90% homologous thereto.

[0391] H16. A TI host cell according to H4, wherein the first locus is an integration site within the UBAP2 gene, and the second locus is an integration site within a gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, MTMR2, XP_003512331.2, and sequences at least about 90% homologous thereto.

[0392] H17. A TI host cell according to H4, wherein the first locus is an integration site within the MTMR2 gene, and the second locus is an integration site within a gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, XP_003512331.2, and sequences at least about 90% homologous thereto.

[0393] H18. A TI host cell according to H4, wherein the first locus is an integration site within the XP_003512331.2 gene, and the second locus is an integration site within a gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and sequences at least about 90% homologous thereto.

[0394] H19. The TI host cell according to H4-H18, wherein at least one exogenous nucleotide sequence integrated into the first locus comprises a regulatable promoter.

[0395] H20. The TI host cell according to H4-H18, wherein at least one exogenous nucleotide sequence integrated into the second locus comprises a regulatable promoter.

[0396] H21. The TI host cell according to H4-H18, wherein at least one exogenous nucleotide sequence integrated at the first locus and at least one exogenous nucleotide sequence integrated at the second locus comprise a regulatable promoter.

[0397] J. A method for preparing a TI host cell expressing at least one polypeptide of interest, the method comprising: providing a TI host cell, the TI host cell comprising at least one targeted integration exogenous nucleotide sequence, the at least one targeted integration exogenous nucleotide sequence being integrated at a site within one or more loci of the genome of the TI host cell, wherein the one or more loci are at least about 90% homologous to a sequence selected from SEQ ID No. 1 to SEQ ID No. 7, and the TI host cell comprising a randomly integrated exogenous nucleic acid sequence, wherein the randomly integrated sequence is integrated at least once into the genome of the host cell, wherein the at least one targeted integration exogenous nucleotide sequence comprises two RRSs, and the two RRSs are flanked by at least one first selection marker; introducing a vector into the cell provided in a), the vector comprising two RRSs, the two RRSs matching the two RRSs on the integrated targeted integration exogenous nucleotide sequence and flanked by at least one targeted integration exogenous SOI and at least one second selection marker; introducing a recombinase or a nucleic acid encoding a recombinase, wherein the recombinase recognizes the RRS; and selecting TI cells expressing the second selection marker, thereby isolating TI host cells expressing the at least one polypeptide.

[0398] J1. A method for preparing a TI host cell expressing at least one first target polypeptide and a second target polypeptide, the method comprising: providing a TI host cell, the TI host cell comprising at least one targeted integration exogenous nucleotide sequence integrated into a site within one or more loci of the host cell's genome, wherein the one or more loci are selected from SEQ ID No. 1 to SEQ ID No.7 is at least about 90% homologous, and the TI host cell comprises a randomly integrated exogenous nucleic acid sequence, wherein the randomly integrated sequence is integrated into the genome of the host cell at least once, wherein the targeted integration exogenous nucleotide sequence comprises a first RRS and a second RRS flanked by at least one first selection marker and a third RRS located between the first RRS and the second RRS, and all RRS have heterologous specificity; introducing a first vector into the cell provided in a), the first vector comprising two RRSs, the two RRSs matching the first RRS and the third RRS on the at least one integrated targeted integration exogenous nucleotide sequence and flanked by at least one integrated first targeted integration exogenous SOI and at least one second selection marker; introducing a second vector into the cell provided in a), the second vector comprising two RRSs, the two RRSs matching the second RRS and the third RRS on the at least one integrated targeted integration exogenous nucleotide sequence and flanked by at least one second targeted integration exogenous SOI; introducing one or more recombinases or one or more nucleic acids encoding one or more recombinases, wherein the one or more recombinases recognize RRS; and selecting TI cells expressing the second selection marker, thereby isolating TI host cells expressing the at least one first target polypeptide and the second target polypeptide.

[0399] J2. A method for preparing a TI host cell expressing a target polypeptide, the method comprising: a) providing a TI host cell, the TI host cell comprising at least one targeted integration exogenous nucleotide sequence, the at least one targeted integration exogenous nucleotide sequence being integrated at a site within one or more loci of the genome of the TI host cell, wherein the one or more loci are at least about 90% homologous to a sequence selected from SEQ ID No.1 to SEQ ID No.7, and the TI host cell comprising a randomly integrated exogenous nucleic acid sequence, wherein the randomly integrated sequence is integrated into the genome of the host cell at least once, wherein the targeted integration exogenous nucleotide sequence comprises one or more RRS; b) introducing a vector into the cell provided in a), the vector comprising one or more RRS, the one or more RRS matching the one or more RRS on the integrated targeted integration exogenous nucleotide sequence and flanked by at least one first targeted integration exogenous SOI operably linked to at least one regulatable promoter; c) introducing a recombinase or a nucleic acid encoding a recombinase, wherein the recombinase recognizes the RRS; and d) selecting a TI cell expressing the exogenous target polypeptide in the presence of an inducer, thereby isolating the TI host cell expressing the target polypeptide.

[0400] J3. A method for expressing a target polypeptide, the method comprising: a) providing a host cell, the host cell comprising at least one targeted integration exogenous SOI integrated into a locus of the host cell's genome, the at least one targeted integration exogenous SOI flanked by two RRSs and an adjustable promoter, wherein the locus is at least about 90% homologous to a sequence selected from SEQ ID No.1 to SEQ ID No.7, and the host cell comprises a randomly integrated exogenous nucleic acid sequence, wherein the randomly integrated sequence is integrated at least once into the host cell's genome; and b) culturing the cell under conditions suitable for expressing the targeted integration SOI, and recovering the target polypeptide therefrom.

[0401] J4. A method for preparing a TI host cell expressing a first polypeptide of interest and a second polypeptide of interest, the method comprising: a) providing a TI host cell, the TI host cell comprising a targeted integration exogenous nucleotide sequence integrated into a site within a locus of the genome of the host cell, wherein the locus is at least about 90% homologous to a sequence selected from SEQ ID No. 1 to SEQ ID No. 7, and the TI host cell comprises a randomly integrated exogenous nucleic acid sequence, wherein the randomly integrated sequence is integrated at least once into the genome of the host cell, wherein the exogenous nucleotide sequence comprises a first RRS, a second RRS, and a third RRS located between the first RRS and the second RRS, and all RRSs have heterologous specificity; b) introducing a first vector into the cell provided in a), the first vector comprising two RRSs that match the first RRS and the third RRS on the integrated targeted integration exogenous nucleotide sequence and are flanked by at least one first targeted integration exogenous SO operably linked to a regulatable promoter. I; c) introducing a second vector into the cell provided in a), the second vector comprising two RRSs, which match the second RRS and the third RRS on the integrated targeted integration exogenous nucleotide sequence and are flanked by at least one second targeted integration exogenous SOI operably linked to a regulatable promoter; d) introducing one or more recombinases or one or more nucleic acids encoding one or more recombinases, wherein the one or more recombinases recognize RRS; and e) selecting TI cells that express a first targeted integration exogenous target polypeptide and a second targeted integration exogenous target polypeptide in the presence of an inducer, thereby isolating TI host cells that express the first target polypeptide and the second target polypeptide.

[0402] J5. The method according to any one of J, J2 and J3, wherein the integration of the targeted integration exogenous nucleic acid comprising at least one targeted integration SOI is promoted by an exogenous nuclease.

[0403] J6. A method according to claim J1 or J4, wherein the first SOI encodes a single-chain antibody, an antibody light chain, an antibody heavy chain, a single-chain Fv fragment (scFv) or an Fc fusion protein; and the second SOI encodes a single-chain antibody, an antibody light chain, an antibody heavy chain, a single-chain Fv fragment (scFv) or an Fc fusion protein.

[0404] J7. A method according to J5, wherein the exogenous nuclease is selected from the group consisting of zinc finger nucleases (ZFNs), ZFN dimers, transcription activator-like effector nucleases (TALENs), TAL effector domain fusion proteins, RNA-guided DNA endonucleases, engineered large-range nucleases and clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) endonucleases.

[0405] J8. A method according to any one of claims J-J4, wherein the regulatable promoter is selected from the group consisting of SV40 and CMV promoters.

[0406] J9. The method according to any one of claims J2 and J4, wherein: the first vector further comprises a promoter sequence, which is operably linked to the codon ATG with a first targeted integration SOI on the upstream side and an RRS on the downstream side; and the second vector further comprises a selection marker, which lacks the ATG transcription start codon with an RRS on the upstream side and a second targeted integration SOI on the downstream side.

[0407] In certain embodiments, the host cells of the present invention can be used to target additional genomic sites through random integration of exogenous SOIs. In certain embodiments, the TI cells of the present disclosure are used in combination with random integration of exogenous SOIs to produce polypeptides of interest, including but not limited to antibodies, enzymes, cytokines, growth factors, hormones, viral proteins, bacterial proteins, vaccine proteins, or proteins with therapeutic functions.

[0408] K. A host cell capable of expressing a polypeptide of interest, the host cell comprising: a) a targeted integration exogenous nucleic acid sequence of interest (SOI) encoding a first polypeptide of interest flanked by two recombination recognition sequences (RRS) and a first selection marker, wherein the targeted integration exogenous SOI is integrated into a targeted locus of the genome of the host cell; and b) a randomly integrated exogenous nucleic acid SOI encoding a second polypeptide of interest and a second selection marker, wherein the randomly integrated SOI is integrated at least once into the genome of the host cell.

[0409] K1. A host cell according to K, wherein the first target polypeptide and the second target polypeptide are the same.

[0410] K2. A host cell according to K, wherein the first selection marker and the second selection marker are the same.

[0411] K3. The host cell according to K, comprising one to ten randomly integrated exogenous nucleic acid SOIs.

[0412] K4. A host cell according to K, wherein the targeted locus is at least about 90% homologous to a sequence selected from SEQ ID No.1 to SEQ ID No.7.

[0413] K4.1. A host cell according to K4, wherein the targeted locus comprises a sequence that is at least 90% homologous to all or part of SEQ ID No.1.

[0414] K4.2. A host cell according to K4, wherein the targeted locus comprises a sequence that is at least 90% homologous to all or part of SEQ ID No.2.

[0415] K4.3. A host cell according to K4, wherein the targeted locus comprises a sequence that is at least 90% homologous to all or part of SEQ ID No.3.

[0416] K4.4. A host cell according to K4, wherein the targeted locus comprises a sequence that is at least 90% homologous to all or part of SEQ ID No.4.

[0417] K4.5. A host cell according to K4, wherein the targeted locus comprises a sequence that is at least 90% homologous to all or part of SEQ ID No.5.

[0418] K4.6. A host cell according to K4, wherein the targeted locus comprises a sequence that is at least 90% homologous to all or part of SEQ ID No.6.

[0419] K4.7. A host cell according to K4, wherein the targeted locus comprises a sequence that is at least 90% homologous to all or part of SEQ ID No.7.

[0420] K.5. A host cell according to any one of K-K4, wherein the host cell comprises: at least one first targeted integrated exogenous nucleic acid SOI, which is integrated into a targeted locus of the genome of the host cell; and at least one second targeted integrated exogenous nucleic acid SOI, which encodes a second target polypeptide SOI integrated into one or more second loci of the genome of the host cell.

[0421] K6. A host cell according to K5, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.1, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of the following sequences: SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6 or SEQ ID No.7.

[0422] K6.1. A host cell according to K5, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.1, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of SEQ ID No.2.

[0423] K6.2. A host cell according to K5, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.1, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of SEQ ID No.3.

[0424] K6.3. A host cell according to K5, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.1, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of SEQ ID No.4.

[0425] K6.4. A host cell according to K5, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.1, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of SEQ ID No.5.

[0426] K6.5. A host cell according to K5, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.1, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of SEQ ID No.6.

[0427] K6.6. A host cell according to K5, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.1, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of SEQ ID No.7.

[0428] K7. A host cell according to K5, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.2, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of the following sequences: SEQ ID No.1, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6 or SEQ ID No.7.

[0429] K7.1. A host cell according to K5, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.2, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of SEQ ID No.1.

[0430] K7.2. A host cell according to K5, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.2, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of SEQ ID No.3.

[0431] K7.3. A host cell according to K5, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.2, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of SEQ ID No.4.

[0432] K7.4. A host cell according to K5, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.2, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of SEQ ID No.5.

[0433] K7.5. A host cell according to K5, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.2, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of SEQ ID No.6.

[0434] K7.6. A host cell according to K5, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.2, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of SEQ ID No.7.

[0435] K8. A host cell according to K5, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.3, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of the following sequences: SEQ ID No.1, SEQ ID No.2, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6 or SEQ ID No.7.

[0436] K8.1. A host cell according to K5, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.3, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of SEQ ID No.1.

[0437] K8.2. A host cell according to K5, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.3, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of SEQ ID No.2.

[0438] K8.3. A host cell according to K5, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.3, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of SEQ ID No.4.

[0439] K8.4. A host cell according to K5, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.3, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of SEQ ID No.5.

[0440] K8.5. A host cell according to K5, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.3, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of SEQ ID No.6.

[0441] K8.6. A host cell according to K5, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.3, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of SEQ ID No.7.

[0442] K9. A host cell according to K5, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.4, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of the following sequences: SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.5, SEQ ID No.6 or SEQ ID No.7.

[0443] K9.1. A host cell according to K5, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.4, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of SEQ ID No.1.

[0444] K9.2. A host cell according to K5, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.4, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of SEQ ID No.2.

[0445] K9.3. A host cell according to K5, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.4, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of SEQ ID No.3.

[0446] K9.4. A host cell according to K5, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.4, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of SEQ ID No.5.

[0447] K9.5. A host cell according to K5, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.4, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of SEQ ID No.6.

[0448] K9.6. A host cell according to K5, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.4, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of SEQ ID No.7.

[0449] K10. A host cell according to K5, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.5, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of the following sequences: SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.6 or SEQ ID No.7.

[0450] K10.1. A host cell according to K5, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.5, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of SEQ ID No.1.

[0451] K10.2. A host cell according to K5, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.5, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of SEQ ID No.2.

[0452] K10.3. A host cell according to K5, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.5, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of SEQ ID No.3.

[0453] K10.4. A host cell according to K5, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.5, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of SEQ ID No.4.

[0454] K10.5. A host cell according to K5, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.5, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of SEQ ID No.6.

[0455] K10.6. A host cell according to K5, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.5, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of SEQ ID No.7.

[0456] K11. A host cell according to K5, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.6, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of the following sequences: SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5 or SEQ ID No.7.

[0457] K11.1. A host cell according to K5, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.6, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of SEQ ID No.1.

[0458] K11.2. A host cell according to K5, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.6, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of SEQ ID No.2.

[0459] K11.3. A host cell according to K5, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.6, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of SEQ ID No.3.

[0460] K11.4. A host cell according to K5, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.6, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of SEQ ID No.4.

[0461] K11.5. A host cell according to K5, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.6, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of SEQ ID No.5.

[0462] K11.6. A host cell according to K5, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.6, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of SEQ ID No.7.

[0463] K12. A host cell according to K5, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.7, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of the following sequences: SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5 or SEQ ID No.6.

[0464] K12.1. A host cell according to K5, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.7, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of SEQ ID No.1.

[0465] K12.2. A host cell according to K5, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.7, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of SEQ ID No.2.

[0466] K12.3. A host cell according to K5, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.7, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of SEQ ID No.3.

[0467] K12.4. A host cell according to K5, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.7, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of SEQ ID No.4.

[0468] K12.5. A host cell according to K5, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.7, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of SEQ ID No.5.

[0469] K12.6. A host cell according to K5, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.7, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of SEQ ID No.6.

[0470] K13. A host cell according to K5, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI is an integration site within the LOC107977062 gene, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI is an integration site within a gene selected from the following group: LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, XP_003512331.2 and sequences at least about 90% homologous thereto.

[0471] K14. A host cell according to K5, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI is an integration site within the LOC100768845 gene, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI is an integration site within a gene selected from the following group: LOC107977062, ITPR2, ERE67000.1, UBAP2, MTMR2, XP_003512331.2 and sequences at least about 90% homologous thereto.

[0472] K15. A host cell according to K5, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI is an integration site within the ITPR2 gene, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI is an integration site within a gene selected from the following group: LOC107977062, LOC100768845, ERE67000.1, UBAP2, MTMR2, XP_003512331.2 and sequences at least about 90% homologous thereto.

[0473] K16. A host cell according to K5, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI is an integration site within the ERE67000.1 gene, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI is an integration site within a gene selected from the following group: LOC107977062, LOC100768845, ITPR2, UBAP2, MTMR2, XP_003512331.2 and sequences at least about 90% homologous thereto.

[0474] K17. A host cell according to K5, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI is an integration site within the UBAP2 gene, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI is an integration site within a gene selected from the following group: LOC107977062, LOC100768845, ITPR2, ERE67000.1, MTMR2, XP_003512331.2 and sequences at least about 90% homologous thereto.

[0475] K18. A host cell according to K5, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI is an integration site within the MTMR2 gene, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI is an integration site within a gene selected from the following group: LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, XP_003512331.2 and sequences at least about 90% homologous thereto.

[0476] K19. A host cell according to K5, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI is an integration site within the XP_003512331.2 gene, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI is an integration site within a gene selected from the following group: LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and sequences at least about 90% homologous thereto.

[0477] K20. A host cell according to any one of K-K19, wherein the polypeptide of interest is selected from the group consisting of: a single-chain antibody, an antibody light chain, an antibody heavy chain, a single-chain Fv fragment (scFv) and an Fc fusion protein.

[0478] K21. A host cell according to any one of K-K19, wherein the host cell is a mammalian host cell.

[0479] K22. The host cell according to K21, wherein the host cell is a hamster host cell, a human host cell, a rat host cell or a mouse host cell.

[0480] K23. The host cell according to K228, wherein the host cell is a CHO host cell, a CHO K1 host cell, a CHO K1SV host cell, a DG44 host cell, a DUKXB-11 host cell, a CHOK1S host cell or a CHO K1M host cell.

[0481] K24. A host cell according to any one of K-K20, wherein the targeted integration of SOI and the selection marker is promoted by an exogenous nuclease.

[0482] K25. A host cell according to K24, wherein the exogenous nuclease is selected from the group consisting of zinc finger nucleases (ZFNs), ZFN dimers, transcription activator-like effector nucleases (TALENs), TAL effector domain fusion proteins, RNA-guided DNA endonucleases, engineered large-range nucleases, and clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) endonucleases.

[0483] L. A method for expressing a polypeptide of interest, the method comprising: a) providing a host cell comprising an exogenous nucleotide sequence integrated at a targeted locus in the genome of the host cell, wherein the exogenous nucleotide sequence comprises two RRSs flanking a first selection marker; b) introducing a nucleic acid into the cell provided in (a), the nucleic acid comprising two RRSs matching the two RRSs of the integrated exogenous nucleotide sequence and flanking a first exogenous SOI encoding a first polypeptide of interest and a second selection marker; c) introducing a recombinase or a nucleic acid encoding a recombinase, wherein the recombinase recognizes the RRSs; d) selecting cells expressing the second selection marker; e) introducing a second exogenous SOI encoding a second polypeptide of interest and a third selection marker into the genome of the host cell by random integration; f) selecting cells expressing the third selection marker; and g) culturing the host cell under conditions sufficient to express the first polypeptide of interest and the second polypeptide of interest.

[0484] L1. The method according to L further comprises recovering the first target polypeptide and the second target polypeptide from the host cell culture.

[0485] L2. A method according to L, wherein the first target polypeptide and the second target polypeptide are the same.

[0486] L3. A method according to L, wherein the targeted locus is at least about 90% homologous to a sequence selected from SEQ ID No. 1 to SEQ ID No. 7.

[0487] L3.1. A method according to L3, wherein the targeted locus comprises a sequence that is at least 90% homologous to all or part of SEQ ID No.1.

[0488] L3.2. A method according to L3, wherein the targeted locus comprises a sequence that is at least 90% homologous to all or part of SEQ ID No.2.

[0489] L3.3. A method according to L3, wherein the targeted locus comprises a sequence that is at least 90% homologous to all or part of SEQ ID No.3.

[0490] L3.4. A method according to L3, wherein the targeted locus comprises a sequence that is at least 90% homologous to all or part of SEQ ID No.4.

[0491] L3.5. A method according to L3, wherein the targeted locus comprises a sequence that is at least 90% homologous to all or part of SEQ ID No.5.

[0492] L3.6. A method according to L3, wherein the targeted locus comprises a sequence that is at least 90% homologous to all or part of SEQ ID No.6.

[0493] L3.7. A method according to L3, wherein the targeted locus comprises a sequence that is at least 90% homologous to all or part of SEQ ID No.7.

[0494] L4. A host cell according to any one of L-L3, wherein the host cell comprises: at least one first targeted integrated exogenous nucleic acid SOI, which is integrated into a targeted locus of the genome of the host cell; and at least one second targeted integrated exogenous nucleic acid SOI, which encodes a second target polypeptide SOI integrated into one or more second loci of the genome of the host cell.

[0495] L5. A method according to L4, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.1, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of the following sequences: SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6 or SEQ ID No.7.

[0496] L5.1. A method according to L4, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.1, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of SEQ ID No.2.

[0497] L5.2. A method according to L4, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.1, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of SEQ ID No.3.

[0498] L5.3. A method according to L4, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.1, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of SEQ ID No.4.

[0499] L5.4. A method according to L4, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.1, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of SEQ ID No.5.

[0500] L5.5. A method according to L4, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.1, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of SEQ ID No.6.

[0501] L5.6. A method according to L4, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.1, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of SEQ ID No.7.

[0502] L6. A method according to L4, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.2, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of the following sequences: SEQ ID No.1, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6 or SEQ ID No.7.

[0503] L6.1. A method according to L4, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.2, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of SEQ ID No.1.

[0504] L6.2. A method according to L4, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.2, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of SEQ ID No.3.

[0505] L6.3. The method according to L4, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI comprises a sequence at least 90% homologous to all or a portion of SEQ ID No. 2, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI comprises at least one sequence at least 90% homologous to all or a portion of SEQ ID No. 4. K7.4. The method according to L4, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI comprises a sequence at least 90% homologous to all or a portion of SEQ ID No. 2, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI comprises at least one sequence at least 90% homologous to all or a portion of SEQ ID No. 5.

[0506] L6.5. A method according to L4, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.2, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of SEQ ID No.6.

[0507] L6.6. A method according to L4, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.2, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of SEQ ID No.7.

[0508] L7. A method according to L4, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.3, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of the following sequences: SEQ ID No.1, SEQ ID No.2, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6 or SEQ ID No.7.

[0509] L7.1. A method according to L4, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.3, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of SEQ ID No.1.

[0510] L7.2. A method according to L4, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.3, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of SEQ ID No.2.

[0511] L7.3. A method according to L4, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.3, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of SEQ ID No.4.

[0512] L7.4. A method according to L4, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.3, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of SEQ ID No.5.

[0513] L7.5. A method according to L4, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.3, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of SEQ ID No.6.

[0514] L7.6. A method according to L4, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.3, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of SEQ ID No.7.

[0515] L8. A method according to L4, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.4, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of the following sequences: SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.5, SEQ ID No.6 or SEQ ID No.7.

[0516] L8.1. A method according to L4, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.4, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of SEQ ID No.1.

[0517] L8.2. A method according to L4, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.4, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of SEQ ID No.2.

[0518] L8.3. A method according to L4, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.4, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of SEQ ID No.3.

[0519] L8.4. A method according to L4, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.4, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of SEQ ID No.5.

[0520] L8.5. A method according to L4, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.4, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of SEQ ID No.6.

[0521] L8.6. A method according to L4, wherein the targeted locus of the first targeted integrated exogenous nucleic acid SOI contains a sequence that is at least 90% homologous to all or part of SEQ ID No.4, and the targeted locus of the second targeted integrated exogenous nucleic acid SOI contains at least one sequence that is at least 90% homologous to all or part of SEQ ID No.7.

[0522] L9. A method ...

Claims

1. A host cell capable of expressing a target polypeptide, the host cell comprising: a) a targeted integration exogenous nucleic acid sequence of interest (SOI) encoding a first polypeptide of interest flanked by two recombination recognition sequences (RRS) and a first selection marker, wherein the targeted integration exogenous nucleic acid SOI is integrated into a targeted locus of the genome of the host cell, wherein the targeted locus is at least 99% homologous to a sequence selected from SEQ ID No. 1 to SEQ ID No. 7, wherein (i) The nucleotide immediately adjacent to the 5' end of the targeted integration exogenous nucleic acid SOI is nucleotide 45269 of NW_006874047.1, The nucleotide immediately adjacent to the 3' end of the targeted integration exogenous nucleic acid SOI is nucleotide 45270 of NW_006874047.1, The nucleotide sequence of at least 300 base pairs immediately adjacent to the 5' end of the targeted integration exogenous nucleic acid SOI is identical to the nucleic acid sequence of the same length in the 5' direction starting from nucleotide 45269 of NW_006874047.1, and The nucleotide sequence of at least 300 base pairs immediately adjacent to the 3' end of the targeted integration exogenous nucleic acid SOI is identical to the nucleic acid sequence of the same length in the 3' direction starting from nucleotide 45270 of NW_006874047.1; (ii) The nucleotide immediately adjacent to the 5' end of the targeted integration exogenous nucleic acid SOI is nucleotide 207911 of NW_006884592.1, The nucleotide immediately adjacent to the 3' end of the targeted integration exogenous nucleic acid SOI is nucleotide 207912 of NW_006884592.1, and the nucleotide sequence of at least 300 base pairs immediately adjacent to the 5' end of the targeted integration exogenous nucleic acid SOI is identical to the nucleic acid sequence of the same length in the 5' direction starting from nucleotide 207911 of NW_006884592.1, and The nucleotide sequence of at least 300 base pairs immediately adjacent to the 3' end of the targeted integration exogenous nucleic acid SOI is identical to the nucleic acid sequence of the same length in the 3' direction starting from nucleotide 207912 of NW_006884592.1; (iii) The nucleotide immediately adjacent to the 5' end of the targeted integration exogenous nucleic acid SOI is nucleotide 491909 of NW_006881296.1, The nucleotide immediately adjacent to the 3' end of the targeted integration exogenous nucleic acid SOI is nucleotide 491910 of NW_006881296.1, The nucleotide sequence of at least 300 base pairs immediately adjacent to the 5' end of the targeted integration exogenous nucleic acid SOI is identical to the nucleic acid sequence of the same length in the 5' direction starting from nucleotide 491909 of NW_006881296.1, and The nucleotide sequence of at least 300 base pairs immediately adjacent to the 3' end of the targeted integration exogenous nucleic acid SOI is identical to the nucleic acid sequence of the same length in the 3' direction starting from nucleotide 491910 of NW_006881296.1; (iv) The nucleotide immediately adjacent to the 5' end of the targeted integration exogenous nucleic acid SOI is nucleotide 79768 of NW_003616412.1, The nucleotide immediately adjacent to the 3' end of the targeted integration exogenous nucleic acid SOI is nucleotide 79769 of NW_003616412.1, The nucleotide sequence of at least 300 base pairs immediately adjacent to the 5' end of the targeted integration exogenous nucleic acid SOI is identical to the nucleic acid sequence of the same length in the 5' direction starting from nucleotide 79768 of NW_003616412.1, and The nucleotide sequence of at least 300 base pairs immediately adjacent to the 3' end of the targeted integration exogenous nucleic acid SOI is identical to the nucleic acid sequence of the same length in the 3' direction starting from nucleotide 79769 of NW_003616412.1; (v) The nucleotide immediately adjacent to the 5' end of the targeted integration exogenous nucleic acid SOI is nucleotide 315265 of NW_003615063.1, The nucleotide immediately adjacent to the 3' end of the targeted integration exogenous nucleic acid SOI is nucleotide 315266 of NW_003615063.1, The nucleotide sequence of at least 300 base pairs immediately adjacent to the 5' end of the targeted integration exogenous nucleic acid SOI is identical to the nucleic acid sequence of the same length in the 5' direction starting from nucleotide 315265 of NW_003615063.1, and The nucleotide sequence of at least 300 base pairs immediately adjacent to the 3' end of the targeted integration exogenous nucleic acid SOI is identical to the nucleic acid sequence of the same length in the 3' direction starting from nucleotide 315266 of NW_003615063.1; (vi) The nucleotide immediately adjacent to the 5' end of the targeted integration exogenous nucleic acid SOI is nucleotide 2662054 of NW_006882936.1, The nucleotide immediately adjacent to the 3' end of the targeted integration exogenous nucleic acid SOI is nucleotide 2662055 of NW_006882936.1, The nucleotide sequence of at least 300 base pairs immediately adjacent to the 5' end of the targeted integration exogenous nucleic acid SOI is identical to the nucleic acid sequence of the same length in the 5' direction starting from nucleotide 2662054 of NW_006882936.1, and The nucleotide sequence of at least 300 base pairs immediately adjacent to the 3' end of the targeted integration exogenous nucleic acid SOI is identical to the nucleic acid sequence of the same length in the 3' direction starting from nucleotide 2662055 of NW_006882936.1; or (vii) The nucleotide immediately adjacent to the 5' end of the targeted integration exogenous nucleic acid SOI is nucleotide 97705 of NW_003615411.1, The nucleotide immediately adjacent to the 3' end of the targeted integration exogenous nucleic acid SOI is nucleotide 97706 of NW_003615411.1, The nucleotide sequence of at least 300 base pairs immediately adjacent to the 5' end of the targeted integration exogenous nucleic acid SOI is identical to the nucleic acid sequence of the same length in the 5' direction starting from nucleotide 97705 of NW_003615411.1, and The nucleotide sequence of at least 300 base pairs immediately adjacent to the 3' end of the targeted integration exogenous nucleic acid SOI is identical to the nucleic acid sequence of the same length in the 3' direction starting from nucleotide 97706 of NW_003615411.1; and b) a randomly integrated exogenous nucleic acid SOI encoding a second polypeptide of interest and a second selection marker, wherein the randomly integrated SOI is integrated at least once into the genome of the host cell, The host cell is a CHO host cell.

2. The host cell according to claim 1, wherein the first polypeptide of interest and the second polypeptide of interest are the same.

3. The host cell of claim 1, wherein the first selection marker and the second selection marker are the same. The host cell according to claim 1 , comprising one to ten randomly integrated exogenous nucleic acid SOIs.

5. The host cell according to claim 1, further comprising a second targeted integrated exogenous nucleic acid SOI encoding a second polypeptide of interest integrated into the targeted locus of the genome of the host cell and a second selection marker, wherein the first targeted integrated exogenous nucleic acid SOI and the first selection marker are flanked by a first RRS and a third RRS, and the second targeted exogenous nucleic acid SOI and the second selection marker are flanked by a second RRS and the third RRS.

6. The host cell according to any one of claims 1-4, wherein the polypeptide of interest is selected from the group consisting of a single-chain antibody, an antibody light chain, an antibody heavy chain, a single-chain Fv fragment (scFv) and an Fc fusion protein.

7. The host cell according to any one of claims 1 to 4, wherein the host cell is a CHO K1 host cell, a CHO K1SV host cell, a DG44 host cell, a DUKXB-11 host cell, a CHOK1S host cell or a CHO K1M host cell.

8. The host cell of any one of claims 1-6, wherein the targeted integration of the SOI and a selectable marker is facilitated by an exogenous nuclease.

9. A host cell according to claim 8, wherein the exogenous nuclease is selected from the group consisting of zinc finger nucleases (ZFNs), ZFN dimers, transcription activator-like effector nucleases (TALENs), TAL effector domain fusion proteins, RNA-guided DNA endonucleases, engineered large-range nucleases, and clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) endonucleases.

10. A method for expressing a target polypeptide, the method comprising: a) providing a host cell comprising an exogenous nucleotide sequence integrated at a targeted locus in the genome of the host cell, wherein the exogenous nucleotide sequence comprises two RRSs flanking a first selection marker, wherein the targeted locus is at least 99% homologous to a sequence selected from SEQ ID No. 1 to SEQ ID No. 7, wherein (i) The nucleotide immediately adjacent to the 5' end of the targeted integration exogenous nucleotide sequence is nucleotide 45269 of NW_006874047.1, The nucleotide immediately adjacent to the 3' end of the targeted integration exogenous nucleotide sequence is nucleotide 45270 of NW_006874047.1, The nucleotide sequence of at least 300 base pairs immediately adjacent to the 5' end of the targeted integration exogenous nucleotide sequence is identical to the nucleic acid sequence of the same length in the 5' direction starting from nucleotide 45269 of NW_006874047.1, and the nucleotide sequence of at least 300 base pairs immediately adjacent to the 3' end of the targeted integration exogenous nucleotide sequence is identical to the nucleic acid sequence of the same length in the 3' direction starting from nucleotide 45270 of NW_006874047.1; (ii) The nucleotide immediately adjacent to the 5' end of the targeted integration exogenous nucleotide sequence is nucleotide 207911 of NW_006884592.1, The nucleotide immediately adjacent to the 3' end of the targeted integration exogenous nucleotide sequence is nucleotide 207912 of NW_006884592.1, The nucleotide sequence of at least 300 base pairs immediately adjacent to the 5' end of the targeted integration exogenous nucleotide sequence is identical to the nucleic acid sequence of the same length in the 5' direction starting from nucleotide 207911 of NW_006884592.1, and The nucleotide sequence of at least 300 base pairs immediately adjacent to the 3' end of the targeted integration exogenous nucleotide sequence is identical to the nucleic acid sequence of the same length in the 3' direction starting from nucleotide 207912 of NW_006884592.1; (iii) The nucleotide immediately adjacent to the 5' end of the targeted integration exogenous nucleotide sequence is nucleotide 491909 of NW_006881296.1, The nucleotide immediately adjacent to the 3' end of the targeted integration exogenous nucleotide sequence is nucleotide 491910 of NW_006881296.1, The nucleotide sequence of at least 300 base pairs immediately adjacent to the 5' end of the targeted integration exogenous nucleotide sequence is identical to the nucleic acid sequence of the same length in the 5' direction starting from nucleotide 491909 of NW_006881296.1, and The nucleotide sequence of at least 300 base pairs immediately adjacent to the 3' end of the targeted integration exogenous nucleotide sequence is identical to the nucleic acid sequence of the same length in the 3' direction starting from nucleotide 491910 of NW_006881296.1; (iv) The nucleotide immediately adjacent to the 5' end of the targeted integration exogenous nucleotide sequence is nucleotide 79768 of NW_003616412.1, The nucleotide immediately adjacent to the 3' end of the targeted integration exogenous nucleotide sequence is nucleotide 79769 of NW_003616412.1, The nucleotide sequence of at least 300 base pairs adjacent to the 5' end of the targeted integration exogenous nucleotide sequence is identical to the nucleic acid sequence of the same length in the 5' direction starting from nucleotide 79768 of NW_003616412.1, and the nucleotide sequence of at least 300 base pairs adjacent to the 3' end of the targeted integration exogenous nucleotide sequence is identical to the nucleic acid sequence of the same length in the 3' direction starting from nucleotide 79769 of NW_003616412.1; (v) The nucleotide immediately adjacent to the 5' end of the exogenous nucleotide sequence targeted for integration is nucleotide 315265 of NW_003615063.1, The nucleotide immediately adjacent to the 3' end of the targeted integration exogenous nucleotide sequence is nucleotide 315266 of NW_003615063.1, The nucleotide sequence of at least 300 base pairs immediately adjacent to the 5' end of the targeted integration exogenous nucleotide sequence is identical to the nucleic acid sequence of the same length in the 5' direction starting from nucleotide 315265 of NW_003615063.1, and The nucleotide sequence of at least 300 base pairs immediately adjacent to the 3' end of the targeted integration exogenous nucleotide sequence is identical to the nucleic acid sequence of the same length in the 3' direction starting from nucleotide 315266 of NW_003615063.1; (vi) The nucleotide immediately adjacent to the 5' end of the targeted integration exogenous nucleotide sequence is nucleotide 2662054 of NW_006882936.1, The nucleotide immediately adjacent to the 3' end of the targeted integration exogenous nucleotide sequence is nucleotide 2662055 of NW_006882936.1, The nucleotide sequence of at least 300 base pairs immediately adjacent to the 5' end of the targeted integration exogenous nucleotide sequence is identical to the nucleic acid sequence of the same length in the 5' direction starting from nucleotide 2662054 of NW_006882936.1, and The nucleotide sequence of at least 300 base pairs immediately adjacent to the 3' end of the exogenous nucleotide sequence targeted for integration is identical to the nucleic acid sequence of the same length in the 3' direction starting from nucleotide 2662055 of NW_006882936.1; or (vii) The nucleotide immediately adjacent to the 5' end of the targeted integration exogenous nucleotide sequence is nucleotide 97705 of NW_003615411.1, The nucleotide immediately adjacent to the 3' end of the targeted integration exogenous nucleotide sequence is nucleotide 97706 of NW_003615411.1, The nucleotide sequence of at least 300 base pairs immediately adjacent to the 5' end of the targeted integration exogenous nucleotide sequence is identical to the nucleic acid sequence of the same length in the 5' direction starting from nucleotide 97705 of NW_003615411.1, and The nucleotide sequence of at least 300 base pairs immediately adjacent to the 3' end of the targeted integration exogenous nucleotide sequence is identical to the nucleic acid sequence of the same length in the 3' direction starting from nucleotide 97706 of NW_003615411.1; b) introducing into the cell provided in (a) a nucleic acid comprising two RRSs, the two RRSs matching the two RRSs of the integrated exogenous nucleotide sequence and flanked by a first exogenous nucleic acid SOI encoding a first polypeptide of interest and a second selection marker; c) introducing a recombinase or a nucleic acid encoding a recombinase, wherein the recombinase recognizes the RRS; d) selecting cells expressing the second selection marker; e) introducing a second exogenous nucleic acid SOI encoding a second polypeptide of interest and a third selection marker into the genome of the host cell by random integration; f) selecting cells expressing said third selection marker; and g) culturing the host cell under conditions sufficient to express the first polypeptide of interest and the second polypeptide of interest, The host cell is a CHO host cell.

11. The method according to claim 10, further comprising recovering the first polypeptide of interest and the second polypeptide of interest from the host cell culture.

12. The method of claim 10, wherein the first polypeptide of interest and the second polypeptide of interest are the same.

13. The method according to any one of claims 10-12, wherein the first polypeptide of interest and the second polypeptide of interest are selected from the group consisting of: single-chain antibodies, antibody light chains, antibody heavy chains, single-chain Fv fragments (scFv) and Fc fusion proteins.

14. The method according to any one of claims 10 to 13, wherein the host cell is a CHO K1 host cell, a CHO K1SV host cell, a DG44 host cell, a DUKXB-11 host cell, a CHOK1S host cell or a CHO K1M host cell.

15. The method of any one of claims 10-14, wherein the targeted integration of any of the SOIs is facilitated by an exogenous nuclease.

16. The method of claim 15, wherein the exogenous nuclease is selected from the group consisting of zinc finger nucleases (ZFNs), ZFN dimers, transcription activator-like effector nucleases (TALENs), TAL effector domain fusion proteins, RNA-guided DNA endonucleases, engineered meganucleases, and clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) endonucleases.

17. The method according to any one of claims 1 to 16, wherein the expression of the SOI is controlled by a regulatable promoter.

18. The method of claim 17, wherein the regulatable promoter is selected from the group consisting of SV40 and CMV promoters.

Images