Targeted integration of nucleic acids

Abstract

Targeted integration (TI) host cells suitable for the expression of recombinant proteins, and methods for making and using the TI host cells, are provided. [Solution] The TI host cell contains an exogenous nucleotide sequence integrated into an integration site within a specific locus in the genome of the host cell, and the nucleotide sequence immediately 5' to the integrated exogenous nucleotide sequence consists of a sequence that is at least 90% homologous to nucleotides 69303-79768 of NW_003616412.1.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 609,806, filed December 22, 2017, and U.S. Provisional Patent Application No. 62 / 711,272, filed July 27, 2018, the entire disclosures of each of which are incorporated herein by reference.

[0002] The subject matter disclosed herein relates to targeted integration (TI) host cells suitable for expression of recombinant proteins, as well as methods for making and using the TI host cells.

[0003] Sequence Listing This specification references a Sequence Listing (submitted electronically as a .txt file named "00B2060779SEQ.txt" on December 21, 2018). The 00B2060779SEQ.txt file was created on December 20, 2018 and is 1,251,240 bytes in size. The entire contents of the Sequence Listing are incorporated herein by reference. [Background technology]

[0004] Rapid advances in cell biology and immunology have driven the demand for developing novel therapeutic recombinant proteins for a variety of diseases, including cancer, cardiovascular disease, and metabolic disorders. These biologics candidates are commonly 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 monoclonal antibodies.

[0005] A conventional strategy for developing commercially available cell lines involves random integration of a nucleotide sequence encoding a polypeptide of interest, followed by selection and isolation of cell lines that produce the polypeptide of interest. However, this approach has several drawbacks. First, not only are such integrations rare events, but given the randomness of where the nucleotide sequence is integrated, these rare events can also result in diverse gene expression and cell growth phenotypes. This variability, known as "position effect variability," is due, at least in part, to the complex gene regulatory networks present in eukaryotic cell genomes and the accessibility of certain genomic loci for integration and gene expression. Second, random integration strategies generally do not control the number of gene copies integrated into the host cell genome. In fact, gene amplification methods are often used to achieve high-producing cells. However, such gene amplification can result in undesirable cell phenotypes, such as unstable cell growth and / or product expression. Third, due to the heterogeneity of integration loci inherent in the random integration process, screening thousands of clones after transfection to isolate cell lines that exhibit desirable expression levels of the polypeptide of interest requires significant time and effort. Even after isolating such a cell line, stable expression of the polypeptide of interest is not guaranteed, and further screening may be required to obtain a stable, commercially available cell line. Finally, polypeptides produced from randomly integrated cell lines exhibit a high degree of sequence variability, which may be due in part to the mutagenicity of the selection agent used to select for high-level expression of the polypeptide of interest. Summary of the Invention

[0006] The presently disclosed subject matter relates to targeted integration (TI) host cells suitable for recombinant protein expression. The presently disclosed subject matter not only provides a TI site in a host cell with high productivity, but also a novel method for introducing multiple sequences of interest into a single TI locus of a host cell by recombinase-mediated cassette exchange (RMCE). Advantages of the RMCE method include improved productivity and the flexibility to co-express multiple polypeptides at different ratios from the same TI locus. For example, but not by way of limitation, the RMCE strategy disclosed herein allows for the insertion of one, two, three, four, five, six, seven, eight, or more sequences (e.g., antibody heavy chain (HC) or light chain (LC) sequences) into a TI locus. Together with the ability to simultaneously target eight or more sequences, two-plasmid RMCE enables improved productivity by adjusting the HC and LC chain ratio of a mAb, expression of complex molecules with multiple chains, and alteration of cellular pathways by targeting transgenes, endogenous genes, or RNAi with antibodies.

[0007] In certain embodiments, the TI host cell comprises an exogenous nucleotide sequence integrated into an integration site within a locus in the host cell genome, the locus being at least about 90% homologous to a sequence selected from SEQ ID NOs: 1-7. In certain embodiments, the nucleotide sequence immediately 5' to the integrated exogenous nucleotide sequence is selected from the group consisting of a sequence at least about 90% homologous to nucleotides 41190-45269 of NW_006874047.1, nucleotides 63590-207911 of NW_006884592.1, nucleotides 253831-491909 of NW_006881296.1, nucleotides 69303-79768 of NW_003616412.1, nucleotides 293481-315265 of NW_003615063.1, nucleotides 2650443-2662054 of NW_006882936.1, and nucleotides 82214-97705 of NW_003615411.1. In certain embodiments, the nucleotide sequence immediately 3' to the integrated exogenous nucleotide sequence is selected from the group consisting of a sequence at least about 90% homologous to nucleotides 45270-45490 of NW_006874047.1, nucleotides 207912-792374 of NW_006884592.1, nucleotides 491910-667813 of NW_006881296.1, nucleotides 79769-100059 of NW_003616412.1, nucleotides 315266-362442 of NW_003615063.1, nucleotides 2662055-2701768 of NW_006882936.1, or nucleotides 97706-105117 of NW_003615411.1. In certain embodiments, the integrated exogenous nucleotide sequence is operably linked to a nucleotide sequence selected from the group consisting of a sequence at least about 90% homologous to a sequence selected from SEQ ID NOs: 1-7.

[0008] In certain embodiments, the TI host cell comprises an exogenous nucleotide sequence integrated into an integration site within an endogenous gene selected from the group consisting of LOC107977062 (SEQ ID NO: 1), LOC100768845 (SEQ ID NO: 2), ITPR2 (SEQ ID NO: 3), ERE67000.1 (SEQ ID NO: 4), UBAP2 (SEQ ID NO: 5), MTMR2 (SEQ ID NO: 6), XP_003512331.2 (SEQ ID NO: 7), and sequences at least about 90% homologous thereto. In certain embodiments, the exogenous nucleotide sequence is integrated into an integration site operably linked to an endogenous gene selected from the group consisting of LOC107977062 (SEQ ID NO: 1), LOC100768845 (SEQ ID NO: 2), ITPR2 (SEQ ID NO: 3), ERE67000.1 (SEQ ID NO: 4), UBAP2 (SEQ ID NO: 5), MTMR2 (SEQ ID NO: 6), XP_003512331.2 (SEQ ID NO: 7), and sequences at least about 90% homologous thereto. In certain embodiments, the integrated exogenous nucleotide sequence is flanked by a nucleotide sequence selected from the group consisting of sequences at least about 90% homologous to a sequence selected from SEQ ID NOs: 1-7. In certain embodiments, the exogenous nucleotide sequence is integrated into an integration site immediately adjacent to all or a portion of a sequence selected from the group consisting of sequences at least about 90% homologous to a sequence selected from SEQ ID NOs: 1-7.

[0009] In certain embodiments, the TI host cell is a mammalian host cell. In certain embodiments, the TI host cell is a hamster host cell, a human host cell, a rat host cell, or a mouse host cell. In certain embodiments, the TI host cell is a Chinese hamster ovary (CHO) host cell, a CHO K1 host cell, a CHO K1SV host cell, a DG44 host cell, a DUKXB-11 host cell, a CHOK1S host cell, or a CHO K1M host cell.

[0010] In certain embodiments, the TI host cell comprises an integrated exogenous nucleotide sequence, wherein the exogenous nucleotide sequence comprises one or more recombination recognition sequences (RRSs). In certain embodiments, the exogenous nucleotide sequence comprises at least two RRSs. The RRSs can be recognized by a recombinase, such as Cre recombinase, FLP recombinase, Bxb1 integrase, or φC31 integrase. The 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.

[0011] In certain embodiments, the exogenous nucleotide sequence comprises a first and a second RRS and at least one selectable marker located between the first and second RRS. In certain embodiments, the exogenous nucleotide sequence further comprises a third RRS, where the third RRS is located between the first and the second RRS and is different from the first or second RRS. In certain embodiments, the exogenous nucleotide sequence comprises a first and a second RRS and a first selectable marker located between the first and the second RRS. In certain embodiments, the exogenous nucleotide sequence further comprises a second selectable marker, where the first and second selectable markers are different. In certain embodiments, the exogenous nucleotide sequence may further comprise a third selectable marker and an internal ribosome entry site (IRES), where the IRES is operably linked to the third selectable marker. The third selectable marker may be different from the first or second selectable marker. The selectable marker may be selected from the group consisting of aminoglycoside phosphotransferase (APH) (e.g., hygromycin phosphotransferase (HYG), neomycin, and G418 APH), dihydrofolate reductase (DHFR), thymidine kinase (TK), glutamine synthetase (GS), asparagine synthetase, tryptophan synthetase (indole), histidinol dehydrogenase (histidinol D), and genes encoding resistance to puromycin, blasticidin, bleomycin, phleomycin, chloramphenicol, zeocin, and mycophenolic acid.The selectable marker may also be selected from the group consisting of a green fluorescent protein (GFP) marker, an improved GFP (eGFP) marker, a synthetic GFP marker, a yellow fluorescent protein (YFP) marker, an improved YFP (eYFP) marker, a cyan fluorescent protein (CFP) marker, an mPlum marker, an mCherry marker, a tdTomato marker, an mStrawberry marker, a J-red marker, a DsRed-monomer marker, an mOrange marker, an mKO marker, an mCitrine marker, a Venus marker, a YPet marker, an Emerald6 marker, a CyPet marker, an mCFPm marker, a Cerulean marker, and a T-Sapphire marker. In some embodiments, the selectable marker is selected from the group consisting of a green fluorescent protein (GFP) marker, an improved GFP (eGFP) marker, and a synthetic GFP marker.

[0012] In certain embodiments, the exogenous nucleotide sequence comprises at least one selectable marker and at least one exogenous sequence of interest (SOI). In certain embodiments, the exogenous nucleotide sequence comprises a first and a second RRS, and at least one selectable marker and at least one exogenous SOI located between the first and second RRS. In certain embodiments, the exogenous nucleotide sequence comprises a first, a second, and a third RRS, at least one selectable marker and at least one exogenous SOI located between the first and third RRS, and at least one exogenous SOI located between the third and second RRS. The sequence of interest can encode any polypeptide of interest, including, but not limited to, the examples listed herein. The SOIs can be the same or different; for example, the SOI can comprise coding sequences for both chains of an antibody. In cases where two or more different polypeptides are expressed, the sequence of interest may contain various ratios of the two polypeptides, for example, where the eight coding sequences encode two different polypeptides, including, but not limited to, 1:7, 2:6, etc. The SOI may encode a single chain antibody, an antibody light chain, an antibody heavy chain, a single chain Fv fragment (scFv), or an Fc fusion protein.

[0013] The presently disclosed subject matter also provides methods for targeted integration of an exogenous nucleic acid into a host cell to promote expression of a polypeptide of interest. In certain embodiments, such methods involve targeted integration of an exogenous nucleic acid into a host cell by recombinase-mediated recombination. In certain embodiments, such methods involve a cell comprising an exogenous nucleotide sequence integrated into a site within an endogenous gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, XP_003512331.2, and sequences at least about 90% homologous thereto. In certain embodiments, such methods involve a cell comprising an exogenous nucleotide sequence integrated into a locus in the host cell genome, the locus comprising a nucleotide sequence at least about 90% homologous to a sequence selected from SEQ ID NOs: 1-7.

[0014] In certain embodiments, the present disclosure provides a method for preparing a TI host cell that expresses a polypeptide of interest, the method comprising: a) providing a TI host cell that includes an exogenous nucleotide sequence integrated into a site within a locus in the TI host cell genome, the locus being at least about 90% homologous to a sequence selected from SEQ ID NOs: 1-7, and the exogenous nucleotide sequence including two RRSs flanking at least one first selectable marker; b) introducing into the cell provided in a) a vector that includes at least one exogenous SOI and two RRSs that match the two RRSs on the integrated exogenous nucleotide sequence and are flanked by at least one second selectable marker; c) introducing a recombinase, wherein the recombinase recognizes the RRSs; and d) isolating the TI host cell that expresses the polypeptide of interest by selecting for TI cells that express the second selectable marker.

[0015] In certain embodiments, the present disclosure provides methods for preparing a TI host cell that expresses first and second polypeptides of interest (wherein the first and second polypeptides can be the same or different), the method comprising: a) providing a TI host cell comprising an exogenous nucleotide sequence integrated into a site within a locus of the host cell genome, the locus being at least about 90% homologous to a sequence selected from SEQ ID NOs: 1-7, the exogenous nucleotide sequence comprising first and second RRSs flanking at least one first selectable marker, and a third RRS located between the first and second RRSs, wherein the first, second, and third RRSs are heterospecific, i.e., the first, second, and third RRSs are non-identical RRSs, such that any two of the first, second, and third RRSs are: b) introducing into the cells provided in a) a first vector comprising two RRSs that match the first and third RRSs on the integrating exogenous nucleotide sequence and that are flanked by at least one first exogenous SOI and at least one second selectable marker; c) introducing into the cells provided in a) a second vector comprising two RRSs that match the second and third RRSs on the integrating exogenous nucleotide sequence and that are flanked by at least one second exogenous SOI; d) introducing one or more recombinases, wherein the one or more recombinases recognize the RRSs; and e) isolating TI host cells that express the first and second polypeptides of interest by selecting for TI cells that express the second selectable marker.

[0016] In certain embodiments, the present disclosure provides a method for expressing a polypeptide of interest, the method comprising: a) providing a host cell comprising at least one exogenous SOI flanked by two RRSs integrated into a locus in the host cell genome and at least one selectable marker, the locus being at least about 90% homologous to a sequence selected from SEQ ID NOs: 1-7; and b) culturing the cell of a) under conditions suitable for expressing the SOI and recovering the polypeptide of interest therefrom.

[0017] In certain embodiments, the present disclosure provides a method for expressing a polypeptide of interest, the method comprising: a) providing a host cell comprising at least two exogenous SOIs and at least one selectable marker integrated into a locus in the host cell genome, the locus being at least about 90% homologous to a sequence selected from SEQ ID NOs: 1-7, wherein the at least one exogenous SOI and one selectable marker are flanked by first and third RRSs, and the at least one exogenous SOI is flanked by second and third RRSs; and b) culturing the cell of a) under conditions suitable for expressing the SOIs and recovering the polypeptide of interest therefrom.

[0018] The presently disclosed subject matter also provides compositions and methods for producing a polypeptide of interest through the use of TI host cells. Such compositions and methods include, but are not limited to, TI host cells and compositions containing exogenous nucleotide sequences, as well as polynucleotides and vectors that facilitate integration of the exogenous nucleotide sequence into the TI host cells, and methods for using the same. In certain embodiments, the vector may contain a nucleotide sequence at least 50% identical to a sequence selected from SEQ ID NOs: 1-7 flanking a DNA cassette, the DNA cassette containing at least one selectable marker flanked by two RRSs and at least one exogenous SOI. The vector may be selected from the group consisting of adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, retroviral vectors, integrative phage vectors, non-viral vectors, transposon and / or transposase vectors, integrase substrates, and plasmids.

[0019] In certain embodiments, methods of the disclosure involve targeted integration of an exogenous nucleic acid encoding a polypeptide of interest into a host cell via homologous recombination, homology-directed repair (HDR), and / or non-homologous end joining (NHEJ). In certain embodiments, such methods involve integration of the exogenous nucleic acid encoding the polypeptide of interest into a cell at a site within an endogenous gene selected from the group consisting of LOC107977062 (SEQ ID NO: 1), LOC100768845 (SEQ ID NO: 2), ITPR2 (SEQ ID NO: 3), ERE67000.1 (SEQ ID NO: 4), UBAP2 (SEQ ID NO: 5), MTMR2 (SEQ ID NO: 6), XP_003512331.2 (SEQ ID NO: 7), and sequences at least about 90% homologous thereto. In certain embodiments, such methods involve integrating into a cell an exogenous nucleic acid encoding a polypeptide of interest at a site within a locus in the host cell genome, the locus comprising a nucleotide sequence that is at least about 90% homologous to a sequence selected from SEQ ID NOs: 1-7.

[0020] In certain embodiments, the present disclosure provides methods for preparing a TI host cell that expresses a polypeptide of interest, the methods comprising: (a) providing a host cell comprising a locus in the host cell genome, the locus being at least about 90% identical to a sequence selected from SEQ ID NOs: 1-7; (b) introducing a vector into the host cell, the vector comprising a nucleotide sequence at least 50% identical to a sequence selected from SEQ ID NOs: 1-7 flanking a DNA cassette, the DNA cassette comprising at least one selectable marker flanked by two RRSs and at least one exogenous SOI; and (c) selecting the selectable marker and isolating a TI host cell having the SOI integrated into the genomic locus to express the polypeptide of interest. In certain embodiments, such methods comprise the use of an exogenous nuclease to facilitate targeted integration. The exogenous nuclease may be selected from the group consisting of zinc finger nucleases (ZFNs), ZFN dimers, transcription activator-like effector nucleases (TALENs), TAL effector domain fusion proteins, RNA-guided DNA endonucleases, engineered meganucleases, and clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) endonucleases.

[0021] In certain embodiments, the present disclosure provides a method for preparing a TI host cell that expresses a polypeptide of interest, the method comprising: a) providing a TI host cell that includes at least one exogenous nucleotide sequence integrated into a site within one or more loci in the TI host cell genome, the one or more loci being at least about 90% homologous to a sequence selected from SEQ ID NOs: 1-7, and the exogenous nucleotide sequence including one or more RRSs; b) introducing into the cell provided in a) a vector that includes one or more RRSs that match the one or more RRSs on the integrated exogenous nucleotide sequence and are adjacent to at least one exogenous SOI that is operably linked to a regulatable promoter; c) introducing a recombinase or a nucleic acid encoding a recombinase, wherein the recombinase recognizes the RRS; and d) isolating the TI host cell that expresses the polypeptide of interest by selecting for TI cells that express the exogenous SOI in the presence of an inducer.

[0022] In certain embodiments, the present disclosure provides a method for expressing a polypeptide of interest, the method comprising: a) providing a host cell comprising at least one exogenous SOI flanked by two RRSs integrated within a locus in the host cell genome and operably linked to a regulatable promoter, the locus being at least about 90% homologous to a sequence selected from SEQ ID NOs: 1-7; and b) culturing the cell under conditions suitable for expressing the SOI and recovering the polypeptide of interest therefrom.

[0023] In certain embodiments, the present disclosure provides a method for preparing a TI host cell that expresses first and second polypeptides of interest (wherein the first and second polypeptides can be the same or different), the method comprising: a) providing a TI host cell that includes an exogenous nucleotide sequence integrated into a site within a locus of a host cell genome, the locus being at least about 90% homologous to a sequence selected from SEQ ID NOs: 1-7, the exogenous nucleotide sequence including a first RRS, a second RRS, and a third RRS located between the first RRS and the second RRS, wherein the first, second, and third RRSs are heterospecific, i.e., the first, second, and third RRSs are non-identical RRSs, such that any two of the first, second, and third RRSs are inaccessible to recombinase-mediated recombination; and b) providing in the cell provided in a); The method includes: introducing a first vector containing two RRSs flanking at least one first exogenous SOI that matches the first and third RRSs on the integrating exogenous nucleotide sequence and is operably linked to a regulatable promoter; c) introducing into the cells provided in a) a second vector containing two RRSs flanking at least one second exogenous SOI that matches the second and third RRSs on the integrating exogenous nucleotide sequence and is operably linked to a regulatable promoter; d) introducing one or more recombinases, or one or more nucleic acids encoding one or more recombinases, wherein the one or more recombinases recognize the RRSs; and e) isolating TI host cells that express the first and second polypeptides of interest by selecting for TI cells that express the exogenous SOI in the presence of an inducer. [Brief explanation of the drawings]

[0024] [Figure 1] 1 is a schematic diagram outlining the genome-wide screening steps to identify CHO TI loci that allow stable, high-level expression of antibodies. [Figure 2] Figure 1 shows the two double selection schemes utilized to generate populations expressing target antibodies. The transposase-generated TI host is shown in A, and the random integration-generated TI host is shown in B. [Figure 3]Productivity of top clones generated by two specific TI hosts is shown. [Figure 4] A two-plasmid RMCE strategy is shown, involving the use of three RRS sites to simultaneously perform two independent RMCEs. One vector (forward) contains RRS1, the first SOI, and a promoter (P) followed by an initiation codon (ATG) and RRS3. The other vector (reverse) contains RRS3, SOI2, and RRS2 fused to the coding sequence of a marker lacking an initiation codon (ATG). Only when the two plasmids are correctly targeted will the complete expression cassette of the selectable marker be assembled, thereby conferring resistance to selection. [Figure 5] Results are shown using either single-vector or two-vector RMCE in conjunction with the expression of antibodies D, E, F, G, and J. FACS, genomic PCR, and gene copy analysis were evaluated to confirm that both plasmid cassettes accurately targeted the TI site. With two-plasmid RMCE, more total HC and LC copies were targeted to the TI site when compared to single-plasmid RMCE. [Figure 6] The titers of six molecules are shown. [Figure 7] Results are shown for the use of two-vector RMCE to express a composite mAb format. Two bispecific molecules, each requiring four different chains (two HC and two LC), were assayed for production and % bispecific formation. In both cases, cell lines were developed that displayed >1.5 g / L with >80% bispecific content. [Figure 8] Day 14 titers for the four bispecific molecules are shown. [Figure 9A] Figure 1 shows the generation of cell lines expressing mAb-I or mAb-II using the RTI system. The histological results of cell line development are shown in A. [Figure 9B] Figure 1 shows the generation of cell lines expressing mAb-I or mAb-II in the RTI system. A schematic diagram of the RTI cell line development (CLD) process is shown in B. [Figure 10A]Figure 1 shows the titer and specific productivity results of cell lines expressing mAb-I or mAb-II. End-of-production titers are shown in A. [Figure 10B] The titer and specific productivity results of cell lines expressing mAb-I or mAb-II are shown. The end-of-production IVCC is shown in B. [Figure 10C] The titer and specific productivity results of cell lines expressing mAb-I or mAb-II are shown. End-of-production specific productivity is shown in C. [Figure 11] The expression levels of HC and LC mRNA in mAb-I and mAb-II expressing cell lines are shown. The expression level of HC mRNA is shown in A, and the expression level of HL mRNA is shown in B. [Figure 12A] Intracellular levels of HC, HL, and BiP molecules in cell lines expressing mAb-I or mAb-II are shown. A schematic diagram of the effects of cycloheximide and Dox on the RTI system is shown in A. [Figure 12B] Intracellular levels of HC, HL, and BiP molecules in cell lines expressing mAb-I or mAb-II are shown in Figure 1. Intracellular levels of HC, LC, and BiP as well as HC and LC levels in the supernatants of cell lines expressing mAb-A or mAb-B are shown in Figure 2. [Figure 12C] Intracellular levels of HC, HL, and BiP molecules in cell lines expressing mAb-I or mAb-II are shown in Figure 1. Intracellular levels of HC, LC, and BiP in cell lines expressing mAb-I or mAb-II after overnight treatment with CHX are shown in Figure 2. [Figure 12D] Intracellular levels of HC, HL, and BiP molecules in cell lines expressing mAb-I or mAb-II are shown. Intracellular levels of HC, LC, and BiP in cell lines expressing mAb-I or mAb-II after removal of doxycycline from the culture medium are shown in D. [Figure 13A] The correlation between intracellular accumulation of BiP and mAb-I LC expression is shown. End-production titers are shown in A. [Figure 13B] Correlation between intracellular accumulation of BiP and mAb-I LC expression is shown. Accumulation of HC, LC, and BiP is shown in B. [Figure 13C] The correlation between intracellular accumulation of BiP and mAb-I LC expression is shown in Figure 1. A schematic diagram of the strand exchange experiment is shown in Figure 2. [Figure 13D] Correlation between intracellular accumulation of BiP and mAb-I LC expression is shown. Intracellular levels of HC, LC, and BiP of RTI pools expressing mAb-I, mAb-II, or strand-exchanged forms of these molecules are shown in D. [Figure 14A] The contribution of mAb-A HC and HL to the pooled expression of this molecule is shown. End-of-production titers are shown in A. [Figure 14B] The contribution of mAb-A HC and HL to the pool expression of this molecule is shown. The end-of-production IVCC is shown in B. [Figure 14C] The contribution of mAb-A HC and HL to the pooled expression of this molecule is shown. End-production specific productivity is shown in C. [Figure 15] The CDR regions of the LC and HC that may contribute to reduced expression are shown. A schematic representation of the mAb-I and mAb-II LC subunits is shown in A. A schematic representation of the mAb-I and mAb-II HC regions is shown in B. [Figure 16A] The results of using two point mutations in the HC constant region in relation to the expression of HCA are shown in A. The production end titers of various chain exchanges between the HC and LC subunits of mAb-I and mAb-II are shown in A. [Figure 16B] The results of using two point mutations in the HC constant region in relation to the expression of HCA are shown in B. Intracellular levels of HC, LC, and BiP in various RTI pools are shown in B. [Figure 16C] The results of using two point mutations in the HC constant region in relation to the expression of HCA are shown. A schematic diagram for the use of the RTI system as a diagnostic tool for low protein expression is shown in C. [Figure 17-1] Production using two different cell culture processes is shown. [Figure 17-2] Production using two different cell culture processes is shown. [Figure 18]Figure 1 shows titer and specific productivity results for RMCE pools of three mAbs. Titers at day 14 for RMCE pools of three mAbs comparing HL and HLL configurations are shown in A. Specific productivity (Qp) at day 14 for RMCE pools of three mAbs comparing HL and HLL configurations is shown in B. Titers at day 14 for clones generated from pools in A are shown in C. Qp at day 14 for clones generated from pools in A are shown in D. In C and D, six clones were tested per configuration. [Figure 19A] Figure 1 shows titer, Qp, and growth (expressed as integral of viable cell concentration, IVCC), mRNA expression, and protein expression for RMCE pools of mAb Y comparing three different plasmid configurations. Titer, Qp, and growth (expressed as integral of viable cell concentration, IVCC) at day 14 are shown in A, B, and C, respectively. Each bar represents data from a single pool. A is seed train mRNA expression and intracellular antibody protein expression from the pool. [Figure 19B] Figure 1 shows titer, Qp, and growth (expressed as integral of viable cell concentration, IVCC), mRNA expression, and protein expression for RMCE pools of mAb Y comparing three different plasmid configurations. Titer, Qp, and growth (expressed as integral of viable cell concentration, IVCC) at day 14 are shown in A, B, and C, respectively. Each bar represents data from a single pool. [Figure 19C] Figure 1 shows titer, Qp, and growth (expressed as integral of viable cell concentration, IVCC), mRNA expression, and protein expression for RMCE pools of mAb Y comparing three different plasmid configurations. Titer, Qp, and growth (expressed as integral of viable cell concentration, IVCC) at day 14 are shown in A, B, and C, respectively. Each bar represents data from a single pool. [Figure 19D]Figure 1 shows titer, Qp, and proliferation (expressed as integral viable cell concentration, IVCC), mRNA expression, and protein expression for RMCE pools of mAb Y comparing three different plasmid configurations. Heavy and light chain mRNA expression was normalized to the HLL-HL configuration, and each bar is the average of four technical replicates (error bars are standard deviations). Heavy and light chain mRNA expression was normalized to the HLL-HL configuration. [Figure 19E] Figure 1 shows titer, Qp, and proliferation (expressed as integrated viable cell concentration, IVCC), mRNA expression, and protein expression for RMCE pools of mAb Y comparing three different plasmid configurations. Intracellular heavy and light chain protein expression from all pools quantified from Western blots and normalized to the HLL-HL configuration is shown in E; each bar is the average of three technical replicates (error bars are standard deviations). [Figure 19F] Figure 1 shows titer, Qp, and proliferation (expressed as integral viable cell concentration, IVCC), mRNA expression, and protein expression for the RMCE pool of mAb Y comparing three different plasmid configurations. A representative Western blot image of the data shown in E is shown in F. [Figure 20A] The titer, Qp, and proliferation (expressed as integrated viable cell concentration, IVCC) of monoclonals expressing mAb Y or mAb III generated from RMCE pools with three different compositions are shown. The titer at day 14 of the monoclonal expressing mAb Y is shown in A. [Figure 20B] The titer, Qp, and proliferation (expressed as integrated viable cell concentration, IVCC) of monoclonals expressing mAb Y or mAb III generated from RMCE pools with three different compositions are shown in Figure 1. The Qp at day 14 of the monoclonals expressing mAb Y is shown in Figure 2B. [Figure 20C] The titer, Qp, and proliferation (expressed as integrated viable cell concentration, IVCC) of single clones expressing mAb Y or mAb III generated from RMCE pools with three different compositions are shown. The IVCC at day 14 of the clone expressing mAb Y is shown in C. [Figure 20D] The titer, Qp, and proliferation (expressed as integrated viable cell concentration, IVCC) of monoclonals expressing mAb Y or mAb III generated from RMCE pools with three different compositions are shown in Figure 1. The titer of the monoclonal expressing mAb III at day 14 is shown in Figure 2. [Figure 20E] The titer, Qp, and proliferation (expressed as integrated viable cell concentration, IVCC) of monoclonals expressing mAb Y or mAb III generated from RMCE pools with three different compositions are shown. The Qp at day 14 of the monoclonals expressing mAb III is shown in E. [Figure 20F] The titer, Qp, and proliferation (expressed as integrated viable cell concentration, IVCC) of single clones expressing mAb Y or mAb III generated from RMCE pools with three different compositions are shown. The IVCC at day 14 of the clone expressing mAb III is shown in F. [Figure 21A] Expression levels, titers, and Qp of RMCE pools of various configurations are shown. A schematic of one heavy chain, one light chain plasmid transfected to assess the effect of plasmid position on chain expression is shown in A. The antibody heavy chain (abbreviated H) and light chain (abbreviated L) were either expressed on the same plasmid or split between the forward and reverse plasmids. Note the L3, Loxfas, and 2L sites, as well as the pac puromycin resistance gene in the reverse plasmid. [Figure 21B] Expression levels, titers and Qp of RMCE pools of various configurations are shown. Seed train mRNA expression of heavy and light chains from pools with the DNA configurations outlined in A is shown in B. [Figure 21C] Expression levels, titers and Qp for RMCE pools of various configurations are shown. Titers at day 14 for RMCE pools of various configurations are shown in C. [Figure 21D] Expression levels, titers and Qp of RMCE pools of various configurations are shown. Qp at day 14 of various configurations is shown in D. [Figure 21E]Expression levels, titers and Qp of RMCE pools of various configurations are shown. The heavy and light chain DNA copy numbers of the pools are shown in E. [Figure 22A] The effect of the HC:LC ratio on the high molecular weight species (HMWS) percentage of mAbs is independent of the TI site. Day 14 titers for antibody S on host 7 cells are shown in A. [Figure 22B] The effect of the HC:LC ratio on the high molecular weight species (HMWS) percentage of mAb is independent of the TI site. The high molecular weight species (HMWS) percentage of antibody S on host 7 cells is shown in B. [Figure 22C] The effect of the HC:LC ratio on the percentage of high molecular weight species (HMWS) of mAbs is independent of the TI site. Day 14 titers for antibody S on host 4 cells are shown in C. [Figure 22D] The effect of the HC:LC ratio on the high molecular weight species (HMWS) percentage of mAb is independent of the TI site. The high molecular weight species (HMWS) percentage of antibody S on host 4 cells is shown in D. [Figure 23A] Figure 1 shows the effect of HC:LC ratio on antibody IV productivity and high molecular weight species (HMWS). Day 14 titers for antibody IV on host 4 cells are shown in A. [Figure 23B] Figure 1 shows the effect of HC:LC ratio on antibody IV productivity and high molecular weight species (HMWS). The high molecular weight species (HMWS) percentage of antibody IV on host 4 cells is shown in B. [Figure 24A] Figure 1 shows the effect of HC:LC ratio on the productivity and high molecular weight species (HMWS) of antibody VI in two-site TI host cells. Day 14 titers for antibody VI on two-site TI host cells are shown in A. [Figure 24B] Figure 1 shows the effect of HC:LC ratio on the productivity and high molecular weight species (HMWS) of antibody VI in two-site TI host cells. The high molecular weight species (HMWS) percentage of antibody IV on two-site TI host cells is shown in A. [Figure 25]Two clones were shown to have titers of 8.4 and 8.7 g / L and contain 7.4% and 5.6% aggregates, respectively. [Figure 26] Figure 14 shows the titer of antibody Z in different regulated targeted integration cell lines at day 14. [Figure 27] Specific productivity of antibody Z in different regulated targeted integration cell lines is shown. DETAILED DESCRIPTION OF THE INVENTION

[0025] In certain embodiments, the host cells, genetic constructs (e.g., vectors), compositions, and methods described herein may be used in the development and / or use of targeted integration (TI) host cells, hi certain embodiments, such TI host cells comprise an exogenous nucleotide sequence integrated into a specific gene or specific locus in the host cell genome.

[0026] For clarity of disclosure, and not by way of limitation, the detailed description is divided into the following subsections: 1.Definition 2. Integration site 3. Exogenous Nucleotide Sequence 4.Host cells 5. Targeted Integration 6. Preparation and Use of TI Host Cells 7. Products 8. Exemplary Non-Limiting Embodiments

[0027] 1.Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below; however, methods and materials similar or equivalent to those described herein can also 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.

[0028] The terms "comprise(s)," "include(s)," "having," "has," "can," "contain(s)," and variations thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not exclude the possibility of additional acts or structures. The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments that "comprising," "consisting of," and "consisting essentially of" the embodiments or elements presented herein, whether explicitly stated or not.

[0029] For the recitation of numerical ranges herein, each intervening number to the same degree of precision is expressly contemplated. For example, in the range 6 to 9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and in 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.

[0030] As used herein, the term "about" or "approximately" refers to 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 limitations of the measurement system, i.e., how the value is measured or determined. For example, "about" can mean within 3 or more standard deviations, as is customary in the art. Alternatively, "about" can mean within 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 one order of magnitude, preferably within 5-fold, and more preferably within 2-fold of a value.

[0031] As used herein, the term "selection marker" refers to a gene that allows cells carrying the gene to be specifically selected or selected against in the presence of a corresponding selection agent. For example, without intending to be limiting, a selection marker can allow host cells transformed with the selection marker gene to be positively selected in the presence of the gene, while non-transformed host cells will not be able to grow or survive under selective conditions. A selection marker can be positive, negative, or bifunctional. A positive selection marker can allow for the selection of cells carrying the marker, while a negative selection marker can allow for the selective elimination of cells carrying the marker. A selection marker can confer resistance to a drug or complement a metabolic or catabolic disorder in a host cell. In particular, in prokaryotic cells, genes that confer resistance to ampicillin, tetracycline, kanamycin, or chloramphenicol can be used. Resistance genes useful as selectable markers in eukaryotic cells include, but are not limited to, genes for aminoglycoside phosphotransferase (APH) (e.g., hygromycin phosphotransferase (HYG), neomycin, and G418 APH), dihydrofolate reductase (DHFR), thymidine kinase (TK), glutamine synthetase (GS), asparagine synthetase, tryptophan synthetase (indole), histidinol dehydrogenase (histidinol D), and genes encoding resistance to puromycin, blasticidin, bleomycin, phleomycin, chloramphenicol, zeocin, and mycophenolic acid. Additional marker genes are described in WO 92 / 08796 and WO 94 / 28143.

[0032] Alternatively, the selectable marker may be a gene encoding a molecule that facilitates selection in the presence of a corresponding selective agent but is not normally present in the cell, such as green fluorescent protein (GFP), improved GFP (eGFP), synthetic GFP, yellow fluorescent protein (YFP), improved 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 lacking this gene, for example, by detecting the fluorescence emitted by the encoded polypeptide.

[0033] As used herein, the term "operably linked" refers to the juxtaposition of two or more components, where the components are in a relationship permitting them to function in their intended manner. For example, a promoter and / or enhancer is operably linked to a coding sequence if the promoter and / or enhancer functions to regulate the transcription of the coding sequence. In certain embodiments, "operably linked" DNA sequences are contiguous and contiguous on a single chromosome. In certain embodiments, for example, where two protein-encoding regions, such as a secretory leader and a polypeptide, are to be joined, the sequences are contiguous, contiguous, and in the same reading frame. In certain embodiments, an operably linked promoter may be located upstream of and adjacent to the coding sequence. In certain embodiments, for example, with an enhancer sequence regulating expression of a coding sequence, two components may be non-contiguous yet operably linked. An enhancer is operably linked to a coding sequence if the enhancer increases the transcription of the coding sequence. An operably linked enhancer can be located upstream, within, or downstream of the coding sequence, and can be located a considerable distance from the promoter of the coding sequence. Operable linkage can be achieved by recombinant methods known in the art, for example, using PCR techniques and / or by ligation at convenient restriction sites. If convenient restriction sites do not exist, synthetic oligonucleotide adapters or linkers can be used according to conventional methods. An internal ribosome entry site (IRES) is operably linked to an open reading frame (ORF) if it is capable of initiating translation of the ORF at an internal location in a 5'-end-independent manner.

[0034] As used herein, the term "expression" refers to transcription and / or translation. In certain 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 certain embodiments, the protein encoded by a sequence of interest can be quantified by various methods, such as by ELISA, by assaying for the biological activity of the protein, or by using assays that are independent of such activity, such as Western blotting or radioimmunoassays that use antibodies that recognize and bind to the protein.

[0035] The term "antibody" as used herein is used 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, so long as the antibody exhibits the desired antigen-binding activity.

[0036] As used herein, the term "antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen bound by the intact antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and multispecific antibodies formed from antibody fragments. For a review of certain antibody fragments, see Holliger and Hudson, Nature Biotechnology 23:1126-1136 (2005).

[0037] As used herein, the term "variable region" or "variable domain" refers to the domains of an antibody heavy or light chain that are involved in binding the antibody to an antigen. H and V LThe variable domains of IgG1 and IgG2a have generally similar structures, with each domain containing four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, for example, Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)). A single V H or V L A V domain may be sufficient to confer antigen-binding specificity. Furthermore, an antibody that binds to a particular antigen may have a V domain from an antibody that binds to that antigen. H or V L The V domains were isolated and each was complementary to L or V H Libraries of domains may be screened. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0038] As used herein, the term "heavy chain" refers to an immunoglobulin heavy chain.

[0039] As used herein, the term "light chain" refers to an immunoglobulin light chain.

[0040] The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0041] The term "monoclonal antibody," as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., each individual antibody within the population is identical and / or binds to the same epitope, except for variant antibodies that contain, for example, naturally occurring mutations or that may arise during production of a monoclonal antibody preparation, in which such variants are generally present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as obtained from a population of substantially homogeneous antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies in accordance with the present invention may be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci; such methods and other exemplary methods for producing monoclonal antibodies are described herein.

[0042] A "multispecific antibody" is a monoclonal antibody that has binding specificities for at least two different sites, i.e., different epitopes on different antigens or different epitopes on the same antigen. In certain embodiments, a multispecific antibody has three or more binding specificities. Multispecific antibodies may be prepared as full-length antibodies or antibody fragments.

[0043] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a native antibody structure or having a heavy chain containing an Fc region as defined herein.

[0044] An "antibody fragment" 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 scFab), single-domain antibodies (dAbs), and multispecific antibodies formed from antibody fragments. For a review of certain antibody fragments, see Holliger and Hudson, Nature Biotechnology 23:1126-1136 (2005).

[0045] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0046] A "human antibody" is an antibody having an amino acid sequence that corresponds to that of an antibody produced by a human or human cell, or derived from a non-human source that utilizes the human antibody repertoire or other human antibody-encoding sequences. This definition of human antibody specifically excludes humanized antibodies that comprise non-human antigen-binding residues.

[0047] A "humanized" antibody refers to a chimeric antibody comprising amino acid residues of non-human CDRs and human FRs. In certain embodiments, a humanized antibody comprises substantially all of at least one, typically two, variable domains, in which all or substantially all of the CDRs correspond to those of a non-human antibody and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization. The term "monoclonal antibody," as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., each individual antibody in the population is identical and / or binds the same epitope, except for variant antibodies that contain naturally occurring mutations or that may arise during the production of a monoclonal antibody preparation, in which such variants are generally present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.

[0048] The term "therapeutic antibody" refers to an antibody used to treat disease. Therapeutic antibodies can have various mechanisms of action. A therapeutic antibody may bind to an antigen-associated target and neutralize its normal function. For example, a monoclonal antibody that blocks the activity of a protein required for cancer cell survival causes cell death. Another therapeutic monoclonal antibody may bind to an antigen-associated target and activate its normal function. For example, a monoclonal antibody may bind to a protein on a cell and induce an apoptotic signal. Still other monoclonal antibodies may bind to a target antigen expressed only on diseased tissue. Conjugation of a toxic payload (effective drug), such as a chemotherapeutic or radioactive agent, to the monoclonal antibody can create a drug for specifically delivering the toxic payload to diseased tissue, reducing harm to healthy tissue. A "biologically functional fragment" of a therapeutic antibody exhibits at least one, if not some or all, of the biological functions attributed to the intact antibody, including at least specific binding to the target antigen.

[0049] The term "diagnostic antibody" refers to an antibody used as a diagnostic reagent for disease. A diagnostic antibody may bind to a target antigen that is specifically associated with or shows increased expression in a particular disease. A diagnostic antibody may be used, for example, to detect a target in a biological sample from a patient or in diagnostic imaging of a disease site, such as a tumor in a patient. A "biologically functional fragment" of a diagnostic antibody will exhibit at least one, if not some or all, of the biological functions attributable to an intact antibody, including at least specific binding to a target antigen.

[0050] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and its progeny without regard to the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0051] The terms "nucleic acid molecule" or "polynucleotide" include any compound and / or substance comprising a polymer of nucleotides. Each nucleotide consists of a base, specifically a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Nucleic acid molecules are often described by the sequence of bases, whereby the bases represent the primary (linear) structure of the nucleic acid molecule. The sequence of bases is usually represented 5' to 3'. As used herein, the term nucleic acid molecule encompasses, for example, deoxyribonucleic acid (DNA), including complementary DNA (cDNA) and genomic DNA; ribonucleic acid (RNA), particularly messenger RNA (mRNA); synthetic forms of DNA or RNA; and mixed polymers comprising two or more of these molecules. Nucleic acid molecules can be linear or circular. Additionally, the term nucleic acid molecule encompasses both sense and antisense strands, as well as single- and double-stranded forms. Furthermore, the nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases with derivatized sugar or phosphate backbone linkages or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules suitable as vectors for direct expression of the antibodies of the present invention in vitro and / or in vivo, for example, in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors may be unmodified or modified. For example, mRNA may be chemically modified to enhance the stability of the RNA vector and / or the expression of the encoded molecule, such that antibodies can be generated in vivo upon injection of the mRNA into a subject (see, e.g., Stadler et al., Nature Medicine 2017, published online June 12, 2017, doi:10.1038 / nm.4356 or EP 2 101 823 B1).

[0052] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. Isolated nucleic acid includes a nucleic acid molecule contained in a cell that ordinarily contains the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0053] As used herein, the term "vector" 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 as well as vectors that are integrated into the genome of a host cell into which they are introduced. In certain embodiments, vectors induce the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors."

[0054] As used herein, the term "homologous sequences" refers to sequences that share significant sequence similarity as determined by sequence alignment. For example, two sequences may be approximately 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 refer to both DNA and protein sequences.

[0055] As used herein, the term "adjacent" refers to a first nucleotide sequence being located at the 5' end or 3' end, or at both ends, of a second nucleotide sequence. The adjacent nucleotide sequence may be adjacent to the second nucleotide sequence or may be at a specified distance from the second nucleotide sequence. There is no particular limit to the length of the adjacent nucleotide sequence. For example, the adjacent sequence may be a few base pairs or several thousand base pairs. In certain embodiments, the length of the contiguous nucleotide sequence can be about at least 15 base pairs, at least 20 base pairs, at least 30 base pairs, at least 40 base pairs, at least 50 base pairs, at least 75 base pairs, at least 100 base pairs, at least 150 base pairs, at least 200 base pairs, at least 300 base pairs, at least 400 base pairs, at least 500 base pairs, at least 1,000 base pairs, at least 1,500 base pairs, at least 2,000 base pairs, at least 3,000 base pairs, at least 4,000 base pairs, at least 5,000 base pairs, at least 6,000 base pairs, at least 7,000 base pairs, at least 8,000 base pairs, at least 9,000 base pairs, or at least 10,000 base pairs.

[0056] As used herein, the term "exogenous" indicates that a nucleotide sequence is not native to the host cell and is introduced into the host cell by conventional DNA delivery methods, such as transfection, electroporation, or transformation. The term "endogenous" refers to a nucleotide sequence that is derived from the host cell. An "exogenous" nucleotide sequence may have an "endogenous" counterpart that is identical in base composition, but the "exogenous" sequence is introduced into the host cell, for example, by recombinant DNA technology.

[0057] 2. Integration site The presently disclosed subject matter provides host cells suitable for targeted integration of exogenous nucleotide sequences. In certain embodiments, the host cells comprise an exogenous nucleotide sequence integrated into an integration site on the genome of the host cell, i.e., the TI host cell.

[0058] An "integration site" comprises a nucleic acid sequence within a host cell genome into which an exogenous nucleotide sequence is inserted. In certain embodiments, the integration site is located between two adjacent nucleotides on the host cell genome. In certain embodiments, the integration site comprises a stretch of nucleotides between any of the nucleotides into which the exogenous nucleotide sequence may be inserted. In certain embodiments, the integration site is located within a specific locus in the TI host cell genome. In certain embodiments, the integration site is located within an endogenous gene of the TI host cell.

[0059] In certain embodiments, the exogenous nucleotide sequence is integrated into a site within a specific locus of the TI host cell genome. In certain embodiments, the locus into which the exogenous nucleotide sequence is integrated is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or at least about 99.9% homologous to a sequence selected from SEQ ID NOs: 1-7.

[0060] In certain embodiments, the exogenous nucleotide sequence is integrated into a site within a specific locus of the TI host cell genome. In certain embodiments, the locus into which the exogenous nucleotide sequence is integrated is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or at least about 99.9% homologous to a sequence selected from contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1.

[0061] In certain embodiments, the exogenous nucleotide sequence is integrated into an integration site located within a position selected from nucleotides totaling 1 to 1,000 bp, 1,000 to 2,000 bp, 2,000 to 3,000 bp, 3,000 to 4,000 bp, and 4,000 to 4,301 bp of SEQ ID NO:1. In certain embodiments, the exogenous nucleotide sequence is integrated into an integration site located within a position selected from nucleotides of SEQ ID NO:2 that total between 1 and 100,000 bp, 100,000 and 200,000 bp, 200,000 and 300,000 bp, 300,000 and 400,000 bp, 400,000 and 500,000 bp, 500,000 and 600,000 bp, 600,000 and 700,000 bp, and 700,000 and 728,785 bp. In certain embodiments, the exogenous nucleotide sequence is integrated into an integration site located within a position selected from nucleotides that add up to 1 to 100,000 bp, 100,000 to 200,000 bp, 200,000 to 300,000 bp, 300,000 to 400,000 bp, and 400,000 to 413,983 bp of SEQ ID NO: 3. In certain embodiments, the exogenous nucleotide sequence is integrated into an integration site located within a position selected from nucleotides that add up to 1 to 10,000 bp, 10,000 to 20,000 bp, 20,000 to 30,000 bp, and 30,000 to 30,757 bp of SEQ ID NO: 4. In certain embodiments, the exogenous nucleotide sequence is integrated into an integration site located within a position selected from nucleotides that add up to 1 to 10,000 bp, 10,000 to 20,000 bp, 20,000 to 30,000 bp, 30,000 to 40,000 bp, 40,000 to 50,000 bp, 50,000 to 60,000 bp, and 60,000 to 68,962 bp of SEQ ID NO: 5. In certain embodiments, the exogenous nucleotide sequence is integrated into an integration site located within a position selected from nucleotides that add up to 1 to 10,000 bp, 10,000 to 20,000 bp, 20,000 to 30,000 bp, 30,000 to 40,000 bp, 40,000 to 50,000 bp, and 50,000 to 51,326 bp of SEQ ID NO: 6.In certain embodiments, the exogenous nucleotide sequence is integrated into an integration site located within a position selected from nucleotides totaling 1 to 10,000 bp, 10,000 to 20,000 bp, and 20,000 to 22,904 bp of SEQ ID NO:7.

[0062] In certain embodiments, the nucleotide sequence immediately 5' to the integrated exogenous sequence is selected from the group consisting of nucleotides 41190-45269 of NW_006874047.1, nucleotides 63590-207911 of NW_006884592.1, nucleotides 253831-491909 of NW_006881296.1, nucleotides 69303-79768 of NW_003616412.1, nucleotides 293481-315265 of NW_003615063.1, nucleotides 2650443-2662054 of NW_006882936.1, or nucleotides 82214-97705 of NW_003615411.1, and sequences at least 50% homologous thereto. In certain embodiments, the nucleotide sequence immediately 5' to the integrated exogenous sequence is 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 NW_003615063. NW_006882936.1, nucleotides 2650443-2662054 of NW_006882936.1, or nucleotides 82214-97705 of NW_003615411.1.

[0063] In certain embodiments, the nucleotide sequence immediately 3' to the integrated exogenous sequence is selected from the group consisting of nucleotides 45270-45490 of NW_006874047.1, nucleotides 207912-792374 of NW_006884592.1, nucleotides 491910-667813 of NW_006881296.1, nucleotides 79769-100059 of NW_003616412.1, nucleotides 315266-362442 of NW_003615063.1, nucleotides 2662055-2701768 of NW_006882936.1, or nucleotides 97706-105117 of NW_003615411.1, and sequences at least 50% homologous thereto. In certain embodiments, the nucleotide sequence immediately 3′ to the integrated exogenous sequence is 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 80069-90069 of NW_003615063 NW_006882936.1, nucleotides 2662055-2701768 of NW_006882936.1, or nucleotides 97706-105117 of NW_003615411.1, are at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or at least about 99.9% homologous to nucleotides 315266-362442 of NW_006882936.1, nucleotides 2662055-2701768 of NW_006882936.1, or nucleotides 97706-105117 of NW_003615411.1.

[0064] In certain embodiments, the integrated exogenous nucleotide sequence is operably linked to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-7 and sequences at least 50% homologous thereto. In certain embodiments, the nucleotide sequence operably linked to the exogenous nucleotide sequence is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or at least about 99.9% homologous to a sequence selected from SEQ ID NOs: 1-7. In certain embodiments, the integrated exogenous nucleotide sequence comprises at least one SOI. In certain embodiments, the operably linked nucleotide sequence increases the expression level of the SOI compared to a randomly integrated SOI. In certain embodiments, the integrated exogenous SOI is expressed at about 20%, 30%, 40%, 50%, 100%, 2-fold, 3-fold, 5-fold, or 10-fold higher than a randomly integrated SOI.

[0065] In certain embodiments, the integrated exogenous sequence is a nucleotide sequence selected from the group consisting of nucleotides 41190-45269 of NW_006874047.1, nucleotides 63590-207911 of NW_006884592.1, nucleotides 253831-491909 of NW_006881296.1, nucleotides 69303-79768 of NW_003616412.1, nucleotides 293481-315265 of NW_003615063.1, nucleotides 2650443-2662054 of NW_006882936.1, and nucleotides 82214-97705 of NW_003615411.1, and sequences at least 50% homologous thereto. and is adjacent to the 5' side of a nucleotide sequence selected from the group consisting of nucleotides 45270-45490 of NW_006874047.1, nucleotides 207912-792374 of NW_006884592.1, nucleotides 491910-667813 of NW_006881296.1, nucleotides 79769-100059 of NW_003616412.1, nucleotides 315266-362442 of NW_003615063.1, nucleotides 2662055-2701768 of NW_006882936.1, and nucleotides 97706-105117 of NW_003615411.1, and sequences at least 50% homologous thereto.In certain embodiments, the nucleotide sequence flanking the 5′ side of the integrated exogenous nucleotide sequence is 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 79303-79768 of NW_0036 and at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or at least about 99.9% homologous to nucleotides 293481-315265 of NW_006882936.1, nucleotides 2650443-2662054 of NW_006882936.1, and nucleotides 82214-97705 of NW_003615411.1. The nucleotide sequences adjacent to the 3' side of the integrated exogenous nucleotide sequence are: nucleotides 45270-45490 of SEQ ID NO: NW_006874047.1; nucleotides 207912-792374 of NW_006884592.1; nucleotides 491910-667813 of NW_006881296.1; nucleotides 79769-100059 of NW_003616412.1; and NW_0036150 NW_006882936.1, nucleotides 2662055-2701768 of NW_006882936.1, and nucleotides 97706-105117 of NW_003615411.1.

[0066] In certain embodiments, the integrated exogenous nucleotide is integrated into a locus immediately adjacent to all or a portion of a sequence selected from the group consisting of sequences at least about 90% homologous to a sequence selected from SEQ ID NOs: 1-7.

[0067] In certain embodiments, the integrating exogenous nucleotide sequence is flanked by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-7 and sequences at least 50% homologous thereto. In certain embodiments, the integrating exogenous nucleotide sequence is within about 100 bp, about 200 bp, about 500 bp, or about 1 kb of a sequence selected from the group consisting of SEQ ID NOs: 1-7 and sequences at least 50% homologous thereto. In certain embodiments, the nucleotide sequence flanking the exogenous nucleotide sequence is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or at least about 99.9% homologous to a sequence selected from SEQ ID NOs: 1-7.

[0068] In certain embodiments, the exogenous nucleotide sequence is integrated into an integration site adjacent to a position selected from nucleotides that total 1 to 1,000 bp, 1,000 to 2,000 bp, 2,000 to 3,000 bp, 3,000 to 4,000 bp, and 4,000 to 4,301 bp of SEQ ID NO: 1. In certain embodiments, the exogenous nucleotide sequence is integrated into an integration site adjacent to a position selected from nucleotides that total 1 to 100,000 bp, 100,000 to 200,000 bp, 200,000 to 300,000 bp, 300,000 to 400,000 bp, 400,000 to 500,000 bp, 500,000 to 600,000 bp, 600,000 to 700,000 bp, and 700,000 to 728,785 bp of SEQ ID NO: 2. In certain embodiments, the exogenous nucleotide sequence is integrated into an integration site adjacent to a position selected from nucleotides that add up to 1 to 100,000 bp, 100,000 to 200,000 bp, 200,000 to 300,000 bp, 300,000 to 400,000 bp, and 400,000 to 413,983 bp of SEQ ID NO: 3. In certain embodiments, the exogenous nucleotide sequence is integrated into an integration site adjacent to a position selected from nucleotides that add up to 1 to 10,000 bp, 10,000 to 20,000 bp, 20,000 to 30,000 bp, and 30,000 to 30,757 bp of SEQ ID NO: 4. In certain embodiments, the exogenous nucleotide sequence is integrated into an integration site adjacent to a position selected from nucleotides that add up to 1 to 10,000 bp, 10,000 to 20,000 bp, 20,000 to 30,000 bp, 30,000 to 40,000 bp, 40,000 to 50,000 bp, 50,000 to 60,000 bp, and 60,000 to 68,962 bp of SEQ ID NO: 5. In certain embodiments, the exogenous nucleotide sequence is integrated into an integration site adjacent to a position selected from nucleotides that add up to 1 to 10,000 bp, 10,000 to 20,000 bp, 20,000 to 30,000 bp, 30,000 to 40,000 bp, 40,000 to 50,000 bp, and 50,000 to 51,326 bp of SEQ ID NO: 6.In certain embodiments, the exogenous nucleotide sequence is integrated into an integration site adjacent to a position selected from nucleotides totaling 1 to 10,000 bp, 10,000 to 20,000 bp, and 20,000 to 22,904 bp of SEQ ID NO:7.

[0069] In certain embodiments, the locus comprising the integration site of the exogenous nucleotide sequence does not encode an open reading frame (ORF). In certain embodiments, the locus comprising the integration site of the exogenous nucleotide sequence comprises cis-acting elements, such as promoters and enhancers. In certain embodiments, the locus comprising the integration site of the exogenous nucleotide sequence does not comprise any cis-acting elements, such as promoters and enhancers, that enhance gene expression.

[0070] In certain embodiments, the exogenous nucleotide sequence is integrated into an integration site within an endogenous gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2. The endogenous LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2 genes include wild-type and all homologous sequences of the LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2 genes. In certain embodiments, the homologous sequences of the LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2 genes may be at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or at least about 99.9% homologous to the wild-type LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2 genes. In certain embodiments, the LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2 genes are wild-type mammalian LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2 genes. In certain embodiments, the LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2 genes are wild-type human LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2 genes.In certain embodiments, the LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2 genes are wild-type hamster LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2 genes.

[0071] In certain embodiments, the integration site is operably linked to an endogenous gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, XP_003512331.2, and sequences at least about 90% homologous thereto. In certain embodiments, the integration site is adjacent to an endogenous gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, XP_003512331.2, and sequences at least about 90% homologous thereto.

[0072] Table 1 provides exemplary integration sites for TI host cells: Table 1 - Integration sites in TI host cells TIFF2026035601000002.tif147170

[0073] In certain embodiments, the integration site and / or nucleotide sequences adjacent to the integration site can be experimentally identified. In certain embodiments, the integration site and / or nucleotide sequences adjacent to the integration site can be identified by a genome-wide screening approach to isolate host cells that express desired levels of a polypeptide of interest encoded by one or more SOIs integrated into one or more exogenous nucleotide sequences, the exogenous sequences being sequences integrated into one or more loci in the host cell genome. In certain embodiments, the integration site and / or nucleotide sequences adjacent to the integration site can be identified by a genome-wide screening approach after a transposase-based cassette integration event. In certain embodiments, the integration site and / or nucleotide sequences adjacent to the integration site can be identified by brute-force random integration screening. In certain embodiments, the integration site and / or nucleotide sequences adjacent to the integration site can be determined by conventional sequencing approaches, such as targeted locus amplification (TLA) followed by next-generation sequencing (NGS) and whole-genome NGS. In certain embodiments, the location of the integration site on the chromosome can be determined by conventional cell biology approaches, such as, for example, fluorescent in-situ hybridization (FISH) analysis.

[0074] In certain embodiments, the TI host cell comprises a first exogenous nucleotide sequence integrated at a first integration site within a first specific locus in the TI host cell genome and a second exogenous nucleotide sequence integrated at a second integration site within a second specific locus in the genome, hi certain embodiments, the TI host cell comprises multiple exogenous nucleotide sequences integrated at multiple integration sites in the TI host cell genome.

[0075] In certain embodiments, the TI host cell of the present disclosure comprises at least two different exogenous nucleotide sequences, for example, an exogenous nucleotide sequence comprising at least one RRS. In certain embodiments, two or more exogenous nucleotide sequences can be targeted for the introduction of one or more SOIs. In certain embodiments, the SOIs are the same. In certain embodiments, the SOIs are different. In certain embodiments, the parent TI host cell comprising a first exogenous nucleotide sequence can comprise a second exogenous nucleotide sequence at an integration site different from the integration site of the first exogenous nucleotide sequence.

[0076] In certain embodiments, the integration site is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or at least about 99.9% homologous to a sequence selected from SEQ ID NOs: 1-7. In certain embodiments, the integration sites may be located on the same chromosome. In certain embodiments, the integration sites are located within 1 to 1,000 nucleotides, 1,000 to 100,000 nucleotides, 100,000 to 1,000,000 nucleotides or more from each other in the same chromosome. In certain embodiments, the integration sites are located on different chromosomes. In certain embodiments, a TI host cell containing an exogenous nucleotide sequence at one integration site may be used for the insertion of at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or more exogenous nucleotide sequences at the same or different integration sites.

[0077] In certain embodiments, the feasibility of at least two integration sites for recombinase-mediated cassette exchange (RMCE) can be assessed for each site individually. In certain embodiments, the feasibility of at least two integration sites for RMCE can be assessed simultaneously. The feasibility of multiple sites for RMCE can be assessed by methods known in the art, such as measuring polypeptide titer or specific polypeptide production. In certain embodiments, assessment can be performed by methods known in the art, such as assessing the titer and / or specific productivity of a culture of TI host cells expressing the SOI(s). Exemplary culture strategies include, but are not limited to, fed-batch shake flask culture and fed-batch bioreactor culture. The titer and specific productivity of TI host cells expressing a polypeptide of interest can be assessed by methods known in the art, such as, but not limited to, ELISA, FACS, Fluorometric Microvolume Assay Technology (FMAT), Protein A affinity chromatography, and Western blot analysis.

[0078] 3. Exogenous Nucleotide Sequence An exogenous nucleotide sequence is a nucleotide sequence that is not native to a host cell but can be introduced into a host cell by conventional DNA delivery methods, such as transfection, electroporation, or transformation. In certain embodiments, the exogenous nucleotide sequence is a sequence of interest (SOI), e.g., a nucleotide sequence encoding a polypeptide of interest. However, in certain embodiments, the exogenous nucleotide sequence used in the context of the present disclosure includes elements, such as one or more recombination recognition sequences (RRS) and one or more selectable markers, which facilitate the introduction of additional nucleic acid sequences, e.g., SOIs. In certain embodiments, an exogenous nucleotide sequence that facilitates the introduction of additional nucleic acid sequences is referred to herein as a "landing pad." Thus, in certain embodiments, a TI host cell may comprise: (1) an exogenous nucleotide sequence comprising one or more SOIs, e.g., an SOI integrated into a specific locus in the host cell genome by exogenous site-specific nuclease-mediated (e.g., CRISPR / Cas9-mediated) targeted integration; (2) an exogenous nucleotide sequence comprising one or more landing pads; or (3) an exogenous nucleotide sequence comprising one or more landing pads into which one or more SOIs have been integrated.

[0079] In certain embodiments, the TI host cell comprises at least one exogenous nucleotide sequence integrated into one or more integration sites in the TI host cell genome. In certain embodiments, the exogenous nucleotide sequence is integrated into one or more integration sites within a specific locus in the TI host cell genome. For example, and not by way of limitation, the at least one exogenous nucleic acid sequence can be integrated into one or more loci having 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% identity to a sequence selected from SEQ ID NOs: 1-7.

[0080] 3.1 Landing Pad In certain embodiments, the integrating exogenous nucleotide sequence comprises one or more recombination recognition sequences (RRSs), which can be recognized by a recombinase. In certain embodiments, the integrating exogenous nucleotide sequence comprises at least two RRSs. In certain embodiments, the integrating exogenous nucleotide sequence comprises two RRSs, and the two RRSs are the same. In certain embodiments, the integrating exogenous nucleotide sequence comprises two RRSs, and the two RRSs are heterospecific, i.e., not recognized by the same recombinase. In certain embodiments, the integrating exogenous nucleotide sequence comprises three RRSs, and the third RRS is located between the first and second RRSs. In certain embodiments, the first and second RRSs are the same, and the third RRS is different from the first or second RRS. In certain embodiments, all three RRSs are heterospecific. In certain embodiments, the integrating exogenous nucleotide sequence comprises four, five, six, seven, or eight RRSs. In certain embodiments, the integrated exogenous nucleotide sequence comprises multiple RRSs. In certain embodiments, the multiple two or more RRSs are the same. In certain embodiments, the two or more RRSs are heterospecific. In certain embodiments, each RRS can be recognized by a different recombinase. In certain embodiments, a subset of the total number of RRSs are homospecific, i.e., recognized by the same recombinase, and a subset of the total number of RRSs are heterospecific, i.e., not recognized by the same recombinase. In certain embodiments, the RRS or multiple 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.

[0081] In certain embodiments, the integrating exogenous nucleotide sequence comprises at least one selectable marker. In certain embodiments, the integrating exogenous nucleotide sequence comprises one RRS and at least one selectable marker. In certain embodiments, the integrating exogenous nucleotide sequence comprises a first and a second RRS and at least one selectable marker. In certain embodiments, the selectable marker is located between the first RRS and the second RRS. In certain embodiments, two RRSs flank the at least one selectable marker, i.e., the first RRS is located 5' upstream of the selectable marker and the second RRS is located 3' downstream of the selectable marker. In certain embodiments, the first RRS is located adjacent to the 5' end of the selectable marker and the second RRS is located adjacent to the 3' end of the selectable marker.

[0082] In certain embodiments, the selectable marker is located between a first RRS and a second RRS, and the two flanking RRSs are the same. In certain embodiments, both of the two RRSs flanking the selectable marker are LoxP sequences. In certain embodiments, both of the two RRSs flanking the selectable marker are FRT sequences. In certain embodiments, the selectable marker is located between a first RRS and a second RRS, and the two flanking RRSs are heterospecific. In certain embodiments, the first flanking RRS is a LoxP L3 sequence, and the second flanking RRS is a LoxP 2L sequence. In certain embodiments, the LoxP L3 sequence is located on the 5' side of the selectable marker, and the LoxP 2L sequence is located on the 3' side of the selectable marker. In certain embodiments, the first flanking RRS is a wild-type FRT sequence, and the second flanking RRS is a mutant FRT sequence. In certain embodiments, the first flanking RRS is a Bxb1 attP sequence, and the second flanking RRS is a Bxb1 attB sequence. In certain embodiments, the first adjacent RRS is a φC31 attP sequence, and the second adjacent RRS is a φC31 attB sequence. In certain embodiments, the two RRSs are arranged in the same direction. In certain embodiments, the two RRSs are both in the forward or reverse direction. In certain embodiments, the two RRSs are arranged in opposite directions.

[0083] In certain embodiments, the selectable marker can be a gene encoding 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 resistance to puromycin, blasticidin, bleomycin, phleomycin, chloramphenicol, zeocin, or mycophenolic acid. In certain embodiments, the selectable marker can be GFP, eGFP, synthetic GFP, YFP, eYFP, CFP, mPlum, mCherry, tdTomato, mStrawberry, J-red, DsRed-monomer, mOrange, mKO, mCitrine, Venus, YPet, Emerald, CyPet, mCFPm, Cerulean, or T-Sapphire marker. In certain embodiments, the selectable marker can be a fusion construct comprising at least two selectable markers. In certain embodiments, the gene encoding the selectable marker or a fragment of the selectable marker can be fused to a gene encoding a different selectable marker or a fragment thereof.

[0084] In certain embodiments, the integrated exogenous nucleotide sequence comprises two selectable markers flanked by two RRSs, wherein the first selectable marker is different from the second selectable marker. In certain embodiments, the two selectable markers are both selected from the group consisting of a glutamine synthetase selectable marker, a thymidine kinase selectable marker, a HYG selectable marker, and a puromycin resistance selectable marker. In certain embodiments, the integrated exogenous nucleotide sequence comprises a thymidine kinase selectable marker and a HYG selectable marker. In certain embodiments, the first 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 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 GFP, eGFP, synthetic GFP, YFP, eYFP, CFP, mPlum, mCherry, tdTomato, mStrawberry, J-red, DsRed-monomer, mOrange, mKO, mCitrine, Venus, YPet, Emerald, CyPet, mCFPm, Cerulean, and T-Sapphire markers. In certain embodiments, the first selection marker is a glutamine synthetase selection marker, and the second selection marker is a GFP marker. In certain embodiments, the two RRSs flanking both selection markers are the same. In certain embodiments, the two RRSs flanking both selection markers are different.

[0085] In certain embodiments, the selection marker is operably linked to a promoter sequence. In certain embodiments, the selection marker is operably linked to an SV40 promoter. In certain embodiments, the selection marker is operably linked to a cytomegalovirus (CMV) promoter.

[0086] In certain embodiments, the integrating exogenous nucleotide sequence comprises at least one selectable marker and an IRES, wherein the IRES is operably linked to the selectable marker. In certain embodiments, the selectable 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 certain embodiments, the selectable marker operably linked to the IRES is a GFP marker. In certain embodiments, the integrating exogenous nucleotide sequence comprises an IRES flanked by two RRSs and two selectable markers, wherein the IRES is operably linked to a second selectable marker. In certain embodiments, the integrating exogenous nucleotide sequence comprises an IRES flanked by two RRSs and three selectable markers, where the IRES is operably linked to a third selectable marker. In certain embodiments, the integrating exogenous nucleotide sequence comprises an IRES flanked by two RRSs and three selectable markers, where the IRES is operably linked to a third selectable marker. In certain embodiments, the third selectable marker is different from the first or second selectable marker. In certain embodiments, the integrating exogenous nucleotide sequence comprises a first selectable marker operably linked to a promoter and a second selectable marker operably linked to an IRES. In certain embodiments, the integrating 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 certain embodiments, the integrating exogenous nucleotide sequence comprises a thymidine kinase selectable marker and an HYG selectable marker operably linked to a CMV promoter, and a GFP selectable marker operably linked to an IRES.

[0087] In certain embodiments, the integrated exogenous nucleotide sequence comprises three RRSs. In certain embodiments, the third RRS is located between the first and second RRSs. In certain embodiments, all three RRSs are the same. In certain embodiments, the first and second RRSs are the same, and the third RRS is different from the first or second RRS. In certain embodiments, all three RRSs are heterospecific.

[0088] 3.2 Sequence of Interest (SOI) In certain embodiments, the integrated exogenous nucleotide sequence comprises at least one exogenous SOI. In certain embodiments, the integrated exogenous nucleotide sequence comprises at least one selectable marker and at least one exogenous SOI. In certain embodiments, the integrated exogenous nucleotide sequence comprises at least one selectable marker, at least one exogenous SOI, and at least one RRS. In certain embodiments, the integrated exogenous nucleotide sequence comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or more SOIs. In certain embodiments, the SOIs are the same. In certain embodiments, the SOIs are different.

[0089] In certain embodiments, the SOI encodes a single-chain antibody or fragment thereof. In certain embodiments, the SOI encodes an antibody heavy chain sequence or fragment thereof. In certain embodiments, the SOI encodes an antibody light chain sequence or fragment thereof. In certain embodiments, the integrated exogenous nucleotide sequence comprises an SOI encoding an antibody heavy chain sequence or fragment thereof and an SOI encoding an antibody light chain sequence or fragment thereof. In certain embodiments, the integrated exogenous nucleotide sequence comprises an SOI encoding a first antibody heavy chain sequence or fragment thereof, an SOI encoding a second antibody heavy chain sequence or fragment thereof, and an SOI encoding an antibody light chain sequence or fragment thereof. In certain embodiments, the integrated exogenous nucleotide sequence comprises an SOI encoding a first antibody heavy chain sequence or fragment thereof, an SOI encoding a second antibody heavy chain sequence or fragment thereof, an SOI encoding a first antibody light chain sequence or fragment thereof, and a second SOI encoding an antibody light chain sequence or fragment thereof. In certain 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 a desired amount of bispecific antibody production. In certain embodiments, the individual SOIs encoding the heavy and light chain sequences can be integrated, for example, into a single exogenous nucleic acid sequence present at a single integration site, into multiple exogenous nucleic acid sequences present at a single integration site, or into multiple exogenous nucleic acid sequences integrated at different integration sites within the TI host cell.

[0090] In certain embodiments, the integrated exogenous nucleotide sequence comprises at least one selectable marker, at least one exogenous SOI, and one RRS. In certain embodiments, the RRS is located adjacent to the at least one selectable marker or at least one exogenous SOI. In certain embodiments, the integrated exogenous nucleotide sequence comprises at least one selectable marker, at least one exogenous SOI, and two RRSs. In certain embodiments, the integrated exogenous nucleotide sequence comprises at least one selectable marker and at least one exogenous SOI located between the first RRS and the second RRS. In certain embodiments, the two RRSs flanking the selectable marker and the exogenous SOI are the same. In certain embodiments, the two RRSs flanking the selectable marker and the exogenous SOI are different. In certain embodiments, the first flanking RRS is a LoxP L3 sequence, and the second flanking RRS is a LoxP 2L sequence. In certain embodiments, the L3 LoxP sequence is located 5' to the selectable marker and the exogenous SOI, and the LoxP 2L sequence is located 3' to the selectable marker and the exogenous SOI.

[0091] In certain embodiments, the integrated exogenous nucleotide sequence comprises three RRSs and two exogenous SOIs, with the third RRS located between the first and second RRSs. In certain embodiments, the first SOI is located between the first and third RRSs, and the second SOI is located between the third and second RRSs. In certain embodiments, the first and second SOIs are different. In certain embodiments, the first and second RRSs are the same, and the third RRS is different from the first or second RRS. In certain embodiments, all three RRSs are heterospecific. In certain embodiments, the first RRS is a LoxP L3 site, the second RRS is a LoxP 2L site, and the third RRS is a LoxFas site. In certain embodiments, the integrated exogenous nucleotide sequence comprises three RRSs, one exogenous SOI, and one selectable marker. In certain embodiments, the SOI is located between the first and third RRSs, and the selectable marker is located between the third and second RRSs. In certain embodiments, the integrated exogenous nucleotide sequence comprises three RRSs, two exogenous SOIs, and one selectable marker. In certain embodiments, the first SOI and selectable marker are located between the first and third RRSs, and the second SOI is located between the third and second RRSs.

[0092] In certain embodiments, the exogenous SOI encodes a polypeptide of interest. Such a polypeptide of interest may be selected from the group 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 therapeutic function. In certain embodiments, the exogenous SOI encodes an antibody or an antigen-binding fragment thereof. In certain embodiments, the exogenous SOI encodes a single-chain antibody, an antibody light chain, an antibody heavy chain, a single-chain Fv fragment (scFv), or an Fc fusion protein. In certain embodiments, the exogenous SOI(s) encode a standard antibody. In certain embodiments, the exogenous SOI(s) encode a half antibody, such as, but not limited to, antibody B, Q, T, and mAbI of the present disclosure. In certain embodiments, the exogenous SOI(s) encode a composite antibody. In certain embodiments, the composite antibody may be a bispecific antibody, such as, but not limited to, bispecific molecule A, bispecific molecule B, bispecific molecule C, or bispecific molecule D of the present disclosure. In certain embodiments, the exogenous SOI is operably linked to at least one cis-acting element, such as a promoter or enhancer. In certain embodiments, the exogenous SOI is operably linked to a CMV promoter.

[0093] In certain embodiments, the integrated exogenous nucleotide sequence comprises two RRSs and at least two exogenous SOIs located between the two RRSs. In certain embodiments, an SOI encoding one heavy chain and one light chain of an antibody is located between the two RRSs. In certain embodiments, an SOI encoding one heavy chain and two light chains of an antibody is located between the two RRSs. In certain embodiments, an SOI encoding a different combination of heavy and light chain copies of an antibody is located between the two RRSs.

[0094] In certain embodiments, the integrated exogenous nucleotide sequence comprises three RRSs and at least two exogenous SOIs, with the third RRS located between the first and second RRSs. In certain 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 certain embodiments, the first and second RRSs are the same, and the third RRS is different from the first or second RRS. In certain embodiments, all three RRSs are heterospecific. In certain embodiments, an SOI encoding one heavy chain and one light chain of a first antibody is located between the first and third RRSs, and an SOI encoding one heavy chain and one light chain of a second antibody is located between the third and second RRSs. In certain embodiments, the SOI encoding one heavy chain and two light chains 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 certain embodiments, the SOI encoding one heavy chain and three light chains of the first antibody is located between the first and third RRSs, 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 and second RRSs. In certain embodiments, the SOI encoding one heavy chain and three light chains of the first antibody is located between the first and third RRSs, 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 and second RRSs. In certain embodiments, SOIs encoding different combinations of heavy and light chain copies of multiple antibodies are located between the first and third RRSs and between the third and second RRSs.

[0095] In certain embodiments, the number of SOIs is selected to increase the titer and / or specific productivity of a host cell expressing the SOIs. For example, and not by way of limitation, the incorporation of two, three, four, five, six, seven, eight, or more SOIs can result in increased titer and / or specific productivity.

[0096] In the context of antibody expression, the inclusion of an additional heavy or light chain encoding SOI can result in increased titer and / or specific productivity. For example, and not by way of limitation, increased titer and / or specific productivity can be achieved when increasing copy number from a sequence encoding one heavy chain and one light chain (HL) to a sequence encoding one heavy chain and two light chains (HLL). Similarly, as outlined in the Examples below, increasing copy number from HLL (three SOIs) to HLL-HL (five SOIs) or HLL-HLL (six SOIs) can increase titer and / or specific productivity. Furthermore, increasing copy number to HLL-HL (five SOIs) or HLL-HLHL (seven SOIs) can increase titer and / or specific productivity. Additional heavy and light chain SOI copy number options include, but are not limited to, HHL, HHL-H, HLL-H, HHL-HH, HHL-HL, HHL-LL, HLL-HH, HLL-HL, HLL-LL, HHL-HHL, HHL-HHH, HHL-HLL, HHK-LLL, HLL-HHL, HLL-HHH, HLL-LLL, HHL-HHHL, HHL-HHHH, HHL-HHLL, HHL-HLLL, HHL-LLLL, HLL-HHHL, HLL-HHHH, HLL-HLLL, and HLL-LLLL. While in certain embodiments, the inclusion of the additional copy occurs at a single genomic locus, in certain embodiments, the SOI copy may be integrated at more than one locus, e.g., multiple copies may be integrated at a single locus and one or more copies may be integrated at one or more additional loci.

[0097] In certain embodiments, the location of the SOI, e.g., whether one SOI is located 3' or 5' relative to another SOI, is selected to increase the titer and / or specific productivity of a host cell expressing the SOI. For example, but not by way of limitation, in the context of antibody production, the location of integration of the heavy and light chain SOIs can result in increased titer and / or specific productivity. As outlined in Figures 23A-D and the Examples presented below, the relative positions of the heavy and light chain SOIs can affect titer and specific productivity despite the absence of a change in SOI copy number.

[0098] 4.Host cells The presently disclosed subject matter provides host cells suitable for targeted integration of a nucleotide sequence and expression of a polypeptide of interest. In certain embodiments, the host cell comprises an endogenous gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, XP_003512331.2, and sequences at least 50% homologous thereto, or a locus in the host cell genome, wherein the locus comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-7, and sequences at least 50% homologous thereto.

[0099] In certain embodiments, the host cell is a eukaryotic host cell. In certain embodiments, the host cell is a mammalian host cell. In certain embodiments, the host cell is a hamster host cell, a human host cell, a rat host cell, or a mouse host cell. In certain embodiments, the host cell is a Chinese hamster ovary (CHO) host cell, a CHO K1 host cell, a CHO K1SV host cell, a DG44 host cell, a DUKXB-11 host cell, a CHOK1S host cell, or a CHO K1M host cell.

[0100] In certain 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 as described in Graham et al., J. Gen Virol. 36:59 (1977)), baby hamster kidney cells (BHK), mouse Sertoli cells (e.g., TM4 cells as 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 (MMT060562), e.g., Mather et al., Annals The cells are selected from the group consisting of TRI cells, MRC5 cells, FS4 cells, Y0 cells, NS0 cells, Sp2 / 0 cells, and PER.C6® cells, as described in NYAcad. Sci. 383:44-68 (1982).

[0101] In certain embodiments, the host cell is a cell line. In certain embodiments, the host cell is a cell line that has been cultured for a certain number of generations. In certain embodiments, the host cell is a primary cell.

[0102] In certain embodiments, expression of a polypeptide of interest is stable when the expression level is maintained at a certain level and increases or decreases by less than 20% over 10, 20, 30, 50, 100, 200, or 300 generations. In certain embodiments, expression of a polypeptide of interest is stable when the culture can be maintained without any selection. In certain embodiments, expression of a polypeptide of interest is high when 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.

[0103] The presently disclosed subject matter also relates to methods for producing a polypeptide of interest. In certain embodiments, such methods include: a) providing a host cell comprising at least one exogenous SOI and at least one selectable marker integrated into a locus in the host cell genome, the locus being at least about 90% homologous to a sequence selected from SEQ ID NOs: 1-7, wherein the at least one exogenous SOI and one selectable marker are flanked by a first and a third RRS, and the at least one exogenous SOI is flanked by a second and a third RRS; and b) culturing the cell of a) under conditions suitable for expressing the SOI and recovering the polypeptide of interest therefrom. In certain embodiments, such methods include: a) providing a host cell comprising at least two exogenous SOIs and at least one selectable marker integrated into a locus in the host cell genome, the locus being at least about 90% homologous to a sequence selected from SEQ ID NOs: 1-7, wherein the at least one exogenous SOI and one selectable marker are flanked by a first and a third RRS, and the at least one exogenous SOI is flanked by a second and a third RRS; and b) culturing the cell of a) under conditions suitable for expressing the SOI and recovering the polypeptide of interest therefrom. In certain embodiments, such methods include: a) providing a host cell containing at least one exogenous SOI flanked by two RRSs integrated into an endogenous gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, XP_003512331.2, and sequences at least about 90% homologous thereto, and at least one selectable marker; and b) culturing the cell of a) under conditions suitable for expressing the SOI and recovering a polypeptide of interest therefrom.In certain embodiments, such methods include: a) providing a host cell comprising at least two exogenous SOIs and at least one selectable marker integrated into an endogenous gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, XP_003512331.2, and sequences at least about 90% homologous thereto, wherein the at least one exogenous SOI and one selectable marker are flanked by a first and a third RRS, and the at least one exogenous SOI is flanked by a second and a third RRS; and b) culturing the cell of a) under conditions suitable for expressing the SOIs and recovering a polypeptide of interest therefrom.

[0104] In certain embodiments, the polypeptide of interest is produced and secreted into the cell culture medium. In certain embodiments, the polypeptide of interest is expressed and maintained within the host cell. In certain embodiments, the polypeptide of interest is expressed in, inserted into, and maintained in the host cell membrane.

[0105] The exogenous nucleotide or vector of interest can be introduced into host cells by conventional cell biology methods, including, but not limited to, transfection, transduction, electroporation, or injection. In certain embodiments, the exogenous nucleotide or vector of interest is introduced into host cells by chemical-based transfection methods, including lipid-based transfection, calcium phosphate-based transfection, cationic polymer-based transfection, or nanoparticle-based transfection. In certain embodiments, the exogenous nucleotide of interest is introduced into host cells by virus-mediated transduction, including, but not limited to, lentivirus-, retrovirus-, adenovirus-, or adeno-associated virus-mediated transduction. In certain embodiments, the exogenous nucleotide or vector of interest is introduced into host cells by gene gun-mediated injection. In certain embodiments, both DNA and RNA molecules are introduced into host cells using the methods described herein.

[0106] 5. Targeted Integration Targeted integration allows exogenous nucleotide sequences to be integrated into one or more predetermined sites in the genome of a host cell. In certain embodiments, targeted integration is mediated by a recombinase that recognizes one or more RRSs. In certain embodiments, targeted integration is mediated by homologous recombination. In certain embodiments, targeted integration is mediated by exogenous site-specific nucleases, followed by HDR and / or NHEJ.

[0107] 5.1. Targeted Integration by Recombinase-Mediated Recombination A "recombination recognition sequence" (RRS) is a nucleotide sequence recognized by a recombinase and is necessary and sufficient for a recombinase-mediated recombination event. An RRS can be used to define the location at which a recombination event will occur in a nucleotide sequence.

[0108] In certain embodiments, the RRS is selected from the group consisting of a LoxP sequence, a LoxP L3 sequence, a LoxP 2L sequence, a LoxFas sequence, a Lox511 sequence, a Lox2272 sequence, a Lox2372 sequence, a Lox5171 sequence, a Loxm2 sequence, a Lox71 sequence, a Lox66 sequence, an FRT sequence, a Bxb1 attP sequence, a Bxb1 attB sequence, a φC31 attP sequence, and a φC31 attB sequence.

[0109] In certain embodiments, the RRS can be recognized by Cre recombinase. In certain embodiments, the RRS can be recognized by FLP recombinase. In certain embodiments, the RRS can be recognized by Bxb1 integrase. In certain embodiments, the RRS can be recognized by φC31 integrase.

[0110] In certain embodiments, when the RRS is a LoxP site, the host cell requires Cre recombinase to perform recombination. In certain embodiments, when the RRS is a FRT site, the host cell requires FLP recombinase to perform recombination. In certain embodiments, when the RRS is a Bxb1 attP or Bxb1 attB site, the host cell requires Bxb1 integrase to perform recombination. In certain embodiments, when the RRS is a φC31 attP or φC31attB site, the host cell requires φC31 integrase to perform recombination. The recombinase can be introduced into the host cell using an expression vector containing the coding sequence for the enzyme.

[0111] 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 intramolecular and intermolecular recombination between LoxP sequences. The LoxP sequence consists of an 8-bp non-palindromic core region flanked by two 13-bp inverted repeat sequences. Cre recombinase mediates recombination within the 8-bp core region by binding to the 13-bp repeat sequence. Cre-LoxP-mediated recombination is highly efficient and does not require any other host factors. When two LoxP sequences are positioned in the same orientation on the same nucleotide sequence, Cre-mediated recombination results in the excision of the DNA sequence located between the two LoxP sequences as a covalently closed circle. When two LoxP sequences are located at opposite positions on the same nucleotide sequence, Cre-mediated recombination will reverse the orientation of the DNA sequence located between the two sequences.LoxP sequences can also be located on different chromosomes to promote recombination between different chromosomes.When two LoxP sequences are located on two different DNA molecules and one DNA molecule is circular, Cre-mediated recombination will result in the integration of the circular DNA sequence.

[0112] In certain embodiments, the LoxP sequence is a wild-type LoxP sequence. In certain embodiments, the LoxP sequence is a mutant LoxP sequence. Mutant LoxP sequences have been developed to increase the efficiency of Cre-mediated integration or replacement. In certain 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, the Lox71 sequence has a mutated 5-bp in the left 13-bp repeat sequence. The Lox66 sequence has a mutated 5-bp in the right 13-bp repeat sequence. Both wild-type and mutant LoxP sequences can mediate Cre-dependent recombination.

[0113] The FLP-FRT site-specific recombination system is similar to the Cre-Lox system. The system contains flippase (FLP) recombinase, which is derived 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 13-bp palindromic sequences, each flanked by an 8-bp spacer. FLP binds to the 13-bp palindromic sequences and mediates DNA cleavage, 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 certain embodiments, the FRT sequence is a wild-type FRT sequence. In certain embodiments, the FRT sequence is a mutant FRT sequence. Both wild-type and mutant FRT sequences can mediate FLP-dependent recombination. In certain embodiments, the FRT sequence is fused to a responsive receptor domain sequence, such as, but not limited to, a tamoxifen-responsive receptor domain sequence.

[0114] Bxb1 and φC31 belong to the serine recombinase family. Both are derived from bacteriophages and are used by these bacteriophages to establish lysogeny and promote site-specific integration of the phage genome into the bacterial genome. These integrases catalyze site-specific recombination events between short (40-60 bp) DNA substrates, called attP and attB sequences, which are the original attachment sites located on the phage DNA and bacterial DNA, respectively. After recombination, two new sequences are formed, called attL and attR sequences, each containing half sequences derived from attP and attB. Recombination can also occur between the attL and attR sequences to excise the integrated phage from bacterial DNA. Both integrases can catalyze recombination without the assistance of any additional host factors. In the absence of any auxiliary factors, these integrases mediate unidirectional recombination between attP and attB with an efficiency of over 80%. Due to the short DNA sequences that can be recognized by these integrases and the unidirectional recombination, these recombination systems have been developed as a complement to the Cre-LoxP and FRT-FLP systems that are widely used for genetic engineering purposes.

[0115] The terms "matched RRS" and "homospecific RRS" indicate that recombination occurs between two RRSs. In certain embodiments, the two matched RRSs are the same. In certain embodiments, both RRSs are wild-type LoxP sequences. In certain embodiments, both RRSs are mutant LoxP sequences. In certain embodiments, both RRSs are wild-type FRT sequences. In certain embodiments, both RRSs are mutant FRT sequences. In certain embodiments, the two matched RRSs are different sequences but can be recognized by the same recombinase. In certain embodiments, the first matched RRS is a Bxb1 attP sequence and the second matched RRS is a Bxb1 attB sequence. In certain embodiments, the first matched RRS is a φC31 attB sequence and the second matched RRS is a φC31 attB sequence.

[0116] In certain embodiments, the integrating exogenous nucleotide sequence comprises two RRSs, and the vector comprises two RRSs that match the two RRSs on the integrating exogenous nucleotide sequence, i.e., the first RRS on the integrating exogenous nucleotide sequence matches the first RRS on the vector, and the second RRS on the integrating exogenous nucleotide sequence matches the second RRS on the vector. In certain embodiments, the first RRS on the integrating exogenous nucleotide sequence and the first RRS on the vector are the same as the second RRS on the integrating exogenous nucleotide sequence and the second RRS on the vector. A non-limiting example of such a "single-vector RMCE" strategy is provided in Figure 2A. In certain embodiments, the first RRS on the integrating exogenous nucleotide sequence and the first RRS on the vector are different from the second RRS on the integrating exogenous nucleotide sequence and the second RRS on the vector. In certain embodiments, the first RRS on the integrating exogenous nucleotide sequence and the first RRS on the vector are both LoxP L3 sequences, and the second RRS on the integrating exogenous nucleotide sequence and the second RRS on the vector are both LoxP 2L sequences.

[0117] In certain embodiments, a "two-vector RMCE" strategy is used. For example, and not by way of limitation, the integrating exogenous nucleotide sequence may comprise three RRSs, e.g., a first vector may comprise two RRSs that match the first and third RRSs on the integrating exogenous nucleotide sequence, and a second vector may comprise two RRSs that match the third and second RRSs on the integrating exogenous nucleotide sequence, while the third RRS ("RRS3") is located between the first RRS ("RRS1") and the second RRS ("RRS2"). An example of a two-vector RMCE strategy is shown in Figure 4. In such an example, RRS1, RRS2, and RRS3 are heterospecific, e.g., they do not cross-react with each other. In some embodiments, one vector (forward) comprises RRS1, a first SOI, and a promoter, followed by a start codon and RRS3 (in that order). The other vector (rear) contains RRS3, SOI2, and RRS2 (in that order) fused to the coding sequence of a marker lacking an initiation codon (ATG). Additional nucleotides may be inserted between the RRS3 site and the selectable marker sequence to ensure in-frame translation of the fusion protein. In some embodiments, the first SOI encodes an antibody. In some embodiments, the antibody is a single-chain antibody, an antibody light chain, an antibody heavy chain, a single-chain Fv fragment (scFv), or an Fc-fusion protein. In some embodiments, the second SOI encodes an antibody. In some embodiments, the antibody is a single-chain antibody, an antibody light chain, an antibody heavy chain, a single-chain Fv fragment (scFv), or an Fc-fusion protein. In certain embodiments, the antibodies encoded by the first and second SOIs pair to form a multispecific, e.g., bispecific, antibody.

[0118] Such a two-vector RMCE strategy allows the introduction of eight or more SOIs by incorporating an appropriate number of SOIs between each pair of RRSs.

[0119] Both single-vector and two-vector RMCE allow for the unidirectional integration of one or more donor DNA molecules(s) into a predetermined site in the host cell genome and the 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 at least one selectable marker (although in certain two-vector RMCE instances, a "split selectable marker" may be used as outlined herein) and / or two heterospecific RRSs flanking at least one exogenous SOI. RMCE involves a recombinase-catalyzed double recombination crossover event between the target genomic locus and two heterospecific RRSs within the donor DNA molecule. RMCE is designed to introduce a copy of the SOI or selectable marker into a predetermined locus in the host cell genome. Unlike recombination involving only a single crossover event, RMCE is performed in a way that does not introduce prokaryotic vector sequences into the host cell genome, thereby reducing and / or preventing unwanted induction of host immune or defense mechanisms. The RMCE procedure can be repeated with multiple DNA cassettes.

[0120] In certain embodiments, targeted integration is achieved by a single crossover recombination event, whereby one exogenous nucleotide sequence comprising one RRS adjacent to at least one exogenous SOI or at least one selectable marker is integrated into a predetermined site of the host cell genome.In certain embodiments, targeted integration is achieved by a single RMCE, whereby a DNA cassette comprising at least one exogenous SOI or at least one selectable marker adjacent to two heterospecific RRSs is integrated into a predetermined site of the host cell genome.In certain embodiments, targeted integration is achieved by two RMCEs, whereby two different DNA cassettes, each comprising at least one exogenous SOI or at least one selectable marker adjacent to two heterospecific RRSs, are both integrated into a predetermined site of the host cell genome.In certain embodiments, targeted integration is achieved by multiple RMCEs, whereby DNA cassettes from multiple vectors, each comprising at least one exogenous SOI or at least one selectable marker adjacent to two heterospecific RRSs, are all integrated into a predetermined site of the host cell genome. In certain embodiments, the selectable marker can be partially encoded on a first vector and partially encoded on a second vector, such that integration of both RMCEs allows for expression of the selectable marker. An example of such a system is provided in Figure 4.

[0121] In certain embodiments, targeted integration by recombinase-mediated recombination results in the selection marker or one or more exogenous SOIs integrated into the host cell genome at one or more predetermined integration sites along with sequences from the prokaryotic vector. In certain embodiments, targeted integration by recombinase-mediated recombination results in the selection marker or one or more exogenous SOIs integrated into the host cell genome at one or more predetermined integration sites that do not include sequences from the prokaryotic vector.

[0122] 5.2 Targeted integration by homologous recombination, HDR, or NHEJ The presently disclosed subject matter also relates to targeted integration mediated by homologous recombination or by exogenous site-specific nucleases followed by HDR or NHEJ.

[0123] Homologous recombination is the recombination between DNA molecules that share extensive sequence homology. It can be used to induce error-free repair of double-stranded DNA breaks, generating sequence diversity in gametes during meiosis. Because homologous recombination involves the exchange of genetic information between two homologous DNA molecules, it does not change the overall arrangement of genes on chromosomes. During homologous recombination, a nick or break is formed in double-stranded DNA (dsDNA), followed by invasion of the homologous dsDNA molecule by the single-stranded DNA end, pairing of the homologous sequences, branch migration to form a Holliday junction, and eventual dissociation of the Holliday junction.

[0124] Double-strand breaks (DSBs) are the most severe form of DNA damage, and repair of such DNA damage is essential for maintaining genome integrity in all organisms. There are two main repair pathways for DSB repair. The first is the homologous recombination repair (HDR) pathway, and homologous recombination is the most common form of HDR. Because HDR requires the presence of homologous DNA in the cell, this repair pathway is typically active during the S and G2 phases of the cell cycle, when newly replicated sister chromatids are available as homologous templates. HDR is also the primary repair pathway for repairing collapsed replication forks during DNA replication. HDR is considered a relatively error-free repair pathway. The second DSB repair pathway is non-homologous end joining (NHEJ). NHEJ is a repair pathway in which the ends of broken DNA are ligated together without the need for a homologous DNA template.

[0125] Targeted integration can be promoted by HDR followed by exogenous site-specific nucleases. This is because the frequency of homologous recombination can be increased by introducing DSBs at specific target genomic sites. In certain embodiments, the exogenous nuclease can be selected from the group consisting of zinc finger nucleases (ZFNs), ZFN dimers, transcription activator-like effector nucleases (TALENs), TAL effector domain fusion proteins, RNA-guided DNA endonucleases, engineered meganucleases, and clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) endonucleases.

[0126] The CRISPR / Cas and TALEN systems are two genome editing tools that offer the easiest construction and highest efficiency. CRISPR / Cas was identified as a bacterial immune defense mechanism against bacteriophage invasion. Cas is a nuclease that, when guided by a synthetic guide RNA (gRNA), associates with a specific nucleotide sequence in a cell and can edit the DNA in or around that nucleotide sequence by, for example, creating a single-strand break, a DSB, and / or a point mutation. TALENs are engineered site-specific nucleases composed of the DNA-binding domain of a TALE (transcription activator-like effector) and the catalytic domain of the restriction endonuclease FokI. By altering the amino acids present in the hypervariable residue region of the DNA-binding domain monomer, various artificial TALENs can be created to target various nucleotide sequences. The DNA-binding domain then guides the nuclease to the target sequence, creating a DSB.

[0127] Targeted integration by homologous recombination or HDR comprises the presence of a homologous sequence at the integration site.In certain embodiments, the homologous sequence is present on a vector.In certain embodiments, the homologous sequence is present on a polynucleotide.

[0128] In certain embodiments, a vector intended for targeted integration of an exogenous nucleotide sequence into a host cell comprises a nucleotide sequence homologous to an endogenous gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2, or to a sequence selected from SEQ ID NOs: 1-7 adjacent to at least one selectable marker. In certain embodiments, a vector intended for targeted integration of an exogenous nucleotide sequence into a host cell comprises a nucleotide sequence homologous to an endogenous gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2, or to a sequence selected from SEQ ID NOs: 1-7 adjacent to at least one selectable marker and at least one exogenous SOI. In certain embodiments, a vector intended for targeted integration of an exogenous nucleotide sequence into a host cell comprises a nucleotide sequence at least 50% homologous to a sequence selected from SEQ ID NOs: 1-7 flanking a DNA cassette, the DNA cassette comprising at least one selectable marker flanked by two RRSs and at least one exogenous SOI. In certain embodiments, a vector intended for targeted integration of an exogenous nucleotide sequence into a host cell comprises a nucleotide sequence at least 50% homologous to an endogenous gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2 flanking a DNA cassette, the DNA cassette comprising at least one selectable marker flanked by two RRSs and at least one exogenous SOI.In certain embodiments, the vector nucleotide sequence is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or at least about 99.9% homologous to an endogenous gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2, or to a sequence selected from SEQ ID NOs: 1-7. In certain embodiments, the vector is selected from the group consisting of an adenoviral vector, an adeno-associated viral vector, a lentiviral vector, a retroviral vector, an integrating phage vector, a non-viral vector, a transposon and / or transposase vector, an integrase substrate, and a plasmid.

[0129] In certain embodiments, a polynucleotide intended for targeted integration of an exogenous nucleotide sequence into a host cell comprises a nucleotide sequence homologous to an endogenous gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2, or to a sequence selected from SEQ ID NOs: 1-7 adjacent to at least one selectable marker. In certain embodiments, a polynucleotide intended for targeted integration of an exogenous nucleotide sequence into a host cell comprises a nucleotide sequence homologous to an endogenous gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2, or to a sequence selected from SEQ ID NOs: 1-7 adjacent to at least one selectable marker and at least one exogenous SOI. In certain embodiments, a polynucleotide intended for targeted integration of an exogenous nucleotide sequence into a host cell comprises a nucleotide sequence at least 50% identical to a sequence selected from SEQ ID NOs: 1-7 flanking a DNA cassette, the DNA cassette comprising at least one selectable marker flanked by two RRSs and at least one exogenous SOI. In certain embodiments, a polynucleotide intended for targeted integration of an exogenous nucleotide sequence into a host cell comprises a nucleotide sequence at least 50% identical to an endogenous gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2 flanking a DNA cassette, the DNA cassette comprising at least one selectable marker flanked by two RRSs and at least one exogenous SOI.In certain embodiments, the flanking nucleotide sequence is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or at least about 99.9% homologous to an endogenous gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2, or to a sequence selected from SEQ ID NOs: 1-7.

[0130] In certain embodiments, homologous recombination is carried out without the use of any auxiliary factors. In certain embodiments, homologous recombination is facilitated by the presence of an integrating vector. In certain embodiments, the integrating vector is selected from the group consisting of an adeno-associated viral vector, a lentiviral vector, a retroviral vector, and an integrating phage vector.

[0131] 5.3 Regulated targeted integration Suboptimal protein expression levels are often the result of difficult-to-express proteins. Low expression levels of difficult-to-express proteins can have a variety of causes, making them difficult to identify. One possibility is toxicity of the protein expressed in the host cell. In such cases, regulated expression systems can be used to express toxic proteins when the protein-encoding sequence of interest is under the control of an inducible promoter. In these systems, expression of the difficult-to-express protein is promoted only when a regulator, such as a small molecule, including but not limited to tetracycline or its analog doxycycline (DOX), is added to the culture. Regulating the expression of the toxic protein can alleviate toxic effects and allow the culture to achieve the desired cell growth before production. In certain embodiments, a regulated targeted integration (RTI) system includes an SOI that is integrated into a specific locus, such as an exogenous nucleic acid sequence containing one or more RRSs, and transcribed under a regulated promoter operably linked to it. In certain embodiments, the RTI system can be used to determine the underlying cause of low protein expression of difficult-to-express molecules, such as, but not limited to, antibodies. In certain embodiments, the ability to selectively silence expression of an SOI in an RTI system can be used to correlate expression of the SOI with observed adverse effects.

[0132] In certain embodiments, a regulated targeted integration (RTI) system can be used to minimize the effects of transcriptional and cell line variability during root cause analysis of difficult-to-express molecules. For example, but not by way of limitation, expression of an SOI in a TI host can be induced by adding a regulatory factor, such as doxycycline, to the culture. In certain embodiments, an RTI vector utilizes a tetracycline-regulated promoter to express the SOI, which can be integrated, for example, into an exogenous nucleic acid sequence containing an RRS that itself integrates into an integration site in the host cell's genome, thereby allowing regulated expression of the SOI.

[0133] In certain embodiments, the RTI system described in this disclosure can be used to successfully determine the underlying cause(s) for low protein expression of an SOI, e.g., a therapeutic antibody, compared to a control cell line. In certain embodiments, once low relative expression of an SOI, e.g., a therapeutic antibody, in an RTI 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 cycloheximide.

[0134] 5.4 Adjusted Systems The subject matter disclosed herein also relates to regulated systems for use in TI. For example, and not by way of limitation, such regulation can be based on a gene switch for blocking or activating mRNA synthesis through the regulated binding of a transcriptional repressor or activator to a constitutive or minimal promoter. In certain non-limiting embodiments, repression can be achieved, for example, by binding to a repressor protein that sterically blocks transcription initiation, or by actively suppressing transcription through a transcription silencer. In certain non-limiting embodiments, activation of a mammalian or viral enhancerless minimal promoter can be achieved through regulated binding to an activation domain.

[0135] In certain embodiments, the conditional binding of a transcriptional repressor or activator can be achieved by using an allosteric protein that binds to a promoter in response to an external stimulus. In certain embodiments, the conditional binding of a transcriptional repressor or activator can be achieved by using an intracellular receptor that can bind to a target promoter upon release from a sequestering protein. In certain embodiments, the conditional binding of a transcriptional repressor or activator can be achieved by using a chemically induced dimerizer.

[0136] In certain embodiments, the allosteric protein used in the disclosed TI system can be a protein that regulates transcriptional activity in response to antibiotics, bacterial quorum-sensing messengers, catabolites, or culture parameters such as temperature (e.g., cold or heat). In certain embodiments, such RTI systems can be catabolite-based, for example, when a bacterial repressor controlling catabolic genes for alternative carbon sources is introduced into mammalian cells. In certain embodiments, repression of the target promoter can be achieved by coumarate-responsive binding of the repressor CymR. In certain 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 herpes simplex VP16 transactivation domain.

[0137] In certain embodiments, the TI system can be a prokaryotic quorum-sensing-based expression system that manages intra- and inter-population communication through quorum-sensing molecules. These quorum-sensing molecules bind to target cell receptors and modulate the affinity of the receptor for its cognate promoter, resulting in the initiation of specific regulon switches. In certain embodiments, the quorum-sensing molecule can be N-(3-oxo-octanoyl)-homoserine lactone, in the presence of which a TraR-p65 fusion protein activates expression from a minimal promoter fused to a TraR-specific operator sequence. In certain embodiments, the quorum-sensing molecule can be butyrolactone SCB1 (racemic 2-(1'-hydroxy-6-methylheptyl)-3-(hydroxymethyl)-butanolide) in a system based on the Streptomyces coelicolor A3(2) ScbR repressor, which binds to its cognate operator OScbR in the absence of SCB1. In certain embodiments, the quorum sensing molecule can be a homoserine-derived inducer used in the RTI system, in which the Pseudomonas aeruginosa quorum sensing repressors RhlR and LasR are fused to the SV40 T antigen nuclear localization sequence and herpes simplex VP16 domain to activate promoters containing specific operator sequences (las boxes).

[0138] In certain embodiments, the inducer molecule that regulates the allosteric protein used in the RTI system of the present disclosure can be, but is not limited to, coumarate, isopropyl-β-D-galactopyranoside (IPTG), macrolides, 6-hydroxynicotine, doxycycline, streptogramins, NADH, and tetracycline.

[0139] In certain embodiments, the intracellular receptor used in the RTI system of the present disclosure can be a cytoplasmic or nuclear receptor. In certain embodiments, the RTI system of the present disclosure can utilize the use of small molecules to release transcription factors from protein sequestration and inhibition. In certain embodiments, the RTI 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 to the nucleus, and activate a selected promoter. In certain embodiments, mutant receptors are used, which can be regulated by synthetic steroid analogs to avoid crosstalk with endogenous steroid hormones. In certain embodiments, the receptor can be a 4-hydroxytamoxifen-responsive estrogen receptor variant or a RU486-inducible progesterone receptor variant. In certain embodiments, a rosiglitazone-responsive transcriptional switch derived from a nuclear receptor based on the human nuclear peroxisome proliferator-activated receptor gamma (PPARγ) can be used in the RTI system of the present disclosure. In certain 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 several repeats of the Gal4 response element.

[0140] In certain embodiments, the RTI systems disclosed herein may utilize chemically induced dimerization of a DNA-binding protein and a transcriptional activator for activation of a minimal core promoter fused to a cognate operator. In certain embodiments, the RTI systems disclosed herein may utilize rapamycin-regulated dimerization of FKBP and FRB. In this system, FRB is fused to the 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 certain embodiments, the FKBP may be mutated. In certain embodiments, the RTI systems disclosed herein may utilize the bacterial gyrase B subunit (GyrB), which dimerizes in the presence of the antibiotic coumermycin and dissociates with novobiocin.

[0141] In certain embodiments, the RTI system of the present disclosure can be used for regulated siRNA expression. In certain embodiments, the regulated siRNA expression system can be a tetracycline, a macrolide, or an OFF-type and ON-type QuoRex system. In certain embodiments, the RTI system can use a Xenopus terminal oligopyrimidine element (TOP), which blocks translation initiation by forming a hairpin structure in the 5' untranslated region.

[0142] In certain embodiments, the RTI systems described in this disclosure may utilize gas-phase regulated expression, such as the acetaldehyde-inducible regulation (AIR) system. The AIR system employs 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 gaseous or liquid acetaldehyde.

[0143] In certain embodiments, the RTI system of the present disclosure may utilize a Tet-On or Tet-Off system, in which expression of one or more SOIs can be regulated by tetracycline or its analog, doxycycline.

[0144] In certain embodiments, the RTI systems of the present disclosure may utilize PIP-on or PIP-off systems, in which expression of the SOI may be regulated by, for example, pristinamycin, tetracycline, and / or erythromycin.

[0145] 6. Preparation and Use of TI Host Cells The subject matter disclosed herein relates to a method for targeted integration of an exogenous nucleotide sequence into a host cell. In certain embodiments, the method involves integrating an exogenous nucleotide sequence into a host cell to create a host cell suitable for subsequent targeted integration of a SOI. In certain embodiments, the method involves recombinase-mediated recombination. In certain embodiments, the method involves homologous recombination, HDR, and / or NHEJ.

[0146] 6.1 Preparation of TI host cells using recombinase-mediated recombination In certain embodiments, the present disclosure provides a method for preparing a TI host cell for expressing a polypeptide of interest, the method comprising: a) providing a TI host cell comprising an exogenous nucleotide sequence integrated into a site within a locus in the host cell genome, the locus being at least about 90% homologous to SEQ ID NOs: 1-7, the exogenous nucleotide sequence comprising two RRSs flanking at least one first selectable marker; b) introducing into the cell provided in a) a vector comprising at least one exogenous SOI and two RRSs that match the two RRSs on the integrated exogenous nucleotide sequence and that are flanked by at least one second selectable marker; c) introducing a recombinase, wherein the recombinase recognizes the RRSs; and d) isolating the TI host cell that expresses the polypeptide of interest by selecting for TI cells that express the second selectable marker.

[0147] In certain embodiments, the present disclosure provides a method for preparing a TI host cell for expressing a polypeptide of interest, the method comprising: a) providing a TI host cell comprising an exogenous nucleotide sequence integrated into a site within an endogenous gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, XP_003512331.2, and sequences at least about 90% homologous thereto, wherein the exogenous nucleotide sequence comprises two RRSs flanking at least one first selectable marker; b) introducing into the cell provided in a) a vector comprising at least one exogenous SOI and two RRSs that match the two RRSs on the integrated exogenous nucleotide sequence and that are flanked by at least one second selectable marker; c) introducing a recombinase, wherein the recombinase recognizes the RRSs; and d) isolating the TI host cell that expresses the polypeptide of interest by selecting for TI cells that express the second selectable marker.

[0148] In certain embodiments, the present disclosure provides a method for preparing a TI host cell for expressing a polypeptide of interest, the method comprising: a) providing a TI host cell comprising an exogenous nucleotide sequence integrated into a site within a locus in the TI host cell genome, the locus being at least about 90% homologous to SEQ ID NOs: 1-7, the exogenous nucleotide sequence comprising a first DNA cassette comprising two heterologous RRSs flanked by at least one first selectable marker; b) introducing into the cell provided in a) a vector comprising a second DNA cassette, the second DNA cassette matching the two RRSs on the integrated exogenous nucleotide sequence and comprising at least one exogenous SOI and two heterologous RRSs flanked by at least one second selectable marker; c) introducing a recombinase, which recognizes the RRSs and performs a single RMCE; and d) isolating the TI host cell expressing the polypeptide of interest by selecting for TI cells expressing the second selectable marker.

[0149] In certain embodiments, the disclosure provides a method of preparing a TI host cell for expressing a polypeptide of interest, the method comprising: a) providing a TI host cell comprising an exogenous nucleotide sequence integrated into a site within an endogenous gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, XP_003512331.2, and sequences at least about 90% homologous thereto, wherein the exogenous nucleotide sequence comprises two heterospecific nucleotide sequences flanking at least one first selectable marker; b) introducing into the cells provided in a) a vector containing a second DNA cassette containing two heterospecific RRSs that match the two RRSs on the integrated exogenous nucleotide sequence and are flanked by at least one exogenous SOI and at least one second selection marker; c) introducing a recombinase, which recognizes the RRSs and performs a single RMCE; and d) isolating TI host cells that express the polypeptide of interest by selecting TI cells that express the second selection marker.

[0150] In certain embodiments, the disclosure provides methods for preparing a TI host cell for expressing first and second polypeptides of interest (wherein the first and second polypeptides can be the same or different), the methods comprising: a) providing a TI host cell comprising an exogenous nucleotide sequence integrated into a site within a locus of a host cell genome, the locus being at least about 90% homologous to SEQ ID NOs: 1-7, the exogenous nucleotide sequence comprising first and second RRSs flanking at least one first selectable marker, and a third RRS located between the first and second RRSs, wherein all RRSs are heterospecific; and b) generating a TI host cell comprising the integrated exogenous nucleotide sequence in the cell provided in a). The method includes: (a) introducing a first vector containing two RRSs that match the first and third RRSs on the nucleotide sequence and are flanked by at least one first exogenous SOI and at least one second selectable marker; (b) introducing into the cells provided in (a) a second vector containing two RRSs that match the second and third RRSs on the integrating exogenous nucleotide sequence and are flanked by at least one second exogenous SOI; (c) introducing into the cells provided in (a) a second vector containing two RRSs that match the second and third RRSs on the integrating exogenous nucleotide sequence and are flanked by at least one second exogenous SOI; (d) introducing one or more recombinases that recognize the RRSs; and (e) isolating TI host cells that express the first and second polypeptides of interest by selecting for TI cells that express the second selectable marker. In certain embodiments, rather than having the entire selectable marker on the first vector, the first vector contains a promoter sequence operably linked to the ATG codon located upstream of the first SOI and adjacent downstream of the RRS, and the second vector contains a selectable marker lacking an ATG transcription start codon upstream of the RRS and adjacent downstream of the second SOI.

[0151] In certain embodiments, the present disclosure provides a method for preparing a TI host cell for expressing first and second polypeptides of interest (wherein the first and second polypeptides can be the same or different), the method comprising: a) providing a TI host cell comprising an exogenous nucleotide sequence integrated into a site within an endogenous gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, XP_003512331.2, and sequences at least about 90% homologous thereto, wherein the exogenous nucleotide sequence comprises first and second RRSs flanking at least one first selectable marker, and a third RRS located between the first and second RRSs, and wherein all RRSs are integrated into the TI host cell; b) introducing into the cells provided in a) a first vector comprising two RRSs that match the first and third RRSs on the integrating exogenous nucleotide sequence and are flanked by at least one first exogenous SOI and at least one second selectable marker; c) introducing into the cells provided in a) a second vector comprising two RRSs that match the second and third RRSs on the integrating exogenous nucleotide sequence and are flanked by at least one second exogenous SOI; d) introducing one or more recombinases, wherein the one or more recombinases recognize the RRSs; and e) isolating TI host cells that express the first and second polypeptides of interest by selecting for TI cells that express the second selectable marker. In certain embodiments, rather than having the entire selectable marker on a first vector, the first vector comprises a promoter sequence operably linked to the codon ATG located upstream of a first SOI and adjacent downstream of an RRS, and the second vector comprises a selectable marker lacking the ATG transcription start codon upstream of the RRS and adjacent downstream of a second SOI.

[0152] In certain embodiments, the present disclosure provides methods for preparing a TI host cell for expressing first and second polypeptides of interest (wherein the first and second polypeptides can be the same or different), the method comprising: a) providing a TI host cell comprising an exogenous nucleotide sequence integrated into a site within a locus of the host cell genome, the locus being at least about 90% homologous to SEQ ID NOs: 1-7, the exogenous nucleotide sequence comprising a first DNA cassette comprising first and second RRSs flanking at least one first selectable marker, and a third RRS located between the first and second RRSs, wherein all three RRSs are heterospecific; b) introducing into the cell provided in a) a first vector comprising the second DNA cassette; The cassette contains two heterospecific RRSs that match the first and third RRSs of the first DNA cassette and are flanked by at least one first exogenous SOI and at least one second selection marker; c) introducing a second vector containing a third DNA cassette into the cells provided in a), wherein the third DNA cassette contains two heterospecific RRSs that match the second and third RRSs of the first DNA cassette and are flanked by at least one second exogenous SOI; d) introducing one or more recombinases, which recognize the RRSs and perform two rounds of RMCE; and e) isolating TI host cells that express the first and second polypeptides of interest by selecting TI cells that express the second selection marker. In certain embodiments, rather than having the entire selectable marker on a first vector, the first vector comprises a promoter sequence operably linked to the codon ATG located upstream of a first SOI and adjacent downstream of an RRS, and the second vector comprises a selectable marker lacking the ATG transcription start codon upstream of the RRS and adjacent downstream of a second SOI.

[0153] In certain embodiments, the present disclosure provides a method of preparing a TI host cell for expressing first and second polypeptides of interest (wherein the first and second polypeptides can be the same or different), the method comprising: a) providing a TI host cell comprising an exogenous nucleotide sequence integrated into a site within an endogenous gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, XP_003512331.2, and sequences at least about 90% homologous thereto, wherein the exogenous nucleotide sequence comprises a first DNA cassette comprising first and second RRSs flanking at least one first selectable marker, and a third RRS positioned between the first and second RRSs, wherein all three RRSs are heterospecific; and b) providing a TI host cell comprising an exogenous nucleotide sequence integrated into a site within an endogenous gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, XP_003512331.2, and sequences at least about 90% homologous thereto, wherein the exogenous nucleotide sequence comprises a first DNA cassette comprising first and second RRSs flanking at least one first selectable marker, and a third RRS positioned between the first and second RRSs, wherein all three RRSs are heterospecific; c) introducing into the cell a first vector comprising a second DNA cassette, wherein the second DNA cassette comprises two heterospecific RRSs that match the first and third RRSs of the first DNA cassette and are flanked by at least one first exogenous SOI and at least one second selection marker; c) introducing into the cell provided in a) a second vector comprising a third DNA cassette, wherein the third DNA cassette comprises two heterospecific RRSs that match the second and third RRSs of the first DNA cassette and are flanked by at least one second exogenous SOI; d) introducing one or more recombinases, wherein the one or more recombinases recognize the RRSs and perform two rounds of RMCE; and e) isolating TI host cells that express the first and second polypeptides of interest by selecting TI cells that express the second selection marker. In certain embodiments, rather than having the entire selectable marker on a first vector, the first vector comprises a promoter sequence operably linked to the codon ATG located upstream of a first SOI and adjacent downstream of an RRS, and the second vector comprises a selectable marker lacking the ATG transcription start codon upstream of the RRS and adjacent downstream of a second SOI.

[0154] In certain embodiments, the present disclosure provides a method for preparing a TI host cell for expressing a polypeptide of interest, the method comprising: a) providing a TI host cell comprising an exogenous nucleotide sequence integrated into a site within a locus in the host cell genome, the locus being at least about 90% homologous to a sequence selected from SEQ ID NOs: 1-7, the exogenous nucleotide sequence comprising an RRS adjacent to at least one first selection marker; b) introducing into the cell provided in a) a vector comprising at least one exogenous SOI and an RRS that matches the RRS on the integrated exogenous nucleotide sequence and is adjacent to at least one second selection marker; c) introducing a recombinase, wherein the recombinase recognizes the RRS; and d) isolating the TI host cell that expresses the polypeptide of interest by selecting for TI cells that express the second selection marker.

[0155] In certain embodiments, the present disclosure provides a method for preparing a TI host cell for expressing a polypeptide of interest, the method comprising: a) providing a TI host cell comprising an exogenous nucleotide sequence integrated into a site within an endogenous gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, XP_003512331.2, and sequences at least about 90% homologous thereto, wherein the exogenous nucleotide sequence comprises an RRS adjacent to at least one first selection marker; b) introducing into the cell provided in a) a vector that matches the RRS on the integrated exogenous nucleotide sequence and comprises at least one exogenous SOI and an RRS adjacent to at least one second selection marker; c) introducing a recombinase, wherein the recombinase recognizes the RRS; and d) isolating the TI host cell that expresses the polypeptide of interest by selecting for TI cells that express the second selection marker.

[0156] The subject matter disclosed herein also relates to a method for producing a polypeptide of interest, the method comprising: a) providing a TI host cell described herein; and b) culturing the TI host cell of a) under conditions suitable for expressing an SOI and recovering the polypeptide of interest therefrom.

[0157] In certain embodiments, the present disclosure provides a method for preparing a TI host cell suitable for subsequent targeted integration, the method comprising: a) providing a TI host cell comprising an exogenous nucleotide sequence integrated at a site within a locus in the host cell genome, the locus being at least about 90% homologous to a sequence selected from SEQ ID NOs: 1-7, the exogenous nucleotide sequence comprising at least one exogenous SOI and two RRSs flanking at least one first selection marker; b) introducing into the cell provided in a) a vector comprising two RRSs that match the two RRSs on the integrated exogenous nucleotide sequence and that are flanked by at least one second selection marker; c) introducing a recombinase, wherein the recombinase recognizes the RRSs; and d) isolating TI host cells suitable for subsequent targeted integration by selecting for TI cells that express the second selection marker.

[0158] In certain embodiments, the present disclosure provides a method of preparing a TI host cell suitable for subsequent targeted integration, the method comprising: a) providing a TI host cell comprising an exogenous nucleotide sequence integrated into a site within an endogenous gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, XP_003512331.2, and sequences at least about 90% homologous thereto, wherein the exogenous nucleotide sequence is selected from the group consisting of at least one b) introducing into the cells provided in a) a vector containing two RRSs that match the two RRSs on the exogenous nucleotide sequence to be integrated and that are flanked by at least one second selection marker; c) introducing a recombinase, which recombinase recognizes the RRSs; and d) isolating TI host cells suitable for subsequent targeted integration by selecting for TI cells that express the second selection marker.

[0159] In certain embodiments, the present disclosure provides methods for preparing a TI host cell suitable for subsequent targeted integration, the methods comprising: a) providing a TI host cell comprising an exogenous nucleotide sequence integrated at a site within a locus in the host cell genome, the locus being at least about 90% homologous to a sequence selected from SEQ ID NOs: 1-7, the exogenous nucleotide sequence comprising first and second RRSs flanked by at least one exogenous SOI and at least one first selectable marker; b) introducing into the cell provided in a) a vector comprising three RRSs; a first RRS of the vector matches a first RRS on the integrating exogenous nucleotide sequence, a second RRS of the vector matches a second RRS on the integrating exogenous nucleotide sequence, and at least one second selection marker is located between the first and second RRS; c) introducing a recombinase, which recognizes the first and second RRS on both the vector and the integrating exogenous nucleotide sequence; and d) isolating TI host cells suitable for subsequent targeted integration by selecting for TI host cells that express the second selection marker.

[0160] In certain embodiments, the present disclosure provides a method for preparing a TI host cell suitable for subsequent targeted integration, the method comprising: a) providing a TI host cell comprising an exogenous nucleotide sequence integrated into a site within an endogenous gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, XP_003512331.2, and sequences at least about 90% homologous thereto, wherein the exogenous nucleotide sequence comprises first and second RRSs flanked by at least one exogenous SOI and at least one first selectable marker; and b) providing a TI host cell comprising the exogenous nucleotide sequence provided in a). a) introducing into the cell to be integrated a vector containing three RRSs, wherein a first RRS of the vector matches a first RRS on the exogenous nucleotide sequence to be integrated and a second RRS of the vector matches a second RRS on the exogenous nucleotide sequence to be integrated, with at least one second selection marker located between the first and second RRSs; b) introducing a recombinase, wherein the recombinase recognizes the first and second RRSs on both the vector and the exogenous nucleotide sequence to be integrated; and c) isolating TI host cells suitable for subsequent targeted integration by selecting for TI host cells that express the second selection marker.

[0161] 6.2 Methods for Targeted Modification of Host Cells Using Homologous Recombination, HDR, or NHEJ In certain embodiments, the present disclosure provides a method for preparing a TI host cell for expressing a polypeptide of interest, the method comprising: (a) providing a TI host cell comprising a locus in the host cell genome, the locus being at least about 90% identical to SEQ ID NOs: 1-7; (b) introducing a vector into the TI host cell, the vector comprising a nucleotide sequence at least 50% identical to a sequence selected from SEQ ID NOs: 1-7 flanking a DNA cassette, the DNA cassette comprising at least one selectable marker and at least one exogenous SOI; and (c) selecting the selectable marker and isolating a TI host cell having the SOI integrated into the genomic locus to express the polypeptide of interest. In certain embodiments, the DNA cassette of the vector further comprises at least one selectable marker and at least one exogenous SOI flanked by two RRSs.

[0162] In certain embodiments, the present disclosure provides a method for preparing a TI host cell for expressing a polypeptide of interest, the method comprising: (a) providing a TI host cell comprising a locus in the host cell genome, the locus being at least about 90% identical to a sequence selected from SEQ ID NOs: 1-7; (b) introducing a polynucleotide into the host cell, the polynucleotide comprising a nucleotide sequence at least 50% identical to a sequence selected from SEQ ID NOs: 1-7 flanked by a DNA cassette, the DNA cassette comprising at least one selectable marker and at least one exogenous SOI; and (c) selecting the selectable marker and isolating a TI host cell having the SOI integrated into the genomic locus to express the polypeptide of interest. In certain embodiments, the DNA cassette of the vector further comprises at least one selectable marker and at least one exogenous SOI flanked by two RRSs.

[0163] In certain embodiments, homologous recombination is promoted by an integrating vector. In certain embodiments, the vector is selected from the group consisting of an adenoviral vector, an adeno-associated viral vector, a lentiviral vector, a retroviral vector, an integrating phage vector, a non-viral vector, a transposon and / or transposase vector, an integrase substrate, and a plasmid. In certain embodiments, the transposon can be a PiggyBac (PB) transposon system.

[0164] In certain embodiments, integration is facilitated by an exogenous nuclease, hi certain embodiments, the exogenous nuclease is selected from the group consisting of zinc finger nucleases (ZFNs), ZFN dimers, transcription activator-like effector nucleases (TALENs), TAL effector domain fusion proteins, RNA-guided DNA endonucleases, engineered meganucleases, and clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) endonucleases.

[0165] In certain embodiments, the present disclosure provides a method for preparing a TI host cell suitable for subsequent targeted integration, the method comprising: a) providing a TI host cell comprising a locus in the host cell genome, the locus being at least about 90% homologous to a sequence selected from SEQ ID NOs: 1-7; b) introducing a vector into the TI host cell, the vector comprising a nucleotide sequence at least 50% homologous to a sequence selected from SEQ ID NOs: 1-7 flanking a DNA cassette, the DNA cassette comprising at least one selectable marker flanked by two RRSs; and c) selecting for the selectable marker and isolating a TI host cell suitable for subsequent targeted integration.

[0166] In certain embodiments, the present disclosure provides a method for preparing a TI host cell suitable for subsequent targeted integration, the method comprising: a) providing a TI host cell comprising a locus in the host cell genome, the locus being at least about 90% homologous to a sequence selected from SEQ ID NOs: 1-7; b) introducing a polynucleotide into the TI host cell, the polynucleotide comprising a nucleotide sequence at least 50% homologous to a sequence selected from SEQ ID NOs: 1-7 flanking a DNA cassette, the DNA cassette comprising at least one selectable marker flanked by two RRSs; and c) selecting the selectable marker and isolating a TI host cell suitable for subsequent targeted integration.

[0167] In certain embodiments, the present disclosure provides a method for preparing a TI host cell suitable for subsequent targeted integration, the method comprising: a) providing a TI host cell comprising a locus in the host cell genome, the locus being at least about 90% homologous to a sequence selected from SEQ ID NOs: 1-7; b) introducing a vector into the host cell, the vector comprising a nucleotide sequence at least 50% homologous to a sequence selected from SEQ ID NOs: 1-7 flanking a DNA cassette, the DNA cassette comprising three RRSs, the third RRS and at least one selectable marker being located between the first and second RRSs; and c) selecting the selectable markers and isolating a TI host cell suitable for subsequent targeted integration.

[0168] In certain embodiments, the present disclosure provides a method for preparing a TI host cell suitable for subsequent targeted integration, the method comprising: a) providing a TI host cell comprising a locus in the host cell genome, the locus being at least about 90% homologous to a sequence selected from SEQ ID NOs: 1-7; b) introducing a polynucleotide into the host cell, the polynucleotide comprising a nucleotide sequence at least 50% homologous to a sequence selected from SEQ ID NOs: 1-7 flanking a DNA cassette, the DNA cassette comprising three RRSs, the third RRS and at least one selectable marker being located between the first and second RRSs; and c) selecting the selectable markers and isolating a TI host cell suitable for subsequent targeted integration.

[0169] In certain embodiments, the present disclosure provides a method for preparing a TI host cell that expresses at least one polypeptide of interest, the method comprising: a) providing a TI host cell that includes at least one exogenous nucleotide sequence integrated into a site within one or more loci in the TI host cell genome, the one or more loci being at least about 90% homologous to a sequence selected from SEQ ID NOs: 1-7, wherein the at least one exogenous nucleotide sequence includes two RRSs flanking at least one first selectable marker; b) introducing into the cell provided in a) a vector that matches the two RRSs on the integrated exogenous nucleotide sequence and includes at least one exogenous SOI and two RRSs flanking at least one second selectable marker; c) isolating the TI host cell that expresses at least one polypeptide of interest by introducing a recombinase or a nucleic acid encoding a recombinase, wherein the recombinase recognizes the RRSs, and selecting for TI cells that express the second selectable marker.

[0170] In certain embodiments, the present disclosure provides methods for preparing a TI host cell that expresses at least one first and second polypeptide of interest (wherein the first and second polypeptides can be the same or different), the method comprising: a) providing a TI host cell that includes at least one exogenous nucleotide sequence integrated into a site within one or more loci of the host cell genome, the one or more loci being at least about 90% homologous to a sequence selected from SEQ ID NOs: 1-7, the exogenous nucleotide sequence comprising first and second RRSs flanking at least one first selectable marker, and a third RRS located between the first and second RRSs, wherein all RRSs are heterospecific; and b) providing a TI host cell that includes at least one integrated exogenous nucleotide sequence into the cell provided in a). c) introducing into the cells provided in a) a second vector comprising two RRSs that match the second and third RRSs on the at least one integrated exogenous nucleotide sequence and that are flanked by at least one second exogenous SOI; d) introducing one or more recombinases, or one or more nucleic acids encoding one or more recombinases, wherein the one or more recombinases recognize the RRSs; and e) isolating TI host cells that express at least one first and second polypeptide of interest by selecting for TI cells that express the second selectable marker. In certain embodiments, rather than having the entire selectable marker on a first vector, the first vector comprises a promoter sequence operably linked to the codon ATG located upstream of a first SOI and adjacent downstream of an RRS, and the second vector comprises a selectable marker lacking the ATG transcription start codon upstream of the RRS and adjacent downstream of a second SOI.

[0171] In certain embodiments, the present disclosure provides a method for preparing a TI host cell that expresses a polypeptide of interest, the method comprising: a) providing a TI host cell that includes at least one exogenous nucleotide sequence integrated into a site within one or more loci in the TI host cell genome, the one or more loci being at least about 90% homologous to a sequence selected from SEQ ID NOs: 1-7, and the exogenous nucleotide sequence including one or more RRSs; b) introducing into the cell provided in a) a vector that includes one or more RRSs that match the one or more RRSs on the integrated exogenous nucleotide sequence and are adjacent to at least one exogenous SOI that is operably linked to a regulatable promoter; c) introducing a recombinase or a nucleic acid encoding a recombinase, wherein the recombinase recognizes the RRS; and d) isolating the TI host cell that expresses the polypeptide of interest by selecting for TI cells that express the exogenous SOI in the presence of an inducer.

[0172] In certain embodiments, the present disclosure provides a method for expressing a polypeptide of interest, the method comprising: a) providing a host cell comprising at least one exogenous SOI flanked by two RRSs and a regulatable promoter integrated within a locus in the host cell genome, the locus being at least about 90% homologous to a sequence selected from SEQ ID NOs: 1-7; and b) culturing the cell under conditions suitable for expressing the SOI and recovering the polypeptide of interest therefrom.

[0173] In certain embodiments, the present disclosure provides a method for preparing a TI host cell that expresses first and second polypeptides of interest (wherein the first and second polypeptides can be the same or different), the method comprising: a) providing a TI host cell that includes an exogenous nucleotide sequence integrated into a site within a locus of a host cell genome, the locus being at least about 90% homologous to a sequence selected from SEQ ID NOs: 1-7, the exogenous nucleotide sequence including a first RRS, a second RRS, and a third RRS located between the first and second RRS, wherein all RRSs are heterospecific; and b) providing in the cell provided in a) a nucleotide sequence that corresponds to the first and third RRSs on the integrated exogenous nucleotide sequence and is operably linked to a regulatable promoter. The method includes: introducing a first vector containing two RRSs flanking at least one first exogenous SOI to which the exogenous nucleotide sequence is ligated; c) introducing into the cells provided in a) a second vector containing two RRSs matching the second and third RRSs on the integrating exogenous nucleotide sequence and flanking at least one second SOI operably linked to a regulatable promoter; d) introducing one or more recombinases, or one or more nucleic acids encoding one or more recombinases, wherein the one or more recombinases recognize the RRSs; and e) isolating TI host cells expressing the polypeptide of interest by selecting TI cells expressing at least the first and second exogenous SOIs in the presence of an inducer. In certain embodiments, rather than having the entire selectable marker on the first vector, the first vector contains a promoter sequence operably linked to an ATG codon located upstream of the first SOI and adjacent downstream of the RRS, and the second vector contains a selectable marker lacking an ATG transcription start codon upstream of the RRS and adjacent downstream of the second SOI.

[0174] 7. Products The host cells of the present disclosure can be used to express any molecule of interest, e.g., a polypeptide of interest. In certain embodiments, the host cells of the present disclosure can be used to express a polypeptide, e.g., a mammalian polypeptide. Non-limiting examples of such polypeptides include hormones, receptors, fusion proteins, regulatory factors, growth factors, complement system factors, enzymes, clotting factors, anticoagulants, kinases, cytokines, CD proteins, interleukins, therapeutic proteins, diagnostic proteins, and antibodies. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a therapeutic antibody. In some embodiments, the antibody is a diagnostic antibody. In some embodiments, the antibody is a human antibody. In some embodiments, the antibody is a humanized antibody.

[0175] In certain embodiments, exemplary polypeptides encompassed within the definition herein include mammalian polypeptides such as, for example, renin, growth hormones, including human growth hormone and bovine growth hormone, growth hormone releasing factor, parathyroid hormone, thyroid stimulating hormone, lipoproteins, alpha-1-antitrypsin, insulin A chain, insulin B chain, proinsulin, follicle stimulating hormone, calcitonin, luteinizing hormone, glucagon, leptin, clotting factors, e.g., Factor VIIIC, Factor IX, tissue factor, and von Willebrand factor. anticoagulants such as protein C, atrial natriuretic factor, pulmonary surfactant, plasminogen activators such as urokinase or human urinary or tissue-type plasminogen activator (t-PA), bombesin, thrombin, hematopoietic growth factors, tumor necrosis factor-alpha and -beta, tumor necrosis factor receptors such as death receptor 5 and CD120, TNF-related apoptosis-inducing ligand (TRAIL), B-cell maturation antigen (BCMA), B-lymphocyte stimulating factor (BLyS), proliferation-inducing ligand (APRIL), endogenous factor (EF), endogenous factor (ETF ... Cephalinase, RANTES (regulated upon activation and normally expressed and secreted by T cells), human macrophage inflammatory protein (MIP-1-alpha), serum albumins such as human serum albumin, microbial proteins such as Müllerian inhibitory substance, relaxin A chain, relaxin B chain, prorelaxin, mouse gonadotropin-related peptide, beta-lactamase, DNase, IgE, cytotoxic T lymphocyte-associated antigen (CTLA) such as CTLA-4, inhibin, activin, platelet-derived endothelial growth factor (PD-ECGF), vascular endothelial growth factor family proteins (e.g., VEGF-A, VEGF-B, VEGF-C, VEGF-D, and P1GF), platelet-derived growth factor (PDGF) family proteins (e.g., PDGF-A, PDGF-B, PDGF-C, PDGF-D, and dimers thereof), fibroblast growth factor (FGF) family proteins such as aFGF, bFGF, FGF4, and FGF9, epidermal growth factor (EGF), hormone or growth factor receptors, e.g., VEGF receptor(s) (e.g., VEGFR1, VEGFR2, and VEGFR3),Epidermal growth factor (EGF) receptor(s) (e.g., ErbB1, ErbB2, ErbB3, and ErbB4 receptors), platelet-derived growth factor (PDGF) receptor(s) (e.g., PDGFR-α and PDGFR-β), and fibroblast growth factor receptor(s), TIE ligands (angiopoietin, ANGPT1, ANGPT2), angiopoietin receptors, such as TIE1 and TIE2, protein A or D, rheumatoid factor, neurotrophic factors, such as bone-derived neurotrophic factor (BDNF), neurotrophin-3, -4, - nerve growth factors such as NT-3, NT-4, NT-5, or NT-6, or NGF-b; transforming growth factors (TGFs), such as TGF-alpha and TGF-beta, including TGF-β1, TGF-β2, TGF-β3, TGF-β4, or TGF-β5; insulin-like growth factors-I and -II (IGF-I and IGF-II), des(1-3)-IGF-I (brain IGF-I); insulin-like growth factor binding proteins (IGFBPs); CD proteins, such as CD3, CD4, CD8, and CD19. and CD20, erythropoietin, bone morphogenetic factors, immunotoxins, bone morphogenetic proteins (BMPs), chemokines such as CXCL12 and CXCR4, interferons such as interferon-alpha, -beta, and -gamma, colony stimulating factors (CSFs) such as M-CSF, GM-CSF, and G-CSF, cytokines such as interleukins (ILs) such as IL-1 to IL-10, midkine, superoxide dismutase, T cell receptors, surface membrane proteins, decay accelerating factors, viral antigens such as A Parts of the IDS envelope, such as transport proteins, homing receptors, addressins, regulatory proteins, integrins, such as CD11a, CD11b, CD11c, CD18, ICAM, VLA-4 and VCAM, ephrins, Bv8, delta-like ligand 4 (DLL4), Del-1, BMP9, BMP10, follistatin, hepatocyte growth factor (HGF) / scatter factor (SF), Alk1, Robo4, ESM1, perlecan, EGF-like domain multiple 7 (EGFL7), CTGF and its family members, thrombospondins, such asThrombospondin 1 and thrombospondin 2, collagens such as collagen IV and collagen XVIII, neuropilins such as NRP1 and NRP2, pleiotrophin (PTN), progranulin, proliferin, Notch proteins such as Notch1 and Notch4, semaphorins such as Sema3A, Sema3C, and Sema3F, tumor-associated antigens such as CA125 (ovarian cancer antigen), immunoadhesins, and fragments and / or variants of any of the above-listed polypeptides, as well as antibodies, including antibody fragments, that bind to one or more proteins, including, for example, any of the above-listed proteins.

[0176] In certain embodiments, the polypeptide of interest is a bispecific, trispecific, or multispecific polypeptide, such as a bispecific antibody. Various molecular formats of multispecific antibodies are known in the art and are included herein (see, for example, Spiess et al., Mol Immunol 67 (2015) 95-106). A specific type of multispecific antibody further included herein is a bispecific antibody designed to simultaneously bind to a surface antigen on a target cell, such as a tumor cell, and to an activation invariant component of the T cell receptor (TCR) complex, such as CD3, for the purpose of retargeting T cells to kill the target cell. Other examples of bispecific antibody formats include so-called "BiTE" (bispecific T cell derivative) molecules in which two scFv molecules are fused by a flexible linker (see, for example, WO2004 / 106381, WO2005 / 061547, WO2007 / 042261, and WO2008 / 119567; Nagorsen and Bauerle, Exp Cell Res 317, 1255-1260 (2011)), diabodies (Holliger et al., Prot Eng 9, 299-305 (1996)) and their derivatives, such as tandem diabodies ("TandAb", Kipriyanov et al., J Mol Biol 293, 41-56 (1999)), "DART" (dual affinity retargeting) molecules (Johnson et al., J Mol Biol 399, 436-449 (2010)), which are based on the diabody format but feature a C-terminal disulfide bridge for further stabilization, as well as so-called triomab, a fully hybrid mouse / rat IgG molecule (reviewed in Seimetz et al., Cancer Treat Rev 36, 458-467 (2010)). Specific T cell bispecific antibody formats included herein are described in WO2013 / 026833, WO2013 / 026839, WO2016 / 020309, Bacac et al., Oncoimmunology 5(8) (2016) e1203498.

[0177] In certain embodiments, the host cells of the present disclosure may be used to express chaperones, protein-modifying enzymes, shRNAs, gRNAs, or other proteins or peptides, either constitutively or regulated, while expressing a therapeutic protein or molecule of interest.

[0178] In some embodiments, the polypeptide expressed by a host cell of the disclosure is any protein, e.g., 8MPI, 8MP2, 8MP38 (GDF10), 8MP4, 8MP6, 8MP8, CSFI (M-CSF), CSF2 (GM-CSF), CSF3 (G-CSF), EPO, FGF1 (αFGF), FGF2 (βFGF), FGF3 (int-2), FGF4 (HST), FGF5, FGF6 (HST-2), FGF7 (KGF), FGF9, FGF10, FGF11, FGF12, FGF12B, FGF14, FGF16, FGF17 , FGF19, FGF20, FGF21, FGF23, IGF1, IGF2, IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFN81, IFNG, IFNWI, FEL1, FEL1(epsilon), FEL1(zeta), IL1A, IL1B, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL11, IL12A, IL12B, IL13, IL14, IL15, IL16, IL17, IL17B, IL18, IL19, IL20, IL22, IL23, IL24, IL25, IL26 , IL27, IL28A, IL28B, IL29, IL30, PDGFA, PDGFB, TGFA, TGFB1, TGFB2, TGFBb3, LTA (TNF-β), LTB, TNF (TNF-α), TNFSF4 (OX40 ligand), TNFSF5 (CD40 ligand), TNFSF6 (FasL), TNFSF7 (CD27 ligand), TNFSF8 (CD30 ligand), TNFSF9 (4-1BB ligand), TNFSF10 (TRAIL), TNFSF11 (TRANCE), TNFSF12 (APO3L), TNFSF13 (April) , TNFSF13B, TNFSF14(HVEM-L), TNFSF15(VEGI), TNFSF18, HGF(VEGFD), VEGF, VEGFB, VEGFC, IL1R1, IL1R2, IL1RL1, IL1RL2, IL2RA, IL2RB, IL2RG, IL3RA , IL4R, IL5RA, IL6R, IL7R, IL8RA, IL8RB, IL9R, IL10RA, IL10RB, IL11RA, IL12RB1, IL12RB2, IL13RA1, IL13RA2, IL15RA, IL17R, IL18R1, IL20RA, IL21R,It may bind to or interact with proteins including, but not limited to, cytokines, cytokine-related proteins, and cytokine receptors selected from the group consisting of IL22R, IL1HY1, IL1RAP, IL1RAPL1, IL1RAPL2, IL1RN, IL6ST, IL18BP, IL18RAP, IL22RA2, AIF1, HGF, LEP (leptin), PTN, and THPO.k.

[0179] In some embodiments, the polypeptide expressed by a host cell of the disclosure is CCLI(1-309), CCL2 (MCP-1 / MCAF), CCL3 (MIP-Iα), CCL4 (MIP-Iβ), CCL5 (RANTES), CCL7 (MCP-3), CCL8 (mcp-2), CCL11 (eotaxin), CCL13 (MCP-4), CCL15 (MIP-Iδ), CCL16 (HCC-4), CCL17 (TARC), CCL18 (PARC), CCL19 (MDP-3b), CCL20 (MIP-3α), CCL21 (SLC / Exodus-2), CCL22 (MDC / STC-1), CCL23 (MPIF-1), CCL24 (MPIF-2 / Eotaxin-2), CC15 (CC16), CC17 (TARC), CCL18 (PARC), CCL19 (MDP-3b), CCL20 (MIP-3α), CCL21 (SLC / Exodus-2), CCL22 (MDC / STC-1), CCL23 (MPIF-1), CCL24 (MPIF-2 / Eotaxin-2), CC18 (CC19), CC29 (CC29), CC30 (CC30), CC40 (CC40), CC50 (CC50), CC60 (CC60), CC70 (CC70), CC80 (CC80), CC90 (CC90), CC10 (CC10), CC11 (CC11), CC12 (CC12), CC13 (CC14), CC14 (CC15), CC15 (CC16), CC16 (CC17), CC18 (CC19), CC29 (CC19), CC20 (CC19), CC21 (CC19), CC22 (CC19), CC23 (CC19), CC24 (CC19), CC25 (CC19), CC26 (CC1 L25(TECK), CCL26(eotaxin-3), CCL27(CTACK / ILC), CCL28, CXCLI(GROI), CXCL2(GR02), CXCL 3(GR03), CXCL5(ENA-78), CXCL6(GCP-2), CXCL9(MIG), CXCL10(IP10), CXCL11(1-TAC), CXCL1 2(SDFI), CXCL13, CXCL14, CXCL16, PF4(CXCL4), PPBP(CXCL7), CX3CL1(SCYDI), SCYEI, nhotactin), XCL2 (SCM-Iβ), BLRI (MDR15), CCBP2 (D6 / JAB61), CCRI (CKRI / HM145), CCR2 (mcp-IRB IRA), CCR3(CKR3 / CMKBR3), CCR4, CCR5(CMKBR5 / ChemR13), CCR6(CMKBR6 / CKR-L3 / STRL22 / DRY6), CCR7(CKR7 / EBI I), CCR8 (CMKBR8 / TER1 / CKR-L1), CCR9 (GPR-9-6), CCRL1 (VSHK1), CCRL2 (L-CCR), XCR1 (GPR5 / CCXCR1), CMKLR1, CM KOR1(RDC1), CX3CR1(V28), CXCR4, GPR2(CCR10), GPR31, GPR81(FKSG80), CXCR3(GPR9 / CKR-L2), CXCR6(TYMSTR / S TRL33 / Bonzo), HM74, IL8RA (IL8Rα), IL8RB (IL8Rβ), LTB4R (GPR16), TCP10, CKLFSF2, CKLFSF3, CKLFSF4, CKLFSF5,It may bind to or interact with a chemokine, chemokine receptor, or chemokine-related protein selected from the group consisting of CKLFSF6, CKLFSF7, CKLFSF8, BDNF, C5, C5R1, CSF3, GRCC10(C10), EPO, FY(DARC), GDF5, HDF1, HDF1α, DL8, PRL, RGS3, RGS13, SDF2, SLIT2, TLR2, TLR4, TREM1, TREM2, and VHL. In some embodiments, the polypeptide expressed by the host cells of the disclosure is selected from the group consisting of 0772P (CA125, MUC16) (i.e., ovarian cancer antigen), ABCF1, ACVR1, ACVR1B, ACVR2, ACVR2B, ACVRL1, ADORA2A, aggrecan, AGR2, AICDA, AIF1, AIG1, AKAP1, AKAP2, AMH, AMHR2, amyloid beta, ANGPTL, ANGPT2, ANGPTL3, AN GPTL4, ANPEP, APC, APOC1, AR, ASLG659, ASPHD1 (aspartate beta-hydroxylase domain containing 1, LOC253982), AZGP1 (zinc-α-glycoprotein), B7.1, B7.2, BAD, BAFF-R (B cell-activating factor receptor, BLyS receptor 3), BR3, BAG1, BAI1, BCL2, BCL6, BDNF, BLNK, BLRI (MDR15), BMP1, BMP2, BMP3 B (GDF10), BMP4, BMP6, BMP8, BMPR1A, BMPR1B (bone morphogenetic protein receptor type IB), BMPR2, BPAG1 (plectin), BRCA1, brevican, C19orf10 (IL27w), C3, C4A, C5, C5R1, CANT1, CASP1, CASP4, CAV1, CCBP2 (D6 / JAB61), CCL1 (1-309), CCL11 (eotaxin), CCL13 (MCP-4), CCL1 5 (MIP1δ), CCL16 (HCC-4), CCL17 (TARC), CCL18 (PARC), CCL19 (MIP-3β), CCL2 (MCP-1), MCAF, CCL20 (MIP-3α), CCL21 (MTP-2), SLC, Exodus-2, CCL22 (MDC / STC-1), CCL23 (MPIF-1), CCL24 (MPIF-2 / eotaxin-2), CCL25 (TECK), CCL26 (eotaxin-3),CCL27 (CTACK / ILC), CCL28, CCL3 (MTP-Iα), CCL4 (MDP-Iβ), CCL5 (RANTES), CCL7 (MCP-3), CCL8 (mcp-2), CCNA1, CCNA2, CCND1, CCNE1, CCNE2, CCR1 (CKR I / HM145), CCR2(mcp-IRβ / RA), CCR3(CKR / CMKBR3), CCR4, CCR5(CMKBR5 / ChemR13), CCR6(CMKBR6 / CKR-L3 / STRL22 / DRY6), CCR7(CKBR7 / EBI1), CCR8(CM KBR8 / TER1 / CKR-L1), CCR9 (GPR-9-6), CCRL1 (VSHK1), CCRL2 (L-CCR), CD164, CD19, CD1C, CD20, CD200, CD22 (B cell receptor CD22-B isoform), CD24, CD28, CD3, CD37, CD38, CD3E, CD3G, CD3Z, CD4, CD40, CD40L, CD44, CD45RB, CD52, CD69, CD72, CD74, CD79A (CD79α, immunoglobulin-related alpha, B cell-specific protein), CD79B, CDS, CD8 0, CD81, CD83, CD86, CDH1 (E-cadherin), CDH10, CDH12, CDH13, CDH18, CDH19, CDH20, CDH5, CDH7, CDH8, CDH9, CDK2, CDK3, CDK4, CDK5, CDK6, CDK7, CDK9, CD KN1A(p21 / WAF1 / Cip1), CDKN1B(p27 / Kip1), CDKN1C, CDKN2A(P16INK4a), CDKN2B, CDKN2C, CDKN3, CEBPB, CER1, CHGA, CHGB, chitinase, CHST10, CKLFSF2, CK LFSF3, CKLFSF4, CKLFSF5, CKLFSF6, CKLFSF7, CKLFSF8, CLDN3, CLDN7 (claudin-7), CLL-1 (CLEC12A, MICL, and DCAL2), CLN3, CLU (clusterin), CMKLR1, CMKOR1 (RDC1), CNR1, COL18A1, COL1A1, COL4A3, COL6A1, complement factor D, CR2, CRP, CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratoma-derived growth factor), CSFI (M-CSF), CSF2 (GM-CSF),CSF3 (GCSF), CTLA4, CTNNB1 (b-catenin), CTSB (cathepsin B), CX3CL1 (SCYDI), CX3CR1 (V28), CXCL1 (GRO1), CXCL10 (IP-10), CXCL11 (I-TAC / IP-9), CXCL12 (S DF1), CXCL13, CXCL14, CXCL16, CXCL2(GRO2), CXCL3(GRO3), CXCL5(ENA-78 / LIX), CXCL6(GCP-2), CXCL9(MIG), CXCR3(GPR9 / CKR-L2), CXCR4, CXCR5(Bar Kit lymphoma receptor 1, G protein-coupled receptor), CXCR6 (TYMSTR / STRL33 / Bonzo), CYB5, CYC1, CYSLTR1, DAB2IP, DES, DKFZp451J0118, DNCLI, DPP4, E16 (LAT1, SLC7A5), E2F1, ECGF1, EDG1, EFNA1, EFNA3, EFNB2, EGF, EGFR, ELAC2, ENG, ENO1, ENO2, ENO3, EPHB4, EphB2R, EPO, ERBB2 (Her-2), EREG, ERK8, ESR1, ESR2, ETBR (Endo Serine type B receptor), F3 (TF), FADD, FasL, FASN, FCER1A, FCER2, FCGR3A, FcRH1 (Fc receptor-like protein 1), FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain-containing phosphatase anchor protein 1a), SPAP1B, SPAP1C), FGF, FGF1 (αFGF), FGF10, FGF11, FGF12, FGF12B, FGF13, FGF14, FGF16, FGF17, FGF18, FGF19, FGF2 (bFGF), FGF20, FGF21, FGF22, FGF 23, FGF3 (int-2), FGF4 (HST), FGF5, FGF6 (HST-2), FGF7 (KGF), FGF8, FGF9, FGFR, FGFR3, FIGF (VEGFD), FELl (epsilon), FILl (zeta), FLJ12584, FLJ25530, FLRTI (fibronectin), FLT1, FOS, FOSL1 (FRA-1), FY (DARC), GABRP (GABAa), GAGEB1, GAGEC1, GALNAC4S-6ST, GATA3, GDF5, GDNF-Ra1 (GDNF family receptor alpha 1,GFRA1, GDNFR, GDNFRA, RETL1, TRNR1, RET1L, GDNFR-alpha1, GFR-alpha-1), GEDA, GFI1, GGT1, GM-CSF, GNASI, GNRHI, GPR2 (CCR10), GPR19 (G protein-coupled receptor 19, Mm.4787), GPR31, GPR44, GPR54 (KISS1 receptor, KISS1R, GPR54, HOT7T175, AXOR12), GPR81 (FKSG80), GPR172A (G protein-coupled receptor 172A, GPCR41, FLJ11856, D 15Ertd747e), GRCCIO (C10), GRP, GSN (gelsolin), GSTP1, HAVCR2, HDAC4, HDAC5, HDAC7A, HDAC9, HGF, HIF1A, HOP1, histamine and histamine receptor, HLA-A, HLA-DOB (beta subunit of MHC class II molecule (Ia antigen)), HLA-DRA, HM74, HMOXI, HUMCYT2A, ICEBERG, ICOSL, 1D2, IFN-α, IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNB1, IFN-gamma , DFNW1, IGBP1, IGF1, IGF1R, IGF2, IGFBP2, IGFBP3, IGFBP6, IL-l, IL10, IL10RA, IL10RB, IL11, IL11RA, IL-12, IL12A, IL12B, IL12RB1, IL12RB2, IL13 , IL13RA1, IL13RA2, IL14, IL15, IL15RA, IL16, IL17, IL17B, IL17C, IL17R, IL18, IL18BP, IL18R1, IL18RAP, IL19, IL1A, IL1B, ILIF10, IL1F5, IL1F6, I L1F7, IL1F8, IL1F9, IL1HY1, IL1R1, IL1R2, IL1RAP, IL1RAPL1, IL1RAPL2, IL1RL1, IL1RL2, ILIRN, IL2, IL20, IL20Rα, IL21R, IL22, IL-22c, IL22R, IL2 2RA2, IL23, IL24, IL25, IL26, IL27, IL28A, IL28B, IL29, IL2RA, IL2RB, IL2RG, IL3, IL30, IL3RA, IL4, IL4R, IL5, IL5RA, IL6, IL6R, IL6ST (glycoprotein 130),Influenza A, influenza B, EL7, EL7R, EL8, IL8RA, DL8RB, IL8RB, DL9, DL9R, DLK, INHA, INHBA, INSL3, INSL4, IRAK1, IRTA2 (immunoglobulin superfamily receptor translocation associated 2), ERAK2, ITGA1, , ITGA2, ITGA3, ITGA6 (a6 integrin), ITGAV, ITGB3, ITGB4 (b4 integrin), α4β7 and αEβ7 integrin heterodimers, JAG1, JAK1, JAK3, JUN, K6HF, KAI1, KDR, KITLG, KLF5 (GC box BP), KLF6, KLKIO, KLK12, KLK13, KLK14, KLK15, KLK3, KLK4, KLK5, KLK6, KLK9, KRT1, KRT19 (keratin 19), KRT2A, KHTHB6 (hair-specific H-type keratin), LAMAS, LEP ( leptin), LGR5 (leucine-rich repeat-containing G protein-coupled receptor 5, GPR49, GPR67), Lingo-p75, Lingo-Troy, LPS, LTA (TNF-β), LTB, LTB4R (GPR16), LTB4R2, LTBR, ​​LY64 (lymphocyte antigen 64 (RP105), a type I membrane protein of the leucine-rich repeat (LRR) family), Ly6E (lymphocyte antigen 6 complex, locus E, Ly67, RIG-E, SCA-2, TSA-1), Ly6G6D (lymphocyte antigen 6 complex, locus G6D, Ly6-D, MEGT1), L Y6K (lymphocyte antigen 6 complex, locus K, LY6K, HSJ001348, FLJ35226), MACMARCKS, MAG or OMgp, MAP2K7 (c-Jun), MDK, MDP, MIB1, midkine, MEF, MIP-2, MKI67, (Ki-67), MMP2, MMP9, MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin), MS4A1, MSG783 (RNF124, hypothetical protein FLJ20315), MSMB, MT3 (metallothionein-111), MTSS1, MUC1 (mucin), MYC, MY0 88, Napi3b (also known as NaPi2b) (NAPI-3B, NPTIIb, SLC34A2, solute transporter family 34 (sodium phosphate), member 2, type II sodium-dependent phosphate transporter 3b), NCA, NCK2, neurocan, NFKB1, NFKB2, NGFB (NGF), NGFR, NgR-Lingo, NgR-Nogo66 (Nogo), NgR-p75, NgR-Troy, NME1 (NM23A), NOX5, NPPB, NR0B1, NR0B2, NR1D1, NR1D2, NR1H2, NR1H3, NR1H4,NR112, NR113, NR2C1, NR2C2, NR2E1, NR2E3, NR2F1, NR2F2, NR2F6, NR3C1, NR3C2, NR4A1, NR4A2, NR4A3, NR5A1, NR5A2, NR6A1, NRP1, NRP2, NT5E, NTN4, ODZI, OPRD1, OX40, P2RX7, P2X5 (purinergic receptor P2X ligand-gated ion channel 5), PAP, PART1, PATE, PAWR, PCA3, PCNA, PD-L1, PD-L2, PD -1, POGFA, POGFB, PECAM1, PF4 (CXCL4), PGF, PGR, phosphacan, PIAS2, PIK3CG, PLAU (uPA), PLG, PLXDC1, PMEL17 (silver homolog, SILV, D12S53E , PMEL17, SI, SIL), PPBP(CXCL7), PPID, PRI, PRKCQ, PRKDI, PRL, PROC, PROK2, PSAP, PSCAhlg(2700050C12Rik, C530008O16Rik, RIKEN cDNA2700050C12, RIKEN cDNA2700050C12 gene), PTAFR, PTEN, PTGS2 (COX-2), PTN, RAC2 (p21 Rac2), RARB, RET (ret proto-oncogene, MEN2A, HSCR1, MEN2B, MTC1, PTC, CDHF12, Hs.168114, RET51, RET-ELE1), RGSI, RGS13, RGS3, RNF110 (ZNF144), ROBO2, S100A2, SCGB1D2 (lipophilin B), SCGB2A1 (mammaglobin 2), SCGB2A2 (mammaglobin 1), SCYEI (endothelial monocyte-activating cytokine), SDF2, Sema5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, semaphorin 5b) Hlog, sema domain, seven thrombospondin repeats (type 1 and type 1-like), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5B), SERPINA1, SERPINA3, SERP1NB5 (maspin), SERPINE1 (PAI-1), SERPDMF1, SHBG, SLA2, SLC2A2, SLC33A1, SLC43A1, SLIT2, SPPI, SPRR1B (Sprl), ST6GAL1, STABI, STAT6,STEAP (six-transmembrane epithelial antigen of the prostate), STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer-associated gene 1, prostate cancer-associated protein 1, six-transmembrane epithelial antigen 2 of the prostate, six-transmembrane prostate protein), TB4R2, TBX21, TCPIO, TOGFI, TEK, TENB2 (putative transmembrane proteoglycan), TGFA, TGFBI, TGFB1II, TGFB2, TGFB3, TGFBI, TGFBRI, TGFBR2, TGFBR3, THIL, THBSI (thrombospondin-like protein TMP3, tissue factor, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TMEFF1 (transmembrane protein 1 with EGF-like and two follistatin-like domains, tomoregulin-1), TMEM46 (shisa homolog 2), TNF, TNF-α, TNFAEP2 (B94), TNFAIP3, TNFRSFIIA, TNFRSF1A, TNFRSF1B, TNFRSF21, TNFRSF5, TNFRSF6 (Fas), TNFRSF7, TNFRSF8, TNFRSF9, TNFSF10 (TRAIL), TNFSF11 (TRANCE), TNFSF12 (AP03L), TNFSF13 (April), TNFSF13B, TNFSF14 (HVEM-L), TNFSF15 (VEGI), TNFSF18, TNFSF4 (OX40 ligand), TNFSF5 (CD40 ligand), TNFSF6 (FasL), TNFSF7 (CD27 ligand), TNFSFS (CD30 ligand), TNFSF9 (4-1BB ligand), TOLLIP, Toll-like receptor receptor, TOP2A (topoisomerase Ea), TP53, TPM1, TPM2, TRADD, TMEM118 (ring finger protein, transmembrane 2, RNFT2, FLJ14627), TRAF1, TRAF2, TRAF3, TRAF4, TRAF5, TRAF6, TREM1, TREM2, TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channel, subfamily M, member 4), TRPC6, TSLP, TWEAK, tyrosinase (TYR, OCAIA, OCA1A, tyrosinase,It may bind to or interact with SHEP3), VEGF, VEGFB, VEGFC, versican, VHLC5, VLA-4, XCL1 (lymphotactin), XCL2 (SCM-1b), XCRI (GPR5 / CCXCRI), YY1, and / or ZFPM2.

[0180] In certain embodiments, target molecules for antibodies (or bispecific antibodies) produced according to the methods disclosed herein include CD proteins such as CD3, CD4, CDS, CD16, CD19, CD20, CD21 (CR2 (complement receptor 2) or C3DR (C3d / Epstein-Barr virus receptor) or Hs.73792), CD33, CD34, CD64, CD72 (B cell differentiation antigen CD72, Lyb-2), CD79b (CD79B, CD79β, IGb (immunoglobulin-related beta), B29), CD200 members of the ErbB receptor family, such as EGF receptor, HER2, HER3, or HER4 receptor, cell adhesion molecules such as LFA-1, Mac1, p150.95, VLA-4, ICAM-1, VC AM, alpha4 / beta7 integrin, and alphav / beta3 integrin, including either the alpha or beta subunits thereof (e.g., anti-CD11a, anti-CD18, or anti-CD11b antibodies), growth factors such as VEGF-A, VEGF-C, tissue factor (TF), alpha interferon (alpha IFN), TNF alpha, interleukins such as IL-1 beta, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-13, IL17AF, IL-1S, IL-13R alpha 1, IL13R alpha 2, IL-4R, IL-5R, IL-9R, IgE, blood group antigens, flk2 / flt3 receptor, obesity (OB) receptor, mpl receptor, CTLA-4, RANKL, RANK, RSV F protein, protein C, and the like. In certain embodiments, the methods provided herein can be used to produce antibodies (or multispecific antibodies, such as bispecific antibodies) that specifically bind to complement protein C5 (e.g., anti-C5 agonist antibodies that specifically bind to human C5).

[0181] In certain embodiments, the methods provided herein can be used to produce antibodies (or multispecific antibodies, such as bispecific antibodies) that specifically bind to influenza virus B hemagglutinin, i.e., "fluB" (e.g., antibodies that bind to hemagglutinin from the Yamagata lineage of influenza B virus, antibodies that bind to hemagglutinin from the Victoria lineage of influenza B virus, antibodies that bind to hemagglutinin from an ancestral lineage of influenza B virus, or antibodies that bind to hemagglutinin from the Yamagata lineage, Victoria lineage, and ancestral lineage of influenza B virus in vitro and / or in vivo). Further details regarding anti-FluB antibodies are described in WO2015 / 148806, which is incorporated herein by reference in its entirety.

[0182] In certain embodiments, the antibody (or bispecific antibody) produced according to the methods provided herein binds to low-density lipoprotein receptor-related protein (LRP)-1 or LRP-8 or transferrin receptor, and to at least one target selected from the group consisting of beta-secretase (BACE1 or BACE2), alpha-secretase, gamma-secretase, tau-secretase, amyloid precursor protein (APP), death receptor 6 (DR6), amyloid beta peptide, alpha-synuclein, parkin, huntingtin, p75NTR, CD40, and caspase-6.

[0183] In certain embodiments, the antibody produced according to the methods provided herein is a human IgG2 antibody against CD40. In certain embodiments, the anti-CD40 antibody is RG7876.

[0184] In certain embodiments, the polypeptide produced according to the methods provided herein is a target immunocytokine. In certain embodiments, the target immunocytokine is a CEA-IL2v immunocytokine. In certain embodiments, the CEA-IL2v immunocytokine is RG7813. In certain embodiments, the target immunocytokine is a FAP-IL2v immunocytokine. In certain embodiments, the FAP-IL2v immunocytokine is RG7461.

[0185] In certain embodiments, multispecific antibodies (such as bispecific antibodies) produced according to the methods provided herein bind to CEA and at least one additional target molecule. In certain embodiments, multispecific antibodies (such as bispecific antibodies) produced according to the methods provided herein bind to a tumor-targeting cytokine and at least one additional target molecule. In certain embodiments, multispecific antibodies (such as bispecific antibodies) produced according to the methods provided herein are fused to IL2v (i.e., an interleukin-2 variant) and bind to an IL1-based immunocytokine and at least one additional target molecule. In certain embodiments, multispecific antibodies (such as bispecific antibodies) produced according to the methods provided herein are T cell bispecific antibodies (i.e., bispecific T cell derivatives or BiTEs).

[0186] In certain embodiments, multispecific antibodies (such as bispecific antibodies) produced according to the methods provided herein are directed to antibodies against IL-1 alpha and IL-1 beta, IL-12 and IL-1S, IL-13 and IL-9, IL-13 and IL-4, IL-13 and IL-5, IL-5 and IL-4, IL-13 and IL-1 beta, IL-13 and IL-25, IL-13 and TARC, IL-13 and MDC, IL-13 and MEF, IL-13 and TGF-β, IL-13 and an LHR agonist, IL-12 and TWEAK, IL-13 and CL25, IL-13 and IL-16, IL-16 and IL-18, IL-16 and IL-19, IL-16 and IL-20, IL-16 and IL-19, IL-16 and IL-21, IL-16 and IL-18, IL-16 and IL-19, IL-16 and IL-22, IL-16 and IL-19, IL-16 and IL-19, IL-16 and IL-23, IL-16 and IL-19 ... -13 and SPRR2a, IL-13 and SPRR2b, IL-13 and ADAMS, IL-13 and PED2, IL17A and IL17F, CEA and CD3, CD3 and CD19, CD138 and CD20, CD138 and CD40, CD19 and CD20, CD20 and CD3, CD3S and CD13S, CD3S and CD20, CD3S and CD40, CD40 and CD20, CD-S and IL-6, CD20 and BR3, TNF alpha and TGF-beta, TNF alpha and IL-1 beta, TNF alpha and IL-2, TNF alpha and and IL-3, TNF-alpha and IL-4, TNF-alpha and IL-5, TNF-alpha and IL-6, TNF-alpha and IL-8, TNF-alpha and IL-9, TNF-alpha and IL-10, TNF-alpha and IL-11, TNF-alpha and IL-12, TNF-alpha and IL-13, TNF-alpha and IL-14, TNF-alpha and IL-15, TNF-alpha and IL-16, TNF-alpha and IL-17, TNF-alpha and IL-18, TNF-alpha and IL-19, TNF-alpha and IL-20, TNF-alpha and IL-1 -23, TNF alpha and IFN alpha, TNF alpha and CD4, TNF alpha and VEGF, TNF alpha and MIF, TNF alpha and ICAM-1, TNF alpha and PGE4, TNF alpha and PEG2, TNF alpha and RANK ligand, TNF alpha and Te38, TNF alpha and BAFF, TNF alpha and CD22, TNF alpha and CTLA-4, TNF alpha and GP130, TNFa and IL-12p40, VEGF and angiopoietin, VEGF and HER2, VEGF-A and HER2,VEGF-A and PDGF, HER1 and HER2, VEGFA and ANG2, VEGF-A and VEGF-C, VEGF-C and VEGF-D, HER2 and DR5, VEGF and IL-8, VEGF and MET, VEGFR and MET receptor, EGFR and MET, VEGFR and EGFR, HER2 and CD64, HER2 and CD3, HER2 and CD16, HER2 and HER3, EGFR (HER1) and HER2, EGFR and HER3, EGFR and HER4, IL-14 and IL-13, IL-13 and CD40L, IL4 and CD40L, TNFR1 and IL-1R, TNFR1 and IL-6R, and TNFR1 and IL-18R, EpCAM and CD3, MAPG and CD28, EGFR and CD64, CSPGs and RGM A, CTLA-4 and BTN02, IGF1 and IGF2, IGF1 / 2 and Erb2B, MAG and RGM A, NgR and RGM A, NogoA and RGM A, OMGp and RGM A, POL-1 and CTLA-4, and RGM A and RGM B.

[0187] In certain embodiments, the multispecific antibody (such as a bispecific antibody) is an anti-CEA / anti-CD3 bispecific antibody. In certain embodiments, the anti-CEA / anti-CD3 bispecific antibody is RG7802. Further details regarding anti-CEA / anti-CD3 bispecific antibodies are provided in WO2014 / 121712, which is incorporated herein by reference in its entirety.

[0188] In certain embodiments, the multispecific antibody (e.g., bispecific antibody) is an anti-VEGF / anti-angiopoietin bispecific antibody. In certain embodiments, the anti-VEGF / anti-angiopoietin bispecific antibody is a crossmAb. In certain embodiments, the anti-VEGF / anti-angiopoietin bispecific antibody is RG7716.

[0189] In certain embodiments, the multispecific antibody (such as a bispecific antibody) is an anti-Ang2 / anti-VEGF bispecific antibody. In certain embodiments, the anti-Ang2 / anti-VEGF bispecific antibody is RG7221. In certain embodiments, the anti-Ang2 / anti-VEGF bispecific antibody is CAS number 1448221-05-3.

[0190] Many other antibodies and / or other proteins may be expressed by host cells in the present disclosure, and the above list is not intended to be limiting.

[0191] The host cells of the present disclosure may be used for manufacturing-scale production of a molecule of interest. "Manufacturing-scale" production of therapeutic or other proteins utilizes cell cultures ranging from about 400 L to about 80,000 L, depending on the protein being produced and the needs. Typically, such manufacturing-scale production utilizes cell culture sizes ranging from about 400 L to about 25,000 L. Within this range, specific cell culture sizes may be utilized, such as, for example, 4,000 L, about 6,000 L, about 8,000 L, about 10,000 L, about 12,000 L, about 14,000 L, or about 16,000 L.

[0192] The host cells of the present disclosure can be used to produce large quantities of a molecule of interest in a shorter time frame compared to non-TI cells used in current cell culture methods. In certain embodiments, the host cells of the present disclosure can be used to improve the quality of a molecule of interest compared to non-TI cells used in current cell culture methods. In certain embodiments, the host cells of the present disclosure can be used to enhance seed train stability by preventing chronic toxicity that can be caused by products that can cause cellular stress and clonal instability over time. In certain embodiments, the host cells of the present disclosure can be used for optimal expression of acutely toxic products.

[0193] In certain embodiments, the host cell, TI system of the present disclosure may be used for cell culture process optimization and / or process development.

[0194] In certain embodiments, the host cells of the present disclosure can be used to accelerate the production of a molecule of interest by about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, or about 10 weeks when compared to non-TI cells used in conventional cell culture methods. In certain embodiments, the host cells of the present disclosure can be used to accelerate the harvest of a molecule of interest by about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, or about 10 weeks when compared to non-TI cells used in conventional cell culture methods.

[0195] In certain embodiments, the host cells of the present embodiments may be used to reduce aggregation levels of a molecule of interest when compared to non-TI cells used in conventional cell culture methods.

[0196] In certain embodiments, the host cells of the present disclosure may be used to achieve increased expression of a polypeptide (or polypeptides) of interest compared to randomly integrated host cells. For example, and not intended to be limiting, host cells of the present disclosure may achieve expression of standard antibodies and half antibodies at titers of at least 3 g / L, 3.5 g / L, 4 g / L, 4.5 g / L, 5 g / L, 5.5 g / L, 6 g / L, 6.5 g / L, 7 g / L, 7.5 g / L, 8 g / L, 8.5 g / L, 9 g / L, 9.5 g / L, 10 g / L, 10.5 g / L, 11 g / L, or more, and expression of multispecific antibodies, e.g., bispecific antibodies, at titers of at least 1.5 g / L, 2 g / L, 2.5 g / L, 3 g / L, 3.5 g / L, 4 g / L, 4.5 g / L, 5 g / L, 5.5 g / L, 6 g / L, or more. In certain embodiments, host cells of the present disclosure may achieve increased bispecific content compared to randomly integrated host cells. For example, and not by way of limitation, host cells of the present disclosure may achieve a bispecific content of at least 80%, 85%, 90%, 95%, 96%, 98%, 99% or more.

[0197] In certain embodiments, the host cells of the present disclosure can be used for the constitutive expression of selected subunits of a therapeutic molecule and the regulated expression of other, different subunits of the same therapeutic molecule. In certain embodiments, the therapeutic molecule can be a fusion protein. In certain embodiments, the host cells of the present disclosure can be used to understand the role and effect of each antibody subunit in the expression and secretion of a fully assembled antibody molecule.

[0198] In certain embodiments, the host cells of the present disclosure can be used as a research tool. In certain embodiments, the host cells of the present disclosure can be used as a diagnostic tool to resolve the root cause of low protein expression of problematic molecules in various cells. In certain embodiments, the host cells of the present disclosure can be used to directly correlate observed phenomena or cellular behavior with transgene expression in the cell. The host cells of the present disclosure can also be used to demonstrate whether observed behavior is reversible in the cell. In certain embodiments, the host cells of the present disclosure can be utilized to identify and alleviate problems with the transcription and expression of transgene(s) in cells.

[0199] In certain embodiments, the host cells of the present disclosure can be used to exchange transgene subunits of difficult-to-express molecules, such as, but not limited to, the HC and LC subunits of an antibody, for subunits of an average molecule in a TI system to identify the problematic subunit(s). In certain embodiments, amino acid sequence analysis can then be used to narrow down and focus on amino acid residues or regions that may be responsible for low protein expression.

[0200] 8. Exemplary Non-Limiting Embodiments A. A targeted integration (TI) host cell, comprising an exogenous nucleotide sequence integrated into an integration site within a specific locus in the host cell genome, wherein the locus is at least about 90% homologous to a sequence selected from SEQ ID NOs: 1-7.

[0201] A1. The nucleotide sequence immediately 5' to the integrated 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, and nucleotides 253831-491909 of NW_003616412.1. nucleotides 69303-79768 of NW_003615063.1, nucleotides 293481-315265 of NW_006882936.1, nucleotides 2650443-2662054 of NW_006882936.1, or nucleotides 82214-97705 of NW_003615411.1.

[0202] A2. the nucleotide sequence immediately 5' to the integrated exogenous nucleotide sequence is nucleotide 45269 of NW_006874047.1, nucleotide 207911 of NW_006884592.1, nucleotide 491909 of NW_006881296.1, nucleotide 79768 of NW_003616412.1, nucleotide 315265 of NW_003615063.1, nucleotide 2662054 of NW_006882936.1, or nucleotide 2662054 of NW_003615411.1 97705, wherein the sequence is selected from the group consisting of at least 15 base pairs, at least 20 base pairs, at least 30 base pairs, at least 40 base pairs, at least 50 base pairs, at least 75 base pairs, at least 100 base pairs, at least 150 base pairs, at least 200 base pairs, at least 300 base pairs, at least 400 base pairs, at least 500 base pairs, at least 1,000 base pairs, at least 1,500 base pairs, at least 2,000 base pairs, and at least 3,000 base pairs.

[0203] A3. The nucleotide sequence immediately 3' to the integrated 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, and nucleotides 501910-501913 of NW_003616412.1. nucleotides 79769-100059 of NW_003615063.1, nucleotides 315266-362442 of NW_006882936.1, nucleotides 2662055-2701768 of NW_006882936.1, or nucleotides 97706-105117 of NW_003615411.1.

[0204] A4. the nucleotide sequence immediately 3' to the integrated exogenous nucleotide sequence is: nucleotide 45270 of NW_006874047.1; nucleotide 207912 of NW_006884592.1; nucleotide 491910 of NW_006881296.1; nucleotide 79769 of NW_003616412.1; nucleotide 315266 of NW_003615063.1; nucleotide 2662055 of NW_006882936.1; or nucleotide 97706, wherein the TI host cell is selected from the group consisting of at least 15 base pairs, at least 20 base pairs, at least 30 base pairs, at least 40 base pairs, at least 50 base pairs, at least 75 base pairs, at least 100 base pairs, at least 150 base pairs, at least 200 base pairs, at least 300 base pairs, at least 400 base pairs, at least 500 base pairs, at least 1,000 base pairs, at least 1,500 base pairs, at least 2,000 base pairs, and at least 3,000 base pairs.

[0205] A5. The TI host cell of any one of A-A4, wherein the integrated exogenous nucleotide sequence is operably linked to a nucleotide sequence selected from the group consisting of sequences at least about 90% homologous to a sequence selected from SEQ ID NOs: 1-7.

[0206] A6. A TI host cell comprising an exogenous nucleotide sequence integrated into an integration site within an endogenous gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, XP_003512331.2, and sequences at least about 90% homologous thereto.

[0207] A7. A TI host cell comprising an exogenous nucleotide sequence integrated into an integration site operably linked to an endogenous gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, XP_003512331.2, and sequences at least about 90% homologous thereto.

[0208] A8. A TI host cell comprising an exogenous nucleotide sequence integrated into an integration site immediately adjacent to all or part of a sequence selected from the group consisting of sequences at least about 90% homologous to a sequence selected from SEQ ID NOs: 1-7.

[0209] A9. The TI host cell according to any one of A to A8, wherein the TI host cell is a mammalian host cell.

[0210] A10. The TI host cell according to A9, wherein the TI host cell is a hamster host cell, a human host cell, a rat host cell, or a mouse host cell.

[0211] A11. The TI host cell of A9 or A10, wherein the TI host cell is a Chinese hamster ovary (CHO) host cell, a CHO K1 host cell, a CHO K1SV host cell, a DG44 host cell, a DUKXB-11 host cell, a CHOK1S host cell, or a CHO K1M host cell.

[0212] A12. The TI host cell of any one of A-A11, wherein the exogenous nucleotide sequence comprises one or more recombination recognition sequences (RRS), wherein the RRS can be recognized by a recombinase.

[0213] A13. The TI host cell of A12, wherein the exogenous nucleotide sequence comprises at least two RRSs.

[0214] A14. The TI host cell according to A12 or A13, wherein the recombinase is Cre recombinase or FLP recombinase.

[0215] A15. The TI host cell of A12 or A13, wherein the recombinase is Bxb1 integrase or φC31 integrase.

[0216] A16. The TI host cell according to any one of A12 to A15, wherein 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.

[0217] A17. A TI host cell described in any one of A13 to A16, wherein the exogenous nucleotide sequence comprises a first and a second RRS, and at least one selectable marker located between the first and second RRS.

[0218] A18. The TI host cell of A17, comprising a first selection marker, wherein the first selection marker is selected from the group consisting of aminoglycoside phosphotransferase (APH), hygromycin phosphotransferase (HYG), neomycin, G418 APH), dihydrofolate reductase (DHFR), thymidine kinase (TK), glutamine synthetase (GS), asparagine synthetase, tryptophan synthetase (indole), histidinol dehydrogenase (histidinol D), blasticidin, bleomycin, phleomycin, chloramphenicol, zeocin, and mycophenolic acid.

[0219] A19. The TI host cell of A17 or A18, further comprising a second selectable marker, wherein said first and said second selectable markers are different.

[0220] A20. The TI host cell of A19, wherein the second selectable 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.

[0221] A21. The TI host cell of A19 or A20, further comprising a third selection marker and an internal ribosome entry site (IRES), wherein the IRES is operably linked to the third selection marker.

[0222] A22. The TI host cell of A21, wherein the third selection marker is different from the first or second selection marker.

[0223] A23. The TI host cell of A21 or A22, wherein the third selection marker is selected from the group consisting of a green fluorescent protein (GFP) marker, an improved GFP (eGFP) marker, a synthetic GFP marker, a yellow fluorescent protein (YFP) marker, an improved YFP (eYFP) marker, a cyan fluorescent protein (CFP) marker, an mPlum marker, an mCherry marker, a tdTomato marker, an mStrawberry marker, a J-red marker, a DsRed-monomer marker, an mOrange marker, an mKO marker, an mCitrine marker, a Venus marker, a YPet marker, an Emerald6 marker, a CyPet marker, an mCFPm marker, a Cerulean marker, and a T-Sapphire marker.

[0224] A24. A TI host cell described in any one of A17 to A23, further comprising a third RRS, wherein the third RRS is located between the first RRS and the second RRS, and the third RRS is heterospecific compared to the first or second RRS.

[0225] A25. The TI host cell of any one of A12 to A24, wherein the exogenous nucleotide sequence comprises at least one selectable marker and at least one exogenous sequence of interest (SOI).

[0226] A26. The TI host cell of any one of A12 to A25, wherein the exogenous nucleotide sequence further comprises at least one exogenous SOI.

[0227] A27. The TI host cell of A26, wherein the exogenous SOI is located between the first RRS and the second RRS.

[0228] A28. The TI host cell of A26 or A27, 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.

[0229] A29. A TI host cell described in any one of A24 to A28, wherein the exogenous nucleotide sequence further comprises at least one exogenous SOI located between the first RRS and the third RRS, and at least one exogenous SOI located between the third RRS and the second RRS.

[0230] A30. The TI host cell of A29, 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.

[0231] A31. The TI host cell of A26, wherein the SOI encodes an antibody that is expressed at a higher level compared to a randomly integrated host cell.

[0232] A32. The TI host cell of A31, wherein the antibody is a standard antibody or half antibody expressed at a titer of at least 3 g / L, 3.5 g / L, 4 g / L, 4.5 g / L, 5 g / L, 5.5 g / L, 6 g / L, 6.5 g / L, 7 g / L, 7.5 g / L, 8 g / L, 8.5 g / L, 9 g / L, 9.5 g / L, 10 g / L, 10.5 g / L, 11 g / L, or more.

[0233] A33. The TI host cell of A31, wherein the antibody is a multispecific antibody, e.g., a bispecific antibody, expressed at a titer of at least 1.5 g / L, 2 g / L, 2.5 g / L, 3 g / L, 3.5 g / L, 4 g / L, 4.5 g / L, 5 g / L, 5.5 g / L, 6 g / L.

[0234] A34. The TI host cell of A31, wherein the antibody is a bispecific antibody and the bispecific content is increased compared to a random integration host cell.

[0235] A35. The TI host cell of A1, wherein the antibody is a bispecific antibody and the bispecific content is at least 80%, 85%, 90%, 95%, 96%, 98%, 99% or more.

[0236] B. A method for preparing a TI host cell that expresses a polypeptide of interest, comprising: providing a TI host cell comprising an exogenous nucleotide sequence integrated into a site within a locus in the TI host cell genome, the locus being at least about 90% homologous to a sequence selected from SEQ ID NOs: 1-7, the exogenous nucleotide sequence comprising two RRSs flanking at least one first selection marker; introducing into the cell provided in a) a vector comprising at least one exogenous SOI and at least one second selection marker, the two RRSs matching the two RRSs on the integrated exogenous nucleotide sequence; introducing a recombinase or a nucleic acid encoding a recombinase, wherein the recombinase recognizes the RRSs; and isolating TI host cells that express the polypeptide by selecting for TI cells that express the second selection marker.

[0237] B1. The method according to B, wherein the recombinase is Cre recombinase or FLP recombinase.

[0238] B2. The method of B, wherein the recombinase is Bxb1 integrase or φC31 integrase.

[0239] B3. The method of any one of B-B2, 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.

[0240] B4. The method of any one of claims B-B3, wherein the TI host cell is a mammalian host cell.

[0241] B5. The method of B4, wherein the TI host cell is a hamster host cell, a human host cell, a rat host cell, or a mouse host cell.

[0242] B6. The method of B4 or B5, wherein the TI host cell is a CHO host cell, a CHO K1 host cell, a CHO K1SV host cell, a DG44 host cell, a DUKXB-11 host cell, a CHOK1S host cell, or a CHO K1M host cell.

[0243] C. A method for expressing a polypeptide of interest, comprising providing a TI host cell comprising at least one exogenous SOI and at least one selectable marker, wherein the at least one exogenous SOI and at least one selectable marker are flanked by two RRSs integrated into a locus in the genome of the TI host cell, the locus being at least about 90% homologous to a sequence selected from SEQ ID NOs: 1-7, and culturing the cell of a) under conditions suitable for expressing the polypeptide of interest and recovering the polypeptide of interest therefrom.

[0244] C1. The method of C, wherein the at least one 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.

[0245] D. A method for preparing a TI host cell that expresses at least a first and a second polypeptide of interest, comprising providing a TI host cell comprising an exogenous nucleotide sequence integrated into a site within a locus of the host cell genome, the locus being at least about 90% homologous to a sequence selected from SEQ ID NOs: 1-7, the exogenous nucleotide sequence comprising first and second RRSs flanking at least one first selectable marker, and a third RRS located between the first and second RRSs, all of which RRSs being heterospecific; and providing in the cell provided in a) at least one first exogenous nucleotide sequence that matches the first and third RRSs on the integrated exogenous nucleotide sequence. a) introducing into the cells provided in a) a first vector comprising two RRSs flanked by a heterologous SOI and at least one second selectable marker; b) introducing into the cells provided in a) a second vector comprising two RRSs that match the second and third RRSs on the integrated exogenous nucleotide sequence and are flanked by at least one second exogenous SOI; c) introducing one or more recombinases, or one or more nucleic acids encoding one or more recombinases, wherein the one or more recombinases recognize the RRSs; and d) isolating TI host cells that express the first and second polypeptides of interest by selecting for TI cells that express the second selectable marker.

[0246] D1. The method of D, wherein the recombinase is Cre recombinase or FLP recombinase.

[0247] D2. The method of D, wherein the recombinase is Bxb1 integrase or φC31 integrase.

[0248] D3. The method of D, wherein the first vector further comprises a promoter sequence operably linked to the codon ATG located upstream of the first SOI and adjacent to the downstream RRS, and the second vector further comprises a selectable marker lacking an ATG transcription start codon upstream of the RRS and adjacent to the downstream RRS of the second SOI.

[0249] D4. The method of any one of D-D3, wherein the first SOI encodes a single chain antibody, an antibody light chain, an antibody heavy chain, a single chain Fv fragment (scFv), or an Fc fusion protein.

[0250] D5. The method of any one of D-D3, wherein the second SOI encodes a single chain antibody, an antibody light chain, an antibody heavy chain, a single chain Fv fragment (scFv), or an Fc fusion protein.

[0251] D6. The method of any one of C or D-D5, wherein the TI host cell is a mammalian host cell.

[0252] D7. The method of D6, wherein the TI host cell is a hamster host cell, a human host cell, a rat host cell, or a mouse host cell.

[0253] D8. The method of D7, wherein the TI host cell is a CHO host cell, a CHO K1 host cell, a CHO K1SV host cell, a DG44 host cell, a DUKXB-11 host cell, a CHOK1S host cell, or a CHO K1M host cell.

[0254] E. A method for expressing a polypeptide of interest, comprising providing a host cell comprising at least two exogenous SOIs and at least one selectable marker integrated into a locus in the host cell genome, the locus being at least about 90% homologous to a sequence selected from SEQ ID NOs: 1-7, wherein the at least one exogenous SOI and one selectable marker are flanked by a first and a third RRS, and the at least one exogenous SOI is flanked by a second and the third RRS, and culturing the cell of a) under conditions suitable for expressing the polypeptide of interest and recovering the polypeptide of interest therefrom.

[0255] E1. The method of E, wherein the first SOI encodes a single chain antibody, an antibody light chain, an antibody heavy chain, a single chain Fv fragment (scFv), or an Fc fusion protein.

[0256] E2. The method of E or E1, wherein the second SOI encodes a single chain antibody, an antibody light chain, an antibody heavy chain, a single chain Fv fragment (scFv), or an Fc fusion protein.

[0257] E3. The method of any one of E-E2, wherein the TI host cell is a mammalian host cell.

[0258] E4. The method of E3, wherein the TI host cell is a hamster host cell, a human host cell, a rat host cell, or a mouse host cell.

[0259] E5. The method of E4, wherein the TI host cell is a CHO host cell, a CHO K1 host cell, a CHO K1SV host cell, a DG44 host cell, a DUKXB-11 host cell, a CHOK1S host cell, or a CHO K1M host cell.

[0260] F. A vector comprising: a) two reference sequences selected from any portion of SEQ ID NO: 1; b) two reference sequences selected from any portion of SEQ ID NO: 2; c) two reference sequences selected from any portion of SEQ ID NO: 3; d) two reference sequences selected from any portion of SEQ ID NO: 4; e) two reference sequences selected from any portion of SEQ ID NO: 5; f) two reference sequences selected from any portion of SEQ ID NO: 6; or g) two reference sequences selected from any portion of SEQ ID NO: 7 wherein the sequences flank a DNA cassette, and the DNA cassette comprises at least one selectable marker flanked by two RRSs and at least one exogenous SOI.

[0261] F1. The vector of F, wherein the vector comprises two nucleotide sequences that are at least 60% homologous to the two reference sequences.

[0262] F2. The vector of F, wherein the vector comprises two nucleotide sequences that are at least 70% homologous to the two reference sequences.

[0263] F3. The vector of F, wherein the vector comprises two nucleotide sequences that are at least 80% homologous to the two reference sequences.

[0264] F4. The vector of F, wherein the vector comprises two nucleotide sequences that are at least 90% homologous to the two reference sequences.

[0265] F5. The vector of F, wherein the vector comprises two nucleotide sequences that are at least 95% homologous to the two reference sequences.

[0266] F6. The vector of F, wherein the vector comprises two nucleotide sequences that are at least 99% homologous to the two reference sequences.

[0267] F7. The vector of any one of F to F6, 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.

[0268] F8. The vector of any one of F-F7, 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.

[0269] G. A method for preparing a TI host cell that expresses a polypeptide of interest, comprising: providing a host cell comprising a locus in the host cell genome, the locus being at least about 90% homologous to a sequence selected from SEQ ID NOs: 1-7; introducing a vector into the host cell, the vector comprising a nucleotide sequence at least 50% homologous to a sequence selected from SEQ ID NOs: 1-7 flanking a DNA cassette, the DNA cassette comprising at least one selectable marker flanked by two RRSs and at least one exogenous SOI; and selecting for the selectable marker; isolating a TI host cell having the SOI integrated into the locus in the genome and expressing the polypeptide of interest.

[0270] G1. The method of G, wherein the vector is selected from the group consisting of an adenoviral vector, an adeno-associated viral vector, a lentiviral vector, a retroviral vector, an integrating phage vector, a non-viral vector, a transposon and / or transposase vector, an integrase substrate, and a plasmid.

[0271] G2. The method of G or G1, wherein the at least one 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.

[0272] G3. The method of any one of G-G2, wherein the TI host cell is a mammalian host cell.

[0273] G4. The method of G3, wherein the TI host cell is a hamster host cell, a human host cell, a rat host cell, or a mouse host cell.

[0274] G5. The method of G3 or G4, wherein the TI host cell is a CHO host cell, a CHO K1 host cell, a CHO K1SV host cell, a DG44 host cell, a DUKXB-11 host cell, a CHOK1S host cell, or a CHO K1M host cell.

[0275] G6. The method of any one of G1 to G5, wherein the integration of the nucleic acid comprising the at least one SOI and a selectable marker is facilitated by an exogenous nuclease.

[0276] G7. The method of G6, wherein the exogenous nuclease is selected from the group consisting of zinc finger nucleases (ZFNs), ZFN dimers, transcription activator-like effector nucleases (TALENs), TAL effector domain fusion proteins, RNA-guided DNA endonucleases, engineered meganucleases, and clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) endonucleases.

[0277] G8. The method of G, wherein said polypeptide of interest is a multispecific antibody.

[0278] G9. The method of G8, wherein the multispecific antibody is a bispecific antibody.

[0279] H. A TI host cell comprising at least one exogenous nucleotide sequence integrated into an integration site within one or more specific loci of the host cell genome, the loci being at least about 90% homologous to a sequence selected from SEQ ID NOs: 1-7.

[0280] An H1.TI host cell comprising at least one exogenous nucleotide sequence integrated into one or more integration sites within an endogenous gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, XP_003512331.2, and sequences at least about 90% homologous thereto.

[0281] H2. The TI host cell of any one of H-H1, wherein the at least one exogenous nucleotide sequence comprises one or more recombination recognition sequences (RRS), wherein the RRS can be recognized by a recombinase.

[0282] H3. The TI host cell of H2, wherein said at least one exogenous nucleotide sequence further comprises at least one exogenous SOI.

[0283] H4. The TI host cell of any one of H-H3, wherein the host cell comprises at least one exogenous nucleotide sequence at a first locus in the genome of the host cell and at least one exogenous nucleotide sequence at two or more second loci in the genome of the host cell.

[0284] H5. The TI host cell of H4, wherein the first locus comprises a sequence at least 90% identical to all or a portion of SEQ ID NO:1, and the second locus comprises at least one sequence at least 90% identical to all or a portion of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7.

[0285] H5.1. The TI host cell of H4, wherein the first locus comprises a sequence at least 90% identical to all or a portion of SEQ ID NO:1, and the second locus comprises at least one sequence at least 90% identical to all or a portion of SEQ ID NO:2.

[0286] H5.2. The TI host cell of H4, wherein the first locus comprises a sequence at least 90% identical to all or a portion of SEQ ID NO:1, and the second locus comprises at least one sequence at least 90% identical to all or a portion of SEQ ID NO:3.

[0287] H5.3. The TI host cell of H4, wherein the first locus comprises a sequence at least 90% identical to all or a portion of SEQ ID NO:1, and the second locus comprises at least one sequence at least 90% identical to all or a portion of SEQ ID NO:4.

[0288] H5.4. The TI host cell of H4, wherein the first locus comprises a sequence at least 90% identical to all or a portion of SEQ ID NO:1, and the second locus comprises at least one sequence at least 90% identical to all or a portion of SEQ ID NO:5.

[0289] H5.5. The TI host cell of H4, wherein the first locus comprises a sequence at least 90% identical to all or a portion of SEQ ID NO:1, and the second locus comprises at least one sequence at least 90% identical to all or a portion of SEQ ID NO:6.

[0290] H5.6. The TI host cell of H4, wherein the first locus comprises a sequence at least 90% identical to all or a portion of SEQ ID NO:1, and the second locus comprises at least one sequence at least 90% identical to all or a portion of SEQ ID NO:7.

[0291] H6. The TI host cell of H4, wherein the first locus comprises a sequence at least 90% identical to all or a portion of SEQ ID NO:2, and the second locus comprises at least one sequence at least 90% identical to all or a portion of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7.

[0292] H6.1. The TI host cell of H4, wherein the first locus comprises a sequence at least 90% identical to all or a portion of SEQ ID NO:2, and the second locus comprises at least one sequence at least 90% identical to all or a portion of SEQ ID NO:1.

[0293] H6.2. The TI host cell of H4, wherein the first locus comprises a sequence at least 90% identical to all or a portion of SEQ ID NO:2, and the second locus comprises at least one sequence at least 90% identical to all or a portion of SEQ ID NO:3.

[0294] H6.3. The TI host cell of H4, wherein the first locus comprises a sequence at least 90% identical to all or a portion of SEQ ID NO:2, and the second locus comprises at least one sequence at least 90% identical to all or a portion of SEQ ID NO:4.

[0295] H6.4. The TI host cell of H4, wherein the first locus comprises a sequence at least 90% identical to all or a portion of SEQ ID NO:2, and the second locus comprises at least one sequence at least 90% identical to all or a portion of SEQ ID NO:5.

[0296] H6.5. The TI host cell of H4, wherein the first locus comprises a sequence at least 90% identical to all or a portion of SEQ ID NO:2, and the second locus comprises at least one sequence at least 90% identical to all or a portion of SEQ ID NO:6.

[0297] H6.6. The TI host cell of H4, wherein the first locus comprises a sequence at least 90% identical to all or a portion of SEQ ID NO:2, and the second locus comprises at least one sequence at least 90% identical to all or a portion of SEQ ID NO:7.

[0298] H7. The TI host cell of H4, wherein the first locus comprises a sequence at least 90% homologous to all or a portion of SEQ ID NO:3, and the second locus comprises at least one sequence at least 90% homologous to all or a portion of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7.

[0299] H7.1. The TI host cell of H4, wherein the first locus comprises a sequence at least 90% identical to all or a portion of SEQ ID NO:3, and the second locus comprises at least one sequence at least 90% identical to all or a portion of SEQ ID NO:1.

[0300] H7.2. The TI host cell of H4, wherein the first locus comprises a sequence at least 90% identical to all or a portion of SEQ ID NO:3, and the second locus comprises at least one sequence at least 90% identical to all or a portion of SEQ ID NO:2.

[0301] H7.3. The TI host cell of H4, wherein the first locus comprises a sequence at least 90% identical to all or a portion of SEQ ID NO:3, and the second locus comprises at least one sequence at least 90% identical to all or a portion of SEQ ID NO:4.

[0302] H7.4. The TI host cell of H4, wherein the first locus comprises a sequence at least 90% identical to all or a portion of SEQ ID NO:3, and the second locus comprises at least one sequence at least 90% identical to all or a portion of SEQ ID NO:5.

[0303] H7.5. The TI host cell of H4, wherein the first locus comprises a sequence at least 90% identical to all or a portion of SEQ ID NO:3, and the second locus comprises at least one sequence at least 90% identical to all or a portion of SEQ ID NO:6.

[0304] H7.6. The TI host cell of H4, wherein the first locus comprises a sequence at least 90% identical to all or a portion of SEQ ID NO:3, and the second locus comprises at least one sequence at least 90% identical to all or a portion of SEQ ID NO:7.

[0305] H8. The TI host cell of H4, wherein the first locus comprises a sequence at least 90% identical to all or a portion of SEQ ID NO:4, and the second locus comprises at least one sequence at least 90% identical to all or a portion of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7.

[0306] H8.1. The TI host cell of H4, wherein the first locus comprises a sequence at least 90% identical to all or a portion of SEQ ID NO:4, and the second locus comprises at least one sequence at least 90% identical to all or a portion of SEQ ID NO:1.

[0307] H8.2. The TI host cell of H4, wherein the first locus comprises a sequence at least 90% identical to all or a portion of SEQ ID NO:4, and the second locus comprises at least one sequence at least 90% identical to all or a portion of SEQ ID NO:2.

[0308] H8.3. The TI host cell of H4, wherein the first locus comprises a sequence at least 90% identical to all or a portion of SEQ ID NO:4, and the second locus comprises at least one sequence at least 90% identical to all or a portion of SEQ ID NO:3.

[0309] H8.4. The TI host cell of H4, wherein the first locus comprises a sequence at least 90% identical to all or a portion of SEQ ID NO:4, and the second locus comprises at least one sequence at least 90% identical to all or a portion of SEQ ID NO:5.

[0310] H8.5. The TI host cell of H4, wherein the first locus comprises a sequence at least 90% identical to all or a portion of SEQ ID NO:4, and the second locus comprises at least one sequence at least 90% identical to all or a portion of SEQ ID NO:6.

[0311] H8.6. The TI host cell of H4, wherein the first locus comprises a sequence at least 90% identical to all or a portion of SEQ ID NO:4, and the second locus comprises at least one sequence at least 90% identical to all or a portion of SEQ ID NO:7.

[0312] H9. The TI host cell of H4, wherein the first locus comprises a sequence at least 90% identical to all or a portion of SEQ ID NO:5, and the second locus comprises at least one sequence at least 90% identical to all or a portion of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:7.

[0313] H9.1. The TI host cell of H4, wherein the first locus comprises a sequence at least 90% identical to all or a portion of SEQ ID NO:5, and the second locus comprises at least one sequence at least 90% identical to all or a portion of SEQ ID NO:1.

[0314] H9.2. The TI host cell of H4, wherein the first locus comprises a sequence at least 90% identical to all or a portion of SEQ ID NO:5, and the second locus comprises at least one sequence at least 90% identical to all or a portion of SEQ ID NO:2.

[0315] H9.3. The TI host cell of H4, wherein the first locus comprises a sequence at least 90% identical to all or a portion of SEQ ID NO:5, and the second locus comprises at least one sequence at least 90% identical to all or a portion of SEQ ID NO:3.

[0316] H9.4. The TI host cell of H4, wherein the first locus comprises a sequence at least 90% identical to all or a portion of SEQ ID NO:5, and the second locus comprises at least one sequence at least 90% identical to all or a portion of SEQ ID NO:4.

[0317] H9.5. The TI host cell of H4, wherein the first locus comprises a sequence at least 90% identical to all or a portion of SEQ ID NO:5, and the second locus comprises at least one sequence at least 90% identical to all or a portion of SEQ ID NO:6.

[0318] H9.6. The TI host cell of H4, wherein the first locus comprises a sequence at least 90% identical to all or a portion of SEQ ID NO:5, and the second locus comprises at least one sequence at least 90% identical to all or a portion of SEQ ID NO:7.

[0319] H10. The TI host cell of H4, wherein the first locus comprises a sequence at least 90% identical to all or a portion of SEQ ID NO:6, and the second locus comprises at least one sequence at least 90% identical to all or a portion of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:7.

[0320] H10.1. The TI host cell of H4, wherein the first locus comprises a sequence at least 90% identical to all or a portion of SEQ ID NO:6, and the second locus comprises at least one sequence at least 90% identical to all or a portion of SEQ ID NO:1.

[0321] H10.2. The TI host cell of H4, wherein the first locus comprises a sequence at least 90% identical to all or a portion of SEQ ID NO:6, and the second locus comprises at least one sequence at least 90% identical to all or a portion of SEQ ID NO:2.

[0322] H10.3. The TI host cell of H4, wherein the first locus comprises a sequence at least 90% identical to all or a portion of SEQ ID NO:6, and the second locus comprises at least one sequence at least 90% identical to all or a portion of SEQ ID NO:3.

[0323] H10.4. The TI host cell of H4, wherein the first locus comprises a sequence at least 90% identical to all or a portion of SEQ ID NO:6, and the second locus comprises at least one sequence at least 90% identical to all or a portion of SEQ ID NO:4.

[0324] H10.5. The TI host cell of H4, wherein the first locus comprises a sequence at least 90% identical to all or a portion of SEQ ID NO:6, and the second locus comprises at least one sequence at least 90% identical to all or a portion of SEQ ID NO:5.

[0325] H10.6. The TI host cell of H4, wherein the first locus comprises a sequence at least 90% identical to all or a portion of SEQ ID NO:6, and the second locus comprises at least one sequence at least 90% identical to all or a portion of SEQ ID NO:7.

[0326] H11. The TI host cell of H4, wherein the first locus comprises a sequence at least 90% identical to all or a portion of SEQ ID NO:7, and the second locus comprises at least one sequence at least 90% identical to all or a portion of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6.

[0327] H11.1. The TI host cell of H4, wherein the first locus comprises a sequence at least 90% identical to all or a portion of SEQ ID NO:7, and the second locus comprises at least one sequence at least 90% identical to all or a portion of SEQ ID NO:1.

[0328] H11.2. The TI host cell of H4, wherein the first locus comprises a sequence at least 90% identical to all or a portion of SEQ ID NO:7, and the second locus comprises at least one sequence at least 90% identical to all or a portion of SEQ ID NO:2.

[0329] H11.3. The TI host cell of H4, wherein the first locus comprises a sequence at least 90% identical to all or a portion of SEQ ID NO:7, and the second locus comprises at least one sequence at least 90% identical to all or a portion of SEQ ID NO:3.

[0330] H11.4. The TI host cell of H4, wherein the first locus comprises a sequence at least 90% identical to all or a portion of SEQ ID NO:7, and the second locus comprises at least one sequence at least 90% identical to all or a portion of SEQ ID NO:4.

[0331] H11.5. The TI host cell of H4, wherein the first locus comprises a sequence at least 90% identical to all or a portion of SEQ ID NO:7, and the second locus comprises at least one sequence at least 90% identical to all or a portion of SEQ ID NO:5.

[0332] H11.6. The TI host cell of H4, wherein the first locus comprises a sequence at least 90% identical to all or a portion of SEQ ID NO:7, and the second locus comprises at least one sequence at least 90% identical to all or a portion of SEQ ID NO:6.

[0333] H12. The TI host cell of H4, wherein the first locus is an integration site within the LOC107977062 gene and the second locus is an integration site within a gene selected from the following group: LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, XP_003512331.2, and sequences at least about 90% homologous thereto.

[0334] H13. The TI host cell of H4, wherein the first locus is an integration site within the LOC100768845 gene and the second locus is an integration site within a gene selected from the following group: LOC107977062, ITPR2, ERE67000.1, UBAP2, MTMR2, XP_003512331.2, and sequences at least about 90% homologous thereto.

[0335] H14. The TI host cell of H4, wherein the first locus is an integration site within the ITPR2 gene and the second locus is an integration site within a gene selected from the following group: LOC107977062, LOC100768845, ERE67000.1, UBAP2, MTMR2, XP_003512331.2, and sequences at least about 90% homologous thereto.

[0336] H15. The TI host cell of H4, wherein the first locus is an integration site within the ERE67000.1 gene and the second locus is an integration site within a gene selected from the following group: LOC107977062, LOC100768845, ITPR2, UBAP2, MTMR2, XP_003512331.2, and sequences at least about 90% homologous thereto.

[0337] H16. The TI host cell of H4, wherein the first locus is an integration site within the UBAP2 gene and the second locus is an integration site within a gene selected from the following group: LOC107977062, LOC100768845, ITPR2, ERE67000.1, MTMR2, XP_003512331.2, and sequences at least about 90% homologous thereto.

[0338] H17. The TI host cell of H4, wherein the first locus is an integration site within the MTMR2 gene and the second locus is an integration site within a gene selected from the following group: LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, XP_003512331.2, and sequences at least about 90% homologous thereto.

[0339] H18. The TI host cell of H4, wherein the first locus is an integration site within the XP_003512331.2 gene and the second locus is an integration site within a gene selected from the following group: LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and sequences at least about 90% homologous thereto.

[0340] H19. The TI host cell of any one of H4 to H18, wherein the at least one exogenous nucleotide sequence integrated into the first locus comprises a regulatable promoter.

[0341] H20. The TI host cell of any one of H4 to H18, wherein the at least one exogenous nucleotide sequence integrated into the second locus comprises a regulatable promoter.

[0342] H21. The TI host cell of any one of H4 to H18, wherein the at least one exogenous nucleotide sequence integrated into the first locus and the at least one nucleotide sequence integrated into the second locus comprise a regulatable promoter.

[0343] J. A method for preparing a TI host cell that expresses at least one polypeptide of interest, comprising: providing a TI host cell comprising at least one exogenous nucleotide sequence integrated into a site within one or more loci in the genome of the TI host cell, the one or more loci being at least about 90% homologous to a sequence selected from SEQ ID NOs: 1-7, and the at least one exogenous nucleotide sequence comprising two RRSs flanking at least one first selectable marker; introducing into the cell provided in a) a vector comprising at least one exogenous SOI and at least one second selectable marker, the two RRSs matching the two RRSs on the integrated exogenous nucleotide sequence; introducing a recombinase or a nucleic acid encoding a recombinase, wherein the recombinase recognizes the RRSs; and isolating the TI host cell that expresses the at least one polypeptide of interest by selecting for TI cells that express the second selectable marker.

[0344] J1. A method for preparing a TI host cell that expresses at least one first and second polypeptide of interest, comprising providing a TI host cell comprising at least one exogenous nucleotide sequence integrated into a site within one or more loci of the host cell genome, wherein the one or more loci are at least about 90% homologous to a sequence selected from SEQ ID NOs: 1-7, the exogenous nucleotide sequence comprising first and second RRSs flanking at least one first selectable marker, and a third RRS located between the first and second RRSs, all of which are heterospecific; and providing in the cell provided in a) a sequence identical to the first and third RRSs on the at least one integrated exogenous nucleotide sequence and at least The method comprises introducing a first vector comprising two RRSs flanked by a first exogenous SOI and at least one second selectable marker; introducing into the cells provided in a) a second vector comprising two RRSs that match the second and third RRSs on the at least one integrated exogenous nucleotide sequence and are flanked by at least one second exogenous SOI; introducing one or more recombinases or one or more nucleic acids encoding one or more recombinases, wherein the one or more recombinases recognize the RRSs; and isolating TI host cells that express the at least one first and second polypeptide of interest by selecting TI cells that express the second selectable marker.

[0345] J2. A method for preparing a TI host cell that expresses a polypeptide of interest, comprising: a) providing a TI host cell comprising at least one exogenous nucleotide sequence integrated into a site within one or more loci in the genome of the TI host cell, wherein the one or more loci are at least about 90% homologous to a sequence selected from SEQ ID NOs: 1-7, and the exogenous nucleotide sequence comprises one or more RRSs; b) introducing into the cell provided in a) a vector comprising at least one exogenous SOI that matches one or more RRSs on the integrated exogenous nucleotide sequence and that is operably linked to at least one regulatable promoter; c) introducing a recombinase or a nucleic acid encoding a recombinase, wherein the recombinase recognizes the RRSs; and d) isolating TI host cells that express the exogenous polypeptide of interest by selecting for TI cells that express the exogenous polypeptide of interest in the presence of an inducer.

[0346] J3. A method for expressing a polypeptide of interest, comprising: a) providing a host cell comprising at least one exogenous SOI flanked by two RRSs and a regulatable promoter integrated into a locus in the host cell genome, the locus being at least about 90% homologous to a sequence selected from SEQ ID NOs: 1-7; and b) culturing the cell under conditions suitable for expressing the SOI and recovering the polypeptide of interest therefrom.

[0347] J4. A method for preparing a TI host cell that expresses a first and a second polypeptide of interest, comprising: a) providing a TI host cell comprising an exogenous nucleotide sequence integrated into a site within a locus of the host cell genome, the locus being at least about 90% homologous to a sequence selected from SEQ ID NOs: 1-7, the exogenous nucleotide sequence comprising a first RRS, a second RRS, and a third RRS located between the first RRS and the second RRS, all of the RRSs being heterospecific; and b) providing in the cell provided in a) two RRSs flanking at least one first exogenous SOI that matches the first and third RRSs on the integrated exogenous nucleotide sequence and is operably linked to a regulatable promoter. c) introducing into the cell provided in a) a second vector comprising two RRSs flanking at least one second exogenous SOI that matches the second and third RRSs on the integrated exogenous nucleotide sequence and is operably linked to a regulatable promoter; d) introducing one or more recombinases, or one or more nucleic acids encoding one or more recombinases, wherein the one or more recombinases recognize the RRSs; and e) isolating TI host cells that express the first and second polypeptides of interest by selecting for TI cells that express the first and second exogenous polypeptides of interest in the presence of an inducer.

[0348] J5. The method of any one of claims J, J2, and J3, wherein the integration of nucleic acid comprising at least one SOI is facilitated by an exogenous nuclease.

[0349] J6. The method of Claim J1 or J4, wherein the first SOI encodes a single-chain antibody, an antibody light chain, an antibody heavy chain, a single-chain Fv fragment (scFv), or an Fc fusion protein, and the second SOI encodes a single-chain antibody, an antibody light chain, an antibody heavy chain, a single-chain Fv fragment (scFv), or an Fc fusion protein.

[0350] J7. The method of J5, wherein the exogenous nuclease is selected from the group consisting of zinc finger nucleases (ZFNs), ZFN dimers, transcription activator-like effector nucleases (TALENs), TAL effector domain fusion proteins, RNA-guided DNA endonucleases, engineered meganucleases, and clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) endonucleases.

[0351] J8. The method of any one of claims J-J4, wherein the regulatable promoter is selected from the group consisting of SV40 and CMV promoters.

[0352] J9. The method of any one of claims J2 and J4, wherein the first vector further comprises a promoter sequence operably linked to the codon ATG located upstream of the first SOI and adjacent to the downstream RRS, and the second vector further comprises a selectable marker lacking an ATG transcription start codon upstream of the RRS and adjacent to the downstream RRS of the second SOI.

[0353] K. A vector a) two reference sequences selected from any portion of SEQ ID NO: 1; b) two reference sequences selected from any portion of SEQ ID NO: 2; c) two reference sequences selected from any portion of SEQ ID NO: 3; d) two reference sequences selected from any portion of SEQ ID NO: 4; e) two reference sequences selected from any portion of SEQ ID NO: 5; f) two reference sequences selected from any portion of SEQ ID NO: 6; or g) two reference sequences selected from any portion of SEQ ID NO: 7 wherein the two nucleotide sequences are flanking a DNA cassette, and the DNA cassette is operably linked to a regulatable promoter and comprises at least one exogenous SOI flanked by two RRSs.

[0354] K1. The vector of K, wherein the vector comprises two nucleotide sequences that are at least 60% homologous to the two reference sequences.

[0355] K2. The vector of K, wherein the vector comprises two nucleotide sequences that are at least 70% homologous to the two reference sequences.

[0356] K3. The vector of K, wherein the vector comprises two nucleotide sequences that are at least 80% homologous to the two reference sequences.

[0357] K4. The vector of K, wherein the vector comprises two nucleotide sequences that are at least 90% homologous to the two reference sequences.

[0358] K5. The vector of K, wherein the vector comprises two nucleotide sequences that are at least 95% homologous to the two reference sequences.

[0359] K6. The vector of K, wherein the vector comprises two nucleotide sequences that are at least 99% homologous to the two reference sequences.

[0360] K7. The vector of any one of K to K6, 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.

[0361] K8. The vector of any one of K-K7, 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. [Example]

[0362] The following examples are merely illustrative of the subject matter disclosed herein and should not be construed as limiting in any way.

[0363] Example 1: Discovery of productive targeted integration sites in CHO host cells for clinical and commercial cell line development This example describes a method for identifying targeted integration loci in the genome of highly productive CHO cells. Conventional cell line development (CLD) is based on random integration (RI) of a plasmid carrying a sequence of interest (SOI). This process is unpredictable and labor-intensive. Therefore, identifying high-producing RI clones requires significant effort. Unlike conventional RI CLD, targeted integration (TI) CLD introduces a transgene with a defined copy number (usually 1–2 copies) into a predefined "hot spot" in the CHO genome. Given the low copy number and pretested integration site, TI cell lines should have superior stability compared to RI lines. Furthermore, because the selection marker is used only to select cells with the appropriate TI and not to select cells with high levels of transgene expression, applying a low-mutagenicity marker may minimize the possibility of sequence variants (SVs), which are partially due to the mutagenicity of selection agents such as methotrexate (MTX) or methionine sulfoximine (MSX).

[0364] Figure 1 is a schematic diagram outlining the genome-wide screening steps to identify CHO TI loci that enable stable, high-level antibody expression. To screen for transcriptionally active sequences in the CHO genome, two approaches were utilized to introduce antibody cassettes expressing either antibody A or antibody B, flanked by antibody sequences RRS1 and RRS2, into the CHO genome. One approach was a conventional random integration method, while the other was a transposase-based integration method, which required cotransfection of a transposase expression plasmid and an antibody plasmid. 15k to 20k transfectants were screened for each method. A total of approximately 300 clones with high antibody titers and low HC gene copy numbers, based on intact IgG ELISA assays and gene copy analysis, were expanded in shake flasks for fed-batch evaluation. Forty clones representing 40 different potential highly transcriptionally active integration sites identified by the two methods and with acceptable titer and product quality characteristics, were then selected for GFP landing pad exchange to generate TI hosts.

[0365] To replace the antibody cassette at the transcriptionally active locus, a landing pad encoding a GFP gene flanked by the same RRS1 and RRS2 genes and an appropriate selection marker was constructed for RMCE. To initiate RMCE, the recombinase and GFP landing pad were cotransfected into each of the top 40 clones. Two selection markers were used to enhance targeted integration events. If RMCE is successful, the GFP landing pad should target the transcriptionally active locus and replace the antibody cassette, resulting in increased GFP expression and loss of antibody expression. The phenotypic change from GFP- / mAB+ to GFP+ / mAb- should be easily detectable using FACS analysis. GFP+ / mAb- enrichment was detected in 14 of the 40 RMCE pools using FACS analysis. Each of these 14 RMCE pools was then single-cell cloned to isolate individual TI host candidates. A total of 90 potential TI host candidates were selected from the 14 pools based on FACS and genomic PCR confirmation, in which the original antibody cassette was removed and replaced with a GFP landing pad at the intended integration site. Several of these candidate hosts were subsequently evaluated for their RMCE capacity and efficiency using test antibody C, which flanks the same RRS1 and RRS2. Depending on how transcriptionally active loci were identified, two dual selection schemes were utilized to generate populations expressing the target antibody (Figures 2A and 2B). The composition of the landing pad differed slightly between the two screening methods. Based on antibody productivity as well as RMCE efficiency of the TI transfection pools, seven final hosts were selected, representing seven unique integration sites in the CHOK1M host for high antibody expression.

[0366] These seven hosts were analyzed by targeted locus amplification / next-generation sequencing (TLA / NGS) to identify CHO genomic sequences flanking the integration site, thereby providing gene sequences where the desired integration site may reside. Table 2 - Integration sites in TI host cells TIFF2026035601000003.tif147170

[0367] Based on the integration site, the 5' flanking sequence can include 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.

[0368] Based on the integration site, the 3' flanking sequence can include 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.

[0369] Further analysis of two of the hosts, hosts 4 and 7, showed that a single GFP landing pad had been inserted into the CHO host genome. No antibody sequences were found in these two hosts, indicating the complete removal of the initial antibody cassette used to identify transcriptionally active loci. Additionally, we performed in-house whole-genome sequencing of genomic DNA isolated from these two TI hosts. While no antibody-specific sequences were detected in these two TI hosts, GFP sequences (from the landing pad) were readily detected. We achieved greater than 63x genome coverage in the TI hosts. To further ensure the absence of antibodies A and B in the TI hosts, multiple HCCF samples from TI cell lines expressing antibodies other than antibodies A and B were evaluated using LC-MS experiments. The LC-MS data were analyzed using a similar approach to sequence variant analysis by LC-MS / MS, which has the ability to detect variant peptides at levels greater than 0.5%. No antibody A or B peptides were detected in any samples collected from the TI cell lines. Finally, fluorescent in-situ hybridization (FISH) analysis was also performed by Chrombios to determine the chromosomal location of the GFP landing pad in these two TI host genomes.

[0370] These two well-characterized TI hosts were further evaluated for the robustness of their RMCE in clinical cell line development using a total of 16 different standard antibodies. Figure 3 shows the productivity of the top clones generated by these TI hosts. Historically, the average productivity of cell lines generated by random integration was approximately 3 g / L. In most cases tested, the TI cell lines showed productivity that was comparable to or better than that of the RI clones, on average.

[0371] We expected that TI cell lines would be more stable and have lower SV than RI cell lines due to their lower antibody gene copy number. To ensure clonal stability, monthly fed-batch production was set up for four months, and productivity was monitored. Table 3 shows that only 10% of TI cell lines generated by one of the TI hosts experienced a titer drop of more than 20% 120 days after PSB. Historically, 60% of RI cell lines have had a similar titer drop. This strongly suggests that the identified integration site is highly suitable for stable antibody expression. We also subjected TI cell lines to NAT-based sequence variant analysis. The frequency of SV above 5% was significantly lower in TI cell lines compared to RI cell lines (4% vs. 15%, Table 3). Table 3. Advantages of TI vs. RI TIFF2026035601000004.tif85170

[0372] Example 2: Two-plasmid RMCE strategy To address the challenges of achieving high-titer expression of standard antibodies and expressing multi-chain complexes, we developed an innovative two-plasmid RMCE strategy. The two-plasmid RMCE method allows for eight or more chains to simultaneously target the TI site. This approach not only improves productivity by providing greater flexibility in adjusting the HC and LC chain ratio of antibodies, but also allows for the creation of multi-chain complexes, as well as the targeting of transgenes, endogenous genes, or RNAi with antibodies to alter cellular pathways.

[0373] The two-plasmid RMCE strategy involves using three RRS sites to simultaneously perform two independent RMCEs (Figure 4). Therefore, the GFP landing pad in the TI host described above was replaced with a third RRS site (RRS3) that does not cross-react with either the RRS1 or RRS2 sites. The two targeting expression plasmids require the same flanking RRS sites for efficient targeting: one expression plasmid (forward) is flanked by RRS1 and RRS3, and the other (reverse) is flanked by RRS3 and RRS2. Because we expect the efficiency of two-plasmid RMCE to be low, a stringent selection scheme is useful to enhance rare RMCE-specific events. Two-plasmid RMCE requires at least two selectable markers. In certain embodiments, three selectable markers are used, splitting the single selectable marker expression cassette into two parts (see, e.g., Figure 4). In certain embodiments involving split selectable marker expression cassettes, the forward plasmid will contain a promoter followed by a start codon and an RRS3 sequence. The rear plasmid will have the RRS3 sequence fused to the N-terminus of the selectable marker coding region, excluding the ATG start. Additional nucleotides may need to be inserted between the RRS3 site and the selectable marker sequence to ensure in-frame translation of the fusion protein. Only if the two plasmids are correctly targeted will the complete expression cassette of the selectable marker be assembled, thus conferring resistance to selection to the cells. Figure 4 is a schematic diagram illustrating the two-plasmid RMCE strategy. Of course, single-plasmid RMCE can subsequently be performed using RRS1 and RRS2, if desired.

[0374] To test the robustness of the two-plasmid RMCE approach, the original GFP landing pad was replaced with a new GFP landing pad containing three RRS sites using a previously identified TI host, i.e., host 4. Compared to single-plasmid RMCE, we observed a more severe reduction in viability and a longer recovery time with two-plasmid RMCE, consistent with a lower initial RMCE efficiency with two-plasmid RMCE. Once pools were harvested, they were assessed using FACS, genomic PCR, and gene copy analysis to confirm that both plasmid cassettes accurately targeted the TI site. In total, five standard antibodies (Q, R, S, T, and U) were tested for productivity using both single-plasmid and two-plasmid RMCE (Figure 5). With two-plasmid RMCE, more total HC and LC copies were targeted to the TI site compared to single-plasmid RMCE. In all five cases, the productivity of the two-plasmid TI transfection pools was consistently higher, increasing by up to 200% compared to the productivity of the single-plasmid pools. The titer increase in two-plasmid RMCE was mainly due to specific productivity, which was found to be a much higher improvement of 300% compared to that of single-plasmid RMCE.

[0375] Well-characterized TI hosts, e.g., Host 4, were further evaluated in different cell culture platforms for their ability to achieve high titers with five different antibodies. Figure 6 shows the productivities of these antibodies by these TI hosts. For most antibodies, productivities greater than 10 g / L were achieved by day 14, and greater than 12 g / L by day 16.

[0376] To evaluate the expression of a composite mAb format using two-plasmid RMCE, two bispecific molecules were tested that required the expression of four different chains (two HC and two LC) in the same cells for bispecific assembly. By allowing for simultaneous targeting with two separate expression plasmids, the plasmid configuration of the different chains could be manipulated to achieve optimal chain ratios for balanced expression. In both cases, two-plasmid RMCE was used to develop cell lines derived from host 4 with >1.5 g / L bispecific content at >80% (Figure 7).

[0377] We also evaluated day 14 titers for four bispecific molecules that required the expression of four different chains (two HC and two LC) in the same cell for bispecific assembly. All cell lines with titers above 1.5 g / L (Figure 8) with bispecific content above 80% were developed using two-plasmid RMCE and were derived from host 4.

[0378] Example 3: Expression and proliferation analysis of RTI cell lines expressing mAb-I (difficult to express) versus mAb-II (average expressing) antibody molecules A difficult-to-express molecule is defined as one in which all cell lines generated after several CLD attempts achieve titers below those typically expected from a standard CLD platform process. For one previously identified difficult-to-express antibody (mAb-I), for example, over 120 cell lines from four separate CLD attempts were evaluated in fed-batch production cultures. Nevertheless, compared with an average antibody molecule (mAb-II), for which only 24 cell lines were screened, even the highest mAb-I-expressing cell line achieved only 50% of the normal titer (Table 9A, Figure 9A). Unfortunately, investigating the underlying cause(s) that may make a molecule difficult to express in a random integration system is extremely difficult, primarily due to differences in transgene copy number and gene integration site among different cell lines.

[0379] To identify the underlying factor(s) that make mAb-I difficult to express, we initiated CLD for both mAb-I and mAb-II molecules using the RTI system. Both the HC and LC constructs were cloned into expression vectors under the control of the inducible CMV-TO promoter, and these vectors were transfected into TI hosts to increase the likelihood of isolating cell lines with similar transgene transcription levels. An overall schematic of the RTI CLD approach for mAb-I and mAb-II is shown in Figure 9B. Transfected cell lines were subjected to selection using two separate conditions: 1) induced in the presence of Dox (induced) or 2) induced in the absence of Dox (uninduced) throughout the CLD process. Cell lines induced in the absence of Dox were only transiently induced and ranked based on seed train titer or during production assays, where indicated. This allowed for evaluation of the role of antibody expression (expression pressure) during the selection process (Figure 9B).

[0380] Production assays were performed using two representative mAb-I and mAb-II cell lines derived from host 7 from both the "induced" and "uninduced" seed train populations in the presence or absence of doxycycline. The latter were used to assess promoter leakiness and basal antibody production levels in the absence of induction. In the absence of doxycycline, fairly tightly regulated antibody expression was observed, suggesting that the regulated expression system functioned effectively in these cell lines (Figures 10A and 10C). The growth (Figure 10B) profiles of all cell lines in the presence or absence of doxycycline were comparable, although the growth profiles appeared slightly better in the absence of doxycycline. Cell lines induced under "induced" or "uninduced" seed train conditions exhibited relatively comparable titers and specific productivity during production (Figures 10A and 10C, comparing mAb-I or mAb-II cell lines 1 and 2 with 3 and 4), indicating that constitutive antibody expression during selection did not play a major role in the isolation of high-titer cell lines (Figure 10). In addition, similar to the RI CLD trials, the titers and specific productivity of the mAb-I RTI cell line were an additional 50% lower than those of the mAb-II RTI cell line (Figures 10A and 10C). This observation ruled out chronic toxicity as a cause of low expression of mAb-I, as both the "induced" and "uninduced" RTI cultures of mAb-I exhibited similarly low titers compared to the mAb-II control cell line.

[0381] Example 4: Lower antibody expression in the mAb-I cell line was not due to lower transcription To analyze the lower antibody expression observed in the mAb-I-expressing cell line, we performed qRT-PCR experiments to measure the mRNA levels of antibody HC and LC in RTI cell lines expressing mAb-I or mAb-II under doxycycline-induced and non-induced conditions. In the absence of Dox, the mAb-I or mAb-II cell lines expressed little or no HC or LC mRNA, indicating relatively tight transcriptional regulation by the inducible promoter (Figure 11). In the presence of Dox, antibody HC and LC transcript levels in the mAb-I-expressing cell line were comparable to or even higher than those in the mAb-II-expressing cell line (Figures 11A and 11B). These results confirmed that reduced transgene transcript levels were not the reason for the reduced antibody expression in the mAb-I-expressing cell line. Furthermore, the presence or absence of doxycycline in seed train cultures during the CLD process had no effect on transgene transcription rates, as cell lines isolated under "induced" or "uninduced" conditions had comparable HC or LC mRNA levels (Figures 11A and 11B).

[0382] Example 5: Induction of mAb-I expression resulted in reversible accumulation of intracellular BiP and delayed antibody HC degradation To identify the underlying factors affecting mAb-I expression, we investigated antibody secretion, folding, and degradation using Dox induction along with cycloheximide (CHX) treatment and removal. In these experiments, "induced" cultures were treated with CHX to halt protein synthesis, and after 5 h, cells were washed and resuspended in medium containing Dox alone (no CHX) to resume protein synthesis (Figure 12A). Approximately 60 min after CHX removal, no HC or LC was detected in the supernatants of these cultures; only a gradual increase in antibody secretion was detected at a similar rate after 120 min (Figure 12B, top panel). This suggested that secretion of properly folded and assembled antibody molecules was comparable between the mAb-I and mAb-II cell lines after CHX removal. Intracellular levels of antibody LC were also fairly comparable between the mAb-I- and mAb-II-expressing cell lines (Figure 12B, LC panel). However, even 5 h after CHX treatment, we detected significantly higher levels of intracellular antibody HC in the mAb-I-expressing cell line compared with the mAb-II-expressing line (Figure 12B, HC panel). This was related to delayed or problematic degradation and clearance of unfolded or misfolded antibody HC in the mAb-I-expressing cell line. Interestingly, we detected significantly higher levels of intracellular BiP in the mAb-I-expressing cell line compared with the mAb-II-expressing cell line both before and after CHX treatment (Figure 12B, bottom panel, BiP). BiP, as a chaperone, is involved in the folding of antibody HCs or LCs in addition to the unfolded protein response (UPR), and its presence in seed train cultures could be an indicator of ER-associated cellular stress.

[0383] To further investigate the correlation between mAb-I expression and intracellular BiP accumulation, we treated "induced" seed train cultures overnight with or without CHX in medium containing doxycycline, and assessed the intracellular HC, LC, and BiP levels in these samples. Without CHX treatment, the basal levels of intracellular antibody HC molecules were higher in the mAb-I compared with the mAb-II cell line, whereas the levels of intracellular LC were comparable across all sample sets. On the other hand, BiP levels were significantly higher in the mAb-I compared with the mAb-II-expressing cell line (Figure 12C, -CHX sample). Treatment of these cultures overnight with CHX reduced the levels of HC and LC in all cell lines, as well as BiP levels, particularly in the mAb-I-expressing cell line (Figure 12C, +CHX sample). This was consistent with previous findings (Figure 12B). Because cycloheximide indiscriminately inhibits the synthesis of all eukaryotic proteins, we were unable to directly correlate the higher BiP levels in the mAb-I cell line with the accumulation of antibody HC or LC subunits. However, the regulated expression system provided the ability to directly test the relationship between intracellular BiP and antibody HC or LC levels. To do so, we removed doxycycline from the medium for 6 days, specifically shutting off antibody expression without affecting the expression of other endogenous proteins, and monitored intracellular HC, LC, and BiP levels. We observed a direct and specific relationship between antibody (HC and LC) expression and intracellular BiP accumulation in the mAb-I cell line, whereas expression of mAb-II showed no effect on intracellular BiP levels (Figure 12D).

[0384] Example 6: mAb-I LC expression induced intracellular BiP accumulation, but mAb-I HC also clearly contributed to the low expression of this antibody To determine whether the mAb-I LC played a role in intracellular BiP accumulation, we generated a construct expressing the mAb-I HC under a regulated promoter while constitutively expressing the LC. This construct was co-transfected into CHO cells with constructs expressing the regulated HC / LC of mAb-I or mAb-II (control), yielding two separate pools from each transfection. Pool titers were assessed in production cultures (Figure 13A), while seed train cultures were assessed by Western blot analysis in the presence and absence of doxycycline (Figure 13B). During production assays, each pool was either induced with Dox or left uninduced. The uninduced pool expressed little antibody during production, indicating tight expression regulation (Figure 13A, -Dox). Analysis of Dox-induced cultures during production revealed that the regulated HC / constitutive LC mAb-I expression pool derived from the "uninduced" seed train culture had significantly lower titers than the regulated HC / LC mAb-I pool and the regulated HC / constitutive LC mAb-I pool "induced" during seed train (Figure 13A). Western blot analysis of the "uninduced" cultures showed that the antibody HC was expressed only when Dox was added to the medium under all conditions. In addition, constitutive expression of the mAb-I LC alone, without the mAb-I HC, induced the accumulation of intracellular BiP (Figure 13B). These results suggested a direct link between the expression of the mAb-I LC subunit and the accumulation of intracellular BiP. When constitutively expressed in the absence of the HC, the mAb-I LC molecule may form unfolded aggregates in the ER that interact with BiP chaperones, leading to increased intracellular levels of BiP in these cells. Expression of the mAb-I HC during the production phase further burdened these cells, increasing the demand for intracellular BiP responsible for folding both the HC and LC molecules, which could result in inefficient antibody assembly, folding, and secretion efficiency (Figure 13A).

[0385] Although these findings indicated a link between mAb-I LC expression and cellular ER stress, as indicated by the accumulation of intracellular BiP, a direct link between mAb-I LC expression and the low titer of the mAb-I cell line needed to be established. To this end, an antibody molecule was generated that swapped the heavy and light chains of mAb-I and mAb-II to produce the HC I LC I , H.C. II LC II , H.C. I LC II , and H.C. II LC I A combination of antibodies was generated. I LC II The construct expressed an antibody with mAb-I HC and mAb-II LC, but not HC II LC I The constructs expressed antibodies with mAb-II HC and mAb-I LC. Antibody HC and LC expression for all these constructs was under the control of the CMV-TO promoter in the RTI vector system (Figure 13C). These constructs were then transfected into our CHO TI host cells to generate two separate RTI pools for each construct, and the expression of mAb-I, mAb-II, or hybrid forms of these antibodies was tested. As mentioned previously (Figure 13B), we observed a clear and direct correlation between mAb-I LC expression and the accumulation of intracellular BiP (Figure 13D, LC and BiP blots). Interestingly, intracellular BiP also expressed HC II LC II Compared to HC I LC II Although not very strong, they accumulated in a significant manner in the pools expressing hybrid antibodies, and in these pools, HC I This suggested a certain level of ER stress, which may be due to the expression of II LC II and H.C. I LC I (Compare long-term exposure BiP levels in the samples). Ablation of antibody expression by Dox removal resulted in the loss of mAb-II (HC B LC BAll RTI pools except the ) pool resulted in a decrease in intracellular BiP levels, and the initial levels of both intracellular HC and BiP were significantly reduced even before Dox removal (Fig. 13D, HC II LC II (Compare HC and BiP levels on day 0 between the pool and the other pool).

[0386] To assess the role of mAb-I HC and LC on overall antibody expression, HC I LC I , H.C. II LC II , H.C. I LC II , and H.C. II LC I The RTI pools were evaluated in production assays. In the absence of doxycycline, all RTI pools expressed little antibody, indicating tight regulation of antibody expression (Figure 14A, -Dox). The growth rates of all RTI pools were similar, with induced cultures having slightly lower growth rates compared to non-induced cultures (Figure 14B). As previously observed, the mAb-I and mAb-II RTI pools had the lowest and highest titers and specific productivity, respectively (Figures 14A and 14C). Interestingly, HC II LC I and H.C. I LC II Both RTI pools were I LC I was higher than that, but HC II LC II The mAb-I pool showed lower titers and specific productivity than the RTI pool (Figures 14A and 14C). These results indicated that both the mAb-I HC and LC subunits individually contributed to the reduced expression of this antibody compared to mAb-II. Contrary to our expectations, the LC I The hybrid RTI pool expressing the subunits was IThe mAb-I HC had slightly higher titers and specific productivity compared to the pools expressing the mAb-I LC subunit (Figures 14A and 14C). This indicated that the mAb-I HC played a slightly more important role in the reduced expression of this antibody compared to the LC subunit. Use of the antibody chain exchange approach in combination with the RTI system revealed negative effects associated with the expression of the mAb-I HC subunit, even when clear indicators of biological stress (such as intracellular BiP accumulation) could not be identified.

[0387] Example 7: Amino acid sequence differences between the HC and LC subunits of mAb-I and mAb-II To assess whether certain amino acid residues may contribute to the problematic expression of mAb-I HC and LC, the amino acid sequences of the LC (FIG. 15A) and HC (FIG. 15B) subunits of mAb-I and mAb-II were aligned. The amino acid compositions of all CDR regions for the HC and LC molecules of mAb-I and mAb-II were different from each other because they were developed to target different antigens. In the LC subunit, there are no amino acid sequence differences between the mAb-I and mAb-II molecules for the remainder of the variable region and all constant regions. I The CDR1 segment of LC II Although it is 6 amino acids longer than LC II The CDR3 segment of LC I As for the antibody HC subunit, except for the CDR segments, the HC subunit is one amino acid longer than the CDR segments, except for four amino acid residues. I and H.C. II The remainder of the variable and constant regions of HC are identical (Figure 15B). Two of these amino acid changes are fairly conservative (T→S and L→A), and therefore HC I It is unlikely that this contributes to the low expression of HC. I The N→G change in HC prevents antibody HC glycosylation. I HC on the expression of II and H.C. IThe effects of N → G and Y → V amino acid differences between HC and HC were investigated. Two point mutations were generated, and HC I The G and V amino acid residues of HC1 were changed to N and Y, respectively. A Double mutant or HC I dm). HC I dm to LC I and L.C. II RTI pools were generated using HC and antibody expression in these pools was assessed in production cultures (Fig. 16A). I dm to LC I By co-expressing with HC II LC II This indicates that these mutations increased the expression of HC A This suggests that the low expression level of the antibody can be restored (Fig. 16A). II When co-expressed with HC I dm is HC II LC II This indicates that the titer achieved was higher than that of HC I dm is HC II This suggests that HC has superior expression and folding properties compared to HC (Fig. 16A). I LC I had the lowest titer, while HC I LC II showed an intermediate antibody expression profile (Fig. 16A). Western blot analysis revealed that HC I Even when co-expressed with dm, LC I It was revealed that expression of β-lactamase inhibitors (β-lactamase inhibitors) was still associated with increased intracellular BiP accumulation (Figure 16B). Figure 16C shows a diagnostic roadmap of how the RTI system can be used to analyze and identify the cause of problems in cell lines expressing difficult-to-express molecules.

[0388] Example 8: Generation of TI host cells containing two distinct and independent landing pads. Host 8 is a targeted integration (TI) host containing two distinct and independent landing pads, which can target the expression of antibody genes individually or simultaneously. The parent cell line for Host 8 is Host 7 (see Table 2), which contains a single landing pad. A second landing pad was inserted into Host 8 at the genomic location where the landing pad was located in the Host 4TI host (see Table 2). This insertion was performed by CRISPR / Cas9-based knock-in of the new landing pad into the Host 4TI site location within the Host 7TI host.

[0389] To achieve this, the genomic region of the host 4TI site was characterized in TI host 4 and TI host 7. Based on this information, a guide RNA was constructed to target this specific genomic region to facilitate CRISPR / Cas9-based knock-in. Separately, a donor plasmid containing the newly created landing pad was generated to knock-in into this site, constructed with homology arms corresponding to the genomic regions upstream and downstream of the targeted knock-in site to facilitate knock-in, as well as additional genes to enable screening of knock-in clones. Co-transfection of the plasmid containing the gRNA and Cas9 genes and the donor plasmid was performed to facilitate CRISPR / Cas9-based knock-in of the new landing pad.

[0390] After selection and screening of clones that underwent CRISPR / Cas9-based knock-in, host 8 was finally identified and further evaluated. The existing host 7 landing pad was determined to be intact, and the newly knocked-in landing pad at the host 4 site was also confirmed to be present, intact, and correctly positioned.

[0391] The feasibility of recombinase-mediated cassette exchange (RMCE) at both sites was then evaluated for each site individually and then simultaneously. Clones were generated by RMCE on the landing pad of host 7 only, the landing pad of host 4 only, and both sites simultaneously, and then evaluated in a 14-day fed-batch shake flask evaluation. The titers and specific productivity of clones expressing antibody simultaneously at both sites were higher compared to expression at each site individually. Further evaluation was performed on cell culture platform B, a different cell culture process, and showed further increases in titers for all clones compared to cell culture platform A (Figure 17). The average titer at day 14 for the host 4 site was 3.0 g / L in platform A cell culture, 1.9 g / L for the host 7 site, and 3.9 g / L for both sites simultaneously.

[0392] Finally, TI host 8 was generated from TI host 7 by adding a second landing pad located at the genomic location of the landing pad of TI host 4. Further investigations confirm the feasibility of landing pad knock-in and RMCE at both existing and novel landing pads. Furthermore, evaluation of clones generated from host 8 at each site individually and at both sites simultaneously demonstrates the productivity of antibody expression. Expressing antibody genes at both sites simultaneously, versus at individual sites, has an additive effect on productivity seen in both cell culture platform A and cell culture platform B (Figure 17).

[0393] method Antibody Plasmid DNA Construct Construction: To construct the 1-plasmid antibody constructs, the antibody HC and LC fragments were cloned into a vector backbone containing the L3 and 2L sequences plus a puromycin N-acetyltransferase (pac) selectable marker. To construct the 2-plasmid antibody constructs, the antibody HC and LC fragments were cloned into a forward vector backbone containing the L3 and LoxFAS sequences, and a reverse vector containing the LoxFAS and 2L sequences and a pac selectable marker. The Cre recombinase plasmid pOG231 (Wong ET et al., Nucleic Acids Res 2005, 33, (17), e147; O'Gorman S et al., Proc Natl Acad Sci USA 1997, 94, (26), 14602-7, the contents of each of which are incorporated herein by reference) was used for all RMCE processes.

[0394] Cell culture: CHO cells were cultured in a proprietary DMEM / F12-based medium in 125 mL shake flasks with shaking at 150 rpm, 37°C and 5% CO2. Cells were cultured at 3 x 10 5 The cells were passaged at a seeding density of 1000 cells / mL.

[0395] RMCE stable cell line development: Expression plasmids were transfected into TI hosts by MaxCyte STX® electroporation (MaxCyte, Gaithersburg, MD). RMCE transfection pools were then selected with puromycin and 1-(2'-deoxy-2'-fluoro-1-beta-D-arabinofuranosyl-5-iodo)uracil (FIAU) (Moravek). After pool selection, single-cell cloning (SCC) was performed to generate TI cell lines for further evaluation.

[0396] Fed-batch production assay: Fed-batch production cultures were performed in shake flasks or ambr15 vessels (Sartorius Stedim) using a proprietary chemically defined production medium. Cells were cultured at 1 × 10 on day 0. 6Cells were seeded at 0.5 cells / ml and the temperature was shifted from 37°C to 35°C on day 3. Cultures received proprietary feed medium on days 3, 7, and 10. Viable cell count (VCC) and percent viability of cultured cells were measured on days 0, 3, 7, 10, and 14 using a Vi-Cell™ XR instrument (Beckman Coulter). Glucose and lactate concentrations were measured on days 7, 10, and 14 using a Bioprofile 400 Analyzer (Nova Biomedical). Titers on day 14 were determined using Protein A affinity chromatography with UV detection.

[0397] Determination of gene copy number by droplet PCR: Droplet PCR assays were performed using the ddPCR™ Supermix kit (Bio-Rad). Each ddPCR reaction contained ddPCR master mix, 900 nM forward primer, 900 nM reverse primer, 250 μM probe, 3 units of HaeIII restriction enzyme, and sample DNA. After 10 minutes of incubation at room temperature, droplets were generated using an Automatic Droplet Generator (Bio-Rad). The PCR thermal cycling conditions were 95°C for 10 minutes, followed by 40 cycles of 94°C for 30 seconds and 60°C for 1 minute, followed by 98°C for 10 minutes to inactivate the enzyme. After the PCR reaction, droplets were read on a QX200™ Droplet reader (Bio-Rad). Data were collected and analyzed using Quanta software. HC and LC gene copy numbers were normalized based on the defined copy numbers of the reference genes Bax, albumin, Hprt, and β-microglobulin. The primers used in this experiment were designed using Primer Express v3.0 (Life Technologies). The primer sequences are listed in Table 4. Table 4. Droplet PCR primer sequences. TIFF2026035601000005.tif255170

[0398] Quantitative real-time PCR (qRT-PCR or TaqMan) analysis: RNA samples from seed train cultures were purified using the Qiagen RNeasy plus mini kit according to the manufacturer's instructions. TaqMan RT-PCR assays were performed using the TaqMan One® Step RT-PCR Master mix kit (Life Technologies). Each RT-TaqMan PCR reaction contained RT-PCR master mix, reverse transcriptase, 300 nM forward primer, 300 nM reverse primer, 100 nM probe, and 10 ng of purified RNA sample. Thermal cycling conditions were 48°C for 30 minutes and 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. All reactions were processed on a 7900 HT Fast Real-Time PCR System (Life Technologies). Data were analyzed after TaqMan RT-PCR amplification using SDS v2.4 software (Life Technologies). The relative expression levels of HC and LC were determined based on delta Ct analysis. The expression level of the housekeeping gene cyclophilin was used as a reference gene to normalize the different RNA samples in each reaction. The primers used in this experiment were designed using Primer Express v3.0 (Life Technologies). The primer sequences are listed in Table 5. Table 5. Primer sequences for quantitative real-time PCR TIFF2026035601000006.tif135170

[0399] Western blot analysis: Cell pellets from seed train cultures were washed with PBS and resuspended in NP-40 lysis buffer containing a mini cOmplete™ cocktail of protease inhibitors (Roche). Cell lysates were centrifuged at 13,000 RPM for 10 minutes, and the supernatants were collected. The protein concentration of each lysate was determined using the Pierce BCA Protein Assay Kit (Thermo Scientific). Equal protein concentrations of each lysate were mixed with SDS-PAGE buffer and NuPage Sample Reducing Agent (Invitrogen) and then heat-denatured at 95°C for 5 minutes. Lysates were then loaded onto NuPage precast 4-12% Bis-Tris gels (Invitrogen) and electrophoresed. Proteins were transferred to nitrocellulose membranes using the iBlot system (Invitrogen). The membranes were blocked for 1 hour at room temperature using a 5% nonfat milk solution in Tris-buffered saline (TBS-T) containing 0.1% Tween 20. After blocking, membranes were incubated with specific primary antibodies for 1 hour at room temperature, followed by three 10-minute washes with TBS-T. Secondary horseradish peroxidase-conjugated antibodies were added for 1 hour at room temperature, followed by three 15-minute washes with TBS-T. Membranes were developed using either Amersham ECL detection reagent or Amersham ECL Prime detection reagent (GE Life Sciences). Image Lab 5.1 (Biorad) was used to quantify band signals. The following primary antibodies were used: goat anti-human IgG-HRP (MP Biomedicals) at 1:3000 and mouse anti-β-actin (Sigma Aldrich) at 1:2500. The following secondary antibodies were used: sheep anti-mouse (GE Healthcare UK) at 1:10000.

[0400] Example 9: Addition of an additional light chain to a one heavy, one light chain plasmid DNA construct improves antibody titer and specific productivity. To investigate the effect of antibody copy number on the productivity of TI hosts, RMCE pools were generated by transfecting a single plasmid containing either one heavy chain and one light chain (HL configuration) or one heavy chain and two light chains (HLL configuration). After selection, recovery, and validation of RMCE by flow cytometry, the productivity of the pools was assessed in a 14-day fed-batch production assay. For three distinct antibodies (mAb-Q, mAb-S, and mAb-T), we observed an approximately 2-fold increase in titer in the HLL pool compared to the HL pool, primarily driven by a 1.5- to 2.5-fold increase in specific productivity (Figures 18A and 18B). Additional single-cell clones were generated from the transfected pools using limiting dilution, confirming that the titer and specific productivity were also higher for clones derived from the HLL configuration than for those from the HL configuration (Figures 18C and 18D).

[0401] Example 10: Transfection with up to seven antibody chains in RMCE pools increases antibody specific productivity and titer. The effect of increasing the number of antibody chains on antibody expression was evaluated. For five antibodies, the HLL single plasmid construct was compared with the HLL-HL or HLL-HLL double plasmid constructs.

[0402] We compared the HLL-HL (5-chain), HLL-HLL (6-chain), and HLL-HLHL (7-chain) configurations of mAb Y. The titers and Qp of RMCE pools during production generally increased from the HLL-HL configuration to the HLL-HLL and HLL-HLHL configurations (Figures 19A and 19B), but growth (expressed as the integrated viable cell count, IVCC) was relatively comparable among the different pools (Figure 19C). When examining seed-train antibody mRNA expression in these pools, we observed an increase in light chain mRNA from HLL-HL to HLL-HLL and HLL-HLHL, as well as an increase in heavy chain mRNA in the HLL-HLHL pool compared to the HLL-HL and HLL-HLL pools (Figure 19D). Intracellular protein levels of heavy and light chains from these RMCE pools were also measured using Western blot. Intracellular light chain protein levels increased from five to six and seven chain configurations, in close agreement with that observed for light chain mRNA (FIGS. 19E, 19F).

[0403] Example 11: Single cell clones with a seven-chain HLL-HLHL configuration have high titers and specific productivity for two test antibodies. To assess whether the high titer and productivity of the seven-chain HLL-HLHL construct were maintained after single-cell cloning, single-cell monoclones were generated from the HLL-HL, HLL-HLL, and HLL-HLHL RMCE pools of mAb Y. These monoclones were then evaluated in a 14-day fed-batch production assay. Looking at the top six clones for each construct, it was found that titer and Qp increased slightly from the HLL-HL construct to HLL-HLL and HLL-HLHL (Figures 20A and 20B), while growth remained similar (Figure 20C).

[0404] To confirm that the HLL-HLHL configuration resulted in high monoclonal titers, a different mAb, mAb III, was used to generate single-cell clones derived from host 4 from HLL-HL (5-chain) and HLL-HLHL (7-chain) RMCE pools. After evaluation in a 14-day fed-batch production assay, we observed a clear titer advantage of HLL-HLHL clones over HLL-HL clones (Figure 20D), which was higher than that seen with mAb U. The increased titer appeared to be primarily driven by the higher Qp of the HLL-HLHL clones (Figure 20E), with little difference in mean IVCC between the two configurations (Figure 20F).

[0405] Example 12: The position of antibody chains in the transfecting plasmid affects their productivity. The e...

Claims

1. 1. A targeted integration (TI) host cell comprising an exogenous nucleotide sequence integrated into an integration site within a specific locus in the genome of the host cell, the locus being at least about 90% homologous to a sequence selected from SEQ ID NOs: 1-7.

2. 2. The TI host cell of claim 1, wherein the nucleotide sequence immediately 5' to the integrated exogenous nucleotide sequence is selected from the group consisting of a sequence at least about 90% homologous to nucleotides 41190-45269 of NW_006874047.1, nucleotides 63590-207911 of NW_006884592.1, nucleotides 253831-491909 of NW_006881296.1, nucleotides 69303-79768 of NW_003616412.1, nucleotides 293481-315265 of NW_003615063.1, nucleotides 2650443-2662054 of NW_006882936.1, or nucleotides 82214-97705 of NW_003615411.

1.

3. the nucleotide sequence immediately 5' to the integrated exogenous nucleotide sequence is nucleotide 45269 of NW_006874047.1, nucleotide 207911 of NW_006884592.1, nucleotide 491909 of NW_006881296.1, nucleotide 79768 of NW_003616412.1, nucleotide 315265 of NW_003615063.1, nucleotide 2662054 of NW_006882936.1, or nucleotide 977 of NW_003615411.1 2. The TI host cell of claim 1, wherein the TI host cell is selected from the group consisting of a sequence of at least 15 base pairs, at least 20 base pairs, at least 30 base pairs, at least 40 base pairs, at least 50 base pairs, at least 75 base pairs, at least 100 base pairs, at least 150 base pairs, at least 200 base pairs, at least 300 base pairs, at least 400 base pairs, at least 500 base pairs, at least 1,000 base pairs, at least 1,500 base pairs, at least 2,000 base pairs, or at least 3,000 base pairs from 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,

4. the nucleotide sequence immediately 3' to the integrated 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 79 769-100059 of NW_003615063.1, nucleotides 315266-362442 of NW_006882936.1, nucleotides 2662055-2701768 of NW_006882936.1, or nucleotides 97706-105117 of NW_003615411.

1.

5. the nucleotide sequence immediately 3' to the integrated exogenous nucleotide sequence is nucleotide 45270 of NW_006874047.1, nucleotide 207912 of NW_006884592.1, nucleotide 491910 of NW_006881296.1, nucleotide 79769 of NW_003616412.1, nucleotide 315266 of NW_003615063.1, nucleotide 2662055 of NW_006882936.1, or nucleotide 977 of NW_003615411.1 2. The TI host cell of claim 1, wherein the TI host cell is selected from the group consisting of a sequence of at least 15 base pairs, at least 20 base pairs, at least 30 base pairs, at least 40 base pairs, at least 50 base pairs, at least 75 base pairs, at least 100 base pairs, at least 150 base pairs, at least 200 base pairs, at least 300 base pairs, at least 400 base pairs, at least 500 base pairs, at least 1,000 base pairs, at least 1,500 base pairs, at least 2,000 base pairs, or at least 3,000 base pairs from nucleotides 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,

6. The TI host cell of any one of claims 1 to 5, wherein the integrated exogenous nucleotide sequence is operably linked to a nucleotide sequence selected from the group consisting of sequences at least about 90% homologous to a sequence selected from SEQ ID NOs: 1 to 7.

7. A targeted integration (TI) host cell comprising an exogenous nucleotide sequence integrated into an integration site within an endogenous gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, XP_003512331.2, and sequences at least about 90% homologous thereto.

8. A TI host cell comprising an exogenous nucleotide sequence integrated into an integration site operably linked to an endogenous gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, XP_003512331.2, and sequences at least about 90% homologous thereto.

9. A TI host cell comprising an exogenous nucleotide sequence integrated into an integration site immediately adjacent to all or part of a sequence selected from the group consisting of sequences at least about 90% homologous to a sequence selected from SEQ ID NOs: 1-7.

10. The TI host cell according to any one of claims 1 to 9, wherein the TI host cell is a mammalian host cell.

11. The TI host cell of claim 10, wherein the TI host cell is a hamster host cell, a human host cell, a rat host cell, or a mouse host cell.

12. 12. The TI host cell of claim 10 or 11, wherein the TI host cell is a Chinese hamster ovary (CHO) host cell, a CHO K1 host cell, a CHO K1SV host cell, a DG44 host cell, a DUKXB-11 host cell, a CHOK1S host cell, or a CHO K1M host cell.

13. The TI host cell according to any one of claims 1 to 12, wherein the exogenous nucleotide sequence comprises one or more recombination recognition sequences (RRS), wherein the RRS can be recognized by a recombinase.

14. The TI host cell of claim 13 , wherein the exogenous nucleotide sequence comprises at least two RRSs.

15. 15. The TI host cell of claim 13 or 14, wherein the recombinase is Cre recombinase or FLP recombinase.

16. The TI host cell of claim 13 or 14, wherein the recombinase is Bxb1 integrase or φC31 integrase.

17. The TI host cell according to any one of claims 13 to 16, wherein 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.

18. 18. The TI host cell of any one of claims 14 to 17, wherein the exogenous nucleotide sequence comprises a first and a second RRS, and at least one selectable marker located between the first and second RRS.

19. 19. The TI host cell of claim 18, comprising a first selection marker, wherein the first selection marker is selected from the group consisting of aminoglycoside phosphotransferase (APH), hygromycin phosphotransferase (HYG), neomycin, G418 APH), dihydrofolate reductase (DHFR), thymidine kinase (TK), glutamine synthetase (GS), asparagine synthetase, tryptophan synthetase (indole), histidinol dehydrogenase (histidinol D), blasticidin, bleomycin, phleomycin, chloramphenicol, zeocin, and mycophenolic acid.

20. 20. The TI host cell of claim 18 or 19, further comprising a second selection marker, wherein the first and second selection markers are different.

21. 21. The TI host cell of claim 20, wherein the second selectable 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.

22. 22. The TI host cell of claim 20 or 21, further comprising a third selection marker and an internal ribosome entry site (IRES), wherein the IRES is operably linked to the third selection marker.

23. 23. The TI host cell of claim 22, wherein the third selection marker is different from the first or second selection marker.

24. The TI host cell of claim 22 or 23, wherein the third selection marker is selected from the group consisting of a green fluorescent protein (GFP) marker, an improved GFP (eGFP) marker, a synthetic GFP marker, a yellow fluorescent protein (YFP) marker, an improved YFP (eYFP) marker, a cyan fluorescent protein (CFP) marker, an mPlum marker, an mCherry marker, a tdTomato marker, an mStrawberry marker, a J-red marker, a DsRed-monomer marker, an mOrange marker, an mKO marker, an mCitrine marker, a Venus marker, a YPet marker, an Emerald6 marker, a CyPet marker, an mCFPm marker, a Cerulean marker, and a T-Sapphire marker.

25. The TI host cell of any one of claims 18 to 24, further comprising a third RRS, wherein the third RRS is located between the first RRS and the second RRS, and the third RRS is heterospecific compared to the first or second RRS.

26. 26. The TI host cell of any one of claims 13 to 25, wherein the exogenous nucleotide sequence comprises at least one selectable marker and at least one exogenous sequence of interest (SOI).

27. The TI host cell of any one of claims 13 to 26, wherein the exogenous nucleotide sequence further comprises at least one exogenous SOI.

28. 28. The TI host cell of claim 27, wherein the exogenous SOI is located between the first RRS and the second RRS.

29. 29. The TI host cell of claim 27 or 28, 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.

30. 30. The TI host cell of any one of claims 25 to 29, wherein the exogenous nucleotide sequence further comprises at least one exogenous SOI located between the first RRS and the third RRS, and at least one exogenous SOI located between the third RRS and the second RRS.

31. 31. The TI host cell of claim 30, wherein the SOI encodes a single-chain antibody, an antibody light chain, an antibody heavy chain, a single-chain Fv fragment (scFv), or an Fc-fusion protein.

32. 1. A method for preparing a TI host cell that expresses a polypeptide of interest, comprising: a) providing a TI host cell comprising an exogenous nucleotide sequence integrated at a site within a locus of the TI host cell genome, the locus being at least about 90% homologous to a sequence selected from SEQ ID NOs: 1-7, and the exogenous nucleotide sequence comprising two RRSs flanking at least one first selectable marker; b) introducing into the cell provided in a) a vector comprising two RRSs that match the two RRSs on the integrated exogenous nucleotide sequence and that are flanked by at least one exogenous SOI and at least one second selectable marker; c) introducing a recombinase or a nucleic acid encoding a recombinase, wherein said recombinase recognizes said RRS; and d) isolating TI host cells that express said polypeptide by selecting for TI cells that express said second selectable marker.

33. 33. The method of claim 32, wherein the recombinase is Cre recombinase or FLP recombinase.

34. 33. The method of claim 32, wherein the recombinase is Bxb1 integrase or φC31 integrase.

35. 35. The method of any one of claims 32 to 34, 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.

36. 36. The method of any one of claims 32 to 35, wherein the TI host cell is a mammalian host cell.

37. 37. The method of claim 36, wherein the TI host cell is a hamster host cell, a human host cell, a rat host cell, or a mouse host cell.

38. 38. The method of claim 36 or 37, wherein the TI host cell is a CHO host cell, a CHO K1 host cell, a CHO K1SV host cell, a DG44 host cell, a DUKXB-11 host cell, a CHOK1S host cell, or a CHO K1M host cell.

39. 1. A method for expressing a polypeptide of interest, comprising: a) providing a TI host cell comprising at least one exogenous SOI and at least one selectable marker, wherein the at least one exogenous SOI and the at least one selectable marker are flanked by two RRSs integrated into a locus in the TI host cell genome, the locus being at least about 90% homologous to a sequence selected from SEQ ID NOs: 1-7; b) culturing the cells of a) under conditions suitable for expressing and recovering the polypeptide of interest therefrom.

40. 1. A method for preparing a TI host cell that expresses at least a first and a second polypeptide of interest, comprising: a) providing a TI host cell comprising an exogenous nucleotide sequence integrated at a site within a locus of the host cell genome, said locus being at least about 90% homologous to a sequence selected from SEQ ID NOs: 1-7, said exogenous nucleotide sequence comprising first and second RRSs flanking at least one first selectable marker, and a third RRS located between said first and second RRSs, wherein all said RRSs are heterospecific; b) introducing into the cell provided in a) a first vector comprising two RRSs that match the first and third RRSs on the integrated exogenous nucleotide sequence and that are flanked by at least one first exogenous SOI and at least one second selectable marker; c) introducing into the cell provided in a) a second vector comprising two RRSs that match the second and third RRSs on the integrated exogenous nucleotide sequence and flank at least one second exogenous SOI; d) introducing one or more recombinases or one or more nucleic acids encoding one or more recombinases, wherein said one or more recombinases recognize said RRS; and e) isolating TI host cells that express said first and second polypeptides of interest by selecting for TI cells that express said second selectable marker.

41. 41. The method of claim 40, wherein the recombinase is Cre recombinase or FLP recombinase.

42. 41. The method of claim 40, wherein the recombinase is Bxb1 integrase or φC31 integrase.

43. 41. The method of claim 40, wherein the first vector further comprises a promoter sequence operably linked to the codon ATG located upstream of the first SOI and adjacent to the downstream RRS, and the second vector further comprises a selectable marker lacking an ATG transcription start codon upstream of the RRS and adjacent to the downstream RRS of the second SOI.

44. 40. The method of claim 39, wherein the at least one 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.

45. 44. The method of any one of claims 40 to 43, wherein the first SOI encodes a single chain antibody, an antibody light chain, an antibody heavy chain, a single chain Fv fragment (scFv), or an Fc fusion protein.

46. 44. The method of any one of claims 40 to 43, wherein the second SOI encodes a single chain antibody, an antibody light chain, an antibody heavy chain, a single chain Fv fragment (scFv), or an Fc fusion protein.

47. 47. The method of any one of claims 39 to 46, wherein the TI host cell is a mammalian host cell.

48. 48. The method of claim 47, wherein the TI host cell is a hamster host cell, a human host cell, a rat host cell, or a mouse host cell.

49. 49. The method of claim 48, wherein the TI host cell is a CHO host cell, a CHO K1 host cell, a CHO K1SV host cell, a DG44 host cell, a DUKXB-11 host cell, a CHOK1S host cell, or a CHO K1M host cell.

50. 1. A method for expressing a polypeptide of interest, comprising: a) providing a host cell comprising at least two exogenous SOIs and at least one selectable marker integrated into a locus of the host cell genome, the locus being at least about 90% homologous to a sequence selected from SEQ ID NOs: 1-7, wherein the at least one exogenous SOI and one selectable marker are flanked by first and third RRSs, and the at least one exogenous SOI is flanked by a second and the third RRS; and b) culturing the cells of a) under conditions suitable for expressing and recovering the polypeptide of interest therefrom.

51. A vector comprising: a. two reference sequences selected from any portion of SEQ ID NO: 1; b. two reference sequences selected from any portion of SEQ ID NO: 2; c. two reference sequences selected from any portion of SEQ ID NO: 3; d. two reference sequences selected from any portion of SEQ ID NO: 4; e. two reference sequences selected from any portion of SEQ ID NO: 5; f. two reference sequences selected from any portion of SEQ ID NO: 6, or g. Two reference sequences selected from any portion of SEQ ID NO: 7 wherein the sequences flank a DNA cassette, and the DNA cassette comprises at least one selectable marker flanked by two RRSs and at least one exogenous SOI.

52. 52. The vector of claim 51, 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.

53. 53. The vector of claim 51 or 52, 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.

54. 1. A method for preparing a TI host cell that expresses a polypeptide of interest, comprising: a) providing a host cell comprising a locus in the host cell genome, wherein the locus is at least about 90% homologous to a sequence selected from SEQ ID NOs: 1-7; b) introducing a vector into the host cell, wherein the vector comprises a nucleotide sequence at least 50% identical to a sequence selected from SEQ ID NOs: 1-7 flanking a DNA cassette, the DNA cassette comprising at least one selectable marker flanked by two RRSs and at least one exogenous SOI; and c) selecting for said selectable marker and isolating TI host cells having said SOI integrated into said locus in said genome and expressing said polypeptide of interest.

55. 55. The method of claim 54, 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.

56. 56. The method of claim 54 or 55, wherein the at least one 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.

57. 57. The method of any one of claims 53 to 56, wherein the TI host cell is a mammalian host cell.

58. 58. The method of claim 57, wherein the TI host cell is a hamster host cell, a human host cell, a rat host cell, or a mouse host cell.

59. 59. The method of claim 57 or 58, wherein the TI host cell is a CHO host cell, a CHO K1 host cell, a CHO K1SV host cell, a DG44 host cell, a DUKXB-11 host cell, a CHOK1S host cell, or a CHO K1M host cell.

60. 58. The method of any one of claims 54 to 57, wherein the integration of the nucleic acid comprising the at least one SOI and a selectable marker is facilitated by an exogenous nuclease.

61. 58. The method of claim 57, wherein the exogenous nuclease is selected from the group consisting of zinc finger nucleases (ZFNs), ZFN dimers, transcription activator-like effector nucleases (TALENs), TAL effector domain fusion proteins, RNA-guided DNA endonucleases, engineered meganucleases, and clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) endonucleases.

62. A TI host cell comprising at least one exogenous nucleotide sequence integrated into an integration site within one or more specific loci of the host cell genome, the loci being at least about 90% homologous to a sequence selected from SEQ ID NOs: 1-7.

63. A TI host cell comprising at least one exogenous nucleotide sequence integrated into one or more integration sites within an endogenous gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, XP_003512331.2, and sequences at least about 90% homologous thereto.

64. 64. The TI host cell of any one of claims 62 to 63, wherein the at least one exogenous nucleotide sequence comprises one or more recombination recognition sequences (RRS), wherein the RRS can be recognized by a recombinase.

65. 65. The TI host cell of claim 64, wherein the at least one exogenous nucleotide sequence further comprises at least one exogenous SOI.

66. 1. A method for preparing a TI host cell that expresses at least one polypeptide of interest, comprising: e) providing a TI host cell comprising at least one exogenous nucleotide sequence integrated into a site within one or more loci of the TI host cell genome, wherein the one or more loci are at least about 90% homologous to a sequence selected from SEQ ID NOs: 1-7, and wherein the at least one exogenous nucleotide sequence comprises two RRSs flanking at least one first selectable marker; f) introducing into the cell provided in a) a vector comprising two RRSs that match the two RRSs on the integrated exogenous nucleotide sequence and that are flanked by at least one exogenous SOI and at least one second selectable marker; g) introducing a recombinase or a nucleic acid encoding a recombinase, wherein said recombinase recognizes said RRS; and and isolating TI host cells that express said at least one polypeptide of interest by selecting for TI cells that express said second selectable marker.

67. 1. A method for preparing a TI host cell that expresses at least one first and second polypeptide of interest, comprising: a) providing a TI host cell comprising at least one exogenous nucleotide sequence integrated into a site within one or more loci of the host cell genome, wherein the one or more loci are at least about 90% homologous to a sequence selected from SEQ ID NOs: 1-7, and wherein the exogenous nucleotide sequence comprises first and second RRSs flanking at least one first selectable marker, and a third RRS located between the first and second RRSs, and wherein all of the RRSs are heterospecific; b) introducing into the cell provided in a) a first vector comprising two RRSs that match the first and third RRSs on the at least one integrated exogenous nucleotide sequence and that flank at least one first exogenous SOI and at least one second selectable marker; c) introducing into the cell provided in a) a second vector comprising two RRSs that match the second and third RRSs on the at least one integrated exogenous nucleotide sequence and flank at least one second exogenous SOI; d) introducing one or more recombinases or one or more nucleic acids encoding one or more recombinases, wherein said one or more recombinases recognize said RRS; and e) isolating TI host cells that express said at least one first and second polypeptide of interest by selecting for TI cells that express said second selectable marker.

68. 1. A method for preparing a TI host cell that expresses a polypeptide of interest, comprising: a) providing a TI host cell comprising at least one exogenous nucleotide sequence integrated into a site within one or more loci of the TI host cell genome, wherein the one or more loci are at least about 90% homologous to a sequence selected from SEQ ID NOs: 1-7, and wherein the exogenous nucleotide sequence comprises one or more RRSs; b) introducing into the cell provided in a) a vector comprising one or more RRSs flanked by at least one exogenous SOI that matches one or more RRSs on the integrated exogenous nucleotide sequence and that is operably linked to at least one regulatable promoter; c) introducing a recombinase or a nucleic acid encoding a recombinase, wherein said recombinase recognizes said RRS; and d) isolating TI host cells that express the exogenous polypeptide of interest by selecting for TI cells that express the exogenous polypeptide of interest in the presence of an inducer.

69. 1. A method for expressing a polypeptide of interest, comprising: a) providing a host cell comprising at least one exogenous SOI flanked by two RRSs and a regulatable promoter integrated into a locus in the host cell genome, the locus being at least about 90% homologous to a sequence selected from SEQ ID NOs: 1-7; and b) culturing said cells under conditions suitable for expressing said SOI and recovering the polypeptide of interest therefrom.

70. 1. A method for preparing a TI host cell that expresses a first and a second polypeptide of interest, comprising: a) providing a TI host cell comprising an exogenous nucleotide sequence integrated at a site within a locus of the host cell genome, wherein the locus is at least about 90% homologous to a sequence selected from SEQ ID NOs: 1-7, wherein the exogenous nucleotide sequence comprises a first RRS, a second RRS, and a third RRS located between the first RRS and the second RRS, and wherein all of the RRSs are heterospecific; b) introducing into the cell provided in a) a first vector comprising two RRSs flanking at least one first exogenous SOI that matches the first and third RRSs on the integrated exogenous nucleotide sequence and is operably linked to a regulatable promoter; c) introducing into the cell provided in a) a second vector comprising two RRSs flanking at least one second exogenous SOI that matches the second and third RRSs on the integrated exogenous nucleotide sequence and is operably linked to a regulatable promoter; d) introducing one or more recombinases or one or more nucleic acids encoding one or more recombinases, wherein said one or more recombinases recognize said RRS; and e) isolating TI host cells that express the first and second exogenous polypeptides of interest by selecting for TI cells that express the first and second exogenous polypeptides of interest in the presence of an inducer.

71. 70. The method of any one of claims 66, 68, and 69, wherein the integration of nucleic acid comprising at least one SOI is facilitated by an exogenous nuclease.

72. 71. The method of claim 67 or 70, wherein the first SOI encodes a single-chain antibody, an antibody light chain, an antibody heavy chain, a single-chain Fv fragment (scFv), or an Fc-fusion protein, and the second SOI encodes a single-chain antibody, an antibody light chain, an antibody heavy chain, a single-chain Fv fragment (scFv), or an Fc-fusion protein.

73. 72. The method of Claim 71, wherein the exogenous nuclease is selected from the group consisting of zinc finger nucleases (ZFNs), ZFN dimers, transcription activator-like effector nucleases (TALENs), TAL effector domain fusion proteins, RNA-guided DNA endonucleases, engineered meganucleases, and clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) endonucleases.

74. 71. The method of any one of claims 66 to 70, wherein the regulatable promoter is selected from the group consisting of SV40 and CMV promoters.

75. a. the first vector further comprises a promoter sequence operably linked to the codon ATG located upstream of the first SOI and adjacent downstream of the RRS; b) The method of any one of claims 67 and 70, wherein the second vector further comprises a selectable marker lacking an ATG transcription start codon upstream of the RRS and adjacent downstream of the second SOI.

76. A vector comprising two sequences selected from any part of SEQ ID NOs: 1 to 7, namely: a. two reference sequences selected from any portion of SEQ ID NO: 1; b. two reference sequences selected from any portion of SEQ ID NO: 2; c. two reference sequences selected from any portion of SEQ ID NO: 3; d. two reference sequences selected from any portion of SEQ ID NO: 4; e. two reference sequences selected from any portion of SEQ ID NO: 5; f. two reference sequences selected from any portion of SEQ ID NO: 6, or g. Two reference sequences selected from any portion of SEQ ID NO: 7 wherein the sequences flank a DNA cassette, and the DNA cassette comprises at least one exogenous SOI operably linked to a regulatable promoter and flanked by two RRSs.

77. 77. The vector of claim 76, 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.

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