Multi-vector recombinase mediated cassette exchange

Abstract

The presently disclosed subject matter relates to multi-vector recombinase-mediated cassette exchange methods to achieve targeted integration of sequences of interest for producing host cells that express recombinant proteins such as monoclonal antibodies, as well as components derived therefrom such as bispecific antibodies, such as bispecific antibodies. And other complex forms of proteins such as membrane protein complexes, and other molecules that are difficult to express.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application Serial No. 63 / 292,869, filed on December 22, 2021, the disclosure of each of which is hereby incorporated by reference in its entirety.

[0003] Sequence Listing

[0004] A sequence listing that complies with the rules of WIPO Standard ST.26 is hereby incorporated by reference. The sequence listing has been submitted as an electronic file by EFS-Web in ASCII form encoded as XML. The electronic file was created on December 21, 2022, is named "00B206_1310_ST26.xml", and is 983,880 bytes in size. Technical Field

[0005] The presently disclosed subject matter relates to cell line development employing a multi-vector recombinase-mediated cassette exchange approach to achieve targeted integration of sequences of interest for generating host cells that express recombinant proteins such as monoclonal antibodies, and compositions derived therefrom such as bispecific antibodies, and other complex forms of proteins such as membrane protein complexes, as well as other difficult to express molecules. Background Art

[0006] Due to the rapid development of cell biology and immunology, there is an increasing demand for the development of novel therapeutic recombinant proteins (e.g., monoclonal antibodies, bispecific antibodies, and complex forms of proteins) for a variety of 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, in recent years, Chinese hamster ovary (CHO) cells have been widely used to produce therapeutic monoclonal antibodies or bispecific antibodies and more complex forms of proteins.

[0007] Conventional strategies for developing commercial cell lines typically involve ongoing efforts to integrate nucleotide sequences encoding a polypeptide of interest (either randomly or at specific ("targeted") locations, followed by selection and isolation of cell lines that produce the polypeptide. However, there is a need in the art for new cell line development strategies that not only conserve resources but also generate cell lines that exhibit improved expression levels, product quality attributes, and production culture performance relative to conventional methods. Summary of the invention

[0008] The presently disclosed subject matter relates to a multivector recombinase-mediated cassette exchange (multivector RMCE) method to achieve targeted integration of target sequences for generating host cells that express recombinant proteins such as monoclonal antibodies, and compositions derived therefrom such as bispecific antibodies, and other complex forms of proteins such as membrane protein complexes, as well as other difficult to express molecules.

[0009] In certain embodiments, the present disclosure relates to a TI host cell comprising an exogenous nucleotide sequence integrated at an integration site within the following sequences: a) all or part of nucleotides 41190 to 45269 of NW_006874047.1, all or part of nucleotides 63590 to 207911 of NW_006884592.1, all or part of nucleotides 253831 to 491909 of NW_006881296.1, at least about 90% of all or part of nucleotides 69303 to 79768 of NW_003616412.1, all or part of nucleotides 293481 to 315265 of NW_003615063.1, all or part of nucleotides 2650443 to 2662054 of NW_006882936.1, or all or part of nucleotides 82214 to 97705 of NW_003615411.1 or b) all or part of nucleotides 45270 to 45490 of NW_006874047.1, all or part of nucleotides 207912 to 792374 of NW_006884592.1, all or part of nucleotides 491910 to 667813 of NW_006881296.1, all or part of nucleotides 79769 to 100059 of NW_003616412.1, _003615063.1, all or part of nucleotides 315266 to 362442 of NW_006882936.1, all or part of nucleotides 2662055 to 2701768 of NW_003615411.1, or all or part of nucleotides 97706 to 105117 of NW_003615411.1; and wherein the exogenous nucleotide sequence comprises four or more incompatible recombination recognition sequences (RRS). In certain embodiments, the RRS is selected from the group consisting of: LoxP sequence, LoxP L3 sequence, LoxP 2L sequence, LoxFas sequence, Lox5 1 1 sequence, Lox2272 sequence, Lox2372 sequence, Lox5 l7l sequence, Loxm2 sequence, Lox7l sequence, Lox66 sequence, FRT sequence, Bxbl attP sequence, Bxbl attB sequence, attP sequence and attB sequence. In certain embodiments, the RRS is recognized by a recombinase selected from the group consisting of: Cre recombinase, FLP recombinase, Bxbl integrase and Integrase. In certain embodiments, the exogenous nucleotide sequence comprises a selection marker between the most 5' RRS and the next RRS in the 3' direction. In certain embodiments, the 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. In certain embodiments, the cell comprises a second selection marker, wherein the first selection marker and the second selection marker are different. In certain embodiments, 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. In certain embodiments, the cell comprises a third selection marker and an internal ribosome entry site (IRES), wherein the IRES is operably linked 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 third selection marker is selected from the group consisting of a green fluorescent protein (GFP) marker, an enhanced GFP (eGFP) marker, In some embodiments, the TI host cell is a mammalian host cell. In some embodiments, the TI host cell is a hamster host cell, a human host cell, a rat host cell, or a mouse host cell. In some embodiments, the TI host cell is a Chinese hamster ovary (CHO) host cell, a CHO K1 host cell, a CHOK1 SV host cell, a DG44 host cell, a DUKXB-1 1 host cell, a CHOK1 S host cell, or a CHO K1M host cell.

[0010] In certain embodiments, the present disclosure relates to a method for preparing a TI host cell expressing SOI, the method comprising: a) providing a TI host cell comprising an exogenous nucleotide sequence integrated at an integration site within the following sequence: (A) with all or part of nucleotides 41190 to 45269 of NW_006874047.1, all or part of nucleotides 63590 to 207911 of NW_006884592.1, all or part of nucleotides 253831 to 491909 of NW_006881296.1, and nucleotides 3616412.1 of NW_006874047.1; or (B) is at least about 90% homologous to all or part of nucleotides 45270 to 45490 of NW_006874047.1, all or part of nucleotides 293481 to 315265 of NW_003615063.1, all or part of nucleotides 2650443 to 2662054 of NW_006882936.1, or all or part of nucleotides 82214 to 97705 of NW_003615411.1; or (C) is at least about 90% homologous to all or part of nucleotides 45270 to 45490 of NW_006874047.1, all or part of nucleotides 207912 to 2662054 of NW_006882936.1, or all or part of nucleotides 82214 to 97705 of NW_003615411.1; to 792374, all or part of nucleotides 491910 to 667813 of NW_006881296.1, all or part of nucleotides 79769 to 100059 of NW_003616412.1, all or part of nucleotides 315266 to 362442 of NW_003615063.1, all or part of nucleotides 2662055 to 2701768 of NW_006882936.1, or all or part of nucleotides 97706 to 105117 of NW_003615411.1, at least about 90% homologous; and wherein the exogenous nucleotide sequence comprises four or more incompatible RRSs; b) introducing at least three vectors into the cells provided in a), each vector comprising: (A) two RRSs, which match the two sequentially oriented RRSs on the integrated exogenous nucleotide sequence; and (B) each pair of RRSs, which 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 d) selecting TI cells expressing the selection marker, thereby isolating TI host cells expressing the SOI. In certain embodiments, the recombinase is selected from Cre recombinase, FLP recombinase, Bxbl integrase and Integrase. In certain embodiments, 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 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 CHO host cell, a CHO K1 host cell, a CHO K1SV host cell, a DG44 host cell, a DUKXB-1 1 host cell, a CHOK1S host cell, or a CHO K1M host cell. 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.

[0011] In certain embodiments, the present disclosure relates to a method of expressing an SOI, the method comprising: a) providing a TI host cell comprising an exogenous nucleotide sequence integrated at an integration site within the following sequence: (A) with all or part of nucleotides 41190 to 45269 of NW_006874047.1, all or part of nucleotides 63590 to 207911 of NW_006884592.1, all or part of nucleotides 253831 to 491909 of NW_006881296.1, all or part of nucleotides 69303 to 79768 of NW_003616412.1; or (B) is at least about 90% homologous to all or part of nucleotides 45270 to 45490 of NW_006874047.1, all or part of nucleotides 207912 to 792374 of NW_006884592.1, or all or part of nucleotides 2650443 to 2662054 of NW_006882936.1; or all or part of nucleotides 82214 to 97705 of NW_003615411.1; or (C) is at least about 90% homologous to all or part of nucleotides 45270 to 45490 of NW_006874047.1, all or part of nucleotides 207912 to 792374 of NW_006884592.1, or all or part of nucleotides 2650443 to 2662054 of NW_006882936.1. 296.1, all or part of nucleotides 491910 to 667813 of NW_003616412.1, all or part of nucleotides 79769 to 100059 of NW_003615063.1, all or part of nucleotides 315266 to 362442 of NW_003615063.1, all or part of nucleotides 2662055 to 2701768 of NW_006882936.1, or all or part of nucleotides 97706 to 105117 of NW_003615411.1; and wherein the exogenous nucleotide sequence comprises four or more different b) introducing at least three vectors into the cells provided in a), each vector comprising: (A) two RRSs, which match the two sequentially oriented RRSs on the integrated exogenous nucleotide sequence; and (B) each pair of RRSs, which is 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; d) selecting TI cells expressing the selection marker, thereby isolating TI host cells expressing the SOI; and e) culturing the cells in d) under conditions suitable for expressing the SOI and recovering the expressed protein therefrom. In certain embodiments, the recombinase is selected from Cre recombinase, FLP recombinase, Bxbl integrase and Integrase. In certain embodiments, 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 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 CHO host cell, a CHO K1 host cell, a CHOK1SV host cell, a DG44 host cell, a DUKXB-1 1 host cell, a CHOK1S host cell, or a CHO K1M host cell. 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.

[0012] In certain embodiments, the present disclosure relates to a method for producing a recombinant mammalian cell comprising a nucleic acid encoding an antibody, the method comprising: a) providing a mammalian cell comprising at least a single exogenous nucleic acid comprising four or more incompatible RRSs, the single exogenous nucleic acid incorporated into a predetermined locus in the genome of the mammalian cell; b) introducing at least three vectors into the recombinant mammalian cell of a), each vector comprising an incompatible RRS pair that matches two of the incompatible RRSs contained in the exogenous nucleic acid, the exogenous nucleic acid incorporated into the genome of the mammalian cell at a predetermined locus, and each incompatible RRS pair is flanked by one or more SOIs and / or one or more selection markers, wherein the SOI encodes an antibody; c) introducing one or more recombinases simultaneously or sequentially with the introduction of the at least three vectors comprising the SOIs and / or selection markers; and d) selecting cells expressing one or more of the SOIs and / or selection markers, thereby producing a recombinant mammalian cell comprising a nucleic acid SOI encoding the antibody. In certain embodiments, the exogenous nucleic acid is incorporated at the following loci: (A) with all or part of nucleotides 41190 to 45269 of NW_006874047.1, all or part of nucleotides 63590 to 207911 of NW_006884592.1, all or part of nucleotides 253831 to 491909 of NW_006881296.1, all or part of nucleotides 69303 to 79768 of NW_003616412.1, all or part of nucleotides 293481 to 315265 of NW_003615063.1, all or part of nucleotides 2650443 to 2662054 of NW_006882936.1, or all or part of nucleotides 82214 to 97705 of NW_003615411.1 or (B) all or part of nucleotides 45270 to 45490 of NW_006874047.1, all or part of nucleotides 207912 to 792374 of NW_006884592.1, all or part of nucleotides 491910 to 667813 of NW_006881296.1, or all or part of nucleotides 491910 to 667813 of NW_006881296.1. 79769 to 100059 of NW_003615063.1, all or part of nucleotides 315266 to 362442 of NW_006882936.1, all or part of nucleotides 2662055 to 2701768 of NW_003615411.1, or all or part of nucleotides 97706 to 105117 of NW_003615411.1. In certain embodiments, the recombinase is selected from the group consisting of Cre recombinase, FLP recombinase, Bxbl integrase, and Integrase. In certain embodiments, 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 mammalian cell is a hamster host cell, a human host cell, a rat host cell, or a mouse host cell. In certain embodiments, the mammalian cell is a CHO host cell, a CHO K1 host cell, a CHO K1SV host cell, a DG44 host cell, a DUKXB-1 1 host cell, a CHOK1S host cell, or a CHO K1M host cell. 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.

[0013] In certain embodiments, the present disclosure relates to a method of producing a recombinant mammalian cell comprising a nucleic acid encoding one or more antibodies, the method comprising: a) providing a mammalian cell comprising at least two exogenous nucleic acids, the at least two exogenous nucleic acids incorporated at a predetermined locus in the genome of the mammalian cell, each exogenous nucleic acid comprising four or more incompatible RRSs, wherein the exogenous nucleic acids may comprise the same or different RRSs; b) introducing at least three vectors into the recombinant mammalian cell of a), each vector comprising an incompatible RRS pair, the incompatible RRS pair matching two of the incompatible RRSs contained in one or both of the exogenous nucleic acids, the exogenous nucleic acids incorporated at a predetermined locus in the genome of the mammalian cell, and each incompatible RRS pair is flanked by one or more SOIs and / or one or more selection markers, wherein the SOI encodes an antibody; c) introducing one or more recombinases simultaneously or sequentially with the introduction of the at least three vectors comprising the SOIs and / or selection markers; and d) selecting cells expressing one or more of the SOIs and / or selection markers,

[0014] Thus, a recombinant mammalian cell comprising a nucleic acid SOI encoding the antibody is produced. In certain embodiments, the exogenous nucleic acid is incorporated at a different locus selected from the following loci: (A) all or part of nucleotides 41190 to 45269 of NW_006874047.1, all or part of nucleotides 63590 to 207911 of NW_006884592.1, all or part of nucleotides 253831 to 491909 of NW_006881296.1 , all or part of nucleotides 69303 to 79768 of NW_003616412.1, all or part of nucleotides 293481 to 315265 of NW_003615063.1, all or part of nucleotides 2650443 to 2662054 of NW_006882936.1, or all or part of nucleotides 82214 to 97705 of NW_003615411.1 or (B) is at least about 90% homologous to all or part of nucleotides 45270 to 45490 of NW_006874047.1, all or part of nucleotides 207912 to 792374 of NW_006884592.1, all or part of nucleotides 491910 to 667813 of NW_006881296.1, all or part of nucleotides 491910 to 667813 of NW_003616412.1 In some embodiments, the recombinase is selected from the group consisting of Cre recombinase, FLP recombinase, Bxbl integrase, and NW_003615063.1. Integrase. In certain embodiments, 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 mammalian cell is a hamster host cell, a human host cell, a rat host cell, or a mouse host cell. In certain embodiments, the mammalian cell is a CHO host cell, a CHO K1 host cell, a CHO K1SV host cell, a DG44 host cell, a DUKXB-1 1 host cell, a CHOK1S host cell, or a CHO K1M host cell. 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1An exemplary multi-carrier RMCE strategy of the present disclosure is depicted.

[0016] Figure 2 An exemplary multi-carrier RMCE strategy of the present disclosure is depicted.

[0017] Figure 3 An exemplary multi-carrier RMCE strategy of the present disclosure is depicted.

[0018] Figure 4 An exemplary multi-carrier RMCE strategy of the present disclosure is depicted.

[0019] Figure 5 An exemplary multi-carrier RMCE strategy of the present disclosure is depicted.

[0020] Figure 6 Depicted are the copy number results of CHO cells modified by two-vector RMCE compared to three-vector RMCE. The gene copy number of CHO clones was similar to expectations, with 2HC and 4LC expected for two-vector RMCE clones and 3HC and 6LC expected for three-vector RMCE clones.

[0021] Figure 7 An exemplary multi-carrier RMCE strategy of the present disclosure is depicted.

[0022] FIG. 8A to FIG. 8B Depicted is a comparison of the productivity of TI transfected libraries using dual-plasmid-based RMCE and 2-site multivector plasmid-based RMCE (in this case, three plasmids). Fig. 8A ) and mAb B TI pool (Fig. 8b), both titers (bars) and Qp (dots) were higher.

[0023] FIG. 9A to FIG. 9B Certain CLD parameters assessed for various mAb C TI pools, mAb D TI pools, and mAb E TI pools are graphically depicted, including titer ( Fig. 9A ) and Qp( Fig. 9B ).

[0024] FIG. 10A to FIG. 10C Graphically illustrates the The titers observed for each of the eight mAb C clones in culture ( Fig. 10A ) and the observed Qp ( Fig. 10B ), and also provides a comparison of the average mAb C clone titer and mAb C clone Qp ( Fig. 10C ).

[0025] FIG. 11A to FIG. 11C Graphically illustrates the The titers observed for each of the eight mAb D clones in culture ( Fig.11A ) and the observed Qp ( Fig. 11B ), and also provides a comparison of the mean mAb D clone titer and mAb D clone Qp ( Fig. 11C ).

[0026] FIG. 12A to FIG. 1 B compares the titers of two specific clones (clone A and clone B) from the two-vector method and the multi-vector method, respectively. Fig. 12A ) and Qp( Fig. 12B ). DETAILED DESCRIPTION

[0027] The presently disclosed subject matter relates to a multi-vector RMCE approach to achieve targeted integration of target sequences for generating host cells that express recombinant proteins such as monoclonal antibodies, and compositions derived therefrom such as bispecific antibodies, and other complex forms of proteins such as membrane protein complexes, as well as other difficult to express molecules.

[0028] The subject matter disclosed herein provides specific benefits relative to conventional targeted integration-based methods. For example, if three vectors, such as plasmids, are integrated simultaneously, at least 15 genes (5 chains / donor plasmid) can be introduced simultaneously at a single TI site to improve titer, improve product quality, increase cell adaptability, etc. Similarly, if 4 plasmids are integrated simultaneously, at least 20 genes (5 chains / donor plasmid) can be introduced simultaneously to improve titer, improve product quality, increase cell adaptability, etc.

[0029] The multi-vector RMCE method described herein also allows for the construction of simplified vectors, such as plasmids. In certain embodiments, for mAbs and 1 cell complex formats with up to 9 chains at a single integration site, this construction can be simplified by 1-step 6-way connection. Similarly, the multi-vector RMCE method described herein also allows complex bispecific and trispecific antibodies containing 4 or 5 or more unique genes to be simultaneously integrated into the host genome to generate cell lines expressing high titer and high product quality products. In addition, the three-vector RMCE allows unique plasmids containing all genes required for manufacturing gene therapy products to be simultaneously integrated into the host genome. Finally, additional unique RSS sites can be added via Crispr / Cas or zinc fingers or TALEN or random integration, and if these unique RSS sites correspond to recombinases other than Cre and Flp (e.g. integrase and Bxb1, etc.), which will facilitate the integration of additional genes into one or more landing pads at the same time.

[0030] For the sake of clarity and not limitation, the detailed description is divided into the following subsections:

[0031] 1. Definition

[0032] 2. Exogenous nucleotide sequences

[0033] 3. Host cells

[0034] 4. Targeted Integration

[0035] 5. Products

[0036] 1. Definition

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art. In case 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 methods or materials 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 by reference in their entirety. The materials, methods and examples disclosed herein are illustrative only and are not intended to be limiting.

[0038] As used herein, the terms "comprises," "including," "having," "having," "may," "containing," and variations thereof are intended to be open transitional phrases, terms, or words and 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.

[0039] 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.

[0040] 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, more preferably within 2 times.

[0041] 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 and include, but are not limited to, genes for aminoglycoside phosphotransferases (APHs) (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.

[0042] 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.

[0043] 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 adapters 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.

[0044] 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 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 the protein, using assays unrelated to this activity, such as Western blotting or radioimmunoassay.

[0045] The term "target sequence" is used herein to refer to a polypeptide sequence (or, in some cases, a nucleic acid encoding a polypeptide sequence), wherein the expression of the polypeptide sequence is of interest. In certain embodiments, such polypeptide sequences may comprise a subunit of a multi-subunit protein complex. In certain embodiments, such polypeptide sequences may comprise a fragment of such a subunit. In certain embodiments, such polypeptide sequences may comprise an antibody sequence, such as an antibody heavy chain or light chain sequence. In certain embodiments, such polypeptide sequences may comprise a fragment of such an antibody sequence.

[0046] The term "antibody" is used herein in the broadest sense and encompasses 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.

[0047] As used herein, "standard antibody" and "standard monoclonal antibody" are antibodies or antibody fragments with a single binding specificity. In certain embodiments, the single binding specificity of a standard antibody is the result of pairing a heavy chain sequence or fragment thereof with a light chain sequence or fragment thereof.

[0048] As used herein, a "bispecific antibody" or "BsAb" is an antibody that can simultaneously bind to two different epitopes, such as two different epitopes on two different antigens or two different epitopes on a single antigen. BsAb encompasses many different structures, including variable heavy (V) antibodies that comprise a pairing of two different parent monoclonal antibodies. H ) domain and light (V L ) domains, thereby generating one "arm" of the BsAb (i.e., a paired V H and V L ), which has the binding specificity of the first parent antibody, and the second "arm" of the BsAb, which has the binding specificity of the second parent antibody. BsAb is a subset of multispecific antibodies, where multispecific antibodies contain at least two binding specificities (i.e., BsAb), but also includes trispecific antibodies and antibodies with a greater number of specificities.

[0049] 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.

[0050] 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 LThe 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).

[0051] 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 are incorporated into the genome of a host cell into which they have 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."

[0052] 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.

[0053] As used herein, the term "flanking" refers to a first nucleotide sequence being located at the 5' end or the 3' end or both ends of a second nucleotide sequence. The flanking nucleotide sequence may be adjacent to or at a defined 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 several thousand base pairs.

[0054] 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.

[0055] As used herein, 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 nucleotide sequence. 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.

[0056] As used herein, a "recombinase recognition sequence" (RRS) is a nucleotide sequence 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. As used herein, an "incompatible" RRS is an RRS recognized by a different recombinase.

[0057] As used herein, the term "TI host cell" refers to a cell comprising one or more genomic loci (i.e., integration sites) for expressing a sequence of interest. In certain embodiments, the presence of an exogenous nucleotide sequence at one or more integration sites comprising four or more RRSs (e.g., four or more incompatible RRSs) facilitates integration of the SOI into the TI host cell.

[0058] 2. Exogenous nucleotide sequences

[0059] The currently disclosed subject matter provides a host cell suitable for integrating an exogenous nucleotide sequence. In certain embodiments, the exogenous nucleotide sequence serves as an integration site for use in a multi-vector (i.e., three or more vectors) RMCE strategy, for example, by comprising four or more recombinase recognition sequences. In certain embodiments, the exogenous nucleotide sequence serves as an integration site for use in a multi-vector RMCE strategy comprising four or more incompatible recombinase recognition sequences. In certain embodiments, the exogenous nucleotide sequence encodes a target sequence. Therefore, in certain embodiments, the host cell will comprise one or more exogenous nucleotide sequences, which will promote the targeted integration of one or more exogenous nucleotide sequences encoding one or more target sequences. In certain embodiments, a host cell comprising an exogenous nucleotide sequence at an integration site on the genome of the host cell is referred to as a TI host cell. The exogenous nucleotide sequence encoding one or more target sequences can then be introduced into the TI host cell, and integration is targeted to the integration site. As outlined below, TI host cells may contain multiple integration sites defined by the presence of exogenous nucleotide sequences comprising elements that facilitate integration of the exogenous nucleotide sequences encoding one or more sequences of interest (eg, recombinase recognition sequences).

[0060] 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.

[0061] In certain embodiments, the host cell comprises a first exogenous nucleotide sequence integrated at a first integration site within a specific first locus in the host cell genome and a second exogenous nucleotide sequence integrated at a second integration site within a specific second locus in the genome. In certain embodiments, the host cell comprises a plurality of exogenous nucleotide sequences integrated at a plurality of integration sites in the host cell genome.

[0062] 2.1 Exogenous sequences containing four or more RRS

[0063] In certain embodiments, the integrated exogenous nucleotide sequence comprises four or more RRS, wherein the RRS can be recognized by a recombinase. In certain embodiments, the integrated exogenous nucleotide sequence comprises four or more incompatible RRS. In certain embodiments, the integrated exogenous nucleotide sequence comprises four, five, six, seven or eight or more RRS. In certain embodiments, one or more RRS can 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, attP sequence and attB sequence.

[0064] In certain embodiments, the exogenous nucleotide sequence of integration comprises at least one selection marker. In certain embodiments, the exogenous nucleotide sequence through integration comprises at least four RRS and at least one selection marker. In certain embodiments, the exogenous nucleotide sequence through integration comprises four incompatible 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 of the four RRS are flanked by at least one selection marker, i.e., the first RRS is located at 5' upstream, and the second RRS is located at 3' 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.

[0065] In certain embodiments, the selection marker is located between the first RRS and the second RRS, and the two flanking RRSs 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 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 certain embodiments, the first flanking RRS is attP sequence, and the second flanking RRS is attB sequence. In certain embodiments, the two RRS are positioned in the same orientation. In certain embodiments, the two RRS are both in a forward or reverse orientation. In certain embodiments, the two RRS are positioned in opposite orientations.

[0066] In certain embodiments, the selection marker can 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 can 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.

[0067] In certain embodiments, the integrated exogenous nucleotide sequence comprises two selection markers flanked by at least two of four or more 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, eY 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 of the two selection markers on the side are the same. In certain embodiments, the two RRSs of the two selection markers on the side are different.

[0068] 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.

[0069] 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 through integration includes IRES and two selection markers with at least two of four or more RRS on the side, wherein the IRES is operably connected to the second selection marker. In certain embodiments, the exogenous nucleotide sequence integrated comprises IRES and three selection markers of at least two of at least four RRS, wherein the IRES is operably connected to the third selection marker. In certain embodiments, the exogenous nucleotide sequence integrated comprises IRES and three selection markers of at least two of at least four 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 integrated comprises the first selection marker operably connected to the promoter and the second selection marker operably connected to IRES. In certain embodiments, the exogenous nucleotide sequence integrated comprises the glutamine synthetase selection marker operably connected to the SV40 promoter and the GFP selection marker operably connected to IRES. In certain embodiments, the exogenous nucleotide sequence integrated comprises the thymidine kinase selection marker operably connected to the CMV promoter and the HYG selection marker and the GFP selection marker operably connected to IRES.

[0070] In certain embodiments, the exogenous nucleotide sequence used as an integration site will be present at a site within a specific locus of the genome of the TI host cell. Exemplary TI host cells and strategies for their use are described in detail in U.S. Patent Application Publication No. US20210002669, the contents of which are incorporated by reference in their entirety.

[0071] In certain embodiments employing targeted integration, 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 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 the group consisting of Contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1.

[0072] 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, Nucleotides 253831-491909 of W_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.

[0073] 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.

[0074] 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 sequences at least 50% homologous thereto. In certain embodiments, the integrated exogenous sequence 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, 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 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 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.

[0075] In certain embodiments, the integrated exogenous nucleotide sequence is operably linked to a nucleotide sequence selected from the group consisting of Contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1, 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 the group consisting of: Contig NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1.

[0076] In certain embodiments, the nucleic acid encoding the product of interest can be integrated into the host cell genome using transposase-based integration. Transposase-based integration techniques are disclosed in, for example, Trubitsyna et al., Nucleic Acids Res. 45(10): e89(2017), Li et al., PNAS 110(25): E2279-E2287(2013) and WO 2004 / 009792, which are incorporated herein by reference in their entirety.

[0077] Table 1 provides exemplary TI host cell integration sites:

[0078] Table 1 - TI host cell integration sites

[0079]

[0080] 2.2 Exogenous nucleotide sequences containing SOI

[0081] 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, at least nine, at least ten or more SOIs. In certain embodiments, the SOIs are the same. In certain embodiments, the SOIs are different.

[0082] As described above, in some embodiments, SOI encodes one or more subunits of a multi-subunit protein complex. In some embodiments, such polypeptide sequences may comprise fragments of such subunit sequences. In some embodiments, the target sequence may comprise a combination of such subunit sequences. For example, but not by way of limitation, such a combination may comprise one, two, three, four, five, six, seven, eight, nine, ten or more of a first subunit sequence and / or one, two, three, four, five, six, seven, eight, nine, ten or more sequences of a second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth or more subunit sequence. In addition, in some embodiments, such a combination may comprise one, two, three, four, five, six, seven, eight, nine, ten or more significant variations of a first subunit sequence and / or one, two, three, four, five, six, seven, eight, nine, ten or more significant variations of a second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth or more subunit sequence.

[0083] 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 SOIs encoding heavy and light chain sequences can be integrated into, for example, a single exogenous nucleic acid sequence present at a single integration site, multiple exogenous nucleic acid sequences present at a single integration site, or multiple exogenous nucleic acid sequences integrated at different integration sites within a TI host cell.

[0084] 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 5' of the selection marker and the exogenous SOI, and the LoxP 2L sequence is located at 3' of the selection marker and the exogenous SOI.

[0085] 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 are different. 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.

[0086] In certain embodiments, the exogenous SOI encodes a target polypeptide, including but not limited to an antibody, an enzyme, a cytokine, a growth factor, a hormone, a viral protein, a bacterial protein, a vaccine protein, or a protein with a therapeutic function. 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 is operably linked to at least one cis-acting element, such as a promoter or an enhancer. In certain embodiments, the exogenous SOI is operably linked to a CMV promoter.

[0087] 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.

[0088] 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 are different. 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.

[0089] 3. Host cells

[0090] 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.

[0091] 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, e.g., described in Graham et al., J. Gen Virol. 36:59 (1977)), baby hamster kidney cells (BHK), mouse Sertoli cells (TM4 cells, e.g., described in Mather, 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 (MMT 060562), TRI cells (e.g., described 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.

[0092] 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.

[0093] 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.

[0094] 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.

[0095] Conventional cell biology methods can be used to introduce the target exogenous nucleotides or vectors into host cells, including but not limited to transfection, transduction, electroporation or injection. In certain embodiments, the target exogenous nucleotides or vectors are introduced into host cells using chemical-based transfection methods, including lipid-based transfection methods, calcium phosphate-based transfection methods, cationic polymer-based transfection methods, or nanoparticle-based transfection methods. In certain embodiments, viral-mediated transduction is used to introduce the target exogenous nucleotides or vectors into host cells, including but not limited to transduction mediated by lentivirus, retrovirus, adenovirus or adeno-associated virus. In certain embodiments, the target exogenous nucleotides or vectors are introduced into host cells by gene gun-mediated injection. In certain embodiments, DNA and RNA molecules are introduced into host cells using the methods described herein.

[0096] 4. Targeted Integration

[0097] Targeted integration methods allow exogenous nucleotide sequences 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.

[0098] 4.1. Targeted integration via multi-vector RMCE

[0099] Multi-vector RMCE allows one or more donor DNA molecules to be unidirectionally integrated into a predetermined site of the host cell genome, and precisely exchanges 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 four heterospecific RRSs (although in some RMCE examples, "separate selection markers" can be used) and at least one exogenous SOI, at least two of which are flanked by at least one selection marker. RMCE involves double recombination crossover events between two heterospecific RRSs within the target genomic locus and donor DNA molecules, which are catalyzed by recombinases. RMCE is designed to introduce a copy of SOI or a 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 immune or defense mechanisms. The RMCE method can be repeated through multiple DNA cassettes. For example, the RMCE procedure can be used to facilitate the introduction of multiple different "front" cassettes and multiple different "back" cassettes. However, as described above, RMCE (and other integration strategies) can be used to introduce as few as one cassette and as many as ten or more cassettes into a single predetermined site in the host genome. Figures 1 to 5As outlined in , all three vectors can be used in a multi-vector RMCE approach to introduce three or more SOIs into a single locus. In certain embodiments, multi-RMCE can be used to introduce as few as one cassette and as many as ten or more cassettes simultaneously or sequentially into two, three, four, five, six, seven or more different loci.

[0100] 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, attP sequence and attB sequence.

[0101] 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 Integrase recognition.

[0102] 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 Bxb1attB site, the host cell requires Bxb1 integrase to perform recombination. In certain embodiments, when the RRS is a attP or attB site, the host cell requires Integrase to perform recombination. The recombinase can be introduced into the host cell using an expression vector containing the coding sequence of the enzyme.

[0103] The Cre-LoxP site-specific recombination system has been widely used in many biological experimental systems. Cre is a 38kDa 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 positions, 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.

[0104] 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 the efficiency of 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 13bp repeat sequence on the left. The Lox66 sequence has 5bp mutations in the 13bp repeat sequence on the right. Both the wild-type LoxP sequence and the mutant LoxP sequence can mediate Cre-dependent recombination.

[0105] 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.

[0106] Bxb1 and Belong to the serine recombinase family. They all originate from bacteriophages and are used by these bacteriophages 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 (40-60bp) DNA substrates called attP and attB sequences, which are originally located at attachment sites on phage DNA and bacterial DNA, respectively. After recombination, two new sequences are formed, called attL and attR sequences, each of which contains half sequences from attP and attB. Recombination can also occur between attL and attR sequences, cutting the integrated phage from 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 and unidirectional recombination that can be recognized by these integrases, these recombination systems have been developed as a supplement to the widely used Cre-LoxP and FRT-FLP systems for genetic engineering purposes.

[0107] The term "matched RRS" means that recombination has occurred between the two RRS. In certain embodiments, the two matched RRS are identical. In certain embodiments, both RRS are wild-type LoxP sequences. In certain embodiments, both RRS are mutant LoxP sequences. In certain embodiments, both RRS are wild-type FRT sequences. In certain embodiments, both RRS are mutant FRT sequences. In certain embodiments, the two RRS are different sequences but can be recognized by the same recombinase. In certain embodiments, the first matched RRS is a Bxb1 attP sequence and the second matched RRS is a Bxb1 attB sequence. In certain embodiments, the first matched RRS is attB sequence, and the second matching RRS is attB sequence.

[0108] In certain embodiments, targeted integration is achieved by multiple RMCEs, wherein DNA cassettes from multiple vectors, each of which comprises at least one exogenous SOI flanked by two heterologous specific RRSs or at least one selection marker, are all integrated into a predetermined site of the host cell genome. In certain embodiments, the selection marker may be partially encoded on the first vector and partially encoded on the second, third, or subsequent vectors, such that the integration of multiple RMCEs allows the expression of the selection marker.

[0109] 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 containing 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. In certain embodiments of multi-vector RMCE, the selection marker and two SOIs can be incorporated into a first locus, while an additional segment marker and an additional SOI are incorporated into a second locus. In addition, or alternatively, the methods described herein provide for the incorporation of multiple SOIs at multiple loci, wherein each locus can be an incorporation site for one or more SOIs.

[0110] In certain embodiments, the target sequence comprises the sequence of one or more subunits of a multi-subunit protein complex. In certain embodiments, such polypeptide sequences may comprise fragments of such subunit sequences. In certain embodiments, the target sequence may comprise a combination of such subunit sequences. For example, but not by way of limitation, such combinations may comprise one, two, three, four, five, six, seven, eight, nine, ten or more of a first subunit sequence and / or one, two, three, four, five, six, seven, eight, nine, ten or more sequences of a second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth or more subunit sequence. In addition, in certain embodiments, such combinations may comprise one, two, three, four, five, six, seven, eight, nine, ten or more significant variations of a first subunit sequence and / or one, two, three, four, five, six, seven, eight, nine, ten or more significant variations of a second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth or more subunit sequence.

[0111] In certain embodiments, the plurality of target sequences comprises one or more heavy chain sequences ("H") and / or one or more antibody light chain sequences ("L"). As used herein, such "H" and "L" sequences may be full-length heavy or light chain sequences and heavy or light chain fragments, including but not limited to variable region fragments and complementary determining region fragments. In certain embodiments, the target sequence may comprise a combination of such H and L sequences. For example, but not limiting, such combinations may comprise one, two, three, four, five, six, seven, eight, nine, ten or more H sequences and / or one, two, three, four, five, six, seven, eight, nine, ten or more L sequences. In addition, in certain embodiments, such combinations may comprise one, two, three, four, five, six, seven, eight, nine, ten or more of the same or different apparent H' sequences and / or one, two, three, four, five, six, seven, eight, nine, ten or more of the same or different apparent L' sequences. The presently disclosed subject matter also encompasses sequences comprising from one to ten (or more) additional H and L sequences, e.g., H", H"', L", and L"'. Exemplary embodiments include, but are not limited to, sequences comprising any combination of various apparent H sequences and various apparent L sequences, such as: HL; LH; H'L; LH'; H'L'; L'H'; HLL; HHL; LLH; LHH; H'LL; H'HL; L'HH; L'LH; H'H'L; LH'H'; HLLL; LLLH; HLHL; LHLH; H'LLL; L'LLH; H'LHL; L'HLH; etc.

[0112] In certain embodiments, the present disclosure relates to a method for expressing an SOI, the method comprising: providing a plurality of TI host cells; contacting the plurality of TI host cells with a plurality of vectors comprising one or more SOIs and at least one marker; introducing the one or more SOIs into one or more of the plurality of TI host cells; selecting TI cells expressing the target sequence; and culturing the cells under conditions suitable for expressing the target sequence and recovering the target sequence therefrom.

[0113] In certain embodiments, the transfected host cell will contain one or more target sequences, wherein the target sequence comprises the sequence of one or more subunits of a multi-subunit protein complex. In certain embodiments, such polypeptide sequences may comprise fragments of such subunit sequences. In certain embodiments, the target sequence may comprise a combination of such subunit sequences. For example, but not limiting, such a combination may comprise one, two, three, four, five, six, seven, eight, nine, ten or more of a first subunit sequence and / or one, two, three, four, five, six, seven, eight, nine, ten or more sequences of a second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth or more subunit sequence. Furthermore, in certain embodiments, such combinations may be intended to include one, two, three, four, five, six, seven, eight, nine, ten or more of the same or different distinct variations of a first subunit sequence, and / or one, two, three, four, five, six, seven, eight, nine, ten or more of the same or different distinct variations of a second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth or more subunit sequences.

[0114] In certain embodiments, the transfected host cells will contain one or more target sequences, wherein the target sequence comprises a combination of H and L sequences. For example, but not by way of limitation, such combinations may comprise one, two, three, four, five, six, seven, eight, nine, ten or more H sequences and / or one, two, three, four, five, six, seven, eight, nine, ten or more L sequences. In addition, in certain embodiments, such combinations may comprise one, two, three, four, five, six, seven, eight, nine, ten or more distinct H' sequences and / or one, two, three, four, five, six, seven, eight, nine, ten or more distinct L' sequences. The presently disclosed subject matter also encompasses the inclusion of one to ten (or more) additional H and L sequences, such as H", H"', L", and L"'. Exemplary embodiments include, but are not limited to, sequences comprising any combination of various apparent H sequences and various apparent L sequences, such as: HL; LH; H'L; LH'; H'L'; L'H'; HLL; HHL; LLH; LHH; H'LL; H'HL; L'HH; L'LH; H'H'L; LH'H'; HLLL; LLLH; HLHL; LHLH; H'LLL; L'LLH; H'LHL; L'HLH; etc.

[0115] 4.2 Adjustable system

[0116] The subject matter disclosed herein also relates to a regulatory system used in a multi-vector RMCE to generate TI host cells. For example, there are many situations where protein expression levels are not optimal, mainly because the encoded protein is difficult to express. The low expression level of a protein that is difficult to express may have a variety of reasons that are difficult to identify. One possibility is the toxicity of the protein expressed in the host cell. In such cases, an adjustable expression system can be used to express the toxic protein, in which the target sequence encoding the protein is under the control of an inducible promoter. In these systems, the expression of the difficult-to-express protein is promoted only when a regulatory factor (e.g., a small molecule, such as but not limited to tetracycline or its analogs, doxycycline (DOX)) is added to the culture medium. Regulating the expression of toxic proteins can reduce toxic effects and enable the culture to achieve the desired cell growth before production. In certain embodiments, the TI expression system prepared according to the present disclosure comprises an SOI that is integrated into a specific locus (e.g., an exogenous nucleic acid sequence comprising one or more RRSs) and transcribed under a regulatory promoter operably connected thereto. In certain embodiments, the TI expression system prepared according to the present disclosure can be used to determine the root cause of low protein expression of molecules that are difficult to express (such as but not limited to antibodies). In certain embodiments, the ability to selectively shut down SOI expression in a TI system can be used to correlate the expression of the SOI with observed adverse effects.

[0117] In certain embodiments, to minimize the effects of transcription and cell line variability during root cause analysis of difficult-to-express molecules, a TI system can be used. For example, but not by way of limitation, expression of an SOI in a TI host can be triggered by adding a regulatory factor (e.g., doxycycline) to the culture medium. In certain embodiments, a TI vector utilizes a promoter regulated by tetracycline to express an SOI that 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 tunable expression of the SOI.

[0118] In certain embodiments, the TI 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 TI 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.

[0119] For example, but not by way of limitation, such regulation can be based on a gene switch for blocking or activating mRNA synthesis by regulatable coupling of a transcriptional repressor or activator to a constitutive or minimal promoter. In certain non-limiting embodiments, repression can be achieved by binding to a repressor protein, for example, where the protein spatially blocks transcription initiation, or actively represses transcription by a transcriptional silencer. In certain non-limiting embodiments, activation of a mammalian or viral-free enhancer minimal promoter can be achieved by regulatable coupling to an activation domain.

[0120] 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.

[0121] 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 TI 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 reaction binding of inhibitor CymR. In certain embodiments, a system based on catabolites can rely on the 6-hydroxynicotine reaction binding activation of a chimeric promoter by the prokaryotic inhibitor HdnoR fused to the herpes simplex VP16 transactivation domain.

[0122] 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 TI system, in which the Pseudomonas aeruginosa quorum sensing suppressors RhlR and LasR are fused to the SV40 T antigen nuclear localization sequence and the herpes simplex VP16 domain, and can activate a promoter containing a specific operator gene sequence (las box).

[0123] In certain embodiments, the inducing molecule that regulates the allosteric protein used in the TI 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.

[0124] In certain embodiments, the intracellular receptor used in the TI system of the present disclosure may be a cytoplasmic or nuclear receptor. In certain embodiments, the TI system of the present disclosure may use small molecules to release transcription factors from chelated and inhibited proteins. In certain embodiments, the TI 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 TI 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.

[0125] In certain embodiments, the TI system 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 TI system 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 TI system disclosed herein may utilize bacterial gyrase B subunit (GyrB), wherein GyrB dimerizes in the presence of the antibiotic coumermycin and dissociates from neomycin.

[0126] In certain embodiments, the TI 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 TI system can utilize the Xenopus terminal oligopyrimidine element (TOP), which blocks translation initiation by forming a hairpin structure in the 5' untranslated region.

[0127] In certain embodiments, the TI system 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.

[0128] In certain embodiments, the TI 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.

[0129] In certain embodiments, the TI 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.

[0130] 5. Products

[0131] Host cells of the present disclosure can be used to express any target molecule. 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, anticoagulant factors, kinases, cytokines, CD proteins, interleukins, therapeutic proteins, diagnostic proteins, and antibodies. In certain embodiments, host cells of the present disclosure can be used to express molecular chaperones, protein modifying enzymes, shRNA, gRNA, or other proteins or peptides, while constitutively or adjustably expressing target therapeutic proteins or molecules.

[0132] In certain embodiments, the polypeptide of interest is a bispecific antibody, a trispecific antibody, or a multispecific antibody (eg, a bispecific antibody).

[0133] Compared to cells used in conventional cell culture methods (e.g., non-TI cells), host cells of the present disclosure can be used to produce a large number of target molecules in a shorter period of time. In certain embodiments, compared to cells used in conventional cell culture methods (e.g., non-TI cells), host cells of the present disclosure can be used to improve the quality of target molecules. In certain embodiments, host cells of the present disclosure can enhance the stability of seed series by preventing chronic toxicity caused by unstable products that may cause cell stress and clone over time. In certain embodiments, host cells of the present disclosure can be used to optimize the expression of acute toxic products.

[0134] In certain embodiments, the host cells and systems of the present disclosure can be used for cell culture process optimization and / or process development.

[0135] 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.

[0136] 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.

[0137] In certain embodiments, the host cells of the present disclosure can be used to exchange transgenic subunits of molecules that are difficult to express (such as, but not limited to, HC and LC subunits of antibodies) with transgenic subunits of common molecules in the 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.

[0138] Examples

[0139] Example 1: Comparison between two-carrier RMCE and multi-carrier RMCE

[0140] In this example, differences in various CLD parameters were identified when multi-carrier RMCE (in this example, three-carrier RMCE) was employed compared to two-carrier RMCE.

[0141] Plastid construction

[0142] For two-vector-based RMCE, a pre-antibody targeting vector and a post-antibody targeting vector were constructed. The HC and LC cDNAs of the antibody were cloned into the pre-vector backbone containing L3, a promoter and a start codon (ATG) followed by a LoxFAS sequence, and the post-vector backbone containing LoxFAS, a Pac lacking a start codon, and a 2L sequence. The Cre recombinase plasmid (pOG231) was used for all RMCE processes.

[0143] For the three-vector-based RMCE, three antibody targeting vectors (plasmids 1, 2, and 3) were constructed. Plasmid 1 is the same as the provector in the two-vector-based RMCE described above. The backbone of plasmid 2 contains LoxFAS, Pac lacking a start codon, a CMV promoter, and Frt3 sequences. The backbone of plasmid 3 contains Frt3, NeoR, and Frt sequences. The coding sequence for the FlpO recombinase was synthesized based on a mouse codon-optimized sequence. The sequence was cloned into a proprietary expression vector driven by a CMV promoter and containing a polyadenylation sequence.

[0144] Transfection and RMCE

[0145] For two-vector-based RMCE, the pre- and post-antibody targeting vectors containing HC and LC and pOG231 were transfected into TI host cells by electroporation using Maxcyte STX (Maxcyte, Gaithersburg, MD).

[0146] For three-vector-based RMCE, plasmids 1, 2, 3 antibody targeting vectors containing HC and LC, and pOG231 and FlpO expression plasmids were transfected into TI host cells by electroporation using Maxcyte STX (Maxcyte, Gaithersburg, MD).

[0147] 48 hours after transfection, cells were pelleted by centrifugation. The pellet was then resuspended in a proprietary DMEM / F12-based medium containing selection and incubated at 37°C and 5% CO2 until the transfected pool was recovered.

[0148] Fed-batch shake flask evaluation of the RMCE library

[0149] Fed-batch production cultures were performed in shake flasks with proprietary chemically defined media and bolus feeds on days 1, 3, and 4. The temperature was maintained at 35°C throughout the run. Titers on day 7 were determined using protein A affinity chromatography with UV detection. Viability percentage and viable cell counts were determined using a Vi-Cell XR instrument (Beckman Coulter).

[0150] Copy number analysis by digital droplet PCR (ddPCR)

[0151] 1×10 50 μL of DNA was purified using the MagNA Pure 96 DNA and Viral NA Small Volume Kit (Roche Molecular Systems, catalog number 06543588001) on a MagNA Pure 96 instrument (Roche, Molecular Systems). 6The genomic DNA of each cell pellet was collected. The copy numbers of Chinese hamster Bcl-2-associated X protein (Bax) and albumin (Alb) were used as references for determining the copies of HC and LC genes, respectively. The ddPCR reactions were prepared according to the manual of the QX200 ddPCR system (Bio-Rad, catalog number 186-3010). In brief, the ddPCR reactions were set up with HEX-labeled HC or LC probes and FAM-labeled Bax or Alb probes, with 20 μl per reaction. The samples were then placed in a QX200 droplet generator, and the droplets were transferred to a 96-well plate, PCR was performed in a thermal cycler, and then droplet reading was performed. The cycling conditions were as follows: 10 minutes at 95°C; 40 cycles of 30 seconds at 94°C and 1 minute at 60°C; then 10 minutes at 98°C to inactivate the enzyme. Primer express v3.0 (Life Technologies) was used to design the primers and probes used in this study.

[0152] mRNA expression by digital droplet reverse transcriptase PCR

[0153] 1×10 50 μL RNA was purified using the MagNA Pure 96 Cellular RNA Large Volume Kit (Roche Molecular Systems, catalog number 05467535001) using the MagNA Pure 96 instrument (Roche, Molecular Systems). 6 Total RNA of each cell pellet was collected and quantified using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, catalog number ND-2000). ddRT-PCR was performed using approximately 20pg to 40pg of RNA using the One-Step RT-ddPCR Advanced Probe Kit (Bio-Rad, catalog number 1864022). The primers and probes for IgG heavy and light chains were the same as those used for gene copy analysis. Droplet generation, thermal cycling, and droplet reading were performed using the Bio-Rad QX200 ddPCR system (Bio-Rad, catalog number 186-3010). Thermal cycling conditions were 1 hour at 50°C; 10 minutes at 95°C; then 40 cycles of 30 seconds at 95°C and 1 minute at 58°C; followed by 10 minutes at 98°C to inactivate the enzyme. The resulting concentration was normalized to the number of copies per 20pg of total RNA.

[0154] Table 2. Differences identified in various CLD parameters when multi-carrier RMCE (in this example, three-carrier RMCE) is employed compared to two-carrier RMCE.

[0155]

[0156] Example 2: Two-carrier RMCE and multi-carrier RMCE 250 Comparison

[0157] In this example, the comparison 250 system (TAP Biosystem) between the gene copy number and mRNA expression of the cell lines expressing antibodies, but one of the cell lines was prepared using a two-plasmid RMCE method and the second cell line was prepared using a multi-vector RMCE method. In the two-plasmid method, each plasmid contains one copy of the heavy chain coding sequence and two copies of the light chain coding sequence ("HLL-HLL"). In the multi-vector method, each plasmid similarly contains one copy of the heavy chain coding sequence and two copies of the light chain coding sequence. However, in view of the presence of a third plasmid in the multi-vector RMCE, this method produces a "HLL-HLL-HLL" configuration.

[0158] exist Twelve clones were evaluated in the TAP Biosystem 250 system (TAP Biosystem). The 250 system is a high-throughput automated method that uses a disposable 250 mL micro bioreactor, which is used in this example as recommended by the manufacturer. In brief, 30 million cells were inoculated into a proprietary chemical composition-defined production medium with a pH of 7.15, and the culture temperature was maintained at 37°C within the first 48 hours. The culture temperature was then converted to 35°C for the remainder of the culture. A 14-day fed-batch culture was cultured with five bolus feeds on days 1, 3, 6, 9, and 12. Cell viability and viable cell counts were monitored using BioProfile Flex2 (Nova Biomedical) by trypan blue dye exclusion.

[0159] Example 3: Comparison of two-vector RMCE and two-site multi-vector RMCE

[0160] In this example, differences in various CLD parameters were identified when multi-vector RMCE (in this example, three-vector RMCE, where two vectors were integrated at the first site and the third vector was integrated at the second site) was employed compared to two-vector RMCE. Figure 7Except as shown, antibody expression constructs were prepared as outlined in Example 1. Briefly, while the green fluorescent protein (GFP) landing pad at the "G" site was used as shown for incorporation into the first two plasmids as described above and in the related figures, the blue fluorescent protein (BFP) landing pad at the "B" site consisted of the zeocin resistance gene (ZeoR) and (BFP) (flanked by Frt and Frt3 recombinase sites). The promoter-less Neo was placed downstream of the Frt site. The antibody targeting vector at the "B" site contained the SAT gene and mAb expression cassettes encoding various HC and LC cDNA copies, followed by a promoter, all flanked by Frt3 and Frt.

[0161] FIG. 8A to FIG. 8B Depicted is a comparison of the productivity of TI transfected libraries using dual-plasmid based RMCE and 2-site multi-vector plasmid (in this case, triple-plasmid) based RMCE. Fig. 8A ) and mAb B TI pool (Fig. 8b), both titers (bars) and Qp (dots) were higher.

[0162] Example 4: Comparison between two-carrier RMCE and multi-carrier RMCE

[0163] In this example, two-vector and multi-vector RMCE (in this case, three-plasmid RMCE) shake flask cultures of mAb C, mAb D, and mAb E were prepared as described above and cultured for seven days. After the seven-day culture was completed, certain CLD parameters of various TI pools of mAb C, mAb D, and mAb E were evaluated, as reported in Table 3. The results are also graphically depicted in Fig. 9A (titer) and Fig. 9B (Qp) in.

[0164] Table 3.

[0165]

[0166] Example 5: Two-carrier RMCE and multi-carrier RMCE Compare

[0167] In this example, cell lines expressing mAb C or mAb D were prepared using two- or multi-vector RMCE (in this case, three-plasmid RMCE) as outlined above. Separate cloning of each of mAb C and mAb D was performed. The 2-vector RMCE clones were cultured (TAP Biosystem) (four clones each of two-vector RMCE and multi-vector RMCE for each antibody), and the multi-vector RMCE clones showed higher mean titers and higher mean Qp relative to the two-vector RMCE clones, as reported in Table 4.

[0168] Table 4.

[0169]

[0170] Fig. 10A The titers observed for each of the eight mAb C clones are graphically illustrated. Fig. 10B The Qp observed for each of those eight mAb C clones is illustrated graphically. Fig. 10C The mean mAb C clone titers and mAb C clone Qp were compared. Fig.11A The titers observed for each of the eight mAb D clones are graphically illustrated. Fig. 11B The Qp observed for each of those eight mAb D clones is illustrated graphically. Fig. 11C The mean mAb D clone titers and mean mAb D clone Qp were compared. FIG. 12A to FIG. 12B The titers of two specific clones (clone A and clone B) from each of the two-vector and multi-vector approaches were compared ( Fig. 12A ) and Qp( Fig. 12B ). FIG. 12A to FIG. 12B The data presented graphically in is also presented in Table 5 below.

[0171] Table 5.

[0172]

[0173] ****

[0174] The foregoing examples are merely illustrative of the presently disclosed subject matter and should not be construed as limiting in any way.

[0175] In addition to the various embodiments depicted and claimed, the disclosed subject matter also relates to other embodiments having other combinations of features disclosed and claimed herein. Thus, the specific features presented herein may be combined with each other in other ways within the scope of the disclosed subject matter, such that the disclosed subject matter includes any suitable combination of features disclosed herein. For purposes of illustration and description, the foregoing description of specific embodiments of the disclosed subject matter has been presented. It is not intended to be exhaustive or to limit the disclosed subject matter to those embodiments disclosed.

[0176] It is obvious to those skilled in the art that various modifications and changes can be made to the composition and method of the disclosed subject matter without departing from the spirit or scope of the disclosed subject matter. Therefore, it is intended that the disclosed subject matter includes modifications and changes within the scope of the attached claims and their equivalents.

[0177] Various publications, patents, and patent applications are cited herein, the contents of which are incorporated by reference in their entireties.

Claims

1. A targeted integration (TI) host cell comprising an exogenous nucleotide sequence integrated at an integration site within the following sequence: a) is at least about 90% homologous to all or part of nucleotides 41190 to 45269 of NW_006874047.1, all or part of nucleotides 63590 to 207911 of NW_006884592.1, all or part of nucleotides 253831 to 491909 of NW_006881296.1, all or part of nucleotides 69303 to 79768 of NW_003616412.1, all or part of nucleotides 293481 to 315265 of NW_003615063.1, all or part of nucleotides 2650443 to 2662054 of NW_006882936.1, or all or part of nucleotides 82214 to 97705 of NW_003615411.1; or b) all or part of nucleotides 45270 to 45490 of NW_006874047.1, all or part of nucleotides 207912 to 792374 of NW_006884592.1, all or part of nucleotides 491910 to 667813 of NW_006881296.1, all or part of nucleotides 79769 to 667814 of NW_003616412.1 100059, all or part of nucleotides 315266 to 362442 of NW_003615063.1, all or part of nucleotides 2662055 to 2701768 of NW_006882936.1, or all or part of nucleotides 97706 to 105117 of NW_003615411.1; and The exogenous nucleotide sequence comprises four or more incompatible recombination recognition sequences (RRS).

2. The TI host cell according to claim 1, wherein the RRS is selected from the group consisting of: LoxP sequence, LoxP L3 sequence, LoxP 2L sequence, LoxFas sequence, Lox5 11 sequence, Lox2272 sequence, Lox2372 sequence, Lox5171 sequence, Loxm2 sequence, Lox71 sequence, Lox66 sequence, FRT sequence, Bxbl attP sequence, Bxbl attB sequence, attP sequence and attB sequence.

3. The TI host cell according to claim 1, wherein the RRS is recognized by a recombinase selected from the group consisting of: Cre recombinase, FLP recombinase, Bxbl integrase and Integrase.

4. The TI host cell according to any one of claims 1 to 3, wherein the exogenous nucleotide sequence comprises a selection marker located between the 5'-most RRS and the next RRS in the 3' direction.

5. The TI host cell according to claim 4, wherein the 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.

6. The TI host cell of claim 5, further comprising a second selection marker, wherein the first selection marker and the second selection marker are different.

7. The TI host cell of claim 6, wherein 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.

8. The TI host cell of claim 6, further comprising a third selection marker and an internal ribosome entry site (IRES), wherein the IRES is operably linked to the third selection marker.

9. The TI host cell of claim 8, wherein the third selection marker is different from the first selection marker or the second selection marker.

10. The TI host cell according to claim 9, 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, an mOrange marker, an mKO marker, an mCitrine marker, a Venus marker, a YPet marker, an Emerald6 marker, a CyPet marker, an mCFPm marker, a Cerulean marker and a T-Sapphire marker.

11. The TI host cell according to any one of claims 1 to 10, wherein the TI host cell is a mammalian host cell. 12 . The TI host cell according to claim 11 , wherein the TI host cell is a hamster host cell, a human host cell, a rat host cell or a mouse host cell.

13. The TI host cell according to claim 11 or 12, wherein the TI host cell is a Chinese hamster ovary (CHO) host cell, a CHO K1 host cell, a CHO K1 SV host cell, a DG44 host cell, a DUKXB-1 1 host cell, a CHOK1 S host cell or a CHO K1M host cell.

14. A method for preparing a TI host cell expressing a sequence of interest (SOI), the method comprising: a) providing a TI host cell comprising an exogenous nucleotide sequence integrated at an integration site within: a. is at least about 90% homologous to all or part of nucleotides 41190 to 45269 of NW_006874047.1, all or part of nucleotides 63590 to 207911 of NW_006884592.1, all or part of nucleotides 253831 to 491909 of NW_006881296.1, all or part of nucleotides 69303 to 79768 of NW_003616412.1, all or part of nucleotides 293481 to 315265 of NW_003615063.1, all or part of nucleotides 2650443 to 2662054 of NW_006882936.1, or all or part of nucleotides 82214 to 97705 of NW_003615411.1; or b. All or part of nucleotides 45270 to 45490 of NW_006874047.1, all or part of nucleotides 207912 to 792374 of NW_006884592.1, all or part of nucleotides 491910 to 667813 of NW_006881296.1, all or part of nucleotides 79769 to 667813 of NW_003616412.1 100059, all or part of nucleotides 315266 to 362442 of NW_003615063.1, all or part of nucleotides 2662055 to 2701768 of NW_006882936.1, or all or part of nucleotides 97706 to 105117 of NW_003615411.1; and wherein the exogenous nucleotide sequence comprises four or more incompatible RRSs; b) introducing into said cell provided in a) at least three vectors, each vector comprising: a. two RRSs that match two sequentially oriented RRSs on the integrated exogenous nucleotide sequence; as well as b. Each RRS pair is 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; as well as d) selecting TI cells expressing the selection marker, thereby isolating TI host cells expressing the SOI.

15. The method according to claim 14, wherein the recombinase is selected from the group consisting of Cre recombinase, FLP recombinase, Bxbl integrase and Integrase.

16. The method of any one of claims 14 to 15, wherein the 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.

17. The method according to any one of claims 14 to 16, wherein the TI host cell is a mammalian host cell.

18. The method of claim 17, wherein the TI host cell is a hamster host cell, a human host cell, a rat host cell or a mouse host cell.

19. The method according to claim 17 or 18, 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 CHOK1M host cell.

20. The method according to any one of claims 14 to 19, 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.

21. A method for expressing SOI, the method comprising: a) providing a TI host cell comprising an exogenous nucleotide sequence integrated at an integration site within the following sequence: a. is at least about 90% homologous to all or part of nucleotides 41190 to 45269 of NW_006874047.1, all or part of nucleotides 63590 to 207911 of NW_006884592.1, all or part of nucleotides 253831 to 491909 of NW_006881296.1, all or part of nucleotides 69303 to 79768 of NW_003616412.1, all or part of nucleotides 293481 to 315265 of NW_003615063.1, all or part of nucleotides 2650443 to 2662054 of NW_006882936.1, or all or part of nucleotides 82214 to 97705 of NW_003615411.1; or b. All or part of nucleotides 45270 to 45490 of NW_006874047.1, all or part of nucleotides 207912 to 792374 of NW_006884592.1, all or part of nucleotides 491910 to 667813 of NW_006881296.1, all or part of nucleotides 79769 to 667813 of NW_003616412.1 100059, all or part of nucleotides 315266 to 362442 of NW_003615063.1, all or part of nucleotides 2662055 to 2701768 of NW_006882936.1, or all or part of nucleotides 97706 to 105117 of NW_003615411.1; and wherein the exogenous nucleotide sequence comprises four or more incompatible RRSs; b) introducing into said cell provided in a) at least three vectors, each vector comprising: a. two RRSs that match two sequentially oriented RRSs on the integrated exogenous nucleotide sequence; as well as b. Each RRS pair is 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; d) selecting TI cells expressing the selection marker, thereby isolating TI host cells expressing the SOI; as well as e) culturing the cells of d) under conditions suitable for expressing the SOI and recovering the expressed protein therefrom.

22. The method according to claim 21, wherein the recombinase is selected from the group consisting of Cre recombinase, FLP recombinase, Bxbl integrase and Integrase.

23. The method of any one of claims 21 to 22, wherein the 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.

24. The method of any one of claims 21 to 23, wherein the TI host cell is a mammalian host cell.

25. The method of claim 24, wherein the TI host cell is a hamster host cell, a human host cell, a rat host cell or a mouse host cell.

26. The method according to claim 24 or 25, 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 CHOK1M host cell.

27. The method according to any one of claims 21 to 26, 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.

28. A method for producing a recombinant mammalian cell comprising a nucleic acid encoding an antibody, the method comprising: a) providing a mammalian cell, said mammalian cell comprising at least a single exogenous nucleic acid comprising four or more incompatible RRSs incorporated into a predetermined locus in the genome of said mammalian cell; b) introducing at least three vectors into the recombinant mammalian cell of a), each vector comprising an incompatible RRS pair that matches two of the incompatible RRSs contained in the exogenous nucleic acid incorporated at a predetermined locus in the genome of the mammalian cell, and each incompatible RRS pair is flanked by one or more SOIs and / or one or more selection markers, wherein the SOI encodes an antibody; c) introducing one or more recombinases simultaneously or sequentially with the introduction of said at least three vectors comprising said SOI and / or selection marker; and d) selecting cells expressing one or more of the SOI and / or the selection marker, thereby generating recombinant mammalian cells comprising the nucleic acid SOI encoding the antibody.

29. The method of claim 28, wherein the exogenous nucleic acid is incorporated at the following locus: a. is at least about 90% homologous to all or part of nucleotides 41190 to 45269 of NW_006874047.1, all or part of nucleotides 63590 to 207911 of NW_006884592.1, all or part of nucleotides 253831 to 491909 of NW_006881296.1, all or part of nucleotides 69303 to 79768 of NW_003616412.1, all or part of nucleotides 293481 to 315265 of NW_003615063.1, all or part of nucleotides 2650443 to 2662054 of NW_006882936.1, or all or part of nucleotides 82214 to 97705 of NW_003615411.1; or b. All or part of nucleotides 45270 to 45490 of NW_006874047.1, all or part of nucleotides 207912 to 792374 of NW_006884592.1, all or part of nucleotides 491910 to 667813 of NW_006881296.1, and all or part of nucleotides 79769 to 667813 of NW_003616412.1 to 100059, all or part of nucleotides 315266 to 362442 of NW_003615063.1, all or part of nucleotides 2662055 to 2701768 of NW_006882936.1, or all or part of nucleotides 97706 to 105117 of NW_003615411.1 are at least about 90% homologous.

30. The method according to claims 28 to 29, wherein the recombinase is selected from the group consisting of Cre recombinase, FLP recombinase, Bxbl integrase and Integrase.

31. The method of any one of claims 28 to 30, wherein the 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.

32. The method of claim 28, wherein the mammalian cell is a hamster host cell, a human host cell, a rat host cell, or a mouse host cell.

33. The method of claim 32, wherein the mammalian 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.

34. The method of any one of claims 28 to 33, 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.

35. A method for producing a recombinant mammalian cell comprising a nucleic acid encoding one or more antibodies, the method comprising: a) providing a mammalian cell, the mammalian cell comprising at least two exogenous nucleic acids incorporated into a predetermined locus of the genome of the mammalian cell, each exogenous nucleic acid comprising four or more incompatible RRSs, wherein the exogenous nucleic acids can comprise the same or different RRSs; b) introducing at least three vectors into the recombinant mammalian cell of a), each vector comprising an incompatible RRS pair that matches two of the incompatible RRSs contained in one or both of the exogenous nucleic acids incorporated at a predetermined locus in the genome of the mammalian cell, and each incompatible RRS pair is flanked by one or more SOIs and / or one or more selectable markers, wherein the SOI encodes an antibody; c) introducing one or more recombinases simultaneously or sequentially with the introduction of said at least three vectors comprising said SOI and / or selection marker; and d) selecting cells expressing one or more of the SOI and / or the selection marker, thereby generating recombinant mammalian cells comprising the nucleic acid SOI encoding the antibody.

36. The method of claim 35, wherein the exogenous nucleic acid is incorporated at a different locus selected from the group consisting of: a. is at least about 90% homologous to all or part of nucleotides 41190 to 45269 of NW_006874047.1, all or part of nucleotides 63590 to 207911 of NW_006884592.1, all or part of nucleotides 253831 to 491909 of NW_006881296.1, all or part of nucleotides 69303 to 79768 of NW_003616412.1, all or part of nucleotides 293481 to 315265 of NW_003615063.1, all or part of nucleotides 2650443 to 2662054 of NW_006882936.1, or all or part of nucleotides 82214 to 97705 of NW_003615411.1; or b. All or part of nucleotides 45270 to 45490 of NW_006874047.1, all or part of nucleotides 207912 to 792374 of NW_006884592.1, all or part of nucleotides 491910 to 667813 of NW_006881296.1, and all or part of nucleotides 79769 to 667813 of NW_003616412.1 to 100059, all or part of nucleotides 315266 to 362442 of NW_003615063.1, all or part of nucleotides 2662055 to 2701768 of NW_006882936.1, or all or part of nucleotides 97706 to 105117 of NW_003615411.1 are at least about 90% homologous.

37. The method according to claim 35 to 36, wherein the recombinase is selected from the group consisting of Cre recombinase, FLP recombinase, Bxbl integrase and Integrase.

38. The method of any one of claims 35 to 37, wherein the 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.

39. The method of claim 35, wherein the mammalian cell is a hamster host cell, a human host cell, a rat host cell, or a mouse host cell.

40. The method of claim 39, wherein the mammalian 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.

41. The method of any one of claims 35 to 40, 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.

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