Combined transposon-mediated and targeted integration of nucleic acids into host cells

Abstract

The subject matter of the present disclosure relates to targeted integration (TI) host cells suitable for expression of recombinant proteins, which TI host cells are also subject to transposon-mediated genomic integration of one or more exogenous nucleic acids, and the subject matter of the present disclosure also relates to methods of producing and using combined transposon-mediated genomic integration and TI host cells.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 383,885, filed November 15, 2022, which is incorporated herein by reference in its entirety.

[0002] Sequence Listing A sequence listing conforming to the rules of WIPO Standard ST.26 is incorporated herein by reference. The sequence listing has been submitted to the Patent Center as an electronic document encoded in XML format. The electronic document, created on November 15, 2023, is titled "00B206_1384.xml" and is 983,930 bytes in size.

[0003] Technical Field The subject matter of the present disclosure relates to targeted integration (TI) host cells suitable for expression of recombinant proteins, which TI host cells are also subject to transposon-mediated genomic integration of one or more exogenous nucleic acids, and to methods for producing and using combined transposon-mediated genomic integration and TI host cells. [Background technology]

[0004] Rapid advances in cell biology and immunology have driven the demand for the development of novel therapeutic recombinant proteins for a variety of diseases, including cancer, cardiovascular disease, and metabolic disorders. These biopharmaceutical candidates are generally produced using commercially available cell lines capable of expressing the protein of interest. For example, Chinese hamster ovary (CHO) cells have been widely adapted for the production of monoclonal antibodies.

[0005] Traditional strategies for developing commercially available cell lines involve randomly integrating a nucleotide sequence encoding a polypeptide of interest, followed by selection and isolation of cell lines that produce the polypeptide of interest. However, these approaches have several drawbacks. First, such integration is not only a rare event, but given the random location of nucleotide sequence integration, such rare events can result in diverse gene expression and cell growth phenotypes. This diversity, known as "position effect variability," is at least in part due to the complex gene regulatory networks present in eukaryotic cell genomes and the ease of integration and gene expression at specific genomic loci. Second, random integration strategies generally cannot control the number of gene copies integrated into the host cell genome. In fact, gene amplification methods are often used to obtain high-producing cells. However, such gene amplification can result in undesirable cellular 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 the desired level of expression of the polypeptide of interest is time-consuming and labor-intensive. 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 in purely randomly integrated cell lines exhibit a high degree of sequence diversity, 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 subject matter of the present disclosure relates, in part, to TI host cells suitable for expressing recombinant proteins, which TI host cells are also subject to transposon-mediated genomic integration of one or more exogenous nucleic acids, and to methods for producing and using combined transposon-mediated genomic integration and TI host cells. The subject matter of the present disclosure not only provides highly productive host cell TI sites, but also provides for achieving increased expression of a sequence of interest by subjecting the cell to transposon-mediated genomic integration of one or more exogenous nucleic acids into the TI host.

[0007] In certain embodiments, the present disclosure provides a host cell comprising: a) a targeted integration exogenous nucleic acid sequence (SOI) flanked by two recombination recognition sequences (RRSs), the SOI encoding a first polypeptide of interest and a first selectable marker, wherein the SOI is integrated into a targeted locus in the genome of the host cell; and b) a transposon-mediated genomic integration exogenous nucleic acid SOI encoding a second polypeptide of interest and a second selectable marker, wherein the transposon-mediated genomic integration exogenous nucleic acid SOI is integrated into the genome of the host cell at least once, the targeted integration exogenous nucleic acid SOI is constitutively or inducibly expressed, and the transposon-mediated genomic integration exogenous nucleic acid SOI is constitutively or inducibly expressed. In certain embodiments, the first polypeptide of interest and the second polypeptide of interest can be the same. In certain embodiments, the first selectable marker and the second selectable marker can be the same. In certain embodiments, the host cell can include 1 to 10 transposon-mediated genome-integrated exogenous nucleic acid SOIs. In certain embodiments, the targeted locus can be all or a portion of a contig selected from NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1, or a sequence selected from SEQ ID NOs: 1-7.

[0008] In certain embodiments, the host cell of the present disclosure further comprises a second targeted integrating exogenous nucleic acid SOI encoding a second polypeptide of interest and a second selectable marker integrated within a targeted locus in the genome of the host cell, wherein the first targeted integrating exogenous nucleic acid SOI and the first selectable marker can be flanked by a first RRS and a third RRS, and the second targeted integrating exogenous SOI and the second selectable marker can be flanked by a second RRS and a third RRS.

[0009] In certain embodiments, the polypeptide of interest may be selected from the group consisting of a single chain antibody, an antibody light chain, an antibody heavy chain, a single chain Fv fragment (scFv), and an Fc fusion protein.

[0010] In certain embodiments, the host cell may be a mammalian host cell. In certain embodiments, the host cell may be a hamster host cell, a human host cell, a rat host cell, or a mouse host cell. In certain embodiments, the host cell may be a CHO host cell, a CHO K1 host cell, a CHO K1SV host cell, a DG44 host cell, a DUKXB-11 host cell, a CHOK1S host cell, or a CHO K1M host cell.

[0011] In certain embodiments, targeted integration of the SOI and selectable marker may be facilitated by an exogenous nuclease, which 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.

[0012] In certain embodiments, the targeted integration exogenous nucleic acid SOI is constitutively expressed. In certain embodiments, the targeted integration exogenous nucleic acid SOI is inducibly expressed. In certain embodiments, the transposon-mediated genome integration exogenous nucleic acid SOI is constitutively or inducibly expressed.

[0013] The presently disclosed subject matter also provides a method for expressing a polypeptide of interest. In certain embodiments, the present disclosure provides a method for expressing a polypeptide of interest, comprising: a) providing a host cell comprising an exogenous nucleotide sequence integrated into a targeted locus in the genome of the host cell, the exogenous nucleotide sequence comprising two RRSs flanking a first selectable marker; b) introducing into the cell provided in (a) a nucleic acid comprising two RRSs that match the two RRSs of the integrated exogenous nucleotide sequence and that flank a first exogenous SOI encoding a first polypeptide of interest and a second selectable marker; and c) introducing a recombinase that recognizes the RRSs or a nucleic acid encoding the recombinase. d) selecting cells that express a second selection marker; e) introducing a second exogenous SOI encoding a second polypeptide of interest and a third selection marker into the genome of the host cell via transposon-mediated genomic integration; f) constitutively or inducibly expressing the exogenous nucleotide sequence integrated at the targeted locus in the genome of the host cell and constitutively or inducibly expressing the second exogenous SOI; g) selecting cells that express the third selection marker; and h) culturing the host cells under conditions sufficient to express the first polypeptide of interest and the second polypeptide of interest. In certain embodiments, such methods may further include recovering the first and second polypeptides of interest from the host cell culture. In certain embodiments, the first and second polypeptides of interest may be the same. In certain embodiments, the targeted locus can be all or a portion of a contig selected from NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1, or a sequence selected from SEQ ID NOs: 1-7. In certain embodiments, the first polypeptide of interest and the second polypeptide of interest can be selected from the group consisting of a single-chain antibody, an antibody light chain, an antibody heavy chain, a single-chain Fv fragment (scFv), and an Fc-fusion protein. In certain embodiments, the host cell can be a mammalian host cell.In certain embodiments, the host cell may be a hamster host cell, a human host cell, a rat host cell, or a mouse host cell. In certain embodiments, the host cell may be a CHO host cell, a CHO K1 host cell, a CHO K1SV host cell, a DG44 host cell, a DUKXB-11 host cell, a CHOK1S host cell, or a CHO K1M host cell. In certain embodiments, targeted integration of any SOI may be facilitated by an exogenous nuclease. In certain embodiments, the exogenous nuclease may be selected from the group consisting of zinc finger nucleases (ZFNs), ZFN dimers, transcription activator-like effector nucleases (TALENs), TAL effector domain fusion proteins, RNA-guided DNA endonucleases, engineered meganucleases, and clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) endonucleases. In certain embodiments, expression of the SOI may be controlled by a regulatable promoter. In certain embodiments, the regulatable promoter may be selected from the group consisting of SV40 and CMV promoters. In certain embodiments, the exogenous nucleotide sequence integrated into the targeted locus of the host cell genome is constitutively expressed. In certain embodiments, the exogenous nucleotide sequence integrated into the targeted locus of the host cell genome is inducibly expressed. In certain embodiments, the second exogenous SOI is constitutively expressed. In certain embodiments, the second exogenous SOI is inducibly expressed. [Brief explanation of the drawings]

[0014] [Figure 1] Representative recovery curves after selection of TI hosts with piggyBac alone (PB), targeted integration alone (TI), co-transfection of targeted integration and piggyBac (TIP), sequential transfection with targeted integration and piggyBac after 2 days (TI+PB), and sequential transfection with piggyBac and targeted integration after 2 days (PB+TI). [Figures 2A-2C]Figure 2A shows the titer (Figure 2A), specific productivity (Qp) (Figure 2B), and integration viability (IVCC) (Figure 2C) at day 7 for TI host cell pools transfected with piggyBac alone (PB), with targeted integration alone (TI), with targeted integration and piggyBac cotransfected (TIP), with targeted integration and piggyBac transfected sequentially after 2 days (TI+PB), and with piggyBac and targeted integration transfected sequentially after 2 days (PB+TI). Data shown are for two PB pools, one TI pool, four TIP pools, seven TI+PB pools, and one PB+TI pool. The lower TIP titers and Qp are likely due to the use of less PB plasmid in TIP transfections to improve post-transfection survival. All error bars indicate the standard deviation of the pools in each transfection group. [Figure 3A-3B] Figure 3A shows the heavy and light chain gene copy numbers per cell (Figure 3A) and mRNA transcript levels (Figure 3B) for TI host cell pools transfected with piggyBac alone (PB), targeted integration alone (TI), cotransfected with targeted integration and piggyBac (TIP), sequentially transfected with targeted integration followed by piggyBac 2 days later (TI+PB), and sequentially transfected with piggyBac followed by targeted integration 2 days later (PB+TI). Data shown here are for two PB pools, one TI pool, four TIP pools, seven TI+PB pools, and one PB+TI pool. All error bars indicate the standard deviation of the pools in each transfection group. DETAILED DESCRIPTION OF THE INVENTION

[0015] The subject matter of the present disclosure relates to TI host cells suitable for expression of recombinant proteins, which TI host cells are also subject to transposon-mediated genomic integration of one or more exogenous nucleic acids, and to methods for producing and using combined transposon-mediated genomic integration and TI host cells. In certain embodiments, the host cells, genetic constructs (e.g., vectors), compositions, and methods described herein may be employed in the development and / or use of combined transposon-mediated genomic integration and TI host cells.

[0016] For purposes of clarity of disclosure and not of limitation, this detailed description is divided into the following subsections: 1.Definition 2. Integration site 3. Exogenous Nucleotide Sequence 4.Host cells 5. Methods of targeted integration and transposon-mediated genomic integration 6. Preparation and Use of TI Host Cells 7.Product 8. Working Example

[0017] 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 be used in the practice or testing of the subject matter of this disclosure. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and are not intended to be limiting.

[0018] As used herein, "comprise," "include," "having," "has," "can," "contain," and variations thereof are intended to be open-ended transitional phrases, terms, or words that do not exclude the possibility of additional acts or constructs. 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.

[0019] When numerical ranges are recited herein, each intervening number is expressly contemplated to the same degree of precision. 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.

[0020] As used herein, the term "about" or "approximately" means within an acceptable error range of a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 3 or more than 3 standard deviations, in accordance with practice in the art. Alternatively, "about" can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably even up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold of a value.

[0021] As used herein, the term "selection marker" refers to a gene that allows cells carrying a gene to be specifically selected or specifically eliminated in the presence of a corresponding selection agent. For example, but not limited to, a selection marker may allow host cells transformed with the selection marker gene to be positively selected in the presence of the gene, while untransformed host cells cannot grow or survive under selective conditions. Selection markers can be positive, negative, or bifunctional. A positive selection marker may allow for the selection of cells carrying the marker, whereas a negative selection marker may allow for the selective elimination of cells carrying the marker. A selection marker may confer resistance to a drug in a host cell or complement a metabolic or catabolic defect. In prokaryotic cells, genes that confer resistance to ampicillin, tetracycline, kanamycin, or chloramphenicol, among others, may be used. Resistance genes useful as selectable markers in eukaryotic cells include, but are not limited to, genes for aminoglycoside phosphotransferase (APH) (e.g., hygromycin phosphotransferase (HYG), neomycin, and G418 APH), dihydrofolate reductase (DHFR), thymidine kinase (TK), glutamine synthetase (GS), asparagine synthetase, tryptophan synthase (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.

[0022] In addition to facilitating selection in the presence of the corresponding selection agent, a selectable marker may alternatively provide a gene encoding a molecule not normally present in the cell, such as green fluorescent protein (GFP), enhanced GFP (eGFP), synthetic GFP, yellow fluorescent protein (YFP), enhanced YFP (eYFP), cyan fluorescent protein (CFP), mPlum, mCherry, tdTomato, mStrawberry, J-red, DsRed-monomer, mOrange, mKO, mCitrine, Venus, YPet, Emerald, CyPet, mCFPm, Cerulean, and T-Sapphire. Cells carrying such a gene can be distinguished from cells not carrying the gene by, for example, detection of fluorescence emitted by the encoded polypeptide.

[0023] As used herein, the term "operably linked" refers to the juxtaposition of two or more components, in a relationship permitting them to function in a desired manner. For example, a promoter and / or enhancer is operably linked to a coding sequence if it acts to modulate the transcription of the coding sequence. In certain embodiments, "operably linked" DNA sequences are contiguous and adjacent on a single chromosome. In certain embodiments, when it is necessary to join two protein coding regions, e.g., a secretory leader and a polypeptide, these sequences are contiguous, adjacent, 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 that regulates expression of a coding sequence, two components may be operably linked even though they are not adjacent. An enhancer is operably linked to a coding sequence if it increases the transcription of the coding sequence. An operably linked enhancer can be located upstream, within, or downstream of a 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 methodology and / or by ligation at convenient restriction sites. Where convenient restriction enzyme recognition sites are not present, synthetic oligonucleotide adapters or linkers can be used according to the practice. An internal ribosome entry site (IRES) is operably linked to an open reading frame (ORF) if it enables translation to be initiated at a location internal to the ORF independent of the 5' end.

[0024] As used herein, the term "expression" refers to transcription and / or translation. In certain embodiments, the level of transcription of a desired product may be determined based on the amount of corresponding mRNA present. For example, mRNA transcribed from a sequence of interest may be quantified by PCR or by Northern hybridization. In certain embodiments, the protein encoded by a sequence of interest may be quantified by various methods, e.g., by ELISA, by assaying for biological activity of the protein, or by using assays independent of such activity, e.g., Western blotting or radioimmunoassays that use antibodies that recognize and bind to the protein.

[0025] The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), half antibodies, and antibody fragments, so long as the antibody exhibits the desired antigen-binding activity.

[0026] 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 to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. For a review of specific antibody fragments, see Holliger and Hudson, Nature Biotechnology 23:1126-1136 (2005).

[0027] As used herein, the term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The heavy and light chain variable domains (VH and VL, respectively) of native antibodies generally have similar structures, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). See, for example, Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a specific antigen can be isolated by screening a library of complementary VL or VH domains, respectively, using the VH or VL domain of an antibody that binds to the antigen. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0028] As used herein, the term "heavy chain" refers to an immunoglobulin heavy chain.

[0029] As used herein, the term "light chain" refers to an immunoglobulin light chain.

[0030] 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, some of which can 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.

[0031] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind to the same epitope, except for variant antibodies that contain, for example, naturally occurring mutations or that may arise during production of the 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 should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies according to the present invention can 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, as well as other exemplary methods for producing monoclonal antibodies, are described herein.

[0032] 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 can be prepared as full-length antibodies or antibody fragments.

[0033] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein and refer to an antibody having a structure substantially similar to a native antibody structure, or an antibody having a heavy chain that includes an Fc region as defined herein.

[0034] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds 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 specific antibody fragments, see Holliger and Hudson, Nature Biotechnology 23:1126-1136 (2005).

[0035] 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.

[0036] A "human antibody" is an antibody having an amino acid sequence corresponding to an antibody produced by a human or human cell, or an antibody of non-human origin that utilizes human antibody-encoding sequences, such as the human antibody repertoire. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.

[0037] A "humanized" antibody refers to a chimeric antibody comprising amino acid residues derived from non-human CDRs and human FRs. In certain embodiments, a humanized antibody comprises substantially all of at least one, typically two, variable domains, with all or substantially all of the CDRs corresponding to those of a non-human antibody and all or substantially all of the FRs corresponding 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. As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind the same epitope, except for variant antibodies that contain, for example, 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.

[0038] The term "therapeutic antibody" refers to an antibody used to treat disease. Therapeutic antibodies can have various mechanisms of action. A therapeutic antibody can bind to an antigen-associated target and neutralize its normal function. For example, a monoclonal antibody that blocks the activity of a protein necessary for cancer cell survival can cause cell death. Another therapeutic monoclonal antibody can bind to an antigen-associated target and activate its normal function. For example, a monoclonal antibody can bind to a protein on a cell and trigger an apoptotic signal. Another monoclonal antibody can 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 a monoclonal antibody can create an agent 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 a target antigen.

[0039] 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 exhibits at least one, if not some or all, of the biological functions attributed to an intact antibody, including at least specific binding to a target antigen.

[0040] 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, regardless of the number of passages. The progeny may be completely identical in nucleic acid content to the parent cell or 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.

[0041] The terms "nucleic acid molecule" or "polynucleotide" include any compound and / or substance comprising a polymer of nucleotides. Each nucleotide is composed 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 typically represented 5' to 3'. As used herein, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including, for example, 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. In addition, the term nucleic acid molecule includes both sense and antisense strands, and both single- and double-stranded forms. Furthermore, the nucleic acid molecules described herein can 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 directing expression of the antibodies of the invention in vitro and / or in vivo, e.g., in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors can be unmodified or modified. For example, mRNA can be chemically modified to increase the stability of the RNA vector and / or the expression of the encoded molecule, such that the mRNA can be injected into a subject to produce antibodies in vivo (see, e.g., Stadler et al., Nature Medicine 2017, published online 12 June 2017, doi:10.1038 / nm.4356 or EP 2101823 B1).

[0042] An "isolated" nucleic acid refers to a nucleic acid molecule that is separated from a component of its natural environment. Isolated nucleic acid includes a nucleic acid molecule contained within a cell that normally 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.

[0043] 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 direct the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors."

[0044] 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 about 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 99.9% homologous. Alignment is performed by algorithms and computer programs, including but not limited to BLAST, FASTA, and HMME, that 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.

[0045] As used herein, the term "adjacent" refers to a first nucleotide sequence being located at either the 5' or 3' end, or both ends, of a second nucleotide sequence. An adjacent nucleotide sequence may be located next to the second nucleotide sequence or at a predetermined distance from it. 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.

[0046] As used herein, the term "exogenous" indicates that a nucleotide sequence is not native to the host cell but 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 an "exogenous" sequence is introduced into the host cell by, for example, recombinant DNA techniques.

[0047] 2. Integration site The presently disclosed subject matter provides host cells suitable for targeted integration of an exogenous nucleotide sequence. In certain embodiments, the host cell comprises an exogenous nucleotide sequence integrated into an integration site on the genome of the host cell, i.e., the TI host cell.

[0048] An "integration site" comprises a nucleic acid sequence within the genome of a host cell into which an exogenous nucleotide sequence is inserted. In certain embodiments, the integration site is between two adjacent nucleotides on the genome of the host cell. 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 genome of the TI host cell. In certain embodiments, the integration site is within an endogenous gene of the TI host cell.

[0049] In certain embodiments, the exogenous nucleotide sequence is integrated into a site within a specific locus in the genome of the TI host cell. In certain embodiments, the locus into which the exogenous nucleotide sequence is integrated is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or at least about 99.9% homologous to a sequence selected from SEQ ID NOs: 1-7.

[0050] In certain embodiments, the locus into which the exogenous nucleotide sequence has been incorporated 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 all or a portion of 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.

[0051] In certain embodiments, the exogenous nucleotide sequence is integrated into an integration site located within a position selected from nucleotides numbered 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 numbered 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 728785 bp of SEQ ID NO:2. In certain embodiments, the exogenous nucleotide sequence is integrated at an integration site located within a position selected from nucleotides numbered 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 of SEQ ID NO: 3. In certain embodiments, the exogenous nucleotide sequence is integrated at an integration site located within a position selected from nucleotides numbered 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 numbered 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 numbered 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 numbered 1 to 10,000 bp, 10,000 to 20,000 bp, and 20,000 to 22,904 bp of SEQ ID NO:7.

[0052] In certain embodiments, the nucleotide sequence immediately 5' to the integrated exogenous sequence is a nucleotide within the sequence: 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. In certain embodiments, the nucleotide immediately 5' of the integrated exogenous nucleotide sequence is a nucleotide within a nucleotide sequence that is at least 50% 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.In certain embodiments, the nucleotides immediately 5' to the integrated exogenous sequence are nucleotides 41190 to 45269 of NW_006874047.1, nucleotides 63590 to 207911 of NW_006884592.1, nucleotides 253831 to 491909 of NW_006881296.1, nucleotides 69303 to 79768 of NW_003616412.1, nucleotide 293 nucleotides 481 to 315265 of NW_006882936.1, nucleotides 2650443 to 2662054 of NW_006882936.1, or nucleotides 82214 to 97705 of NW_003615411.1 that 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 481 to 315265 of NW_006882936.1, nucleotides 2650443 to 2662054 of NW_006882936.1, or nucleotides 82214 to 97705 of NW_003615411.1.

[0053] In certain embodiments, the nucleotide immediately 3' to the integrated exogenous sequence is a nucleotide within the sequence: 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 nucleotide sequence immediately 3' of the integrated exogenous sequence is a nucleotide within a sequence of nucleotides that is at least 50% 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 nucleotides immediately 3′ of the integrated exogenous sequence are nucleotides 45270 to 45490 of NW_006874047.1, nucleotides 207912 to 792374 of NW_006884592.1, nucleotides 491910 to 667813 of NW_006881296.1, nucleotides 79769 to 100059 of NW_003616412.1, nucleotides 80069 to 90059 of NW_003615063.1, NW_006882936.1, nucleotides 2662055 to 2701768 of NW_006882936.1, or nucleotides 97706 to 105117 of NW_003615411.1.

[0054] 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.

[0055] 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. adjacent to the 5' side of the sequence and adjacent to the 3' side of a nucleotide sequence selected from the group consisting of nucleotides 45270 to 45490 of NW_006874047.1, nucleotides 207912 to 792374 of NW_006884592.1, nucleotides 491910 to 667813 of NW_006881296.1, nucleotides 79769 to 100059 of NW_003616412.1, nucleotides 315266 to 362442 of NW_003615063.1, nucleotides 2662055 to 2701768 of NW_006882936.1, and nucleotides 97706 to 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 selected from the group consisting of nucleotides 41190 to 45269 of NW_006874047.1, nucleotides 63590 to 207911 of NW_006884592.1, nucleotides 253831 to 491909 of NW_006881296.1, nucleotides 69303 to 79768 of NW_003616412.1, and nucleotides 10303 to 10768 of NW_003616412.1. NW_006882936.1; nucleotides 2650443 to 2662054 of NW_006882936.1; and nucleotides 82214 to 97705 of NW_003615411.1. The nucleotide sequences flanking the 3' side of the integrated exogenous nucleotide sequence are: nucleotides 45270 to 45490 of SEQ ID NO: NW_006874047.1; nucleotides 207912 to 792374 of NW_006884592.1; nucleotides 491910 to 667813 of NW_006881296.1; nucleotides 79769 to 100059 of NW_003616412.1; NW_006882936.1, nucleotides 2662055 to 2701768 of NW_006882936.1, and nucleotides 97706 to 105117 of NW_003615411.1.

[0056] In certain embodiments, the incorporated exogenous nucleotide is incorporated within a 20-nucleotide sequence that is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or at least about 99.9% homologous to the 20-nucleotide sequence of NW_006874047.1 comprising position 45269; NW_006884592.1 comprising position 207911; NW_006881296.1 comprising position 491909; NW_003616412.1 comprising position 79768; NW_003615063.1 comprising position 315265; NW_006882936.1 comprising position 2662054; and NW_003615411.1 comprising position 97705.

[0057] In certain embodiments, the incorporated exogenous nucleotide is incorporated within a 50-nucleotide sequence that is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or at least about 99.9% homologous to the 50-nucleotide sequence of NW_006874047.1 comprising position 45269; NW_006884592.1 comprising position 207911; NW_006881296.1 comprising position 491909; NW_003616412.1 comprising position 79768; NW_003615063.1 comprising position 315265; NW_006882936.1 comprising position 2662054; and NW_003615411.1 comprising position 97705.

[0058] In certain embodiments, the incorporated exogenous nucleotide is incorporated within a 100 nucleotide sequence that is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or at least about 99.9% homologous to the 100 nucleotide sequence of NW_006874047.1 comprising position 45269; NW_006884592.1 comprising position 207911; NW_006881296.1 comprising position 491909; NW_003616412.1 comprising position 79768; NW_003615063.1 comprising position 315265; NW_006882936.1 comprising position 2662054; and NW_003615411.1 comprising position 97705.

[0059] In certain embodiments, the incorporated exogenous nucleotide is incorporated within a 200 nucleotide sequence that is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or at least about 99.9% homologous to the 200 nucleotide sequence of NW_006874047.1 comprising position 45269; NW_006884592.1 comprising position 207911; NW_006881296.1 comprising position 491909; NW_003616412.1 comprising position 79768; NW_003615063.1 comprising position 315265; NW_006882936.1 comprising position 2662054; and NW_003615411.1 comprising position 97705.

[0060] In certain embodiments, the incorporated exogenous nucleotide is incorporated within a 500 nucleotide sequence that is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or at least about 99.9% homologous to the 500 nucleotide sequence of NW_006874047.1 comprising position 45269; NW_006884592.1 comprising position 207911; NW_006881296.1 comprising position 491909; NW_003616412.1 comprising position 79768; NW_003615063.1 comprising position 315265; NW_006882936.1 comprising position 2662054; and NW_003615411.1 comprising position 97705.

[0061] In certain embodiments, the incorporated exogenous nucleotide is incorporated within a 1000 nucleotide sequence that is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or at least about 99.9% homologous to the 1000 nucleotide sequence of NW_006874047.1 comprising position 45269; NW_006884592.1 comprising position 207911; NW_006881296.1 comprising position 491909; NW_003616412.1 comprising position 79768; NW_003615063.1 comprising position 315265; NW_006882936.1 comprising position 2662054; and NW_003615411.1 comprising position 97705.

[0062] In certain embodiments, the integrated exogenous nucleotide sequence 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.

[0063] In certain embodiments, the integrated 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 integrated 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.

[0064] In certain embodiments, the exogenous nucleotide sequence is integrated into an integration site adjacent to a position selected from nucleotides numbered 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 numbered 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 728785 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 numbered 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 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 numbered 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 numbered 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 numbered 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 numbered 1 to 10,000 bp, 10,000 to 20,000 bp, and 20,000 to 22,904 bp of SEQ ID NO:7.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] Table 1 shows the integration sites for exemplary TI host cells. TIFF2025539936000002.tif69170

[0069] In certain embodiments, the integration site and / or nucleotide sequences flanking the integration site may be identified experimentally. In certain embodiments, the integration site and / or nucleotide sequences flanking the integration site may be identified by genome-wide screening techniques 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, where the exogenous sequences are themselves integrated into one or more loci within the genome of the host cell. In certain embodiments, the integration site and / or nucleotide sequences flanking the integration site may be identified by genome-wide screening techniques following a transposase-based cassette integration event. In certain embodiments, the integration site and / or nucleotide sequences flanking the integration site may be identified by brute-force random integration screening. In certain embodiments, the integration site and / or nucleotide sequences flanking the integration site may be determined by conventional sequencing techniques, 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 may be determined by conventional cell biology techniques such as fluorescence in situ hybridization (FISH) analysis.

[0070] In certain embodiments, the TI host cell comprises a first exogenous nucleotide sequence integrated at a first integration site within a specific first locus within the genome of the TI host cell and a second exogenous nucleotide sequence integrated at a second integration site within a specific second locus within the genome, hi certain embodiments, the TI host cell comprises multiple exogenous nucleotide sequences integrated at multiple integration sites within the genome of the TI host cell.

[0071] In certain embodiments, the TI host cell of the present disclosure comprises at least two different exogenous nucleotide sequences, for example, exogenous nucleotide sequences comprising at least one RRS. In certain embodiments, two or more exogenous nucleotide sequences can be targeted for introduction of one or more SOIs. In certain embodiments, the SOIs are identical. In certain embodiments, the SOIs are distinct. 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.

[0072] 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 to insert at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or more exogenous nucleotide sequences at the same or different integration sites.

[0073] In certain embodiments, the feasibility of recombinase-mediated cassette exchange (RMCE) of at least two integration sites may be assessed for each site individually. In certain embodiments, the feasibility of RMCE of at least two integration sites may be assessed simultaneously. The feasibility of RMCE at multiple sites may be assessed by methods known in the art, for example, by measuring polypeptide titer or polypeptide-specific production. In certain embodiments, the assessment may be performed by methods known in the art, for example, by assessing the titer and / or specific productivity of a culture of TI host cells expressing one or more SOIs. Exemplary culture strategies include, but are not limited to, fed-batch shake flask culture and fed-batch bioreactor culture. The titer and specific productivity of TI host cells expressing a polypeptide of interest may be assessed by methods known in the art, for example, but not limited to, ELISA, FACS, fluorescence microvolume assay technology (FMAT), protein A affinity chromatography, and Western blot analysis.

[0074] 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, for example, transfection, electroporation, or transformation. In certain embodiments, the exogenous nucleotide sequence is a nucleotide sequence encoding a sequence of interest (SOI), e.g., a polypeptide of interest. However, in certain embodiments, an exogenous nucleotide sequence used in the context of the present disclosure includes elements that facilitate the introduction of additional nucleic acid sequences, e.g., SOIs, such as one or more recombination recognition sequences (RRSs) and one or more selectable markers. 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, the 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 genome of the host cell via 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.

[0075] In certain embodiments, the TI host cell comprises at least one exogenous nucleotide sequence integrated into one or more integration sites in the genome of the TI host cell. In certain embodiments, the exogenous nucleotide sequence is integrated into one or more integration sites within specific loci in the genome of the TI host cell. For example, without limitation, the at least one exogenous nucleic acid sequence may be integrated into one or more loci that 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 a sequence selected from SEQ ID NOs: 1-7.

[0076] 3.1 Landing Pad In certain embodiments, the integrated exogenous nucleotide sequence comprises one or more recombination recognition sequences (RRSs), wherein the RRSs can be recognized by a recombinase. In certain embodiments, the integrated exogenous nucleotide sequence comprises at least two RRSs. In certain embodiments, the integrated exogenous nucleotide sequence comprises two RRSs, wherein the two RRSs are identical. In certain embodiments, the integrated exogenous nucleotide sequence comprises two RRSs, wherein the two RRSs are heterospecific, i.e., not recognized by the same recombinase. In certain embodiments, the integrated exogenous nucleotide sequence comprises three RRSs, wherein the third RRS is located between the first and second RRSs. In certain embodiments, the first and second RRSs are identical, and the third RRS is different from both the first and second RRSs. In certain embodiments, all three RRSs are heterospecific. In certain embodiments, the integrated 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, two or more of the multiple RRSs are identical. In certain embodiments, 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 RRSs may 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.

[0077] In certain embodiments, the integrated exogenous nucleotide sequence comprises at least one selectable marker. In certain embodiments, the integrated exogenous nucleotide sequence comprises one RRS and at least one selectable marker. In certain embodiments, the integrated exogenous nucleotide sequence comprises a first RRS, 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, the two RRSs are adjacent to 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 adjacent to the 5' end of the selectable marker and the second RRS is adjacent to the 3' end of the selectable marker.

[0078] In certain embodiments, the selectable marker is located between a first RRS and a second RRS, and the two flanking RRSs are identical. 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 5' of the selectable marker, and the LoxP 2L sequence is located 3' 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 oriented in the forward or reverse direction. In certain embodiments, the two RRSs are positioned in opposite orientations.

[0079] In certain embodiments, the selectable marker may be an aminoglycoside phosphotransferase (APH) (e.g., hygromycin phosphotransferase (HYG), neomycin, and G418 APH), dihydrofolate reductase (DHFR), thymidine kinase (TK), glutamine synthetase (GS), asparagine synthetase, tryptophan synthetase (indole), histidinol dehydrogenase (histidinol D), and a gene encoding resistance to puromycin, blasticidin, bleomycin, phleomycin, chloramphenicol, zeocin, or mycophenolic acid. In certain embodiments, the selectable marker can be a GFP, eGFP, synthetic GFP, YFP, eYFP, CFP, mPlum, mCherry, tdTomato, mStrawberry, J-red, DsRed-monomer, mOrange, mKO, mCitrine, Venus, YPet, Emerald, CyPet, mCFPm, Cerulean, or T-Sapphire marker. In certain embodiments, the selectable marker can be a fusion construct comprising at least two selectable markers. In certain embodiments, a gene encoding a selectable marker or a fragment of a selectable marker can be fused to a gene encoding a different selectable marker or fragment thereof.

[0080] In certain embodiments, the integrated exogenous nucleotide sequence comprises two selectable markers flanked by two RRSs, the first selectable marker being different from the second selectable marker. In certain embodiments, both selectable markers are 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). The first selectable marker is selected from the group consisting of genes encoding resistance to glutamine synthetase (APH), dihydrofolate reductase (DHFR), thymidine kinase (TK), glutamine synthetase (GS), asparagine synthetase, tryptophan synthase (indole), histidinol dehydrogenase (histidinol D), and puromycin, blasticidin, bleomycin, phleomycin, chloramphenicol, zeocin, and mycophenolic acid, and the second selectable 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. In certain embodiments, the first selectable marker is a glutamine synthetase selectable marker and the second selectable marker is a GFP marker. In certain embodiments, the two RRSs flanking both selectable markers are identical. In certain embodiments, the two RRSs flanking both selectable markers are different.

[0081] In certain embodiments, the selectable marker is operably linked to a promoter sequence. In certain embodiments, the selectable marker is operably linked to an SV40 promoter. In certain embodiments, the selectable marker is operably linked to a cytomegalovirus (CMV) promoter.

[0082] In certain embodiments, the integrated 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 integrated exogenous nucleotide sequence comprises two selectable markers flanked by two RRSs and an IRES, wherein the IRES is operably linked to a second selectable marker. In certain embodiments, the integrated exogenous nucleotide sequence comprises three selectable markers flanked by two RRSs and an IRES, where the IRES is operably linked to a third selectable marker. In certain embodiments, the integrated exogenous nucleotide sequence comprises three selectable markers flanked by two RRSs and an IRES, 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 integrated 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 integrated 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 integrated 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.

[0083] In certain embodiments, the integrated exogenous nucleotide sequence comprises three RRSs. In certain embodiments, the third RRS is located between the first RRS and the second RRS. In certain embodiments, all three RRSs are identical. In certain embodiments, the first RRS and the second RRS are identical, and the third RRS is different from the first RRS or the second RRS. In certain embodiments, all three RRSs are heterospecific.

[0084] 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 identical. In certain embodiments, the SOIs are different.

[0085] In certain embodiments, the SOI encodes a single-chain antibody or a fragment thereof. In certain embodiments, the SOI encodes an antibody heavy chain sequence or a fragment thereof. In certain embodiments, the SOI encodes an antibody light chain sequence or a fragment thereof. In certain embodiments, the incorporated exogenous nucleotide sequence comprises an SOI encoding an antibody heavy chain sequence or a fragment thereof, and an SOI encoding an antibody light chain sequence or a fragment thereof. In certain embodiments, the incorporated exogenous nucleotide sequence comprises an SOI encoding a first antibody heavy chain sequence or a fragment thereof, an SOI encoding a second antibody heavy chain sequence or a fragment thereof, and an SOI encoding an antibody light chain sequence or a fragment thereof. In certain embodiments, the incorporated exogenous nucleotide sequence comprises an SOI encoding a first antibody heavy chain sequence or a fragment thereof, an SOI encoding a second antibody heavy chain sequence or a fragment thereof, an SOI encoding a first antibody light chain sequence or a fragment thereof, and a second SOI encoding an antibody light chain sequence or a fragment thereof. In 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 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.

[0086] 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 exogenous SOI and at least one selectable marker located between the first RRS and the second RRS. In certain embodiments, the two RRSs flanking the selectable marker and the exogenous SOI are identical. 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, a LoxP L3 sequence is located 5' of the selectable marker and a LoxP 2L sequence is located 3' of the selectable marker and the exogenous SOI.

[0087] In certain embodiments, the integrated exogenous nucleotide sequence comprises three RRSs and two exogenous SOIs, with the third RRS located between the first RRS and the second RRS. In certain embodiments, the first SOI is located between the first RRS and the third RRS, and the second SOI is located between the third RRS and the second RRS. In certain embodiments, the first SOI and the second SOI are different. In certain embodiments, the first RRS and the second RRS are the same, and the third RRS is different from both the first RRS and the 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 RRS and the third, and the selectable marker is located between the third RRS and the second RRS. 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 the selectable marker are located between the first RRS and the third RRS, and the second SOI is located between the third RRS and the second RRS.

[0088] 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, hi certain embodiments, the exogenous SOI is operably linked to a CMV promoter.

[0089] In certain embodiments, the incorporated 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 copies of the heavy and light chains of an antibody is located between the two RRSs.

[0090] In certain embodiments, the incorporated exogenous nucleotide sequence comprises three RRSs and at least two exogenous SOIs, with the third RRS located between the first RRS and the second RRS. In certain embodiments, at least one SOI is located between the first RRS and the third RRS, and at least one SOI is located between the third RRS and the second RRS. In certain embodiments, the first RRS and the second RRS are the same, and the third RRS is different from both the first RRS and the second RRS. In certain embodiments, all three RRSs are heterospecific. In certain embodiments, an SOI encoding one heavy chain and one light chain of the first antibody is located between the first RRS and the third RRS, and an SOI encoding one heavy chain and one light chain of the second antibody is located between the third RRS and the second RRS. In certain embodiments, the SOI encoding one heavy chain and two light chains of the first antibody is located between the first RRS and the third RRS, and the SOI encoding one heavy chain and one light chain of the second antibody is located between the third RRS and the second RRS. In certain embodiments, the SOI encoding one heavy chain and three light chains of the first antibody is located between the first RRS and the third RRS, and the SOI encoding one light chain of the first antibody and one heavy chain and one light chain of the second antibody are located between the third RRS and the second RRS. In certain embodiments, the SOI encoding one heavy chain and three light chains of the first antibody is located between the first RRS and the third RRS, and the SOI encoding two light chains of the first antibody and one heavy chain and one light chain of the second antibody are located between the third RRS and the second RRS. In certain embodiments, SOIs encoding different combinations of copies of the heavy and light chains of multiple antibodies are located between the first and third RRSs and between the third and second RRSs.

[0091] 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, but not limited to, the incorporation of two, three, four, five, six, seven, eight, or more SOIs can increase the titer and / or specific productivity.

[0092] In the context of antibody expression, the inclusion of additional heavy or light chain encoding SOIs can increase titer and / or specific productivity. For example, without limitation, increasing the 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) can achieve increased titer and / or specific productivity. Similarly, as outlined in the Examples below, increasing the copy number from HLL (3 SOIs) to HLL-HL (5 SOIs) or HLL-HLL (6 SOIs) can provide increased titer and / or specific productivity. Additionally, increasing the copy number to HLL-HL (5 SOIs) or HLL-HLHL (7 SOIs) can provide increased titer and / or specific productivity. Further options for heavy and light chain SOI copy numbers include, but are not limited to, HHL;HHL-H;HLL-H;HHL-HH;HHL-HL;HHL-LL;HLL-HH;HLL-HL;HLL-LL;HHL-HHL;HHL-HHH;HHL-HLL;HHL-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 additional copies occurs at a single genomic locus, in certain embodiments, the SOI copy may integrate at more than one locus, e.g., multiple copies may integrate at a single locus and one or more copies may integrate at one or more additional loci.

[0093] In certain embodiments, the location of the SOIs, e.g., whether one SOI is located 3' or 5' relative to another SOI, is selected to enhance the titer and / or specific productivity of host cells expressing the SOIs. For example, but not by way of limitation, in the context of antibody production, the location of integration of heavy and light chain SOIs can result in increased titer and / or specific productivity. In certain embodiments, the relative locations of the heavy and light chain SOIs can affect titer and specific productivity without altering the copy number of the SOIs.

[0094] 4.Host cells The presently disclosed subject matter provides host cells suitable for combining transposon-mediated genomic integration, targeted integration of a nucleotide sequence, and expression of a polypeptide of interest. For example, but not limited to, the host cells of the present disclosure contain 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. In certain embodiments, a host cell of the present disclosure comprises a locus comprising a nucleotide sequence selected from the group consisting of all or at least a portion of a contig sequence selected from NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1, and sequences at least 50%, 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 thereto.

[0095] 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.

[0096] In certain embodiments, the host cells are selected from the group consisting of SV40-transformed monkey kidney CV1 cells (COS-7), human embryonic kidney cells (e.g., 293 or 293 cells described in Graham et al., J. Gen Virol. 36:59 (1977)), baby hamster kidney cells (BHK), mouse Sertoli cells (e.g., TM4 cells described in Mather, Biol. Reprod. 23:243-251 (1980)), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK; buffalo rat hepatocytes (BRL 3A), human lung cells (W138), human hepatocytes (Hep G2), mouse mammary tumor (MMT 060562), TRI cells, e.g., as described in Mather et al., Annals The cells are selected from the group consisting of TRI cells, MRC 5 cells, FS4 cells, Y0 cells, NS0 cells, Sp2 / 0 cells, and PER.C6 (registered trademark) cells, as described in NYAcad.Sci.383:44-68 (1982).

[0097] 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 number of generations. In certain embodiments, the host cell is a primary cell.

[0098] In certain embodiments, expression of a polypeptide of interest is stable if the expression level is maintained at a particular 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 if the culture can be maintained without any selection. In certain embodiments, expression of a polypeptide of interest is high if the polypeptide product of the gene of interest reaches about 1 g / L, about 2 g / L, about 3 g / L, about 4 g / L, about 5 g / L, about 10 g / L, about 12 g / L, about 14 g / L, or about 16 g / L.

[0099] In certain embodiments, the expression level of the combination of the transposon-mediated genomic integrated SOI and the TI SOI is increased compared to a randomly integrated SOI, hi 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 the randomly integrated SOI.

[0100] 5. Methods of targeted integration and transposon-mediated genomic integration In certain embodiments, targeted integration may be combined with transposon-mediated genomic integration. In certain embodiments, targeted integration may be followed by transposon-mediated genomic integration. In certain embodiments, targeted integration may be performed simultaneously with transposon-mediated genomic integration. In certain embodiments, targeted integration may be followed by transposon-mediated genomic integration.

[0101] 5.1. Targeted Integration via Recombinase-Mediated Recombination A "recombination recognition sequence" (RRS) is a nucleotide sequence that is recognized by a recombinase and is necessary and sufficient for a recombinase-mediated recombination event. An RRS can be used to define the location at which a recombination event occurs within a nucleotide sequence.

[0102] 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.

[0103] 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.

[0104] 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 an 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. Recombinases can be introduced into host cells using expression vectors containing the coding sequences for the enzymes.

[0105] 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 nonpalindromic core region flanked by two 13-bp inverted repeats. Cre recombinase binds to the 13-bp repeats, thereby mediating recombination within the 8-bp core region. Cre-LoxP-mediated recombination occurs with high efficiency and does not require any other host factors. When two LoxP sequences are located in the same nucleotide sequence and in the same orientation, Cre-mediated recombination excises the DNA sequence located between the two LoxP sequences into a covalently closed circle. If two LoxP sequences are located in opposite orientations on the same nucleotide sequence, Cre-mediated recombination will reverse the orientation of the DNA sequence located between the two sequences. LoxP sequences can be located on different chromosomes to facilitate recombination between different chromosomes. If two LoxP sequences are located on two different DNA molecules and one of the DNA molecules is circular, Cre-mediated recombination will result in the integration of the circular DNA sequence.

[0106] 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, LoxP 2L, LoxFas, Lox511, Lox2272, Lox2372, Lox5171, Loxm2, Lox71, and Lox66 sequences. For example, the Lox71 sequence has a 5-bp mutation in the left 13-bp repeat. The Lox66 sequence has a 5-bp mutation in the right 13-bp repeat. Both wild-type and mutant LoxP sequences can mediate Cre-dependent recombination.

[0107] The FLP-FRT site-specific recombination system is similar to the Cre-Lox system. It contains flippase (FLP) recombinase 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 breakage, exchange, and ligation within the 8-bp spacer. As with Cre recombinase, the position and orientation of the two FRT sequences determine the outcome of FLP-mediated recombination. In 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.

[0108] 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, excising the integrated phage from the bacterial DNA. Both integrases can catalyze recombination without the aid of 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 were developed as a complement to the Cre-LoxP and FRT-FLP systems that are widely used for genetic engineering purposes.

[0109] 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.

[0110] In certain embodiments, the integrated exogenous nucleotide sequence comprises two RRSs, and the vector comprises two RRSs that match the two RRSs on the integrated exogenous nucleotide sequence, i.e., the first RRS on the integrated exogenous nucleotide sequence matches the first RRS on the vector, and the second RRS on the integrated exogenous nucleotide sequence matches the second RRS on the vector. In certain embodiments, the first RRS on the integrated exogenous nucleotide sequence and the first RRS on the vector are identical to the second RRS on the integrated exogenous nucleotide sequence and the second RRS on the vector. A non-limiting example of such a "single-vector RMCE" strategy is shown in Figure 2A of PCT Application Publication No. U.S. Patent Application Publication No. 2018 / 067070 (International Publication No. WO 2019126634). In certain embodiments, the first RRS on the integrated exogenous nucleotide sequence and the first RRS on the vector are different from the second RRS on the integrated exogenous nucleotide sequence and the second RRS on the vector, hi certain embodiments, the first RRS on the integrated exogenous nucleotide sequence and the first RRS on the vector are both LoxP L3 sequences, and the second RRS on the integrated exogenous nucleotide sequence and the second RRS on the vector are both LoxP 2L sequences.

[0111] In certain embodiments, a "two-vector RMCE" strategy is used. For example, but not limited to, the integrated exogenous nucleotide sequence can include three RRSs, e.g., a third RRS ("RRS3") located between the first RRS ("RRS1") and the second RRS ("RRS2"), where the first vector includes two RRSs that match the first and third RRSs on the integrated exogenous nucleotide sequence, and the second vector includes two RRSs that match the third and second RRSs on the integrated exogenous nucleotide sequence. Figure 4 of PCT U.S. Patent Application Publication No. 2018 / 067070 (International Publication No. 2019126634) shows an example of a two-vector RMCE strategy. 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) contains RRS1, a first SOI, and a promoter followed by a start codon and RRS3 (in that order). The other vector (reverse) contains RRS3, SOI2, and RRS2 (in that order) fused to a coding sequence for a marker that does not contain a start 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 SOI and the second SOI pair to form a multispecific, e.g., bispecific, antibody.

[0112] 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.

[0113] Both single-vector and two-vector RMCE allow for the unidirectional integration of one or more donor DNA molecules into a predetermined site in the genome of a host cell and the precise exchange of a DNA cassette present on the donor DNA with a DNA cassette on the host genome that remains at the integration site. The DNA cassette features at least one selectable marker (although certain two-vector RMCE examples may use a "split selectable marker" as outlined herein) and / or two heterospecific RRSs flanking at least one exogenous SOI. RMCE involves a double recombination crossover event, catalyzed by a recombinase between the two heterospecific RRSs and the donor DNA molecule within the target genomic locus. RMCE is designed to introduce a copy of the SOI or selectable marker into a predetermined locus in the genome of the host cell. Unlike recombination involving only a single crossover event, RMCE can be performed in a way that prevents the introduction of prokaryotic vector sequences into the host cell genome, thereby reducing and / or preventing the undesired triggering of host immune or defense mechanisms. The RMCE procedure can be repeated for multiple DNA cassettes.

[0114] In certain embodiments, targeted integration is achieved by a single crossover recombination event in which a single exogenous nucleotide sequence comprising an RRS flanked by at least one exogenous SOI or at least one selectable marker is integrated into a predetermined site in the genome of the host cell. In certain embodiments, targeted integration is achieved by a single RMCE in which a DNA cassette comprising at least one exogenous SOI or at least one selectable marker flanked by two heterospecific RRSs is integrated into a predetermined site in the genome of the host cell. In certain embodiments, targeted integration is achieved by two RMCEs in which two different DNA cassettes, each comprising at least one exogenous SOI or at least one selectable marker flanked by two heterospecific RRSs, are integrated into a predetermined site in the genome of the host cell. In certain embodiments, targeted integration is achieved by multiple RMCEs in which DNA cassettes from multiple vectors, each comprising at least one exogenous SOI or at least one selectable marker flanked by two heterospecific RRSs, are integrated into a predetermined site in the genome of the host cell. In certain embodiments, the selectable marker can be encoded partially on a first vector and partially on a second vector, such that integration of both RMCEs allows expression of the selectable marker. An example of such a system is shown in Figure 4 of PCT US Patent Application Publication No. 2018 / 067070 (International Publication No. 2019126634).

[0115] In certain embodiments, targeted integration via recombinase-mediated recombination results in the selection marker or one or more exogenous SOIs integrated into one or more predetermined integration sites in the genome of the host cell along with sequences derived from the prokaryotic vector. In certain embodiments, targeted integration via recombinase-mediated recombination results in the selection marker or one or more exogenous SOIs integrated into one or more predetermined integration sites in the genome of the host cell that do not contain sequences derived from the prokaryotic vector.

[0116] 5.2 Targeted integration via homologous recombination, HDR, or NHEJ The subject matter of this disclosure relates to targeted integration via homologous recombination, or exogenous site-specific nucleases followed by HDR or NHEJ.

[0117] 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. Homologous recombination involves the exchange of genetic information between two homologous DNA molecules, so it does not change the overall arrangement of genes on chromosomes. During homologous recombination, a nick or break is formed in the 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 Holliday junctions, and eventual separation of the Holliday junctions.

[0118] 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 homology-directed repair (HDR) pathway, and homologous recombination is the most common form of HDR. Because HDR requires the presence of homologous DNA within the cell, this repair pathway is usually 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 repair pathway for DSBs is non-homologous end joining (NHEJ). NHEJ is a repair pathway in which the ends of broken DNA are joined together without the need for a homologous DNA template.

[0119] Targeted integration can be promoted by exogenous site-specific nuclease followed by HDR. This can increase the frequency of homologous recombination by introducing DSB at a specific target genomic site. 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.

[0120] 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 invading bacteriophages. Cas is a nuclease that, when guided by a synthetic guide RNA (gRNA), associates with specific nucleotide sequences within a cell and can edit the DNA within or surrounding that nucleotide sequence by, for example, creating one or more single-strand breaks, DSBs, and / or point mutations. 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 highly variable residue region of the DNA-binding domain monomer, different artificial TALENs can be created that target various nucleotide sequences. The DNA-binding domain then directs the nuclease to the target sequence, creating DSBs.

[0121] Targeted integration by homologous recombination or HDR involves the presence of a homologous sequence to 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.

[0122] In certain embodiments, a vector for targeted integration of an exogenous nucleotide sequence into a host cell comprises an endogenous sequence comprising a portion of one of the contig sequences of contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1, or a gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2, or a nucleotide sequence homologous to a sequence selected from SEQ ID NOs: 1-7 adjacent to at least one selectable marker. In certain embodiments, a vector for targeted integration of an exogenous nucleotide sequence into a host cell comprises an endogenous sequence comprising a portion of the contig sequence of one of the contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1, or a gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2, or a nucleotide sequence homologous 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 for targeted integration of an exogenous nucleotide sequence into a host cell comprises a portion of the contig sequence of one of contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1, and a nucleotide sequence at least 50% homologous to a sequence selected from SEQ ID NOs: 1-7 flanking a DNA cassette, wherein the DNA cassette comprises at least one selectable marker flanked by two RRSs and at least one exogenous SOI.In certain embodiments, a vector for targeted integration of an exogenous nucleotide sequence into a host cell comprises an endogenous sequence of a portion of the contig sequence of one of the contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1. , or a nucleotide sequence at least 50% homologous to a gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2 flanking the DNA cassette, wherein the DNA cassette comprises at least one selectable marker flanked by two RRSs and at least one exogenous SOI. In certain embodiments, the vector nucleotide sequence is the endogenous sequence of a portion of one of the contig sequences of contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1, or The vector 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 gene selected from the group consisting of 100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2, or a sequence selected from SEQ ID NOs: 1 to 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.

[0123] In certain embodiments, the polynucleotide for targeted integration of an exogenous nucleotide sequence into a host cell comprises a nucleotide sequence homologous to an endogenous sequence of a portion of one of the contig sequences of contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1, or a gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2, or a sequence selected from SEQ ID NOs: 1-7 adjacent to at least one selectable marker. In certain embodiments, the polynucleotide for targeted integration of an exogenous nucleotide sequence into a host cell comprises a nucleotide sequence homologous to an endogenous sequence of a portion of one of the contig sequences of contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1, or a gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2, or 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 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, wherein the DNA cassette comprises at least one selectable marker flanked by two RRSs and at least one exogenous SOI.In certain embodiments, the polynucleotide for targeted integration of an exogenous nucleotide sequence into a host cell is at least 50% identical to the endogenous sequence of a portion of the contig sequence of one of contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1. The DNA cassette comprises a homologous nucleotide sequence or a nucleotide sequence at least 50% homologous to a gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2, flanking the DNA cassette, and the DNA cassette comprises at least one exogenous SOI flanked by at least one selectable marker and two RRSs. In certain embodiments, the flanking nucleotide sequence is an endogenous sequence of a portion of one of the contig sequences of contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1, or 100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2, or a sequence selected from SEQ ID NOs: 1 to 7, which 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 gene selected from the group consisting of:

[0124] 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.

[0125] 5.3 Transposon-mediated genomic integration The subject matter of the present disclosure relates to TI host cells that are also subjected to transposon-mediated genomic integration of one or more exogenous nucleic acids, and to the methods for producing and using the combination of transposon-mediated genomic integration and TI host cells.As outlined herein, in certain embodiments, targeted integration can be performed simultaneously with transposon-mediated genomic integration.In certain embodiments, targeted integration can be performed after transposon-mediated genomic integration.In certain embodiments, targeted integration can be performed before transposon-mediated genomic integration.

[0126] Transposons useful in connection with the methods described herein are known in the art and include, but are not limited to: piggyBac transposon (see, e.g., Wilson et al., Molecular Therapy, 15(1):139-145 (2007)); sleeping beauty transposon (see, e.g., Ivics et al., Cell 91:501-510 (1997)); and Tol2 transposon (e.g., Balciunas et al., PLoS Genent. Nov 10;2(11):e169. doi:10.1371 / journal.pgen.0020169. Epub 2006 Aug 28).

[0127] Generally, transposons useful in the method of the present disclosure translocate via a non-replicative "cut and paste" mechanism. For example, without being bound by theory, the transposition catalyzed by a transposon useful in the method described herein can proceed by the recognition of two terminal inverted repeats (TIRs) by a DNA transposase, which cuts its target and thereby releases the DNA transposon from its donor sequence (e.g., donor plasmid). Once excised, the transposon can be integrated into the host cell genome by cutting the corresponding sequence in the genome with the same transposase.

[0128] 5.4 Controlled SOI expression 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 that are 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 regulatory factor, such as a small molecule such as, 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, the regulated expression system includes at least one SOI transcribed under a regulated promoter operably linked thereto. In certain embodiments, the regulated expression system can 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 a regulated expression system can be used to link expression of the SOI to observed adverse effects.

[0129] In certain embodiments, a regulated expression system may be used to minimize the effects of transcriptional and cell line variability during analysis of the root cause of difficult-to-express molecules. For example, but not limited to, expression of a SOI in a transposon-mediated genomic integration host and a TI host combination may be induced by the addition of a regulatory factor, such as doxycycline, to the culture. In certain embodiments, the regulated expression vector utilizes a tetracycline-regulated promoter to express the SOI, allowing for regulated expression of the SOI.

[0130] In certain embodiments, the controlled expression system described in the present disclosure may be used to better determine one or more underlying causes of low protein expression of an SOI, e.g., a therapeutic antibody, compared to a control cell line. In certain embodiments, once relatively low expression of an SOI, e.g., a therapeutic antibody, in a controlled expression cell line is confirmed, the intracellular accumulation and secretion levels of the SOI may be assessed by utilizing protein translation inhibitor treatment, e.g., Dox and cycloheximide.

[0131] As outlined in detail herein, regulated expression can be based on gene switches for blocking or activating mRNA synthesis through the controlled binding of transcriptional repressors or activators to constitutive or minimal promoters. 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 via a transcriptional silencer. In certain non-limiting embodiments, activation of a mammalian or viral enhancer-less minimal promoter can be achieved by regulated coupling to an activation domain.

[0132] In certain embodiments, conditional coupling of a transcriptional repressor or transcriptional activator can be achieved by using an allosteric protein that binds to the promoter in response to an external stimulus. In certain embodiments, conditional coupling of a transcriptional repressor or transcriptional activator can be achieved by using an intracellular receptor that can be released from a sequestering protein and thus bind to the target promoter. In certain embodiments, conditional coupling of a transcriptional repressor or transcriptional activator can be achieved by using a chemically derived dimerizing agent.

[0133] In certain embodiments, the allosteric protein used in the regulated expression system of the present disclosure can be an antibiotic, a bacterial quorum-sensing messenger, a catabolite, or a protein that regulates transcriptional activity in response to a culture parameter such as temperature, e.g., cold or heat. In certain embodiments, such regulated expression systems can be catabolite-based, for example, in which a bacterial repressor controlling the catabolic genes of an alternative carbon source has been introduced into mammalian cells. In certain embodiments, repression of the target promoter can be achieved by cumate-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 transcription activation domain.

[0134] In certain embodiments, the disclosed regulated expression system can be used in prokaryotic quorum-sensing-based expression systems that govern intra- and inter-population communication via quorum-sensing molecules. These quorum-sensing molecules bind to receptors in target cells, modulating the receptor's affinity for its cognate promoter and initiating specific regulon switches. In certain embodiments, the quorum-sensing molecule can be N-(3-oxo-octanoyl)-homoserine lactone, the presence of which 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 an 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).

[0135] In certain embodiments, inducer molecules that regulate allosteric proteins used in the regulated expression systems of the present disclosure may be, but are not limited to, coumarate, isopropyl-β-D-galactopyranoside (IPTG), macrolides, 6-hydroxynicotine, doxycycline, streptogramins, NADH, and tetracycline.

[0136] In certain embodiments, the intracellular receptor used in the controlled expression system of the present disclosure may be a cytoplasmic or nuclear receptor. In certain embodiments, the controlled expression system of the present disclosure may utilize protein sequestration and release of transcription factors from inhibition using small molecules. In certain embodiments, the controlled expression system of the present disclosure may rely on steroid regulation, where a hormone receptor is fused to a natural or artificial transcription factor that can be released from HSP90 in the cytosol, translocate into the nucleus, and activate a selected promoter. In certain embodiments, mutant receptors regulated by synthetic steroid analogs may be used to avoid crosstalk with endogenous steroid hormones. In certain embodiments, the receptor may be a 4-hydroxytamoxifen-responsive estrogen receptor variant or a RU486-inducible progesterone receptor variant. In certain embodiments, a nuclear receptor-derived rosiglitazone-responsive transcriptional switch based on the human nuclear peroxisome proliferator-activated receptor gamma (PPARγ) may be used in the controlled expression system of the present disclosure. In certain embodiments, the steroid-responsive receptor variant can be RheoSwitch, which is based on a modified spruce budworm (Choristoneura fumiferana) ecdysone receptor and a 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 a Gal4 response element.

[0137] In certain embodiments, the regulated expression systems disclosed herein may utilize chemically induced dimerization of DNA-binding proteins and transcriptional activators for activation of a minimal core promoter fused to its cognate operator. In certain embodiments, the regulated expression systems disclosed herein may utilize rapamycin-regulated dimerization of FRB and FKBP. 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 regulated expression systems disclosed herein may utilize the bacterial gyrase B subunit (GyrB), which dimerizes in the presence of the antibiotic coumermycin and dissociates with novobiocin.

[0138] In certain embodiments, the controlled expression system of the present disclosure can be used for controlled siRNA expression. In certain embodiments, the controlled 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 utilize a Xenopus terminal oligopyrimidine element (TOP), which blocks translation initiation by forming a hairpin structure in the 5' untranslated region.

[0139] In certain embodiments, the regulated expression systems described herein may utilize gas-phase regulated expression, such as the acetaldehyde-inducible regulation (AIR) system. The AIR system may use the Aspergillus nidulans AlcR transcription factor, which specifically activates a PAIR promoter constructed from an AlcR-specific operator fused to a minimal human cytomegalovirus promoter in the presence of non-toxic concentrations of gaseous or liquid acetaldehyde.

[0140] In certain embodiments, the regulated expression of the present disclosure may utilize a Tet-on or Tet-off system, in which expression of one or more SOIs may be controlled by tetracycline or its analog, doxycycline.

[0141] In certain embodiments, the regulated expression systems of the present disclosure may utilize a PIP-on or PIP-off system, in which expression of the SOI may be controlled by, for example, pristinamycin, tetracycline, and / or erythromycin.

[0142] 6. Preparation and Use of Transposon-Mediated Genomic Integration and Combination with TI Host Cells The subject matter of the present disclosure relates to methods for targeted integration of an exogenous nucleotide sequence into a host cell. In certain embodiments, the methods involve integration of an exogenous nucleotide sequence into a host cell to generate a host cell suitable for subsequent targeted integration of a SOI. In certain embodiments, the methods involve recombinase-mediated recombination. In certain embodiments, the methods involve homologous recombination, HDR, and / or NHEJ.

[0143] In certain embodiments, the presently disclosed subject matter relates to a method for targeted integration of an exogenous nucleotide sequence into a host cell in combination with transposon-mediated genomic integration of the exogenous nucleotide sequence into the same host cell. In certain embodiments, the method relates to integration of an exogenous nucleotide sequence into a host cell to generate a host cell suitable for subsequent targeted integration of a SOI in combination with transposon-mediated genomic integration of the same or a different SOI. In certain embodiments, the method comprises recombinase-mediated recombination. In certain embodiments, the method comprises homologous recombination, HDR, and / or NHEJ.

[0144] 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, inserted into, and maintained in the host cell membrane.

[0145] 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 viral 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 injection via a gene gun. In certain embodiments, both DNA molecules and RNA molecules are introduced into host cells using the methods described herein.

[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 that expresses 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 genome of the TI host cell, wherein the locus is at least about 90% homologous to SEQ ID NOs: 1-7, and the exogenous nucleotide sequence comprises 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 comprises at least one exogenous SOI and two RRSs flanking at least one second selectable marker; c) introducing a recombinase that recognizes the RRSs; and d) selecting TI cells that express the second selectable marker, thereby isolating the TI host cell that expresses the polypeptide of interest.

[0147] 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 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 that recognizes the RRSs; and d) selecting for TI cells that express the second selectable marker, thereby isolating the TI host cell that expresses the polypeptide of interest.

[0148] 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 comprising an exogenous nucleotide sequence integrated into a site within a locus in the genome of the TI host cell, the locus being at least about 90% homologous to SEQ ID NOs: 1-7, and 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 that matches the two RRSs on the integrated exogenous nucleotide sequence and comprises at least one exogenous SOI and two heterologous RRSs flanked by at least one second selectable marker; c) introducing a recombinase that recognizes the RRSs and performs a single RMCE; and d) selecting TI cells that express the second selectable marker, thereby isolating the TI host cell that expresses the polypeptide of interest.

[0149] In certain embodiments, the disclosure provides a method for preparing a TI host cell that expresses a polypeptide of interest, the method comprising: a) inserting an exogenous nucleotide into a site within an endogenous sequence comprising all or a portion of the contig sequence of one of contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1, or into a 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. a) providing a TI host cell comprising an exogenous nucleotide sequence, wherein the exogenous nucleotide sequence comprises a first DNA cassette comprising two heterologous RRSs flanked by at least one first selection marker; b) introducing into the cell provided in a) a vector comprising a second DNA cassette that matches the two RRSs on the integrated exogenous nucleotide sequence and comprises at least one exogenous SOI and two heterologous RRSs flanked by at least one second selection marker; c) introducing a recombinase that recognizes the RRSs and performs a single RMCE; and d) selecting TI cells that express the second selection marker, thereby isolating TI host cells that express the polypeptide of interest.

[0150] In certain embodiments, the present disclosure provides a method for preparing a TI host cell that expresses a first polypeptide of interest and a 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 comprises an exogenous nucleotide sequence integrated into a site within a locus in the genome of the host cell, the locus being located within contig NW_006874047.1, N and NW_003615411.1, or a sequence that is at least about 90% homologous to all or a portion of the contig sequence of one of SEQ ID NOs: 1 to 7, wherein the exogenous nucleotide sequence comprises a first RRS and a second RRS flanking at least one first selectable marker, and a first and a third RRS located between the first RRS and the second RRS, wherein all RRSs are heterospecific; b) introducing into the cells provided in a) a first vector comprising two RRSs that match the first and third RRSs on the integrated 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 integrated 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) selecting TI cells that express the second selectable marker, thereby isolating TI host cells that express the first polypeptide of interest and the second polypeptide of interest. In certain embodiments, rather than having the entire selection marker on a first vector, the first vector comprises a promoter sequence operably linked to an ATG codon flanked upstream by a first SOI and downstream by an RRS; and the second vector comprises a selection marker lacking an ATG transcription start codon flanked upstream by an RRS and downstream by a second SOI.

[0151] In certain embodiments, the present disclosure provides a method for preparing a TI host cell that expresses a first polypeptide of interest and a second polypeptide of interest (wherein the first polypeptide and the second polypeptide can be the same or different), the method comprising: a) preparing a TI host cell from contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003616412.1; and a TI host cell comprising an exogenous sequence nucleotide sequence integrated into a site within an endogenous sequence comprising all or a portion of a contiguous sequence of one of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, XP_003512331.2, and sequences at least about 90% homologous thereto, wherein the exogenous nucleotide sequence comprises at least The method includes: providing a TI host cell comprising a first RRS and a second RRS flanking a first selectable marker, and a third RRS located between the first RRS and the second RRS, all of the RRSs being heterologous; b) introducing into the cell provided in a) a first vector comprising two RRSs that match the first RRS and the third RRS on the integrated exogenous nucleotide sequence and are flanked by at least one first exogenous SOI and at least one second selectable marker; c) introducing into the cell provided in a) a second vector comprising two RRSs that match the second RRS and the third RRS on the integrated exogenous nucleotide sequence and are flanked by at least one second exogenous SOI; d) introducing one or more recombinases that recognize the RRSs; and e) selecting TI cells that express the second selectable marker, thereby isolating TI host cells that express the first polypeptide of interest and the second polypeptide of interest.In certain embodiments, rather than having the entire selection marker on a first vector, the first vector comprises a promoter sequence operably linked to an ATG codon flanked upstream by a first SOI and downstream by an RRS; and the second vector comprises a selection marker lacking an ATG transcription start codon flanked upstream by an RRS and downstream by a second SOI.

[0152] In certain embodiments, the present disclosure provides a method for preparing a TI host cell that expresses a first polypeptide of interest and a second polypeptide of interest (wherein the first polypeptide and the second polypeptide 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 in the genome of the TI host cell, the locus being selected from the contigs NW_006874047.1, NW_006884592.1, NW_006881296 .1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1, or a sequence comprising all or a portion of one of the contig sequences of SEQ ID NOs: 1 to 7, and the exogenous nucleotide sequence comprises a first DNA cassette comprising a first RRS and a second RRS adjacent to at least one first selectable marker, and a third RRS located between the first RRS and the second RRS, wherein all three RRSs are different. b) introducing into the cells provided in a) 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 cells 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 that recognize the RRSs and perform two rounds of RMCE; and e) selecting TI cells that express the second selection marker, thereby isolating TI host cells that express the first polypeptide of interest and the second polypeptide of interest.In certain embodiments, rather than having the entire selection marker on a first vector, the first vector comprises a promoter sequence operably linked to an ATG codon flanked upstream by a first SOI and downstream by an RRS; and the second vector comprises a selection marker lacking an ATG transcription start codon flanked upstream by an RRS and downstream by a second SOI.

[0153] In certain embodiments, the present disclosure provides a method for preparing a TI host cell that expresses a first polypeptide of interest and a second polypeptide of interest (wherein the first polypeptide and the second polypeptide can be the same or different), the method comprising: a) subcloning the entire contig sequence of one of contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1; or a portion thereof, or into a gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, XP_003512331.2, and sequences at least about 90% homologous thereto, wherein the exogenous nucleotide sequence comprises a first RRS and a second RRS adjacent to at least one first selectable marker, and a third RRS located between the first RRS and the second RRS. b) introducing into the cells provided in a) a first vector comprising a second DNA cassette, wherein the second DNA 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 selectable marker; c) introducing into the cells provided in a) a third DNA cassette, wherein the second DNA 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 selectable marker; 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 that recognize the RRSs and perform two rounds of RMCE; and e) selecting TI cells that express a second selection marker, thereby isolating TI host cells that express the first polypeptide of interest and the second polypeptide of interest.In certain embodiments, rather than having the entire selection marker on a first vector, the first vector comprises a promoter sequence operably linked to an ATG codon flanked upstream by a first SOI and downstream by an RRS; and the second vector comprises a selection marker lacking an ATG transcription start codon flanked upstream by an RRS and downstream by a second SOI.

[0154] In certain embodiments, the 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 comprising an exogenous nucleotide sequence integrated into a site within a locus of the TI host cell genome, wherein the locus is a sequence selected from a sequence comprising all or a portion of the contig sequence of one of contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1, or ... The method includes providing a TI host cell that is at least about 90% homologous to a sequence of any one of Nos. 1 to 7, wherein the exogenous nucleotide sequence comprises an RRS flanked by at least one first selectable marker; b) introducing into the cell provided in a) a vector that contains an RRS that matches the RRS on the integrated exogenous nucleotide sequence and that is flanked by at least one exogenous SOI and at least one second selectable marker; c) introducing a recombinase that recognizes the RRS; and d) selecting for TI cells that express the second selectable marker, thereby isolating TI host cells that express the polypeptide of interest.

[0155] In certain embodiments, the disclosure provides a method for preparing a TI host cell to express a polypeptide of interest, the method comprising: a) locating a site within an endogenous sequence comprising all or a portion of the contig sequence of one of contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1, or LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, XP_003512331.2, and sequences at least about 90% homologous thereto; The method includes the steps of: providing a TI host cell comprising an exogenous nucleotide sequence integrated into a gene selected from the group consisting of sequences, wherein the exogenous nucleotide sequence comprises an RRS adjacent to at least one first selection marker; b) introducing into the cells 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 that recognizes the RRS; and d) isolating TI host cells that express the polypeptide of interest by selecting for TI cells that express the second selection marker.

[0156] The subject matter of the present disclosure 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 into a site within a locus of the TI host cell genome, wherein the locus comprises a sequence comprising all or a portion of the contig sequence of one of contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1, or a sequence selected from SEQ ID NOs: 1-7. a) providing a TI host cell that is at least about 90% homologous to a sequence expressed in a target vector, wherein the exogenous nucleotide sequence comprises two RRSs flanked by at least one exogenous SOI and at least one first selection marker; b) introducing into the cell provided in a) a vector that comprises two RRSs that match the two RRSs on the integrated exogenous nucleotide sequence and are flanked by at least one second selection marker; c) introducing a recombinase that recognizes the RRSs; and d) selecting TI cells that express the second selection marker, thereby isolating TI host cells suitable for subsequent targeted integration.

[0158] In certain embodiments, the present disclosure provides a method for preparing a TI host cell suitable for subsequent targeted integration, the method comprising: a) targeting a TI host cell to a site within an endogenous sequence comprising all or a portion of the contig sequence of one of contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1, or a sequence selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, XP_003512331.2, and a sequence at least about 90% homologous thereto; a) providing a TI host cell comprising an exogenous nucleotide sequence integrated into a gene to be selected, wherein the exogenous nucleotide sequence comprises two 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 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 that recognizes the RRSs; and d) selecting TI cells that express the second selectable marker, thereby isolating TI host cells suitable for subsequent targeted integration.

[0159] In certain embodiments, the present disclosure provides methods 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 genome of the TI host cell, wherein the locus is at least about 90% homologous to a sequence comprising all or a portion of the contig sequence of one of contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1, or a sequence selected from SEQ ID NOs: 1-7, wherein the exogenous nucleotide sequence is 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, wherein the first RRS of the vector matches the first RRS on the integrated exogenous nucleotide sequence, the second RRS of the vector matches the second RRS on the integrated exogenous nucleotide sequence, and at least one second selection marker is located between the first and second RRSs; c) introducing a recombinase that recognizes the first and second RRSs in both the vector and the integrated exogenous nucleotide sequence; and d) selecting TI host cells that express the second selection marker, thereby isolating TI host cells suitable for subsequent targeted integration.

[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) cloning the entire contig sequence of one of contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1; and a sequence at least about 90% homologous thereto, wherein the exogenous nucleotide sequence is integrated into a site within an endogenous sequence comprising a portion or part of the endogenous sequence, or into a 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 integrated into at least one The method includes: providing a TI host cell comprising a first RRS and a second RRS flanked by an exogenous SOI and at least one first selection marker; b) introducing into the cell provided in a) a vector comprising three RRSs, wherein the first RRS of the vector matches the first RRS on the integrated exogenous nucleotide sequence, the second RRS of the vector matches the second RRS on the integrated exogenous nucleotide sequence, and at least one second selection marker is located between the first and second RRSs; c) introducing a recombinase that recognizes the first RRS and the second RRS on both the vector and the integrated exogenous nucleotide sequence; and d) selecting TI host cells that express the second selection marker, thereby isolating TI host cells suitable for subsequent targeted integration.

[0161] 6.2 Methods for Targeted Modification of Host Cells Using Homologous Recombination, HDR, or NHEJ 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 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 for 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 that expresses 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 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.

[0163] In certain embodiments, homologous recombination is facilitated 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 the 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 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 genome of the TI host cell, wherein the locus is at least about 90% homologous to a sequence comprising all or a portion of a contig sequence of one of contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1, or a sequence selected from SEQ ID NOs: 1-7; b) introducing into the TI host cell a vector and NW_003615411.1, or a nucleotide sequence at least 50% homologous to a portion of the contig sequence of one of contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1, or a sequence selected from SEQ ID NOs: 1-7 flanking the DNA cassette, wherein the DNA cassette comprises at least one selectable marker flanked by two RRSs; c) selecting the selectable marker and isolating a TI host cell suitable for subsequent targeted integration.

[0166] 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 a locus in the genome of the TI host cell, wherein the locus is at least about 90% homologous to a sequence comprising all or a portion of the contig sequence of one of contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1, or a sequence selected from SEQ ID NOs: 1-7; b) introducing a polynucleotide into the TI host cell. and NW_003615411.1, or a nucleotide sequence at least 50% homologous to a sequence selected from SEQ ID NOs: 1-7 adjacent to a DNA cassette, wherein the DNA cassette comprises at least one selectable marker flanked by two RRSs; c) selecting the selectable marker and isolating a suitable TI host cell 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 genome of the TI host cell, wherein the locus is at least about 90% homologous to a sequence comprising all or a portion of a contig sequence of one of contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1, or a sequence selected from SEQ ID NOs: 1-7; b) introducing a vector into the host cell, wherein the vector , a sequence comprising all or a portion of one of the contig sequences of contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1, or a nucleotide sequence at least 50% homologous to a sequence selected from SEQ ID NOs: 1-7 adjacent to the DNA cassette, wherein the DNA cassette comprises three RRSs, and the third RRS and at least one selectable marker are located between the first RRS and the second RRS; 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 genome of the TI host cell, wherein the locus is at least about 90% homologous to a sequence comprising all or a portion of the contig sequence of one of contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1, or a sequence selected from SEQ ID NOs: 1-7; b) introducing a polypeptide into the host cell, wherein the polypeptide a) introducing a polypeptide into a TI host cell comprising a sequence comprising all or a portion of the contig sequence of one of contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1, or a nucleotide sequence at least 50% homologous to a sequence selected from SEQ ID NOs: 1-7 adjacent to the DNA cassette, wherein the DNA cassette comprises three RRSs, and a third RRS and at least one selectable marker are located between the first RRS and the second RRS; and b) 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 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 comprise a sequence comprising all or a portion of the contig sequence of one of contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1, or a sequence selected from SEQ ID NOs: 1-7. The method includes: providing a TI host cell that is at least about 90% homologous to the sequence, wherein at least one exogenous nucleotide sequence comprises two RRSs flanked by 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 comprises at least one exogenous SOI and two RRSs flanked by at least one second selectable marker; c) introducing a recombinase that recognizes the RRSs, or a nucleic acid encoding the recombinase, and selecting TI cells that express the second selectable marker, thereby isolating TI host cells that express at least one polypeptide of interest.

[0170] In certain embodiments, the present disclosure provides a method for preparing a TI host cell that expresses at least one first polypeptide of interest and a second polypeptide of interest (wherein the first polypeptide and the second polypeptide can be the same or different), the method 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 selected from the group consisting of contig NW_00687404 7.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1, or a sequence selected from SEQ ID NOs: 1 to 7, wherein the exogenous nucleotide sequence comprises a first RRS and a second RRS flanking at least one first selectable marker, and a second RRS flanking the first RRS and a second RRS flanking the first selectable marker. b) introducing into the cells 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 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 at least one integrated exogenous nucleotide sequence and that are flanked by at least one second exogenous SOI; d) introducing one or more recombinases that recognize the RRSs, or one or more nucleic acids encoding the recombinases; and e) selecting TI cells that express the second selectable marker, thereby isolating TI host cells that express at least one first polypeptide of interest and a second polypeptide of interest.In certain embodiments, rather than having the entire selection marker on a first vector, the first vector comprises a promoter sequence operably linked to an ATG codon flanked upstream by a first SOI and downstream by an RRS; and the second vector comprises a selection marker lacking an ATG transcription start codon flanked upstream by an RRS and downstream by a second SOI.

[0171] In certain embodiments, the 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 comprising at least one exogenous nucleotide sequence integrated into a site within one or more loci of the genome of the TI host cell, wherein the one or more loci comprise a sequence comprising all or a portion of the contig sequence of one of contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1, or a sequence comprising all or a portion of the contig sequence of one of SEQ ID NOs: 1-7. b) introducing into the cell provided in a) a vector containing 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 operably linked to a regulatable promoter; c) introducing a recombinase that recognizes the RRS or a nucleic acid encoding the recombinase; and d) selecting TI cells that express the exogenous SOI in the presence of an inducer, thereby isolating TI host cells that express the polypeptide of interest.

[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 genome of the host cell, wherein the locus comprises a sequence comprising all or a portion of the contig sequence of one of contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1, or a sequence that is at least about 90% homologous to a sequence selected from SEQ ID NOs: 1-7; and b) culturing the cell under conditions suitable for expression of 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 a first polypeptide of interest and a second polypeptide of interest (wherein the first polypeptide and the second polypeptide 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 TI host cell genome, the locus being selected from the contigs NW_006874047.1, NW_00688459 2.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1, or a sequence selected from SEQ ID NOs: 1 to 7, wherein the exogenous nucleotide sequence comprises a first RRS, a second RRS, and a third RRS located between the first RRS and the second RRS, and all The method includes providing a TI host cell in which the RRS is 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 are adjacent to at least one first exogenous SOI operably linked to a regulatable promoter; 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 are adjacent to at least one second SOI operably linked to a regulatable promoter; d) introducing one or more recombinases that recognize the RRSs, or one or more nucleic acids encoding the recombinases; and e) selecting TI cells that express at least the first and second exogenous SOIs in the presence of an inducer, thereby isolating TI host cells that express the polypeptide of interest. In certain embodiments, rather than having the entire selection marker on a first vector, the first vector comprises a promoter sequence operably linked to an ATG codon flanked upstream by a first SOI and downstream by an RRS; and the second vector comprises a selection marker lacking an ATG transcription start codon flanked upstream by an RRS and downstream by a second SOI.

[0174] 7.Product The host cells of the present disclosure may be used for the expression of any molecule of interest, e.g., a polypeptide of interest. In certain embodiments, the host cells of the present disclosure may be used for the expression of 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 by the definition herein include, for example, mammalian polypeptides such as 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, e.g., protein C; atrial natriuretic factor; pulmonary surfactant; plasminogen activators, e.g., urokinase or human urinary or tissue plasminogen activator (t-PA); bombesin; thrombin; hematopoietic growth factors; tumor necrosis factor-alpha and -beta; tumor necrosis factor receptors, e.g., death receptor 5 and CD120; TNF-related apoptosis-inducing ligand (TRAIL); B-cell maturation antigen (BCMA); B-lymphocyte stimulating factor (BLy); S); proliferation-inducing ligand (APRIL); enkephalinase; RANTES (regulated upon activation and normally expressed and secreted by T cells); human macrophage inflammatory protein (MIP-1-alpha); serum albumin, e.g., human serum albumin; Müllerian inhibitory substance; relaxin A chain; relaxin B chain; prorelaxin; mouse gonadotropin-related peptide; microbial proteins, e.g., beta-lactamase; DNase; IgE; cytotoxic T lymphocyte-associated antigen (CTLA), e.g., CTLA-4; inhibin; activin; platelet-derived endothelial cell growth factor (PD-ECGF); vascular endothelial growth factor family proteins (e.g., VEGF-A, VEGF-B, VEGF-C, VEGF-D, and P1GF); platelet-derived growth factor (PDGF) family proteins (e.g., PDGF-A, PDGF-B, PDGF-C, PDGF-D, and their dimers); fibroblast growth factor (FGF) family, e.g., aFGF, bFGF, FGF4, and FGF9; epidermal growth factor (EGF);Hormone or growth factor receptors, for example, one or more VEGF receptors (e.g., VEGFR1, VEGFR2, and VEGFR3), one or more epidermal growth factor (EGF) receptors (e.g., ErbB1, ErbB2, ErbB3, and ErbB4 receptors), one or more platelet-derived growth factor (PDGF) receptors (e.g., PDGFR-α and PDGFR-β), and one or more fibroblast growth factor receptors; TIE ligands (angiopoietin, ANGPT1, ANGPT2), angiopoietin receptors, for example, TIE1 and TIE2; protein A or D; rheumatoid factor; neurotrophic factors, for example, bone-derived neurotrophic factor (BDNF), neurotrophin-3, neurotrophin-4, neurotrophin-5 , or neurotrophin-6 (NT-3, NT-4, NT-5, or NT-6), or nerve growth factor, e.g., NGF-b; transforming growth factors (TGFs), e.g., 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 protein (IGF BP); CD proteins, e.g., CD3, CD4, CD8, CD19, and CD20; erythropoietin; osteoinductive factors; immunotoxins; bone morphogenetic proteins (BMPs); chemokines, e.g., CXCL12 and CXCR4; interferons, e.g., interferon-alpha, interferon-beta, and interferon-gamma; colony-stimulating factors (CSFs), e.g., M-CSF, GM-CSF, and G-CSF; cytokines, e.g., interleukins (ILs), e.g., For example, IL-1 to IL-10; midkine; superoxide dismutase; T cell receptors; surface membrane proteins; decay-accelerating factors; viral antigens, e.g., portions of the AIDS envelope; transport proteins; homing receptors; addressins; regulatory proteins; integrins, e.g., CD11a, CD11b, CD11c, CD18, ICAM, VLA-4, and VCAM; ephrins; Bv8; delta-like ligand 4 (DLL4); Del-1; BMP9; BMP10; follistatin;Included are hepatocyte growth factor (HGF) / scatter factor (SF); Alk1; Robo4; ESM1; perlecan; EGF-like domain multiple 7 (EGFL7); CTGF and its family members; thrombospondins, e.g., thrombospondin 1 and thrombospondin 2; collagens, e.g., collagen IV and collagen XVIII; neuropilins, e.g., NRP1 and NRP2; pleiotrophin (PTN); progranulin; proliferin; Notch proteins, e.g., Notch1 and Notch4; semaphorins, e.g., Sema3A, Sema3C, and Sema3F; tumor-associated antigens, e.g., CA125 (ovarian cancer antigen); immunoadhesins; and fragments and / or variants of any of the above polypeptides, as well as antibodies, including, for example, antibody fragments, that bind to one or more proteins, including any of the above proteins.

[0176] In certain embodiments, the polypeptide of interest is a bispecific, trispecific, or multispecific polypeptide, e.g., a bispecific antibody. Various molecular formats of multispecific antibodies are known in the art and are included herein (see, e.g., Spiess et al., Mol Immunol 67 (2015) 95-106). A particular type of multispecific antibody also included herein is a bispecific antibody designed to simultaneously bind to a surface antigen on a target cell, e.g., a tumor cell, and to an active, invariant component of the T cell receptor (TCR) complex, e.g., CD3, to retarget T cells and kill the target cell. Further examples of bispecific antibody formats include so-called "BiTEs" (bispecific T cell engagers), in which two scFv molecules are fused by a flexible linker (see, e.g., WO 2004 / 106381, WO 2005 / 061547, WO 2007 / 042261 and WO 2008 / 119567; Nagorsen and Baeuerle, Exp Cell Res 317, 1255-1260 (2011)); diabodies (Holliger et al., Prot Eng 9, 299-305 (1996)) and their derivatives, such as tandem diabodies ("TandAbs"; Kipriyanov et al., J Mol Biol 293, 41-56 (1999); "DART" (dual affinity retargeting) molecules, which are based on the diabody format but feature a C-terminal disulfide bridge for further stabilization (Johnson et al., J Mol Biol 399, 436-449 (2010)), and the so-called triomabs, which are all-hybrid mouse / rat IgG molecules (reviewed in Seimetz et al., Cancer Treat Rev 36, 458-467 (2010)). Specific T cell bispecific antibody formats included herein are described in WO 2013 / 026833, WO 2013 / 026839, WO 2016 / 020309; Bacac et al., Oncoimmunology 5(8) (2016) e1203498.

[0177] In certain embodiments, the host cells of the present disclosure may be used for the expression of 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 the host cell of the disclosure is selected from the group consisting of, for example, 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, FGF1 O 1A, IL 1B, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL1 0, IL 11, IL 12A, IL 12B, IL 13, IL 14, IL 15, IL 16, IL 17, IL 17B, IL 18, IL 19, 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 (T RAIL), 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, ILThe antibody may bind to or interact with any protein, including, but not limited to, cytokines, cytokine-related proteins, and cytokine receptors selected from the group consisting of IL11RA, IL12RB1, IL12RB2, IL13RA1, IL13RA2, IL15RA, IL17R, IL18R1, IL20RA, IL21R, IL22R, IL1HY1, IL1RAP, IL1RAPL1, IL1RAPL2, IL1RN, IL6ST, IL18BP, IL18RAP, IL22RA2, AIF1, HGF, LEP (leptin), PTN, and THPO.k.

[0179] 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);α4β αEβ7 and αEβ7 integrin heterodimer; JAG1; JAK1; JAK3; JUN; K6HF; KAI1; KDR; KITLG; KLF5 (GCBoxBP); KLF6; KLKIO; KLK12; KLK13; KLK14; KLK15; KLK3; KLK4; KLK5; KLK6; KLK9; KRT1; KRT19 (keratin 19); KRT2A; KHTHB6 (hair-specific H-type keratin); Llama; LEP (leptin); LGR5 (leucine-rich repeat-containing G protein-coupled receptor 5); GPR49, GPR 67); 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); LY6K (lymphocyte antigen 6 complex, locus K; L Y6K; 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 (metallothionectin-111); MTSS1; MUC1 (mucin); MYC; MY088;Napi3b (also known as NaPi2b) (NAPI-3B, NPTIIb, SLC34A2, solute carrier 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; NR 1H3;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;POG FB;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, RIKENcDNA2700050C12, RIKENcDNA2700050C12 gene); PTAFR;PTEN;PTGS2(COX-2);PTN;RAC2(p21Rac2);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 5bHlog, 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); ST6 GAL1;STABI;STAT6;STEAP (six transmembrane epithelial antigens 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 antigens of the prostate 2, six transmembrane prostate protein);TB4R2;TBX21;TCPIO;TOGFI;TEK;TENB2 (putative transmembrane proteoglycan);TGFA;TGFBI;TGFB1II;TGFB2;TGFB3;TGFBI;TGFBRI;TGFB R2;TGFBR3;THIL;THBSI (thrombospondin-1);THBS2;THBS4;THPO;TIE (Tie-1);TMP3;tissue factor;TLR1;TLR2;TLR3;TLR4;TLR5;TLR6;TLR7;TLR8;TLR9;TLR10;TMEFF1 (transmembrane protein with EGF-like and two follistatin-like domains 1; tomoregulin-1);TMEM46 (shisa homolog 2);TNF;TNF-a;TNFAEP2 (B94);TNFAIP3;TNFRSFIIA;TNFRSF1A;TNFR SF1B;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; 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 receptor potential cation channel, subfamily M, member 4); TRPC6; TSLP; TWEAK; tyrosinase (TYR; OCAIA; OCA1A; tyrosinase; 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 by the cells and methods disclosed herein include CD proteins, e.g., CD3, CD4, CD5, 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, e.g., EGF receptor, HER2, HER3, or HER4 receptor; cell adhesion molecules, e.g., LFA-1, Mac1, p150.95, VLA-4, ICAM-1, VC integrin, alpha4 / beta7 integrin, and alphav / beta3 integrin, including 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 may be used to generate antibodies (or multispecific antibodies, such as bispecific antibodies) that specifically bind to complement protein C5 (e.g., anti-C5 agonist antibodies that specifically bind to humans).

[0181] In certain embodiments, the methods provided herein may be used to generate 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 in vitro and / or in vivo to hemagglutinin from the Yamagata lineage, Victoria lineage, and ancestral lineage of influenza B virus). Further details regarding anti-FluB antibodies are described in WO 2015 / 148806, which is incorporated herein by reference in its entirety.

[0182] In certain embodiments, the antibody (or bispecific antibody) produced by the methods provided herein binds to low-density lipoprotein receptor-related protein (LRP)-1 or LRP-8 or transferrin receptor and at least one target selected from the group consisting of 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, p75 NTR, CD40, and caspase-6.

[0183] In certain embodiments, the antibody produced by 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 by the methods provided herein is a targeted immunocytokine. In certain embodiments, the targeted immunocytokine is a CEA-IL2v immunocytokine. In certain embodiments, the CEA-IL2v immunocytokine is RG7813. In certain embodiments, the targeted immunocytokine is a FAP-IL2v immunocytokine. In certain embodiments, the FAP-IL2v immunocytokine is RG7461.

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

[0186] In certain embodiments, multispecific antibodies (such as bispecific antibodies) produced according to the methods provided herein are directed to: 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-1 3 and CL25; IL-13 and SPRR2a; IL-13 and SPRR2b; IL-13 and ADAMS; IL-13 and PED2; IL17A and IL17F; CEA and CD3; CD3 and CD19; CD138 and CD20; CD138 and CD40; CD19 and CD20; CD20 and CD3; CD3S and CD13S; CD3S and CD20; CD3S and CD40; CD40 and CD20; CD-S and IL-6; CD20 and BR3; TNF alpha and TGF-beta, TNF alpha and IL-1 beta;TNFalpha and IL-2, TNFalpha and IL-3, TNFalpha and IL-4, TNFalpha and IL-5, TNFalpha and IL-6, TNFalpha and IL-8, TNFalpha and IL-9, TNFalpha and IL-10, TNFalpha and IL-11, TNFalpha and IL-12, TNFalpha and IL-13, TNFalpha and IL-14, TNFalpha and IL-15, TNFalpha and IL-16, TNFalpha and IL-17, TNFalpha and IL-18, TNFalpha and IL-19, TNFalpha and IL-20, TNFalpha and IL-23, TNFalpha and IFNalpha, TNFalpha and CD4, TNFalpha and VEGF, TNFalpha and MIF, TNFalpha and ICAM-1, TNFalpha and PGE4, TNFalpha and PEG2, TNFalpha and RANK ligand, TNFalpha and Te38, TNFalpha and BAFF, TNFalpha and CD22, TNF alpha and CTLA-4, TNF alpha and GP130, TNFα 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, V EGFR 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 binds to at least two target molecules selected from 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 WO 2014 / 121712, which is incorporated herein by reference in its entirety.

[0188] In certain embodiments, the multispecific antibody (such as a 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 meant to be limiting.

[0191] The host cells of the present disclosure can be used for manufacturing-scale production of molecules 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 from about 400 L to about 25,000 L. Within this range, specific cell culture sizes such as 4,000 L, about 6,000 L, about 8,000 L, about 10,000 L, about 12,000 L, about 14,000 L, or about 16,000 L can be utilized.

[0192] In certain embodiments, the polypeptide of interest is a bispecific, trispecific, or multispecific polypeptide, such as a bispecific antibody.

[0193] The host cells of the present disclosure may 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 may be used to improve the quality of the molecule of interest compared to non-TI cells used in current cell culture methods. In certain embodiments, the host cells of the present disclosure may 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 may be used for optimal expression of acutely toxic products.

[0194] In certain embodiments, the host cells, TI systems of the present disclosure may be used for cell culture process optimization and / or process development.

[0195] In certain embodiments, the host cells of the present disclosure may 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 compared to non-TI cells used in conventional cell culture methods. In certain embodiments, the host cells of the present disclosure may 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 compared to non-TI cells used in conventional cell culture methods.

[0196] In certain embodiments, the host cells of the present embodiments may be used to reduce aggregation levels of a molecule of interest compared to non-TI cells used in conventional cell culture methods.

[0197] 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 host cells into which an exogenous sequence expressing the polypeptide (or polypeptides) of interest has been randomly integrated. For example, without limitation, host cells of the present disclosure may be used to 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, the host cells of the present disclosure may achieve increased bispecific production compared to host cells into which the exogenous sequence expressing the bispecific product has been randomly integrated, for example, but not limited to, the host cells of the present disclosure may achieve at least 80%, 85%, 90%, 95%, 96%, 98%, 99% or more bispecific production.

[0198] In certain embodiments, the host cells of the present disclosure may 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 may be a fusion protein. In certain embodiments, the host cells of the present disclosure may be used to understand the role and effect of each antibody subunit in the expression and secretion of a fully assembled antibody molecule.

[0199] In certain embodiments, the host cells of the present disclosure may be used as an investigative tool. In certain embodiments, the host cells of the present disclosure may be used as a diagnostic tool to determine the root cause of low protein expression of problematic molecules in various cells. In certain embodiments, the host cells of the present disclosure may be used to directly link observed phenomena or cellular behavior to transgene expression in the cell. The host cells of the present disclosure may also be used to demonstrate whether observed behavior is reversible in the cell. In certain embodiments, the host cells of the present disclosure may be used to identify and alleviate problems related to the transcription and expression of one or more transgenes in the cell.

[0200] In certain embodiments, the host cells of the present disclosure may 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, with those of an average molecule in a TI system to identify the problematic subunit or subunits. In certain embodiments, amino acid sequence analysis may then be used to narrow down and focus on amino acid residues or regions that may be responsible for low protein expression. In certain embodiments, the host cells of the present disclosure can be used to express a polypeptide of interest comprising: a) a targeted integration exogenous nucleic acid sequence (SOI) encoding a first polypeptide of interest and a first selectable marker flanked by two recombination recognition sequences (RRS), wherein the targeted integration exogenous SOI is integrated into a targeted locus in the genome of the host cell; b) a transposon-mediated genomic integration exogenous nucleic acid SOI encoding a second polypeptide of interest and a second selectable marker, wherein the transposon-mediated genomic integration exogenous nucleic acid SOI has been integrated at least once into the genome of the host cell; or c) a transposon-mediated genomic integration exogenous nucleic acid SOI encoding the second polypeptide of interest and a second selectable marker, wherein the targeted integration exogenous nucleic acid SOI is constitutively or inducibly expressed and the transposon-mediated genomic integration exogenous nucleic acid SOI is constitutively or inducibly expressed. In certain embodiments, the targeted integration exogenous nucleic acid SOI is constitutively expressed. In certain embodiments, the targeted integration exogenous nucleic acid SOI is inducibly expressed. In certain embodiments, the transposon-mediated genomic integration exogenous nucleic acid SOI is constitutively expressed. In certain embodiments, the targeted integration exogenous nucleic acid SOI is inducibly expressed, and the transposon-mediated genomic integration exogenous nucleic acid SOI is constitutively expressed. In certain embodiments, the targeted integration exogenous nucleic acid SOI is constitutively expressed, and the transposon-mediated genomic integration exogenous nucleic acid SOI is constitutively expressed. In certain embodiments, the targeted integration exogenous nucleic acid SOI is constitutively expressed, and the transposon-mediated genomic integration exogenous nucleic acid SOI is inducibly expressed.

[0201] In certain embodiments, the present disclosure provides a method for expressing a polypeptide of interest, comprising: a) providing a host cell comprising an exogenous nucleotide sequence integrated into a targeted locus in the genome of the host cell, the exogenous nucleotide sequence comprising two RRSs flanking a first selectable marker; b) introducing into the cell provided in a) a nucleic acid comprising two RRSs that match the two RRSs of the integrated exogenous nucleotide sequence and that flank a first exogenous SOI encoding a first polypeptide of interest and a second selectable marker; and c) introducing a recombinase that recognizes the RRSs or a nucleic acid encoding the recombinase. d) selecting cells that express the second selection marker; e) introducing a second exogenous SOI encoding a second polypeptide of interest and a third selection marker into the genome of the host cell via transposon-mediated genomic integration; f) constitutively or inducibly expressing the exogenous nucleotide sequence integrated at the targeted locus in the genome of the host cell and constitutively or inducibly expressing the second exogenous SOI; g) selecting cells that express the third selection marker; and h) culturing the host cell under conditions sufficient to express the first polypeptide of interest and the second polypeptide of interest. In certain embodiments, the exogenous nucleotide sequence integrated at the targeted locus in the genome of the host cell is constitutively expressed. In certain embodiments, the exogenous nucleotide sequence integrated at the targeted locus in the genome of the host cell is inducibly expressed. In certain embodiments, the second exogenous SOI is constitutively expressed. In certain embodiments, the transposon-mediated genomic integrated exogenous SOI is inducibly expressed. In certain embodiments, the exogenous nucleotide sequence integrated into the targeted locus of the genome of the host cell is inducibly expressed and the transposon-mediated genomic integrated exogenous SOI is constitutively expressed. In certain embodiments, the exogenous nucleotide sequence integrated into the targeted locus of the genome of the host cell is constitutively expressed and the transposon-mediated genomic integrated exogenous SOI is constitutively expressed.In certain embodiments, the exogenous nucleotide sequence integrated into the targeted locus in the genome of the host cell is constitutively expressed, and the transposon-mediated genome-integrated exogenous SOI is inducibly expressed.

[0202] 8. Exemplary Implementations In certain embodiments, the present disclosure provides a) a targeted integration exogenous nucleic acid sequence (SOI) encoding a first polypeptide of interest and a first selectable marker flanked by two recombination recognition sequences (RRS), wherein the targeted integration exogenous SOI is integrated into a targeted locus in the genome of a host cell; b) a transposon-mediated genomic integration exogenous nucleic acid SOI encoding a second polypeptide of interest and a second selectable marker, the SOI having been integrated at least once into the genome of the host cell; and c) a transposon-mediated genomic integration exogenous nucleic acid SOI encoding a second polypeptide of interest and a second selectable marker, wherein the targeted integration exogenous nucleic acid SOI is constitutively or inducibly expressed, and the transposon-mediated genomic integration exogenous nucleic acid SOI is constitutively or inducibly expressed; A host cell comprising the

[0203] Additionally, or alternatively, in certain embodiments of the host cells of the present disclosure, the first polypeptide of interest and the second polypeptide are the same. Additionally, or alternatively, in certain such embodiments, the first selection marker and the second selection marker are the same.

[0204] Additionally or alternatively, in certain embodiments of the host cells of the present disclosure, the host cell comprises between 1 and 10 transposon-mediated genome-integrated exogenous nucleic acid SOIs.

[0205] Additionally, or alternatively, in certain embodiments of the host cells of the present disclosure, the targeted locus is at least about 90% homologous to a sequence comprising all or part of the contig sequences of contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1, or a sequence selected from SEQ ID NOs: 1-7.

[0206] Additionally or alternatively, in certain embodiments of the host cells of the present disclosure, the host cell may comprise a second targeted integrating exogenous nucleic acid SOI encoding a second polypeptide of interest and a second selectable marker integrated into a targeted locus in the genome of the host cell, wherein the first targeted integrating exogenous nucleic acid SOI and the first selectable marker are flanked by first and third RRSs, and the second targeted integrating exogenous nucleic acid SOI and the second selectable marker are flanked by second and third RRSs.

[0207] Additionally or alternatively, in certain embodiments of the host cells of the present disclosure, the polypeptide of interest is selected from the group consisting of a single chain antibody, an antibody light chain, an antibody heavy chain, a single chain Fv fragment (scFv), and an Fc fusion protein.

[0208] Additionally or alternatively, in certain embodiments of the host cells of the present disclosure, 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 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.

[0209] Additionally or alternatively, in certain embodiments of the host cell of the present disclosure, targeted integration of the SOI and selectable marker is facilitated by an exogenous nuclease. In certain embodiments, the exogenous nuclease is selected from the group consisting of zinc finger nucleases (ZFNs), ZFN dimers, transcription activator-like effector nucleases (TALENs), TAL effector domain fusion proteins, RNA-guided DNA endonucleases, engineered meganucleases, and clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) endonucleases. In certain embodiments, the targeted integration exogenous nucleic acid SOI is constitutively expressed. In certain embodiments, the targeted integration exogenous nucleic acid SOI is inducibly expressed. In certain embodiments, the transposon-mediated genomic integration exogenous nucleic acid SOI is constitutively expressed. In certain embodiments, the transposon-mediated genomic integration exogenous nucleic acid SOI is inducibly expressed. In certain embodiments, the targeted integration exogenous nucleic acid SOI is inducibly expressed, and the transposon-mediated genome integration exogenous nucleic acid SOI is constitutively expressed. In certain embodiments, the targeted integration exogenous nucleic acid SOI is inducibly expressed, and the transposon-mediated genome integration exogenous nucleic acid SOI is inducibly expressed. In certain embodiments, the targeted integration exogenous nucleic acid SOI is constitutively expressed, and the transposon-mediated genome integration exogenous nucleic acid SOI is constitutively expressed. In certain embodiments, the targeted integration exogenous nucleic acid SOI is constitutively expressed, and the transposon-mediated genome integration exogenous nucleic acid SOI is inducibly expressed.

[0210] In certain embodiments, the present disclosure provides a method for expressing a polypeptide of interest, comprising: a) providing a host cell comprising an exogenous nucleotide sequence integrated into a targeted locus in the genome of the host cell, wherein the exogenous nucleotide sequence comprises two RRSs flanking a first selectable marker; b) introducing into the cell provided in (a) a nucleic acid comprising two RRSs that match the two RRSs of the integrated exogenous nucleotide sequence and that flank a first exogenous SOI encoding a first polypeptide of interest and a second selectable marker; c) introducing a recombinase that recognizes the RRSs or a nucleic acid encoding the recombinase; d) selecting cells that express a second selection marker; e) introducing a second exogenous SOI encoding a second polypeptide of interest and a third selection marker into the genome of the host cell via transposon-mediated genomic integration; f) constitutively or inducibly expressing the exogenous nucleotide sequence integrated at the targeted locus in the genome of the host cell, thereby constitutively or inducibly expressing the transposon-mediated genomic-integrated exogenous SOI; g) selecting cells that express the third selection marker; and h) culturing the host cells under conditions sufficient to express the first polypeptide of interest and the second polypeptide of interest. In certain embodiments, the method further comprises recovering the first and second polypeptides of interest from the host cell culture. In certain embodiments, the first and second polypeptides of interest may be the same. In certain embodiments, the targeted locus is at least about 90% homologous to a sequence comprising all or part of one of the contig sequences of contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1, or a sequence selected from SEQ ID NOs: 1-7. In certain embodiments, the first polypeptide of interest and the second polypeptide of interest are selected from the group consisting of a single-chain antibody, an antibody light chain, an antibody heavy chain, a single-chain Fv fragment (scFv), and an Fc-fusion protein. In certain embodiments, the host cell 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 CHO host cell, a CHO K1 host cell, a CHO K1SV host cell, a DG44 host cell, a DUKXB-11 host cell, a CHOK1S host cell, or a CHO K1M host cell. In certain embodiments, targeted integration of any SOI is facilitated by an exogenous nuclease. In certain embodiments, the exogenous nuclease is selected from the group consisting of zinc finger nucleases (ZFNs), ZFN dimers, transcription activator-like effector nucleases (TALENs), TAL effector domain fusion proteins, RNA-guided DNA endonucleases, engineered meganucleases, and clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) endonucleases. In certain embodiments, expression of the SOI is controlled by a regulatable promoter. In certain embodiments, the regulatable promoter is selected from the group consisting of SV40 and CMV promoters. In certain embodiments, the exogenous nucleotide sequence integrated into the targeted locus of the host cell genome is constitutively expressed. In certain embodiments, the exogenous nucleotide sequence integrated into the targeted locus of the host cell genome is inducibly expressed. In certain embodiments, the transposon-mediated genomic integrated exogenous SOI is constitutively expressed. In certain embodiments, the transposon-mediated genomic integrated exogenous SOI is inducibly expressed. In certain embodiments, the exogenous nucleotide sequence integrated into the targeted locus of the host cell genome is inducibly expressed and the transposon-mediated genomic integrated exogenous SOI is constitutively expressed. In certain embodiments, the exogenous nucleotide sequence integrated into the targeted locus of the host cell genome is inducibly expressed and the transposon-mediated genomic integrated exogenous SOI is inducibly expressed. In certain embodiments, the exogenous nucleotide sequence integrated into the targeted locus in the genome of the host cell is constitutively expressed, and the transposon-mediated genome-integrated exogenous SOI is constitutively expressed.In certain embodiments, the exogenous nucleotide sequence integrated into the targeted locus in the genome of the host cell is constitutively expressed, and the transposon-mediated genome-integrated exogenous SOI is inducibly expressed.

[0211] 9. Working Example 9.1 Comparison of cell pools produced by transfection of targeted integration plasmids, piggyBac plasmids, and their combinations In the instant example, four cell populations expressing mAb1 were compared for cell viability, titer, specific productivity, number of integrated viable cells, gene copy number, and recovery of mRNA expression. These four cell populations are comprised of TI host cells. Each of these TI host cells contains a pre-inserted landing pad, and the presence of a pair of recombination sites, e.g., cre-lox recombination sites, flanking the landing pad and the expression cassette in the TI plasmid facilitates integration of the expression cassette by recombinase-mediated cassette exchange (RMCE). However, the four cell populations differ based on the specific plasmids used to provide the mAb1 heavy and light chain coding sequences, in some cases a plasmid that facilitates transposon-mediated genomic integration of the exogenous sequence of interest (in this case, utilizing the piggyBac transposon). As outlined below, the "PB cell pool" was transfected with a piggyBac ("PB") plasmid, the "TI cell pool" was transfected with a targeted integration ("TI") plasmid, and the "TI+PB cell pool" and "PB+TI cell pool" were transfected with both PB and TI plasmids.

[0212] 9.1.1 Vector construction Two different expression plasmids were used to express the mAb1 heavy chain (HC) and light chain (LC). Briefly, the mAb1 HC and LC cDNAs were cloned into the FrontTI plasmid, which contains the L3, promoter, and start codon (ATG) followed by the LoxFAS sequence, and the BackTI plasmid, which contains Pac, lacking the LoxFAS, start codon, and 2L sequences. A Cre recombinase plasmid (pOG231, developed by Dr. Geoffrey Wahl's group) was used for all RMCE processes. Furthermore, the PB plasmid (transposon) consists of a CMV promoter driving the expression of mAb1 LC, followed by an internal ribosome entry site (IRES) and a Zeocin (Zeo) resistance marker, and another CMV promoter driving the expression of mAb1 HC downstream of the Zeo marker. All elements described above in relation to the PB plasmid are flanked by two inverted terminal repeat (ITR) sites.

[0213] 9.1.2 Transfection and RMCE To facilitate transfection of the mAb1 HC and LC sequences, TI host cells were cultured at 4 × 10 5 Three different transfection schedules were tested: co-transfecting the TI host with the TI and PB plasmids ("TIP"); Transfect the TI plasmid into the TI host, followed two days later by transfection with the PB plasmid ("TI+PB"); and The PB plasmid was transfected into the TI host, followed two days later by the TI plasmid ("PB+TI").

[0214] For TI transfection, 12.5 μg of TI front plasmid, 12.5 μg of TI back plasmid, and 5 μg of Cre recombinase plasmid or 1 μg of Cre recombinase mRNA were used. For PB transfection, 2–30 μg of PB transposase mRNA and 22–33 μg of PB plasmid were used. Each transfection contained 30 × 10 6 Cells were used and electroporated using MaxCyte. All pools were transferred to selection medium 5 days after the initial transfection.

[0215] 9.1.3 Selection of transfected cell pools The transfected pool was 6 x 10 5 At a seeding density of 1000 cells / ml, selection was performed with 5 μg / mL puromycin, 0.25–0.5 μM FIAU, and 150–300 μg / mL Zeocin. Flow cytometry analysis after viability recovery confirmed homogeneous Ab-expressing populations in all pools. Figure 1 shows representative recovery curves after selection of TI hosts with piggyBac alone (PB), targeted integration alone (TI), co-transfection of targeted integration and piggyBac (TIP), sequential transfection of targeted integration followed by piggyBac two days later (TI+PB), and sequential transfection of piggyBac followed by targeted integration two days later (PB+TI). Data shown here are from two PB cell pools, one TI cell pool, four TIP cell pools, seven TI+PB cell pools, and one PB+TI cell pool. The TI and TIP timelines are nearly identical, while the TI+PB timeline shows a delay of approximately two days to account for the additional transfection. The timeline for PB+TI is long, but recovery will occur eventually.

[0216] 9.1.4 Antibody Production and Assays A 7-day enriched fed-batch shake flask production assay was used to assess the productivity of the various transfection pools. On day 0, 20 ml of cells were seeded into a 125 ml shake flask (starting cell density in 20 ml was 30 × 10). 6 The cells were cultured at 13%, 13%, and 10% v / v on days 1, 3, and 4, respectively. A second feed was also added at 0.34% v / v on all feeding days. Glucose feed was added as needed to avoid glucose depletion. Cells were shaken at 150 rpm, 35°C, and 5% CO2 throughout the 7-day period. Cell samples from day 4 were collected for RNA isolation and mRNA analysis by droplet PCR. Supernatants were collected on days 3, 4, and 7 and titered using a ProA-based assay. Gene copy number was measured by droplet PCR using genomic DNA isolated from seed-train cells. A ViCell XR was used as a cell counter throughout all steps.

[0217] Figures 2A–2C show the day 7 titers (Figure 2A), specific productivity (Qp) (Figure 2B), and number of viable integrated cells (IVCC) (Figure 2C) for TI host cell pools transfected with piggyBac alone (PB), with targeted integration alone (TI), with targeted integration and piggyBac cotransfected (TIP), with targeted integration and piggyBac transfected sequentially 2 days later (TI+PB), and with piggyBac and targeted integration transfected sequentially 2 days later (PB+TI). Data shown here are for two PB pools, one TI pool, four TIP pools, seven TI+PB pools, and one PB+TI pool. The lower TIP titers and Qp may be due to the use of less PB plasmid in TIP transfections to improve post-transfection survival. All error bars indicate the standard deviation of the pools in each transfection group.

[0218] Figures 3A-B show the heavy and light chain gene copy numbers per cell (Figure 3A) and mRNA transcript levels (Figure 3B) for TI host cell pools transfected with piggyBac alone (PB), targeted integration alone (TI), co-transfected with targeted integration and piggyBac (TIP), sequentially transfected with targeted integration followed by piggyBac two days later (TI+PB), and sequentially transfected with piggyBac followed by targeted integration two days later (PB+TI). Data shown here are for two PB pools, one TI pool, four TIP pools, seven TI+PB pools, and one PB+TI pool. All error bars indicate the standard deviation of the pools in each transfection group.

Claims

1. A host cell capable of expressing a polypeptide of interest, a) a targeted integration exogenous nucleic acid sequence of interest (SOI) encoding a first polypeptide of interest and a first selectable marker flanked by two recombination recognition sequences (RRS), the SOI being integrated into a targeted locus in the genome of the host cell; b) a transposon-mediated genomic integration exogenous nucleic acid SOI encoding a second polypeptide of interest and a second selectable marker, the transposon-mediated genomic integration exogenous nucleic acid SOI being integrated at least once into the genome of the host cell; and Including, c) the targeted integration exogenous nucleic acid SOI is constitutively or inducibly expressed, and the transposon-mediated genome integration exogenous nucleic acid SOI is constitutively or inducibly expressed; host cell.

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

3. The host cell of claim 1 , wherein the first and second selection markers are the same.

4. 2. The host cell of claim 1, comprising 1 to 10 transposon-mediated genome-integrated exogenous nucleic acid SOIs.

5. 2. The host cell of claim 1, wherein the targeted locus is at least about 90% homologous to a sequence comprising all or a portion of one of the contig sequences of contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1, or a sequence selected from SEQ ID NOs: 1-7.

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

7. 6. The host cell of claim 1, wherein the polypeptide of interest is selected from the group consisting of a single chain antibody, an antibody light chain, an antibody heavy chain, a single chain Fv fragment (scFv), and an Fc fusion protein.

8. The host cell according to any one of claims 1 to 5, wherein the host cell is a mammalian host cell.

9. The host cell of claim 8 , wherein the host cell is a hamster host cell, a human host cell, a rat host cell, or a mouse host cell.

10. 10. The host cell of claim 9, wherein the host cell is a CHO host cell, a CHO K1 host cell, a CHO K1SV host cell, a DG44 host cell, a DUKXB-11 host cell, a CHOK1S host cell, or a CHO K1M host cell.

11. The host cell of any one of claims 1 to 7, wherein the targeted integration of the SOI and selectable marker is facilitated by an exogenous nuclease.

12. 12. The host cell of claim 11, 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.

13. The host cell of claim 1 , wherein the targeted integration exogenous nucleic acid SOI is constitutively expressed.

14. The host cell of claim 1 , wherein the targeted integration exogenous nucleic acid SOI is inducibly expressed.

15. The host cell of claim 1 , wherein the transposon-mediated genome-integrated exogenous nucleic acid SOI is constitutively expressed.

16. The host cell of claim 1 , wherein the transposon-mediated genome-integrated exogenous nucleic acid SOI is inducibly expressed.

17. The host cell of claim 1, wherein the targeted integration exogenous nucleic acid SOI is inducibly expressed and the transposon-mediated genome integration exogenous nucleic acid SOI is constitutively expressed.

18. The host cell of claim 1 , wherein the targeted integration exogenous nucleic acid SOI is inducibly expressed and the transposon-mediated genome integration exogenous nucleic acid SOI is inducibly expressed.

19. The host cell of claim 1, wherein the targeted integration exogenous nucleic acid SOI is constitutively expressed and the transposon-mediated genome integration exogenous nucleic acid SOI is constitutively expressed.

20. The host cell of claim 1 , wherein the targeted integration exogenous nucleic acid SOI is constitutively expressed and the transposon-mediated genome integration exogenous nucleic acid SOI is inducibly expressed.

21. 1. A method for expressing a polypeptide of interest, comprising: a) providing a host cell comprising an exogenous nucleotide sequence integrated into a targeted locus in the genome of the host cell, wherein the exogenous nucleotide sequence comprises two RRSs flanking a first selectable marker; b) introducing into the cell provided in (a) a nucleic acid comprising two RRSs that match the two RRSs of the integrated exogenous nucleotide sequence and flank a first exogenous SOI encoding a first polypeptide of interest and a second selectable marker; c) introducing a recombinase that recognizes the RRS or a nucleic acid encoding the recombinase; d) selecting cells that express the second selection marker; e) introducing a second exogenous SOI encoding a second polypeptide of interest and a third selectable marker into the genome of the host cell via transposon-mediated genomic integration; f) the exogenous nucleotide sequence integrated into the targeted locus of the genome of the host cell is constitutively or inducibly expressed, and the transposon-mediated genome-integrated exogenous SOI is constitutively or inducibly expressed; g) selecting cells expressing said third selectable marker; h) culturing said host cell under conditions sufficient to express said first polypeptide of interest and said second polypeptide of interest; A method comprising:

22. 22. The method of claim 21, further comprising recovering the first polypeptide of interest and the second polypeptide of interest from the host cell culture.

23. 22. The method of claim 21, wherein the first polypeptide of interest and the second polypeptide of interest are the same.

24. 22. The method of claim 21, wherein the targeted locus is at least about 90% homologous to a sequence comprising all or a portion of the contig sequence of one of contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1, or a sequence selected from SEQ ID NOs: 1-7.

25. 25. The method of any one of claims 21 to 24, wherein the first polypeptide of interest and the second polypeptide of interest are selected from the group consisting of a single chain antibody, an antibody light chain, an antibody heavy chain, a single chain Fv fragment (scFv), and an Fc fusion protein.

26. The method of any one of claims 21 to 25, wherein the host cell is a mammalian host cell.

27. 27. The method of claim 26, wherein the host cell is a hamster host cell, a human host cell, a rat host cell, or a mouse host cell.

28. 28. The method of claim 27, wherein the host cell is a CHO host cell, a CHO K1 host cell, a CHO K1SV host cell, a DG44 host cell, a DUKXB-11 host cell, a CHOK1S host cell, or a CHO K1M host cell.

29. 29. The method of any one of claims 21 to 28, wherein the targeted integration of any of the SOIs is facilitated by an exogenous nuclease.

30. 30. The method of claim 29, 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.

31. The method of any one of claims 21 to 30, wherein the expression of the SOI is controlled by a regulatable promoter.

32. 32. The method of claim 31 , wherein the regulatable promoter is selected from the group consisting of SV40 and CMV promoters.

33. 22. The method of claim 21, wherein the exogenous nucleotide sequence integrated into the targeted locus of the genome of the host cell is constitutively expressed.

34. 22. The method of claim 21, wherein the exogenous nucleotide sequence integrated into the targeted locus of the genome of the host cell is inducibly expressed.

35. 22. The method of claim 21, wherein the transposon-mediated genomic integrated exogenous SOI is constitutively expressed.

36. 22. The method of claim 21, wherein the transposon-mediated genomic integrated exogenous SOI is inducibly expressed.

37. 22. The method of claim 21, wherein the exogenous nucleotide sequence integrated into the targeted locus of the genome of the host cell is inducibly expressed and the transposon-mediated genome-integrated exogenous SOI is constitutively expressed.

38. 22. The method of claim 21, wherein the exogenous nucleotide sequence integrated into the targeted locus of the genome of the host cell is inducibly expressed, and the transposon-mediated genome-integrated exogenous SOI is inducibly expressed.

39. 22. The method of claim 21, wherein the exogenous nucleotide sequence integrated into the targeted locus of the genome of the host cell is constitutively expressed and the transposon-mediated genome-integrated exogenous SOI is constitutively expressed.

40. 22. The method of claim 21, wherein the exogenous nucleotide sequence integrated into the targeted locus of the genome of the host cell is constitutively expressed and the transposon-mediated genome-integrated exogenous SOI is inducibly expressed.

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