Multiple vector recombinase-mediated cassette exchange

Abstract

The subject matter disclosed herein relates to a multiple vector recombinase-mediated cassette exchange approach to achieve targeted incorporation of sequences of interest for production host cells expressing recombinant proteins, such as monoclonal antibodies, and compositions derived therefrom, such as bispecific 5 antibodies, and other complex format proteins, such as membrane protein complexes, and other difficult to express molecules.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 292,869, filed December 22, 2021, the disclosure of which is incorporated by reference in its entirety into this specification.

[0002] Sequence Listing A sequence listing conforming to the rules of WIPO standard ST.26 is incorporated herein by reference. The sequence listing was submitted as an electronic document via EFS-Web in ASCII format coded as XML. The electronic document, created on December 21, 2022, is named "00B206_1310_ST26.xml" and is 983,880 bytes in size.

[0003] Technical Field The presently disclosed subject matter relates to the development of cell lines that use a multiple vector recombinase-mediated cassette exchange approach to achieve targeted incorporation of sequences of interest for production host cells expressing recombinant proteins, e.g., monoclonal antibodies, and compositions derived therefrom, e.g., bispecific antibodies, and other complex format proteins, e.g., membrane protein complexes, and other difficult to express molecules. [Background technology]

[0004] Rapid advances in cell biology and immunology have created an increasing demand for the development of novel therapeutic recombinant proteins, such as monoclonal antibodies, bispecific antibodies, and proteins in complex formats, for a variety of diseases, including cancer, cardiovascular disease, and metabolic disease. These biopharmaceutical candidates are generally produced by commercially available cell lines capable of expressing the protein of interest. For example, Chinese Hamster Ovary (CHO) cells have been widely adapted to produce therapeutic monoclonal or bispecific antibodies, as well as proteins in more complex formats in recent years.

[0005] Conventional strategies for developing commercial cell lines typically involve repeated efforts aimed at incorporating nucleotide sequences encoding a polypeptide of interest, either at random or specific ("targeted") locations, followed by selection and isolation of cell lines that produce such polypeptides. However, there is a need in the art for novel 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 compared to conventional methods. Summary of the Invention

[0006] The presently disclosed subject matter relates to a multiple vector recombinase-mediated cassette exchange (multiple vector RMCE) approach to achieve targeted incorporation of sequences of interest for production host cells expressing recombinant proteins, e.g., monoclonal antibodies, and compositions derived therefrom, e.g., bispecific antibodies, and other complex format proteins, e.g., membrane protein complexes, and other difficult to express molecules.

[0007] In some embodiments, the disclosure provides a method for the preparation of a nucleic acid sequence comprising: a) all or a portion of nucleotides 41190-45269 of NW_006874047.1; all or a portion of nucleotides 63590-207911 of NW_006884592.1; all or a portion of nucleotides 253831-491909 of NW_006881296.1; all or a portion of nucleotides 69303-79768 of NW_003616412.1 or b) is at least about 90% homologous to all or a portion of nucleotides 293481-315265 of NW_003615063.1, all or a portion of nucleotides 2650443-2662054 of NW_006882936.1, or all or a portion of nucleotides 82214-97705 of NW_003615411.1; or all or a portion of nucleotides 207912-792374 of NW_006884592.1, all or a portion of nucleotides 491910-667813 of NW_006881296.1, all or a portion of nucleotides 79769-100059 of NW_003616412.1, all or a portion of nucleotides 315266-362442 of NW_003615063.1, NW The present invention relates to a TI host cell comprising an exogenous nucleotide sequence integrated into an integration site within a sequence that is at least about 90% homologous to all or a portion of nucleotides 2662055-2701768 of NW_006882936.1 or all or a portion of nucleotides 97706-105117 of NW_003615411.1, wherein the exogenous nucleotide sequence comprises four or more incompatible recombination recognition sequences (RRS). In some 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 Lox5 11 sequence, a Lox2272 sequence, a Lox2372 sequence, a Lox5 171 sequence, a Loxm2 sequence, a Lox71 sequence, a Lox66 sequence, a FRT sequence, a Bxbl attP sequence, a Bxbl attB sequence, a φC31 attP sequence, and a φC31 attB sequence.In some embodiments, the RRS is recognized by a recombinase selected from the group consisting of Cre recombinase, FLP recombinase, Bxbl integrase, and φC31 integrase. In some embodiments, the exogenous nucleotide sequence comprises a selection marker located between the 5'-most RRS and the next RRS in the 3' direction. In some 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 some embodiments, the cells comprise a second selection marker, which is different from the first selection marker and the second selection marker. In some 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 some embodiments, the cells comprise a third selection marker and an internal ribosome entry site (IRES), the IRES being operably linked to the third selection marker. In some embodiments, the third selection marker is different from the first selection marker or the second selection marker.In some embodiments, 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, a mStrawberry marker, a J-red marker, a DsRed-monomer marker, a mOrange marker, a mKO marker, a mCitrine marker, a Venus marker, a YPet marker, an Emerald6 marker, a CyPet marker, a mCFPm marker, a Cerulean marker, and a T-Sapphire marker. 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 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.

[0008] In some embodiments, the disclosure is directed to a method of preparing a TI host cell that expresses a SOI, the method comprising: a) selecting a TI host cell that is a TI host cell that expresses a SOI comprising: (A) all or a portion of nucleotides 41190-45269 of NW_006874047.1, all or a portion of nucleotides 63590-207911 of NW_006884592.1, all or a portion of nucleotides 253831-491909 of NW_006881296.1, all or a portion of nucleotides 69303-79768 of NW_003616412.1, all or a portion of nucleotides 293481-315265 of NW_003615063.1, all or a portion of nucleotides 2650443-2662054 of NW_006882936.1, or NW_003615411. or (B) is at least about 90% homologous to all or a portion of nucleotides 82214-97705 of NW_006874047.1; or (C) is at least about 90% homologous to all or a portion of nucleotides 45270-45490 of NW_006874047.1, all or a portion of nucleotides 207912-792374 of NW_006884592.1, nucleotides 491910 of NW_006881296.1, or (D) is at least about 90% homologous to all or a portion of nucleotides 82214-97705 of NW_006874047.1; -667813, all or part of nucleotides 79769-100059 of NW_003616412.1, all or part of nucleotides 315266-362442 of NW_003615063.1, all or part of nucleotides 2662055-2701768 of NW_006882936.1, or NW_003615411.and (B) a respective pair of RRSs flanking at least one exogenous SOI and at least one second selectable marker; c) introducing a recombinase or a nucleic acid encoding a recombinase, wherein the recombinase recognizes an RRS; and d) selecting TI cells that express the selectable marker, thereby isolating TI host cells that express the SOI. In some embodiments, the recombinase is selected from Cre recombinase, FLP recombinase, Bxbl integrase, and φC31 integrase. In some embodiments, 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. 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 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 some 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 uptake phage vector, a non-viral vector, a transposon and / or transposase vector, an integrase substrate, and a plasmid.

[0009] In some embodiments, the disclosure is directed to a method for expressing a SOI, the method comprising: a) selecting from: (A) all or a portion of nucleotides 41190-45269 of NW_006874047.1, all or a portion of nucleotides 63590-207911 of NW_006884592.1, all or a portion of nucleotides 253831-491909 of NW_006881296.1, all or a portion of nucleotides 69303-79768 of NW_003616412.1, all or a portion of nucleotides 293481-315265 of NW_003615063.1, all or a portion of nucleotides 2650443-2662054 of NW_006882936.1, or all or a portion of nucleotides 2650443-2662054 of NW_006882936.1, or all or a portion of nucleotides 2650443-2662054 of NW_003615411.1; or (B) is at least about 90% homologous to all or a portion of nucleotides 45270-45490 of NW_006874047.1, all or a portion of nucleotides 207912-792374 of NW_006884592.1, or nucleotides 491910-6 of NW_006881296.1. 67813, all or a portion of nucleotides 79769-100059 of NW_003616412.1, all or a portion of nucleotides 315266-362442 of NW_003615063.1, all or a portion of nucleotides 2662055-2701768 of NW_006882936.1, or NW_003615411.and (B) a respective pair of RRSs flanking at least one exogenous SOI and at least one second selectable marker; c) introducing a recombinase or a nucleic acid encoding a recombinase, wherein the recombinase recognizes an RRS; d) selecting TI cells that express the selectable marker, thereby isolating TI host cells that express the SOI; and e) culturing the cells of d) under conditions suitable for expression of the SOI and recovering the expressed protein therefrom. In some embodiments, the recombinase is selected from Cre recombinase, FLP recombinase, Bxbl integrase, and φC31 integrase. In some embodiments, 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. 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 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 some 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 uptake phage vector, a non-viral vector, a transposon and / or transposase vector, an integrase substrate, and a plasmid.

[0010] In some embodiments, the present disclosure is directed to a method for generating 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 integrated into a predetermined locus of the genome of the mammalian cell comprising four or more incompatible RRSs; b) introducing at least three vectors into the recombinant mammalian cell of a), each vector comprising a pair of incompatible RRSs that match two of the incompatible RRSs comprised in the exogenous nucleic acid integrated into the predetermined locus of the genome of the mammalian cell, each pair of incompatible RRSs flanking one or more SOIs, the SOIs encoding an antibody and / or one or more selectable markers; c) introducing one or more recombinases simultaneously or sequentially with the introduction of the at least three vectors comprising the SOI and / or selectable markers; and d) selecting cells that express one or more of the SOIs and / or selectable markers, thereby generating a recombinant mammalian cell comprising a nucleic acid SOI encoding an antibody.In some embodiments, the exogenous nucleic acid is selected from the group consisting of: (A) all or a portion of nucleotides 41190-45269 of NW_006874047.1; all or a portion of nucleotides 63590-207911 of NW_006884592.1; all or a portion of nucleotides 253831-491909 of NW_006881296.1; is at least about 90% homologous to all or a portion of nucleotides 69303-79768 of NW_003615063.1, all or a portion of nucleotides 293481-315265 of NW_003615063.1, all or a portion of nucleotides 2650443-2662054 of NW_006882936.1, or all or a portion of nucleotides 82214-97705 of NW_003615411.1; or (B) all or a portion of nucleotides 45270-45490 of NW_006874047.1, all or a portion of nucleotides 207912-792374 of NW_006884592.1, all or a portion of nucleotides 491910-667813 of NW_006881296.1, or all of nucleotides 79769-100059 of NW_003616412.1. The SOI is integrated into a locus that is at least about 90% homologous to a portion or portion of NW_003615063.1, all or a portion of nucleotides 315266-362442 of NW_003615063.1, all or a portion of nucleotides 2662055-2701768 of NW_006882936.1, or all or a portion of nucleotides 97706-105117 of NW_003615411.1. In some embodiments, the recombinase is selected from Cre recombinase, FLP recombinase, Bxbl integrase, and φC31 integrase. In some embodiments, 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. In some embodiments, the mammalian cell is a hamster host cell, a human host cell, a rat host cell, or a mouse host cell. In some 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 some embodiments, the vector is selected from the group consisting of an adenoviral vector, an adeno-associated viral vector, a lentiviral vector, a retroviral vector, an integrating phage vector, a non-viral vector, a transposon and / or transposase vector, an integrase substrate, and a plasmid.

[0011] In some embodiments, the disclosure is directed to a method for generating 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 integrated into a predetermined locus in the genome of the mammalian cell, where each exogenous nucleic acid comprises four or more incompatible RRSs, where the exogenous nucleic acids may comprise the same or different RRSs; and b) introducing into the recombinant mammalian cell of a) at least three vectors, where each vector comprises one or more of the exogenous nucleic acids integrated into the predetermined locus in the genome of the mammalian cell. a) introducing at least three vectors comprising a pair of incompatible RRSs that match two of the incompatible RRSs contained in both vectors, each pair of incompatible RRSs flanking one or more SOIs, the SOIs encoding an antibody and / or one or more selectable markers; b) introducing one or more recombinases simultaneously or sequentially with the introduction of the at least three vectors comprising the SOI and / or selectable markers; and d) selecting cells that express one or more of the SOI and / or selectable markers, thereby generating a recombinant mammalian cell comprising a nucleic acid SOI encoding an antibody.In some embodiments, the exogenous nucleic acid is selected from the group consisting of (A) all or a portion of nucleotides 41190-45269 of NW_006874047.1, all or a portion of nucleotides 63590-207911 of NW_006884592.1, all or a portion of nucleotides 253831-491909 of NW_006881296.1, nucleotides 693 of NW_003616412.1, and / or (B) all or a portion of nucleotides 41190-45269 of NW_006874047.1, all or a portion of nucleotides 63590-207911 of NW_006884592.1, all or a portion of nucleotides 253831-491909 of NW_006881296.1, nucleotides 693 of NW_003616412.1, and / or (C) all or a portion of nucleotides 41190-45269 of NW_006874047.1, all or a portion of nucleotides 63590-207911 of NW_006884592.1, all or a portion of nucleotides 253831-491909 of NW_003616412.1, and / or (D) all or a portion of nucleotides 41190-45269 of NW_006874047.1, all or a portion of nucleotides 41190-45269 of NW_006874047.1, all or a portion of nucleotides 63590-207911 of (B) is at least about 90% homologous to all or a portion of NW_003615063.1, all or a portion of nucleotides 293481-315265 of NW_003615063.1, all or a portion of nucleotides 2650443-2662054 of NW_006882936.1, or all or a portion of nucleotides 82214-97705 of NW_003615411.1; all or a portion of nucleotides 45270-45490 of NW_006874047.1, all or a portion of nucleotides 207912-792374 of NW_006884592.1, all or a portion of nucleotides 491910-667813 of NW_006881296.1, all or a portion of nucleotides 79769-100059 of NW_003616412.1, The recombinase is integrated into a separate locus selected from a locus that is at least about 90% homologous to all or a portion of nucleotides 315266-362442 of W_003615063.1, all or a portion of nucleotides 2662055-2701768 of NW_006882936.1, or all or a portion of nucleotides 97706-105117 of NW_003615411.1. In some embodiments, the recombinase is selected from Cre recombinase, FLP recombinase, Bxbl integrase, and φC31 integrase. In some embodiments, 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. In some embodiments, the mammalian cell is a hamster host cell, a human host cell, a rat host cell, or a mouse host cell. In some 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 some embodiments, the vector is selected from the group consisting of an adenoviral vector, an adeno-associated viral vector, a lentiviral vector, a retroviral vector, an integrating phage vector, a non-viral vector, a transposon and / or transposase vector, an integrase substrate, and a plasmid. [Brief description of the drawings]

[0012] [Figure 1] 1 shows an exemplary multi-vector RMCE strategy of the present disclosure. [Diagram 2] 1 shows an exemplary multi-vector RMCE strategy of the present disclosure. [Diagram 3] 1 shows an exemplary multi-vector RMCE strategy of the present disclosure. [Figure 4] 1 shows an exemplary multi-vector RMCE strategy of the present disclosure. [Diagram 5] 1 shows an exemplary multi-vector RMCE strategy of the present disclosure. [Figure 6] Copy number results for CHO cells modified with 2-vector RMCE compared to 3-vector RMCE are shown. Gene copy numbers for CHO clones were close to expected, with 2 HC and 4 LC expected for 2-vector RMCE clones and 3 HC and 6 LC expected for 3-vector RMCE clones. [Figure 7] 1 shows an exemplary multi-vector RMCE strategy of the present disclosure. [Figure 8] A and B show a comparison of the productivity of TI transfection pools using two-plasmid-based RMCE and two-site multiple vector plasmid (in this case, three plasmids)-based RMCE. Both the titer (bars) and Qp (dots) are higher for the two-site multiple vector plasmid-based TI pool of mAb A (A) and the TI pool of mAb B (B). [Figure 9] A and B graphically depict certain CLD parameters evaluated for various TI pools of mAb C, mAb D, and mAb E, including titer (A) and Qp (B). [Figure 10] AC graphically show the titers observed for each of the eight mAb C clones in Ambr15® cultures (A) and the Qp observed for each of the eight mAb C clones (B), providing a comparison of the average mAb C clone titers and mAb C clone Qp (C). [Figure 11] AC graphically show the titers observed for each of the eight mAb D clones in Ambr15® cultures (A) and the Qp observed for each of the eight mAb D clones (B), providing a comparison of the average mAb D clone titers and mAb D clone Qp (C). [Figure 12] A and B compare the titer (A) and Qp (B) of two specific clones (clone A and clone B) from the two-vector and multiple-vector approaches, respectively. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] The subject matter disclosed herein relates to a multi-vector RMCE approach to achieve targeted uptake of sequences of interest for production host cells expressing recombinant proteins, e.g., monoclonal antibodies, and compositions derived therefrom, e.g., bispecific antibodies, and other complex format proteins, e.g., membrane protein complexes, and other difficult to express molecules.

[0014] The subject matter disclosed herein offers certain advantages over traditional targeted integration-based approaches. For example, when three vectors, e.g., plasmids, are integrated simultaneously, at least 15 genes (5 strands / donor plasmid) can be simultaneously introduced into a single TI site for titer enhancement, improved product quality, improved cell compatibility, etc. Similarly, when four plasmids are integrated simultaneously, at least 20 genes (5 strands / donor plasmid) can be simultaneously introduced for titer enhancement, improved product quality, improved cell compatibility, etc.

[0015] The multi-vector RMCE approach described herein also allows for streamlined vector, e.g., plasmid, construction. In some embodiments, such construction can be streamlined using one-step 6-way ligation for both mAb and one-cell composite formats containing up to nine chains in a single incorporation site. Similarly, the multi-vector RMCE approach described herein also allows composite bispecific and trispecific antibodies containing four or five or more unique genes to be simultaneously integrated into the host genome to generate cell lines expressing products with high titer and high product quality. In addition, three-vector RMCE allows for simultaneous integration of unique plasmids containing all the genes required to make a gene therapy product into the host genome. Finally, additional unique RSS sites can be added via Crispr / Cas or zinc finger or TALEN or random integration, and if these unique RSS sites correspond to recombinases other than Cre and Flp (e.g. φC31 integrase & Bxb1 etc.), this will facilitate simultaneous integration of additional genes into one or more landing pads.

[0016] For purposes of clarity of disclosure, and not by way of limitation, the detailed description is divided into the following subsections: 1.Definition 2. Exogenous Nucleotide Sequences 3.Host cells 4. Targeted Uptake 5.Product

[0017] 1.Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. In case of conflict, the present specification, including definitions, will control. Although preferred methods and materials are described below, methods and materials similar or equivalent to those described herein can be used in the practice or testing of the subject matter disclosed herein. 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.

[0018] The terms "comprise," "include," "contain," "having," "has," "can," and variations thereof, as used herein, are intended to be open transitional phrases, terms, or words that do not exclude the possibility of additional features or structures. The singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments that "comprise," "consist," and "consist essentially of" the embodiments or elements presented herein, whether or not expressly indicated.

[0019] When numerical ranges are recited herein, each intervening value is expressly contemplated with the same precision. For example, for the range 6 to 9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0 to 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.

[0020] The term "about" or "approximately" as used herein means within an acceptable error range of a particular value as determined by one of ordinary skill in the art, which will depend in part on the method of measuring or determining the value, i.e., the limitations of the measurement system. For example, "about" can mean within 3 standard deviations or more than 3 standard deviations depending on the field of art. Alternatively, "about" can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and even 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, preferably within 5-fold, more preferably within 2-fold, of a value.

[0021] The term "selection marker" as used herein can be a gene that allows cells carrying the gene to be specifically selected or not selected in the presence of a corresponding selection agent. For example, but not limited to, a selection marker can allow host cells transformed with the selection marker gene to be positively selected in the presence of the gene; non-transformed host cells will not be able to grow or survive under the selection conditions. Selection markers can be positive, negative, or bifunctional. Positive selection markers can allow for the selection of cells carrying the marker, and negative selection markers allow for the selective elimination of cells carrying the marker. Selection markers can confer resistance to drugs or complement metabolic or catabolic defects of the host cell. In prokaryotic cells, genes that confer resistance to ampicillin, tetracycline, kanamycin, or chloramphenicol, among others, can be used. Resistance genes useful as selectable markers in eukaryotic cells include, but are not limited to, genes for aminoglycoside phosphotransferases (APH) (e.g., hygromycin phosphotransferase (HYG), neomycin and G418 (APH), dihydrofolate reductase (DHFR), thymidine kinase (TK), glutamine synthetase (GS), asparagine synthetase, tryptophan synthetase (indole), histidinol dehydrogenase (histidinol D), as well as genes encoding resistance to puromycin, blasticidin, bleomycin, phleomycin, chloramphenicol, zeocin, and mycophenolic acid. Further marker genes are described in WO 92 / 08796 and WO 94 / 28143.

[0022] In addition to facilitating selection in the presence of a corresponding selection agent, a selection marker can 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 a gene can be distinguished from cells not carrying this gene, for example, by detection of the fluorescence emitted by the encoded polypeptide.

[0023] The term "operably linked" as used herein refers to the juxtaposition of two or more components, where the components are in a relationship that allows them to function in their intended manner. For example, a promoter and / or enhancer is operably linked to a coding sequence if it acts to regulate the transcription of the coding sequence. In some embodiments, "operably linked" DNA sequences are contiguous and adjacent on a single chromosome. In some embodiments, for example, when necessary to join two protein coding regions, such as a secretory leader and a polypeptide, the sequences are contiguous, adjacent, and in the same reading frame. In some embodiments, an operably linked promoter may be located upstream of the coding sequence and adjacent to it. In some embodiments, for example, with respect to an enhancer sequence that regulates expression of a coding sequence, the two components may not be contiguous, but may be operably linked. An enhancer is operably linked to a coding sequence if it increases the transcription of the coding sequence. An operably linked enhancer may be located upstream, within, or downstream of the coding sequence, and may be located at a significant distance from the promoter of the coding sequence. Operable linking can be achieved by recombinant methods known in the art, for example, using PCR methodology and / or by ligation at a convenient restriction enzyme recognition site. If a convenient restriction enzyme recognition site does not exist, synthetic oligonucleotide adaptors or linkers can be used according to the 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 in a 5'-end independent manner.

[0024] The term "expression" as used herein refers to transcription and / or translation. In some embodiments, the level of transcription of a desired product can be determined based on the amount of corresponding mRNA present. For example, mRNA transcribed from a sequence of interest can be quantified by PCR or by Northern hybridization. In some embodiments, the protein encoded by a sequence of interest can be quantified by various methods, such as by ELISA, by assaying the biological activity of the protein, or by using an assay independent of such activity, such as Western blotting or radioimmunoassay, using an antibody that recognizes and binds to the protein.

[0025] The term "sequence of interest" is used herein to refer to a polypeptide sequence (or, in some cases, a nucleic acid encoding a polypeptide sequence), for which expression of the polypeptide sequence is desired. Such a polypeptide sequence, in some embodiments, comprises a subunit of a multi-subunit protein complex. In some embodiments, such a polypeptide sequence may comprise a fragment of such a subunit. Such a polypeptide sequence, in some embodiments, comprises an antibody sequence, e.g., an antibody heavy or light chain sequence. In some embodiments, such a polypeptide sequence may comprise a fragment of such an antibody sequence.

[0026] The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), half antibodies, and antibody fragments that exhibit the desired antigen-binding activity.

[0027] As used herein, a "reference antibody" and a "reference monoclonal antibody" are antibodies or antibody fragments that have a single binding specificity. In some embodiments, the single binding specificity of a reference antibody is the result of pairing a heavy chain sequence, or a fragment thereof, with a light chain sequence, or a fragment thereof.

[0028] As used herein, a "bispecific antibody" or "BsAb" is an antibody that can simultaneously bind to two distinct epitopes, e.g., two distinct epitopes on two distinct antigens or two distinct epitopes on a single antigen. BsAbs encompass a number of distinct structures, including structures that combine the variable heavy (V) domains of two distinct parent monoclonal antibodies. H ) domain and light (V L ) domains, resulting in one "arm" of the BsAb having the binding specificity of the first parent antibody (i.e., a pair of V H and V L ) and those which provide a second "arm" of the BsAb with the binding specificity of a second parent antibody. BsAbs are a subset of multispecific antibodies, which not only contain at least two binding specificities (i.e., BsAbs), but also trispecific antibodies, as well as antibodies with even greater numbers of specificities.

[0029] The term "antibody fragment" as used herein refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0030] The term "variable region" or "variable domain" as used herein refers to the domain of an antibody heavy 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 ) usually have a similar structure, each containing four conserved framework regions (FR) and three hypervariable regions (HVR). See, e.g., Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007). A single V H Or V LThe V domain may be sufficient to confer antigen-binding specificity. Moreover, an antibody that binds to a particular antigen may have a complementary V domain. L Or V H To screen the domain libraries, we used V from an antibody that binds to the antigen. H Or V L Domains can be isolated using, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0031] The term "vector" as used herein refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors as self-replicating nucleic acid structures and vectors that are integrated into the genome of a host cell into which they are introduced. In some embodiments, vectors direct the expression of a nucleic acid to which they are operably linked. Such vectors are referred to herein as "expression vectors."

[0032] The term "homologous sequence" as used herein refers to sequences that share significant sequence similarity as determined by sequence alignment. For example, two sequences can be about 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 compare sequences and calculate the statistical significance of matches based on factors such as sequence length, sequence identity and similarity, and the presence and length of sequence mismatches and gaps. Homologous sequences can be referred to as DNA or protein sequences.

[0033] The term "flanking" as used herein refers to a first nucleotide sequence being located at the 5' end or 3' end, or both, of a second nucleotide sequence. The flanking nucleotide sequence can be adjacent to the second nucleotide sequence or at a defined distance from the second nucleotide sequence. There is no particular limitation on the length of the flanking nucleotide sequence. For example, the flanking sequence can be a few base pairs or several thousand base pairs.

[0034] The term "exogenous" as used herein refers to a nucleotide sequence that is not native to the host cell and has been introduced into the host cell by traditional DNA delivery methods, such as by transfection, electroporation, or transformation methods. The term "endogenous" refers to a nucleotide sequence that is native to the host cell. An "exogenous" nucleotide sequence may have an "endogenous" counterpart that is identical in base composition, but an "exogenous" sequence is introduced into the host cell, for example, via recombinant DNA technology.

[0035] As used herein, an "integration site" comprises a nucleic acid sequence within the genome of a host cell into which an exogenous nucleotide sequence is inserted. In some embodiments, the integration site is between two adjacent nucleotides on the genome of a host cell. In some embodiments, the integration site comprises a stretch of a nucleotide sequence. In some embodiments, the integration site is located within a specific locus of the genome of a TI host cell. In some embodiments, the integration site is within an endogenous gene of a TI host cell.

[0036] As used herein, a "recombinase recognition sequence" (RRS) is a nucleotide sequence that is recognized by a recombinase and is necessary and sufficient for a recombinase-mediated recombination event. An RRS can be used to define the location within a nucleotide sequence where a recombination event will occur. As used herein, a "non-compatible" RRS is an RRS that is recognized by a distinct recombinase.

[0037] As used herein, the term "TI host cell" refers to a cell that contains one or more genomic loci, i.e., one or more integration sites, for use in expressing a sequence of interest. In some embodiments, integration of an SOI into a TI host cell is facilitated by the presence of an exogenous nucleotide sequence at one or more integration sites that contain four or more RRSs, e.g., four or more incompatible RRSs.

[0038] 2. Exogenous Nucleotide Sequences The presently disclosed subject matter provides host cells suitable for the incorporation of exogenous nucleotide sequences. In some embodiments, the exogenous nucleotide sequence serves as an incorporation site for use in a multiple vector (i.e., three or more vectors) RMCE strategy, for example, by including four or more recombinase recognition sequences. In some embodiments, the exogenous nucleotide sequence serves as an incorporation site for use in a multiple vector RMCE strategy including four or more incompatible recombinase recognition sequences. In some embodiments, the exogenous nucleotide sequence encodes a sequence of interest. Thus, in some embodiments, the host cell will include one or more exogenous nucleotide sequences that will facilitate targeted incorporation of one or more exogenous nucleotide sequences encoding one or more sequences of interest. In some embodiments, the host cell that includes the exogenous nucleotide sequence incorporated at an incorporation site on the genome of the host cell is referred to as a TI host cell. In this case, the exogenous nucleotide sequence encoding one or more sequences of interest can be introduced into the TI host cell, and incorporation can be targeted to the incorporation site. As outlined below, a TI host cell may contain multiple integration sites defined by the presence of elements that facilitate integration of exogenous nucleotide sequences encoding one or more sequences of interest, e.g., exogenous nucleotide sequences that contain recombinase recognition sequences.

[0039] In some embodiments, the incorporation site and / or the nucleotide sequence flanking the incorporation site can be identified by experiment. In some embodiments, the incorporation site and / or the nucleotide sequence flanking the incorporation site can be identified by genome-wide screening techniques and host cells can be isolated that express at a desired level a polypeptide of interest encoded by one or more SOIs incorporated into one or more exogenous nucleotide sequences, the exogenous sequences themselves being incorporated into one or more loci in the genome of the host cell. In some embodiments, the incorporation site and / or the nucleotide sequence flanking the incorporation site can be identified by genome-wide screening techniques after a transposase-based cassette incorporation event. In some embodiments, the incorporation site and / or the nucleotide sequence flanking the incorporation site can be identified by brute-force random incorporation screening. In some embodiments, the incorporation site and / or the nucleotide sequence flanking the incorporation site can be determined by conventional screening techniques such as targeted locus amplification (TLA) followed by next generation sequencing (NGS) and whole genome NGS. In some embodiments, the location of the incorporation site on the chromosome can be determined by conventional cell biology techniques such as fluorescent in-situ hybridization (FISH) analysis.

[0040] In some embodiments, the host cell comprises a first exogenous nucleotide sequence integrated at a first integration site within a specific first locus of the genome of the host cell and a second exogenous nucleotide sequence integrated at a second integration site within a specific second locus of the genome, hi some embodiments, the host cell comprises multiple exogenous nucleotide sequences integrated at multiple integration sites of the genome of the host cell.

[0041] 2.1 Exogenous sequences containing four or more RRSs In some embodiments, the incorporated exogenous nucleotide sequence comprises four or more RRSs, and the RRSs can be recognized by a recombinase. In some embodiments, the incorporated exogenous nucleotide sequence comprises four or more incompatible RRSs. In some embodiments, the incorporated exogenous nucleotide sequence comprises four, five, six, seven, or eight or more RRSs. In some embodiments, the one or more RRSs can be 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, an FRT sequence, a Bxb1 attP sequence, a Bxb1 attB sequence, a φC31 attP sequence, and a φC31 attB sequence.

[0042] In some embodiments, the incorporated exogenous nucleotide sequence comprises at least one selection marker. In some embodiments, the incorporated exogenous nucleotide sequence comprises at least four RRSs and at least one selection marker. In some embodiments, the incorporated exogenous nucleotide sequence comprises four incompatible RRSs and at least one selection marker. In some embodiments, the selection marker is located between the first RRS and the second RRS. In some embodiments, two of the four RRSs flank at least one selection marker, i.e., the first RRS is located 5' upstream of the selection marker and the second RRS is located 3' downstream of the selection marker. In some 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.

[0043] In some embodiments, the selection marker is located between a first RRS and a second RRS, and the two flanking RRSs are different. In some embodiments, the first flanking RRS is a LoxP L3 sequence and the second flanking RRS is a LoxP 2L sequence. In some embodiments, the LoxP L3 sequence is located 5' of the selection marker and the LoxP 2L sequence is located 3' of the selection marker. In some embodiments, the first flanking RRS is a wild type FRT sequence and the second flanking RRS is a mutant FRT sequence. In some embodiments, the first flanking RRS is a Bxb1 attP sequence and the second flanking RRS is a Bxb1 attB sequence. In some embodiments, the first flanking RRS is a φC31 attP sequence and the second flanking RRS is a φC31 attB sequence. In some embodiments, the two RRSs are positioned in the same orientation. In some embodiments, both of the two RRSs are in a forward or reverse orientation. In some embodiments, the two RRSs are positioned in opposite orientations.

[0044] In some embodiments, the selectable marker is 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 puromycin, blasticidin, bleomycin, phleomycin, It can be a gene encoding resistance to chloramphenicol, zeocin, or mycophenolic acid. In some 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.

[0045] In some embodiments, the incorporated exogenous nucleotide sequence comprises two selection markers flanked by at least two of the four or more RRSs, the first selection marker being different from the second selection marker. In some embodiments, both selection markers are selected from the group consisting of a glutamine synthetase selection marker, a thymidine kinase selection marker, a HYG selection marker, and a puromycin resistance selection marker. In some embodiments, the incorporated exogenous nucleotide sequence comprises a thymidine kinase selection marker and a HYG selection marker. In some 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, and mycophenolic acid, and the second selection marker is selected from the group consisting of genes encoding resistance to 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 some embodiments, the first selection marker is a glutamine synthetase selection marker and the second selection marker is a GFP marker. In some embodiments, the two RRSs flanking both selection markers are the same. In some embodiments, the two RRSs flanking both selection markers are different.

[0046] In some embodiments, the selection marker is operably linked to a promoter sequence. In some embodiments, the selection marker is operably linked to an SV40 promoter. In some embodiments, the selection marker is operably linked to a cytomegalovirus (CMV) promoter.

[0047] In some embodiments, the incorporated exogenous nucleotide sequence comprises at least one selection marker and an IRES, the IRES being operably linked to the selection marker. In some embodiments, the selection marker operably linked to the IRES is selected from the group consisting of GFP, eGFP, synthetic GFP, YFP, eYFP, CFP, mPlum, mCherry, tdTomato, mStrawberry, J-red, DsRed-monomer, mOrange, mKO, mCitrine, Venus, YPet, Emerald, CyPet, mCFPm, Cerulean, and T-Sapphire markers. In some embodiments, the selection marker operably linked to the IRES is a GFP marker. In some embodiments, the incorporated exogenous nucleotide sequence comprises an IRES and two selection markers flanked by at least two of the four or more RRSs, the IRES being operably linked to a second selection marker. In some embodiments, the incorporated exogenous nucleotide sequence comprises an IRES and three selectable markers flanked by at least two of the at least four RRSs, the IRES operably linked to a third selectable marker. In some embodiments, the incorporated exogenous nucleotide sequence comprises an IRES and three selectable markers flanked by at least two of the at least four RRSs, the IRES operably linked to a third selectable marker. In some embodiments, the third selectable marker is different from the first selectable marker or the second selectable marker. In some embodiments, the incorporated exogenous nucleotide sequence comprises a first selectable marker operably linked to a promoter and a second selectable marker operably linked to an IRES. In some embodiments, the incorporated exogenous nucleotide sequence comprises a glutamine synthetase selectable marker operably linked to an SV40 promoter and a GFP selectable marker operably linked to an IRES. In some embodiments, the incorporated exogenous nucleotide sequence comprises a thymidine kinase selection marker and a HYG selection marker operably linked to a CMV promoter, and a GFP selection marker operably linked to an IRES.

[0048] In some embodiments, the exogenous nucleotide sequence that serves as the integration site will be present at a site within a specific locus in 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. 20210002669, the contents of which are incorporated by reference in their entirety.

[0049] In some embodiments using targeted integration, the exogenous nucleotide sequence is integrated into a site within a specific locus of the genome of the TI host cell. In some embodiments, the locus into which the exogenous nucleotide sequence is integrated is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or at least about 99.9% homologous to a sequence selected from contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1.

[0050] In some embodiments, the nucleotide sequence immediately 5' to the incorporated 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 some embodiments, the nucleotide sequence immediately 5' to the incorporated 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_0036150 NW_006882936.1, nucleotides 2650443-2662054 of NW_006882936.1, or nucleotides 82214-97705 of NW_003615411.1.

[0051] In some embodiments, the nucleotide sequence immediately 3' to the incorporated 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 some embodiments, the nucleotide sequence immediately 3' to the incorporated 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_0036150 NW_006882936.1, nucleotides 2662055-2701768 of NW_006882936.1, or nucleotides 97706-105117 of NW_003615411.1.

[0052] In some embodiments, the incorporated exogenous sequence is flanked 5' 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, or a sequence that is at least 50% homologous thereto. In some embodiments, the incorporated exogenous sequence is flanked 3' 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, or a sequence that is at least 50% homologous thereto.In some embodiments, the nucleotide sequence flanking the 5' end of the incorporated exogenous nucleotide 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, and nucleotides 70111-70112 of NW_003616412.1. NW_006882936.1; and NW_003615411.1. In some embodiments, the nucleotide sequence flanking the 3' end of the incorporated exogenous nucleotide 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, and nucleotides 80111-80159 of NW_003616412.1. NW_006882936.1; and NW_003615411.1.

[0053] In some embodiments, the incorporated 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 that are at least 50% homologous thereto. In some 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 contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1.

[0054] In some embodiments, the nucleic acid encoding the product of interest can be incorporated into the genome of the host cell using transposase-based integration. Transposase-based integration techniques are disclosed, for example, in 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.

[0055] Table 1 shows exemplary uptake sites in TI host cells: Table 1 - TI host cell uptake sites TIFF2025500443000002.tif60170

[0056] 2.2 Exogenous nucleotide sequence containing SOI In some embodiments, the incorporated exogenous nucleotide sequence comprises at least one exogenous SOI. In some embodiments, the incorporated exogenous nucleotide sequence comprises at least one selectable marker and at least one exogenous SOI. In some embodiments, the incorporated exogenous nucleotide sequence comprises at least one selectable marker, at least one exogenous SOI, and at least one RRS. In some embodiments, the incorporated 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 some embodiments, the SOIs are the same. In some embodiments, the SOIs are different.

[0057] As noted above, in some embodiments, the SOI encodes one or more subunits of a multi-subunit protein complex. In some embodiments, such polypeptide sequences can include fragments of such subunit sequences. In some embodiments, the sequence of interest can include a combination of such subunit sequences. For example, and without limitation, such combinations can include one, two, three, four, five, six, seven, eight, nine, ten, or more first subunit sequences and / or one, two, three, four, five, six, seven, eight, nine, ten, or more second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, or more subunit sequences. Further, in some embodiments, such combinations can include one, two, three, four, five, six, seven, eight, nine, ten or more distinct variations of a first subunit sequence and / or one, two, three, four, five, six, seven, eight, nine, ten or more distinct variations of a second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth or more subunit sequences.

[0058] In some embodiments, the SOI encodes a single chain antibody or a fragment thereof. In some embodiments, the SOI encodes an antibody heavy chain sequence or a fragment thereof. In some embodiments, the SOI encodes an antibody light chain sequence or a fragment thereof. In some embodiments, the incorporated 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 some embodiments, the incorporated 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 some embodiments, the incorporated 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 some embodiments, the number of SOIs encoding heavy and light chain sequences can be selected to achieve a desired expression level of heavy and light chain polypeptides, e.g., to achieve production of a desired amount of a bispecific antibody. In some embodiments, individual SOIs encoding heavy and light chain sequences can be incorporated within the TI host cell, e.g., into a single exogenous nucleic acid sequence present at a single incorporation site, into multiple exogenous nucleic acid sequences present at a single incorporation site, or into multiple exogenous nucleic acid sequences incorporated at separate incorporation sites.

[0059] In some embodiments, the incorporated exogenous nucleotide sequence comprises at least one selectable marker, at least one exogenous SOI, and one RRS. In some embodiments, the RRS is located adjacent to the at least one selectable marker or the at least one exogenous SOI. In some embodiments, the incorporated exogenous nucleotide sequence comprises at least one selectable marker, at least one exogenous SOI, and two RRSs. In some embodiments, the incorporated exogenous nucleotide sequence comprises at least one selectable marker and at least one exogenous SOI located between a first RRS and a second RRS. In some embodiments, the two RRSs flanking the selectable marker and the exogenous SOI are the same. In some embodiments, the two RRSs flanking the selectable marker and the exogenous SOI are different. In some embodiments, the first flanking RRS is a LoxP L3 sequence and the second flanking RRS is a LoxP 2L sequence. In some embodiments, the L3 LoxP sequence is located 5' of the exogenous SOI and the selectable marker, and the LoxP 2L sequence is located 3' of the exogenous SOI and the selectable marker.

[0060] In some embodiments, the incorporated exogenous nucleotide sequence comprises three RRSs and two exogenous SOIs, and the third RRS is located between the first RRS and the second RRS. In some 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 some embodiments, the first SOI and the second SOI are different. In some embodiments, the first RRS and the second RRS are the same, and the third RRS is different from the first RRS or the second RRS. In some embodiments, all three RRSs are different. In some 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 some embodiments, the incorporated exogenous nucleotide sequence comprises three RRSs, an exogenous SOI, and a selection marker. In some embodiments, the SOI is located between the first and third RRS, and the selection marker is located between the third and second RRS. In some embodiments, the incorporated exogenous nucleotide sequence comprises three RRSs, two exogenous SOIs, and a selection marker. In some embodiments, the first SOI and the selection marker are located between the first and third RRS, and the second SOI is located between the third and second RRS.

[0061] In some embodiments, the exogenous SOI encodes a polypeptide of interest, 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 some embodiments, the exogenous SOI encodes an antibody or an antigen-binding fragment thereof. In some 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 some embodiments, the exogenous SOI is operably linked to at least one cis-acting element, such as a promoter or enhancer. In some embodiments, the exogenous SOI is operably linked to a CMV promoter.

[0062] In some embodiments, the incorporated exogenous nucleotide sequence comprises two RRSs and at least two exogenous SOIs located between the two RRSs. In some embodiments, the SOI encoding one heavy chain and one light chain of the antibody is located between the two RRSs. In some embodiments, the SOI encoding one heavy chain and two light chains of the antibody is located between the two RRSs. In some embodiments, the SOI encoding different combinations of copies of the heavy and light chains of the antibody is located between the two RRSs.

[0063] In some embodiments, the incorporated exogenous nucleotide sequence comprises three RRSs and at least two exogenous SOIs, with the third RRS located between the first and second RRSs. In some embodiments, at least one SOI is located between the first and third RRSs, and at least one SOI is located between the third and second RRSs. In some embodiments, the first and second RRSs are the same, and the third RRS is different from the first or second RRS. In some embodiments, all three RRSs are different. In some embodiments, the SOI encoding one heavy chain and one light chain of the first antibody is located between the first and third RRSs, and the SOI encoding one heavy chain and one light chain of the second antibody is located between the third and second RRSs. In some 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 some 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 some 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 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 some embodiments, the SOIs encoding different combinations of copies of the heavy and light chains of the multiple antibodies are located between the first RRS and the third RRS, and between the third RRS and the second RRS.

[0064] 3.Host cells In some embodiments, the host cell is a eukaryotic host cell. In some embodiments, the host cell is a mammalian host cell. In some embodiments, the host cell is a hamster host cell, a human host cell, a rat host cell, or a mouse host cell. In some 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.

[0065] In some embodiments, the host cell is an SV40 transformed monkey kidney CV1 line (COS-7), a human embryonic kidney line (e.g., 293 or 293 cells described in Graham et al., J. Gen Virol. 36:59 (1977)), baby hamster kidney cells (BHK), mouse Sertoli cells (e.g., TM4 cells 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), e.g., Mather et al., Annals NY Acad. Sci. 383:44-68 (1982), TRI cells, MRC 5 cells, FS4 cells, Y0 cells, NS0 cells, Sp2 / 0 cells, and PER.C6® cells.

[0066] In some embodiments, the host cell is a cell line. In some embodiments, the host cell is a cell line that has been cultured for a certain number of generations. In some embodiments, the host cell is a primary cell.

[0067] In some embodiments, expression of a polypeptide of interest is stable if the expression level is maintained at a constant level or increases or decreases by less than 20% over 10, 20, 30, 50, 100, 200, or 300 generations. In some embodiments, expression of a polypeptide of interest is stable if the culture can be maintained without selection. In some embodiments, expression of a 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.

[0068] In some embodiments, the polypeptide of interest is produced and secreted into the cell culture medium. In some embodiments, the polypeptide of interest is expressed and retained inside the host cell. In some embodiments, the polypeptide of interest is expressed and inserted into and retained in the host cell membrane.

[0069] The exogenous nucleotide or vector of interest can be introduced into the host cell by conventional cell biology methods, including but not limited to transfection, transduction, electroporation, or injection. In some embodiments, the exogenous nucleotide or vector of interest is introduced into the host cell by 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 some embodiments, the exogenous nucleotide of interest is introduced into the host cell by virus-mediated transduction, including but not limited to lentivirus, retrovirus, adenovirus, or adeno-associated virus-mediated transduction. In some embodiments, the exogenous nucleotide or vector of interest is introduced into the host cell via gene gun-mediated injection. In some embodiments, both DNA and RNA molecules are introduced into the host cell using the methods described herein.

[0070] 4. Targeted Uptake Targeted integration techniques allow exogenous nucleotide sequences to be integrated into one or more predetermined sites of the genome of a host cell. In some embodiments, targeted integration is mediated by a recombinase that recognizes one or more RRSs. In some embodiments, targeted integration is mediated by homologous recombination.

[0071] 4.1. Targeted uptake via multivector RMCE Multivector RMCE allows for unidirectional integration of one or more donor DNA molecules into a predefined site in the host cell genome and precise exchange of a DNA cassette present on the donor DNA with a DNA cassette on the host genome where the integration site resides. The DNA cassette features four heterospecific RRSs, at least two of which are flanked by at least one selectable marker (although in some RMCE examples a "split selectable marker" can be used) and at least one exogenous SOI. RMCE involves a recombinase-catalyzed double recombination crossover event between the donor DNA molecule and two heterospecific RRSs within the target genomic locus. RMCE is designed to introduce a copy of the SOI or selectable marker into a predefined locus in the host cell genome. Unlike recombinations involving only one crossover event, RMCE can be performed in such a way that no prokaryotic vector sequences are introduced into the host cell genome, thus reducing and / or preventing unwanted triggering of the host's immune or defense mechanisms. The RMCE procedure can be repeated with multiple DNA cassettes. For example, the RMCE procedure can be used to facilitate the introduction of multiple separate "front" and multiple separate "back" cassettes. However, as described above, RMCE (and other integration strategies) can be used to introduce as little as one cassette and as many as 10 or more cassettes into a single predefined site in the genome of a host cell. For example, as outlined in Figures 1-5, all three vectors can be used in a multiple vector RMCE approach to introduce three or more SOIs into a single locus. In some embodiments, multiplex RMCE can be used to introduce as little as one cassette and as many as 10 or more cassettes into two, three, four, five, six, seven, or more separate loci, either simultaneously or sequentially.

[0072] In some 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, an FRT sequence, a Bxb1 attP sequence, a Bxb1 attB sequence, a φC31 attP sequence, and a φC31 attB sequence.

[0073] In some embodiments, the RRS may be recognized by Cre recombinase. In some embodiments, the RRS may be recognized by FLP recombinase. In some embodiments, the RRS may be recognized by Bxb1 integrase. In some embodiments, the RRS may be recognized by φC31 integrase.

[0074] In some embodiments, when the RRS is a LoxP site, the host cell requires Cre recombinase to perform recombination. In some embodiments, when the RRS is a FRT site, the host cell requires FLP recombinase to perform recombination. In some embodiments, when the RRS is a Bxb1 attP or Bxb1 attB site, the host cell requires Bxb1 integrase to perform recombination. In some embodiments, when the RRS is a φC31 attP or φC31attB site, the host cell requires φC31 integrase to perform recombination. The recombinase can be introduced into the host cell using an expression vector that includes a coding sequence for the enzyme.

[0075] The Cre-LoxP site-specific recombination system is widely used in many biological experimental systems. Cre is a 38-kDa site-specific DNA recombinase that recognizes 34-bp LoxP sequences. Cre is derived from bacteriophage P1 and belongs to the tyrosine family of site-specific recombinases. Cre recombinase can mediate both intra- and intermolecular recombination between LoxP sequences. The LoxP sequence consists of an 8-bp non-palindromic core region flanked by two 13-bp inverted repeats. Cre recombinase binds to the 13-bp repeats, thereby mediating recombination within the 8-bp 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 orientation on the same nucleotide sequence, Cre-mediated recombination will excise the DNA sequence located between the two LoxP sequences as a covalently closed circle. If two LoxP sequences are placed in inverse positions on the same nucleotide sequence, Cre-mediated recombination will invert the orientation of the DNA sequence located between the two sequences. LoxP sequences can also be placed on different chromosomes to facilitate recombination between different chromosomes. If two LoxP sequences are on two different DNA molecules and one DNA molecule is circular, Cre-mediated recombination will result in the incorporation of a circular DNA sequence.

[0076] In some embodiments, the LoxP sequence is a wild-type LoxP sequence. In some embodiments, the LoxP sequence is a mutant LoxP sequence. The mutant LoxP sequence was developed to increase the efficiency of Cre-mediated integration or replacement. In some embodiments, the mutant LoxP sequence is selected from the group consisting of 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, in the Lox71 sequence, 5 bp are mutated in the left 13 bp repeat. In the Lox66 sequence, 5 bp are mutated in the right 13 bp repeat. Both wild-type and mutant LoxP sequences can mediate Cre-dependent recombination.

[0077] The FLP-FRT site-specific recombination system is similar to the Cre-Lox system. This system includes flippase (FLP) recombinase obtained from the 2 μm plasmid of the yeast Saccharomyces cerevisiae. FLP also belongs to the tyrosine family of site-specific recombinases. The FRT sequence is a 34 bp sequence consisting of two palindromic sequences of 13 bp each flanking an 8 bp spacer. FLP binds to the 13 bp palindromic sequence and mediates DNA breakage, exchange and ligation within the 8 bp spacer. As with Cre recombinase, the position and orientation of the two FRT sequences determine the outcome of FLP-mediated recombination. In some embodiments, the FRT sequence is a wild-type FRT sequence. In some embodiments, the FRT sequence is a mutant FRT sequence. Both the wild-type and mutant FRT sequences can mediate FLP-dependent recombination. In some embodiments, the FRT sequence is fused to a responsive receptor domain sequence, such as, but not limited to, a tamoxifen responsive receptor domain sequence.

[0078] Bxb1 and φC31 belong to the serine recombinase family. Both of them are derived from bacteriophages and are used by these bacteriophages to establish lysogeny and facilitate site-specific integration of the phage genome into the bacterial genome. These integrases catalyze site-specific recombination events between short (40-60 bp) DNA substrates, called attP and attB sequences, which are binding sites originally located on the phage DNA and bacterial DNA, respectively. After recombination, two new sequences, called attL and attR sequences, are formed, each of which contains half sequences derived from attP and attB. Recombination can also occur between attL and attR sequences, excising the integrated phage from the bacterial DNA. Both integrases can catalyze recombination without the aid of additional host factors. In the absence of auxiliary factors, these integrases mediate unidirectional recombination between attP and attB with an efficiency of over 80%. Due to the short DNA sequences that can be recognized by these integrases and the unidirectional recombination, these recombination systems were developed as a complement to the Cre-LoxP and FRT-FLP systems that are widely used for genetic engineering purposes.

[0079] The term "matching RRS" indicates that recombination occurs between two RRSs. In some embodiments, the two matching RRSs are the same. In some embodiments, both RRSs are wild-type LoxP sequences. In some embodiments, both RRSs are mutant LoxP sequences. In some embodiments, both RRSs are wild-type FRT sequences. In some embodiments, both RRSs are mutant FRT sequences. In some embodiments, the two matching RRSs are different sequences but can be recognized by the same recombinase. In some embodiments, the first matching RRS is a Bxb1 attP sequence and the second matching RRS is a Bxb1 attB sequence. In some embodiments, the first matching RRS is a φC31 attB sequence and the second matching RRS is a φC31 attB sequence.

[0080] In some embodiments, targeted integration is achieved by multiple RMCEs, where DNA cassettes from multiple vectors, each containing at least an exogenous SOI or at least one selection marker flanked by two heterospecific RRSs, are all integrated into a predefined site in the host cell genome. In some embodiments, the selection marker can be partially encoded on a first vector and partially encoded on a second, third, or more vectors, such that integration of multiple RMCEs allows expression of the selection marker. In some embodiments, targeted integration via recombinase-mediated recombination integrates the selection marker or one or more exogenous SOIs into one or more predefined integration sites in the host cell genome, together with sequences from the prokaryotic vector.

[0081] In some embodiments, targeted integration via recombinase-mediated recombination integrates the selection marker or one or more exogenous SOIs into one or more predefined integration sites of the host cell genome that does not have sequences from the prokaryotic vector. In some embodiments of multi-vector RMCE, the selection marker and two SOIs can be integrated into a first locus, and an additional intercept marker and another SOI is integrated into a second locus. Additionally or alternatively, the methods described herein provide for integration of multiple SOIs at multiple loci, where each locus can be a site of integration for one or more of the SOIs.

[0082] In some embodiments, the sequence of interest comprises the sequence of one or more subunits of a multi-subunit protein complex. In some embodiments, such polypeptide sequences can comprise fragments of such subunit sequences. In some embodiments, the sequence of interest can comprise a combination of such subunit sequences. For example, without limitation, such combinations can comprise one, two, three, four, five, six, seven, eight, nine, ten, or more first subunit sequences and / or one, two, three, four, five, six, seven, eight, nine, ten, or more second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, or more subunit sequences. Further, in some embodiments, such combinations can include one, two, three, four, five, six, seven, eight, nine, ten or more distinct variations of a first subunit sequence and / or one, two, three, four, five, six, seven, eight, nine, ten or more distinct variations of a second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth or more subunit sequences.

[0083] In some embodiments, the plurality of sequences of interest comprises one or more antibody heavy chain sequences ("H") and / or one or more antibody light chain sequences ("L"). As used herein, such "H" and "L" sequences can be full-length heavy or light chain sequences, as well as heavy or light chain fragments, including, but not limited to, variable region fragments and complementarity determining region fragments. In some embodiments, the sequences of interest can comprise combinations of such H and L sequences. For example, but not limited to, such combinations can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more H sequences and / or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more L sequences. Moreover, in some embodiments, such combinations can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more distinct H' sequences that are the same or different, and / or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more distinct L' sequences that are the same or different. The inclusion of 1 to 10 (or more) additional H and L sequences, e.g., H", H'", L", and L'", is also encompassed by the presently disclosed subject matter. Exemplary embodiments include, but are not limited to, sequences that include any combination of various distinct H and various distinct 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.

[0084] In some embodiments, the present disclosure is directed to a method of 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 the TI cells that express a sequence of interest; and culturing the cells under conditions suitable for expression of the sequence of interest and recovering the sequence of interest therefrom.

[0085] In some embodiments, the transfected host cell comprises one or more sequences of interest, the sequences of interest comprising sequences of one or more subunits of a multi-subunit protein complex. In some embodiments, such polypeptide sequences can comprise fragments of such subunit sequences. In some embodiments, the sequences of interest can comprise combinations of such subunit sequences. For example, and without limitation, such combinations can comprise one, two, three, four, five, six, seven, eight, nine, ten, or more first subunit sequences and / or one, two, three, four, five, six, seven, eight, nine, ten, or more second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, or more subunit sequences. Further, in some embodiments, such combinations can include one, two, three, four, five, six, seven, eight, nine, ten or more distinct variations of the same or different first subunit sequences and / or one, two, three, four, five, six, seven, eight, nine, ten or more distinct variations of the same or different second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth or more subunit sequences.

[0086] In some embodiments, the transfected host cell comprises one or more sequences of interest, where the sequences of interest can comprise a combination of H and L sequences. For example, and without limitation, such combinations can 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. Furthermore, in some embodiments, such combinations can 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 inclusion of 1 to 10 (or more) additional H and L sequences, e.g., H", H'", L", and L'", is also encompassed by the presently disclosed subject matter. Exemplary embodiments include, but are not limited to, sequences that include any combination of various distinct H and various distinct 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.

[0087] 4.2 Control System The subject matter disclosed herein also relates to a control system for use in multiple vector RMCE for the generation of TI host cells. For example, there are many cases where protein expression levels are not optimal, mainly because the encoded protein is difficult to express. Low expression levels of difficult-to-express proteins can have a variety of causes that are difficult to identify. One possibility is toxicity of the protein expressed in the host cell. In such cases, a controlled expression system can be used to express a toxic protein, where the sequence of interest encoding the protein is under the control of an inducible promoter. In such a system, expression of the difficult-to-express protein is promoted only when a regulator, such as a small molecule, for example, but not limited to, tetracycline or its analog, doxycycline (DOX), is added to the culture. By regulating the expression of the toxic protein, the toxic effect is alleviated and the culture can achieve the desired cell growth prior to production. In some embodiments, the TI expression system prepared according to the present disclosure includes an exogenous nucleic acid sequence, such as a SOI, including one or more RRSs, incorporated into a specific locus and transcribed under a controlled promoter operably linked thereto. In some embodiments, TI expression systems prepared according to the present disclosure can be used to determine the underlying causes of low protein expression of difficult to express molecules such as, but not limited to, antibodies, In some embodiments, the ability to selectively turn off expression of the SOI in the TI system can be used to link expression of the SOI to observed adverse effects.

[0088] In some embodiments, a TI system can be used to minimize the effects of transcriptional and cell line variability during the characterization of difficult-to-express molecules. For example, but not limited to, expression of the SOI in a TI host can be triggered by the addition of a control factor, such as doxycycline, to the culture. In some embodiments, the TI vector utilizes a tetracycline-regulated promoter to express the SOI, which can be incorporated into an exogenous nucleic acid sequence, such as an RRS, which itself is incorporated into an integration site in the genome of the host cell, allowing for controlled expression of the SOI.

[0089] In some embodiments, the TI system described in the present disclosure can be used to successfully determine one or more underlying causes of low protein expression of the SOI, e.g., therapeutic antibodies, as compared to a control cell line. In some embodiments, once relatively low expression of the SOI, e.g., therapeutic antibodies, in the TI cell line is confirmed, the intracellular accumulation and secretion levels of the SOI can be assessed by utilizing protein translation inhibitor treatments, e.g., Dox and cyclohexamide.

[0090] For example, but not by way of limitation, such control can be based on a gene switch to block or activate mRNA synthesis by controlled binding of a transcriptional repressor or activator to a constitutive or minimal promoter. In some non-limiting embodiments, repression can be achieved by binding to a repressor protein, for example, when the protein sterically blocks the initiation of transcription, or by actively repressing transcription through a transcription silencer. In some non-limiting embodiments, activation of a mammalian or viral enhancer-less minimal promoter can be achieved by controlled binding to an activation domain.

[0091] In some embodiments, conditional binding of a transcriptional repressor or activator can be achieved by using an allosteric protein that binds to the promoter in response to an external stimulus. In some embodiments, conditional binding of a transcriptional repressor or activator can be achieved by using an intracellular receptor that is released from a sequestering protein and can thus bind to the target promoter. In some embodiments, conditional binding of a transcriptional repressor or activator can be achieved by using a chemically derived dimerizing agent.

[0092] In some embodiments, the allosteric protein used in the TI system of the present disclosure can be a protein that regulates transcriptional activity in response to an antibiotic, a bacterial quorum sensing messenger, a catabolite, or in response to a culture parameter, such as temperature, for example, cold or warm temperature. In some embodiments, such a TI system can be catabolite-based, for example, when a bacterial repressor that controls the catabolic genes of alternative carbon sources is transferred to a mammalian cell. In some embodiments, repression of the target promoter can be achieved by cumate-responsive binding of the repressor CymR. In some embodiments, a catabolite-based system can rely on activation of a chimeric promoter by 6-hydroxynicotine-responsive binding of the prokaryotic repressor HdnoR fused to the VP16 transactivation domain of herpes simplex.

[0093] In some embodiments, the TI system can be a quorum sensing-based expression system of prokaryotic origin that governs intra- and inter-population communication by quorum sensing molecules. Such quorum sensing molecules bind to receptors in target cells and modulate the affinity of the receptor for its cognate promoter leading to the initiation of a specific regulon switch. In some 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 from a minimal promoter fused to a TraR-specific operator sequence. In some embodiments, the quorum sensing molecule can be butyrolactone SCB1 (racemic 2-(1'-hydroxy-6-methylheptyl)-3-(hydroxymethyl)-butanolide) of a system based on the Streptomyces coelicolor A3(2) ScbR repressor that binds to its cognate operator OScbR in the absence of SCB1. In some embodiments, the quorum sensing molecule can be a homoserine-derived inducer used in a TI system in which the P. aeruginosa quorum sensing repressors RhlR and LasR are fused to the SV40 T-antigen nuclear localization sequence and the herpes simplex VP16 domain, and can activate promoters containing specific operator sequences (las boxes).

[0094] In some embodiments, the inducer molecule that regulates the allosteric protein used in the TI system of the present disclosure can be, but is not limited to, cumate, isopropyl-β-D-galactopyranoside (IPTG), macrolides, 6-hydroxynicotine, doxycycline, streptogramin, NADH, tetracycline.

[0095] In some embodiments, the intracellular receptors used in the TI system of the present disclosure can be cytoplasmic or nuclear receptors. In some embodiments, the TI system of the present disclosure can utilize the release of transcription factors from sequestering and inhibitory proteins by using small molecules. In some embodiments, the TI system of the present disclosure can rely on steroid regulation, where a hormone receptor is fused to a natural or artificial transcription factor that can be released from HSP90 in the cytosol, translocate into the nucleus, and activate a selected promoter. In some embodiments, mutant receptors controlled by synthetic steroid analogs can be used to avoid crosstalk with endogenous steroid hormones. In some embodiments, the receptor can be an estrogen receptor variant responsive to 4-hydroxytamoxifen or a progesterone receptor mutant inducible by RU486. In some embodiments, a nuclear receptor-derived rosiglitazone-responsive transcriptional switch based on the human nuclear peroxisome proliferator-activated receptor gamma (PPARγ) can be used in the TI system of the present disclosure. In some embodiments, the steroid-responsive receptor variant can 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 to and activate a minimal promoter fused to multiple repeats of the Gal4 response element.

[0096] In some embodiments, the TI system disclosed herein can utilize chemically induced dimerization of a DNA binding protein and a transcription activator for activation of a mini-malecore promoter fused to a cognate operator. In some embodiments, the TI system disclosed herein can utilize rapamycin-regulated dimerization of FKBP and FRB. In this system, FRB is fused to a p65 transactivator and FKBP is fused to a zinc finger domain specific for the cognate operator site placed upstream of an engineered minimal interleukin-12 promoter. In some embodiments, the FKBP can be mutated. In some embodiments, the TI system disclosed herein can utilize the bacterial gyrase B subunit (GyrB), which dimerizes in the presence of the antibiotic coumermycin and dissociates with novobiocin.

[0097] In some embodiments, the TI system of the present disclosure can be used for controlled siRNA expression. In some embodiments, the controlled siRNA expression system can be a tetracycline, a macrolide, or an OFF and ON QuoRex system. In some embodiments, the TI system can utilize Xenopus terminal oligopyrimidine elements (TOPs), which block translation initiation by forming a hairpin structure in the 5' untranslated region.

[0098] In some embodiments, the TI system described in this disclosure can utilize gas-phase regulated expression, such as the acetaldehyde-inducible regulated (AIR) system. The AIR system can use the Aspergillus nidulans AlcR transcription factor, which specifically activates a PAIR promoter assembled from an AlcR-specific operator fused to a minimal human cytomegalovirus promoter in the presence of non-toxic concentrations of gas- or liquid-phase acetaldehyde.

[0099] In some embodiments, the TI system of the present disclosure can utilize the Tet-On or Tet-Off system, in which expression of one or more SOIs can be controlled by tetracycline or its analog, doxycycline.

[0100] In some embodiments, the TI system of the present disclosure can utilize a PIP-on or PIP-off system, in which expression of the SOI can be controlled by, for example, pristinamycin, tetracycline, and / or erythromycin.

[0101] 5.Product The host cells of the present disclosure can be used for the expression of any molecule of interest. In some embodiments, the host cells of the present disclosure can be used for the expression of polypeptides, for example, mammalian polypeptides. Non-limiting examples of such polypeptides include hormones, receptors, fusion proteins, regulators, growth factors, complement system factors, enzymes, coagulation factors, anticoagulants, kinases, cytokines, CD proteins, interleukins, therapeutic proteins, diagnostic proteins, and antibodies. In some embodiments, the host cells of the present disclosure can be used for the expression of chaperones, protein-modifying enzymes, shRNA, gRNA, or other proteins or peptides, and can express therapeutic proteins or molecules of interest constitutively or under regulated conditions.

[0102] In some embodiments, the polypeptide of interest is a bispecific, trispecific or multispecific polypeptide, such as a bispecific antibody.

[0103] The host cells of the present disclosure can be used to produce large amounts of a molecule of interest in a shorter time frame compared to cells used in conventional cell culture methods, e.g., non-TI cells. In some embodiments, the host cells of the present disclosure can be used to improve the quality of a molecule of interest compared to cells used in conventional cell culture methods, e.g., non-TI cells. In some embodiments, the host cells of the present disclosure can be used to enhance seed train stability by preventing chronic toxicity that can result from products that can cause cellular stress and clonal instability over time. In some embodiments, the host cells of the present disclosure can be used for optimal expression of acutely toxic products.

[0104] In some embodiments, the host cells and systems of the present disclosure can be used for cell culture process optimization and / or process development.

[0105] In some embodiments, the host cells of the present disclosure can be used for constitutive expression of selected subunits of a therapeutic molecule and controlled expression of other different subunits of the same therapeutic molecule. In some embodiments, the therapeutic molecule can be a fusion protein. In some embodiments, the host cells of the present disclosure can be used to understand the role and effect of each antibody subunit in the expression and secretion of a fully assembled antibody molecule.

[0106] In some embodiments, the host cells of the present disclosure can be used as a research tool. In some embodiments, the host cells of the present disclosure can be used as a diagnostic tool to pinpoint the cause of low protein expression of molecules of interest in various cells. In some embodiments, the host cells of the present disclosure can be used to directly link observed phenomena or cellular dynamics to the expression of transgenes in cells. The host cells of the present disclosure can also be used to demonstrate whether observed dynamics are reversible in cells. In some embodiments, the host cells of the present disclosure can be utilized to identify and alleviate problems with the transcription and expression of one or more transgenes in cells.

[0107] In some embodiments, the host cells of the present disclosure can be used to swap transgenic subunits of difficult-to-express molecules, such as, but not limited to, the HC and LC subunits of an antibody, with the same subunits of an average molecule in the system to identify one or more problematic subunits. In some 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. EXAMPLES

[0108] Example 1: Comparison of two-vector RMCE and multiple-vector RMCE In this example, differences in various CLD parameters are identified when using multi-vector RMCE (in this example, 3-vector RMCE) compared to 2-vector RMCE.

[0109] Plasmid construction For two-vector-based RMCE, front and back antibody targeting vectors were constructed. Antibody HC and LC cDNAs were cloned into the front vector backbone containing L3, promoter and start codon (ATG) followed by LoxFAS sequence, and the back vector backbone containing LoxFAS, Pac and 2L sequences lacking the start codon. Cre recombinase plasmid (pOG231) was used for all RMCE processes.

[0110] For the three-vector-based RMCE, three antibody targeting vectors (plasmids 1, 2 and 3) were constructed. Plasmid 1 is identical to the front vector of the two-vector-based RMCE described above. The backbone of plasmid 2 contains LoxFAS, Pac lacking a start codon, CMV promoter and Frt3 sequence. The backbone of plasmid 3 contains Frt3, NeoR and Frt sequence. The coding sequence of FlpO recombinase was synthesized based on the mouse codon-optimized sequence. The sequence was cloned into a proprietary expression vector driven by the CMV promoter and containing a polyadenylation sequence.

[0111] Transfection and RMCE For two-vector-based RMCE, the front and back antibody targeting vectors containing the HC and LC, and pOG231 were transfected into TI host cells by electroporation using Maxcyte STX (Maxcyte, Gaithersburg, MD).

[0112] For three-vector-based RMCE, plasmids 1, 2, 3 antibody targeting vectors containing the HC and LC, as well as pOG231 and FlpO expression plasmids, were transfected into TI host cells by electroporation using Maxcyte STX (Maxcyte, Gaithersburg, MD).

[0113] 48 hours after transfection, cells were pelleted by centrifugation, the pellet was then suspended in a proprietary DMEM / F12-based selection-containing medium and cultured at 37° C. and 5% CO2 until the transfection pool was harvested.

[0114] Fed-batch shake flask evaluation of RMCE pools Fed-batch production cultures were performed in shake flasks using a bolus feed of proprietary defined medium on days 1, 3, and 4. Temperature was maintained at 35° C. throughout the run. Titers on day 7 were determined using Protein A affinity chromatography with UV detection. Percent viability and viable cell counts were determined using a ViCell XR instrument (Beckman Coulter).

[0115] Copy number analysis by digital PCR (ddPCR) 1x10 6Genomic DNA from the cell pellet was purified by MagNA Pure 96 instrument (Roche, Molecular Systems) using MagNA Pure 96 DNA and Viral NA Small Volume Kit (Roche Molecular Systems, Cat. No. 06543588001). The copy numbers of Chinese hamster Bcl-2-associated X protein (Bax) and albumin (Alb) were used as standards for the determination of gene copies of HC and LC, respectively. ddPCR reactions were prepared according to the manual of QX200 ddPCR system (Bio-Rad, Cat. No. 186-3010). Briefly, ddPCR reactions were set up in 20 μl per reaction with HC or LC probes labeled with HEX and Bax or Alb probes labeled with FAM. Samples were then placed in the QX200 droplet generator, droplets were transferred to a 96-well plate to perform PCR in a thermal cycler, and the droplets were subsequently read. Cycling conditions were as follows: 10 min at 95° C., 40 cycles of 30 s at 94° C., and 1 min at 60° C., followed by enzyme inactivation for 10 min at 98° C. Primers and probes used in this study were designed using Primer Express v3.0 (Life Technologies).

[0116] mRNA expression by digital reverse transcriptase PCR 1x10 6Total RNA from cell pellets was purified with a MagNA Pure 96 instrument (Roche, Molecular Systems) using the MagNA Pure 96 Cellular RNA Large Volume Kit (Roche Molecular Systems, Cat. No. 05467535001) and quantified using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Cat. No. ND-2000). Approximately 20-40 pg of RNA was used to perform ddRT-PCR using the One-Step RT-ddPCR Advanced Kit for Probes (Bio-Rad, Cat. No. 1864022). Primers and probes for IgG heavy and light chains were identical to those used for gene copy analysis. Droplet generation, temperature cycling, and droplet reading were performed using a Bio-Rad QX200 ddPCR system (Bio-Rad, Cat. No. 186-3010). Thermal cycling conditions were 50° C. for 1 h, 95° C. for 10 min, then 40 cycles of 95° C. for 30 s and 58° C. for 1 min, followed by 98° C. for 10 min for enzyme inactivation. Resulting concentrations were normalized to copies per 20 pg of total RNA.

[0117] Table 2. Identifies differences in various CLD parameters when using multiple vector RMCE (in this example, 3 vector RMCE) compared to two vector RMCE. TIFF2025500443000003.tif40170

[0118] Example 2: Comparison of two-vector RMCE and multiple-vector RMCE with Ambr® 250 In this example, gene copy number and mRNA expression are compared between antibody-expressing cell lines cultured on the Ambr® 250 system (TAP Biosystem), one cell line prepared using a two-plasmid RMCE approach and a second cell line prepared using a multiple-vector RMCE approach. In the two-plasmid approach, each plasmid contains one copy of the heavy chain coding sequence and two copies of the light chain coding sequence ("HLL-HLL"). In the multiple-vector approach, each plasmid also contains one copy of the heavy chain coding sequence and two copies of the light chain coding sequence. However, when a third plasmid is present in the multiple-vector RMCE, this approach results in an "HLL-HLL-HLL" configuration.

[0119] Twelve clones are evaluated in the Ambr® 250 system (TAP Biosystem). The Ambr® 250 system is a high-throughput automated approach using single-use 250 mL mini-bioreactors, which are used in this example as recommended by the manufacturer. Briefly, 30 million cells are seeded in a proprietary synthetic production medium with pH 7.15, and the culture temperature is maintained at 37° C. for the first 48 hours. The culture temperature is then shifted to 35° C. for the remainder of the culture. The 14-day fed-batch culture uses five bolus feeds on days 1, 3, 6, 9, and 12. Cell viability and viable cell count are monitored by trypan blue dye exclusion using a BioProfile Flex2 (Nova Biomedical).

[0120] Example 3: Comparison of two-vector RMCE and two-site multiple vector RMCE This example identifies differences in various CLD parameters when using multi-vector RMCE (in this example, three-vector RMCE, where two vectors are integrated at the first site and a third vector is integrated at the second site) compared to two-vector RMCE. Except as indicated in FIG. 7, antibody expressing constructs were prepared as described in Example 1. Briefly, the green fluorescent protein (GFP) landing pad at the "G" site was used as shown for the integration of the first two plasmids described above and shown in the accompanying drawings, and the blue fluorescent protein (BFP) landing pad at the "B" site consists of a zeocin resistance gene (ZeoR) and (BFP) flanked by Frt and Frt3 recombinase sites. Promoterless Neo is placed downstream of the Frt site. The antibody targeting vector for the "B" site contains the SAT gene and a mAb expression cassette encoding various copies of the HC and LC cDNAs followed by a promoter, all flanked by Frt3 and Frt.

[0121] Figure 8, A and B, show a comparison of the productivity of TI transfection pools using two-plasmid-based RMCE and two-site multiple vector plasmid (in this case, three plasmids)-based RMCE. Both the titer (bars) and Qp (dots) are higher for the two-site multiple vector plasmid-based TI pool of mAb A (A) and the TI pool of mAb B (B).

[0122] Example 4: Comparison of two-vector RMCE and multiple-vector RMCE 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 7 days. Upon completion of the 7-day culture, specific CLD parameters were evaluated for the various TI pools of mAb C, mAb D, and mAb E, and are shown in Table 3. The results are also presented graphically in Figure 9A (titer) and Figure 9B (Qp).

[0123] Table 3. TIFF2025500443000004.tif117170

[0124] Example 5: Ambr15® Comparison of Two-Vector RMCE and Multiple-Vector RMCE In this example, cell lines expressing mAb C or mAb D were prepared using two-vector or multi-vector RMCE (in this case, three-plasmid RMCE) as described above. Ambr15® cultures (TAP Biosystem) of individual clones of each of mAb C and mAb D (four two-vector RMCE and four multi-vector RMCE for each antibody) were performed, and the multi-vector RMCE clones exhibited higher average titers and higher average Qp compared to the two-vector RMCE clones, as shown in Table 4.

[0125] Table 4. TIFF2025500443000005.tif40170

[0126] FIG. 10A graphically depicts the titers observed for each of the eight mAb C clones, and FIG. 10B graphically depicts the Qp observed for each of the eight mAb C clones. FIG. 10C compares the average mAb C clone titer to the mAb C clone Qp. Similarly, FIG. 11A graphically depicts the titers observed for each of the eight mAb D clones, and FIG. 11B graphically depicts the Qp observed for each of the eight mAb D clones. FIG. 11C compares the average mAb D clone titer to the average mAb D clone Qp. In contrast, FIGS. 12A and B compare the titers (FIG. 12A) and Qp (FIG. 12B) of two specific clones (clone A and clone B) for each of the two-vector and multiple-vector approaches. The data shown graphically in FIGS. 12A and B are also shown in Table 5 below.

[0127] Table 5 TIFF2025500443000006.tif66170

[0128] The above examples are merely illustrative of the presently disclosed subject matter and should not be construed as limiting in any way.

[0129] In addition to the various embodiments shown and claimed, the disclosed subject matter is also directed to other embodiments having other combinations of the features disclosed and claimed herein. Thus, the specific features presented herein can be combined in other ways with each of the other features within the scope of the disclosed subject matter, and thus the disclosed subject matter includes any suitable combination of the features disclosed herein. The foregoing description of specific embodiments of the disclosed subject matter has been presented for purposes of illustration and description. The description is not intended to be exhaustive or to limit the disclosed subject matter to those disclosed embodiments.

[0130] Those skilled in the art will recognize that various modifications and variations can be made to the compositions and methods of the disclosed subject matter without departing from the spirit or scope of the disclosed subject matter. Accordingly, it is intended that the disclosed subject matter includes modifications and variations that come within the scope of the claims, and their equivalents.

[0131] Various publications, patents, and patent applications are cited herein, the contents of which are incorporated by reference in their entireties.

Claims

1. 1. A method for preparing a TI host cell that expresses a sequence of interest (SOI), comprising: a) providing a TI host cell comprising an exogenous nucleotide sequence integrated into an integration site, the integration site comprising: a. is at least about 90% homologous to all or a portion of nucleotides 41190-45269 of NW_006874047.1, all or a portion of nucleotides 63590-207911 of NW_006884592.1, all or a portion of nucleotides 253831-491909 of NW_006881296.1, all or a portion of nucleotides 69303-79768 of NW_003616412.1, all or a portion of nucleotides 293481-315265 of NW_003615063.1, all or a portion of nucleotides 2650443-2662054 of NW_006882936.1, or all or a portion of nucleotides 82214-97705 of NW_003615411.1; or b. or is at least about 90% homologous to all or a portion of nucleotides 45270-45490 of NW_006874047.1, all or a portion of nucleotides 207912-792374 of NW_006884592.1, all or a portion of nucleotides 491910-667813 of NW_006881296.1, all or a portion of nucleotides 79769-100059 of NW_003616412.1, all or a portion of nucleotides 315266-362442 of NW_003615063.1, all or a portion of nucleotides 2662055-2701768 of NW_006882936.1, or all or a portion of nucleotides 97706-105117 of NW_003615411.

1. providing a TI host cell, wherein the exogenous nucleotide sequence comprises four or more incompatible recombination recognition sequences (RRS); b) introducing into the cells provided in a) at least three vectors, each vector comprising: a. two RRSs that match two consecutively oriented RRSs on the incorporated exogenous nucleotide sequence; and b. Each pair of RRS flanked by at least one exogenous SOI and at least one second selection marker. introducing at least three vectors into the cells provided in a); c) introducing a recombinase or a nucleic acid encoding a recombinase, wherein the recombinase recognizes an RRS; d) selecting for TI cells that express the selectable marker, thereby isolating TI host cells that express the SOI; A method comprising:

2. 1. A method for developing an SOI, comprising: a) providing a TI host cell comprising an exogenous nucleotide sequence integrated into an integration site, the integration site comprising: a. is at least about 90% homologous to all or a portion of nucleotides 41190-45269 of NW_006874047.1, all or a portion of nucleotides 63590-207911 of NW_006884592.1, all or a portion of nucleotides 253831-491909 of NW_006881296.1, all or a portion of nucleotides 69303-79768 of NW_003616412.1, all or a portion of nucleotides 293481-315265 of NW_003615063.1, all or a portion of nucleotides 2650443-2662054 of NW_006882936.1, or all or a portion of nucleotides 82214-97705 of NW_003615411.1; or b. or is at least about 90% homologous to all or a portion of nucleotides 45270-45490 of NW_006874047.1, all or a portion of nucleotides 207912-792374 of NW_006884592.1, all or a portion of nucleotides 491910-667813 of NW_006881296.1, all or a portion of nucleotides 79769-100059 of NW_003616412.1, all or a portion of nucleotides 315266-362442 of NW_003615063.1, all or a portion of nucleotides 2662055-2701768 of NW_006882936.1, or all or a portion of nucleotides 97706-105117 of NW_003615411.

1. providing a TI host cell, wherein the exogenous nucleotide sequence comprises four or more incompatible RRSs; b) introducing into the cells provided in a) at least three vectors, each vector comprising: a. two RRSs that match two consecutively oriented RRSs on the incorporated exogenous nucleotide sequence; and b. Each pair of RRS flanked by at least one exogenous SOI and at least one second selection marker. and introducing into at least three vectors comprising: c) introducing a recombinase or a nucleic acid encoding a recombinase, wherein the recombinase recognizes an RRS; d) selecting for TI cells that express the selectable marker, thereby isolating TI host cells that express the SOI; e) culturing the cells of d) under conditions suitable for expression of the SOI and recovering the expressed protein therefrom; A method comprising:

3. 3. The method of claim 1, wherein the recombinase is selected from Cre recombinase, FLP recombinase, Bxbl integrase, and φC31 integrase.

4. 3. The method of claim 1 or 2, 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.

5. 3. The method of claim 1 or 2, wherein the TI host cell is a mammalian host cell, particularly wherein the TI host cell is a hamster host cell, a human host cell, a rat host cell, or a mouse host cell, more particularly 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-1 1 host cell, a CHOK1S host cell, or a CHO K1M host cell.

6. 3. The method of claim 1 or 2, 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.

7. 1. A method for producing a recombinant mammalian cell comprising nucleic acid encoding an antibody, comprising: a) providing a mammalian cell comprising at least one exogenous nucleic acid integrated into a predetermined locus in the genome of the mammalian cell, the exogenous nucleic acid comprising four or more incompatible RRSs; b) introducing into the recombinant mammalian cell of a) at least three vectors, each vector comprising a pair of incompatible RRSs that match two of the incompatible RRSs contained in the exogenous nucleic acid integrated at the predetermined locus in the genome of the mammalian cell, and each pair of incompatible RRSs flanking one or more SOIs, wherein the SOIs encode an antibody and / or one or more selectable markers; c) introducing one or more recombinases simultaneously or sequentially with the introduction of at least three vectors containing an SOI and / or a selectable marker; d) selecting cells that express one or more of the SOI and / or selectable markers; thereby generating a recombinant mammalian cell comprising a nucleic acid SOI encoding an antibody.

8. The exogenous nucleic acid is a. is at least about 90% homologous to all or a portion of nucleotides 41190-45269 of NW_006874047.1, all or a portion of nucleotides 63590-207911 of NW_006884592.1, all or a portion of nucleotides 253831-491909 of NW_006881296.1, all or a portion of nucleotides 69303-79768 of NW_003616412.1, all or a portion of nucleotides 293481-315265 of NW_003615063.1, all or a portion of nucleotides 2650443-2662054 of NW_006882936.1, or all or a portion of nucleotides 82214-97705 of NW_003615411.1; or b. or is at least about 90% homologous to all or a portion of nucleotides 45270-45490 of NW_006874047.1, all or a portion of nucleotides 207912-792374 of NW_006884592.1, all or a portion of nucleotides 491910-667813 of NW_006881296.1, all or a portion of nucleotides 79769-100059 of NW_003616412.1, all or a portion of nucleotides 315266-362442 of NW_003615063.1, all or a portion of nucleotides 2662055-2701768 of NW_006882936.1, or all or a portion of nucleotides 97706-105117 of NW_003615411.

1. The method of claim 7, wherein the gene is integrated into a locus.

9. 1. A method for generating a recombinant mammalian cell comprising nucleic acid encoding one or more antibodies, comprising: a) providing a mammalian cell comprising at least two exogenous nucleic acids integrated into a predetermined locus in the genome of the mammalian cell, wherein each exogenous nucleic acid comprises four or more incompatible RRSs, and the exogenous nucleic acids can comprise the same or different RRSs; b) introducing into the recombinant mammalian cell of a) at least three vectors, each vector comprising a pair of incompatible RRSs that match two of the incompatible RRSs contained in one or both of the exogenous nucleic acids integrated at the predetermined locus in the genome of the mammalian cell, and each pair of incompatible RRSs flanking one or more SOIs, wherein the SOIs encode an antibody and / or one or more selectable markers; c) introducing one or more recombinases simultaneously or sequentially with the introduction of at least three vectors containing an SOI and / or a selectable marker; d) selecting cells that express one or more of the SOI and / or selectable markers; thereby generating a recombinant mammalian cell comprising a nucleic acid SOI encoding an antibody.

10. The exogenous nucleic acid is a. is at least about 90% homologous to all or a portion of nucleotides 41190-45269 of NW_006874047.1, all or a portion of nucleotides 63590-207911 of NW_006884592.1, all or a portion of nucleotides 253831-491909 of NW_006881296.1, all or a portion of nucleotides 69303-79768 of NW_003616412.1, all or a portion of nucleotides 293481-315265 of NW_003615063.1, all or a portion of nucleotides 2650443-2662054 of NW_006882936.1, or all or a portion of nucleotides 82214-97705 of NW_003615411.1; or b. or is at least about 90% homologous to all or a portion of nucleotides 45270-45490 of NW_006874047.1, all or a portion of nucleotides 207912-792374 of NW_006884592.1, all or a portion of nucleotides 491910-667813 of NW_006881296.1, all or a portion of nucleotides 79769-100059 of NW_003616412.1, all or a portion of nucleotides 315266-362442 of NW_003615063.1, all or a portion of nucleotides 2662055-2701768 of NW_006882936.1, or all or a portion of nucleotides 97706-105117 of NW_003615411.

1. The method of claim 9, wherein the integration occurs at a separate locus selected from the loci.

11. 11. The method of claim 9 or 10, wherein the recombinase is selected from Cre recombinase, FLP recombinase, Bxbl integrase, and φC31 integrase.

12. 11. The method of claim 9 or 10, 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.

13. 11. The method of claim 9 or 10, wherein the mammalian cell is a hamster host cell, a human host cell, a rat host cell, or a mouse host cell, in particular 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.

14. The method of claim 9 or 10, 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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