Combinatorial vector cloning and transfection strategies for targeted integration of nucleic acids

Abstract

The subject matter of the present disclosure relates to combinatorial vector cloning and transfection strategies for generating targeted integration host cells suitable for expression of recombinant proteins, as well as targeted integration host cells generated by said strategies and compositions comprising said targeted integration host cells.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 479,675, filed January 12, 2023, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application contains an electronically submitted Sequence Listing, which is incorporated herein by reference in its entirety. The XML copy, created on January 12, 2024, is named 0B206.1419.xml and is 987,136 bytes in size.

[0003] The subject matter of the present disclosure relates to combinatorial vector cloning and transfection strategies for generating targeted integration (TI) host cells suitable for expressing recombinant proteins, as well as TI host cells generated by said strategies and compositions comprising said TI host cells. [Background technology]

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

[0005] The traditional strategy for developing commercial cell lines involves random integration of a nucleotide sequence encoding a polypeptide of interest, followed by selection and isolation of cell lines that produce the polypeptide of interest. However, this approach has several drawbacks. First, such integration is not only a rare event, but given the random location of the nucleotide sequence integration, these rare events can result in a variety of gene expression and cell growth phenotypes. Such diversity, known as "position effect variability," is at least in part due to the complex gene regulatory networks present in eukaryotic genomes and the availability of specific genomic loci for integration and gene expression. 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 approaches 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 from randomly integrated cell lines exhibit a high degree of sequence variation, 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] In certain embodiments, the present disclosure relates to a method for generating a library of TI host cells comprising a plurality of sequences of interest (SOIs), the method comprising: generating a first vector library by contacting a first plurality of vectors with the first plurality of SOIs, wherein the first plurality of vectors comprises a plurality of sequences configured to facilitate introduction of the SOIs of the first plurality of SOIs; generating a second vector library by contacting a second plurality of vectors with the second plurality of SOIs, wherein the second plurality of vectors comprises a plurality of sequences configured to facilitate introduction of the SOIs of the second plurality of SOIs; and contacting a plurality of TI host cells with the plurality of vectors of the first vector library and the second vector library, thereby introducing the plurality of SOIs into the plurality of TI host cells, thereby generating a library of TI host cells comprising a plurality of different SOIs.

[0007] In certain embodiments, the present disclosure relates to a method for screening a library of TI host cells expressing multiple SOIs for TI host cells exhibiting a particular cellular or product attribute, the method comprising: generating a first vector library by contacting a first plurality of vectors with the first plurality of SOIs, the first plurality of vectors comprising a plurality of sequences configured to facilitate introduction of SOIs of the first plurality of SOIs; generating a second vector library by contacting a second plurality of vectors with the second plurality of SOIs, the second plurality of vectors comprising a plurality of sequences configured to facilitate introduction of SOIs of the second plurality of SOIs; contacting a plurality of TI host cells with the plurality of vectors of the first vector library and the second vector library, thereby introducing the plurality of SOIs into the plurality of TI host cells, thereby generating a library of TI host cells; separating the plurality of TI host cells of the library of TI host cells into single clones; and screening the clones for the particular cellular or product attribute.

[0008] In certain embodiments of the methods described herein, the sequence configured to facilitate the introduction of an SOI into a vector is a restriction site. In certain embodiments, the first plurality of vectors comprises a pair of distinct restriction sites, the pair of distinct restriction sites flanking a plurality of the first plurality of SOIs in the same order. In certain embodiments, the first plurality of vectors comprises multiple non-overlapping pairs of distinct restriction sites, the non-overlapping pairs of distinct restriction sites flanking a plurality of SOIs of the first plurality of SOIs in the same order for each pair. In certain embodiments, the first plurality of vectors comprises three non-overlapping pairs of distinct restriction sites, the three non-overlapping pairs of distinct restriction sites flanking a plurality of SOIs of the first plurality of SOIs in the same order for each pair. In certain embodiments, the first plurality of vectors comprises four non-overlapping pairs of distinct restriction sites, the four non-overlapping pairs of distinct restriction sites flanking a plurality of SOIs of the first plurality of SOIs in the same order for each pair. In certain embodiments, the second plurality of vectors comprises a pair of distinct restriction sites, the pair of distinct restriction sites flanking a plurality of the second plurality of SOIs in the same order. In certain embodiments, the second plurality of vectors comprises a plurality of non-overlapping pairs of distinct restriction sites, the non-overlapping pairs of distinct restriction sites flanking a plurality of SOIs of the second plurality of SOIs in the same order for each pair. In certain embodiments, the second plurality of vectors comprises three non-overlapping pairs of distinct restriction sites, the three non-overlapping pairs of distinct restriction sites flanking a plurality of SOIs of the second plurality of SOIs in the same order for each pair. In certain embodiments, the second plurality of vectors comprises four non-overlapping pairs of distinct restriction sites, the four non-overlapping pairs of distinct restriction sites flanking a plurality of SOIs of the second plurality of SOIs in the same order for each pair.

[0009] In certain embodiments described herein, the first plurality of vectors comprises a pair of distinct restriction sites, which pair of distinct restriction sites flank a plurality of the first plurality of SOIs in the same order; the second plurality of vectors comprises a pair of distinct restriction sites, which pair of distinct restriction sites flank a plurality of the second plurality of SOIs in the same order. In certain embodiments, the first plurality of vectors comprises a plurality of non-overlapping pairs of distinct restriction sites, which non-overlapping pairs of distinct restriction sites flank a plurality of SOIs of the first plurality of SOIs in the same order for each pair; the second plurality of vectors comprises a plurality of non-overlapping pairs of distinct restriction sites, which non-overlapping pairs of distinct restriction sites flank a plurality of SOIs of the second plurality of SOIs in the same order for each pair. In certain embodiments, the first plurality of vectors comprises three non-overlapping pairs of distinct restriction sites, the three non-overlapping pairs of distinct restriction sites flanking the three SOIs of the first plurality of SOIs in the same order for each pair; the second plurality of vectors comprises three non-overlapping pairs of distinct restriction sites, the three non-overlapping pairs of distinct restriction sites flanking the three SOIs of the second plurality of SOIs in the same order for each pair. In certain embodiments, the first plurality of vectors comprises four non-overlapping pairs of distinct restriction sites, the four non-overlapping pairs of distinct restriction sites flanking the four SOIs of the first plurality of SOIs in the same order for each pair; the second plurality of vectors comprises four non-overlapping pairs of distinct restriction sites, the four non-overlapping pairs of distinct restriction sites flanking the four SOIs of the second plurality of SOIs in the same order for each pair.

[0010] In certain embodiments of the methods described herein, the plurality of first plurality of SOIs comprise distinct SOIs. In certain embodiments, the plurality of first plurality of SOIs comprise the same SOI. In certain embodiments, the plurality of second plurality of SOIs comprise distinct SOIs. In certain embodiments, the plurality of second plurality of SOIs comprise the same SOI. In certain embodiments, the plurality of first plurality of SOIs comprise distinct SOIs; the plurality of second plurality of SOIs comprise distinct SOIs. In certain embodiments, the plurality of first plurality of SOIs comprise the same SOI; the plurality of second plurality of SOIs comprise the same SOI. In certain embodiments, the plurality of first plurality of SOIs comprise distinct SOIs; the plurality of second plurality of SOIs comprise the same SOI. In certain embodiments, the plurality of first plurality of SOIs comprise the same SOI; the plurality of second plurality of SOIs comprise distinct SOIs. In certain embodiments, the plurality of first plurality of SOIs comprise the same SOI; the plurality of second plurality of SOIs comprise distinct SOIs.

[0011] In certain embodiments described herein, the SOI is operably linked to one or more regulatable promoters. In certain embodiments described above, the one or more regulatable promoters are selected from the group consisting of an SV40 promoter and a CMV promoter.

[0012] In certain embodiments described herein, the multiple SOIs are introduced into one or more of the multiple TI host cells by recombinase-mediated integration.

[0013] In certain embodiments described herein, the multiple SOIs are introduced into one or more of the multiple TI host cells by gene editing-mediated integration.

[0014] In certain embodiments described herein, the TI host cell is a mammalian host cell. In certain embodiments, the TI host cell is a hamster host cell, a human host cell, a rat host cell, or a mouse host cell. In certain embodiments, the TI host cell is a CHO host cell, a CHO K1 host cell, a CHO K1SV host cell, a DG44 host cell, a DUKXB-11 host cell, a CHOK1S host cell, or a CHO K1M host cell.

[0015] In certain embodiments described herein, each SOI encodes a polypeptide subunit of a multi-subunit protein, or a fragment thereof, hi certain embodiments, each SOI independently encodes a single-chain antibody, an antibody light chain, an antibody heavy chain, a single-chain Fv fragment (scFv), or an Fc-fusion protein.

[0016] In certain embodiments described herein, the particular cell attribute is selected from cell growth, cell titer, specific productivity, volumetric productivity, clonal stability, and % intact product expression.

[0017] In certain embodiments described herein, the particular product attribute is selected from the level of glycosylation, the level of charge dispersion, reduced mismatches, reduced protein / peptide aggregation, and protein sequence heterogeneity.

[0018] In certain embodiments described herein, the SOI is introduced into one or more loci that are at least about 90% homologous to a sequence selected from SEQ ID NOs: 1-12; 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. [Brief explanation of the drawings]

[0019] [Figure 1A] An overview of the combinatorial vector cloning and transfection strategy for generating TI host cells is shown, including: (1A) an exemplary vector library in which the front cassette vector backbone and back cassette vector backbone consist of an expression cassette flanked by recombinase recognition sequences. [Figure 1B]FIG. 1 shows an overview of combinatorial vector cloning and transfection strategies for generating TI host cells, including: (1B) A flow chart comparing an exemplary targeted integration method, in which each vector construct is constructed separately and used to generate a single transfected pool, with an exemplary combinatorial vector cloning and transfection strategy as outlined herein. [Figure 1C] An overview of combinatorial vector cloning and transfection strategies for generating TI host cells is shown, including: (1C) A table comparing the top clones obtained from standard vector cloning and combinatorial vector cloning and transfection strategies is shown. [Figure 2A] Alternative combinatorial vector cloning and transfection strategies to those shown in Figures 1A-1B are shown, including: (2A) a strategy in which only half of the mAb is expressed in the front cassette and the other half in the back cassette. [Figure 2B] Alternative combinatorial vector cloning and transfection strategies to those shown in Figures 1A-1B are shown, including: (2B) a strategy in which both the antibody chain composition and the promoter for each chain are randomized as either constitutive (e.g., CMV promoter) or inducible (e.g., CMVTO promoter). DETAILED DESCRIPTION OF THE INVENTION

[0020] In certain embodiments, the host cells, genetic constructs (e.g., vectors), compositions, and methods described herein can be used in the development and / or use of combinatorial vector cloning and transfection strategies to generate TI host cells.

[0021] For purposes of clarity of disclosure, and not by way of limitation, this detailed description is divided into the following subsections. 1.Definition 2. Vector Library Preparation and Screening 3. Integration site 4.Host cells 5. Targeted Integration 6. Preparation and Use of TI Host Cells 7.Product

[0022] 1.Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. In case of conflict, the present specification, including definitions, will prevail. Preferred methods and materials are described below, but methods and materials similar or equivalent to those described herein can be used to practice or test the presently disclosed subject matter. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are merely illustrative and are not intended to be limiting.

[0023] As used herein, the terms "comprise(s) / include(s)," "having / has," "can," "contain(s)," and variations thereof are intended to be open-ended transitional phrases, terms, or words that do not exclude the possibility of additional acts or structures. The singular forms "a," "an," and "the" include plural references 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 or not expressly stated.

[0024] When numerical ranges are recited herein, each intervening number is expressly contemplated with the same degree of precision, e.g., 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.

[0025] As used herein, the term "about" or "approximately" refers to an acceptable error range for a particular value as determined by one skilled in the art, which will depend in part on the method of measurement or determination of the value, i.e., the limitations of the measurement system. For example, "about" can mean within 3 or more standard deviations, according to the practice in the art. Alternatively, "about" can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and even more preferably up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within one order of magnitude of a value, preferably within 5-fold, and more preferably within 2-fold.

[0026] As used herein, the term "selection marker" refers to a gene that allows cells carrying the 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 can allow host cells transformed with the selection marker gene to be positively selected in the presence of the gene. Untransformed host cells cannot grow or survive under selective conditions. A selection marker can be positive, negative, or bifunctional. A positive selection marker can allow for the selection of cells carrying the marker, while a negative selection marker can allow for the selective elimination of cells carrying the marker. A selection marker can confer resistance to a drug 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, can be used. Resistance genes useful as selectable markers in eukaryotic cells include, but are not limited to, genes for aminoglycoside phosphotransferase (APH) (e.g., hygromycin phosphotransferase (HYG), neomycin, and G418APH), dihydrofolate reductase (DHFR), thymidine kinase (TK), glutamine synthetase (GS), asparagine synthetase, tryptophan synthase (indole), histidinol dehydrogenase (histidinol D)), as well as 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.

[0027] A selectable marker not only facilitates selection in the presence of the corresponding selection agent, but 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.

[0028] As used herein, the term "operably linked" refers to the juxtaposition of two or more components, wherein the components are in a relationship permitting them to function in their intended manner. For example, a promoter and / or enhancer is operably linked to a coding sequence if it plays a role in regulating the transcription of the coding sequence. In certain embodiments, "operably linked" DNA sequences are contiguous and adjacent on a single chromosome. In certain embodiments, for example, where necessary to link coding regions for two proteins, such as a secretory leader and a polypeptide, the sequences are contiguous, contiguous, and in the same reading frame. In certain embodiments, an operably linked promoter may be located upstream of and adjacent to the coding sequence. In certain embodiments, for example, with respect to an enhancer sequence that regulates expression of a coding sequence, two components may be operably linked even if they are not adjacent. An enhancer is operably linked to a coding sequence if the enhancer increases transcription of the coding sequence. An operably linked enhancer may be located upstream of, within, or downstream of the coding sequence, and may be located at 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. If no convenient restriction sites exist, synthetic oligonucleotide adapters or linkers can be used according to conventional practice. An internal ribosome entry site (IRES) is operably linked to an open reading frame (ORF) if it allows translation initiation of the ORF at an internal location independent of the 5' end.

[0029] As used herein, the term "expression" refers to transcription and / or translation. In certain embodiments, the level of transcription of a desired product can be determined based on the amount of corresponding mRNA present. For example, mRNA transcribed from a sequence of interest can be quantified by PCR or Northern hybridization. In certain embodiments, the protein encoded by a sequence of interest can be quantified by various methods, such as by ELISA, by assaying the biological activity of the protein, or by assays independent of such activity, such as Western blotting or radioimmunoassays using antibodies that recognize and bind to the protein.

[0030] As used herein, the term "antibody" is used in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), half antibodies, and antibody fragments, so long as the antibody exhibits the desired antigen-binding activity.

[0031] As used herein, the term "antibody fragment" refers to a molecule other than an intact antibody that contains a portion of the 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).

[0032] 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 variable domains of the heavy and light chains of a naturally occurring antibody (V H and V L) generally have a similar structure, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). (See, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)). A single V H or V L The domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a specific antigen can be isolated by isolating the V from the antibody that binds the antigen. H or V L The V domains were isolated using the complementary V L or V H Libraries of domains may be screened. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0033] As used herein, the term "heavy chain" refers to an immunoglobulin heavy chain.

[0034] As used herein, the term "light chain" refers to an immunoglobulin light chain.

[0035] 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 may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy-chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0036] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies (i.e., each antibody comprising the population is identical and / or binds to the same epitope). This excludes possible variant antibodies, e.g., including naturally occurring mutations or arising during the production of a monoclonal antibody preparation; such variants are generally present in minor amounts. In contrast to polyclonal antibody preparations, which typically contain 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.

[0037] 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.

[0038] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein and refer to an antibody having a heavy chain that has a structure substantially similar to a native antibody structure or that contains an Fc region as defined herein.

[0039] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv and scFab); single-domain antibodies (dAbs); and multispecific antibodies formed from antibody fragments. For a review of specific antibody fragments, see Holliger and Hudson, Nature Biotechnology 23:1126-1136 (2005).

[0040] 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.

[0041] A "human antibody" is an antibody having an amino acid sequence that corresponds to that of an antibody produced by a human or human cell, or an antibody derived from the human antibody repertoire or other non-human source that utilizes human antibody coding sequences. This definition of a human antibody specifically excludes humanized antibodies, which contain non-human antigen-binding residues.

[0042] 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), with the exception of possible variant antibodies that contain, for example, naturally occurring mutations or that arise during the production of a monoclonal antibody preparation; 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.

[0043] 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. Still other monoclonal antibodies 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 the monoclonal antibody can create a drug that delivers the toxic payload specifically to diseased tissue, reducing harm to healthy tissue. A "biologically functional fragment" of a therapeutic antibody exhibits at least one, if not some or all, of the biological functions attributed to the intact antibody, including at least specific binding to the target antigen.

[0044] The term "diagnostic antibody" refers to an antibody used as a diagnostic reagent for disease. A diagnostic antibody can bind to a target antigen that is specifically associated with or shows increased expression in a particular disease. A diagnostic antibody can 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.

[0045] 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 not be completely identical in nucleic acid content to the parent cell and 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.

[0046] 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 may be linear or circular. Furthermore, the term nucleic acid molecule includes both sense and antisense strands, and both single-stranded and double-stranded forms. Furthermore, the nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases with derivatized sugar or phosphate backbone linkages or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules suitable as vectors for directly expressing the antibodies of the present invention in vitro and / or in vivo, for example, in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors may be unmodified or modified. For example, mRNA can be chemically modified to enhance the stability of the RNA vector and / or the expression of the encoded molecule, allowing the mRNA to be injected into a subject to generate 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).

[0047] 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 that is contained in cells that ordinarily contain 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.

[0048] 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 some embodiments, vectors direct the expression of a nucleic acid to which they are operably linked. Such vectors are referred to herein as "expression vectors."

[0049] The term "homologous sequence" as used herein refers to sequences that share significant sequence similarity as determined by sequence alignment.For example, two sequences can be about 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 99.9% homologous.Alignment is performed by algorithms and computer programs, including but not limited to BLAST, FASTA, and HMME, that compare sequences and calculate the statistical significance of matching based on factors such as sequence length, sequence identity and similarity, and the presence and length of sequence mismatches and gaps.Homologous sequence can refer to both DNA sequences and protein sequences.

[0050] 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 can be adjacent to the second nucleotide sequence or can be at a specified distance from the second nucleotide sequence.There is no particular limit to the length of an adjacent nucleotide sequence.For example, an adjacent sequence can 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.

[0051] As used herein, the term "exogenous" refers to a nucleotide sequence that is not native to the host cell and is introduced into the host cell by conventional DNA delivery methods, such as transfection, electroporation, or transformation. The term "endogenous" refers to a nucleotide sequence that is derived from the host cell. An "exogenous" nucleotide sequence may have an "endogenous" counterpart with identical base composition, but the "exogenous" sequence is introduced into the host cell, for example, by recombinant DNA technology.

[0052] 2. Vector Library Preparation and Screening This disclosure provides a new strategy for expressing bispecific or complex molecules using combinatorial vector cloning and transfection in TI hosts. Unlike targeted integration strategies, in which each vector construct is constructed separately and used to generate a single transfected pool, the strategy outlined herein can involve generating a transfection pool (e.g., a library of TI host cells expressing multiple sequences of interest) by cotransfecting a mixture of combinatorially cloned first and second vector libraries. For example, each vector library can be constructed by randomly inserting subunit genes of a complex or bispecific molecule into predefined slots within one or more expression vectors (e.g., as shown in Figure 1A). Thus, while a transfection pool can potentially consist of hundreds of different vector constructs, each cell will only have one first and one second vector construct at the same TI integration site in its genome.

[0053] In certain embodiments of the methods described herein, a first vector library can be generated by contacting a first plurality of vectors with a first plurality of SOIs, wherein the first plurality of vectors comprises a plurality of sequences configured to facilitate introduction of the SOIs of the first plurality of SOIs.

[0054] In certain embodiments of the methods described herein, a second vector library can be generated by contacting a second plurality of vectors with a second plurality of SOIs, wherein the second plurality of vectors comprises a plurality of sequences configured to facilitate introduction of the SOIs of the second plurality of SOIs.

[0055] In certain embodiments of the methods described herein, a third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth vector library can be generated by contacting a respective plurality of vectors with a respective plurality of SOIs, wherein each of the plurality of vectors comprises a plurality of sequences configured to facilitate introduction of the SOI of a respective plurality of SOIs. In certain embodiments, additional vector libraries beyond the tenth vector library can be generated and used in the methods described herein.

[0056] Additionally or alternatively, as shown in Figure 2A, the disclosed combinatorial vector cloning and transfection strategies can include limiting the variability available to the first or second vector. For example, without limitation, Figure 2A shows an example in which a particular SOI (e.g., half mAb1 or half mAb2) is anchored to the first ("front") or second ("back") vector, respectively. This approach can reduce the number of potential vector combinations but can improve the number of isolated clones capable of expressing a complete bispecific molecule, complete complex molecule, or mAb.

[0057] In certain embodiments of the methods described herein, the sequence configured to facilitate the introduction of an SOI into a vector is a restriction site. In certain embodiments, the first plurality of vectors comprises a pair of distinct restriction sites, the pair of distinct restriction sites flanking a plurality of the first plurality of SOIs in the same order. In certain embodiments, the first plurality of vectors comprises multiple non-overlapping pairs of distinct restriction sites, the non-overlapping pairs of distinct restriction sites flanking a plurality of SOIs of the first plurality of SOIs in the same order for each pair. In certain embodiments, the first plurality of vectors comprises three non-overlapping pairs of distinct restriction sites, the three non-overlapping pairs of distinct restriction sites flanking a plurality of SOIs of the first plurality of SOIs in the same order for each pair. In certain embodiments, the first plurality of vectors comprises four non-overlapping pairs of distinct restriction sites, the four non-overlapping pairs of distinct restriction sites flanking a plurality of SOIs of the first plurality of SOIs in the same order for each pair. In certain embodiments, the second plurality of vectors comprises a pair of distinct restriction sites, the pair of distinct restriction sites flanking a plurality of the second plurality of SOIs in the same order. In certain embodiments, the second plurality of vectors comprises a plurality of non-overlapping pairs of distinct restriction sites, the non-overlapping pairs of distinct restriction sites flanking a plurality of SOIs of the second plurality of SOIs in the same order for each pair. In certain embodiments, the second plurality of vectors comprises three non-overlapping pairs of distinct restriction sites, the three non-overlapping pairs of distinct restriction sites flanking a plurality of SOIs of the second plurality of SOIs in the same order for each pair. In certain embodiments, the second plurality of vectors comprises four non-overlapping pairs of distinct restriction sites, the four non-overlapping pairs of distinct restriction sites flanking a plurality of SOIs of the second plurality of SOIs in the same order for each pair.

[0058] In certain embodiments of the methods described herein, the first plurality of vectors comprises a pair of distinct restriction sites, the pair of distinct restriction sites flanking a plurality of the first plurality of SOIs in the same order. The second plurality of vectors comprises a pair of distinct restriction sites, the pair of distinct restriction sites flanking a plurality of the second plurality of SOIs in the same order. In certain embodiments, the first plurality of vectors comprises multiple non-overlapping pairs of distinct restriction sites, the non-overlapping pairs of distinct restriction sites flanking a plurality of SOIs of the first plurality of SOIs in the same order for each pair. The second plurality of vectors comprises multiple non-overlapping pairs of different restriction sites, the multiple non-overlapping pairs of different restriction sites flanking a plurality of SOIs of the second plurality of SOIs in the same order for each pair. In certain embodiments, the first plurality of vectors comprises three non-overlapping pairs of different restriction sites, the three non-overlapping pairs of different restriction sites flanking a plurality of SOIs of the first plurality of SOIs in the same order for each pair. The second plurality of vectors comprises three non-overlapping pairs of different restriction sites, the three non-overlapping pairs of different restriction sites flanking three SOIs of the second plurality of SOIs in the same order for each pair. In certain embodiments, the first plurality of vectors comprises four non-overlapping pairs of different restriction sites, the four non-overlapping pairs of different restriction sites flanking four SOIs of the first plurality of SOIs in the same order for each pair. The second plurality of vectors comprises four non-overlapping pairs of different restriction sites, the four non-overlapping pairs of different restriction sites flanking four SOIs of the second plurality of SOIs in the same order for each pair.

[0059] In certain embodiments of the methods described herein, the plurality of first plurality of SOIs comprise distinct SOIs. In certain embodiments, the plurality of first plurality of SOIs comprise the same SOI. In certain embodiments, the plurality of second plurality of SOIs comprise distinct SOIs. In certain embodiments, the plurality of second plurality of SOIs comprise the same SOI. In certain embodiments, the plurality of first plurality of SOIs comprise distinct SOIs. The plurality of second plurality of SOIs comprise distinct SOIs. In certain embodiments, the plurality of first plurality of SOIs comprise the same SOI. The plurality of second plurality of SOIs comprise the same SOI. In certain embodiments, the plurality of first plurality of SOIs comprise distinct SOIs. The plurality of second plurality of SOIs comprise the same SOI. In certain embodiments, the plurality of first plurality of SOIs comprise the same SOI. The plurality of second plurality of SOIs comprise distinct SOIs.

[0060] In certain embodiments described herein, the SOI is operably linked to one or more promoters. In certain embodiments, the one or more promoters are selected from the group consisting of an SV40 promoter and a CMV promoter. Alternative promoters that find use in the methods described herein can be derived from the genomes of viruses such as polyomavirus, fowlpox virus, adenovirus (such as adenovirus 2), bovine papillomavirus, avian sarcoma virus, retrovirus, and hepatitis B virus, or from heterologous mammalian promoters, such as actin promoters, immunoglobulin promoters, and heat shock promoters, provided that such promoters are compatible with the host cell system. Additionally or alternatively, as shown in Figure 2B, constitutive or inducible promoters can be used to increase the diversity of chain ratio expression. For example, Figure 2B shows a strategy for randomizing both the antibody chain composition and the promoter for each chain, either constitutive (e.g., CMV promoter) or inducible (e.g., CMVTO promoter). Vectors can be cloned using the 7-way ligation method, with the back or front vector consisting of the TetR gene, which represses transcription from the CMVTO promoter. CMVTO denotes the CMV promoter with the tet operon (TO) inserted before the CMV promoter.

[0061] In certain embodiments of the methods described herein, a library of TI host cells expressing SOIs is created by contacting a plurality of TI host cells with a plurality of vectors from a first and second vector library, thereby introducing a plurality of SOIs into a plurality of TI host cells. In certain embodiments described herein, the plurality of SOIs are introduced into one or more of the plurality of TI host cells by recombinase-mediated integration. In certain embodiments described herein, the plurality of SOIs are introduced into one or more of the plurality of TI host cells by gene editing-mediated integration.

[0062] In certain embodiments described herein, the TI host cell is a mammalian host cell. In certain embodiments, the TI host cell is a hamster host cell, a human host cell, a rat host cell, or a mouse host cell. In certain embodiments, the TI host cell is a CHO host cell, a CHO K1 host cell, a CHO K1SV host cell, a DG44 host cell, a DUKXB-11 host cell, a CHOK1S host cell, or a CHO K1M host cell.

[0063] In certain embodiments of the methods described herein, single-cell cloning is performed from a library of TI host cells expressing an SOI. After single-cell cloning and clonal screening, the top clones generated from the single library of TI host cells expressing an SOI can be evaluated for specific cellular and / or product attributes. For example, without limitation, cells can be evaluated for whether they express the most stoichiometrically balanced ratio of bispecific or complex molecular subunits, resulting in the highest titer and / or best product quality. In certain embodiments described herein, the specific cellular attribute is selected from cell growth, cell titer, specific productivity, volumetric productivity, clonal stability, and % intact product expression, or a combination of such attributes. In certain embodiments described herein, the specific product attribute is selected from the level of glycosylation, the level of charge dispersion, reduced mismatches, reduced protein / peptide aggregation, protein sequence heterogeneity, or a combination of such attributes.

[0064] The strategy described herein can reduce the timeline, cost, and resources required for cell line development. For example, a single ligation and transfection is sufficient to generate a complex library of front and back vectors, eliminating the need to generate and evaluate multiple vector constructs in separate pools. Furthermore, such an approach can increase the diversity of clones obtained from the TI-CLD approach.

[0065] 3. Integration site The presently disclosed subject matter provides host cells suitable for targeted integration of exogenous nucleotide sequences. In certain embodiments, the host cells comprise an exogenous nucleotide sequence integrated into an integration site on the genome of the host cell, i.e., the TI host cell.

[0066] 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 host cell genome. In certain embodiments, the integration site comprises a stretch of nucleotides between either of which the exogenous nucleotide sequence can 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.

[0067] 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.

[0068] In certain embodiments, the exogenous nucleotide sequence is integrated into 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 contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1.

[0069] 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 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, and 400,000 to 413,983 of SEQ ID NO: 3. In certain embodiments, the exogenous nucleotide sequence is integrated into an integration site located within a position selected from nucleotides 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.

[0070] In certain embodiments, the nucleotide sequence immediately 5' to the integrated exogenous sequence is selected from the group consisting of nucleotides 41190-45269 of NW_006874047.1, nucleotides 63590-207911 of NW_006884592.1, nucleotides 253831-491909 of NW_006881296.1, nucleotides 69303-79768 of NW_003616412.1, nucleotides 293481-315265 of NW_003615063.1, nucleotides 2650443-2662054 of NW_006882936.1, or nucleotides 82214-97705 of NW_003615411.1, and sequences at least 50% homologous thereto. In certain embodiments, the nucleotide sequence immediately 5' to the integrated exogenous sequence is nucleotides 41190-45269 of NW_006874047.1, nucleotides 63590-207911 of NW_006884592.1, nucleotides 253831-491909 of NW_006881296.1, nucleotides 69303-79768 of NW_003616412.1, and NW_003615063. NW_006882936.1, nucleotides 2650443-2662054 of NW_006882936.1, or nucleotides 82214-97705 of NW_003615411.1.

[0071] In certain embodiments, the nucleotide sequence immediately 5' to the integrated exogenous sequence is nucleotide 45269 of NW_006874047.1, nucleotide 207911 of NW_006884592.1, nucleotide 491909 of NW_006881296.1, nucleotide 79768 of NW_003616412.1, nucleotide 315265 of NW_003615063.1, nucleotide 2662054 of NW_006882936.1, or nucleotide 2662054 of NW_003615411.1 a sequence of at least 15 base pairs, at least 20 base pairs, at least 30 base pairs, at least 40 base pairs, at least 50 base pairs, at least 75 base pairs, at least 100 base pairs, at least 150 base pairs, at least 200 base pairs, at least 300 base pairs, at least 400 base pairs, at least 500 base pairs, at least 1,000 base pairs, at least 1,500 base pairs, at least 2,000 base pairs, or at least 3,000 base pairs of nucleotide 97705 of

[0072] In certain embodiments, the nucleotide sequence immediately 3' to the integrated exogenous sequence is selected from the group consisting of nucleotides 45270-45490 of NW_006874047.1, nucleotides 207912-792374 of NW_006884592.1, nucleotides 491910-667813 of NW_006881296.1, nucleotides 79769-100059 of NW_003616412.1, nucleotides 315266-362442 of NW_003615063.1, nucleotides 2662055-2701768 of NW_006882936.1, or nucleotides 97706-105117 of NW_003615411.1, and a sequence at least 50% homologous thereto. In certain embodiments, the nucleotide sequence immediately 3′ of the integrated exogenous sequence is nucleotides 45270-45490 of NW_006874047.1, nucleotides 207912-792374 of NW_006884592.1, nucleotides 491910-667813 of NW_006881296.1, nucleotides 79769-100059 of NW_003616412.1, nucleotides 80069-90069 of NW_003615063 NW_006882936.1, nucleotides 2662055-2701768 of NW_006882936.1, or nucleotides 97706-105117 of NW_003615411.1, are at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or at least about 99.9% homologous to nucleotides 315266-362442 of NW_006882936.1, nucleotides 2662055-2701768 of NW_006882936.1, or nucleotides 97706-105117 of NW_003615411.1.

[0073] In certain embodiments, the nucleotide sequence immediately 3′ of the integrated exogenous sequence is nucleotide 45270 of NW_006874047.1, nucleotide 207912 of NW_006884592.1, nucleotide 491910 of NW_006881296.1, nucleotide 79769 of NW_003616412.1, nucleotide 315266 of NW_003615063.1, nucleotide 2662055 of NW_006882936.1, or nucleotide 2662055 of NW_003615411.1 a sequence of at least 15 base pairs, at least 20 base pairs, at least 30 base pairs, at least 40 base pairs, at least 50 base pairs, at least 75 base pairs, at least 100 base pairs, at least 150 base pairs, at least 200 base pairs, at least 300 base pairs, at least 400 base pairs, at least 500 base pairs, at least 1,000 base pairs, at least 1,500 base pairs, at least 2,000 base pairs, or at least 3,000 base pairs of nucleotide 97706 of

[0074] In certain embodiments, the integrated exogenous nucleotide sequence is operably linked to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-7 and sequences at least 50% homologous thereto. In certain embodiments, the nucleotide sequence operably linked to the exogenous nucleotide sequence is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or at least about 99.9% homologous to a sequence selected from SEQ ID NOs: 1-7. In certain embodiments, the integrated exogenous nucleotide sequence comprises at least one SOI. In certain embodiments, the operably linked nucleotide sequence increases the expression level of the SOI compared to a randomly integrated SOI. In certain embodiments, the integrated exogenous SOI is expressed at about 20%, 30%, 40%, 50%, 100%, 2-fold, 3-fold, 5-fold, or 10-fold higher than a randomly integrated SOI.

[0075] In certain embodiments, the integrated exogenous sequence is selected from the group consisting of nucleotides 41190-45269 of NW_006874047.1, nucleotides 63590-207911 of NW_006884592.1, nucleotides 253831-491909 of NW_006881296.1, nucleotides 69303-79768 of NW_003616412.1, nucleotides 293481-315265 of NW_003615063.1, nucleotides 2650443-2662054 of NW_006882936.1, and nucleotides 82214-97705 of NW_003615411.1, and a sequence at least 50% homologous thereto. Thus, it is flanked on the 5' side and on the 3' side by a nucleotide sequence selected from the group consisting of nucleotides 45270-45490 of NW_006874047.1, nucleotides 207912-792374 of NW_006884592.1, nucleotides 491910-667813 of NW_006881296.1, nucleotides 79769-100059 of NW_003616412.1, nucleotides 315266-362442 of NW_003615063.1, nucleotides 2662055-2701768 of NW_006882936.1, and nucleotides 97706-105117 of NW_003615411.1 and a sequence at least 50% homologous thereto.In certain embodiments, the nucleotide sequence flanking the 5' end of the integrated exogenous nucleotide sequence is selected from the group consisting of nucleotides 41190-45269 of NW_006874047.1, nucleotides 63590-207911 of NW_006884592.1, nucleotides 253831-491909 of NW_006881296.1, nucleotides 69303-79768 of NW_003616412.1, and nucleotides 79303-79768 of NW_003616412.1. nucleotides 293481-315265 of NW_006882936.1, nucleotides 2650443-2662054 of NW_006882936.1, and nucleotides 82214-97705 of NW_003615411.1. The nucleotide sequences flanking the 3' end of the integrated exogenous nucleotide sequence are: nucleotides 45270-45490 of SEQ ID NO: NW_006874047.1; nucleotides 207912-792374 of NW_006884592.1; nucleotides 491910-667813 of NW_006881296.1; nucleotides 79769-100059 of NW_003616412.1; and nucleotides 80069-90059 of NW_0036150 NW_006882936.1, nucleotides 2662055-2701768 of NW_006882936.1, and nucleotides 97706-105117 of NW_003615411.1.

[0076] In certain embodiments, the integrated exogenous nucleotide is integrated into a locus immediately adjacent to all or a portion of a sequence selected from the group consisting of sequences at least about 90% homologous to a sequence selected from SEQ ID NOs: 1-7.

[0077] 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 located 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.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] 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.

[0082] Table 1 provides exemplary TI host cell integration sites. TIFF2026502534000002.tif55170

[0083] In certain embodiments, the integration site and / or nucleotide sequences adjacent to the integration site can be identified experimentally. In certain embodiments, the integration site and / or nucleotide sequences adjacent to the integration site can 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 adjacent to the integration site can be identified by genome-wide screening techniques after a transposase-based cassette integration event. In certain embodiments, the integration site and / or nucleotide sequences adjacent to the integration site can be identified by brute-force random integration screening. In certain embodiments, the integration site and / or nucleotide sequences adjacent to the integration site can be determined by conventional sequencing approaches, such as targeted locus amplification (TLA) followed by next-generation sequencing (NGS) and whole-genome NGS. In certain embodiments, the location of the integration site on the chromosome may be determined by conventional cell biology approaches, such as, for example, fluorescent in-situ hybridization (FISH) analysis.

[0084] In certain embodiments, the TI host cell comprises a first exogenous nucleotide sequence integrated at a first integration site within a specific first locus in the TI host cell genome and a second exogenous nucleotide sequence integrated at a second integration site within a specific second locus in the genome, hi certain embodiments, the TI host cell comprises multiple exogenous nucleotide sequences integrated at multiple integration sites in the TI host cell genome.

[0085] In certain embodiments, the TI host cell of the present disclosure comprises at least two distinct exogenous nucleotide sequences, for example, an exogenous nucleotide sequence comprising at least one RRS. In certain embodiments, two or more exogenous nucleotide sequences can be targeted for introduction of one or more SOIs. In certain embodiments, the SOIs are the same. 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.

[0086] 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, of 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.

[0087] In certain embodiments, the feasibility of recombinase-mediated cassette exchange (RMCE) of at least two integration sites can be assessed for each site individually. In certain embodiments, the feasibility of RMCE of at least two integration sites can be assessed simultaneously. The feasibility of RMCE at multiple sites can be assessed by methods known in the art, for example, by measuring polypeptide titer or polypeptide-specific production. In certain embodiments, the assessment can 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 can 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.

[0088] 4. Exogenous Nucleotide Sequence An exogenous nucleotide sequence is a nucleotide sequence that is not native to a host cell but can be introduced into a host cell by conventional DNA delivery methods, such as transfection, electroporation, or transformation. In certain embodiments, the exogenous nucleotide sequence comprises a sequence of interest (SOI), such as a nucleotide sequence encoding a polypeptide of interest. However, in certain embodiments, the exogenous nucleotide sequence used in the context of the present disclosure includes elements such as one or more recombination recognition sequences (RRSs), one or more sequence pairs (e.g., restriction sites) that facilitate the introduction of the SOI, and one or more selectable markers that facilitate the introduction of additional nucleic acid sequences, such as the SOI. 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.

[0089] 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, hi 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, and without limitation, 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 all or a portion of NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, or NW_003615411.1.

[0090] 4.1 Landing Pad In certain embodiments, the integrated exogenous nucleotide sequence comprises one or more recombination recognition sequences (RRSs), where 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, where the two RRSs are the same. In certain embodiments, the integrated exogenous nucleotide sequence comprises two RRSs, where the two RRSs are heterospecific, i.e., not recognized by the same recombinase. In certain embodiments, the integrated exogenous nucleotide sequence comprises three RRSs, where the third RRS is located between the first and second RRSs. In certain embodiments, the first and second RRSs are the same, and the third RRS is different from the first or second 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 the same. In certain embodiments, the two or more RRSs are heterospecific. In certain embodiments, each RRS can be recognized by a different recombinase. In certain embodiments, a subset of the total number of RRSs is homospecific, i.e., recognized by the same recombinase, and a subset of the total number of RRSs is 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.

[0091] 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.

[0092] In certain embodiments, the selectable marker is located between a first and a second RRS, and the two flanking RRSs are the same. In certain embodiments, both of the two RRSs flanking the selectable marker are LoxP sequences. In certain embodiments, both of the two RRSs flanking the selectable marker are FRT sequences. In certain embodiments, the selectable marker is located between a first RRS and a second RRS, and the two flanking RRSs are heterospecific. In certain embodiments, the first flanking RRS is a LoxP L3 sequence, and the second flanking RRS is a LoxP 2L sequence. In certain embodiments, the LoxP L3 sequence is located 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 in the forward or reverse direction. In some embodiments, the two RRSs are arranged in opposite orientations.

[0093] In some embodiments, selectable markers can be genes for aminoglycoside phosphotransferase (APH) (e.g., hygromycin phosphotransferase (HYG), neomycin and G418 (APH), dihydrofolate reductase (DHFR), thymidine kinase (TK), glutamine synthetase (GS), asparagine synthetase, tryptophan synthetase (indole), histidinol dehydrogenase (histidinol D), and genes encoding resistance to puromycin, blasticidin, bleomycin, phleomycin, chloramphenicol, zeocin, or mycophenolic acid. In certain embodiments, The selectable marker may be GFP, eGFP, synthetic GFP, YFP, eYFP, CFP, mPlum, mCherry, tdTomato, mStrawberry, J-red, DsRed-monomer, mOrange, mKO, mCitrine, Venus, YPet, Emerald, CyPet, mCFPm, Cerulean, or T-Sapphire. In certain embodiments, the selectable marker may 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 may be fused to a gene encoding a different selectable marker or a fragment thereof.

[0094] 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 a selectable marker selected from the group consisting of an aminoglycoside phosphotransferase (APH) (e.g., hygromycin phosphotransferase (HYG), neomycin, and G418APH), dihydrofolate reductase (DHFR), thymidine kinase (TK), glutamine synthetase (GS), asparagine synthetase, tryptophan synthetase (indole), histidinol dehydrogenase (histidinol D), and a selectable marker selected from the group consisting of puromycin, blasticidin, bleomycin, and phleomycin. The first selectable marker is selected from the group consisting of genes encoding resistance to cysteine, 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 the same. In some embodiments, the two RRSs flanking both selectable markers are different.

[0095] 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 some embodiments, the selectable marker is operably linked to a cytomegalovirus (CMV) promoter.

[0096] 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 an IRES and two selectable markers flanked by two RRSs, wherein the IRES is operably linked to the second selectable marker. In certain embodiments, the integrated exogenous nucleotide sequence comprises an IRES flanked by two RRSs and three selectable markers, where the IRES is operably linked to a third selectable marker. In certain embodiments, the integrated exogenous nucleotide sequence comprises an IRES flanked by two RRSs and three selectable markers, where the IRES is operably linked to a third selectable marker. In certain embodiments, the third selectable marker is different from the first or second selectable marker. In certain embodiments, the 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 a HYG selectable marker operably linked to a CMV promoter and a GFP selectable marker operably linked to an IRES.

[0097] In certain embodiments, the incorporated 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 the same. In certain embodiments, the first RRS and the second RRS are the same, and the third RRS is different from the first RRS or the second RRS. In certain embodiments, all three RRSs are heterospecific.

[0098] 4.2 Sequence of Interest (SOI) In certain embodiments, the integrated exogenous nucleotide sequence comprises at least one exogenous SOI. In certain embodiments, the integrated exogenous nucleotide sequence comprises at least one selectable marker and at least one exogenous SOI. In certain embodiments, the integrated exogenous nucleotide sequence comprises at least one selectable marker, at least one exogenous SOI, and at least one RRS. In certain embodiments, the integrated exogenous nucleotide sequence comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or more SOIs. In certain embodiments, the SOIs are the same. In certain embodiments, the SOIs are different.

[0099] In certain embodiments, the SOI encodes a single-chain antibody or fragment thereof. In certain embodiments, the SOI encodes an antibody heavy chain sequence or fragment thereof. In certain embodiments, the SOI encodes an antibody light chain sequence or fragment thereof. In certain embodiments, the incorporated exogenous nucleotide sequence comprises an SOI encoding an antibody heavy chain sequence or fragment thereof, and an SOI encoding an antibody light chain sequence or fragment thereof. In certain embodiments, the incorporated exogenous nucleotide sequence comprises an SOI encoding a first antibody heavy chain sequence or fragment thereof, an SOI encoding a second antibody heavy chain sequence or fragment thereof, and an SOI encoding an antibody light chain sequence or fragment thereof. In certain embodiments, the incorporated exogenous nucleotide sequence comprises an SOI encoding a first antibody heavy chain sequence or fragment thereof, an SOI encoding a second antibody heavy chain sequence or fragment thereof, an SOI encoding a first antibody light chain sequence or fragment thereof, and a second SOI encoding an antibody light chain sequence or fragment thereof. In certain embodiments, the number of SOIs encoding heavy and light chain sequences can be selected to achieve a desired expression level of heavy and light chain polypeptides, e.g., to achieve a desired amount of bispecific antibody production. In certain embodiments, the individual SOIs encoding the heavy and light chain sequences can be integrated, for example, into a single exogenous nucleic acid sequence present at a single integration site, into multiple exogenous nucleic acid sequences present at a single integration site, or into multiple exogenous nucleic acid sequences integrated into separate integration sites within the TI host cell.

[0100] In certain embodiments, the integrated exogenous nucleotide sequence comprises at least one selectable marker, at least one exogenous SOI, and one RRS. In certain embodiments, the RRS is located adjacent to the at least one selectable marker or at least one exogenous SOI. In certain embodiments, the integrated exogenous nucleotide sequence comprises at least one selectable marker, at least one exogenous SOI, and two RRSs. In certain embodiments, the integrated exogenous nucleotide sequence comprises at least one selectable marker and at least one exogenous SOI located between the first RRS and the second RRS. In certain embodiments, the two RRSs flanking the selectable marker and the exogenous SOI are the same. In certain embodiments, the two RRSs flanking the selectable marker and the exogenous SOI are different. In certain embodiments, the first flanking RRS is a LoxP L3 sequence, and the second flanking RRS is a LoxP 2L sequence. In certain embodiments, a sequenced L3 LoxP is located 5' of the selectable marker and the exogenous SOI, and a LoxP 2L sequence is located 3' of the selectable marker and the exogenous SOI.

[0101] In certain embodiments, the incorporated 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 the first RRS or 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.

[0102] In certain embodiments, the exogenous SOI encodes a polypeptide of interest. Such a polypeptide of interest may be selected from the group including, but not limited to, an antibody, an enzyme, a cytokine, a growth factor, a hormone, a viral protein, a bacterial protein, a vaccine protein, or a protein with therapeutic function. In certain embodiments, the exogenous SOI encodes an antibody or an antigen-binding fragment thereof. In certain embodiments, the exogenous SOI encodes a single-chain antibody, an antibody light chain, an antibody heavy chain, a single-chain Fv fragment (scFv), or an Fc fusion protein. In certain embodiments, the exogenous SOI(s) encode a standard antibody. In certain embodiments, the exogenous SOI(s) encode a half antibody, such as, but not limited to, antibody B, Q, T, and mAbI of the present disclosure. In certain embodiments, the exogenous SOI(s) encode a composite antibody. In certain embodiments, the composite antibody may be a bispecific antibody, such as, but not limited to, bispecific molecule A, bispecific molecule B, bispecific molecule C, or bispecific molecule D of the present disclosure. In certain embodiments, the exogenous SOI is operably linked to at least one cis-acting element, such as a promoter or enhancer. In certain embodiments, the exogenous SOI is operably linked to a CMV promoter.

[0103] 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.

[0104] 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 and second RRSs. In certain embodiments, at least one SOI is located between the first and third RRSs, and at least one SOI is located between the third and second RRSs. In certain embodiments, the first and second RRSs are the same, and the third RRS is different from either the first or second RRS. In certain embodiments, all three RRSs are heterospecific. In certain embodiments, an SOI encoding one heavy chain and one light chain of a first antibody is located between the first and third RRSs, and an SOI encoding one heavy chain and one light chain of a second antibody is located between the third and second RRSs. In certain embodiments, the SOI encoding one heavy chain and two light chains of the first antibody is located between the first RRS and the third RRS, and the SOI encoding one heavy chain and one light chain of the second antibody is located between the third RRS and the second RRS. In certain embodiments, the SOI encoding one heavy chain and three light chains of the first antibody is located between the first RRS and the third RRS, and the SOI encoding one light chain of the first antibody and one heavy chain and one light chain of the second antibody is located between the third RRS and the second RRS. In certain embodiments, the SOI encoding one heavy chain and three light chains of the first antibody is located between the first RRS and the third RRS, and the SOI encoding two light chains of the first antibody and one heavy chain and one light chain of the second antibody is located between the third RRS and the second RRS. In certain embodiments, SOIs encoding different combinations of copies of multiple antibody heavy and light chains are located between the first and third RRSs and between the third and second RRSs.

[0105] 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 result in increased titer and / or specific productivity.

[0106] In the context of antibody expression, the inclusion of an additional heavy or light chain encoding SOI can result in increased titer and / or specific productivity. For example, but not by way of 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 improved titer and / or specific productivity. Similarly, as outlined in the examples below, increasing the copy number from HLL (three SOIs) to HLL-HL (five SOIs) or HLL-HLL (six SOIs) can provide increased titer and / or specific productivity. Furthermore, increasing the copy number to HLL-HL (five SOIs) or HLL-HLHL (seven SOIs) can result in increased titer and / or specific productivity. Further options for the SOI copy numbers for the heavy and light chains include, but are not limited to, HHL;HHL-H;HLL-H;HHL-HH;HHL-HL;HHL-LL;HLL-HH;HLL-HL;HHL-HHH;HHL-HLL;HHK-LLL;HLL-HHL;HLL-HHH;HLL-LLL;HHL-HHHL;HHL-HHHH;HHL-HHLL;HHL-HLLL;HHL-LLLL;HLL-HHHL;HLL-HHHH;HLL-HLLL; and HLL-LLLL. In certain embodiments, the inclusion of additional copies occurs at a single genomic locus, while in certain embodiments, the SOI copies can be integrated at more than one locus, e.g., multiple copies can be integrated at a single locus and one or more copies can be integrated at one or more additional loci.

[0107] In certain embodiments, the location of the SOI, e.g., whether one SOI is located 3' or 5' relative to another SOI, is selected to increase the titer and / or specific productivity of a host cell expressing the SOI. For example, but not by way of limitation, in the context of antibody production, the location of integration of the heavy and light chain SOIs can result in increased titer and / or specific productivity. 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.

[0108] 5.Host cells The presently disclosed subject matter provides host cells suitable for targeted integration of nucleotide sequences and expression of polypeptides of interest. In certain embodiments, the host cell comprises an endogenous gene or a locus in the genome of the host cell selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, XP_003512331.2 and sequences at least 50% homologous thereto, wherein the locus 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 selected from the group consisting of SEQ ID NOs: 1-7 and sequences at least 50% homologous thereto.

[0109] 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 TI host cell is a Chinese hamster ovary (CHO) host cell, a CHO K1 host cell, a CHO K1SV host cell, a DG44 host cell, a DUKXB-11 host cell, a CHOK1S host cell, or a CHO K1M host cell.

[0110] In certain embodiments, the host cell is an SV40-transformed monkey kidney CV1 line (COS-7), a human embryonic kidney line (e.g., 293 or 293 cells as described in Graham et al., J. Gen Virol. 36:59 (1977)), baby hamster kidney cells (BHK), mouse Sertoli cells (e.g., TM4 cells as described in Mather, Biol. Reprod. 23:243-251 (1980)), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK), buffalo rat liver cells (BRL3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor (MMT060562), e.g., Mather et al., Annals The cells are selected from the group consisting of TRI cells, MRC5 cells, FS4 cells, Y0 cells, NS0 cells, Sp2 / 0 cells, and PER.C6® cells as described in NYAcad. Sci. 383:44-68 (1982).

[0111] 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.

[0112] 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.

[0113] The subject matter of the present disclosure also relates to a method for producing a polypeptide of interest. In certain embodiments, such a method includes: a) providing a host cell comprising at least one exogenous SOI flanked by two RRSs integrated into a locus in the genome of the host cell and at least one selectable marker, wherein the locus is at least about 90% 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, and a sequence selected from SEQ ID NOs: 1-7; and b) culturing the cell of a) under conditions suitable for expressing the SOI, and recovering the polypeptide of interest therefrom. In certain embodiments, such methods comprise the steps of: a) providing a host cell comprising at least two exogenous SOIs and at least one selectable marker integrated within a locus in the genome of the host cell, wherein the locus is selected from the contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_00361541 1.1 and a sequence selected from SEQ ID NOs: 1-7, and wherein at least one exogenous SOI and one selectable marker are flanked by a first and a third RRS, and at least one exogenous SOI is flanked by a second and a third RRS; and b) culturing the cells of a) under conditions suitable for expressing the SOI and recovering the polypeptide of interest therefrom.In certain embodiments, such methods include: a) providing a host cell containing at least one exogenous SOI flanked by two RRSs integrated into an endogenous gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, XP_003512331.2, and sequences at least about 90% homologous thereto, and at least one selectable marker; and b) culturing the cell of a) under conditions suitable for expression of the SOI, and recovering a polypeptide of interest therefrom. In certain embodiments, such methods include: a) providing a host cell containing at least two exogenous SOIs and at least one selectable marker integrated within an endogenous gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2, and sequences at least about 90% homologous thereto, wherein the at least one exogenous SOI and one selectable marker are flanked by first and third RRSs, and the at least one exogenous SOI is flanked by second and third RRSs; and b) culturing the cell of a) under conditions suitable for expression of the SOIs and recovering the polypeptide of interest therefrom.

[0114] 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 retained within the host cell. In certain embodiments, the polypeptide of interest is expressed, inserted into, and retained in the host cell membrane.

[0115] The exogenous nucleotide or vector of interest can be introduced into host cells by conventional cell biology methods, including but not limited to transfection, transduction, electroporation, or injection. In certain embodiments, the exogenous nucleotide or vector of interest is introduced into host cells by chemical-based transfection methods, including lipid-based transfection, calcium phosphate-based transfection, cationic polymer-based transfection, or nanoparticle-based transfection. In certain embodiments, the exogenous nucleotide of interest is introduced into host cells by virus-mediated transduction, including but not limited to lentivirus, retrovirus, adenovirus, or adeno-associated virus-mediated transduction. In certain embodiments, the exogenous nucleotide or vector of interest is introduced into host cells by gene gun-mediated injection. In certain embodiments, both DNA molecules and RNA molecules are introduced into host cells using the methods described herein.

[0116] 6. Targeted Integration Targeted integration allows exogenous nucleotide sequence to be integrated into one or more predetermined sites of the genome of host cell.In certain embodiments, targeted integration is mediated by the recombinase that recognizes one or more RRS.In certain embodiments, targeted integration is mediated by homologous recombination.In certain embodiments, targeted integration is mediated by exogenous site-specific nuclease, followed by HDR and / or NHEJ.

[0117] In certain embodiments, targeted integration can be combined with random integration. In certain embodiments, targeted integration can be followed by random integration. In certain embodiments, random integration can be mediated by any method or system known in the art. In certain embodiments, random integration is mediated by the MaxCyte STX® electroporation system.

[0118] 6.1. Targeted Integration via Recombinase-Mediated Recombination A "recombination recognition sequence" (RRS) is a nucleotide sequence recognized by a recombinase and is necessary and sufficient for a recombinase-mediated recombination event. An RRS can be used to define the location at which a recombination event occurs within a nucleotide sequence.

[0119] 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.

[0120] In certain embodiments, the RRS can be recognized by Cre recombinase. In certain embodiments, the RRS can be recognized by FLP recombinase. In some embodiments, the RRS can be recognized by Bxb1 integrase. In certain embodiments, the RRS can be recognized by φC31 integrase.

[0121] 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 φC31 attB site, the host cell requires φC31 integrase to perform recombination. The recombinase can be introduced into the host cell using an expression vector containing the coding sequence for the enzyme.

[0122] The Cre-LoxP site-specific recombination system is widely used in many biological experimental systems. Cre is a 38 kDa site-specific DNA recombinase that recognizes 34-bp LoxP sequences. Cre is derived from bacteriophage P1 and belongs to the tyrosine family of site-specific recombinases. Cre recombinase can mediate both intramolecular and intermolecular recombination between LoxP sequences. The LoxP sequence consists of an 8-bp non-palindromic core region flanked by two 13-bp inverted 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 other host factors. When two LoxP sequences are positioned in the same orientation on the same nucleotide sequence, Cre-mediated recombination excises the DNA sequence located between the two LoxP sequences into a covalently closed circle. When two LoxP sequences are located in opposite positions on the same nucleotide sequence, Cre-mediated recombination reverses the orientation of the DNA sequence located between these two sequences.LoxP sequences can also be located on different chromosomes to promote recombination between different chromosomes.When two LoxP sequences are located on two different DNA molecules and one DNA molecule is circular, Cre-mediated recombination leads to the integration of the circular DNA sequence.

[0123] In certain embodiments, the LoxP sequence is a wild-type LoxP sequence. In certain embodiments, the LoxP sequence is a mutant LoxP sequence. The mutant LoxP sequence was developed to increase the efficiency of Cre-mediated integration or replacement. In certain embodiments, the mutant LoxP sequence is selected from the group consisting of LoxP L3 sequence, LoxP 2L sequence, LoxFas sequence, Lox511 sequence, Lox2272 sequence, Lox2372 sequence, Lox5171 sequence, Loxm2 sequence, Lox71 sequence, and Lox66 sequence. For example, the Lox71 sequence has a 5-bp mutation in the left 13-bp repeat. The Lox66 sequence has a 5-bp mutation in the right 13-bp repeat. Both the wild-type LoxP sequence and the mutant LoxP sequence can mediate Cre-dependent recombination.

[0124] The FLP-FRT site-specific recombination system is similar to the Cre-Lox system. It involves 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 cleavage, exchange, and ligation within the 8-bp spacer. As with Cre recombinase, the position and orientation of the two FRT sequences determine the outcome of FLP-mediated recombination. In certain embodiments, the FRT sequence is a wild-type FRT sequence. In certain embodiments, the FRT sequence is a mutant FRT sequence. Both wild-type and mutant FRT sequences can mediate FLP-dependent recombination. In certain embodiments, the FRT sequence is fused to a responsive receptor domain sequence, such as, but not limited to, a tamoxifen-responsive receptor domain sequence.

[0125] Bxb1 and φC31 belong to the serine recombinase family. Both originate 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 binding sites originally located on the phage DNA and bacterial DNA, respectively. After recombination, two new sequences, called attL and attR, are formed, each containing half of the sequence derived from attP and attB. Recombination also occurs between the attL and attR sequences, allowing the integrated phage to be excised 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, which are widely used for genetic engineering purposes.

[0126] The terms "matched RRS" and "homonymous 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.

[0127] 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 the same as the second RRS on the integrated exogenous nucleotide sequence and the second RRS on the vector. A non-limiting example of such a "single-vector RMCE" strategy is shown in Figure 2A. In certain embodiments, the first RRS on the integrated exogenous nucleotide sequence and the first RRS on the vector are different from the second RRS on the integrated exogenous nucleotide sequence and the second RRS on the vector. In certain embodiments, the first RRS on the integrated exogenous nucleotide sequence and the first RRS on the vector are both LoxP L3 sequences, and the second RRS on the integrated exogenous nucleotide sequence and the second RRS on the vector are both LoxP 2L sequences.

[0128] 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, such as an arrangement in which the third RRS ("RRS3") is 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. 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 (front) includes RRS1, a first SOI (or a series of SOIs), and a promoter, followed by a start codon and RRS3 (in that order). The other vector (back) contains RRS3, another SOI (or series of SOIs), and RRS2 (in that order) fused to the coding sequence of a marker lacking the initiation codon (ATG). Additional nucleotides may be inserted between the RRS3 site and the selectable marker sequence to ensure in-frame translation of the fusion protein. This two-vector RMCE strategy allows for the introduction of eight or more SOIs by incorporating the appropriate number of SOIs between each pair of RRSs and flanking them with appropriate introduction sequences (e.g., paired restriction sites).

[0129] 6.2 Targeted integration via homologous recombination, HDR, or NHEJ The subject matter of the present disclosure also relates to targeted integration mediated by homologous recombination or by exogenous site-specific nucleases followed by HDR or NHEJ.

[0130] 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 variation 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, migration of the fork to form a Holliday junction, and eventual resolution of the Holliday junction.

[0131] Double-strand breaks (DSBs) are the most severe form of DNA damage, and repair of such DNA damage is essential for maintaining genome integrity in all organisms. There are two main repair pathways for DSB repair. The first is the homologous recombination repair (HDR) pathway, and homologous recombination is the most common form of HDR. Because HDR requires the presence of homologous DNA within the cell, this repair pathway is typically active during the S and G2 phases of the cell cycle, when newly replicated sister chromatids are available as homologous templates. HDR is also the primary repair pathway for repairing collapsed replication forks during DNA replication. HDR is considered a relatively error-free repair pathway. The second repair pathway for DSBs is non-homologous end joining (NHEJ). NHEJ is a repair pathway in which the ends of broken DNA are ligated together without the need for a homologous DNA template.

[0132] Targeted integration can be promoted by exogenous site-specific nucleases followed by HDR. This is because introducing DSBs at specific target genomic sites can increase the frequency of homologous recombination. 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.

[0133] 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, for example, by 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 transcription activator-like effector (TALE) and the catalytic domain of the restriction endonuclease FokI. By varying 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.

[0134] Targeted integration by homologous recombination or HDR involves the presence of a homologous sequence at the integration site. In certain embodiments, the homologous sequence is present on a vector. In certain embodiments, the homologous sequence is present on a polynucleotide.

[0135] 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 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, and a DNA cassette comprising 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 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, LOC1 00768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2, or a sequence selected from SEQ ID NOs: 1-7. In certain embodiments, the vector is selected from the group consisting of an adenoviral vector, an adeno-associated viral vector, a lentiviral vector, a retroviral vector, an integrating phage vector, a non-viral vector, a transposon and / or transposase vector, an integrase substrate, and a plasmid.

[0136] 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 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 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 selected from the group consisting of the 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. The sequence or DNA cassette comprises 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, and the DNA cassette comprises at least one selectable marker flanked by two RRSs and at least one exogenous SOI. In certain embodiments, the flanking nucleotide sequence is an 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, or LOC107977062, LOC1 00768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2, or a sequence selected from SEQ ID NOs: 1-7.

[0137] In certain embodiments, homologous recombination is carried out without the use of auxiliary factors. In certain embodiments, homologous recombination is promoted by the presence of an integrating vector. In certain embodiments, the integrating vector is selected from the group consisting of an adeno-associated virus vector, a lentivirus vector, a retrovirus vector, and an integrating phage vector.

[0138] 6.3 Regulated Targeted Integration Suboptimal protein expression levels are often due primarily to the encoded protein being difficult to express. When expression levels of difficult-to-express proteins are low, the causes can be diverse and 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 tetracycline or its analog, doxycycline (DOX), is added to the culture. Regulating the expression of the toxic protein can alleviate toxic effects and allow the culture to achieve the desired cell growth before production. In certain embodiments, a regulated targeted integration (RTI) system includes an SOI that is integrated into a specific locus, such as an exogenous nucleic acid sequence containing one or more RRSs, and transcribed under a regulated promoter operably linked to it. In certain embodiments, the RTI system can be used to determine the underlying cause of low protein expression of difficult-to-express molecules, such as, but not limited to, antibodies. In certain embodiments, the ability to selectively silence expression of the SOI in an RTI system can be used to link expression of the SOI to observed adverse effects.

[0139] In certain embodiments, a regulated targeted integration (RTI) system can 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 TI host can be induced by adding a regulatory factor, such as doxycycline, to the culture. In certain embodiments, the RTI vector utilizes a tetracycline-regulated promoter to express the SOI, which can be integrated into an exogenous nucleic acid sequence containing, for example, an RRS, which itself integrates into an integration site within the host cell's genome, allowing for regulated expression of the SOI.

[0140] In certain embodiments, the RTI system described in the present disclosure can be used to successfully determine the underlying cause 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 an RTI cell line is confirmed, the intracellular accumulation and secretion levels of the SOI can be assessed by utilizing protein translation inhibitor treatment, e.g., Dox and cycloheximide.

[0141] 6.4 Regulated Systems The subject matter of the present disclosure also relates to regulated systems for use in TI. For example, but not limited to, such regulation can be based on a gene switch to block or activate mRNA synthesis by regulated coupling of a transcriptional repressor or activator to a constitutive or minimal promoter. In certain non-limiting embodiments, repression can be achieved, for example, by binding to a repressor protein that sterically blocks transcription initiation, or by actively suppressing transcription via a transcriptional silencer. In certain non-limiting embodiments, activation of a mammalian or viral enhancerless minimal promoter can be achieved by regulated coupling to an activation domain.

[0142] 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.

[0143] In certain embodiments, the allosteric protein used in the disclosed TI system can be a protein that regulates transcriptional activity in response to a culture parameter, such as an antibiotic, a bacterial quorum-sensing messenger, a catabolite, or temperature, e.g., cold or heat. In certain embodiments, such an RTI system can be catabolite-based, e.g., in which a bacterial repressor that represses catabolic genes of alternative carbon sources 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.

[0144] In certain embodiments, the TI system can be a prokaryotic quorum-sensing-based expression system that manages intra- and inter-population communication through quorum-sensing molecules. These quorum-sensing molecules bind to 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 of a TraR-p65 fusion protein 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 may be a homoserine-derived inducer used in the RTI system, in which the Pseudomonas aeruginosa quorum sensing repressors RhlR and LasR are fused to the SV40 T antigen nuclear localization sequence and herpes simplex VP16 domain and can activate promoters containing specific operator sequences (las boxes).

[0145] In certain embodiments, the inducer molecule that regulates the allosteric protein used in the RTI system of the present disclosure can be, but is not limited to, coumarate, isopropyl-β-D-galactopyranoside (IPTG), macrolides, 6-hydroxynicotine, doxycycline, streptogramins, NADH, and tetracycline.

[0146] In certain embodiments, the intracellular receptor used in the disclosed RTI system can be a cytoplasmic or nuclear receptor. In certain embodiments, the disclosed RTI system can utilize the release of transcription factors from protein capture and inhibition using small molecules. In certain embodiments, the disclosed RTI system can rely on steroid regulation, where a hormone receptor is fused to a natural or artificial transcription factor that can be released from HSP90 in the cytosol, translocate to the nucleus, and activate a selected promoter. In certain embodiments, mutant receptors regulated by synthetic steroid analogs can be used to avoid crosstalk with endogenous steroid hormones. In certain embodiments, the receptor can be a 4-hydroxytamoxifen-responsive estrogen receptor mutant or a RU486-inducible progesterone receptor mutant. In certain embodiments, a nuclear receptor-derived rosiglitazone-responsive transcriptional switch based on the human nuclear peroxisome proliferator-activated receptor γ (PPARγ) can be used in the disclosed RTI system. In certain embodiments, the steroid-responsive receptor variant can be RheoSwitch, which is based on a modified Choristoneura fumiferana ecdysone receptor and the mouse retinoid X receptor (RXR) fused to a Gal4 DNA-binding domain and a VP16 transactivator. In the presence of synthetic ecdysone, the RheoSwitch variant can bind to and activate a minimal promoter fused to several repeats of the Gal4 response element.

[0147] In certain embodiments, the RTI systems disclosed herein can utilize chemically induced dimerization of DNA-binding proteins and transcriptional activators to activate a minimal core promoter fused to a cognate operator. In certain embodiments, the RTI systems disclosed herein can 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, FKBP can be mutated. In certain embodiments, the RTI systems disclosed herein can utilize the bacterial gyrase B subunit (GyrB), which dimerizes in the presence of the antibiotic coumermycin and dissociates with novobiocin.

[0148] In certain embodiments, the RTI system of the present disclosure can be used for regulated siRNA expression. In certain embodiments, the regulated siRNA expression system can be a tetracycline, a macrolide, or an OFF-type and ON-type QuoRex system. In certain embodiments, the RTI system can utilize a Xenopus terminal oligopyrimidine element (TOP), which blocks translation initiation by forming a hairpin structure in the 5' untranslated region.

[0149] In certain embodiments, the RTI system described in this disclosure may utilize gas-phase regulated expression, such as the acetaldehyde-inducible regulation (AIR) system. The AIR system utilizes the Aspergillus nidulans AlcR transcription factor. This transcription factor specifically activates the 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.

[0150] In certain embodiments, the RTI system of the present disclosure can utilize a Tet-on or Tet-off system, in which expression of one or more SOIs can be regulated by tetracycline or its analog, doxycycline.

[0151] In certain embodiments, the RTI system of the present disclosure can utilize a PIP on or PIP off system, in which expression of the SOI can be regulated by, for example, pristinamycin, tetracycline, and / or erythromycin.

[0152] 7. Preparation and Use of TI Host Cells The subject matter of the present disclosure relates to a method for targeted integration of an exogenous nucleotide sequence into a host cell. In certain embodiments, the method relates to the integration of an exogenous nucleotide sequence into a host cell to generate a host cell suitable for subsequent targeted integration of one or more SOIs. In certain embodiments, the method comprises recombinase-mediated recombination. In certain embodiments, the method comprises homologous recombination, HDR, and / or NHEJ.

[0153] 7.1 Preparation of TI host cells using recombinase-mediated recombination 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 in the genome of the host cell, wherein the locus is at least about 90% homologous to all or a portion of NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, or NW_003615411.1; and The method includes the steps of: providing a TI host cell, the nucleotide sequence of which comprises two RRSs flanked by at least one first selection 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 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 that express the polypeptide of interest.

[0154] In certain embodiments, the present disclosure provides a method for preparing a TI host cell that expresses a polypeptide of interest, the method comprising the steps of: 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 flanked by at least one first selection 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 selection marker; c) introducing a recombinase that recognizes the RRSs; and d) selecting TI cells that express the second selection marker, thereby isolating the TI host cell that expresses the polypeptide of interest.

[0155] In certain embodiments, the present disclosure provides a method for preparing a TI host cell that expresses a polypeptide of interest, the method comprising the steps of: 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 comprising two heterologous RRSs that match the two RRSs on the integrated exogenous nucleotide sequence and are flanked by at least one exogenous SOI and at least one second selectable marker; c) introducing a recombinase that recognizes the RRSs and performs a single round of RMCE; and d) selecting TI cells that express the second selectable marker, thereby isolating TI host cells that express the polypeptide of interest.

[0156] In certain embodiments, the disclosure provides a method for preparing a TI host cell that expresses a polypeptide of interest, the method comprising: a) integrating an exogenous nucleotide sequence into a site within 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 a gene 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 containing an exogenous nucleotide sequence comprising a first DNA cassette containing two heterospecific RRSs flanked by at least one first selection marker; b) introducing into the cell provided in a) a vector containing a second DNA cassette containing two heterospecific RRSs that match the two RRSs on the integrated exogenous nucleotide sequence and are flanked by at least one exogenous SOI and at least one second selection marker; c) introducing a recombinase that recognizes the RRSs and performs a single round of RMCE; and d) selecting TI cells that express the second selection marker, thereby isolating TI host cells that express the polypeptide of interest.

[0157] 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 that comprises an exogenous nucleotide sequence integrated into a site within a locus of the host cell genome, the locus being located within contig NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1, or at least about 90% homologous to a portion of the contig sequence 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 and a third RRS located between 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 selectable marker on a first vector, the first vector comprises a promoter sequence operably linked to an ATG codon positioned adjacent to a first SOI upstream and adjacent to an RRS downstream, and the second vector comprises a selectable marker lacking the ATG transcription start codon adjacent to an RRS upstream and adjacent to a second SOI downstream.

[0158] 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) selecting 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; providing a TI host cell comprising an exogenous nucleotide sequence integrated into a site within an endogenous sequence of a portion of a contig sequence of one of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, XP_003512331.2, or a gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, XP_003512331.2, and a sequence at least about 90% homologous thereto, wherein the exogenous nucleotide sequence is selected from the group consisting of at least one first selection matrix. The method includes the steps of: providing a TI host cell comprising a first RRS and a second RRS flanked by a selectable marker, and a third RRS located between the first and second RRS, all of the RRSs being 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 flanked by at least one first exogenous SOI and at least one second selectable marker; c) introducing into the cell provided in a) a second vector comprising two RRSs that match the second and third RRSs on the integrated exogenous nucleotide sequence and 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 selectable marker on a first vector, the first vector comprises a promoter sequence operably linked to an ATG codon positioned adjacent to a first SOI upstream and adjacent to an RRS downstream, and the second vector comprises a selectable marker lacking the ATG transcription start codon adjacent to an RRS upstream and adjacent to a second SOI downstream.

[0159] 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 at a site within a locus in the genome of the TI host cell, wherein the locus is located within the contigs NW_006874047.1, NW_006884592.1, NW_006881296 ... the exogenous nucleotide sequence is at least about 90% homologous to a portion of the contig sequence of one of NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1, or to SEQ ID NOs: 1 to 7, and the exogenous nucleotide sequence comprises a first DNA cassette including 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 heterologous; The method includes the steps of: providing a TI host cell; b) introducing a first vector containing a second DNA cassette into the cell provided in a), 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 selection marker; c) introducing a second vector containing a third DNA cassette into the cell provided in a), wherein the third DNA cassette contains two heterospecific RRSs that match the second and third RRSs of the first DNA cassette and are flanked by at least one second exogenous SOI; d) introducing one or more recombinases that recognize the RRSs and perform RMCE twice; 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 selectable marker on a first vector, the first vector comprises a promoter sequence operably linked to an ATG codon positioned adjacent to a first SOI upstream and adjacent to an RRS downstream, and the second vector comprises a selectable marker lacking the ATG transcription start codon adjacent to an RRS upstream and adjacent to a second SOI downstream.

[0160] 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) selecting 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; providing an exogenous nucleotide sequence integrated at a site within a gene sequence 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 flanking at least one first selectable marker, and a third RRS located between the first and second RRSs; wherein all three RRSs are heterospecific; b) introducing into the 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 selectable marker; c) introducing into the cells provided in a) a second vector comprising the third DNA cassette. The method includes the steps of: d) introducing a second vector, wherein the third DNA cassette contains two heterospecific RRSs that match the second and third RRSs of the first DNA cassette and are flanked by at least one second exogenous SOI; d) introducing one or more recombinases 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 selectable marker on a first vector, the first vector comprises a promoter sequence operably linked to an ATG codon positioned adjacent to a first SOI upstream and adjacent to an RRS downstream, and the second vector comprises a selectable marker lacking the ATG transcription start codon adjacent to an RRS upstream and adjacent to a second SOI downstream.

[0161] 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 at a site within a locus of the TI host cell genome, wherein the locus is a sequence selected from the 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, or a sequence selected from the sequence of a portion of the contig sequence of one of SEQ ID NOs: 1-7. The method includes the steps of: b) providing a TI host cell that is at least about 90% homologous to the exogenous nucleotide sequence, wherein the exogenous nucleotide sequence comprises one RRS flanked by at least one first selection marker; b) introducing into the cell provided in a) a vector that matches the RRS on the integrated exogenous nucleotide sequence and comprises at least one exogenous SOI and one RRS flanked by at least one second selection marker; c) introducing a recombinase that recognizes the RRS; and d) selecting TI cells that express the second selection marker, thereby isolating TI host cells that express the polypeptide of interest.

[0162] In certain embodiments, the disclosure provides a method for preparing a TI host cell that expresses a polypeptide of interest, the method comprising: a) cloning an 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, or 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; The method includes the steps of: providing TI host cells containing an exogenous nucleotide sequence integrated at a site within the array, wherein the exogenous nucleotide sequence comprises one RRS adjacent to at least one first selection marker; b) introducing into the cells provided in a) a vector containing an RRS that matches the RRS on the integrated exogenous nucleotide sequence and that is adjacent to at least one exogenous SOI and at least one second selection marker; c) introducing a recombinase that recognizes the RRS; and d) selecting TI cells that express the second selection marker, thereby isolating TI host cells that express the polypeptide of interest.

[0163] The subject matter of the present disclosure also relates to a method for producing a polypeptide of interest, comprising the steps of: 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.

[0164] In certain embodiments, the present disclosure provides a method for preparing a TI host cell suitable for subsequent targeted integration, the method comprising: a) providing a TI host cell comprising an exogenous nucleotide sequence integrated at a site within a locus in the genome of the host cell, wherein the locus is selected from the 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, or a sequence selected from SEQ ID NOs: 1-7. The method includes the steps of: b) providing a TI host cell that is at least about 90% homologous, 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 comprising two RRSs that match the two RRSs on the integrated exogenous nucleotide sequence and are flanked by at least one second selection marker; c) introducing a recombinase that recognizes the RRSs; and d) selecting TI cells that express the second selection marker, thereby isolating TI host cells suitable for subsequent targeted integration.

[0165] In certain embodiments, the present disclosure provides a method for preparing a TI host cell suitable for subsequent targeted integration, the method comprising: a) integrating a TI host cell with a target sequence within an 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, or 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 containing an exogenous nucleotide sequence integrated at a site, 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 comprising two RRSs that match the two RRSs on the integrated exogenous nucleotide sequence and that are flanked by at least one second selection marker; c) introducing a recombinase that recognizes the RRSs; and d) selecting TI cells that express the second selection marker, thereby isolating TI host cells that are suitable for subsequent targeted integration.

[0166] In certain embodiments, the present disclosure provides a method for preparing a TI host cell suitable for subsequent targeted integration, the method comprising: a) providing a TI host cell comprising 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 of 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, and wherein the exogenous nucleotide sequence comprises a first RRS and a second RRS flanked by at least one exogenous SOI and at least one first selectable marker. and a second RRS; b) introducing into the cell provided in a) a vector comprising three RRSs, wherein a first RRS of the vector matches a first RRS on the integrated exogenous nucleotide sequence, a second RRS of the vector matches a 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.

[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) integrating 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 into a TI host cell; or 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 a site within at least one exogenous SOI. and a first RRS and a second RRS flanked by 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 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.

[0168] 7.2 Methods for targeted modification of host cells using homologous recombination, HDR or NHEJ In certain embodiments, the present disclosure provides a method for preparing a TI host cell 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 SEQ ID NOs: 1-7; (b) introducing a vector into the TI host cell, the vector comprising a nucleotide sequence at least 50% identical to a sequence selected from SEQ ID NOs: 1-7 flanking a DNA cassette, the DNA cassette comprising at least one selectable marker and at least one exogenous SOI; and (c) selecting the selectable marker, isolating a TI host cell having the SOI integrated into the genomic locus, and expressing 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.

[0169] 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, wherein the locus is at least about 90% identical to a sequence selected from SEQ ID NOs: 1-7; (b) introducing a polynucleotide into the host cell, wherein the polynucleotide comprises a nucleotide sequence at least 50% identical to a sequence selected from SEQ ID NOs: 1-7 flanking a DNA cassette, the DNA cassette comprising at least one selectable marker and at least one exogenous SOI; and (c) selecting the selectable marker, isolating a TI host cell having the SOI integrated into the genomic locus, and expressing 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.

[0170] In certain embodiments, homologous recombination is promoted by an integrating vector. In certain embodiments, the vector is selected from the group consisting of an adenovirus vector, an adeno-associated virus vector, a lentivirus vector, a retrovirus vector, an integrating phage vector, a non-viral vector, a transposon and / or a transposase vector, an integrase substrate, and a plasmid. In certain embodiments, the transposon can be a PiggyBac (PB) transposon system.

[0171] In certain embodiments, integration 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.

[0172] In certain embodiments, the present disclosure provides a method for preparing a TI host cell suitable for subsequent targeted integration, the method comprising the steps of: 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 of 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 TI host cell. a) introducing a vector comprising 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; and b) selecting the selectable marker and isolating a TI host cell suitable for subsequent targeted integration.

[0173] In certain embodiments, the present disclosure provides a method for preparing a TI host cell suitable for subsequent targeted integration, the method comprising the steps of: 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 of 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 polynucleotide into the TI host cell. a) introducing a polynucleotide, wherein the polynucleotide comprises 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 a DNA cassette, wherein the DNA cassette comprises at least one selectable marker flanked by two RRSs; and b) selecting the selectable marker and isolating a suitable TI host cell for subsequent targeted integration.

[0174] In certain embodiments, the present disclosure provides a method for preparing a TI host cell suitable for subsequent targeted integration, the method comprising the steps of: a) providing a TI host cell comprising a locus in the host cell's genome, wherein the locus is at least about 90% homologous to a sequence of 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 is and (b) introducing a vector comprising a nucleotide sequence at least 50% homologous to a portion of the contig sequence of one of SEQ ID NOs: 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 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 marker and isolating a TI host cell suitable for subsequent targeted integration.

[0175] In certain embodiments, the present disclosure provides a method for preparing a TI host cell suitable for subsequent targeted integration, the method comprising the steps of: 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 of 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 polypeptide into the host cell, wherein the polypeptide is , 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 marker and isolating a TI host cell suitable for subsequent targeted integration.

[0176] 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 have a sequence similar to at least 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, wherein the at least one exogenous nucleotide sequence is about 90% homologous to the exogenous nucleotide sequence of the TI host cell, and the at least one exogenous nucleotide sequence comprises two RRSs flanked by at least one first selection 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 selection marker; c) introducing a recombinase that recognizes the RRSs, or a nucleic acid encoding the recombinase; and selecting TI cells that express the second selection marker, thereby isolating TI host cells that express at least one polypeptide of interest.

[0177] 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 that comprises at least one exogenous nucleotide sequence integrated into a site within one or more loci of the host cell genome, wherein the one or more loci are located within the contig NW_0068740 47.1, 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 third RRS located between the first and third RRSs on the at least one integrated exogenous nucleotide sequence, 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 at least one integrated exogenous nucleotide sequence and are flanked by at least one first exogenous SOI and at least one second selection marker; c) introducing into the cells provided in a) a second vector comprising two RRSs that match the second and third RRSs on the at least one integrated exogenous nucleotide sequence and are flanked by at least one second exogenous SOI; 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 selection 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 selectable marker on a first vector, the first vector comprises a promoter sequence operably linked to an ATG codon positioned adjacent to a first SOI upstream and adjacent to an RRS downstream, and the second vector comprises a selectable marker lacking the ATG transcription start codon adjacent to an RRS upstream and adjacent to a second SOI downstream.

[0178] 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 at least one exogenous nucleotide sequence integrated into a site within one or more loci of the genome of the TI host cell, wherein the one or more loci are selected from the sequence of 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 SEQ ID NOs: 1-7. The method includes the steps of: b) providing a TI host cell in which the exogenous nucleotide sequence is at least about 90% homologous to the exogenous nucleotide sequence, wherein the exogenous nucleotide sequence comprises one or more RRSs; b) introducing into the cell provided in a) a vector comprising one or more RRSs adjacent to at least one exogenous SOI that matches the one or more RRSs on the integrated exogenous nucleotide sequence and is 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.

[0179] 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 is at least about 90% homologous to a portion of the sequence of one of the contig sequences 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; and b) culturing the cell under conditions suitable for expressing the SOI and recovering the polypeptide of interest therefrom.

[0180] 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 that comprises an exogenous nucleotide sequence integrated into a site within a locus of the 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 RRSs are heterologous. 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 adjacent to at least one first exogenous SOI operably linked to a regulatable promoter; c) introducing into the cells provided in a) a second vector comprising two RRSs 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 selectable marker on a first vector, the first vector comprises a promoter sequence operably linked to an ATG codon positioned adjacent to a first SOI upstream and adjacent to an RRS downstream, and the second vector comprises a selectable marker lacking the ATG transcription start codon adjacent to an RRS upstream and adjacent to a second SOI downstream.

[0181] 8.Product The host cells of the present disclosure can 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 can 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.

[0182] In certain embodiments, exemplary polypeptides encompassed by the definition herein include mammalian polypeptides such as, for example, renin; growth hormones, e.g., 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-type 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 (B LyS); 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, such as VEGF receptors (e.g., VEGFR1, VEGFR2, and VEGFR3), epidermal growth factor (EGF) receptors (e.g., ErbB1, ErbB2, ErbB3, and ErbB4 receptors), platelet-derived growth factor (PDGF) receptors (e.g., PDGFR-α and PDGFR-β), and fibroblast growth factor receptors; TIE ligands (angiopoietin, ANGPT1, ANGPT2), angiopoietin receptors, e.g., TIE1 and TIE2; protein A or D; rheumatoid factor; neurotrophin factors, such as bone-derived neurotrophic factor (BDNF), neurotrophin-3, neurotrophin-4, neurotrophin-5, or neurotrophin-6 (NT-3, NT-4, NT-5, or NT-6), or nerve growth factor, such as NGF-b; transforming growth factors (TGFs), such as TGF-alpha and TGF-beta, including TGF-β1, TGF-β2, TGF-β3, TGF-β4, or TGF-β5; insulin-like growth factors I and II (IGF-I and IGF-II); des(1-3)-IGF- I (brain IGF-I), insulin-like growth factor binding proteins (IGFBPs); CD proteins, 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 (IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-17, IL-18, IL-19, IL-20, IL-19, IL-21, IL-19, IL-22, IL-23, IL-24, IL-25, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-46, IL-47, IL-48, IL-49, IL-50, IL-51, IL-52, IL-53, IL-54, IL-55, IL-56, IL-57, IL-59, IL-60, IL-61, IL-62, IL-63, IL-64, IL-65, IL-66, IL-67, IL-68, IL-69, IL-70, IL-71, IL-72, IL-73, IL-74, IL-75, IL-76, IL-77, IL-78, IL-79, IL-79, ), e.g., 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; 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-mentioned polypeptides, as well as antibodies, including antibody fragments, that bind to one or more proteins, including, for example, any of the above-mentioned proteins.

[0183] In certain embodiments, the polypeptide of interest is a bispecific, trispecific, or multispecific polypeptide, such as a bispecific antibody. Various molecular formats of multispecific antibodies are known in the art and are included herein (see, for example, Spiess et al., Mol Immunol 67 (2015) 95-106). A particular type of multispecific antibody also included herein is a bispecific antibody designed to simultaneously bind to a surface antigen on a target cell, such as a tumor cell, and to an activation invariant component of the T cell receptor (TCR) complex (such as CD3), thereby retargeting T cells and killing the target cell. Further examples of bispecific antibody formats include so-called "BiTE" (bispecific T cell engager) molecules in which two scFv molecules are fused via a flexible linker (see, e.g., WO 2004 / 106381, WO 2005 / 061547, WO 2007 / 042261 and WO 2008 / 119567; N.N. Agorsen and Bauerle, Exp Cell Res 317, 1255-1260 (2011)); diabodies (Holliger et al., Prot Eng 9, 299-305 (1996)) and derivatives thereof, such as tandem diabodies ("TandAb"; Kipriyanov et al., J Mol Biol 293, 41-56 (1999); "DART" (dual affinity retargeting) molecules, which are based on the diabody format but feature a C-terminal disulfide bridge for 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.

[0184] In certain embodiments, the host cells of the present disclosure can be used for the expression of chaperones, protein-modifying enzymes, shRNAs, gRNAs, or other proteins or peptides, while also expressing a therapeutic protein or molecule of interest, either constitutively or regulated.

[0185] In some embodiments, the polypeptide expressed by a host cell of the disclosure is, 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, FGF10, FGF11, FGF12, FGF12B, FGF14, FGF16, FGF17, FGF19, FGF 20, FGF21, FGF23, IGF1, IGF2, IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFN81, IFNG, IFNWI, FEL1, FEL1(EPSELON), FEL1(ZETA), IL1A, IL1B, IL2, IL 3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL11, IL12A, IL12B, IL13, IL14, IL15, IL16, IL17, IL17B, IL18, IL19, IL20, IL22, IL23, IL24, IL25, IL26, IL27, I L28A, IL28B, IL29, IL30, PDGFA, PDGFB, TGFA, TGFB1, TGFB2, TGFBb3, LTA(TNF-β), LTB, TNF(TNF-α), TNFSF4(OX40 ligand), TNFSF5(CD40 ligand), TNFSF6( FasL), TNFSF7 (CD27 ligand), TNFSF8 (CD30 ligand), TNFSF9 (4-1BB ligand), TNFSF10 (TRAIL), TNFSF11 (TRANCE), TNFSF12 (APO3L), TNFSF13 (April), TNFSF1 3B, TNFSF14 (HVEM-L), TNFSF15 (VEGI), TNFSF18, HGF (VEGFD), VEGF, VEGFB, VEGFC, IL1R1, IL1R2, IL1RL1, IL1RL2, IL2RA, IL2RB, IL2RG, IL3RA, IL4R, IL5RA, IL6R, IL7R, IL8RA, IL8RB, IL9R, IL10RA, IL10RB, IL11RA, IL12RB1, IL12RB2, IL13RA1, IL13RA2, IL15RA, IL17R, IL18R1, IL20RA, IL21R, IL22R,It 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 IL1HY1, IL1RAP, IL1RAPL1, IL1RAPL2, IL1RN, IL6ST, IL18BP, IL18RAP, IL22RA2, AIF1, HGF, LEP (leptin), PTN, and THPO.k.

[0186] translocation-associated 2); ERAK2; ITGA1; ITGA2; ITGA3; ITGA6 (a6 integrin); ITGAV; ITGB3; ITGB4 (b4 integrin); α4β7 and αEβ7 integrin heterodimer; JAG1; JAK1; JAK3; JUN; K6HF; KAI1; KDR; KITLG; KLF5 (GC Box BP); KLF6; KLKIO; KLK12; KLK13; KLK14; KLK15; KLK3; KLK4; KLK5; KLK6; KLK9; KRT1; KRT19 (keratin 19); KRT2A; KHTHB6 (hair-specific H-type keratin); Llama; LEP (leptin); LGR5 (leucine-rich repeat-containing G protein-coupled receptor 5; GPR49, GPR67); Lingo-p75; Lingo-Troy; LPS; LTA (TNF-β); LTB; LTB4R( GPR16; LTB4R2; LTBR; LY64 (lymphocyte antigen 64 (RP105), a type I membrane protein of the leucine-rich repeat (LRR) family); Ly6E (lymphocyte antigen 6 complex, locus E; Ly67, RIG-E, SCA-2, TSA-1); Ly6G6D (lymphocyte antigen 6 complex, locus G6D; Ly6-D, MEGT1); LY6K (lymphocyte antigen 6 complex, locus K; LY6K; HSJ001348; FLJ35226); MACMARCK S; 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 (NaPi2 b) (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;NR1H3;NR1H4;NR112;NR113;NR2C1;NR2C2;NR2E1;NR2E3;NR2F1;NR2F2;NR2F6;NR3C1;NR3C2;NR4A1;NR4A2;NR4A3;NR5A1;NR5A2;NR6A1;NRP1;NRP2;NT5E;NTN4;ODZI;OPRD1;OX40;P2RX7;P2X5 (purinergic receptor P2X ligand-gated ion channel 5);PAP;PART 1;PATE;PAWR;PCA3;PCNA;PD-L1;PD-L2;PD-1;POGFA;POGFB;PECAM1;PF4(CXCL4);PGF;PGR;phosphacan;PIAS2;PIK3CG;PLAU(uPA);P LG;PLXDC1;PMEL17(silver homologue;SILV;D12S53E;PMEL17;SI;SIL);PPBP(CXCL7);PPID;PRI;PRKCQ;PRKDI;PRL;PROC;PROK2;PSAP;PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA2700050C12, RIKEN cDNA 2700050C12 gene); PTAFR; PTEN; PTGS2 (COX-2); PTN; RAC2 (p21 Rac2); RARB; RET (ret proto-oncogene; MEN2A; HSCR1; MEN2B; MTC1; PTC; CDHF12; Hs.168114; RET51; RET-ELE1); RGSI; RGS13; RGS3; RNF110 (ZNF144); ROBO2; S100A2; SCGB1D2 (lipophilin B); SCGB2A1 (mammaglobin 2); SCGB2A2 (mammaglobin 1); SCYEI (endothelial-monocyte-activating cytokine); SDF2; Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, semaphorin 5b Hlog, sema domain, seven thrombospondin repeats (type 1 and type 1-like), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5B); SERPINA1; SERPINA3; SERP1NB5 (maspin); SERPINE1 (PAI-1); SERPDMF1; SHBG; SLA2; SLC2A2; SLC33A1; SLC43A1; SLIT2; SPPI;SPRR1B (Sprl); ST6GAL1; STABI; STAT6; STEAP (six transmembrane epithelial antigen of the prostate); STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer-associated gene 1, prostate cancer-associated protein 1, six transmembrane epithelial antigen of the prostate 2, six transmembrane prostate protein); TB4R2; TBX21; TCPIO; TOGFI; TEK; TENB2 (putative transmembrane proteoglycan); TGFA; TGFBI; TGFB1II; TGFB2; TGFB3; TGF BI;TGFBRI;TGFBR2;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-α;TNFAEP2 (B94);TNFAIP3;TNFRSFIIA;T NFRSF1A;TNFRSF1B;TNFRSF21;TNFRSF5;TNFRSF6(Fas);TNFRSF7;TNFRSF8;TNFRSF9;TNFSF10(TRAIL);TNFSF11(TRANCE);TNFSF12(AP03L);TNFSF13 (April);TNFSF13B;TNFSF14(HVEM-L);TNFSF15(VEGI);TNFSF18;TNFSF4(OX40 ligand);TNFSF5(CD40 ligand);TNFSF6(FasL);TNFSF7(CD27 ligand);TNFSFS (CD30 ligand); TNFSF9 (4-1BB ligand); TOLLIP; Toll-like receptor; TOP2A (topoisomerase Ea); TP53; TPM1; TPM2; TRADD; TMEM118 (ring finger protein, transmembrane 2; RNFT2; FLJ14627); TRAF1; TRAF2; TRAF3; TRAF4; TRAF5; TRAF6; TREM1; TREM2; TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channel, subfamily M, member 4); TRPC6; TSLP;TWEAK; tyrosinase (TYR; OCAIA; OCA1A; tyrosinase; SHEP3); VEGF; VEGFB; VEGFC; versican; VHL C5; VLA-4; XCL1 (lymphotactin); XCL2 (SCM-1b); XCRI (GPR5 / CCXCRI); YY1; and / or ZFPM2.

[0187] 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, V CAM, 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 can be used to generate antibodies (or multispecific antibodies, such as bispecific antibodies) that specifically bind to complement protein C5 (e.g., anti-C5 agonist antibodies that specifically bind to human C5).

[0188] In certain embodiments, the methods provided herein can be used to produce antibodies (or multispecific antibodies, such as bispecific antibodies) that specifically bind to influenza virus B hemagglutinin, i.e., "fluB" (e.g., antibodies that bind to hemagglutinin from the Yamagata lineage of influenza B virus, antibodies that bind to hemagglutinin from the Victoria lineage of influenza B virus, antibodies that bind to hemagglutinin from an ancestral lineage of influenza B virus, or antibodies that bind to hemagglutinin from the Yamagata lineage, Victoria lineage, and ancestral lineage of influenza B virus in vitro and / or in vivo). Further details regarding anti-FluB antibodies are described in WO 2015 / 148806, which is incorporated herein by reference in its entirety.

[0189] In certain embodiments, the antibody (or bispecific antibody) produced by the methods provided herein binds to at least one target selected from the group consisting of low-density lipoprotein receptor-related protein (LRP)-1 or LRP-8 or transferrin receptor, and 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.

[0190] In certain embodiments, the antibody produced according to the methods provided herein is a human IgG2 antibody against CD40. In certain embodiments, the anti-CD40 antibody is RG7876.

[0191] 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.

[0192] In certain embodiments, multispecific antibodies (such as bispecific antibodies) produced according to the methods provided herein bind to CEA and at least one additional target molecule. In certain embodiments, multispecific antibodies (such as bispecific antibodies) produced according to the methods provided herein bind to a tumor-targeting cytokine and at least one additional target molecule. In certain embodiments, multispecific antibodies (such as bispecific antibodies) produced according to the methods provided herein are fused to IL2v (i.e., an interleukin-2 variant) and bind to an IL1-based immune cytokine and at least one additional target molecule. In certain embodiments, multispecific antibodies (such as bispecific antibodies) produced according to the methods provided herein are T cell bispecific antibodies (i.e., bispecific T cell engagers or BiTEs).

[0193] In certain embodiments, multispecific antibodies (such as bispecific antibodies) produced according to the methods provided herein bind to at least two target molecules selected from the following: 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 LHR agonist; IL-12 and TWEAK; IL-13 and CL25; IL-13 and SPRR2a; IL-13 and SPRR2b; IL-13 and ADAMS; IL-13 and PED2; IL17A and IL17F; CEA and CD3; CD3 and CD19; CD138 and CD20; CD138 and CD40; CD19 and CD20; CD20 and CD3; CD3S and CD13S; CD3S and CD20; CD3S and CD40; CD40 and 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 IL6, TNFalpha and IL8, 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, TNFa and IL-12p40, VEGF and angiopoietin, VEGF and HER2, VEGF-A and HER2, VEGF-A and PDGF, HER1 and HER2, VEGFA and ANG2, VEGF-A and VEGF-C, VEGF-C and VEGF-D, HER2 and DR5, VEGF and IL-8, VEGF and MET, VEGFR and MET receptor, EGFR and MET, VEGF R and EGFR, HER2 and CD64, HER2 and CD3, HER2 and CD16, HER2 and HER3, EGFR (HER1) and HER2, EGFR and HER3, EGFR and HER4, IL-14 and IL-13, IL-13 and CD40L, IL4 and CD40L, TNFR1 and IL-1R, TNFR1 and IL-6R and TNFR1 and IL-18R, EpCAM and CD3, MAPG and CD28, EGFR and CD64, CSPGs and RGM A; CTLA-4 and BTN02; IGF1 and IGF2; IGF1 / 2 and Erb2B; MAG and RGM A; NgR and RGM A;NogoA and RGM A; OMGp and RGM A; POL-1 and CTLA-4; and RGM A and RGM B.

[0194] 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.

[0195] 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.

[0196] 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.

[0197] The above list is not meant to be limiting, as many other antibodies and / or other proteins may be expressed by the host cells in the present disclosure.

[0198] 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 ranging from about 400 L to about 25,000 L. Within this range, specific cell culture sizes may be utilized, 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.

[0199] In certain embodiments, the polypeptide of interest is a bispecific, trispecific, or multispecific polypeptide, eg, a bispecific antibody.

[0200] The host cells of the present disclosure can be used to produce large quantities of a molecule of interest in a shorter time frame than non-TI cells used in current cell culture methods. In certain embodiments, the host cells of the present disclosure can be used to improve the quality of 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 can be used to enhance seed train stability by preventing chronic toxicity that can be caused by products that can cause cellular stress and clonal instability over time. In certain embodiments, the host cells of the present disclosure can be used for optimal expression of acutely toxic products.

[0201] In certain embodiments, the host cells, TI systems of the present disclosure can be used for cell culture process optimization and / or process development.

[0202] In certain embodiments, the host cells of the present disclosure can be used to accelerate the production of a molecule of interest by about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, or about 10 weeks compared to non-TI cells used in conventional cell culture methods. In certain embodiments, the host cells of the present disclosure can be used to accelerate the recovery 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.

[0203] In certain embodiments, the host cells of the present embodiments can be used to reduce aggregation levels of a molecule of interest compared to non-TI cells used in conventional cell culture methods.

[0204] In certain embodiments, host cells of the present disclosure can be used to achieve increased expression of one or more polypeptides of interest relative to randomly integrated host cells. For example, and without limitation, host cells of the present disclosure can 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, host cells of the present disclosure can achieve increased bispecific content relative to randomly integrated host cells, for example, but not limited to, host cells of the present disclosure can achieve at least 80%, 85%, 90%, 95%, 96%, 98%, 99% or more bispecific content.

[0205] In certain embodiments, the host cells of the present disclosure can be used for the constitutive expression of selected subunits of a therapeutic molecule and the regulated expression of other, different subunits of the same therapeutic molecule. In certain embodiments, the therapeutic molecule can be a fusion protein. In certain embodiments, the host cells of the present disclosure can be used to understand the role and effect of each antibody subunit in the expression and secretion of a fully assembled antibody molecule.

[0206] In certain embodiments, the host cells of the present disclosure can be used as research tools. In certain embodiments, the host cells of the present disclosure can be used as diagnostic tools to determine the root cause of low protein expression of problematic molecules in various cells. In certain embodiments, the host cells of the present disclosure can be used to directly link observed phenomena or cellular behavior to transgene expression in cells. The host cells of the present disclosure can also be used to demonstrate whether observed behavior is reversible in cells. In certain embodiments, the host cells of the present disclosure can be used to identify and alleviate problems with the transcription and expression of one or more transgenes in cells. [Example]

[0207] The following examples are merely illustrative of the subject matter of this disclosure and are not intended to be limiting in any way.

[0208] Example 1: Combinatorial vector cloning and transfection strategies for targeted integration To compare our combinatorial vector cloning and transfection strategy with a TI strategy in which each vector construct is constructed separately and used to generate a single transfected pool, we conducted a case study to express the bispecific molecule "mAb1 / mAb2" in a TI CHO host (Figure 1B). In the TI strategy in which each vector construct is constructed separately and used to generate a single transfected pool, three front vectors and four back vectors were constructed. Five pools, each transfected with a different set of vector constructs, were evaluated in pool production to determine the two optimal configurations for single-cell cloning. In conjunction with our combinatorial vector cloning and transfection strategy, one randomized front library and one randomized back library were constructed (Figure 1A). A single pool was generated by co-transfecting the front and back libraries into a TI host followed by single-cell cloning. The top two clones generated from the standard TI strategy, in which each vector construct was constructed separately and used to generate a single transfected pool ("X1" and "X2"), and the top six clones generated from the combinatorial vector cloning and transfection strategy were evaluated in production cultures for bispecific antibody expression. Clones from both strategies were evaluated together using the same production medium and process. Culture titers, percentage of correct pairing, and effective titers were measured (Figure 1C). In general, clones generated from the new strategy covered a wider range of titers and percentage of correct pairing. However, the top clones obtained from the new strategy had comparable percentage of correct pairing and higher titers than clones obtained from the standard strategy, indicating that the use of the new combinatorial vector cloning and transfection strategy can generate clones with equal or even higher effective titers for bispecific antibody expression.

[0209] Example 2: Use of fixed half mAb constructs and constitutive / inducible promoters in combinatorial vector cloning and transfection strategies in TI for bispecific molecules As shown in Figure 2A, the disclosed combinatorial vector cloning and transfection strategy can involve anchoring half-mAb1 or half-mAb2 constructs in the front or back vector, respectively. This approach reduces the number of potential vector combinations but may improve the number of isolated clones capable of expressing the complete bispecific molecule. Furthermore, as shown in Figure 2B, constitutive or inducible promoters can be added to the mixture to further increase the diversity of chain ratio expression. For example, Figure 2B illustrates a strategy in which both the antibody chain configuration and the promoter for each chain are randomized as either constitutive (e.g., CMV promoter) or inducible (e.g., CMVTO promoter). Vectors can be cloned using a seven-way ligation technique, with the back vector configured with a TetR gene to repress transcription from the CMVTO promoter. CMVTO indicates the CMV promoter with a tet operon (TO) inserted before the CMV promoter.

[0210] In addition to the various embodiments depicted and claimed, the presently disclosed subject matter is directed to other embodiments having other combinations of the features disclosed and claimed herein. Thus, specific features presented herein may be combined with each other in other ways within the scope of the presently disclosed subject matter, such that the presently disclosed subject matter includes any suitable combination of the features disclosed herein. The foregoing description of specific embodiments of the presently disclosed subject matter has been presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the disclosed subject matter to the disclosed embodiments.

[0211] It will be apparent to those skilled in the art that various modifications and variations can be made in the compositions and methods of the disclosed subject matter without departing from the spirit or scope of the disclosed subject matter. Accordingly, it is intended that the presently disclosed subject matter cover modifications and variations that come within the scope of the appended claims and their equivalents.

[0212] Various publications, patents, and patent applications are cited herein, the contents of which are incorporated herein by reference in their entireties.

Claims

1. 1. A method for screening a library of targeted integration (TI) host cells expressing a plurality of sequences of interest (SOIs) for TI host cells that exhibit particular cellular or product attributes, comprising: a. generating a first vector library by contacting a first plurality of vectors with a first plurality of SOIs, wherein the first plurality of vectors comprises a plurality of sequences configured to facilitate introduction of an SOI of the first plurality of SOIs; b. generating a second vector library by contacting a second plurality of vectors with a second plurality of SOIs, wherein the second plurality of vectors comprises a plurality of sequences configured to facilitate introduction of an SOI of the second plurality of SOIs; c. contacting a plurality of TI host cells with a plurality of vectors of the first vector library and the second vector library, thereby introducing a plurality of SOIs into a plurality of the TI host cells, thereby generating the library of TI host cells; d. separating a plurality of TI host cells of said library of TI host cells into single clones; e. screening the clones for a particular cellular or product attribute; A method comprising:

2. The method of claim 1 , wherein the sequence configured to facilitate the introduction of an SOI is a restriction site.

3. 3. The method of claim 2, wherein the first plurality of vectors comprises a pair of distinct restriction sites, the pair of distinct restriction sites flanking a plurality of the first plurality of SOIs in the same order.

4. 3. The method of claim 2, wherein the first plurality of vectors comprises multiple non-overlapping pairs of distinct restriction sites, the non-overlapping pairs of distinct restriction sites flanking multiple SOIs of the first plurality of SOIs in the same order for each pair.

5. 5. The method of claim 4, wherein the first plurality of vectors comprises three non-overlapping pairs of distinct restriction sites, the three non-overlapping pairs of distinct restriction sites flanking three SOIs of the first plurality of SOIs in the same order for each pair.

6. 5. The method of claim 4, wherein the first plurality of vectors comprises four non-overlapping pairs of distinct restriction sites, the four non-overlapping pairs of distinct restriction sites flanking four SOIs of the first plurality of SOIs in the same order for each pair.

7. 3. The method of claim 2, wherein the second plurality of vectors comprises a pair of distinct restriction sites, the pair of distinct restriction sites flanking a plurality of the second plurality of SOIs in the same order.

8. 3. The method of claim 2, wherein the second plurality of vectors comprises multiple non-overlapping pairs of distinct restriction sites, the multiple non-overlapping pairs of distinct restriction sites flanking multiple SOIs of the second plurality of SOIs in the same order for each pair.

9. 9. The method of claim 8, wherein the second plurality of vectors comprises three non-overlapping pairs of distinct restriction sites, the three non-overlapping pairs of distinct restriction sites flanking three SOIs of the second plurality of SOIs in the same order for each pair.

10. 9. The method of claim 8, wherein the second plurality of vectors comprises four non-overlapping pairs of distinct restriction sites, the four non-overlapping pairs of distinct restriction sites flanking four SOIs of the second plurality of SOIs in the same order for each pair.

11. a. the first plurality of vectors comprises a pair of distinct restriction sites, the pair of distinct restriction sites flanking a plurality of the first plurality of SOIs in the same order; b. the second plurality of vectors comprises a pair of distinct restriction sites, the pair of distinct restriction sites flanking a plurality of the second plurality of SOIs in the same order; The method of claim 2.

12. a. the first plurality of vectors comprises a plurality of non-overlapping pairs of distinct restriction sites, the non-overlapping pairs of distinct restriction sites flanking a plurality of SOIs of the first plurality of SOIs in the same order for each pair; b. the second plurality of vectors comprises a plurality of non-overlapping pairs of distinct restriction sites, the non-overlapping pairs of distinct restriction sites flanking a plurality of SOIs of the second plurality of SOIs in the same order for each pair; The method of claim 2.

13. a. the first plurality of vectors comprises three non-overlapping pairs of distinct restriction sites, the three non-overlapping pairs of distinct restriction sites flanking three SOIs of the first plurality of SOIs in the same order for each pair; b. the second plurality of vectors comprises three non-overlapping pairs of distinct restriction sites, the three non-overlapping pairs of distinct restriction sites flanking three SOIs of the second plurality of SOIs in the same order for each pair; The method of claim 12.

14. a. the first plurality of vectors comprises four non-overlapping pairs of distinct restriction sites, the four non-overlapping pairs of distinct restriction sites flanking four SOIs of the first plurality of SOIs in the same order for each pair; b. the second plurality of vectors comprises four non-overlapping pairs of distinct restriction sites, the four non-overlapping pairs of distinct restriction sites flanking four SOIs of the second plurality of SOIs in the same order for each pair; The method of claim 12.

15. The method of claim 3 , wherein the first plurality of SOIs comprise separate SOIs.

16. The method of claim 3 , wherein the first plurality of SOIs comprise the same SOI.

17. The method of claim 7 , wherein the second plurality of SOIs comprise separate SOIs.

18. The method of claim 7 , wherein the second plurality of SOIs comprise the same SOI.

19. a. the first plurality of SOIs comprise separate SOIs; b. the second plurality of SOIs comprise separate SOIs; 15. The method according to any one of claims 11 to 14.

20. a. the first plurality of SOIs comprise the same SOI; b. the second plurality of SOIs comprise the same SOI; 15. The method according to any one of claims 11 to 14.

21. a. the first plurality of SOIs comprise separate SOIs; b. the second plurality of SOIs comprise the same SOI; 15. The method according to any one of claims 11 to 14.

22. a. the first plurality of SOIs comprise the same SOI; b. the second plurality of SOIs comprise separate SOIs; 15. The method according to any one of claims 11 to 14.

23. 23. The method of any one of claims 1 to 22, comprising generating third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, or additional vector libraries by contacting a respective number of vectors with a respective number of SOIs, wherein the respective number of vectors comprise a plurality of sequences configured to facilitate the introduction of SOIs for the respective number of SOIs; and introducing a plurality of SOIs into a plurality of TI host cells by contacting the plurality of TI host cells with a respective plurality of the vectors of the vector library, thereby generating the library of TI host cells.

24. 24. The method of any one of claims 1 to 23, wherein the SOI is operably linked to one or more regulatable promoters.

25. 25. The method of claim 24, wherein the one or more regulatable promoters are selected from the group consisting of an SV40 promoter and a CMV promoter.

26. 26. The method of any one of claims 1 to 25, wherein the plurality of SOIs are introduced into one or more of the plurality of TI host cells by recombinase-mediated integration.

27. 26. The method of any one of claims 1-25, wherein the plurality of SOIs are introduced into one or more of the plurality of TI host cells by gene editing-mediated integration.

28. The method of claim 1 , wherein the TI host cell is a mammalian host cell.

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

30. 30. The method of claim 29, wherein the TI host cell is a CHO host cell, a CHO K1 host cell, a CHO K1SV host cell, a DG44 host cell, a DUKXB-11 host cell, a CHOK1S host cell, or a CHO K1M host cell.

31. 31. The method of any one of claims 1 to 30, wherein each SOI encodes a polypeptide subunit of a multi-subunit protein or a fragment thereof.

32. 31. The method of any one of claims 1 to 30, wherein each SOI independently encodes a single chain antibody, an antibody light chain, an antibody heavy chain, a single chain Fv fragment (scFv), or an Fc fusion protein.

33. 33. The method of any one of claims 1 to 32, wherein the specific cell attribute is selected from cell growth, cell titer, specific productivity, volumetric productivity, clonal stability, % intact product expression, or a combination of such attributes.

34. 34. The method of any one of claims 1 to 33, wherein the particular product attribute is selected from the level of glycosylation, the level of charge dispersion, reduced mismatches, reduced protein / peptide aggregation, protein sequence heterogeneity, or a combination of such attributes.

35. 28. The method of claim 26 or claim 27, wherein the SOI is introduced into one or more loci that are at least about 90% homologous to a sequence selected from SEQ ID NOs: 1-12; 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.

36. 1. A method for generating a library of TI host cells comprising a plurality of SOIs, comprising: a. generating a first vector library by contacting a first plurality of vectors with a first plurality of SOIs, wherein the first plurality of vectors comprises a plurality of sequences configured to facilitate introduction of an SOI of the first plurality of SOIs; b. generating a second vector library by contacting a second plurality of vectors with a second plurality of SOIs, wherein the second plurality of vectors comprises a plurality of sequences configured to facilitate introduction of an SOI of the second plurality of SOIs; c. contacting a plurality of TI host cells with a plurality of the vectors of the first vector library and the second vector library, thereby introducing a plurality of SOIs into a plurality of the TI host cells, thereby generating the library of TI host cells comprising a plurality of SOIs; A method comprising:

37. 37. The method of claim 36, wherein the sequence configured to facilitate the introduction of an SOI is a restriction site.

38. 38. The method of claim 37, wherein the first plurality of vectors comprises a pair of distinct restriction sites, the pair of distinct restriction sites flanking a plurality of the first plurality of SOIs in the same order.

39. 38. The method of claim 37, wherein the first plurality of vectors comprises multiple non-overlapping pairs of distinct restriction sites, the non-overlapping pairs of distinct restriction sites flanking multiple SOIs of the first plurality of SOIs in the same order for each pair.

40. 40. The method of claim 39, wherein the first plurality of vectors comprises three non-overlapping pairs of distinct restriction sites, the three non-overlapping pairs of distinct restriction sites flanking three SOIs of the first plurality of SOIs in the same order for each pair.

41. 40. The method of claim 39, wherein the first plurality of vectors comprises four non-overlapping pairs of distinct restriction sites, the four non-overlapping pairs of distinct restriction sites flanking four SOIs of the first plurality of SOIs in the same order for each pair.

42. 38. The method of claim 37, wherein the second plurality of vectors comprises a pair of distinct restriction sites, the pair of distinct restriction sites flanking a plurality of the second plurality of SOIs in the same order.

43. 38. The method of claim 37, wherein the second plurality of vectors comprises multiple non-overlapping pairs of distinct restriction sites, the multiple non-overlapping pairs of distinct restriction sites flanking multiple SOIs of the second plurality of SOIs in the same order for each pair.

44. 44. The method of Claim 43, wherein the second plurality of vectors comprises three non-overlapping pairs of distinct restriction sites, the three non-overlapping pairs of distinct restriction sites flanking three SOIs of the second plurality of SOIs in the same order for each pair.

45. 44. The method of Claim 43, wherein the second plurality of vectors comprises four non-overlapping pairs of distinct restriction sites, the four non-overlapping pairs of distinct restriction sites flanking four SOIs of the second plurality of SOIs in the same order for each pair.

46. a. the first plurality of vectors comprises a pair of distinct restriction sites, the pair of distinct restriction sites flanking a plurality of the first plurality of SOIs in the same order; b. the second plurality of vectors comprises a pair of distinct restriction sites, the pair of distinct restriction sites flanking a plurality of the second plurality of SOIs in the same order; 38. The method of claim 37.

47. a. the first plurality of vectors comprises a plurality of non-overlapping pairs of distinct restriction sites, the non-overlapping pairs of distinct restriction sites flanking a plurality of SOIs of the first plurality of SOIs in the same order for each pair; b. the second plurality of vectors comprises a plurality of non-overlapping pairs of distinct restriction sites, the non-overlapping pairs of distinct restriction sites flanking a plurality of SOIs of the second plurality of SOIs in the same order for each pair; 38. The method of claim 37.

48. a. the first plurality of vectors comprises three non-overlapping pairs of distinct restriction sites, the three non-overlapping pairs of distinct restriction sites flanking three SOIs of the first plurality of SOIs in the same order for each pair; b. the second plurality of vectors comprises three non-overlapping pairs of distinct restriction sites, the three non-overlapping pairs of distinct restriction sites flanking three SOIs of the second plurality of SOIs in the same order for each pair; 48. The method of claim 47.

49. a. the first plurality of vectors comprises four non-overlapping pairs of distinct restriction sites, the four non-overlapping pairs of distinct restriction sites flanking four SOIs of the first plurality of SOIs in the same order for each pair; b. the second plurality of vectors comprises four non-overlapping pairs of distinct restriction sites, the four non-overlapping pairs of distinct restriction sites flanking four SOIs of the second plurality of SOIs in the same order for each pair; 48. The method of claim 47.

50. 42. The method of any one of claims 38 to 41, wherein the first plurality of SOIs comprise separate SOIs.

51. 42. The method of any one of claims 38 to 41, wherein the first plurality of SOIs comprise the same SOI.

52. 46. ​​The method of any one of claims 42 to 45, wherein the second plurality of SOIs comprise separate SOIs.

53. 47. The method of any one of claims 42 to 46, wherein the second plurality of SOIs comprise the same SOI.

54. a. the first plurality of SOIs comprise separate SOIs; b. the second plurality of SOIs comprise separate SOIs; 50. The method of any one of claims 46 to 49.

55. a. the first plurality of SOIs comprise the same SOI; b. the second plurality of SOIs comprise the same SOI; 50. The method of any one of claims 46 to 49.

56. a. the first plurality of SOIs comprise separate SOIs; b. the second plurality of SOIs comprise the same SOI; 50. The method of any one of claims 46 to 49.

57. a. the first plurality of SOIs comprise the same SOI; b. the second plurality of SOIs comprise separate SOIs; 50. The method of any one of claims 46 to 49.

58. 58. The method of any one of claims 37 to 57, comprising generating a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, or additional vector libraries by contacting a respective number of vectors with a respective number of SOIs, wherein the respective number of vectors comprise a plurality of sequences configured to facilitate the introduction of SOIs for the respective number of SOIs; and introducing a plurality of SOIs into a plurality of TI host cells by contacting the plurality of TI host cells with a respective plurality of the vectors of the vector library, thereby generating the library of TI host cells.

59. 59. The method of any one of claims 37 to 58, wherein the SOI is operably linked to one or more regulatable promoters.

60. 60. The method of claim 59, wherein the one or more regulatable promoters are selected from the group consisting of an SV40 promoter and a CMV promoter.

61. 61. The method of any one of claims 36 to 60, wherein the plurality of SOIs are introduced into one or more of the plurality of TI host cells by recombinase-mediated integration.

62. 61. The method of any one of Claims 36-60, wherein the plurality of SOIs are introduced into one or more of the plurality of TI host cells by gene editing-mediated integration.

63. 37. The method of claim 36, wherein the TI host cell is a mammalian host cell.

64. 64. The method of claim 63, wherein the TI host cell is a hamster host cell, a human host cell, a rat host cell, or a mouse host cell.

65. 65. The method of claim 64, wherein the TI host cell is a CHO host cell, a CHO K1 host cell, a CHO K1SV host cell, a DG44 host cell, a DUKXB-11 host cell, a CHOK1S host cell, or a CHO K1M host cell.

66. 66. The method of any one of claims 36 to 65, wherein each SOI encodes a polypeptide subunit of a multi-subunit protein or a fragment thereof.

67. 66. The method of any one of claims 36 to 65, wherein each SOI independently encodes a single chain antibody, an antibody light chain, an antibody heavy chain, a single chain Fv fragment (scFv), or an Fc fusion protein.

68. 63. The method of claim 61 or claim 62, wherein the SOI is introduced into one or more loci that are at least about 90% homologous to a sequence selected from SEQ ID NOs: 1-12; 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.

69. the nucleotide sequence immediately 3' to the integrated SOI is nucleotide 45270 of NW_006874047.1, nucleotide 207912 of NW_006884592.1, nucleotide 491910 of NW_006881296.1, nucleotide 79769 of NW_003616412.1, nucleotide 315266 of NW_003615063.1, nucleotide 2662055 of NW_006882936.1, or nucleotide 97706 of NW_003615411.1; 69. The method of claim 68, wherein the sequence is selected from the group consisting of a sequence of at least 15 base pairs, at least 20 base pairs, at least 30 base pairs, at least 40 base pairs, at least 50 base pairs, at least 75 base pairs, at least 100 base pairs, at least 150 base pairs, at least 200 base pairs, at least 300 base pairs, at least 400 base pairs, at least 500 base pairs, at least 1,000 base pairs, at least 1,500 base pairs, at least 2,000 base pairs, or at least 3,000 base pairs from

70. the nucleotide sequence immediately 5' to the integrated SOI is nucleotide 45269 of NW_006874047.1, nucleotide 207911 of NW_006884592.1, nucleotide 491909 of NW_006881296.1, nucleotide 79768 of NW_003616412.1, nucleotide 315265 of NW_003615063.1, nucleotide 2662054 of NW_006882936.1, or nucleotide 97705 of NW_003615411.1; 69. The method of claim 68, wherein the sequence is selected from the group consisting of a sequence of at least 15 base pairs, at least 20 base pairs, at least 30 base pairs, at least 40 base pairs, at least 50 base pairs, at least 75 base pairs, at least 100 base pairs, at least 150 base pairs, at least 200 base pairs, at least 300 base pairs, at least 400 base pairs, at least 500 base pairs, at least 1,000 base pairs, at least 1,500 base pairs, at least 2,000 base pairs, or at least 3,000 base pairs from

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