Mammalian Expression Vectors

Abstract

The expression vector for mammalian cells contains a selection cassette comprising a nucleotide sequence encoding glutamine synthetase operably linked to a PGK promoter and a pA signal. The vector may also contain an EASE element, known to promote stable integration of the expression cassette into the genome. The vector also contains a selection cassette as a bacterial selection marker, comprising a nucleotide sequence encoding an enzyme that confers antibiotic resistance to bacteria, operably linked to a suitable promoter. The vector further contains an expression cassette for a target polypeptide, operably linked to a CMV promoter and a pA signal, containing an insertion site for a nucleotide sequence encoding the target polypeptide. The vector also contains a bacterial origin of replication.

Description

[Technical Field]

[0001] Field of the Disclosure The present disclosure relates to mammalian expression vectors and their uses. The present invention also provides methods for producing polypeptides. Reference to sequence listing This application contains a sequence listing. [Background technology]

[0002] background The following introduction to the background is provided merely to aid in the understanding of the present invention and is not admitted to describe or constitute prior art to the present invention. Mammalian cells have become the workhorse in the bioprocessing of proteins for therapeutic or diagnostic use, such as monoclonal antibodies. Today, mammalian cell expression systems are the most powerful production tools for upstream processing. Desired glycosylation patterns combined with the development of serum-free and protein-free culture media paved the way for this expression platform, characterized by suitable cell lines and expression vectors. The most commonly used cell line, used worldwide, is CHO. Large-scale production combined with an improved understanding of the nutritional requirements of mammalian cells has led to substantial optimization in terms of increased efficiency and yield.

[0003] Because the dosage of therapeutic proteins in therapeutic use typically ranges from a few micrograms to milligrams of protein, further increases in efficiency and yield are desirable. Furthermore, production costs depend in part on the expression level achieved. A key component of mammalian expression platforms is the expression vector employed. Summary of the Invention

[0004] Summary of the Disclosure The present disclosure can be viewed generally as relating to the production of polypeptides, such as mammalian polypeptides. Provided are vectors suitable for expressing the respective polypeptides.

[0005] In a first aspect, an expression vector is provided. The expression vector is a mammalian expression vector. The expression vector comprises a selection cassette comprising a eukaryotic selection marker, a selection cassette comprising a bacterial selection marker, an expression cassette for a target polypeptide, and a bacterial origin of replication. The selection cassette comprising a eukaryotic selection marker comprises a nucleotide sequence encoding glutamine synthetase as a eukaryotic selection marker. The nucleotide sequence encoding glutamine synthetase is operably linked to a 3-phosphoglycerate kinase (PGK) promoter and a polyadenylation (pA) signal. The selection cassette comprising a bacterial selection marker comprises a nucleotide sequence encoding an enzyme that confers antibiotic resistance to a bacterial host as a bacterial selection marker, operably linked to a suitable promoter. The expression cassette for a target polypeptide comprises an insertion site for the nucleotide sequence encoding the target polypeptide. The insertion site is operably linked to a cytomegalovirus (CMV) promoter and a polyadenylation (pA) signal.

[0006] According to some embodiments, the expression vector is a mammalian expression vector. According to some embodiments, the expression vector is a vector for a mouse or hamster cell line, such as a CHO cell line or an NS0 (non-secreting) myeloma cell line. According to some embodiments, the expression vector is an expression vector for CHO cells. The CMV promoter is generally the human cytomegalovirus immediate early promoter, which is found, for example, in the pcDNA3.1 or pCMV vector.

[0007] As described above, the expression vector includes a selection cassette comprising a nucleotide sequence encoding an enzyme that confers resistance to an antibiotic to a bacterial host. The respective antibiotic is, in some embodiments, ampicillin. A suitable enzyme that can be encoded as a bacterial selection marker that confers resistance to ampicillin to a bacterial host is beta-lactamase.

[0008] According to some embodiments of the expression vector, the glutamine synthetase encoded by the sequence contained in the expression vector has a sequence that is 98% or more identical to SEQ ID NO:5 and is capable of catalyzing the ATP-dependent conversion of glutamate and ammonia to glutamine.

[0009] According to some embodiments, the selection cassette comprising a eukaryotic selectable marker comprises a nucleotide sequence encoding a mammalian glutamine synthetase as the eukaryotic selectable marker. In some embodiments, the glutamine synthetase has a nucleic acid sequence at least 90% identical to the sequence of SEQ ID NO:3.

[0010] In some embodiments, the glutamine synthetase comprises a CHO glutamine synthetase as a eukaryotic selectable marker. In some embodiments, the glutamine synthetase has a nucleic acid sequence at least 96% identical to the sequence of SEQ ID NO:3.

[0011] In some embodiments, the expression vector further comprises an expression-enhancing sequence element (EASE). In some embodiments, the expression vector further comprises a selection cassette as a eukaryotic selection marker, the selection cassette comprising a nucleotide sequence encoding an enzyme that confers resistance to the antibiotic puromycin, operably linked to a 3-phosphoglycerate kinase (PGK) promoter and a polyadenylation (pA) signal. In some embodiments, the enzyme that confers resistance to the antibiotic puromycin is puromycin-N-acetyltransferase (pac).

[0012] According to some embodiments of the expression vector, the bacterial origin of replication is a pUC origin of replication. According to some embodiments, the PGK promoter has a sequence at least 98% identical to SEQ ID NO:2 and is a functional promoter. According to some embodiments, the glutamine synthetase is a CHO glutamine synthetase having the sequence of SEQ ID NO:3. According to some embodiments, the PGK promoter comprises the sequence of SEQ ID NO:2. According to some embodiments, the glutamine synthetase is a CHO glutamine synthetase comprising the sequence of SEQ ID NO:3. According to some embodiments, the sequence comprising each CHO glutamine synthetase and the sequence of the 3-phosphoglycerate kinase (PGK) promoter operably linked thereto has the sequence of SEQ ID NO:1. According to some embodiments, the CMV promoter has the sequence of SEQ ID NO:4.

[0013] According to some embodiments, the pA signal is a simian virus 40 pA signal. The simian virus 40 pA signal may in some embodiments be an early pA and a late pA signal. According to some embodiments, the bacterial selectable marker that confers resistance to ampicillin to a bacterial host is a beta-lactamase having 95% identity to the sequence of SEQ ID NO:6.

[0014] In a second aspect, there is provided a recombinant host cell, the host cell comprising an expression vector according to the first aspect. In some embodiments, the recombinant host cell is a CHO cell. In some embodiments, the recombinant host cell is an NS0 cell. In some embodiments, the recombinant host cell is a COS-7 monkey kidney cell. In some embodiments, the recombinant host cell is a 3T3 cell. In some embodiments, the recombinant host cell is a baby hamster kidney (BHK) cell. In some embodiments, the recombinant host cell is a human embryonic kidney 293 cell.

[0015] In a third aspect, there is provided a method of producing a polypeptide. The method comprises culturing a recombinant host cell according to the second aspect under conditions suitable for expressing a heterologous target polypeptide. The recombinant host cell according to the second aspect comprises an expression vector comprising a nucleotide sequence encoding a polypeptide as the heterologous target polypeptide at an insertion site for the nucleotide sequence encoding the target polypeptide.

[0016] In typical embodiments of the method according to the third aspect, a nucleotide sequence encoding a polypeptide as the heterologous target polypeptide is operably linked to a CMV promoter and to a pA signal. In some embodiments, the method according to the third aspect comprises maintaining the host cells, e.g., CHO cells or NS0 cells, in suspension in a suitable medium. In some embodiments, the method according to the third aspect comprises maintaining the host cells, e.g., CHO cells or NS0 cells, at a temperature in the range of 33-38° C. Illustratively, the temperature may be selected to be about 37° C.

[0017] In a fourth aspect, there is provided the use of an expression vector according to the first aspect to express a polypeptide at high levels. In a fifth aspect, there is provided the use of a host cell according to the second aspect to express a polypeptide at high levels. [Brief explanation of the drawings]

[0018] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1A-1B] Figure 1A is a vector map of the known vector pcDNA 3.1(+), which is the parent vector of GA fragment 1. Figure 1B is a vector map of pMBL C-GS. [Figure 1C-1D] Figure 1C is a vector map of pMBL CE-GS. Figure 1D is a linear representation of the vector pMBL CE-GS.

[0019] [Figure 2A-2B]Figure 2A shows the sequence of SEQ ID NO: 1, which contains the sequence of CHO glutamine synthetase operably linked to the PGK promoter as a eukaryotic selectable marker, and Figure 2B shows the sequence of SEQ ID NO: 2, which is the PGK promoter. [Figure 2C-2E(1)] Figure 2C shows the sequence of SEQ ID NO: 3, an open reading frame encoding glutamine synthetase as a eukaryotic selectable marker. Figure 2D shows the sequence of SEQ ID NO: 4, a CMV promoter sequence. Figure 2E shows the sequence of SEQ ID NO: 14, an expression-enhancing sequence element sequence. [Figure 2E(2)] FIG. 2E depicts the sequence of SEQ ID NO: 14, which is the sequence of the expression-enhancing sequence element.

[0020] [Figure 3] FIG. 3 is a vector map of pMBL CE-Puro. [Figure 4] FIG. 4 is a vector map of pMBL C-Puro. [Figure 5A] FIG. 5A shows a gel electrophoresis restriction analysis of the vector pMBL C-GS. [Figure 5B-5C] Figure 5B shows a gel electrophoretic restriction analysis of the vector pMBL CE-GS. Figure 5C shows a gel electrophoretic restriction analysis of the vector pMBL CE-Puro. [Figure 5D] FIG. 5D shows a gel electrophoresis restriction analysis of the vector pMBL C-Puro.

[0021] [Figure 6] FIG. 6 is a table providing a summary of the monoclonal antibodies expressed using vectors according to the present disclosure.

[0022] [Figure 7]Figure 7A shows the titer of the anti-CTLA4 antibody Bmab700 expressed using the vector pMBL CE-GS (a) and vector pMBL C-GS (b), compared with expression after cotransfection of the HC and LC of the same anti-CTLA4 antibody in two separate pCHO 1.0 vectors (c) and expression using a single pCHO 1.0 vector in which both the HC and LC were cloned (d). Figure 7B shows the titers of four different anti-CD20 fusion antibodies expressed using pMBL CE-GS at days 5, 7, 9, 12, and 14. Both the HC and LC were cloned into the vector pMBL CE-GS at the XhoI-NotI sites. Days are indicated above the bars. Transient transfections were performed in cotransfection mode. All transfections were performed using the ExpiCHO™ Expression System (Cat. No. A29133, Thermo Fisher Scientific Inc., Waltham, MA, USA), a high-yield transient expression system based on suspension-adapted Chinese hamster ovary (CHO) cells. ExpiCHO-S cells were thawed according to the ExpiCHO-S Expression System manual. After a minimum of two passages after thawing, cells were seeded for transfection at 3.5 × 10 cells / ml one day prior to transfection. One day after seeding, cell counts were estimated and adjusted to 6.0 × 10 cells / ml by dilution with fresh prewarmed medium for each transfection. Transfections were performed according to the ExpiCHO-S Expression System manual. DNA was transfected at a final concentration of 1 μg / ml in the culture. Expifectamine and feed were added according to the manufacturer's protocol. The maximum titer protocol was followed for all transfections. Transfection flasks were cultured in a humidified atmosphere of 8% CO2 in air in an incubator at 37°C on an orbital shaker. VCD and viability were estimated on days 0, 2, 5, 7, 9, and 12. Cultures were harvested when viability fell below 50% or at D-12.

[0023] [Figures 8A-8C] Figure 8A shows the time course of viable cell density for CHO cells expressing anti-CD20 HC-C-TGF□RII fusion antibodies (Fmab25 / 1 and Fmab25 / 2), anti-CD20 LC-C-TGF□RII fusion antibodies (Fmab26 / 1 and Fmab26 / 2), anti-CD20 HC-N-TGF□RII fusion antibodies (Fmab27 / 1 and Fmab27 / 2), and anti-CD20 LC-N-TGF□RII fusion antibodies (Fmab28 / 1 and Fmab28 / 2), all transiently expressed using the vector pMBL CE-GS. Figure 8B shows the time course of cell viability (in percentage). Figure 8C shows the time course of the titer of the produced product. Measurements were performed on days 0, 2, 5, 7, 9, 12, and 14. Numbers above the bars represent the respective days of culture: a=day 12, b=day 14. [Figure 8D] Figure 8D shows the harvest titer of the produced product on day 14. Harvest titer analysis was performed on the samples on day 14 based on the HPLC method. Transient expression studies were performed as described in paragraph

[0032] .

[0024] [Figure 9A-9B] Figure 9A shows the time course of viable cell density for CHO cells expressing anti-CD20 wild-type antibody, anti-CD20 HC-C-TGFβRII fusion antibody (Fmab25), and anti-CD20 TIM3 LC-C-terminus fusion antibody (Fmab30), and Figure 9B shows the time course of cell viability in percentage. Growth profiles were monitored on days 0, 2, 5, 7, and 9. Numbers above the bars represent the respective days of culture. [Figure 9C-9D] Figure 9C shows the time course of the titer of the produced product. Measurements were taken on days 5, 7, and 9. The numbers above the bars represent the respective days of culture. Figure 9D shows the harvest titer of the produced product on day 9. The numbers above the bars indicate the titer obtained. Analysis was based on HPLC.

[0025] [Figure 10A] Figure 10A shows the time course of viable cell density of CHO cells expressing anti-PDL-1 antibody, and Figure 10B shows the time course of cell viability in percentage. Measurements were taken on days 0, 2, 5, 7, 9, and 12. The letters above the bars indicate the respective antibody chains expressed: a-c: atezolizumab, d-f: anti-PDL-1 antibody. [Figures 10B-10C] Figure 10A shows the time course of viable cell density of CHO cells expressing anti-PDL-1 antibody, and Figure 10B shows the time course of cell viability in percentage. Measurements were taken on days 0, 2, 5, 7, 9, and 12. The letters above the bars indicate the respective antibody chains expressed: a-c: atezolizumab, d-f: anti-PDL-1 antibody. Figure 10C shows the harvest titer of the produced product on day 14. The numbers above the bars indicate the titer obtained. Analysis was based on HPLC.

[0026] [Figure 11A] Figure 11A shows the time course of viable cell density of CHO cells expressing additional anti-PDL-1 antibodies, and Figure 11B shows the time course of cell viability in %. Measurements were taken on days 0, 5, 9, and 12. The numbers above the bars represent the respective days of culture. [Figures 11B-11D] Figure 11A shows the time course of viable cell density of CHO cells expressing additional anti-PDL-1 antibodies, and Figure 11B shows the time course of cell viability in % thereof. Measurements were performed on days 0, 5, 9 and 12. The numbers above the bars represent the respective days of culture. Figure 11C shows the time course of the titer of the produced product. Measurements were performed on days 9 and 12. The numbers above the bars represent the respective days of culture. Figure 11D shows the harvest titer of the produced product on day 12. The numbers above the bars represent the titer obtained. Analysis was based on HPLC.

[0027] [Figure 12]Figure 12A shows the percent viability in six flasks during the selection phase in the absence of glutamine, and Figure 12B shows the viable cell density. Flasks 2A, 2B, 3A, and 3B contained CHO cells from various CHO-S GS knockout hosts stably transfected with pMBL CE-GS by electroporation. Flasks 5 and 6 represent mock cell controls containing CHO cells electroporated without DNA for C12-1 and C#26-2, respectively. Flasks 2A and 2B and flasks 3A and 3B were replicate flasks of the same cell line, respectively. pMBL CE-GS encoded etanacept, a fusion protein of the extracellular ligand-binding domain of the p75 TNF receptor and the human Fc portion of IgG1. Day 0 of each experiment was the day of seeding. Days 7 / 6, 6, 3, and 4 were days of selection, respectively.

[0028] [Figures 13A-13C] Figure 13A shows the time course of viable cell density of the same flask of CHO cells stably expressing etanacept as in Figures 12A and B, and Figure 13B shows the cell viability in %. Glucose was used as a feed in simple fed-batch culture, and data were collected at passage No. 5. Figure 13C shows the time course of the titer of the produced product. Measurements were taken on days 5, 7, 10, and 14. The numbers above the bars indicate the respective days of culture. [Figure 13D] Figure 13D shows the harvest titer of the produced product on day 14. Measurements were taken on days 5, 7, 10 and 14. The titer was estimated using HPLC.

[0029] [Figures 14A-14B]Figure 14A shows the time course of viable cell density for the same pool of CHO cells stably expressing etanacept as in Figures 12A and 12B and 13, and Figure 14B shows the percentage of cell viability. CB4 (Cell Boost-4, Hydroclone., GE Healthcare Life Sciences) and EFC (Efficient feed C, Thermo Fisher Scientific Inc., Waltham, MA, USA) were used as feeds in fed-batch culture, and data were collected at passage No. 7. [Figure 14C-14D] Figure 14C shows the time course of the titer of the produced product. Measurements were taken on days 7 and 12. The numbers above the bars represent the respective days of culture. Figure 14D shows the harvest titer of the produced product on day 12.

[0030] [Figure 15] Figure 15 presents a Western blot analysis of the harvest samples for which data are provided in Figures 13 and 14. Unpurified harvest supernatants from fed-batch (see Figure 14) and simple fed-batch (see Figure 13) were analyzed. DETAILED DESCRIPTION OF THE INVENTION

[0031] Detailed Description of the Invention In order that the description of the nucleic acid molecules, vectors, host cells, methods and uses disclosed herein may be more readily understood, certain terms are first defined. definition Unless otherwise stated, the following terms used in this document, including the description and claims, have the definitions given below.

[0032] As used herein, the term "about" refers to a value within an acceptable error range for a specific value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within one or more standard deviations, according to common practice in the art. The term "about" is also used to indicate that the amount or value in question may be the specified value or some other value that is approximately the same. The phrase is intended to convey that similar values ​​promote equivalent results or effects according to the present invention. In this context, "about" may refer to a range above and / or below by up to 10%. In some embodiments, the word "about" refers to a range above or below a certain value, for example, up to 5%, for example, up to 2%, up to 1%, or up to 0.5%. In one embodiment, "about" refers to a range above and below by up to 0.1% of a given value.

[0033] The term "consisting essentially of" is understood to allow for the presence of additional components in the sample or composition that do not affect the properties of the sample or composition. As an illustrative example, a pharmaceutical composition may contain excipients when it consists essentially of the active ingredient.

[0034] The terms "express" and "expression" with respect to polypeptides are intended to be understood in the ordinary sense used in the art. Polypeptides are expressed by cells through transcription of nucleic acids into mRNA, followed by translation into polypeptides, which are folded and optionally further processed. With respect to each biological process itself, the terms "expression," "gene expression," or "express" refer to the entire regulatory pathway that converts the information encoded in the nucleic acid sequence of a gene first into messenger RNA (mRNA) and then into a protein. Consequently, the expression of a gene includes its transcription into primary hnRNA, the processing of this hnRNA into mature RNA, and the translation of the mRNA sequence into the corresponding amino acid sequence of the polypeptide. In this context, it should be noted that the term "gene product" refers not only to polypeptides, including, for example, the final polypeptide (including its splice variants) and each precursor polypeptide encoded by the gene, but also to each mRNA, which, if applicable, can be considered the "first gene product" in the process of gene expression.

[0035] The term "expression vector" or "expression construct" refers to a nucleic acid vehicle, such as a plasmid, that enables the expression of a desired target polypeptide in a host cell using the host cell's transcription and translation machinery. A nucleic acid molecule can be introduced into a respective host cell and comprises one or more regulatory sequences operably linked to a nucleic acid sequence encoding the target polypeptide.

[0036] "Fragment," in the context of a polypeptide such as an immunoglobulin or proteinaceous binding molecule, refers to any amino acid sequence present in the corresponding polypeptide, so long as it is shorter than the full-length sequence and is capable of performing the polypeptide's function of interest—in the case of an immunoglobulin, specifically binding to a desired target, e.g., an antigen such as PDL-1. The term "immunoglobulin fragment" refers to a portion of an immunoglobulin, often the hypervariable region and portions of the surrounding heavy and light chains, that exhibits specific binding affinity for a particular molecule. The hypervariable region is the portion of the immunoglobulin that physically binds to the polypeptide target.

[0037] As used herein, the term "nucleic acid molecule" refers to any nucleic acid in any possible configuration, such as single-stranded, double-stranded, or a combination thereof. Examples of nucleic acids include, for example, DNA molecules, RNA molecules, DNA or RNA analogs produced using nucleotide analogs or nucleic acid chemistry, locked nucleic acid molecules (LNA), protein nucleic acid molecules (PNA), alkyl phosphonate and alkyl phosphotriester nucleic acid molecules, and tecto-RNA molecules (see, for example, Liu, B., et al., J. Am. Chem. Soc. (2004) 126, 4076-4077). LNA has a modified RNA backbone with a methylene bridge between C4' and O2', providing the respective molecules with high duplex stability and nuclease resistance. Alkyl phosphonate and alkyl phosphotriester nucleic acid molecules can be viewed as DNA or RNA molecules in which the phosphate groups of the nucleic acid backbone have been neutralized by replacing the P-OH group with an alkyl or alkoxy group, respectively. The DNA or RNA may be of genomic or synthetic origin and may be single-stranded or double-stranded. Such nucleic acids may be, for example, mRNA, cRNA, synthetic RNA, genomic DNA, cDNA, synthetic DNA, copolymers of DNA and RNA, oligonucleotides, etc. Each nucleic acid may further contain non-natural nucleotide analogs and / or be linked to an affinity tag or label.

[0038] Many nucleotide analogs are known and can be used in the nucleic acids used in the methods of the present invention. Nucleotide analogs are, for example, nucleotides containing modifications in the base, sugar, or phosphate moiety. As an illustrative example, replacing 2'-OH residues in siRNA with 2'F, 2'O-Me, or 2'H residues is known to improve the in vivo stability of the respective RNA. Modifications in the base moiety can be A, C, G, and T / U, various purine or pyrimidine bases such as uracil-5-yl, hypoxanthine-9-yl, and 2-aminoadenine-9-yl, as well as natural or synthetic modifications of non-purine or non-pyrimidine nucleotide bases. Other nucleotide analogs function as universal bases. Examples of universal bases include 3-nitropyrrole and 5-nitroindole. Universal bases are capable of base pairing with any other base. Base modifications are often combined with sugar modifications, such as 2'-O-methoxyethyl, to achieve unique properties, such as increased duplex stability.

[0039] The terms "polypeptide" and "protein" refer to a polymer of amino acid residues and are not limited to a certain minimum length of the product. When both terms are used simultaneously, this dual nomenclature is intended to explain the side-by-side use of both terms in the art.

[0040] As used herein, the terms "polyadenylation site," "polyA site," or "polyA sequence" refer to a nucleic acid sequence, such as a DNA sequence, that can direct both the termination and polyadenylation of a nascent RNA transcript. Efficient polyadenylation of recombinant transcripts is advantageous because transcripts lacking a polyA tail are often unstable and rapidly degraded. The polyA signal utilized in the vectors disclosed herein may be of any origin and may be "endogenous." An endogenous polyA signal is one that is naturally found at the 3' end of the coding region of a given gene in the genome. A commonly used heterologous polyA signal is the SV40 polyA signal. Examples of suitable polyadenylation sequences also include, but are not limited to, the bovine growth hormone (bGH) polyadenylation signal, the beta-globin polyA site, and the herpes simplex virus thymidine kinase polyA site.

[0041] The term "purified" is understood to be a relative indication compared to the cell's original environment, thereby indicating that the cell is relatively purer than its natural environment. It thus includes, but is not limited to, absolute purity from other cells (such as a homogeneous cell population). Compared to the natural level, the level after purification of cells is generally at least 2-5 times greater (e.g., in terms of cells / ml). Purification of at least one order of magnitude, such as about 2- or 3-fold, including about 4- or 5-fold, is expressly contemplated. It may be desirable to obtain cells at a functionally significant level of purity, e.g., about 90%, about 95%, or 99%, at least essentially free of contaminants, particularly free of other cells. The above applies mutatis mutandis to nucleic acids, peptides, or proteins. In this case, purification of nucleic acids, peptides, or proteins is illustratively generally at least 2- to 5-fold greater (e.g., in terms of mg / ml).

[0042] The term "recombinant" is used in this document to describe a nucleic acid molecule that is not related by its origin, manipulation, or both to all or a portion of the nucleic acid molecule with which it is naturally associated. Generally, a recombinant nucleic acid molecule contains a sequence that does not naturally occur in the respective wild-type organism or cell. Typically, recombinant nucleic acid molecules are obtained by genetic engineering and are often constructed outside a cell. Generally, a recombinant nucleic acid molecule is substantially identical to and / or substantially complementary to at least a portion of a corresponding nucleic acid molecule occurring in nature. Recombinant nucleic acid molecules may be of any origin, such as genomic, cDNA, mammalian, bacterial, viral, semisynthetic, or synthetic origin. The term "recombinant" as used with respect to a protein / polypeptide refers to a polypeptide produced by expression of a recombinant polynucleotide.

[0043] Terms such as "comprising," "including," "containing," "having," and the like are intended to be read expansively or open-ended and without limitation. Singular forms such as "a," "an," or "the" include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to a "vector" includes a single vector and multiple vectors, either the same—e.g., the same operon—or different. Similarly, a reference to a "cell" includes a single cell and multiple cells. Unless otherwise indicated, the term "at least" preceding a series of elements is understood to refer to every element in the series. The terms "at least one" and "at least one of" include, for example, one, two, three, four, or five or more elements. Furthermore, it is understood that slight variations above and below the stated ranges can be used to achieve substantially the same results as values ​​within the range. Also, unless otherwise indicated, the ranges of this disclosure are intended as continuous ranges, including every value between the minimum and maximum values.

[0044] The scope and meaning of any use of a term will be apparent from the specific context in which the term is used. Certain further definitions of selected terms used throughout this document will be given in the appropriate context of the detailed description, where applicable. Unless otherwise defined, all other scientific and technical terms used in the description, figures, and claims have their ordinary meanings as commonly understood by those skilled in the art.

[0045] The present invention provides vectors, i.e., circular nucleic acid molecules, capable of driving high-level heterologous protein expression in mammalian cells, particularly CHO cells, as well as methods and uses based thereon. The vectors are replicable in bacterial cells. Vectors according to the present disclosure that are expression vectors generally include a promoter, a transcription terminator sequence, an origin of replication, a selectable marker, an insertion site for the nucleotide sequence, and regulatory elements.

[0046] A promoter is a region of DNA that initiates the transcription of a specific gene. The cytomegalovirus promoter is an example of a mammalian expression promoter derived from cytomegalovirus. The CMV promoter is a strong promoter and drives the constitutive expression of genes under its control, so it is commonly used in vectors used in genetic engineering work carried out in mammalian cells. The promoter is operably linked to the insertion site for the nucleotide sequence encoding the target polypeptide.

[0047] Transcription terminators are portions of nucleic acid sequences that mark the end of a gene or operon in genomic DNA for the transcription process. Such sequences mediate transcription termination by providing a signal in newly synthesized mRNA that triggers the process of releasing the mRNA from the transcription complex. A transcription terminator sequence may be operably linked to the insertion site for the nucleotide sequence encoding the target polypeptide.

[0048] The origin of replication, also called the replication origin, is a specific sequence in the genome where replication begins. DNA replication typically begins at a single replication origin. In E. coli, the replication origin is called oriC. This region is included in expression vectors that can autonomously replicate in E. coli to form multiple copies. A selectable marker is a gene that is introduced into cells, such as bacteria, that confers a trait suitable for artificial selection.

[0049] Transcription of eukaryotic genes is regulated by a variety of cis- and trans-acting regulatory elements. Two of the best-characterized cis elements are promoters and enhancers. A promoter (see above) is a nucleic acid sequence immediately 5' to the coding sequence of a gene; it contains multiple binding sites for trans-acting transcription factors, forming the basal transcription machinery. Enhancers generally contain multiple binding sites for trans-acting transcription factors, which are found far upstream or downstream of the coding sequence, or even within introns. A cis element included in some embodiments of the vectors disclosed herein is an expression-enhancing sequence element (EASE). EASE elements contribute to vector stability, which is important for stable cell line development.

[0050] As disclosed herein, the expression vector comprises three separate expression cassettes. The first cassette is an expression cassette for expressing a selectable marker protein that allows for the selection of eukaryotic cells containing the vector. This expression cassette is also referred to herein as a selection cassette. The second cassette is an expression cassette for expressing a selectable marker protein that allows for the selection of bacterial cells containing the expression vector. This expression cassette is also referred to herein as a selection cassette. The third cassette is an expression cassette for expressing a target polypeptide. This expression cassette generally comprises a site for inserting a nucleotide sequence encoding the target polypeptide downstream and operably linked to the promoter of the expression cassette.

[0051] The third cassette insertion site typically contains at least one restriction enzyme recognition sequence. It may contain two or more restriction enzyme recognition sequences and define a multiple cloning site. In some embodiments, the insertion site contains a recognition sequence for the Not I enzyme. In some embodiments, the insertion site contains a recognition sequence for the Xho I enzyme. In some embodiments, the insertion site contains a recognition sequence for the BamH I enzyme. In some embodiments, the insertion site contains a recognition sequence for the Nhe I enzyme. In some embodiments, the insertion site contains a recognition sequence for the EcoR I enzyme. In some embodiments, the insertion site contains a recognition sequence for the EcoR V enzyme. In some embodiments, the insertion site contains a recognition sequence for the Pme I enzyme. In some embodiments, the insertion site contains a recognition sequence for the Afl II enzyme. In some embodiments, the insertion site contains a recognition sequence for the Hind III enzyme. In some embodiments, the insertion site contains a recognition sequence for the Kpn I enzyme. In some embodiments, the insertion site contains a recognition sequence for the Xba I enzyme. In some embodiments, the insertion site contains a recognition sequence for the BstX I enzyme. In some embodiments, the insertion site contains a recognition sequence for the Pme I enzyme. Cleavage of the circular vector with either one or two of the enzymes for which the restriction enzyme recognition sequence is present creates a linear vector to which a nucleotide sequence encoding a target polypeptide with appropriate termini can be attached.

[0052] The three expression cassettes may be arranged in any order relative to each other in the vector. In some embodiments, the first expression cassette for expressing a selectable marker protein that allows for the selection of eukaryotic cells and the second expression cassette for expressing a selectable marker protein that allows for the selection of bacterial cells are arranged in opposite directions in the vector. In some embodiments, the first expression cassette and the second expression cassette are arranged in the same direction in the vector. In some embodiments, the first expression cassette for expressing a selectable marker protein that allows for the selection of eukaryotic cells and the third expression cassette for expressing a target polypeptide are arranged in the same direction in the vector. In some embodiments, the first and third expression cassettes are arranged in opposite directions in the vector. In some embodiments, the second expression cassette for expressing a selectable marker protein that allows for the selection of bacterial cells and the third expression cassette for expressing a target polypeptide are arranged in opposite directions in the vector. In some embodiments, the second and third expression cassettes are arranged in the same direction in the vector. Illustrative examples of the order and orientation of three expression cassettes are shown in FIGS. 1B, 1C, 3 and 4.

[0053] The eukaryotic selectable marker present in the vectors of the present disclosure is a nucleic acid sequence encoding glutamine synthetase. The enzyme glutamine synthetase is responsible for the biosynthesis of glutamine from glutamate and ammonia. This enzymatic reaction provides the only route for glutamine synthesis in mammalian cells. In the absence of glutamine in the growth medium, the GS enzyme is essential for the survival of mammalian cells in culture. Mammalian cell lines, such as Chinese hamster ovary (CHO) cell lines, express sufficient GS to survive in the absence of exogenous glutamine. GS is the most commonly used selectable marker for selecting transfectants while creating stable cell lines. In cell lines where the host's endogenous GS is nonfunctional due to an inactivating mutation or deletion, supplementation of exogenous GS through the vector allows for the selection of transfected populations containing the desired vector. The resulting recombinant cell lines can be screened in glutamine-free medium, thereby reducing the time and increasing the probability of obtaining high-titer clones.

[0054] In the vector disclosed herein, the enzyme glutamine synthetase is encoded by a nucleic acid sequence operably linked to a PGK promoter.In some embodiments, PGK is PGK-1.The PGK-1 gene encodes 3-phosphoglycerate kinase, a housekeeping enzyme in the glycolytic pathway.Therefore, it is ubiquitously expressed.This gene is located on the X chromosome in mammals.In female mammalian somatic cells, only the PGK-1 allele on the active X chromosome is transcribed, and the other PGK-1 allele on the inactive X chromosome is inactive.Therefore, PGK-1 is always expressed except when it is silenced together with most other genes on the inactive X chromosome in female somatic cells or male germ cells.Therefore, the PGK-1 promoter is active in almost all somatic cells and germ cell types, and is therefore used for gene expression at a high constitutive level.

[0055] In some embodiments, the PGK-1 promoter has the sequence of SEQ ID NO: 2. The PGK-1 promoter of SEQ ID NO: 2 is contained, for example, in a vector represented in Table 2 of WO2014 / 200557. It is also contained in the cloning vector PGK1p-Csy4-pA, GenBank accession number KJ796485.1, dated August 16, 2014, version 1, or the cloning vector PBDGTV, GenBank accession number KU179219.1, dated January 17, 2017, version 1.

[0056] In some embodiments, the PGK promoter has a sequence that is at least 98% identical to the sequence of SEQ ID NO: 2. As an illustrative example, the mutagenesis vector pMtKCNQ2 DNA, GenBank Accession No. AB535097.1, version 1, dated December 4, 2009, has the PGK promoter sequence at positions 2984-3491 of SEQ ID NO: 2, lacking bases 371-382. As another example, the mPGK1 promoter at positions 6435-6924 of the vector pROSA26-DV3, GenBank Accession No. LT726831.1, version 1, dated February 6, 2017, has a six single base deletion compared to the sequence of SEQ ID NO: 2. In some embodiments, the PGK promoter has a sequence that is at least 99% identical to the sequence of SEQ ID NO: 2. For example, the mouse phosphoglycerate kinase promoter at positions 6566-7056 of the plasmid vector pHM2, GenBank Accession No. X76683.1, version 1, dated February 9, 1994, has five deleted bases and one substitution compared to the sequence of SEQ ID NO: 2. As a further example, the complementary sequence of the PGK-1 promoter at positions 4395-4888 of the cloning vector pGZ-DSB-CO, GenBank Accession No. KY447298.1, version 1, dated February 22, 2017, has a two base deletion compared to the sequence of SEQ ID NO: 2.

[0057] In some embodiments, the sequence encoded as CHO glutamine synthetase is the sequence of SEQ ID NO: 5. The sequence is found in GenPept under accession number AJHYQ dated June 3, 2002. In some embodiments, the sequence encoded as CHO glutamine synthetase is a sequence that is 99% or more identical to SEQ ID NO: 5. The sequence may, for example, be the sequence encoded by SwissProt / UniProt accession number G3HG36, version 1 of the sequence dated November 16, 2011, entry version 30 dated May 23, 2018 (which is the sequence encoded by GenBank accession number RLQ66161.1, version 1 dated October 21, 2018). In some embodiments, the sequence encoded as CHO glutamine synthetase is a sequence that is 97% or more identical to SEQ ID NO: 5. In some embodiments, the sequence encoded as CHO glutamine synthetase is a sequence that is 96% or more identical to SEQ ID NO: 5. As an illustrative example, the sequence may be the sequence of SwissProt / UniProt accession number P04773, version 4 of the sequence as of January 23, 2007, or version 107 of the entry as of December 5, 2018. The sequence encoded as CHO glutamine synthetase, in some embodiments, comprises the sequence of SEQ ID NO: 5. The sequence encoded as CHO glutamine synthetase, in some embodiments, comprises a sequence 99% identical to SEQ ID NO: 5. The sequence encoded as CHO glutamine synthetase, in some embodiments, comprises a sequence 99% or greater identical to SEQ ID NO: 5. The sequence encoded as CHO glutamine synthetase, in some embodiments, comprises a sequence 97% or greater identical to SEQ ID NO: 5. The sequence encoded as CHO glutamine synthetase, in some embodiments, comprises a sequence 96% or greater identical to SEQ ID NO: 5. In one embodiment, the sequence may be the sequence of SwissProt / UniProt accession number G3IH33, version 1 of the sequence dated November 16, 2011, or version 22 of the entry dated December 5, 2018.

[0058] In some embodiments, the sequence encoding the CHO glutamine synthetase has the sequence of SEQ ID NO: 3. The sequence encoding the CHO glutamine synthetase of SEQ ID NO: 3 is contained, for example, as positions 147-1268 in the sequence of the mRNA encoding Chinese hamster glutamine synthetase in GenBank Accession No. X03495.1, database entry version 1 dated April 21, 1993, last updated February 4, 2011. As a further example, the sequence is found as SEQ ID NO: 10 in WO2013 / 186371 or as SEQ ID NO: 1 in EP2 825 641.

[0059] The glutamine synthetase (GS) gene expression system (Birch JR and Racher AJ, Advanced Drug Delivery Reviews 2006; 58:671-685) is one of two expression vector systems commonly used in monoclonal antibody production. Another common expression system is based on the dihydrofolate reductase (DHFR) gene. The GS system is particularly useful for CHO and NS0 cells, which rely on the metabolic pathway of glutamate and ammonium to glutamine for selection of recombinant cells. CHO cells already express endogenous GS. Addition of a selective GS inhibitor, such as methionine sulfoximine (MSX), to glutamine-free culture medium selects cell clones with an integrated gene construct containing the GS gene.

[0060] In some embodiments, a sequence encoding a glutamine synthetase, e.g., a CHO glutamine synthetase, has a sequence at least 97% identical to the sequence of SEQ ID NO: 3. The CHO glutamine synthetase sequence, NCBI accession number NM_001246770.1, version 1, dated October 9, 2011, contains a promoter sequence at positions 1-1116 that differs from the sequence of SEQ ID NO: 3 by 39 base substitutions. In some embodiments, a sequence encoding a CHO glutamine synthetase has a sequence at least 92% identical to the sequence of SEQ ID NO: 3. For example, the mouse glutamine synthetase sequence, GenBank accession number X16314.1, version 1, dated April 4, 1995, has 91 base substitutions compared to the sequence of SEQ ID NO: 3. In some embodiments, a sequence encoding a CHO glutamine synthetase has a sequence 90% identical to the sequence of SEQ ID NO: 3. By way of example, human glutamine synthetase, GenBank Accession No. BC051726, version 1, dated May 14, 2003, has a sequence at positions 1307-2422 that differs by 106 substitutions from the sequence of SEQ ID NO: 3. As a further example, porcine glutamine synthetase, GenBank Accession No. AK390323.1, version 1, dated January 11, 2012, has a sequence at positions 252-1367 that differs by 116 substitutions from the sequence of SEQ ID NO: 3.

[0061] The vectors of the present disclosure include a bacterial selectable marker. The marker is a nucleotide sequence encoding an enzyme that provides antibiotic resistance to the bacterial host. In some embodiments, the enzyme provides resistance to chloramphenicol or to kanamycin and geneticin. In some embodiments, the enzyme provides resistance to ampicillin. In some embodiments, the enzyme provides resistance to streptomycin and spectinomycin.

[0062] An example of an enzyme that confers resistance to ampicillin to a bacterial host is beta-lactamase. In some embodiments, the enzyme beta-lactamase has the amino acid sequence of SEQ ID NO: 6, which is the sequence of Escherichia coli beta-lactamase, specifically, SwissProt / UniProt accession number Q79DR3, version 1 of the sequence dated July 5, 2004, entry version 116 of December 5, 2018. The sequence is also identical to the synthetic beta-lactamase construct of SwissProt / UniProt accession number Q285M4, version 1 of the sequence dated April 4, 2006, entry version 36 of May 10, 2017. In some embodiments, the beta-lactamase can be a protein having 99% or greater identity to the sequence of SEQ ID NO: 6. It may be, for example, the beta-lactamase protein encoded by the plasmid pPV having the amino acid sequence of SwissProt / UniProt accession number Q799Y1, version 1 of the sequence of July 5, 2004, entry version 42 of May 10, 2017. It may also be the class A extended-spectrum beta-lactamase TEM-1 from Bordetella avium having the amino acid sequence of SwissProt / UniProt accession number A0A3A0YVF2, version 1 of the sequence of December 5, 2018, entry version 1 of December 5, 2018.

[0063] In some embodiments, the beta-lactamase may be a protein having 98% or greater identity to the sequence of SEQ ID NO: 6. It may be, for example, a beta-lactamase protein from Escherichia coli having the amino acid sequence of SwissProt / UniProt accession number R9URM7, version 1 of the sequence dated September 18, 2013, entry version 19 of December 5, 2018. In some embodiments, the beta-lactamase may be a protein having 96% or greater identity to the sequence of SEQ ID NO: 6. It may be, for example, a beta-lactamase protein from Serratia marcescens having the amino acid sequence of SwissProt / UniProt accession number O33677, version 1 of the sequence dated January 1, 1998, entry version 74 of December 5, 2018. In some embodiments, the beta-lactamase may be a protein having 95% or greater identity to the sequence of SEQ ID NO: 6. It may be, for example, a beta-lactamase protein from Acinetobacter baumannii having the amino acid sequence of SwissProt / UniProt accession number H9AXM0, version 1 of the sequence of May 16, 2012, version 29 of the entry of December 5, 2018.

[0064] In some embodiments, the beta-lactamase may be a protein having 40% or greater identity to the sequence of SEQ ID NO: 6. As an illustrative example, the enzyme beta-lactamase from the CDC 1551 strain of Mycobacterium tuberculosis, having SwissProt / UniProt accession number P9WKD2, sequence version 1 of the April 16, 2014 entry, sequence version 23 of the December 5, 2018 entry, has a sequence of 381 amino acids, 93 of which are identical to the sequence of SEQ ID NO: 6. In some embodiments, the beta-lactamase may be a protein having 2% or greater identity to the sequence of SEQ ID NO: 6. As an illustrative example, the enzyme beta-lactamase from Enterobacter cloacae, having SwissProt / UniProt accession number P05364, sequence version 1 of the November 1, 1988 entry, and sequence version 109 of the December 5, 2018 entry, has a sequence of 381 amino acids, 5 of which are identical to the sequence of SEQ ID NO:6.

[0065] The vectors disclosed herein may also contain sequences encoding enzymes that confer resistance to the antibiotic puromycin. Each enzyme may be aminoglycoside phosphotransferase (aph). The enzyme that confers resistance to the antibiotic puromycin may also be blasticidin S deaminase (bsd). Each enzyme may also be puromycin N-acetyltransferase (pac).

[0066] In some embodiments, the sequence encoding puromycin-N-acetyltransferase can be the sequence of bases 3094-3954 of the cloning vector pL1F-3, version 1, GenBank Accession No. KF955552.1, dated March 3, 2014. This sequence is SEQ ID NO: 7, and is also found, for example, at base positions 6391-7251 of the vector of SEQ ID NO: 87 in U.S. Patent US 10,113,179, or positions 6565-7425 of the vector of SEQ ID NO: 88 in the same patent, or positions 6979-7839 of the repair donor cassette of SEQ ID NO: 221. In some embodiments, the sequence is the complement of SEQ ID NO: 7, which is SEQ ID NO: 8. In some embodiments, the sequence encoding puromycin-N-acetyltransferase may be the sequence of the extended-spectrum beta-lactamase (TEM) gene of Escherichia coli strain SRT41 having GenBank accession number MG653169.1, version 1, dated May 8, 2018. Both sequences encode the amino acid sequence of SEQ ID NO:6.

[0067] The promoter used for the enzyme that confers resistance to ampicillin to the bacterial host may be the beta-lactamase promoter, for example the promoter of the sequence SEQ ID NO: 9. The complementary sequence is the sequence SEQ ID NO: 10.

[0068] A vector contains an insertion site for a nucleotide sequence encoding a target polypeptide to be expressed. The insertion site contains restriction recognition sites for one or more restriction enzymes. Typically, a vector has a multiple cloning site, also called a polylinker. A multiple cloning site is a short segment of nucleic acid sequence containing multiple restriction sites. It may contain up to about 20 restriction sites. Restriction sites within a multiple cloning site are typically unique and occur only once within a given plasmid. The multiple cloning site allows for the insertion of a gene of interest into the multiple cloning site region. The multiple cloning site is generally located immediately after the promoter and ends before the transcription terminator.

[0069] The target polypeptide expressed by the sequence that may be included in the insertion site may be any desired polypeptide. Two illustrative examples are antibodies, including functional antibody fragments, and enzymes. Two further illustrative examples are growth factors and blood clotting factors.

[0070] The insertion site is operably linked to a CMV promoter. The CMV promoter may be of the sequence of SEQ ID NO: 4. The CMV promoter may have a sequence having 99% or greater identity to SEQ ID NO: 4. The CMV promoter may have or include, for example, the sequence of the hCMV immediate early promoter at positions 8186-8389 of the cloning vector pHR'-CMVLacZ, GenBank Accession No. AF105229.1, sequence version 1 as of December 17, 1998. As a further example, the CMV promoter may have or include the sequence of the CRU5 chimeric CMV promoter at positions 366-569 of the retroviral expression vector L149, GenBank Accession No. EU753858.1, sequence version 1 as of May 31, 2009. In some embodiments, the CMV promoter may have a sequence having 98% or greater identity to SEQ ID NO: 4. It may have or include, for example, the sequence of the promoter at positions 8806-9009 of the expression vector pDEST152, GenBank Accession No. MH107058.1, sequence version 1 as of October 30, 2018. As another example, the CMV promoter may have or include the sequence of the hCMV-IE promoter at positions 1053-1256 of the mammalian expression vector pACCMVpLpA-E-hRac1-DN, GenBank Accession No. LT727056.1, sequence version 1 as of February 6, 2017.

[0071] The vector may contain one or more additional promoters operably linked to restriction recognition sites for restriction enzymes to enable expression of desired proteins into which sequences can be inserted at the respective restriction sites. For example, a promoter of the T7 bacteriophage, such as the promoter of the T7 RNA polymerase, may be included in the vector. The sequence of SEQ ID NO: 16 is an example of a T7 promoter.

[0072] The bacterial origin of replication for the vector may be selected as desired. It is generally a plasmid origin. Typically, the origin of replication is a high copy number origin. In one embodiment, the origin is the pMB1 origin. In one embodiment, the bacterial origin of replication is the high copy number pUC origin. The pUC origin is the most common origin of replication used in bacterial vectors. It is derived from the pMB1 origin of the plasmid pBR322, but contains a point substitution within the origin and a deletion of the Rop / Rom genes. The letter "p" indicates that the origin is that of a plasmid, which is a circular, double-stranded DNA molecule. The letters "UC" stand for "University of California."

[0073] In some embodiments, the vector further contains an expression-enhancing sequence element. Such elements are disclosed in WO1997 / 025420. A DNA sequence formed by ligating two sequences, i.e., nucleotides 8672-12273 of SEQ ID NO: 1 in WO1997 / 025420, and nucleotides 14290-14507 of SEQ ID NO: 1 in WO1997 / 025420, has been described as having expression-enhancing activity; see Example 7 of WO1997 / 025420. The sequence of nucleotides 10100-14293 of SEQ ID NO: 1 in WO1997 / 025420 has also been disclosed as having expression-enhancing activity. Aldrich, TL, et al., Cytotechnology (1998) 28, 1-3, 9-17, discloses an EASE element termed a "truncated EASE element." The nucleic acid sequence corresponding to each DNA sequence may be used as an EASE in the context of the present disclosure.

[0074] The expression-enhancing sequence element may, in some embodiments, be a portion of a known EASE. In some embodiments, the EASE may comprise the sequence of SEQ ID NO: 13. The expression-enhancing sequence element sequence of SEQ ID NO: 13 is illustratively found in U.S. Patent No. US 6,596,514 at positions 8672-12273 of SEQ ID NO: 1 herein and has a length of 14,507 bases. The EASE sequence of SEQ ID NO: 13 is also illustratively contained in the CHO sequence of GenBank Accession No. AF193761.1, version 1 as of November 8, 1998, and has a length of 14,462 bases. In this sequence, the EASE sequence of SEQ ID NO: 13 defines positions 8627-12228 of the sequence of the database entry. In some embodiments, the EASE is defined by SEQ ID NO: 13. In some embodiments, the EASE is defined by SEQ ID NO: 14.

[0075] Inclusion of EASE in a vector containing a PGK promoter, such as the PGK1 promoter, operably linked to a nucleotide sequence encoding glutamine synthetase leads to increased expression of a nucleotide sequence encoding a target polypeptide inserted in-frame at the insertion site of the expression cassette. In some embodiments, the expression is transient. Thus, inclusion of EASE in a vector leads to transient, high expression of the target polypeptide in some embodiments. In this regard, the inventors have found that the ability of EASE to promote stable integration leads to an extended period of transient gene production for the vector compared to the same vector lacking EASE. The longer period during which the plasmid is maintained in the host cell and causes expression of the target polypeptide leads to the production of more target polypeptide, thereby increasing the total amount of target polypeptide produced.

[0076] The target polypeptide that may be expressed by the expression vector according to the present disclosure may be, for example, a therapeutic polypeptide. Two illustrative examples of therapeutic polypeptides are adhesion molecules and cytokines. Two further illustrative examples of therapeutic polypeptides are enzymes and receptors. Another example of a therapeutic polypeptide is a lymphokine. An antibody light chain and / or heavy chain is yet another example of a therapeutic polypeptide. In some embodiments, the expression vector is suitable for expressing two target polypeptides, such as the individual polypeptide chains of a heterodimeric protein.

[0077] In some embodiments, a vector as disclosed herein may contain two target polypeptide expression cassettes. These two target polypeptide expression cassettes may, in some embodiments, be identical in composition. When the sequences encoding each target polypeptide are inserted into the vector, the target polypeptide expression cassettes differ only in the sequences encoding the target polypeptides. In some embodiments, two such target polypeptide expression cassettes are arranged in tandem in the vector.

[0078] The methods and uses employing an expression vector as disclosed herein may be methods and uses, respectively, in which a polypeptide is produced. Such a polypeptide is also referred to herein as a "target polypeptide." The method or use may be a method or use for achieving improved production of a target protein. Such a method or use may include culturing a recombinant host cell, such as a CHO or NS0 host cell, containing an expression vector as disclosed herein. In such embodiments, the expression vector generally includes a sequence encoding the target polypeptide. As explained above, such a sequence is included in an insertion site for the nucleotide sequence encoding the target polypeptide. The respective sequence is typically in frame with a cytomegalovirus (CMV) promoter. In some embodiments, the nucleotide sequence encoding the target polypeptide is in frame with a PGK promoter and a nucleotide sequence encoding glutamine synthetase included in a selection cassette.

[0079] Each method or use may include culturing the host cells in a batch or fed-batch process. Each method or use may also include culturing the host cells in a continuous mode. The cells are generally cultured under conditions that allow or promote expression of the polypeptide. Each method or use may include recovering the polypeptide.

[0080] The respective method or use may also comprise selecting host cells based on the presence of a selection cassette containing a sequence encoding glutamine synthetase, thereby leading to expression of glutamine synthetase. The host cells in this regard may be cultured in the absence of glutamine in the culture medium.

[0081] Any steps of the methods according to the present disclosure, including the overall method, may be performed in an automated manner—and repeatedly, illustratively using commercially available robots. Computer-executable instructions may, illustratively, control data analysis or the mechanical course of action employed in each method. The listing or discussion of a previously published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

[0082] The invention illustratively described herein may suitably be practiced in the absence of any element(s), limitation(ies) not specifically disclosed herein. In addition, the terms and expressions employed herein are used as terms of description and not of limitation, and the use of such terms and expressions is not intended to exclude any equivalents of the features shown, described, and described, or portions thereof, but it is recognized that various modifications are possible within the scope of the invention as claimed. Thus, while the invention has been specifically disclosed by exemplary embodiments and optional features, it should be understood that modifications and variations of the invention embodied therein disclosed herein may be resorted to by those skilled in the art, and that such modifications and variations are deemed to be within the scope of the invention.

[0083] The invention has been described broadly and generically herein. Each narrower species and subgeneric group falling within the generic disclosure also forms part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether the excised material is specifically described herein.

[0084] Other embodiments are within the scope of the following claims. Additionally, when features or aspects of the invention are described in terms of a Markush group, those skilled in the art will recognize that the invention is also thereby described in terms of any individual members or subgroups of members of the Markush group. In order that the present invention may be readily understood and put into practice, specific embodiments will now be described by the following non-limiting examples.

[0085] example The examples illustrate the generation and use of expression vectors based on the vector pcDNA 3.1(+). material [Table 1] [Table 2]

[0086] [Table 3]

[0087] [Table 4] [Table 5]

[0088] procedure To assemble the four vectors, three fragments were designed and synthesized from geneart, named GA fragment 1: pMBL C-GS, GA fragment 2: puromycin gene fragment, and GA fragment 3: EASE. The references / authorities for these three fragments are given below: GA fragment 1: Reference for pMBL C-GS

[0089] [Table 6]

[0090] The vector map of pcDNA 3.1(+), the parent vector of GA fragment 1, is shown in Figure 1A. Modifications were made to the parent in-house vector using Vector NTI Version 11.5.1. The in-house vector map of the resulting vector, pMBL C-GS, is shown in Figure 1B. Specific modifications are captured in the table above.

[0091] Puromycin (GA fragment 2) reference: The puromycin gene cassette was supplied by the pCHO1.0 vector from Invitrogen. The puromycin gene was synthesized from GA with a SalI site at the 5' end and a SmaI site at the 3' end of the sequence.

[0092] Reference for EASE (GA Fragment 3): The EASE fragment was sourced from GenBank accession number AF193761.1-8672 bp to 12274 bp. The sequence was ordered from Geneart with a BglII site at the 5' end and a BamHI site at the 3' end of the sequence.

[0093] Process Workflow Three fragments were synthesized from geneart, and the references for each are given in Table 1 above. [Table 7] Table 2 below provides a summary of the vectors that were constructed.

[0094] [Table 8]

[0095] [Table 9]

[0096] Characterization of vector pMBL C-GS The vector pMBL C-GS was obtained from Geneart (GA fragment 1) and resuspended in 25 μL of sterile water to give a final concentration of 200 ng / μL. 1 μL was transformed into CCB00024 / CHE chemically competent cells. Single colonies were streaked and inoculated for plasmid isolation. Overnight cultures were then stored as glycerol stocks. Plasmids were isolated. Plasmid stocks were analyzed by restriction digestion followed by agarose gel electrophoresis, and Figure 5A depicts the resulting gel.

[0097] Characterization of vector pMBL CE-GS The EASE fragment was obtained from Geneart (GA fragment 3). The EASE element was subcloned upstream of the CMV promoter at the BglII site, thereby obtaining the vector pMBL C-GS. The EASE fragment was resuspended in 50 μL of sterile water to obtain a final concentration of 100 ng / μL. 1 μL was transformed into CCB00024 chemically competent cells. The resulting single colonies were streaked and the plasmid was isolated. The EASE fragment was released from GA fragment 3 by using the BglII+BamHI sites and cloned into pMBL C-GS (PLB00876) at the BglII site. The resulting vector is pMBL CE-GS. The identity of the vector pMBL CE-GS was analyzed by restriction digestion followed by agarose gel electrophoresis. Figure 5B shows the resulting gel.

[0098] Characterization of vector pMBL CE-Puro The puromycin gene (GA fragment 2) was obtained from Geneart. The EASE element was subcloned upstream of the CMV promoter at the BglII site, and the GS ORF was replaced with the puromycin selection marker for CHO at the SmaI / SalI sites described above. The fragment was resuspended in 50 μL of sterile water to give a final concentration of 100 ng / μL. 1 μL of the aliquot was transformed into CCB00024 chemically competent cells. Single colonies were streaked and plasmids were isolated. The puromycin gene from GA fragment 2 was cloned into the vector pMBL CE-GS at the SmaI+SalI sites. The resulting vector is the vector pMBL CE-Puro. The identity of the vector PMBL CE-Puro was analyzed by restriction digestion followed by agarose gel electrophoresis, and Figure 5C represents the resulting gel.

[0099] Characterization of vector pMBL C-Puro The puromycin gene (GA fragment 2) was obtained from Geneart. The GS ORF has been replaced with the puromycin selection marker for CHO at the SmaI / SalI sites described above. The fragment was resuspended in 50 μL of sterile water to give a final concentration of 100 ng / μL. 1 μL of the aliquot was transformed into CCB00024 / CHE chemically competent cells. Single colonies were streaked and plasmids were isolated. The puromycin fragment was released from GA fragment 2 by using the SmaI+SalI sites and cloned into the vector pMBL C-GS at the SmaI+SalI sites. The resulting vector is pMBL C-Puro. The identity of the vector PMBL C-Puro was analyzed by restriction digestion followed by agarose gel electrophoresis. Figure 5D shows the resulting gel.

[0100] Vector Evaluation The constructed vectors were further evaluated based on their ability to drive heterologous gene expression in CHO-based cell lines. First, pMBL CE-GS was selected for evaluation. The following constructs were made in pMBL CE-GS: [Table 10]

[0101] Test 1: GFP was cloned into the vector pMBL CE-GS at the XhoI-NotI site. This plasmid served as a transfection efficiency control for evaluating pMBL CE-GS. The plasmids were evaluated by transient gene expression. Transient transfections were performed in co-transfection mode. All transfections were performed using the ExpiCHO™ Expression System (Cat. No. A29133, Thermo Fisher Scientific Inc., Waltham, MA, USA), a high-yield transient expression system based on suspension-adapted Chinese hamster ovary (CHO) cells. ExpiCHO-S cells were thawed according to the expiCHO-S Expression System instructions. After a minimum of two passages after thawing, 3.5 × 10 cells were transfected one day before transfection. 6 Cells were seeded for transfection at 6.0 x 10 cells / ml. One day after seeding, cell counts were estimated and diluted with fresh, pre-warmed medium for each transfection to yield a total of 6.0 x 10 cells / ml. 6 The cell density was adjusted to 1000 cells / ml. Transfections were performed according to the expiCHO-S Expression System instruction manual. DNA was transfected into the culture at a final concentration of 1 μg / ml. Expifectamine and feed were added according to the manufacturer's protocol. The Max titre protocol was followed for all transfections. Transfection flasks were cultured at ~37°C on an orbital shaker in an incubator with a humidified atmosphere of 8% CO2 in air. VCD and viability were estimated on days 0, 2, 5, 7, 9, and 12. Cultures were harvested when viability dropped below 50%.

[0102] Test 2: The heavy and light chains of the anti-CTLA4 antibody (Bmab700) were cloned into the vectors pMBL CE-GS and pMBL C-GS at the XhoI-NotI sites. The resulting constructs were evaluated in comparison with Bmab700-SSC / pCHO1.0 M (HC and LC were cloned into two individual vectors and cotransfected) and Bmab700-DGC / pCHO1.0 (HC and LC were cloned into a single vector). The four constructs were compared by transient gene expression.

[0103] Transient transfections were performed in cotransfection mode. All transfections were performed using the ExpiCHO™ Expression System (Cat. No. A29133, Thermo Fisher Scientific Inc., Waltham, MA, USA), a high-yield transient expression system based on suspension-adapted CHO cells. ExpiCHO-S cells were thawed according to the expiCHO-S Expression System instruction manual. After at least two passages after thawing, 3.5 × 10 cells were transfected one day before transfection. 6 Cells were seeded for transfection at 6.0 x 10 cells / ml. One day after seeding, cell counts were estimated and diluted with fresh, pre-warmed medium for each transfection to yield a total of 6.0 x 10 cells / ml. 6 The cell density was adjusted to 1000 cells / ml. Transfections were performed according to the expiCHO-S Expression System instruction manual. DNA was transfected into the culture at a final concentration of 1 μg / ml. Expifectamine and feed were added according to the manufacturer's protocol. The Max titre protocol was followed for all transfections. Transfection flasks were cultured at ~37°C on an orbital shaker in an incubator with a humidified atmosphere of 8% CO2 in air. VCD and viability were estimated on days 0, 2, 5, 7, 9, and 12. Cultures were harvested on day 15, and the results are shown in Figure 7A. The cultures were carried out in 125 ml shake flasks, using a running volume of 25 ml. The results are presented in Figure 7A.

[0104] Test 3: The HC and LC of anti-CD20 fusion mabs were cloned into the vector pMBL CE-GS at the XhoI-NotI site. The resulting constructs were evaluated for titer analysis by transient gene expression. Transient transfections were performed in co-transfection mode. All transfections were performed using the ExpiCHO™ Expression System (Cat. No. A29133, Thermo Fisher Scientific Inc., Waltham, MA, USA), a high-yield transient expression system based on suspension-adapted CHO cells. ExpiCHO-S cells were thawed according to the expiCHO-S expression system instructions. After at least two passages after thawing, 3.5 × 10 cells were cultured one day before transfection. 6 Cells were seeded for transfection at 6.0 x 10 cells / ml. One day after seeding, cell counts were estimated and diluted with fresh, pre-warmed medium for each transfection to yield a total of 6.0 x 10 cells / ml. 6 The cell density was adjusted to 1000 cells / ml. Transfections were performed according to the expiCHO-S Expression System instruction manual. DNA was transfected into the culture at a final concentration of 1 μg / ml. Expifectamine and feed were added according to the manufacturer's protocol. The Max titre protocol was followed for all transfections. Transfection flasks were cultured at ~37°C on an orbital shaker in an incubator with a humidified atmosphere of 8% CO2 in air. VCD and viability were estimated on days 0, 2, 5, 7, 9, and 12. Cultures were harvested on day 14, and the results are shown in Figure 7B.

Claims

1. (a) a selection cassette comprising a nucleotide sequence encoding glutamine synthetase operably linked to a 3-phosphoglycerate kinase (PGK) promoter and polyadenylation (pA) signal as a eukaryotic selection marker; (b) a selection cassette comprising, as a bacterial selection marker, a nucleotide sequence encoding an enzyme that confers resistance to an antibiotic to a bacterial host, operably linked to a suitable promoter; (c) an expression cassette for a target polypeptide comprising an insertion site for a nucleotide sequence encoding the target polypeptide, operably linked to a cytomegalovirus (CMV) promoter and polyadenylation (pA) signal; (d) a bacterial origin of replication; and (e) Expression-Enhancing Sequence Element (EASE) wherein the EASE comprises the sequence of SEQ ID NO: 13; Expression vectors for mammalian cells.

2. 2. The expression vector of claim 1, further comprising a selection cassette as a eukaryotic selection marker, the selection cassette comprising a nucleotide sequence encoding an enzyme that confers resistance to the antibiotic puromycin, operably linked to a 3-phosphoglycerate kinase (PGK) promoter and a polyadenylation (pA) signal.

3. 3. The expression vector according to claim 2, wherein the enzyme that confers resistance to the antibiotic puromycin is puromycin-N-acetyltransferase (pac).

4. 4. The expression vector according to claim 1, wherein the antibiotic to which the bacterial selectable marker confers resistance is ampicillin and the enzyme that confers resistance to ampicillin to the bacterial host is beta-lactamase.

5. The expression vector according to any one of claims 1 to 4, wherein the bacterial replication origin is a pUC replication origin.

6. 6. The expression vector according to claim 1, wherein the glutamine synthetase as a selectable marker for eukaryotes is encoded by a sequence having at least 90% identity to the sequence of SEQ ID NO: 3 encoding a glutamine synthetase capable of catalyzing the ATP-dependent conversion of glutamate and ammonia to glutamine, or the glutamine synthetase as a selectable marker for eukaryotes is a mammalian glutamine synthetase.

7. 7. The expression vector of claim 1, wherein the glutamine synthetase has an amino acid sequence that is at least 96% identical to the sequence of SEQ ID NO: 5 and is capable of catalyzing the ATP-dependent conversion of glutamate and ammonia to glutamine.

8. 8. The expression vector according to any one of claims 1 to 7, wherein the 3-phosphoglycerate kinase (PGK) promoter has a sequence that is at least 98% identical to the sequence of SEQ ID NO: 2 and is a functional promoter, and / or the glutamine synthetase as a selectable marker for eukaryotes is CHO glutamine synthetase that is encoded by a sequence that is at least 97% identical to the sequence of SEQ ID NO: 3, which encodes a glutamine synthetase capable of catalyzing the ATP-dependent conversion of glutamate and ammonia to glutamine.

9. The expression vector according to any one of claims 1 to 8, wherein the pA signal is a simian virus 40 pA signal.

10. A recombinant CHO or NS0 host cell comprising an expression vector according to any one of claims 1 to 9.

11. 11. A method for producing a polypeptide, comprising culturing the recombinant CHO or NS0 host cell of claim 10 under conditions suitable for expressing a heterologous target polypeptide, wherein the insertion site for an expression cassette for the target polypeptide comprises a nucleotide sequence encoding the polypeptide as the heterologous target polypeptide.

12. The method of claim 11 , wherein the polypeptide comprises an antibody or a fragment thereof capable of specifically binding to an antigen.

13. Use of the expression vector according to any one of claims 1 to 9 to increase the total amount of polypeptide produced.

14. 14. The use according to claim 13, wherein the polypeptide comprises an antibody or a fragment thereof capable of specifically binding to an antigen.

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