Methods of cell selection

Abstract

To provide methods and compositions for identifying, selecting, or culturing cells comprising specific nucleic acid sequences.SOLUTION: Provided are production cells, and methods for identifying, selecting, or culturing production cells comprising a tyrosine auxotrophy selection marker system based on a combination of a sequence encoding a phenylalanine hydroxylase (PAH) lacking a functional N-terminal regulatory domain and a sequence encoding a GTP cyclohydrolase 1 (GCH1). Also provided are methods of making a production cell and making a product with the production cell.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to methods and compositions for identifying, selecting, or culturing cells that contain the subject nucleic acid sequences. [Background technology]

[0002] Cell expression systems are commonly used for the production of recombinant biological products, such as therapeutic biologics. Development of production line cells involves introducing nucleic acid constructs encoding the desired recombinant product into host cells and selecting for cells containing these nucleic acid constructs. This generally involves exposing the cells to selective pressure to favor cells that have incorporated the foreign nucleic acid. Early selectable marker systems used antibiotic resistance markers, but there has been a trend away from the use of such systems. Some alternative systems are based on complementation of metabolic deficiencies, such as dihydrofolate reductase (DHFR) and glutamine synthetase (GS). However, there remains a need for new selection systems that can be used to select for cells used to produce recombinant biological products. Furthermore, as production host cell engineering strategies are increasingly used, those strategies that involve the introduction of new sequences that modify the characteristics of the host cell would benefit from selection systems that are separate from existing or future systems used to introduce the sequences encoding the biological product. Summary of the Invention

[0003] The present invention relates to a selection system based on tyrosine auxotrophy and the production of recombinant products that do not require the inclusion of tyrosine in the culture medium. It has been found that a system based solely on phenylalanine hydroxylase (PAH—catalyzing the conversion of phenylalanine to tyrosine) is ineffective and requires the inclusion of a second enzyme involved in tyrosine biosynthesis, namely, GTP cyclohydrolase 1 (GCH1). Furthermore, it has been found that using PAH with a truncation that removes the N-terminal regulatory domain offers significant advantages over the full-length enzyme. While full-length CHO PAHs either failed to recover or had much slower recovery times in tyrosine-free medium after transfection, truncated (tPAH) forms of the molecules allowed for better recovery. The combination of PAH and GCH1 allows cells to grow at lower levels of tyrosine (e.g., in the absence of tyrosine) than similar cells that do not express these enzymes.

[0004] Thus, in a first aspect, the present invention provides a vector system comprising one or more nucleic acid vectors, the nucleic acid vector system comprising: a) a first nucleic acid sequence comprising a sequence encoding phenylalanine hydroxylase (PAH) lacking a functional N-terminal regulatory domain, operably linked to a first control sequence that enables expression of PAH in a host cell; b) a second nucleic acid sequence comprising a sequence encoding GTP cyclohydrolase 1 (GCH1) operably linked to a second control sequence that allows expression of GCH1 in a host cell; c) a multiple cloning site for inserting one or more product-encoding sequences operably linked to a third control sequence that allows expression of the product of interest in the host cell.

[0005] In a related aspect, the present invention also provides a vector system comprising one or more nucleic acid vectors, the nucleic acid vector system comprising: a) encoding a phenylalanine hydroxylase (PAH) lacking a functional N-terminal regulatory domain operably linked to a first regulatory sequence that allows expression of the PAH in a host cell; a first nucleic acid sequence comprising a sequence encoding b) a second nucleic acid sequence comprising a sequence encoding GTP cyclohydrolase 1 (GCH1) operably linked to a second control sequence that allows expression of GCH1 in a host cell; c) a third nucleic acid sequence comprising a product-encoding sequence operably linked to a third control sequence that allows expression of the product of interest in the host cell.

[0006] Such vectors may be introduced into host cells, the cells containing the vector being selected under tyrosine-limiting conditions that do not allow efficient growth of non-transformed cells. Thus, in a second aspect, the present invention provides a host cell, the host cell comprising: a) a first exogenous nucleic acid comprising a sequence encoding phenylalanine hydroxylase (PAH) operably linked to a first control sequence that allows expression of the PAH in a host cell; b) a second exogenous nucleic acid encoding GTP cyclohydrolase 1 (GCH1), operably linked to a second control sequence that allows expression of GCH1 in the host cell; and c) a third exogenous nucleic acid encoding the product operably linked to a third control sequence that allows expression of the product of interest in the host cell.

[0007] In one embodiment, the first, second, and third nucleic acid molecules are integrated into the genome of the host cell.

[0008] In one embodiment, the host cell is a mammalian cell, for example, a Chinese hamster ovary (CHO) cell.

[0009] In various embodiments of the present invention, the lack of a functional N-terminal regulatory domain in a PAH can be due to, for example, deletion to form a truncated PAH, which, based on the human and CHO PAH amino acid sequences, is typically a deletion of approximately the first 116 amino acids.

[0010] In one embodiment, the PAH is a CHO PAH or a human PAH.

[0011] In one embodiment, the first and / or second regulatory sequence comprises an SV40 promoter.

[0012] The vector systems of the invention are typically used to select cells that have been successfully transformed with a nucleic acid encoding a product of interest, such as a recombinant polypeptide. Thus, in a third aspect, the invention provides a method for selecting cells that contain a product-encoding nucleic acid sequence, the method comprising: a) contacting a population of cells that are unable to survive or grow in the absence of tyrosine with a vector system of the present invention under conditions that allow for uptake of the vector system by the cells; b) culturing the cells under conditions in which the level of tyrosine is lower than that required for survival or growth of cells that do not express the PAH and GCH1 enzymes encoded by the vector system; c) selecting one or more cells that are able to grow under such conditions to obtain one or more cells that contain the nucleic acid sequence encoding the product.

[0013] The level of tyrosine is chosen to ensure stringent selection and is optionally supplemented with phenylalanine, hi one embodiment, the culture medium does not contain added tyrosine.

[0014] In a related aspect, the invention provides the use of the vector system of the invention to select, from a population of cells, one or more cells which contain a nucleic acid sequence which has been introduced into said cells.

[0015] The selected host cells obtained by the selection method of the invention form a further aspect of the invention. Thus, in a fourth aspect, the invention provides a host cell, the host cell comprising: a) a first exogenous nucleic acid comprising a sequence encoding phenylalanine hydroxylase (PAH) lacking a functional N-terminal regulatory domain, operably linked to a first control sequence that enables expression of PAH in a host cell; b) a second exogenous nucleic acid encoding GTP cyclohydrolase 1 (GCH1), operably linked to a second control sequence that allows expression of GCH1 in the host cell; and c) a third exogenous nucleic acid encoding the product operably linked to a third control sequence that allows expression of the product of interest in the host cell.

[0016] The host cells of the present invention can be genetically modified to inhibit or eliminate any endogenous PAH and / or GCH1 activity. In one embodiment, this can be achieved by mutations (insertions, deletions, and / or substitutions) in the genomic sequences that encode and / or regulate the expression of endogenous PAH and / or GCH1.

[0017] A selected host cell of the invention containing a nucleic acid sequence encoding a product of interest is typically used in manufacturing that product. Thus, in a fifth aspect, the invention provides a method of making a product, the method comprising culturing a host cell of the invention containing a nucleic acid sequence encoding the product under conditions suitable for expressing the product, recovering the product, and optionally subjecting the recovered product to one or more processing or purification steps.

[0018] When cell lines developed using the host cells and selection process of the present invention are used for large-scale production, it may no longer be necessary to apply selection pressure by omitting tyrosine during the cultivation step. However, tyrosine, which is considered an essential amino acid, has the second lowest water solubility of any amino acid, after cysteine. The low solubility of tyrosine can be a challenge to generate a feed solution of sufficient concentration to support cell cultivation under biomanufacturing conditions, such as in a fed-batch bioprocess, e.g., in a bioreactor.

[0019] The host cells of the invention can grow efficiently on low levels of tyrosine (including in the absence of tyrosine), reducing the need for a high concentration tyrosine feed. In one embodiment, the medium is supplemented with phenylalanine, as phenylalanine is consumed by the cells to produce tyrosine.

[0020] The present invention also provides a culture medium, such as a feed, containing a plurality of amino acids, such as at least three or four amino acids, wherein the aqueous solution contains less than 0.01 g / L of tyrosine, such as less than 50, 20, or 10 μM of tyrosine (e.g., no tyrosine), and at least 2, preferably at least 3, 4, 5, 6, 7, 8, or 9 mM of phenylalanine. Typically, the culture medium contains less than 10 mM of phenylalanine. The present invention also provides a culture medium mixture in a substantially dry form (e.g., containing less than 5, 4, 3, 2, or 1% water, e.g., significantly no water) containing a plurality of amino acids, such as at least three or four amino acids, with tyrosine and phenylalanine levels such that the culture medium can be prepared by adding an appropriate volume of water.

[0021] The present invention further provides the use of culture media and culture medium mixtures for selecting and / or growing cells transformed with a vector system of the present invention, e.g., in the expression of a product of interest encoded by the vector system.

[0022] The present invention also provides a mixture comprising a host cell of the present invention and a culture medium of the present invention.

[0023] In another aspect, the invention features a bioreactor containing a population of host cells of the invention. In another aspect, the invention features a bioreactor containing a medium and a population of producer cells of the invention. [Brief explanation of the drawings]

[0024] [Figure 1] The domain structure of PAH enzymes is shown. [Figure 2-1](A) Histogram of mean fluorescence from cell populations after transfection and 3 weeks of recovery of the same CHO cell pool obtained using flow cytometry, and (B) a table of fluorescence data. [Figure 2-2] Continued from Figure 2-1. [Figure 3] Graph of PAH mRNA abundance relative to control as measured by qRT-PCR is shown. [Figure 4] (A) Graph of cell growth over 18 days by viable cell concentration of various cell pools, some of which overexpress truncated PAH, in the absence of tyrosine or glutamine, optionally supplemented with phenylalanine, and (B) graph of culture viability of the same cell pools under the same conditions. [Figure 5] Figure 1 shows the growth characteristics of the tyrosine prototrophic cell pool with various phenylalanine supplements. [Figure 6] Graphs are shown of the growth characteristics of tyrosine prototrophic cell pools in the absence of CD CHO tyrosine with 6 mM phenylalanine. (A) Graphs are shown of the viable cell concentration of various cell pools without tyrosine and optionally supplemented with phenylalanine, and (B) the culture viability of the same pools. [Figure 7] Graphs of growth characteristics of pre-adapted tyrosine prototrophic cell pools in which phenylalanine supplementation occurred prior to cell growth assessment are shown. (A) Viable cell concentration of the cell pools and (B) culture viability of the same cell pools under the same conditions are shown. [Figure 8] Shown is a graph of PAH mRNA levels in various cell pools relative to control cells (top), and a graph of GCH1 mRNA levels in various cell pools relative to control cells (bottom). [Figure 9-1] Graphs of growth characteristics of co-expressing tyrosine and glutamine auxotrophic cell pools are shown: (A) viable cell concentration, (B) viability, and (C) cell diameter. [Figure 9-2] Continued from Figure 9-1. DETAILED DESCRIPTION OF THE INVENTION

[0025] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. Additionally, the materials, methods, and examples are merely illustrative and not intended to be limiting. Headings, subheadings, or numbered or lettered elements, such as (a), (b), (i), etc., are presented merely for ease of reading. The use of headings or numbered or lettered elements in this document does not require that steps or elements be performed in alphabetical order or that steps or elements be necessarily separate from one another. Other features, objects, and advantages of the present invention will be apparent from the description and drawings, and from the claims. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0026] "About" or "approximately," as the term is used herein, applies to one or more values of interest and refers to a value similar to a stated reference value. In certain embodiments, the term "about" or "approximately," unless otherwise stated or otherwise clear from the context, refers to a range of values that fall within 5%, 4%, 3%, 2%, 1% in either direction (greater or lesser) of the stated reference value (except where such number exceeds 100% of possible values).

[0027] As used herein, the term "control element" refers to a nucleic acid suitable for regulating (e.g., increasing or decreasing) expression of a coding sequence, e.g., a gene or sequence encoding a product or enzymatic molecule. A control element can include a promoter sequence, an enhancer sequence, or both a promoter sequence and an enhancer sequence. A control element can include contiguous nucleic acid sequences, discontinuous nucleic acid sequences (sequences interrupted by other coding or non-coding nucleic acid sequences), or both. A single control element can be contained on a single nucleic acid or on more than one nucleic acid. In embodiments, a control element can include sequences 5' or 3' of a coding sequence, e.g., a coding sequence for a recombinant, therapeutic, or repressor polypeptide. In embodiments, a control element can include sequences within one or more introns of a gene, e.g., a gene encoding a recombinant, therapeutic, or repressor polypeptide. In embodiments, a control element can be contained in part or in its entirety within sequences 5' or 3' of a coding sequence, e.g., a coding sequence for a recombinant, therapeutic, or repressor polypeptide. In embodiments, a regulatory element may be contained in part or in its entirety within a coding sequence, e.g., the coding sequence for a recombinant, therapeutic, or repressor polypeptide. In embodiments, a regulatory element may be contained in part or in its entirety within one or more introns of a gene, e.g., a gene encoding a recombinant, therapeutic, or repressor polypeptide. In embodiments, a single regulatory element may comprise a nucleic acid sequence i) proximal to (e.g., adjacent to or contained within) a gene, e.g., a gene encoding a recombinant, therapeutic, or repressor polypeptide, or ii) distal to (e.g., separated by 10 or more, 100 or more, 1000 or more, or 10,000 or more bases, or located on a different, distinct nucleic acid) a gene, e.g., a gene encoding a recombinant, therapeutic, or repressor polypeptide.

[0028] The term "about" when referring to a measurable value, such as an amount, temporal duration, etc., is meant to encompass a variation of ±5%, or in some cases ±1%, or in some cases ±0.1% from the specified value, where such variation is appropriate for performing the disclosed method.

[0029] As used herein, the term "bioreactor" refers to a device in which a biological reaction or process takes place. The process can be carried out at industrial, pilot, and laboratory scales, including micro- and nano-scales.

[0030] As used herein, the term "endogenous" refers to any material that originates from or is naturally produced within an organism, cell, tissue, or system.

[0031] As used herein, the term "exogenous" refers to any material that is introduced into or produced outside of an organism, cell, tissue, or system. Thus, an "exogenous nucleic acid" refers to a nucleic acid that is introduced into or produced outside of an organism, cell, tissue, or system. In some embodiments, the sequence of the exogenous nucleic acid is not naturally produced or cannot be found in nature within the organism, cell, tissue, or system into which the exogenous nucleic acid is introduced. In some embodiments, the sequence of the exogenous nucleic acid is a non-naturally occurring sequence or encodes a non-naturally occurring product. In some embodiments, the sequence of the exogenous nucleic acid can also be found in the organism, cell, tissue, or system into which the exogenous nucleic acid is introduced. For example, an exogenous nucleic acid can be a constitutively expressed nucleic acid. The enzyme may be encoded under the control of an endogenous promoter, and the cell into which the exogenous nucleic acid is introduced contains an endogenous nucleic acid sequence encoding the enzyme (e.g., under the control of an endogenous promoter).

[0032] As used herein, the term "enzyme molecule" refers to a polypeptide having an enzymatic activity of interest. An enzyme molecule may share structural similarity (e.g., sequence homology) with a naturally occurring enzyme having the enzymatic activity of interest. In some cases, an enzyme molecule has at least 80% amino acid sequence identity (e.g., at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to a naturally occurring enzyme having the enzymatic activity of interest. In some embodiments, an enzyme molecule is a variant of a naturally occurring enzyme (e.g., a variant containing one or more amino acid sequence modifications (e.g., substitutions, deletions, or insertions) relative to the amino acid sequence of the naturally occurring enzyme). In some cases, the term "molecule," when used in conjunction with an enzyme identifier (e.g., PAH or GCH1), refers to a polypeptide having the enzymatic activity of the identified enzyme. By way of example, the term "PAH molecule" or "PAH enzyme molecule," as used herein, refers to a polypeptide having the enzymatic activity of PAH. As a further example, the term "GCH1 molecule" or "GCH1 enzyme molecule," as used herein, refers to a polypeptide having the enzymatic activity of GCH1. In some embodiments, the enzyme molecule is or comprises a single polypeptide chain. In some embodiments, the enzyme molecule is or comprises a multi-polypeptide complex, e.g., an oligomer (e.g., a dimer, trimer, tetramer, pentamer, hexamer, octamer, decamer, or dodecamer).

[0033] As used herein, the term "enzymatically active fragment" refers to a portion of an enzyme or enzyme molecule that retains the desired enzymatic activity of the enzyme or enzyme molecule. In some embodiments, an enzymatically active fragment is a variant of the enzyme or enzyme molecule that contains a deletion (e.g., truncation) relative to the enzyme or enzyme molecule. In some embodiments, the desired enzymatic activity of the enzymatically active fragment is reduced by 50, 40, 30, 20, or 10% or less relative to the enzyme or enzyme molecule from which the enzymatically active fragment is derived.

[0034] As used herein, the terms "nucleic acid," "polynucleotide," or "nucleic acid molecule" are used interchangeably and refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), or combinations of DNA or RNA and polymers thereof, in either single-stranded or double-stranded form. The term "nucleic acid" includes, but is not limited to, a gene, cDNA, or RNA sequence (e.g., mRNA). In one embodiment, a nucleic acid molecule is synthetic (e.g., chemically synthesized or artificial) or recombinant. Unless otherwise specified, the term encompasses molecules containing analogs or derivatives of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring or non-naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitution can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). As used herein, a "subject nucleic acid" refers to a sequence that may desirably be introduced into or present in a cell described herein, e.g., a sequence encoding a product described herein, or a production factor described herein (e.g., SCD1 and / or SREBF-1, etc.). By "nucleic acid" is meant any nucleic acid of interest that contains a sequence encoding a lipid metabolism modifier (LMM) of the present invention.

[0035] As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds or by means other than peptide bonds. A protein or peptide must contain at least two amino acids, with no limit on the maximum number of amino acids that a protein or peptide sequence can comprise. In one embodiment, a protein can be composed of more than one polypeptide, e.g., two, three, four, five, or more, where each polypeptide is associated with another polypeptide by either covalent or non-covalent bonds / interactions. Polypeptide includes any peptide or protein comprising two or more amino acids linked to each other by peptide bonds or by means other than peptide bonds. As used herein, the term refers to both short chains, e.g., commonly referred to in the art as peptides, oligopeptides, and oligomers, and long chains, of which there are many varieties, commonly referred to in the art as proteins. "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, and fusion proteins, among others.

[0036] As used herein, the term "plurality" refers to two or more (e.g., two or more) of the grammatical object of the article. By way of example, "a plurality of cells" can mean two cells or more than two cells.

[0037] "Product," as the term is used herein, refers to an entity, e.g., a compound (e.g., a polypeptide (e.g., a glycoprotein), a nucleic acid, a lipid, a sugar, a polysaccharide, or any hybrid thereof), a vesicle, an exosome, or a virus, produced, e.g., produced by, e.g., a cell, e.g., a cell that has been modified or engineered to produce a product, e.g., a production cell. In some embodiments, the product is a protein or polypeptide product. In some embodiments, the product comprises a naturally occurring product. In some embodiments, the product comprises a non-naturally occurring product. In some embodiments, a portion of the product is naturally occurring, while another portion of the product is non-naturally occurring. In some embodiments, the product is a polypeptide, e.g., a recombinant polypeptide. In some embodiments, the product is suitable for diagnostic or preclinical uses. In some embodiments, the product is suitable for therapeutic uses, e.g., treatment of a disease. In some embodiments, the product is a recombinant or therapeutic protein described herein, e.g., in the section below entitled "Polypeptides." In some embodiments, the virus comprises a naturally occurring virus, a recombinant virus, a recombinant viral particle, a virus-like particle (VLP), a viral vector, an inactivated (e.g., dead or non-infectious) virus, multiple viral proteins, a viral capsid, or any fragment, component subset, or variant thereof.

[0038] As used herein, a "production cell" refers to a cell that is capable of producing a product, e.g., a recombinant polypeptide. In some embodiments, a production cell comprises an exogenous nucleic acid encoding a product (e.g., a recombinant polypeptide), e.g., operably linked to a regulatory element that regulates expression of the product in the production cell. When cultured under appropriate conditions, e.g., conditions disclosed herein, e.g., in a bioreactor and in an appropriate medium, the production cell produces, e.g., secretes, the product.

[0039] As used herein, "production factor" refers to a polypeptide or nucleic acid that affects the properties of a production cell with respect to the expression of a recombinant product. For example, a production factor can affect the amount (e.g., cellular The production factors may improve the specific productivity per cell or product titer) or product quality (e.g., correct folding and assembly, solubility, etc.). The production factors may be, for example, proteins involved in lipid metabolism (e.g., lipid metabolism modifiers such as SCD1 and / or SREBF-1), protein synthesis, protein folding, post-translational modification, protein trafficking, and / or protein secretion. They may also be polypeptides or nucleic acids that inhibit the expression or activity of endogenous proteins. For example, the production factors may inhibit the expression of a non-essential endogenous protein that is highly expressed and secreted to improve the production capacity of the cells.

[0040] As used herein, the term "promoter" refers to a sequence having sufficient sequence, e.g., from a naturally occurring or engineered promoter, such that operably linking a coding sequence to the promoter results in expression of the coding sequence. For example, a cytomegalovirus (CMV) promoter comprises all or an active fragment of a CMV promoter, e.g., all or an active fragment of a CMV promoter, optionally including intron A and / or UTR sequences. In embodiments, the CMV promoter differs from a naturally occurring or engineered variant CMV promoter by no more than 5, 10, 20, 30, 50, or 100 nucleotides. In embodiments, the CMV promoter differs from a naturally occurring or engineered variant CMV promoter by no more than 1, 5, 10, or 50% of its nucleotides. As used herein, a promoter may be constitutive, regulated, repressible, inducible, strong, weak, or other characteristic of the promoter sequence it comprises. In embodiments, a promoter may comprise sequences 5' or 3' of a coding sequence, e.g., a coding sequence for a recombinant, therapeutic, or repressor polypeptide. In embodiments, a promoter may comprise a sequence within one or more introns of a gene, e.g., a gene encoding a recombinant, therapeutic, or repressor polypeptide. In embodiments, a promoter may be contained in part or entirely within a sequence 5' or 3' of a coding sequence, e.g., the coding sequence of a recombinant, therapeutic, or repressor polypeptide. In embodiments, a promoter may be contained in part or entirely within a coding sequence, e.g., the coding sequence of a recombinant, therapeutic, or repressor polypeptide. In embodiments, a promoter may be contained in part or entirely within one or more introns of a gene, e.g., a gene encoding a recombinant, therapeutic, or repressor polypeptide.

[0041] As used herein, the term "operably linked" refers to the relationship between a nucleic acid sequence encoding a product (e.g., a polypeptide) or enzyme molecule and a control element; the product- or enzyme-molecule-encoding sequence and the control element are operably linked when they are positioned relative to the control element in a manner suitable for regulating expression of the product- or enzyme-molecule-encoding sequence. Thus, with respect to different control elements, operably linked refers to different placements of the product- or enzyme-molecule-encoding sequence relative to the control element. For example, a product- (e.g., polypeptide)-encoding sequence can be operably linked to a control element containing a promoter element if the promoter element and the product- (e.g., polypeptide)-encoding sequence are positioned proximal to each other and on the same nucleic acid. In another example, a product- (e.g., polypeptide)-encoding sequence can be operably linked to a control element containing a distally acting enhancer sequence if the enhancer sequence and the product- (e.g., polypeptide)-encoding sequence are positioned a suitable number of bases apart on the same nucleic acid, or even on different, separate nucleic acids.

[0042] As used herein, a selectable marker refers to one or more nucleic acid sequences that confer a phenotype that can be used to select cells that contain the one or more nucleic acid sequences. In some embodiments, the one or more nucleic acid sequences comprise a sequence that encodes a polypeptide (e.g., and suitable regulatory elements for expression of the polypeptide). For example, a selectable marker may comprise a gene encoding a protein that confers an antibiotic resistance phenotype. Such a selectable marker may be referred to as an antibiotic selectable marker. In some embodiments, a selectable marker is a low The selectable marker comprises one or more nucleic acid sequences that convey the ability to survive (e.g., grow and divide during) conditions that include reduced (e.g., absent) levels of an essential nutrient, e.g., a level insufficient for the cell to survive without the selectable marker. For example, the selectable marker may comprise a first nucleic acid encoding a PAH enzyme molecule and a second nucleic acid encoding a GCH1 enzyme molecule, where the selectable marker conveys the ability to survive reduced (e.g., absent) levels of tyrosine in the culture medium. Such a selectable marker may be referred to as an auxotrophic marker or an auxotrophic selectable marker. The nutrient for which the selectable marker conveys the ability to survive reduced or absent levels is identified by adding the name of a compound, e.g., an amino acid name, to the auxotrophic marker or auxotrophic selectable marker.

[0043] Vectors and Vector Systems The present invention uses vectors encoding components that enable transformed host cells to express a product of interest, such as a recombinant polypeptide, and to grow at low levels and in the absence of tyrosine, an amino acid essential to the cell, the absence of which would result in cell death and / or poor growth.

[0044] The vector comprises three components: a first nucleic acid sequence encoding a phenylalanine hydroxylase (PAH) enzyme molecule, typically lacking a functional N-terminal regulatory domain, and a second nucleic acid sequence encoding a GTP cyclohydrolase 1 (GCH1) enzyme molecule. These sequences are operably linked to a control sequence that allows expression of the enzyme in a suitable host cell. In one embodiment, the control sequence comprises a CMV promoter or an SV40 promoter; for example, a sequence encoding a human PAH sequence can be operably linked to a control sequence comprising an SV40 promoter, and / or a sequence encoding GCH1 can be operably linked to a control sequence comprising an SV40 promoter.

[0045] The third sequence contains an insertion site, e.g., a multiple cloning site, into which a nucleic acid sequence encoding a desired product can be cloned. This site is positioned and operably arranged to control the sequence so that the desired sequence can be expressed in a suitable host cell when introduced. In one embodiment, the three sequences that can be considered as expression cassettes are present in the same vector. In another embodiment, the first and second nucleic acid sequences can be on separate vectors, provided that the third nucleic acid sequence is on the same vector as one of them to ensure that the selection of the desired sequence is linked to the presence of a selectable marker.

[0046] The vector may contain additional expression cassettes for the desired product; i.e., the vector system may contain insertion sites, such as a fourth, and optionally a fifth, and optionally a sixth nucleic acid sequence, each containing a multiple cloning site, into which a nucleic acid sequence encoding the desired product may be cloned. With respect to the third nucleic acid sequence, these sites are positioned and operably arranged to control the sequence so that, when introduced, the desired sequence can be expressed in a suitable host cell. For example, a bispecific antibody has at least three different, usually at least four, different chains. These expression cassettes are ready for insertion of the desired sequence and can be configured in various ways. When the PAH and GCH1 sequences are on different vectors, each vector may contain one or more expression cassettes with multiple cloning sites, for example, two such expression cassettes. In some embodiments, the expression cassettes, each with multiple cloning sites, may be present in a single vector with only one of the selectable markers. Thus, one vector can have three or four expression cassettes, each with multiple cloning sites for introduction of sequences of interest, such as heavy or light chains for bispecific antibody production.

[0047] In one embodiment, and to take full advantage of the ability to introduce multiple sequences in the same step, all components of the vector system can be introduced into the host cell simultaneously.

[0048] In another embodiment, a suitable host cell may already be engineered to contain one of the first or second nucleic acid sequences. Accordingly, the present invention further provides a selection system, the selection system comprising: a) a first nucleic acid comprising a sequence encoding a phenylalanine hydroxylase (PAH) lacking a functional N-terminal regulatory domain, operably linked to a first control sequence that allows expression of the PAH in a host cell; b) a second nucleic acid encoding GTP cyclohydrolase 1 (GCH1) operably linked to a second control sequence that allows expression of GCH1 in the host cell; and c) (i) a multiple cloning site for inserting a sequence encoding a product of interest operably linked to a third control sequence that allows for expression of the product in the host cell, or (ii) a third nucleic acid encoding a product of interest operably linked to a third control sequence that allows for expression of the product in the host cell; d) a host cell, (a) and (c) are present in the vector and (b) is present in the host cell (typically integrated into the host cell genome), or (b) and (c) are present in the vector and (a) is present in the host cell (typically integrated into the host cell genome).

[0049] The nucleic acid sequences encoding the recombinant product and the PAH and GCH1 enzymes can be cloned into several types of vectors. For example, the nucleic acids can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Vectors of particular interest include expression vectors and replication vectors. In embodiments, the expression vector can be provided to cells in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, NY), as well as other virology and molecular biology manuals. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. Generally, suitable vectors contain functional replication origins in at least one organism (therefore, vectors can be self-replicating), control elements including promoter elements and optionally enhancer elements, convenient restriction endonuclease sites, and one or more selectable markers (for example, WO01 / 96584, WO01 / 29058, and U.S. Patent No. 6,326,193).Virus-derived vectors are suitable tools for achieving long-term gene transfer, because they allow the long-term stable integration of transgene and its propagation in daughter cells.

[0050] Vectors may also include, for example, signal sequences that facilitate secretion, polyadenylation signals, and transcription terminators (e.g., from the bovine growth hormone (BGH) gene), elements that allow episomal replication and replication in prokaryotes (e.g., SV40 origin and ColE1 or others known in the art), and / or elements that allow selection, such as a selectable marker or reporter gene.

[0051] Contemplated vectors may contain insertion sites suitable for inserting sequences encoding polypeptides, e.g., exogenous therapeutic polypeptides. The insertion sites may contain restriction endonuclease sites.

[0052] A sequence encoding the product of interest (described in the section below entitled Recombinant Products) can be introduced into the vector systems described herein using cloning techniques well known in the art. The resulting vector system then contains, in addition to the first and second nucleic acid sequences, at least a third nucleic acid sequence comprising a sequence encoding a product of interest operably linked to a third control sequence that allows for expression of the product in a host cell, where this third sequence is present in the same vector as the first and / or second nucleic acid sequence (ensuring that a selectable marker functions to select for cells containing the third nucleic acid sequence).

[0053] As described above, the vector system of the present invention can be used to express multiple sequences of interest, for example, for proteins with multiple subunits, including antibodies (standard and bispecific antibodies). Thus, the vector can include additional expression cassettes for the products of interest, and the multiple sequences of interest can be introduced into the multiple cloning site to produce a vector ready for introduction into a host cell capable of expressing the multiple products of interest. Thus, after introduction of the sequences of interest, in addition to a third nucleic acid sequence comprising a sequence encoding the product of interest operably linked to a third control sequence enabling expression of the product in the host cell, the vector system can also include a fourth, and optionally a fifth, and optionally a sixth, nucleic acid sequence, etc., each comprising a sequence encoding the product of interest operably linked to a control sequence enabling expression of the product in the host cell. These sequences are present in the same vector as the first and / or second nucleic acid sequence (to ensure their selection as a result of being associated with a selectable marker).

[0054] Again, as mentioned above, these expression cassettes can be configured in a variety of ways. When the PAH and GCH1 sequences are on different vectors, each vector can contain one or more expression cassettes encoding the desired product, for example, each can contain two such expression cassettes. In some embodiments, the expression cassettes can be present in a single vector with only one of the selectable markers. Thus, one vector can have three or four expression cassettes, each with a sequence encoding the desired product, such as a heavy or light chain for producing a bispecific antibody.

[0055] The vector may also contain sequences that facilitate integration into the host cell genome in either a random or site-specific manner, such as the PiggyBac™ system, which uses inverted terminal repeats (ITRs) located at both ends of the vector. Sequence-specific transposases, site-specific integration methods, and sequences involved in the transfection process are also described in WO2013 / 190032 and WO2018 / 150269.

[0056] In some embodiments, the vector containing the nucleic acid sequence encoding the product contains an additional selectable marker, such as glutamine synthetase, as described below. Typically, the vector system includes a separate vector containing the additional selectable marker, as described below, and a multiple cloning site for inserting one or more sequences encoding the product(s) of interest operably linked to regulatory sequences that allow for expression of the product in the host cell. When the sequences of interest are cloned into the multiple cloning site, the vector contains an additional selectable marker and a nucleic acid sequence containing a sequence encoding the product of interest operably linked to regulatory sequences that allow for expression of the product in the host cell, as described below. Such vectors generally do not contain PAH or GCH1 sequences.

[0057] The vector(s) may be provided in a kit, including instructions for use, and optionally transfection reagents and the like.

[0058] Also provided herein are nucleic acids, e.g., the subject nucleic acids, that encode the products described herein, e.g., recombinant polypeptides. Nucleic acid sequences encoding desired recombinant polypeptides can be obtained using recombinant methods known in the art, for example, by screening libraries from cells expressing the desired nucleic acid sequence, e.g., gene, by deriving the nucleic acid sequence from a vector known to contain it, or by isolating it directly from cells and tissues containing it using standard techniques. Alternatively, nucleic acids encoding recombinant polypeptides can be produced synthetically rather than cloned. Recombinant DNA techniques and technologies are highly advanced and well established in the art. Thus, one skilled in the art with knowledge of the amino acid sequences of the recombinant polypeptides described herein can easily envision or generate nucleic acid sequences encoding the recombinant polypeptides.

[0059] GCH1 enzyme molecule Naturally occurring GCH1 enzymes catalyze the conversion of GTP to 7,8-dihydroneopterin 3'-triphosphate (consuming two water molecules and producing acetate), which is the first step in the production of BH4. In some embodiments, the GCH1 enzyme molecule has the same or similar activity as the naturally occurring GCH1 enzyme. In some embodiments, the GCH1 enzyme molecule has increased or decreased activity relative to the naturally occurring GCH1 enzyme.

[0060] In some embodiments, the GCH1 enzyme molecule is a naturally occurring GCH1 enzyme. In some embodiments, the GCH1 enzyme molecule comprises a full-length (e.g., uncleaved) GCH1 enzyme. In some embodiments, the GCH1 molecule has at least 50% amino acid sequence identity (e.g., at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to a mammalian GCH1 enzyme.

[0061] In some embodiments, the GCH1 enzyme molecule is a variant of a naturally occurring GCH1 enzyme or a non-naturally occurring (e.g., synthetic) GCH1 enzyme (e.g., a variant containing one or more amino acid sequence modifications (e.g., substitutions, deletions, or insertions) relative to the amino acid sequence of the naturally occurring or non-naturally occurring enzyme). In some embodiments, the GCH1 enzyme molecule is or contains a deletion mutation, e.g., a truncation, e.g., a truncation of the N-terminal region, relative to a naturally occurring GCH1 enzyme. In some embodiments, the GCH1 enzyme molecule is or contains at least 75, 80, 85, 90, 95, or 99% of the amino acid sequence (and optionally up to 100, 99, 95, 90, 85, 80, 79, 78, 77, 76, or 75% of the amino acid sequence) of a naturally occurring GCH1 enzyme. In some embodiments, the GCH1 enzyme molecule comprises no more than 99, 95, 90, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, or 75% of the amino acid sequence of a naturally occurring GCH1 enzyme.

[0062] In some embodiments, the GCH1 enzyme molecule is a monomer, e.g., an active enzyme as a monomer. In some embodiments, the GCH1 enzyme molecule forms a multimer (e.g., under suitable conditions for enzymatic activity, e.g., cellular or physiological conditions, e.g., during a biomanufacturing process), e.g., an active enzyme as a multimer. In some embodiments, the GCH1 enzyme molecule multimer is a dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, or decamer, e.g., a decamer.

[0063] The sequences for use in the GCH1 enzyme molecules of the present disclosure can be derived from any known GCH1 enzyme sequence. In some embodiments, the GCH1 enzyme molecule comprises a human GCH1 enzyme, a variant thereof, or an enzymatically active fragment thereof. In some embodiments, the GCH1 enzyme molecule comprises a CHO GCH1 enzyme, a variant thereof, or an enzymatically active fragment thereof.

[0064] In some embodiments, the GCH1 enzyme molecule comprises an amino acid sequence encoded by SEQ ID NO: 1, e.g., the amino acid sequence of SEQ ID NO: 2. In some embodiments, the GCH1 enzyme molecule comprises an amino acid sequence encoded by NCBI reference sequence: NM_001024024 (e.g., as of October 6, 2019). In some embodiments, the GCH1 enzyme molecule comprises an amino acid sequence that is at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to the amino acid sequence encoded by SEQ ID NO: 1, e.g., the amino acid sequence of SEQ ID NO: 2. In some embodiments, the exogenous nucleic acid encoding the GCH1 enzyme molecule comprises a nucleic acid sequence that is at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to the nucleic acid sequence of SEQ ID NO:1. NCBI reference sequence: NM_001024024 (query number 1) MEKGPVRAPAEKPRGARCSNGFPERDPPRPGPSRPAEKPPRPEAKSAQPADGWKGERPRSEEDNELNLPNLAAAYSSILSSLGENPQRQGLLKTPWRAASAMQFFTKGYQETISDVLNDAIFDEDHDEMVIVKDIDMFSMCEHHLVPFVGKVHIGYLPNKQVLGLSKLARIVEIYSRRLQVQERLTKQIAVAITEALRPAGVGVVVEATHMCMVMRGVQKMNSKTVTSTMLGVFREDPKTREEFLTLIRS (SEQ ID NO: 2)

[0065] PAH enzyme molecule Naturally occurring PAH enzymes catalyze the conversion of phenylalanine to tyrosine using molecular oxygen and tetrahydrobiopterin (BH4). In some embodiments, the PAH enzyme molecule has the same or similar activity as the naturally occurring PAH enzyme. In some embodiments, the PAH enzyme molecule has increased or decreased activity relative to the naturally occurring PAH enzyme.

[0066] In some embodiments, the PAH enzyme molecule is a naturally occurring PAH enzyme. In some embodiments, the PAH enzyme molecule comprises a full-length (e.g., uncleaved) PAH enzyme.

[0067] In some embodiments, the PAH enzyme molecule is a variant of a naturally occurring PAH enzyme or a non-naturally occurring (e.g., synthetic) PAH enzyme (e.g., a variant containing one or more amino acid sequence modifications (e.g., substitutions, deletions, or insertions) relative to the amino acid sequence of the naturally occurring or non-naturally occurring enzyme). In some embodiments, the PAH enzyme molecule is or contains a deletion mutation, e.g., a truncation, e.g., a truncation of the N-terminal region, relative to a naturally occurring PAH enzyme. In some embodiments, the PAH enzyme molecule is or contains at least 75%, 80%, 85%, 90%, 95%, or 99% of the amino acid sequence (and optionally up to 100%, 99%, 95%, 90%, 85%, 80%, 79%, 78%, 77%, 76%, or 75% of the amino acid sequence) of a naturally occurring PAH enzyme. In some embodiments, the PAH enzyme molecule comprises no more than 99, 95, 90, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, or 75% of the amino acid sequence of a naturally occurring PAH enzyme. In some embodiments, the PAH enzyme molecule is or comprises at least 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 335, or 336 amino acids (and optionally no more than 450, 400, 390, 380, 370, 360, 350, 340, or 336 amino acids) of a naturally occurring PAH enzyme molecule. In some embodiments, a PAH enzyme molecule is or comprises no more than 450, 400, 390, 380, 370, 360, 350, 340, or 336 amino acids (and optionally at least 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 335, or 336 amino acids) of a naturally occurring PAH enzyme molecule. For example, a PAH enzyme molecule can include the first amino acids 1-14 and 37 onward, including a deletion of amino acids 15-37. As a further example, a PAH enzyme molecule can include a deletion of amino acids 1-116. As a further example, a PAH enzyme molecule can include a deletion of amino acids 1-10 and 30-40.As a further example, a PAH enzyme molecule can include 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, or 350 C-terminal amino acids, e.g., 343 C-terminal amino acids, of a naturally occurring PAH enzyme.

[0068] In preferred embodiments, the PAH enzyme molecule lacks part or all of the regulatory domain of a naturally occurring PAH enzyme, e.g., such that the PAH enzyme molecule is constitutively active relative to the naturally occurring PAH enzyme. Without wishing to be bound by theory, it is understood that the PAH enzyme comprises an N-terminal region containing one or more regulatory domains that regulate the enzymatic activity of the PAH, e.g., by regulating access to the enzyme active site. The regulatory domain may include an ACT domain, which is known to enable allosteric regulation of metabolic enzymes, and / or an active site lid, which can conditionally block access to the enzyme active site. We believe that PAH enzyme molecules lacking part or all of the regulatory domain are useful in production cells, e.g., in the selection markers described herein, because such PAH enzyme molecules may be more active (e.g., constitutively active) than full-length PAH enzymes, e.g., PAH enzyme molecules that are subject to allosteric regulation of the regulatory domain. In some embodiments, the PAH enzyme molecule lacks the active site lid. In some embodiments, the PAH enzyme molecule lacks the ACT domain. In some embodiments, the PAH enzyme molecule comprises a modification (e.g., substitution, deletion, or insertion) that eliminates the regulatory (e.g., inhibitory) function of the N-terminal regulatory region (e.g., the active site lid and / or the ACT domain). In some embodiments, the PAH enzyme molecule is not significantly inhibited (e.g., not inhibited) by the presence of phenylalanine. ... The PAH enzyme molecule may comprise a deletion of 10, or 1-116 (e.g., 1-116), or a deletion of residues corresponding to amino acids 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, 1-100, 1-110, or 1-116 (e.g., 1-116). In some embodiments, the PAH enzyme molecule lacks the N-terminal 116 amino acids of a naturally occurring PAH enzyme (e.g., a naturally occurring human PAH enzyme), or the corresponding amino acids of a different naturally occurring PAH enzyme. See Daubner et al., 1997, Arch. Biochem. Biophys 348(2):295 (describing a truncated PAH lacking the regulatory domain (first 116 amino acids)). This truncated PAH expressed in E. coli was more stable, more soluble, did not require pre-incubation with phenylalanine to be active, and had a higher affinity for the substrate.) In some embodiments, the PAH enzyme molecule comprises the C-terminal region of a naturally occurring PAH enzyme, e.g., the catalytic and multimerization portions of the PAH enzyme.

[0069] In some embodiments, the PAH enzyme molecule is a monomer, e.g., an active enzyme as a monomer. In some embodiments, the PAH enzyme molecule forms a multimer (e.g., under appropriate conditions for enzymatic activity, e.g., cellular or physiological conditions, e.g., during a biomanufacturing process), e.g., an active enzyme as a multimer. In some embodiments, the PAH enzyme molecule multimer is a dimer, trimer, tetramer, pentamer, hexamer, heptamer, or octamer, e.g., a tetramer.

[0070] Sequences for use in the PAH enzyme molecules of the present disclosure can be derived from any known PAH enzyme sequence. In some embodiments, the PAH enzyme molecule comprises a human PAH enzyme, a variant thereof, or an enzymatically active fragment thereof. In some embodiments, the PAH molecule has at least 50% amino acid sequence identity (e.g., at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to a human PAH enzyme. In some embodiments, the PAH enzyme molecule comprises a CHO PAH enzyme, a variant thereof, or an enzymatically active fragment thereof. In some cases, the PAH molecule has at least 50% amino acid sequence identity (e.g., at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to a CHO PAH enzyme.

[0071] In some embodiments, the PAH enzyme molecule comprises an amino acid sequence encoded by either of SEQ ID NOs: 3 or 4, e.g., the amino acid sequence of either of SEQ ID NOs: 5 or 6. In some embodiments, the PAH enzyme molecule comprises an amino acid sequence that is at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to the amino acid sequence encoded by either of SEQ ID NOs: 3 or 4, e.g., the amino acid sequence of either of SEQ ID NOs: 5 or 6. In some embodiments, the exogenous nucleic acid encoding the PAH enzyme molecule comprises a nucleic acid sequence that is at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to the nucleic acid sequence of either of SEQ ID NOs: 3 or 4. Exemplary CHO PAH nucleic acid sequence (NCBI Reference Sequence: XM_027434726.1) ATGGTGCCCTGGTTCCCAAGGACCATTCAAGAGCTGGACAGATTTGCCAATCAGATTCTCAGTTATGGAGCAGAACTGGATGCAGACCACCCGGGCTTTAAAGATCCTGTGTACCGGGCGAGGCGAAAGCAGTTTGCTGACATTGCCTACAACTACCGCCATGGGCAGCCCATCCCTCGGGTGGAATACACAGAAGAAGA GAAGAAGACCTGGGGAACAGTGTTCAAGACACTGAAGGCCTTGTATAAAACGCATGCCTGCTATGAACACAACCACATTTTCCCACTTCTGGAAAAGTACTGCGGGTTCCGTGAAGACAACATTCCCCAGCTGGAAGATGTTTCTCAGTTTCTGCAGACTTGTACTGGTTTCCGCCTCCGACCTGTTGCTGGCTTACTGTCCTCTCGAGATTTCTTGGGTGGCCTGGCCTTCCGAGTCTTCCACTGCACACAATACATCAGGCATGGGTCTAAGCCCATGTACACACCTGAACCAGACATTTGTCATGAACTGTTGGGACATGTGCCCTTGTTTTCAGATCGCAGCTTTGCCCAGTTTTCCCAGGAAATCGGACTTGCTTCTCTGGGTGCACCTGACGAATACATCGAGAAATTGGCCACAATTTACTGGTTTACTGTGGAGTTTGGGCTCTGCAAGGAAGGAGATTCCATCAAGGCATATGGTGCTGGGCTTCTGTCATCCTTTGGTGAATTACAGTACTGTTTATCAGACAAGCCGAAGCTCCTGCCCCTGGACCTAGAGAAGACAGCCTCACAGGAGTACAATGTCACAGAGTTCCAGCCCCTGTACTACGTGGCAGAGAGTTTCAATGATGCCAAGGAGAAAGTGAGGGCCTTTGCTGCCACAATCCCCCGGCCCTTCTCGGTTCGCTATGATCCCTACACTCAAAGGGTTGAGGTCCTGGACAACACTCAGCAGTTGAAGATTTTGGCTGACTCCATCAACAGTGAGGTTGGAATCCTTTGCAGTGCCCTGCATAAAATAAAGTCATGA (SEQ ID NO: 3) Exemplary CHO PAH amino acid sequence MVPWFPRTIQELDRFANQILSYGAELDADHPGFKDPVYRARRKQFADIAYNYRHGQPIPRVEYTEEEKKTWGTVFKTLKALYKTHACYEHNHIFPLLEKYCGFREDNIPQLEDVSQFLQTCTGFRLRPVAGLLSSRDFLGGLAFRVFHCTQYIRHGSKPMYTPEPDICHELLGHVPLFSDRSFAQFSQEIGLASLGAPDEYIEKLATIYWFTVEFGLCKEGDSIKAYGAGLLSSFGELQYCLSDKPKLLPLDLEKTASQEYNVTEFQPLYYVAESFNDAKEKVRAFAATIPRPFSVRYDPYTQRVEVLDNTQQLKILADSINSEVGILCSALHKIKS (SEQ ID NO: 5) Exemplary Human PAH Nucleic Acid Sequences (GenBank: K03020.1) GCTAGCATGGTGCCCTGGTTCCCAAGAACCATTCAAGAGCTGGACAGATTTGCCAATCAGATTCTCAGCTATGGAGCGGAACTGGATGCTGACCACCCTGGTTTTAAAGATCCTGTGTACCGTGCAAGACGGAAGCAGTTTGCTGACATTGCCTACAACTACCGCCATGGGCAGCCCATCCCTCGAGTGGAATACATGGAGGAAGAAAAGAAAACATGGGGCACAGTGTTCAAGACTCTGAAGTCCTTGTATAAAACCCATGCTTGCTATGAGTACAATCACATTTTTCCACTTCTTGAAAAGTACTGTGGCTTCCATGAAGATAACATTCCCCAGCTGGAAGACGTTTCTCAATTCCTGCAGACTTGCACTGGTTTCCGCCTCCGACCTGTGGCTGGCCTGCTTTCCTCTCGGGATTTCTTGGGTGGCCTGGCCTTCCGAGTCTTCCACTGCACACAGTACATCAGACATGGATCCAAGCCCATGTATACCCCCGAACCTGACATCTGCCATGAGCTGTTGGGACATGTGCCCTTGTTTTCAGATCGCAGCTTTGCCCAGTTTTCCCAGGAAATTGGCCTTGCCTCTCTGGGTGCACCTGATGAATACATTGAAAAGCTCGCCACAATTTACTGGTTTACTGTGGAGTTTGGGCTCTGCAAACAAGGAGACTCCATAAAGGCATATGGTGCTGGGCTCCTGTCATCCTTTGGTGAATTA CAGTACTGCTTATCAGAGAAGCCAAAGCTTCTCCCCCTGGAGCTGGAGAAGACAGCCATCCAAAATTACACTGTCACGGAGTTCCAGCCCCTGTATTACGTGGCAGAGAGTTTTAATGATGCCAAGGAGAAAGTAAGGAACTTTGCTGCCACAAT ACCTCGGCCCTTCTCAGTTCGCTACGACCCATACACCCAAAGGATTGAGGTCTTGGACAATACCCAGCAGCTTAAGATTTTGGCTGATTCCATTAACAGTGAAATTGGAATCCTTTGCAGTGCCCTCCAGAAAATAAAGTAAAGATCT (SEQ ID NO: 4) Exemplary Human PAH Amino Acid Sequences MVPWFPRTIQELDRFANQILSYGAELDADHPGFKDPVYRARRKQFADIAYNYRHGQPIPRVEYMEEEKKTWGTVFKTLKSLYKTHACYEYNHIFPLLEKYCGFHEDNIPQLEDVSQFLQTCTGFRLRPVAGLLSSRDFLGGLAFRVFHCTQYIRHGSKPMYTPEPDICHELLGHVPLFSDRSFAQFSQEIGLASLGAPDEYIEKLATIYWFTVEFGLCKQGDSIKAYGAGLLSSFGELQYCLSEKPKLLPLELEKTAIQNYTVTEFQPLYYVAESFNDAKEKVRNFAATIPRPFSVRYDPYTQRIEVLDNTQQLKILADSINSEIGILCSALQKIK (SEQ ID NO: 6)

[0072] host cell The present disclosure is directed, in part, to a host cell comprising a tyrosine auxotrophic selectable marker, e.g., a first nucleic acid encoding a phenylalanine hydroxylase (PAH) enzyme molecule, and a second nucleic acid encoding a GTP cyclohydrolase 1 (GCH1) enzyme molecule. At least one of these sequences is exogenous to the host cell, i.e., not naturally occurring. Both sequences may be exogenous to the host cell.

[0073] As described in the above section on vectors, the nucleic acid sequences can be present in the same or different vectors, and therefore they can be present in the host cell in the same or different nucleic acid molecules / vectors. These vectors can be self-replicating vectors, especially when maintained extrachromosomally. In some embodiments, the first and / or second nucleic acid is integrated into the genome of the producer cell.

[0074] After the introduction of the vector system, the host cell typically also contains a third exogenous nucleic acid sequence encoding the desired product, and these cells are also referred to herein as "production cells." The product typically does not naturally occur in the unmodified host cell, e.g., a biotherapeutic protein. The third nucleic acid sequence is present in the same nucleic acid as the first nucleic acid sequence and / or the second nucleic acid sequence, depending on the number of vectors used to produce the cell. In some embodiments, the third exogenous nucleic acid is integrated into the genome of the host cell. Additional exogenous nucleic acids introduced using the vector system of the present invention may also be present.

[0075] The first, second, and / or third exogenous nucleic acids can contain one or more regulatory elements. Regulatory elements, e.g., promoters and / or enhancers, can be operably linked to the PAH enzyme molecule-encoding sequence, the GCH1 molecule-encoding sequence, or the product-encoding sequence. In some embodiments, the first and second exogenous nucleic acids contain one or more regulatory elements sufficient to express the PAH enzyme molecule and the GCH1 enzyme molecule in the production cell. In some embodiments, the third exogenous nucleic acid contains one or more regulatory elements sufficient to express the product, e.g., a polypeptide product, in the production cell. Regulatory elements suitable for use in the present invention are known to those of skill in the art, and examples are also described herein.

[0076] host cell type In one aspect, the host cell of the present disclosure can be, be made from, or be derived from any cell type, strain, or cell line described herein. Generally, the methods herein can be used to produce host cells, e.g., cells or cell lines, that contain a nucleic acid construct (e.g., a genomically integrated vector or heterologous nucleic acid) that includes (i) a subject nucleic acid sequence encoding a product of interest, and (ii) one or more exogenous nucleic acid sequences encoding one or more enzyme molecules involved in an amino acid biosynthetic pathway, where the cell or cell line does not endogenously express the enzyme molecule.

[0077] Host cells can be any suitable cell that can be genetically engineered and grown. Typically, the cells are suitable for large-scale culture to produce the product of interest.

[0078] Before the introduction of the vector system of the present invention, the host cell cannot produce a sufficient level of tyrosine to support cell growth in the absence of tyrosine. This may be because the host cell does not express one or more of the enzymes required for tyrosine biosynthesis to a sufficient level or is engineered to knock out the relevant gene. Thus, in one embodiment, the host cell of the present invention is genetically modified to inhibit or eliminate any endogenous PAH and / or GCH1 activity. This can be achieved, for example, by mutation (insertion, deletion, and / or substitution) in the genomic sequence that encodes and / or regulates the expression of endogenous PAH and / or GCH1.

[0079] In some embodiments, the host cell is a eukaryotic cell, such as a mammalian, yeast, or insect cell.

[0080] In one embodiment, the host cell is a mammalian cell. Examples of species from which the host cell may be derived include human, mouse, rat, Chinese hamster, Syrian hamster, monkey, ape, dog, horse, ferret, and cat.

[0081] In an embodiment, the host cell is a Chinese hamster ovary (CHO) cell. In one embodiment, the host cell is a CHO-K1 cell, a CHOK1SV® cell, a DG44 CHO cell, a DUXB11 CHO cell, a CHO-S, a CHO GS knockout cell (a CHO cell in which all endogenous copies of the glutathione synthetase (GS) gene have been inactivated), a CHOK1SV® FUT 8 knockout cell, a CHOZN, or a CHO-derived cell. A CHO GS knockout cell (e.g., a GS-KO cell) is, for example, a CHOK1SV® GS knockout cell (GS Xceed® cell - CHOK1SV GS-KO®, Lonza Biologics, Inc.). A CHO FUT 8 knockout cell is, for example, a Potelligent® CHOK1SV® FUT 8 knockout (Lonza Biologics, Inc.). Biologics, Inc.

[0082] In embodiments, the host cell is a HeLa, MDCK, Sf9, Sf21, Tn5, HT1080, NB324K, FLYRD18, HEK293, HEK293T, HT1080, H9, HepG2, MCF7, Jurkat, NIH3T3, PC12, PER.C6, BHK (baby hamster kidney), VERO, SP2 / 0, NS0, YB2 / 0, YO, EB66, C127, L cell, COS (e.g., COS1 and COS7), QC1-3, CHOK1, CHOK1SV, Potelligent® (CHOK1SV FUT8-KO), CHO GS knockout, GS Xceed™ (CHOK1SV GS-KO), CHOS, CHO DG44, CHO DXB11, or CHOZN cell, or any cell derived therefrom.

[0083] In other embodiments, the host cell is an avian, fish, insect, plant, fungal, or yeast cell, etc. are cells other than mammalian cells.

[0084] In some embodiments, the host cell or cell line of the host cell is formed by a process involving the fusion of multiple cells (e.g., the fusion of two cells of the same type (e.g., two CHO cells) or two cells of different types (e.g., different species)). Examples of host cells or cell lines formed by a process involving the fusion of multiple cells include, but are not limited to, hybridomas, triomas, and quadromas.

[0085] In some embodiments, derivatives thereof include, but are not limited to, cells described herein that further comprise modifications such as mutations (e.g., substitutions, deletions, or insertions) or additions of nucleic acids (e.g., vectors) (e.g., gene knock-ins, gene knock-outs, or gene multiplicities). In some embodiments, derivatives thereof include cells described herein that have undergone directed evolution. In some embodiments, derivatives thereof include combinations of these exemplary modifications described herein.

[0086] Eukaryotic cells include stem cells, which may be, for example, pluripotent stem cells, including embryonic stem cells (ESCs), adult stem cells, induced pluripotent stem cells (iPSCs), tissue-specific stem cells (e.g., hematopoietic stem cells), and mesenchymal stem cells (MSCs).

[0087] In embodiments, the host cell is a differentiated form of any of the cells described herein. In one embodiment, the host cell is a cell derived from any primary cell in culture.

[0088] In embodiments, the host cell is a hepatocyte, such as a human hepatocyte, an animal hepatocyte, or a non-parenchymal cell.For example, the host cell can be a plateable metabolically limited human hepatocyte, a plateable induction-limited human hepatocyte, a plateable Qualyst Transporter Certified™ human hepatocyte, a suspension-limited human hepatocyte (including 10 donor and 20 donor pooled hepatocytes), a human liver Kupffer cell, a human hepatic stellate cell, a dog hepatocyte (including single and pooled Beagle hepatocytes), a mouse hepatocyte (including CD-1 and C57BI / 6 hepatocytes), a rat hepatocyte (including Sprague-Dawley, Wistar Han, and Wistar hepatocytes), a monkey hepatocyte (including Cynomolgus or Rhesus hepatocytes), a cat hepatocyte (including Domestic Shorthair hepatocytes), and a rabbit hepatocyte (including New Zealand White hepatocytes).Exemplary hepatocytes are commercially available from Triangle Research Labs, LLC, 6 Davis Drive, Research Triangle Park, North Carolina, USA 27709.

[0089] In some embodiments, the host cell comprises a knockout of glutamine synthetase (GS). In embodiments, the host cell does not comprise a functional GS gene. In embodiments, the host cell does not comprise a GS gene. In embodiments, the GS gene in the host cell comprises a mutation that renders the gene unable to encode a functional GS protein.

[0090] In embodiments, the eukaryotic cell is, for example, a yeast cell (e.g., a cell of the Pichia genus (e.g., Pichia pastoris, Pichia methanolica, Pichia kluyveri, and Pichia angusta), the genus Komagataella (e.g., Komagataella pastoris, Komagataella pseudopastoris, or Komagataella phaffii), the genus Saccharomyces (e.g., Saccharomyces cerevisae, cerevisiae, Saccharomyces kluyveri, Saccharomyces uvarum), the genus Kluyveromyces (e.g., Kluyveromyces lactis, Kluyveromyces marxi anus), Candida (e.g., Candida utilis, Candida cacaoi, Candida boidinii), Geotrichum (e.g., Geotrichum fermentans), Hansenula polymorpha, Yarrowia lipolytica, or Schizosaccharomyces pombe. In some embodiments, the eukaryotic cell is of the species Pichia pastoris. Examples of Pichia pastoris strains include, but are not limited to, X33, GS115, KM71, KM71H, and CBS7435.

[0091] In embodiments, the eukaryotic cell is a fungal cell (e.g., Aspergillus sp. (e.g., A. niger, A. fumigatus, A. orzyae, A. nidula), Acremonium sp. (e.g., A. thermophilum), Chaetomium sp. (e.g., C. thermophilum), Chrysosporium sp. (e.g., C. thermophile), Cordyceps sp. (e.g., C. militaris), Corynascus sp., Ctenomyces sp., Fusarium sp. (e.g., F. oxysporum), Glomerella sp. (e.g., G. graminicola), Hypocrea sp. (e.g., H. jecorina), Magnaporthe sp. (e.g., M. orzyae), Myceliophthora sp. (e.g., M. thermophile), Nectria sp. (e.g., N. heamatococca), Neurospora sp. (e.g., N. crassa), Penicillium sp., Sporotrichum sp. (e.g., S. thermophile), Thielavia sp. (e.g., T. terrestris, T. heterothallica), Trichoderma sp. (e.g., T. reesei), or Verticillium sp. (e.g., V. dahlia)).

[0092] In embodiments, the eukaryotic cell is an insect cell (e.g., an Sf9, Mimic™ Sf9, Sf21, High Five™ (BT1-TN-5B1-4), or BT1-Ea88 cell), an algal cell (e.g., of the genus Amphora sp., Bacillariophyceae sp., Dunaliella sp., Chlorella sp., Chlamydomonas sp., Cyanophyta sp. (cyanobacteria), Nannochloropsis sp., Spirulina sp., or Ochromonas sp.), or a plant cell (e.g., a cell from a monocotyledonous plant (e.g., corn, rice, wheat, or Setaria sp.) or a dicotyledonous plant (e.g., cassava, potato, soybean, tomato, tobacco, alfalfa, Physcomitrella patens, or Arabidopsis sp.)).

[0093] In embodiments, the host cell is a prokaryotic cell, such as a bacterial cell.

[0094] In embodiments, the prokaryotic cell is a Gram-positive cell, such as a Bacillus sp., Streptomyces sp., Streptococcus sp., Staphylococcus sp., or Lactobacillus sp. Bacillus sp. that can be used is, for example, B. subtilis, B. amyloliquefaciens, B. licheniformis, B. natto, or B. megaterium. In embodiments, the cell is B. subtilis, for example, B. subtilis 3NA and B. subtilis 168. Bacillus sp. can be obtained, for example, from Bacillus Genetic Stock. Center,Biological Sciences 556,484 West 12 th Available from Avenue, Columbus OH 43210-1214 It is Noh.

[0095] In embodiments, the prokaryotic cell is a Gram-negative cell, such as a Salmonella sp. or Escherichia coli, such as TG1, TG2, W3110, DH1, DHB4, DH5a, HMS174, HMS174(DE3), NM533, C600, HB101, JM109, MC4100, XL1-Blue, and Origami, and those derived from E. coli B strains, such as BL-21 or BL21(DE3), or BL21(DE3)pLysS, all of which are commercially available.

[0096] In some embodiments, the prokaryotic cell is a cyanobacteria cell. In some embodiments, the cyanobacteria cell is a blue-green alga, such as a Synechocystis cell.

[0097] Suitable host cells are, for example, those described in the DSMZ (Deutsche Sammlung von They are commercially available from culture collections such as Mikroorganismen and Zellkulturen GmbH, Braunschweig, Germany or the American Type Culture Collection (ATCC).

[0098] Additional selection markers In some embodiments, the host cell comprises one or more selectable markers in addition to the tyrosine auxotrophic selectable marker. In some embodiments, the second selectable marker is a different auxotrophic selectable marker, such as a different amino acid auxotrophic selectable marker. In one embodiment, the amino acid is proline or glutamine. Examples of nucleic acid sequences required for such selectable markers are sequences encoding glutamine synthetase (for glutamine) and pyrroline-5-carboxylate synthase (P5CS) (for proline).

[0099] Another selectable marker is an exogenous nucleic acid encoding a dihydrofolate reductase (DHFR), e.g., a DHFR enzyme molecule that confers resistance to methotrexate (MTX). In some embodiments, the DHFR selectable marker is also a thymidine auxotrophic selectable marker and / or a hypoxanthine auxotrophic selectable marker. In some embodiments, the host cell does not contain an endogenous functional DHFR gene, e.g., does not contain a mutation that renders the endogenous DHFR gene unable to encode a functional DHFR enzyme.

[0100] Additional selectable markers include hypoxanthine-guanine phosphoribosyltransferase (HPRT) selectable markers, e.g., exogenous nucleic acids encoding an HPRT enzyme molecule. In some embodiments, the producer cells are unable to grow and / or divide in the presence of aminopterin without HPRT (e.g., supplemental HPRT encoded by an exogenous nucleic acid) and supplemental purine, e.g., hypoxanthine. In some embodiments, the HPRT selectable marker is also a purine (e.g., hypoxanthine or guanine) auxotrophic selectable marker. In some embodiments, the producer cells do not contain an endogenous functional HPRT gene, e.g., do not contain a mutation that renders the endogenous HPRT gene incapable of encoding a functional HPRT enzyme.

[0101] In one embodiment, the selectable marker is compatible with the Selexis selection system (eg, SUREtechnology Platform™ and Selexis Genetic Elements™, commercially available from Selexis SA) or the Catalent GPEx® selection system.

[0102] A selectable marker for use in a production cell can be associated with a subject nucleic acid. As used herein with respect to the relationship between a selectable marker and a subject nucleic acid, associated refers to a relationship in which the presence of the selectable marker in a production cell correlates with the presence of the subject nucleic acid. The selectable marker is associated with a subject nucleic acid such that selecting for (e.g., requiring) the presence of the selectable marker in a production cell selects for the presence of the subject nucleic acid. In some embodiments, the selectable marker, e.g., at least one component of the selectable marker, is located on the same nucleic acid molecule as the subject nucleic acid, e.g., on the same vector as the subject nucleic acid. For example, a production cell containing a tyrosine auxotrophic selectable marker comprising a first exogenous nucleic acid encoding a PAH enzyme molecule and a second exogenous nucleic acid encoding a GCH1 enzyme molecule can contain the subject nucleic acid located on the same vector as either the first exogenous nucleic acid or the second exogenous nucleic acid. In a production cell containing two or more selectable markers, each selectable marker can be associated with a different subject nucleic acid. In some embodiments, the production cells comprise a first selectable marker associated with a first subject nucleic acid (e.g., encoding a product) and a second selectable marker associated with a second subject nucleic acid (e.g., encoding a production factor, e.g., a lipid metabolism regulator (LMM) such as SCD1 and / or SREBF-1, as described in WO2017 / 191165 and WO2019 / 152876, which are incorporated by reference herein). Accordingly, the additional selectable marker is used to maintain the exogenous production factor introduced into the host cell (including as previously described for producing stable cell lines). In some embodiments, the production cells comprise a first selectable marker associated with a first subject nucleic acid (e.g., encoding a first product) and a second selectable marker associated with a second subject nucleic acid (e.g., encoding a second product). In some embodiments, the production cell comprises a first selectable marker associated with a first subject nucleic acid (e.g., encoding a first product of a multi-polypeptide product) and a second selectable marker associated with a second subject nucleic acid (e.g., encoding a second product of a multi-polypeptide product).It is understood that additional subject nucleic acids may be included that may be associated with different markers or the same marker.

[0103] inhibitors Host cells and / or cultures comprising host cells may contain one or more enzyme molecule inhibitors (also referred to herein as inhibitors). Enzyme molecule inhibitors can be used to increase the stringency of the selection processes described herein, for example, by reducing or preventing endogenous enzyme molecule activity, such that cells that do not incorporate an exogenous nucleic acid encoding the enzyme molecule (e.g., and including a subject nucleic acid sequence) exhibit reduced or undetectable levels of endogenous enzyme molecule activity. Cells that exhibit reduced or undetectable levels of endogenous enzyme molecule activity may be unable to grow and / or survive in the absence of an external supply of an amino acid (e.g., proline, tyrosine, or glutamine) whose synthesis requires the activity of the enzyme molecule. In some embodiments, the inhibitor binds to the enzyme molecule, e.g., binds to and inhibits the enzyme molecule. In embodiments, the inhibitor is an allosteric inhibitor of the enzyme molecule. In embodiments, the inhibitor is a competitive inhibitor of the enzyme molecule.

[0104] The production cells described herein, in some embodiments, can further comprise an inhibitor of an enzyme molecule (e.g., PAH or GCH1) expressed by an exogenous nucleic acid introduced into the cells. In some embodiments, the level of the inhibitor in the cells is sufficient to reduce the activity of the endogenous enzyme molecule to less than about 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, or 10% of the activity observed in cells lacking the inhibitor. In some embodiments, less than about 0.001%, 0.01%, 0.1%, 1%, 5%, or 10% of the cells selected based on growth in medium lacking amino acids do not contain a subject nucleic acid. In some embodiments, the ratio of enzyme molecules and inhibitor molecules in the cells is about: 1:1000, 1:500, 1:250, 1:200, 1:100, 1:90, 1:80, 1:70, 1:60, 1:50, 1:40, 1:30, 1:20, 1:10, 1:9, 1:8, 1: 7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, 200:1, 250:1, 500:1, or 1000:1.

[0105] The inhibitor can be, for example, an amino acid or analog thereof, a polypeptide, a nucleic acid, or a small molecule. In some embodiments, the inhibitor is an analog of an amino acid produced by a biosynthetic pathway involving an enzyme molecule. In some embodiments, the inhibitor is an analog of a substrate of the enzyme molecule. In some embodiments, the inhibitor is an antibody molecule (e.g., an antibody or antibody fragment described herein), a fusion protein, a hormone, a cytokine, a growth factor, an enzyme, a glycoprotein, a lipoprotein, a reporter protein, a therapeutic peptide, an aptamer, or a structural and / or functional fragment or hybrid of any of these. In some embodiments, the inhibitor is an antisense RNA, siRNA, tRNA, ribosomal RNA, microRNA, piRNA, snoRNA, snRNA, exRNA, scaRNA, RNA aptamer, or long non-coding RNA.

[0106] In some embodiments, the inhibitor inhibits an enzyme molecule in the biosynthetic pathway of proline, tyrosine, or glutamine. In one embodiment, the inhibitor inhibits the activity of PAH (e.g., a phenylalanine analog) or GCH1. In an embodiment, the inhibitor is a tetrahydrobiopterin (BH4) analog. In some embodiments, the inhibitor is a GTP analog. In one embodiment, the inhibitor is selected from α-methyltyrosine (e.g., at 50-100 μM), α-methylphenylalanine, and 2,4-amino-6-hydroxypyrimidine.

[0107] In embodiments, the inhibitor, when used, inhibits the activity of an enzyme that forms the basis of one of the additional selectable markers, such as a pyrroline-5-carboxylate synthase (P5CS) molecule. In embodiments, the inhibitor inhibits the activity of P5CS. In embodiments, the inhibitor is a proline analog. In embodiments, the inhibitor is L-azetidine-2-carboxylic acid, 3,4-dehydro-L-proline, or L-4-thiazolidine carboxylic acid. In some embodiments, the inhibitor inhibits the activity of DHFR, e.g., methotrexate. In some embodiments, the inhibitor inhibits glutamine synthetase (e.g., a glutamine analog, methionine sulfoximine (MSX) or an analog thereof (e.g., alpha-methyl or alpha-ethyl MSX)). In some embodiments, the production cell includes more than one selectable marker and includes an enzyme molecule inhibitor for each selectable marker.

[0108] Introduction of nucleic acid into host cells and selection step Many suitable methods for introducing exogenous nucleic acids into host cells are known in the art, including, for example, transfection, transduction (e.g., viral transduction), or electroporation of nucleic acids, such as vectors, into cells. Examples of physical methods for introducing nucleic acids, such as heterologous nucleic acids or vectors described herein, into host cells include, but are not limited to, calcium phosphate precipitation, particle bombardment, microinjection, electroporation, etc. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, NY). Examples of chemical means for introducing a nucleic acid, e.g., a heterologous nucleic acid or vector described herein, into a host cell include lipofection, colloidal dispersion systems, e.g., macromolecule complexes, nanocapsules, microspheres, beads, as well as oil-in-water emulsions, micelles, mixed micelles, and liposomes. Lipid-based systems include, but are not limited to, the following: (1) Colloidal systems. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is liposome (e.g., artificial membrane vesicle). Other cutting-edge methods for targeted delivery of nucleic acid are available, such as delivery of polynucleotides or other suitable submicron-sized delivery systems in targeted nanoparticles.

[0109] Host cells can be transiently or stably transfected with the nucleic acid.

[0110] Selection of host cells containing the introduced nucleic acid can be achieved by culturing the cells under stringent selective conditions that allow cells containing the introduced nucleic acid to grow, while restricting the ability of non-transformed cells to grow, based on the tyrosine auxotrophic selection system of the present invention (and any other additional selection markers that may have been included).

[0111] A population of transfected / transformed cells is cultured under conditions where the level of tyrosine allows easy selection of cells containing the introduced nucleic acid. Therefore, cells are cultured in the presence of a lower level of tyrosine than that required for the survival or growth of the cells. Typically, this involves using a medium lacking tyrosine, so that cells are cultured in the absence of tyrosine. Nevertheless, low levels of tyrosine can be tolerated as long as the selection conditions are sufficiently stringent, such as a culture medium containing less than 0.01 g / L of tyrosine, or less than 50, 20, or 10 μM of tyrosine. Those skilled in the art can easily determine the desired tyrosine level to achieve sufficient selection stringency.

[0112] Because the enzyme molecules supplied by one or more exogenous nucleic acids provide the activity to convert phenylalanine to tyrosine, it may be desirable to supplement the culture medium with additional phenylalanine to meet the host cell's normal requirement for phenylalanine and provide a precursor for tyrosine production. Thus, a population of cells can be cultured in the presence of a level of phenylalanine higher than that required for survival or growth of producer cells cultured in the presence of a level of tyrosine required for survival or growth. Thus, in some embodiments, phenylalanine is provided (e.g., as part of the culture and / or as a component of the culture medium) at a level of at least 0.035 g / L. Thus, cells can be cultured in the presence of a level of phenylalanine of at least 2, 3, or 4 mM. Because high levels of phenylalanine can inhibit cell growth, typically, the level of phenylalanine is less than 10 mM, e.g., less than 9, 8, 7, or 6 mM.

[0113] For CHO PAH enzymes, in one embodiment, the phenylalanine level in the culture medium is preferably 2-9 mM, e.g., 2 or 3 mM to 6 or 7 mM phenylalanine, while for human PAH enzymes, in one embodiment, the preferred range is 4-9 mM phenylalanine.

[0114] The cells can be subjected to an adaptation step to adjust to higher levels of phenylalanine. This step can involve passage of the cells at one or more progressively higher concentrations of phenylalanine in the cell culture medium, for example, 3 mM for the first or second passage, and then at a final desired concentration, for example, 6 mM. This can be done before or after transfection, for example, to allow the cells to recover before the growth phase.

[0115] In some embodiments, the level of phenylalanine is determined by detecting and / or monitoring the level of phenylalanine in the culture and determining if the detected level is below a threshold value. In response, an automated regulation system is established and / or maintained that provides phenylalanine (e.g., until detected levels are above a threshold). In some embodiments, such automated regulation systems utilize spectroscopy (e.g., Raman spectroscopy) to detect and / or monitor phenylalanine levels. Similar considerations apply when additional selectable markers are used.

[0116] When two selectable markers (e.g., the selectable system of the present invention and the GS selectable system) are used, the relevant vectors may be introduced simultaneously, and the cell culture medium may be formulated to provide stringent selection for both types of markers, i.e., medium lacking tyrosine and glutamine, optionally supplemented with phenylalanine, as described above. Alternatively, selection may be a two-step process in which one vector system is introduced and selected under stringent conditions for the first marker, and the resulting selected cells are then transfected / transformed with a second marker and, optionally, under stringent conditions for the first marker, e.g., medium lacking tyrosine and glutamine, optionally supplemented with phenylalanine. Alternatively, less stringent conditions for the first marker may be used when subsequently selecting for the second marker. The culture conditions described above apply mutatis mutandis to this two selectable marker procedure (and when additional markers are used).

[0117] Functional characteristics of producer cells containing introduced nucleic acid sequences In some embodiments, the host cells comprise a tyrosine auxotrophic selectable marker (e.g., a first exogenous nucleic acid encoding a PAH enzyme molecule and a second exogenous nucleic acid encoding a GCH1 enzyme molecule) and are capable of growing and / or dividing in a culture medium containing reduced levels of tyrosine (e.g., in the absence of tyrosine). Such cells are also referred to herein as producer cells. The ability to grow and / or divide is known to those of skill in the art and can be assessed by methods described herein. In some embodiments, the host cells are capable of growing and / or dividing in a culture medium containing less than 0.01 g / L, or less than 50, 20, or 10 μM tyrosine, e.g., in the absence of tyrosine. In some embodiments, the host cells are capable of growing and / or dividing in a culture medium lacking tyrosine.

[0118] In some embodiments, a host cell comprises a subject nucleic acid associated with a selectable marker (e.g., a tyrosine auxotrophic selectable marker comprising a first exogenous nucleic acid encoding a PAH enzyme molecule and a second exogenous nucleic acid encoding a GCH1 enzyme molecule). In some embodiments, the host cell comprises at least a threshold copy number of a subject nucleic acid (e.g., a copy number sufficient to efficiently produce a product), e.g., at least 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 copies of a subject nucleic acid.

[0119] In some embodiments, a host cell comprises a first exogenous nucleic acid encoding a PAH enzyme molecule and a second exogenous nucleic acid encoding a GCH1 enzyme molecule. In some embodiments, the host cell comprises at least a threshold copy number of the first exogenous nucleic acid (e.g., a copy number sufficient for the host cell to grow and / or divide at reduced levels (e.g., in the absence of tyrosine), e.g., at least 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 copies of the first exogenous nucleic acid). In some embodiments, the host cell comprises at least a threshold copy number of the second exogenous nucleic acid (e.g., a copy number sufficient for the host cell to grow and / or divide at a reduced level (e.g., in the absence of tyrosine)), e.g., at least 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 1000, or 10,000 copies of the second exogenous nucleic acid.

[0120] In some embodiments, a subject nucleic acid persists in a host cell (e.g., or its daughter cells or progeny) for a specified interval due to, for example, its association with a selectable marker (e.g., a tyrosine auxotrophic marker comprising a first exogenous nucleic acid encoding a PAH enzyme molecule and a second exogenous nucleic acid encoding a GCH1 enzyme molecule). In some embodiments, the first exogenous nucleic acid persists in a host cell (e.g., or its daughter cells, progeny, generation, or population doubling) for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months (and optionally, persists indefinitely). In some embodiments, the first exogenous nucleic acid persists in the host cell (e.g., or its daughter cells or progeny) for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, or 300 cell divisions (and optionally, persists indefinitely). In some embodiments, the first exogenous nucleic acid persists in the host cell (e.g., or its daughter cells, progeny, generations, or population doublings), for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, or 300 host cycles, population doublings, or generations (and optionally, persists indefinitely), e.g., in a bioreactor described herein. In some embodiments, the second exogenous nucleic acid persists in the host cell (e.g., or its daughter cells, progeny, generations, or population doublings) for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months (and optionally persists indefinitely).In some embodiments, the second exogenous nucleic acid persists in the host cell (e.g., or its daughter cells, progeny, generations, or population doublings) for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, or 300 cell divisions (and optionally, persists indefinitely). In some embodiments, the second exogenous nucleic acid persists in the host cell (e.g., or its daughter cells, progeny, generations, or population doublings), e.g., for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 host cycles, population doublings, or generations of a bioreactor described herein (and optionally, persists indefinitely). In some embodiments, the first exogenous nucleic acid persists in the host cell (e.g., or its daughter cells, progeny, generations, or population doublings) as long as the host cell is maintained in a medium comprising reduced levels of tyrosine (e.g., no tyrosine). In some embodiments, the second exogenous nucleic acid persists in the host cell (e.g., or its daughter cells, progeny, generations, or population doublings) as long as the host cell is maintained in a medium containing reduced levels of tyrosine (e.g., no tyrosine). In some embodiments, persistence of the first, second, or first and second exogenous nucleic acid in the host cell is assessed functionally, e.g., by whether the host cell grows and produces a product in a medium containing reduced levels of tyrosine (e.g., no tyrosine). In some embodiments, persistence of the first, second, or first and second exogenous nucleic acid in the host cell is assessed (e.g., confirmed) by using RT-PCR.

[0121] In some embodiments, host cells that contain a tyrosine auxotrophic selectable marker (e.g., a first exogenous nucleic acid encoding a PAH enzyme molecule and a second exogenous nucleic acid encoding a GCH1 enzyme molecule) grow faster in culture medium containing reduced levels of tyrosine (e.g., in the absence of tyrosine) than other similar cells that do not contain the tyrosine auxotrophic selectable marker. In some embodiments, the host cells grow and / or divide at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% faster, or 10 times, 10 times, or more in culture medium containing reduced levels of tyrosine (e.g., in the absence of tyrosine), than similar cells that do not contain a tyrosine auxotrophic selectable marker. 2 double, 10 3 double, 10 4 double, 10 5 double, or 10 6 grow and / or divide twice as fast.

[0122] In some embodiments, host cells containing a selectable marker comprising exogenous nucleic acids encoding enzyme molecules (e.g., a first exogenous nucleic acid encoding a PAH enzyme molecule and a second exogenous nucleic acid encoding a GCH1 enzyme molecule) (and optionally, a subject nucleic acid associated with the exogenous nucleic acids) exhibit increased enzyme molecule activity compared to cells lacking the exogenous nucleic acids and / or the subject nucleic acids. In some embodiments, the level of enzyme molecule activity is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, 1000% or more relative to the enzyme molecule activity detectable in cells lacking the exogenous nucleic acids encoding the enzyme molecules and / or the associated subject nucleic acids. In some embodiments, cells with increased activity may grow faster than cells lacking the exogenous nucleic acids encoding the enzyme molecules and / or the associated subject nucleic acids. In some embodiments, the rate of cell growth and / or division is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, 1000% or more relative to similar cells under similar culture conditions lacking the exogenous nucleic acid encoding the enzyme molecule and / or related subject nucleic acid. In some embodiments, the host cells grow at least about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 5000, or 10,000 times faster on medium lacking amino acids than similar cells lacking the exogenous nucleic acid encoding the subject nucleic acid and / or enzymatic molecule.

[0123] Methods for producing recombinant products using host (production) cells The host cells of the present invention, also referred to as producer cells, can be used to express products encoded by introduced nucleic acids. These producer cells are typically stably transfected into the cells with a first, second, and / or third exogenous nucleic acid (and optionally additional exogenous nucleic acids described herein, which produce two or more products of interest, including multiple subunit products) to create the producer cells. In alternative embodiments, the host cells may be transiently transfected into suitable cells with the first, second, and / or third exogenous nucleic acid.

[0124] Recombinant products can be expressed by culturing the production cells of the invention by any method known in the art suitable for producing a product, with consideration given to the methods described below. In some embodiments, the culture medium lacks tyrosine or contains levels of tyrosine of 0.01 g / L or less, or 50, 20, or 10 μM or less (e.g., levels of tyrosine insufficient to culture similar cells that do not contain one or more exogenous nucleic acids encoding one or more enzyme molecules and / or a subject nucleic acid). In some embodiments, culturing involves culturing the production cells in the presence of a level of tyrosine lower than that required for survival or growth of cells (e.g., cells similar to the production cells) that do not contain one or more exogenous nucleic acids, e.g., in the absence of tyrosine. Because it may not be necessary to apply selective pressure to the production cell line during expression of the recombinant product, the culture medium can contain tyrosine at various stages during the growth and production phases. However, because tyrosine is difficult to handle in cell culture medium due to its low solubility, it may be advantageous to omit it entirely from the cell culture medium.

[0125] On the other hand, in the absence (or low levels) of tyrosine, cells consume more phenylalanine, so various media used, such as feed solutions, are supplemented with phenylalanine. For example, phenylalanine can be included at a level of at least 0.035 g / L. Thus, in some embodiments, phenylalanine is provided (e.g., as part of the culture and / or as a component of the culture medium) at a level of at least 0.035 g / L. Thus, cells can be cultured in the presence of phenylalanine at a level of at least 2, 3, 4, 5, 6, 7, 8, or 9 mM. Because high levels of phenylalanine can inhibit cell growth, typically, the level of phenylalanine is less than 10 mM, for example, less than 9, 8, 7, or 6 mM.

[0126] For CHO PAH enzymes, in one embodiment, the phenylalanine level in the culture medium is preferably 2-9 mM, e.g., 2 or 3 mM to 6 or 7 mM phenylalanine, while for human PAH enzymes, in one embodiment, the preferred range is 4-9 mM phenylalanine. Our results show that truncated human PAH enzymes provide superior cell performance when the cell culture medium is supplemented with phenylalanine.

[0127] The cells can be subjected to an adaptation step so that they can adjust to higher levels of phenylalanine. In one embodiment, the cells are already adapted during the selection phase to grow in phenylalanine-supplemented medium. Alternatively, this can be done during the production or pre-production phase, for example, in an N-1 bioreactor that produces inoculum for the N bioreactor. Again, adaptation can be done for another period with increased concentrations of phenylalanine, or the cells can be seeded into the cell culture medium already at the final level of supplementation.

[0128] In some embodiments, phenylalanine levels are established and / or maintained using an automated regulation system that detects and / or monitors phenylalanine levels in the culture and, in response to the detected level being below a threshold, provides phenylalanine (e.g., until the detected level is equal to or greater than the threshold). In some embodiments, such automated regulation systems utilize spectroscopy (e.g., Raman spectroscopy) to detect and / or monitor phenylalanine levels. Similar considerations apply when additional selectable markers are used.

[0129] In embodiments, cell culture is performed as a batch culture, a fed-batch culture, abbreviated fed-batch overgrowth (aFOG), draw-and-fill culture, continuous culture, or semi-continuous culture, including perfusion culture. In some embodiments, the bioreactor is capable of or configured to operate continuously or semi-continuously. In one embodiment, the cell culture is a suspension culture. In one embodiment, the cells or cell culture are placed in vivo for expression of a recombinant polypeptide, e.g., placed in a model organism or a human subject. In some embodiments, the cell culture utilizes solid microcarriers (e.g., growth on the surface of solid microcarriers), porous microcarriers (e.g., growth on and / or within microcarriers), or a support substrate (e.g., growth on and / or within a matrix). In some embodiments, the cell culture is a perfusion culture. In some embodiments, the cell culture is shaken. In some embodiments, the cell culture is a microfluidic culture.

[0130] In one embodiment, the culture medium is serum-free. Serum-free, protein-free, and chemically defined, animal component-free (CDACF) medium is commercially available, for example, from Lonza Bioscience.

[0131] In some embodiments, lipid additives (including, for example, cholesterol, oleic acid, linoleic acid, or combinations thereof) can be added to the culture medium.

[0132] Suitable media and culture methods for mammalian cell lines are described, for example, in U.S. Pat. No. 5,633,162 Examples of standard cell culture media for laboratory flasks or low-density cell culture, and adapted to the needs of specific cell types, are well known in the art, as described in, for example, Roswell Park Memorial Institute (RPMI) 1640 medium (Morre, G., The Journal of the American Medical Association, 199, p. 519). f. 1967), L-15 medium (Leibovitz, A. et al., Amer. J. of Hygiene, 78, 1 p. 173 ff., 1963), Dulbecco's modified Eagle's medium (DMEM), Eagle's minimum essential medium (MEM), Ham's F12 medium (Ham, R. et al., Proc. Natl. Acad. Sc. 53, p. 288 ff., 1965), or Iscoves' modified DMEM lacking albumin, transferrin, and lecithin (Iscoves et al., J. Exp. med. 1, p. 923 ff., 1978). For example, Ham's F10 or F12 medium was specifically designed for CHO cell culture. Other media specifically adapted for CHO cell culture are described in EP 481 791. Such culture media may be supplemented with fetal bovine serum (FBS, also called fetal calf serum FCS), the latter known to provide a natural source of numerous hormones and growth factors. Cell culture of mammalian cells is now a routine operation well documented in scientific textbooks and manuals, see, for example, R. Ian Fresney, "Culture of Animal Cells," a manual, 4 thedition, Wiley-Liss / NY, 2000. Any of the cell culture media described herein can be formulated to lack a particular amino acid, e.g., an amino acid whose biosynthesis can be rescued when cells take up a subject nucleic acid, such as tyrosine.

[0133] Other suitable culture methods are known to those skilled in the art and may depend on the recombinant polypeptide product and the host cell utilized. It is within the skill of one of ordinary skill in the art to determine or optimize conditions suitable for the expression and production of a recombinant or therapeutic polypeptide expressed by a cell.

[0134] In one aspect, the disclosure is directed to a method of making or producing a polypeptide product, the method comprising harvesting the polypeptide product. In some embodiments, harvesting comprises separating the polypeptide product from the producing cells and / or culture medium, e.g., by methods described herein or known in the art.

[0135] Culturing can include different culturing steps. Thus, in some embodiments, the culturing steps include culturing the production cells in a first culture medium and then in a second culture medium (i.e., using different media that may have, for example, different levels of tyrosine and / or phenylalanine).

[0136] Production cells can be cultured in any suitable vessel at various scales. For industrial production, bioreactors, e.g., bioreactors having a volume of at least 10 liters, e.g., at least 50 liters, 50 to 800 liters, or 800 to 200,000 liters, can be used. The bioreactor can be a single-use bioreactor. In embodiments, the bioreactor comprises a bioprocess vessel, a shell, at least one agitator, at least one sparger, at least one gas filter inlet port for the sparger and headspace overlay, at least one fill port, at least one harvest port, at least one sample port, and at least one probe. The bioreactor can also comprise processes and probes for monitoring and maintaining one or more parameters, e.g., pH, dissolved oxygen tension (DOT), phenylalanine level, and / or temperature. The bioreactor can be operably coupled to a harvest vessel. Further details and embodiments are provided in the "Applications" section below.

[0137] Once product biosynthesis by the production cells has progressed to a sufficient point, the product can be harvested, for example, by removing the culture medium and separating the supernatant from the cells and cell debris. The product may be subjected to one or more purification / processing steps to obtain a purified product, such as affinity chromatography, ion exchange chromatography, filtration, and / or viral inactivation. The product may also be combined with one or more pharmaceutically acceptable carriers, excipients, or diluents to produce compositions, such as formulated pharmaceutical compositions containing, for example, one or more of a buffer, a surfactant, a stabilizer (such as trehalose, sucrose, glycerol), an amino acid (such as glycine, histidine, arginine), a metal ion / chelator, a salt, and / or a preservative.

[0138] Recombinant products Provided herein are compositions and methods for identifying, selecting, or culturing production cells or cell lines capable of producing high yields of products, e.g., polypeptides, e.g., therapeutic polypeptides, as well as methods for producing such products. Products encompassed by the present disclosure include, but are not limited to, molecules, nucleic acids (e.g., non-coding nucleic acids, e.g., non-coding RNA molecules, e.g., antisense RNA, siRNA, tRNA, ribosomal RNA, microRNA, piRNA, snoRNA, snRNA, exRNA, scaRNA, or long non-coding RNA, e.g., Xist or HOTAIR), polypeptides (e.g., recombinant polypeptides and / or therapeutic polypeptides), or hybrids thereof, that can be produced by expression in cells. In some embodiments, cells are engineered or modified to produce a product. Such modifications include the introduction of molecules that control or result in the production of a product. For example, cells are modified by introducing an exogenous nucleic acid encoding a polypeptide, e.g., a recombinant polypeptide, and the cells are cultured under conditions suitable for the production, e.g., expression and secretion, of the polypeptide, e.g., a recombinant polypeptide. In another example, a cell is modified by introducing an exogenous nucleic acid that controls, e.g., increases, expression of a polypeptide endogenously expressed by the cell, such that the cell produces a level or amount of the polypeptide that is higher than the level or amount endogenously produced in an unmodified cell, In embodiments, the cells or cell lines identified, selected, or generated by the methods described herein produce a product, e.g., a recombinant polypeptide, that is useful for treating a medical condition, disorder, or disease.

[0139] Polypeptides In some embodiments, the product of interest comprises one or more polypeptides, e.g., recombinant polypeptides, which are typically heterologous polypeptides, i.e., products not naturally expressed by cells. The product may be, for example, a therapeutic or diagnostic protein useful for drug screening. The therapeutic or diagnostic protein may be an antibody molecule, e.g., an antibody or antibody fragment, a fusion protein, a hormone, a cytokine, a growth factor, an enzyme, a glycoprotein, a lipoprotein, a reporter protein, a therapeutic peptide, an aptamer, or a structural and / or functional fragment or hybrid of any of these. In one embodiment, the product comprises multiple polypeptide chains, e.g., an antibody or antibody fragment comprising a heavy chain and a light chain.

[0140] In some embodiments, the product is an antibody molecule. Products encompassed herein are diagnostic antibody molecules, e.g., monoclonal antibodies or antibody fragments thereof, useful in imaging techniques, and therapeutic antibody molecules suitable for administration to a subject, e.g., useful in treating a disease or disorder. An antibody molecule is a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. In embodiments, the antibody molecule is a full-length antibody or an antibody fragment. Antibodies and multiformat proteins can be polyclonal or monoclonal, multi-chain or single-chain, or intact immunoglobulins, and can be derived from natural or recombinant sources. The antibody may be a multimer of immunoglobulin molecules, e.g., a tetramer of immunoglobulin molecules. In embodiments, the antibody is a monoclonal antibody. The antibody may be a human or humanized antibody. In one embodiment, the antibody is an IgA, IgG, IgD, IgM, or IgE antibody. In one embodiment, the antibody is an IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, the antibody molecule is or comprises a multispecific antibody, e.g., a bispecific, trispecific, or tetraspecific antibody, e.g., a BiTE.

[0141] "Antibody fragment" refers to at least a portion of an intact antibody, or a recombinant variant thereof, including an antigen-binding domain, e.g., an antigen-determining variable region of the intact antibody, sufficient to confer recognition and specific binding of the antibody fragment to a target, such as an antigen. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, scFv antibody fragments, linear antibodies, single domain antibodies such as sdAbs (either VL or VH), camelid VHH domains, and multispecific antibodies formed from antibody fragments such as bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region, as well as isolated CDRs or other epitope-binding fragments of antibodies. Antigen-binding fragments can also be incorporated into single-domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, e.g., Hollinger and Hudson, Nature Biotechnology 23:1126-1136, 2005). Antigen-binding fragments can also be grafted onto scaffolds based on polypeptides such as fibronectin type III (Fn3) (see U.S. Patent No. 6,703,199, which describes fibronectin polypeptide minibodies).

[0142] Examples of polypeptides of interest include, but are not limited to, those listed below: Hormones: erythropoietin, epoein-α, darbepoetin-α, growth hormone (GH), somatotropin, human follicle-stimulating hormone (FSH), human chorionic gonadotropin, lutropin-α, glucagon, growth hormone-releasing hormone (GHRH), insulin. Clotting / Coagulation Factors: Factor VIIa, Factor VIII, Factor IX, Antithrombin III (AT-III), Protein C Concentrate Cytokines / growth factors: type I alpha-interferon, interferon-αn3 (IFNαn3), interferon-β1a (rIFN-β), interferon-β1b (rIFN-β), interferon-γ1b (IFNγ), aldesleukin (interleukin 2 (IL2), epidermal thymocyte-activating factor; ETAF), palifermin (keratinocyte growth factor; KGF), becaplumin (platelet-derived growth factor; PDGF), anakinra (recombinant IL1 antagonist).

[0143] Antibodies: bevacizumab (VEGFA mAb), cetuximab (EGFR mAb), panitumumab (EGFR MAb), alemtuzumab (CD52 mAb), rituximab (CD20 chimeric Ab), trastuzumab, adalimumab, infliximab, tositumomab, acritumomab, ranibizumab, abciximab, omalizumab, palivizumab, natalizumab, daclizumab, basiliximab, eculizumab.

[0144] Vaccine antigens: Hepatitis B surface antigen (HBsAg), HPV antigen, HIV antigen, influenza antigen.

[0145] Other: Albumin, Anti-Rhesus (Rh) Immunoglobulin G, Enfuvirtide, Spider Silk Proteins, e.g., Fibrion, Botulinum Toxin Type A, Alglucerase, Imiglucerase, Recombinant Human Hyaluronidase, Palifermin, Anakinra, Dornasea Luffa, synthetic porcine secretin.

[0146] The recombinant polypeptide of interest may be a multispecific protein, for example a bispecific antibody, many formats of which are available, such as BsIgG (Triomab), BiTE, DART, TandB, etc.

[0147] In some embodiments, the polypeptide (e.g., produced by a cell and / or by the methods described herein) is an antigen expressed by a cancer cell. In some embodiments, the recombinant or therapeutic polypeptide is a tumor-associated antigen or a tumor-specific antigen. In some embodiments, the recombinant or therapeutic polypeptide is an antigen selected from the group consisting of HER2, CD20, 9-O-acetyl-GD3, βhCG, A33 antigen, CA19-9 marker, CA-125 marker, calreticulin, carboanhydrase IX (MN / CA), and the like. IX), CCR5, CCR8, CD19, CD22, CD25, CD27, CD30, CD33, CD38, CD44v6, CD63, CD70, CC123, CD138, carcinoembryonic antigen (CEA; CD66e), desmoglein 4, E-cadherin neoepitope, endosialin, ephrin A2 (EphA2), epidermal growth factor receptor (EGFR), epithelial cell adhesion molecule (EpCAM), ErbB2, fetal acetylcholine receptor, fibroblast activation antigen (FAP), fucosyl GM1, GD2, GD3, GM2, gangloside GD3, Globo H, glycoprotein 100, HER2 / neu, HER3, HER4, insulin-like growth factor receptor 1, Lewis-Y, LG, Ly-6, melanoma-specific chondroitin sulfate proteoglycan (MCSCP), mesothelin, MUCl, MUC2, MUC3, MUC4, MUC5 AC , MUC5 B , MUC7, MUC16, Mullerian inhibitory substance (MIS) receptor type II, plasma cell antigen, polySA, PSCA, PSMA, sonic hedgehog (SHH), SAS, STEAP, sTn antigen, TNF-alpha precursor, and combinations thereof.

[0148] In some embodiments, the polypeptide (e.g., produced by a cell and / or by a method described herein) is an activating receptor, such as 2B4 (CD244), α4β1 integrin, β2 integrin, CD2, CD16, CD27, CD38, CD96, CD100, CD160, CD137, CEACAM1 (CD66), CRTAM, CS1 (CD319), DNAM-1 (CD226), GITR (TNFRSF18), activating forms of KIR, NK G2C, NKG2D, NKG2E, one or more natural cytotoxic receptors, NTB-A, PEN-5, and combinations thereof; optionally, the β2 integrin comprises CD11a-CD18, CD11b-CD18, or CD11c-CD18; optionally, the activating form of KIR comprises KlR2DS1, KIR2DS4, or KIR-S; and optionally, the natural cytotoxic receptor comprises NKp30, NKp44, NKp46, or NKp80.

[0149] In some embodiments, the polypeptide (e.g., produced by a cell and / or by a method described herein) is an inhibitory receptor and is selected from KIR, ILT2 / LIR-1 / CD85j, an inhibitory form of KIR, KLRG1, LAIR-1, NKG2A, NKR-P1A, Siglec-3, Siglec-7, Siglec-9, and combinations thereof, and optionally, the inhibitory form of KIR includes KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL1, KIR3DL2, or KIR-L.

[0150] In some embodiments, the polypeptide (e.g., produced by a cell and / or by a method described herein) is an activating receptor, such as CD3, CD2 (LFA2, OX34), CD5, CD27 (TNFRSF7), CD28, CD30 (TNFRSF8), CD40L, CD84 (SLAMF5), CD137 (4-1BB), CD2 26, CD229 (Ly9, SLAMF3), CD244 (2B4, SLAMF4), CD319 (CRACC, BLAME), CD352 (Lyl08, NTBA, SLAMF6), CRTAM (CD355), DR3 (TNFRSF25), GITR (CD357), HVEM (CD270), ICOS, LIGHT, LTβR (TNFRSF3), OX40 (CD134), NKG2D, SLAM (CD150, SLAMF1), TCRα, TCRβ, TCRδγ, TIM1 (HAVCR, KIM1), and combinations thereof.

[0151] In some embodiments, the polypeptide (e.g., produced by a cell and / or by the methods described herein) is an inhibitory receptor and is selected from PD-1 (CD279), 2B4 (CD244, SLAMF4), B71 (CD80), B7H1 (CD274, PD-L1), BTLA (CD272), CD160 (BY55, NK28), CD352 (Ly108, NTBA, SLAMF6), CD358 (DR6), CTLA-4 (CD152), LAG3, LAIR1, PD-1H (VISTA), TIGIT (VSIG9, VSTM3), TIM2 (TIMD2), TIM3 (HAVCR2, KIM3), and combinations thereof.

[0152] Other recombinant protein products (e.g., produced by cells and / or by the methods described herein) include non-antibody or alternative protein scaffolds, such as, but not limited to, DARPins, affibodies, and adnectins. Such non-antibody or alternative protein scaffolds can be engineered to recognize or bind to one or more, e.g., 1, 2, 3, 4, or 5 or more, different targets or antigens.

[0153] Purpose The present disclosure features, among other things, production cells, methods for making or manufacturing polypeptide products using production cells, methods for identifying, selecting, and / or culturing cells (e.g., production cells), and methods for making or producing production cells. The cell identification, selection, and / or culturing methods disclosed herein can be used to generate cells useful for producing various products, e.g., production cells, evaluate various cell lines, or evaluate the production of various cell lines for use in bioreactors or process vessels or tanks, or more generally with any source. The compositions and methods described herein are suitable for culturing any desired cell line, including, for example, prokaryotic and / or eukaryotic cell lines. Furthermore, in embodiments, the compositions and methods described herein are suitable for culturing suspension or anchorage-dependent (adherent) cells, and are suitable for production operations configured for the production of pharmaceuticals and biopharmaceuticals, such as polypeptide products, nucleic acid products (e.g., DNA or RNA), exosomes, vesicles, or cells and / or viruses (such as those used in cell and / or viral therapies or as vaccines).

[0154] In embodiments, the cells, e.g., producer cells, express or produce a product, such as a therapeutic or diagnostic recombinant product. As described in more detail below, examples of products produced by the cells include antibody molecules (e.g., monoclonal antibodies, bispecific antibodies), antibody mimetics (polypeptide molecules that specifically bind to an antigen but are not structurally related to antibodies, such as, e.g., DARPins, affibodies, adnectins, or IgNARs), fusion proteins (e.g., Fc fusion proteins, chimeric cytokines), other recombinant proteins (e.g., glycosylated proteins, enzymes, hormones), viral therapeutics (e.g., anti-cancer oncolytic viruses, viral vectors for gene therapy and viral immunotherapy), cellular therapeutics (e.g., pluripotent stem cells, mesenchymal stem cells, and adult stem cells), vaccines or lipid-encapsulated particles (e.g., exosomes, virus-like particles), RNA (e.g., siRNA, etc.) or DNA (e.g., plasmids). In embodiments, the compositions and methods described herein may be used to produce biosimilars.

[0155] As mentioned, in embodiments, the compositions and methods described herein enable the production of eukaryotic cells, e.g., mammalian cells or lower eukaryotic cells, e.g., yeast cells or filamentous fungal cells, or prokaryotic cells, e.g., gram-positive or gram-negative cells, and / or eukaryotic or prokaryotic products, e.g., proteins, peptides, antibiotics, amino acids, nucleic acids (such as DNA or RNA), which are synthesized by eukaryotic cells in a large-scale manner. Unless otherwise stated herein, the compositions and methods described herein can include any desired volume or production capacity, including, but not limited to, bench-scale, pilot-scale, and full production-scale capacities.

[0156] Additionally, and unless otherwise stated herein, the compositions and methods described herein can be used with any suitable reactor, including, but not limited to, stirred tank, air-lift, fiber, microfiber, hollow fiber, ceramic matrix, fluidized bed, fixed bed, and / or spouted bed bioreactors, with or without solid or porous microcarriers or supports. As used herein, a "reactor" can include a fermentor or fermentation unit, or any other reaction vessel, and the term "reactor" is used synonymously with "fermentor." For example, in some embodiments, a bioreactor unit can perform one or more or all of the following: feeding nutrients and / or carbon sources, injecting a suitable gas (e.g., oxygen), inflow and outflow of fermentation or cell culture medium, separating gas and liquid phases, maintaining temperature, maintaining oxygen and CO2 levels, maintaining pH levels, agitation (e.g., stirring), and / or cleaning / sterilization. An exemplary reactor unit, such as a fermentation unit, may include multiple reactors within the unit; for example, a unit may have 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 or more bioreactors within each unit, and / or a facility may include multiple units with single or multiple reactors within the facility. In various embodiments, the bioreactors may be suitable for batch, semi-fed-batch, fed-batch, perfusion, and / or continuous fermentation processes. Any suitable reactor diameter may be used. In embodiments, the bioreactors may have a volume of from about 100 ml to about 50,000 L. Non-limiting examples include volumes at or about 10 ml, 50 ml, 100 ml, 250 ml, 500 ml, 750 ml, 1 liter, 2 liters, 10 liters, 50 liters, 100 liters, 500 liters, 1000 liters, 2000 liters, 5000 liters, 10,000 liters, 15,000 liters, 20,000 liters, and / or 50,000 liters.In the context of industrial-scale manufacturing, required to generate enough product for clinical or commercial use, the volume is typically at least 10 liters. In some embodiments, the bioreactor is configured to grow microfluidic cultures. Furthermore, suitable reactors can be multi-use, single-use, disposable, or non-disposable and can be formed of any suitable material, including metal alloys such as stainless steel (e.g., 316L or any other suitable stainless steel) and Inconel, plastic, and / or glass. In some embodiments, suitable reactors can be round, e.g., cylindrical. In some embodiments, suitable reactors can be square, e.g., rectangular. Square reactors may, in some cases, offer advantages over circular reactors, such as ease of use (e.g., filling and setting up by one skilled in the art), better mixing and homogeneity of the reactor contents, and a smaller floor footprint.

[0157] In embodiments, and unless otherwise stated herein, the compositions and methods described herein can be used with any suitable unit operations and / or equipment not specifically mentioned, e.g., operations and / or equipment for the separation, purification, and isolation of such products. Any suitable equipment and environment can be used, such as traditional stick-built facilities, modular, mobile, and temporary facilities, or any other suitable construction, equipment, and / or layout. For example, in some embodiments, a modular cleanroom can be used. Additionally, and unless otherwise stated, the compositions and methods described herein can be housed and / or performed in a single location or facility, or alternatively, in separate or multiple locations and / or facilities.

[0158] By way of non-limiting example, and without limitation, U.S. Publication Nos. 2013 / 0280797, 2012 / 0077429, 2011 / 0280797, 2009 / 0305626, and U.S. Patent Nos. 8,298,054, 7,629,167, and 5,656,491, which are incorporated by reference herein in their entireties, describe exemplary equipment, devices, and / or systems that may be suitable for use with the compositions and methods described herein.

[0159] The compositions and methods described herein can utilize a wide variety of cells, as described in the section above regarding host cells. In a preferred embodiment, the mammalian cells are CHO cell lines. Examples include CHO-K1 cells, CHO-K1 SV cells, DG44 CHO cells, DUXB11 CHO cells, CHOS, CHO GS knockout cells, CHO FUT8 GS knockout cells, CHOZN, and CHO-derived cells. CHO GS knockout cells (e.g., GSKO cells) are, for example, CHOK1SV® GS knockout cells. CHO FUT8 knockout cells are, for example, Potelligent® CHOK1SV® (Lonza Biologics, Inc.).

[0160] In one embodiment, the eukaryotic cell is, for example, a yeast cell (e.g., a Pichia cell (e.g., Pichia pastoris, Pichia methanolica, Pichia kluyveri, and Pichia angusta), a Komagataella cell (e.g., Komagataella pastoris, Komagataella pseudopastoris, or Komagataella phaffii), a Saccharomyces cell (e.g., Saccharomyces cerevisae, Saccharomyces kluyveri, Saccharomyces uvarum), a Kluyveromyces cell (e.g., Kluyveromyces lactis, Kluyveromyces marxianus), a Candida cell (e.g., Candida utilis, Candida cacaoi, Candida boidinii), a Geotrichum cell (e.g., Geotrichum fermentans), a Hansenula polymorpha, a Yarrowia lipolytica, or a Schizosaccharomyces cell). and lower eukaryotic cells such as P. pombe. The species Pichia pastoris is preferred. Exemplary Pichia pastoris strains are X33, GS115, KM71, KM71H, and CBS7435.

[0161] In embodiments, the cultured cells are used to produce proteins, e.g., antibodies, e.g., monoclonal antibodies, and / or recombinant proteins, for therapeutic use. In embodiments, the cultured cells produce peptides, amino acids, fatty acids, or other useful biochemical intermediates or metabolites. For example, in embodiments, molecules having molecular weights of about 4000 daltons to greater than about 140,000 daltons can be produced. In embodiments, these molecules may be of varying complexity and may include post-translational modifications, including glycosylation.

[0162] The invention is further illustrated by reference to the following non-limiting examples. [Example]

[0163] Example 1: Materials and Methods cell culture Suspension Lonza CHOK1SV® GS-KO® cells were maintained in CD-CHO medium (Gibco 10743-029) supplemented with 6 mM L-glutamine (Sigma G8540). They were incubated at 37°C, 140 rpm, and in a 5% CO atmosphere. Cells were grown in 125 ml Erlenmeyer flasks at 0.2 x 10 cells per 20 ml. 6 Viable cells / ml were seeded and these were passaged every 3–4 days.

[0164] Reversion assay Lonza CHOK1SV® GS-KO® cells were seeded at 5000 viable cells per well in 200 μl medium in 96-well plates and analyzed for growth after 11 days and 3 weeks. CD CHO without tyrosine and Lonza CM76 (Lonza Biologics plc) without tyrosine but supplemented with 6 mM L-glutamine were used as test media. 7.2 × 10 6 Viable cells were tested in CM76 tyrosine-free medium and 2.4 × 10 6 Cells were tested in CD CHO tyrosine-free medium. Complete medium (CD-CHO+L-glut) was used as a positive control, while medium without L-glutamine (CD-CHO only) was used as a negative control.

[0165] Plasmids and transfection for generating stable cell lines [Table 1]

[0166] The truncated PAH sequence has a deletion of 116 amino acids at the N-terminus that contains the regulatory domain (Daubner SC et al., 1997, ibid.). The various domains of PAH are shown in FIG.

[0167] The plasmid was linearized with PvuI (NEB, R3150L) and purified using an ethanol precipitation protocol. Electroporation was performed in a Biorad Genepulser Xcell electroporator. 100 μl of TE buffer and 1 × 10 7 Live Lonza CHOK1SV GS-KO cells / 20 μg of linearized plasmid in 700 μl of CM76 tyrosine-free (+6 mM L-glut) medium were added to an electroporation cuvette. The DNA-cell mix was electroporated at 300 V and 900 μF with a cuvette diameter of 0.4 mm. Immediately after electroporation, 1 ml of prewarmed medium was added to the cuvette. The cells were then transferred to 2 × 5 ml of CM76 tyrosine-free (+6 mM L-glut) medium in a T25 flask. The flasks were incubated at 37°C in a static incubator with 5% CO2 gas. After 24 hours, an additional 5 ml of CM76 tyrosine-free (+6 mM L-glut) medium was added to the T25 flask. Cell counts were performed using a ViCell instrument 21 days after transfection to assess transfection success. Further confirmation of successful transfection was performed by visualizing cells growing in the T25 flasks under a microscope (Leica MZFLIII with GFP2 filter, ×100 magnification) for eGFP expression.

[0168] Growth curve profile and culture viability Cells were cultured at 0.2 × 10 cells per 20 ml in a 125 ml Erlenmeyer flask. 6 Cells / ml were seeded and shaken at 140 rpm at 37°C in a 5% CO2 environment. Viable cell concentration and cell diameter were determined using a ViCell (Beckman Coulter) instrument using 0.2 ml of sample and 0.8 ml of prewarmed PBS. Readings were recorded every 48 hours for the number of days indicated in the example figures.

[0169] FACS 1×10 5The cells were pelleted in a centrifuge at 1,000 rpm for 5 minutes and resuspended in 350 μl of PBS. The samples were then loaded onto a FACScalibur™ (BD biosciences) probe, and fluorescence intensity was measured relative to cell number. Forward scattering (FSC) was measured using an E-1 amplifier, and side scattering (SSC) was set to 465, while FL1 was recorded at 473, and all settings were converted to a logarithmic scale.

[0170] SDS-PAGE, Western blot 1×10 6 Cells were pelleted in a centrifuge at 1000 rpm for 5 min and lysed in 100 μl of ice-cold lysis buffer consisting of 20 mM HEPES-NaOH, pH 7.2, 100 mM NaCl, 10 mM Na β-glycerophosphate, 0.5% Nonidet-P40 containing 50 mM NaF, 1 mM activated Na3VO4, 10 μg / ml leupeptin, 2 μg / ml pepstatin, and 0.2 mM PMSF added immediately before use.

[0171] Ten μg of reduced protein samples or 10 μl of non-reduced supernatant samples were run on a 10% SDS-PAGE acrylamide gel, and Western transfer to nitrocellulose was performed as previously described (Roobol, Carden et al., 2009, FEBS J. 276:286-302). Antibodies were supplied by Sigma (anti-GCH1, SAB1405858-50 μg; anti-PAH, HPA031642; anti-GS G2781; anti-B-actin A5441; anti-human IgG (γ-chain specific) I9764), and CRUK (eGFP 3E1). Anti-tubulin (Woods, Sherwin et al., 1989, J. Cell Sci. 93:491-500) was a contribution from Professor Keith Gull of the University of Oxford, UK, while anti-L7a was generated against the N-terminal sequence of human L7a (Roobol and Carden, 1999, Eur. J. Cell Biol. 78(1):21-32). Secondary antibodies for immunoblot detection of cell lysate proteins were anti-total IgG (mouse or rabbit)-HRP conjugates (Sigma), followed by ECL (GE Healthcare) detection.

[0172] qRTPCR 1 x 10 for RNA extraction 6 Viable cells were harvested and mRNA levels were determined by qRTPCR on an Eppendorf RealPlex cycler instrument using a Qiagen Quantifast kit with the following primer sets: PAH (qrtPAHtotfwd CATCAAGGCATATGGTGCTG (SEQ ID NO: 7) and qrtPAHtotrvs GGGCTGGAACTCTGTGACAT (SEQ ID NO: 8)), GCH1 (GCH1 forward: CTTCACCAAGGGCTACCAGG (SEQ ID NO: 9); GCH1 reverse: AGGCCAAGGACTTGCTTGTT (SEQ ID NO: 10)), and β-actin (CHObactqF agctgagagggaaattgtgcg (SEQ ID NO: 11) and CHObactqR GCAACGGAACCGCTC ATT (SEQ ID NO: 12).

[0173] Example 2: Reversion assay of GSKO cells grown in either CM76 or CD CHO medium without tyrosine but supplemented with 6 mM L-glutamine This example demonstrates the low reversion rate observed when growing exemplary cells that are unable to grow in the absence of tyrosine.

[0174] CHOK1SV GS-KO® host cells were seeded into 96-well plates in medium lacking tyrosine but supplemented with 6 mM glutamine. The positive control was CHOK1SV GS-KO® host cells growing in medium supplemented with 6 mM glutamine, and the negative control was medium lacking glutamine. The results are shown in Table 2 below. In medium lacking tyrosine, no revertant colonies / cell growth were observed, and the plates appeared similar to the negative control. This suggests that the tyrosine auxotrophy marker is a useful selection marker in production cells. [Table 2]

[0175] Example 3: Growth of exemplary production cells in the absence of tyrosine This example demonstrates that cells lacking exogenous nucleic acids encoding PAH and GCH1 enzyme molecules do not grow in the absence of tyrosine, whereas exemplary production cells containing vectors LMM172 or LMM173, which contain exogenous nucleic acids encoding both PAH and GCH1 enzyme molecules, grow in the absence of tyrosine and express the reporter molecule eGFP. However, when full-length CHO PAH was initially tested with GCH1, it was found that transfected cells recovered very slowly in tyrosine-free medium (CD CHO) or not at all when CM76 medium was used (data not shown). Therefore, a truncated form of PAH was attempted, in which the N-terminal 116 amino acids encoding the regulatory domain were removed.

[0176] The vectors used to generate these pools contained a truncated form of PAH (cassette 1, tPAH derived from either CHO cells or human, with the first 116 amino acids deleted, with sequences driven by an SV40 promoter), GCH1 (cassette 3, driven by an SV40 promoter), and eGFP (cassette 2, driven by a CMV promoter). Two controls were included, in which the first cassette contained the glutamine synthetase (GS) gene driven by an SV40 promoter (vector LMM170). Transfected controls were grown either in the absence (negative control) or presence (positive control) of tyrosine.

[0177] CHOK1SV GS-KO® host cells were transfected via electroporation with the linearized vector and then cultured for 3 weeks in medium lacking tyrosine but supplemented with 6 mM glutamine (except for the positive control, which also contained tyrosine).

[0178] CHOK1SV GS-KO® host engineered cells were shown to grow well in tyrosine-free medium only when truncated PAH and GCH1 were coexpressed. In addition, when these components were transfected individually, the cells did not survive transfection and did not grow in tyrosine-free medium (data not shown). Thus, both PAH enzyme molecules, including truncated PAH and GCH1 enzyme molecules, are required to support the growth of exemplary CHO production cells in the absence of tyrosine.

[0179] Figure 2 shows a histogram of mean fluorescence from cell populations after transfection and 3 weeks of recovery, obtained using flow cytometry, confirming the expression of eGFP in cells grown in tyrosine-free medium. The mean fluorescence from exemplary producer cells containing a truncated CHO cell-derived PAH sequence and GCH1 (vector LMM172) was similar to that from the GS positive control (vector LMM170+ve). Producer cells containing the CHO truncated PAH sequence (LMM172) showed higher GFP expression compared to the human truncated PAH sequence (LMM173). This demonstrates that recombinant proteins can replace eGFP when the PAH and GCH1 combination is used as a selectable marker.

[0180] Example 4: PAH protein and mRNA abundance This example shows that exemplary production cells containing vector LMM173, which includes exogenous nucleic acids encoding human PAH and GCH1 enzyme molecules, exhibit PAH protein and mRNA expression and eGFP protein expression, and this example further shows that exemplary production cells containing vector LMM172, which includes exogenous nucleic acids encoding CHO PAH and GCH1 enzyme molecules, exhibit PAH mRNA expression and eGFP protein expression.

[0181] Western blot analysis of lysates from the cell pools in Figure 2 was performed. Controls were grown in medium containing 6 mM glutamine and tyrosine, and LMM170 cells were grown in medium containing tyrosine but not glutamine. LMM172 and LMM173 were grown in tyrosine-free medium supplemented with 6 mM glutamine. Tubulin and L7a were used as loading controls. The PAH antibody detected only human cleaved PAH (bands at approximately 37 and 50 kDa), but not CHO cleaved PAH (LMM172) (data not shown). eGFP was confirmed to be expressed in the cell pools in which the transfected vector contained the eGFP gene in cassette 2.

[0182] Figure 3 shows qRT-PCR data detecting the expression of truncated CHO PAH and truncated human PAH mRNA. The truncated CHO PAH mRNA was expressed to a much higher amount than the truncated human PAH. Both were increased above the control, confirming exogenous PAH mRNA expression in the exemplary production cells.

[0183] Example 5: Growth profile and culture viability in the absence of tyrosine This example demonstrates that exemplary production cells containing vector LMM172, which contains exogenous nucleic acids encoding CHO PAH and GCH1 enzyme molecules, are capable of growing to higher viable cell concentrations in the absence of tyrosine than similar cells without exogenous nucleic acids, and have extended culture survival times.

[0184] Figure 4 shows growth data for an exemplary production cell pool generated as described in Examples 3 and 4. The cell pool was cultured in 125 ml Erlenmeyer flasks for 18 days in the absence of tyrosine or glutamine. Cells were sampled every two days and viable. Cell counts and culture viability were assessed using a ViCell instrument. No additional feed was introduced. Figure 4 shows (A) viable cell concentration and (B) culture viability. The CHO cell-cleaved PAH cell pool (LMM172) expanded to higher cell numbers and had longer culture viability than the human-cleaved PAH cell pool (LMM173).

[0185] Example 6: Exemplary producer cell growth profile and viable cell concentration when grown in the absence of tyrosine but supplemented with additional phenylalanine This example demonstrates the growth and culture viability characteristics of exemplary production cells containing exogenous nucleic acids encoding PAH and GCH1 enzyme molecules.

[0186] Figure 5 shows growth data for an exemplary production cell pool. Cultures were grown in 125 ml Erlenmeyer flasks for 18 days and supplemented with phenylalanine (Sigma P5482) where indicated. Cells were sampled every two days, and no additional feed was introduced. These cells were analyzed for cell growth and culture viability. The exemplary production cells expressing the truncated human PAH grew and reached higher viable cell concentrations in a shorter time than the exemplary production cells expressing the truncated CHO PAH when supplemented with 6 mM phenylalanine. This experiment also demonstrated that the cell line was truly prototrophic, as the GSKO control died.

[0187] Example 7: Growth profile and viable cell concentration of exemplary producer cells when grown in commercially available CD-CHO medium in the absence of tyrosine but supplemented with additional phenylalanine This example evaluates cell proliferation and culture viability. Figure 6 shows growth data for exemplary production cells transfected and grown in commercially available CD-CHO (ThermoFisher Scientific) medium lacking tyrosine but supplemented with 6 mM glutamine. Transfected CHOK1SV GS-KO™ host cells recovered faster after transfection in CD-CHO medium compared to CM76 medium. The recovery rate was reduced from 21 days to 18 days after transfection when the cells were ready to be transferred to shake flasks. In addition, cells transfected with a plasmid DNA construct containing a truncated human PAH recovered after transfection in a similar time and to a similar viable cell number as observed when using vectors containing truncated CHO PAH cells. This indicated that CD-CHO is a better transfection medium for this system.

[0188] Cells evaluated for growth in CD CHO medium were sampled every two days, and no additional feed was introduced. Cultures were analyzed for cell growth and viability. When tyrosine prototrophic cell pools were grown in CD CHO medium (Figure 6), the truncated human PAH cell pool benefited most from the additional 6 mM phenylalanine.

[0189] Example 8: Pre-adapting cells to phenylalanine supplementation reduces the growth lag phase. This example demonstrates that growth is improved by pre-adapting an exemplary production cell pool to additional supplemental phenylalanine before performing batch culture (Figure 7). Human truncated PAH-expressing cells respond better to phenylalanine supplementation than CHO cells. Human truncated PAH-expressing cells (LMM173) were pre-adapted by passaging the cells with 6 mM phenylalanine before starting a growth curve. Cells were cultured in 125 ml Erlenmeyer flasks for 16 days. These cells were analyzed for cell growth, as measured by viable cell concentration and culture viability. Cell growth was enhanced by the addition of phenylalanine, but the growth lag phase was further reduced when the cells were pre-adapted to growth without CD CHO tyrosine supplemented with 6 mM L-glutamine and 6 mM phenylalanine (Sigma P5482). GS-KO host cells were grown in CD CHO medium with or without supplementation with 6 mM phenylalanine. I was unable to do so.

[0190] Example 9: Dual metabolic selection markers with recombinant protein production This example evaluates the utility of truncated PAH / GCH1 combinatorial selection when combined with a cell line producing recombinant protein under glutamine synthetase selection. The strength of the promoter was varied to drive GCH1 expression and determine whether this affected the growth profile of the resulting cells. To achieve maximum growth (highest viable cell concentration), different promoter and plasmid combinations of truncated PAH and GCH1 were found to be the best combination. The vectors used to generate these pools contained truncated forms of either CHO or human PAH (cassette 1, SV40 promoter), GCH1 (cassette 3 driven by either the PGK, SV40, or mCMV promoter), and eGFP (cassette 2 driven by the CMV promoter). See Table 1.

[0191] These were linearized and transfected into a cell line expressing the model monoclonal antibody cB72.3 under GS selection. Transfections were performed in T25 static flasks in CD-CHO medium without glutamine and tyrosine. After transfection and selection, once the cells had recovered and grown, they were transferred to shake flasks in CM76 medium without glutamine and tyrosine.

[0192] Figure 8 shows the expression of truncated CHO PAHs and truncated human PAHs in the resulting cell pools. qRT-PCR data for mRNA expression are shown and are comparable to those in Example 4. GCH1 expression was also detected, with levels reflecting the strength of the promoter driving the cassette. Analysis confirms PAH mRNA overexpression in the truncated PAH cell pool. As previously observed, truncated CHO PAH was expressed at much higher levels than human PAH. GCH1 mRNA expression levels correlated with the strength of the promoter driving the gene.

[0193] Western blot analysis of lysates from the above cell pools was performed. All double-selected cell pools were grown in CM76 medium without glutamine and without tyrosine. CHOK1SV GS-KO™ control samples were collected from cells grown in complete medium (containing glutamine and tyrosine). Cleaved PAH, GCH1, and GS were detected in all double-selected marker expressing cell lines, except for CHO PAH, as the antibody did not detect it (data not shown), according to Example 4. Using a heavy chain antibody, we also confirmed that the recombinant protein (cB72.3) was secreted into the supernatant (data not shown). Tubulin, β-actin, and L7a served as loading controls.

[0194] Figure 9 shows growth data for an exemplary production cell pool. Cell pools were cultured in 125 ml Erlenmeyer flasks for 18 days in the absence of tyrosine and glutamine and supplemented with additional phenylalanine as indicated. Cells were sampled every two days, and viable cell counts and culture viability were assessed using a ViCell instrument. No additional feed was introduced. Maximum growth (highest viable cell concentration achieved) was observed for LMM186 (SV40 human PAH, SV40 GCH1) when pre-adapted to additional 6 mM phenylalanine-supplemented medium.

[0195] Because two selectable markers were simultaneously utilized, cells were cultured in CM76 without tyrosine or glutamine. The best growing cell pools were generated from LMM186 when supplemented with 6 mM phenylalanine (SV40 PAH human and SV40 GCH1).

[0196] These results demonstrate that two different amino acid-based selection systems can be combined without any adverse effects on cell line performance. The resulting cells exhibit excellent growth characteristics. This provides greater flexibility for expression, for example, because one selection system can be used to generate and maintain an engineered stable cell line with a gene product that modifies cell line performance, while another selection system can be used to introduce and maintain a sequence encoding a desired product.

[0197] The results also support the findings of Example 7 that superior performance is achieved using human PAH with phenylalanine supplementation.

[0198] The disclosures of all patents, patent applications, and publications cited herein are incorporated herein by reference in their entirety. Although the present invention has been disclosed with reference to certain embodiments, it will be apparent that other embodiments and variations of the present invention may be devised by those skilled in the art without departing from the true spirit and scope of the present invention. Features and embodiments in different sections may be combined mutatis mutandis.

Claims

1. 1. A method for selecting a eukaryotic cell containing a nucleic acid sequence encoding a product of interest, comprising: i) contacting a population of cells that cannot survive or grow in the absence of tyrosine with a vector system, said vector system comprising: a) a first nucleic acid sequence comprising a sequence encoding a phenylalanine hydroxylase (PAH) lacking a functional N-terminal regulatory domain, operably linked to a first control sequence that allows expression of said PAH in a host cell; (b) a second nucleic acid sequence comprising a sequence encoding GTP cyclohydrolase 1 (GCH1) operably linked to a second control sequence that allows expression of said GCH1 in a host cell; and (c) a third nucleic acid sequence comprising a sequence encoding a product of interest operably linked to a third control sequence that allows for expression of the product in a host cell, wherein the third sequence is present in the same vector as (a) and / or (b) under conditions that allow for uptake of the vector system by the cell; ii) culturing the cells under conditions in which the level of tyrosine is lower than that required for survival or growth of cells that do not express the PAH and GCH1 enzymes encoded by the vector system; iii) selecting one or more cells that are able to grow under such conditions to obtain one or more cells that contain said nucleic acid sequence encoding said product.

2. 2. The method of claim 1, wherein (a), (b), and (c) are present in the same vector.

3. 3. The method of claim 1 or 2, wherein (c) comprises two vectors, wherein (c) is present in the same vector as (a) or (b).

4. The method of any one of claims 1 to 3, wherein the eukaryotic cell is a mammalian cell, for example a CHO cell.

5. 10. The host cell of claim 1, wherein the PAH has a deletion of the N-terminal regulatory domain.

6. 10. The method of any one of the preceding claims, wherein the cell culture medium lacks tyrosine and is optionally supplemented with phenylalanine.

7. 1. A eukaryotic host cell comprising: a) a first exogenous nucleic acid comprising a sequence encoding phenylalanine hydroxylase (PAH) lacking a functional N-terminal regulatory domain, operably linked to a first control sequence enabling expression of said PAH in a host cell; b) a second exogenous nucleic acid encoding GTP cyclohydrolase 1 (GCH1) operably linked to a second control sequence that allows expression of the GCH1 in the host cell; and c) a third exogenous nucleic acid encoding a product of interest operably linked to a third control sequence that allows expression of said product in said host cell, wherein said third exogenous nucleic acid is present in the same exogenous nucleic acid sequence as said first and / or second exogenous nucleic acid.

8. The host cell of claim 7, wherein the PAH has a deletion of the N-terminal regulatory domain.

9. 9. The host cell of claim 7 or 8, wherein the PAH is CHO or human PAH.

10. The host cell according to any one of claims 7 to 9, which is a mammalian cell, for example a CHO cell.

11. The host cell of any one of claims 7 to 10, wherein the first, second, and third nucleic acid molecules are integrated into the genome of the host cell.

12. The host cell according to any one of claims 7 to 10, wherein the activity of endogenous genes encoding PAH and / or GCH1 in said cell is reduced or eliminated.

13. A vector system comprising one or more nucleic acid vectors, a) a first nucleic acid sequence comprising a sequence encoding a phenylalanine hydroxylase (PAH) lacking a functional N-terminal regulatory domain, operably linked to a first control sequence that allows expression of said PAH in a host cell; b) a second nucleic acid sequence comprising a sequence encoding GTP cyclohydrolase 1 (GCH1) operably linked to a second control sequence allowing expression of said GCH1 in a host cell; and and c) a multiple cloning site for inserting a sequence encoding a product of interest operably linked to a third control sequence that allows expression of said product in a host cell, wherein said multiple cloning site and third control sequence are present in the same vector as (a) and / or (b).

14. A vector system comprising one or more nucleic acid vectors, a) a first nucleic acid sequence comprising a sequence encoding a phenylalanine hydroxylase (PAH) lacking a functional N-terminal regulatory domain, operably linked to a first control sequence that allows expression of said PAH in a host cell; b) a second nucleic acid sequence comprising a sequence encoding GTP cyclohydrolase 1 (GCH1) operably linked to a second control sequence allowing expression of said GCH1 in a host cell; and and c) a third nucleic acid sequence comprising a sequence encoding a product of interest operably linked to a third control sequence that allows expression of said product in a host cell, wherein said third nucleic acid sequence is present in the same vector as (a) and / or (b).

15. 11. A method of making a product, said method comprising culturing a host cell according to any one of claims 7 to 10 under conditions suitable for expressing said product, recovering said product, and optionally subjecting said recovered product to one or more processing or purification steps.

16. 16. The method of claim 15, wherein the cells are cultured under conditions in which the level of tyrosine is lower than the level required for survival or growth of cells that do not express the PAH and GCH1 enzymes encoded by the vector system of any one of claims 1 to 5.

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