Vectors incorporating combinations of promoters driving expression of selectable markers

Abstract

Dual vector systems and mammalian host cells comprising these dual vector systems are provided, wherein each vector employs a different promoter that drives the expression of a selectable marker. A mammalian host cell can be used to produce a recombinant protein of interest in mammalian cell culture.

Description

Technical Field

[0001] This invention generally relates to mammalian host cells comprising vector systems for expressing recombinant proteins having three or more strands. Specifically, the vector system contains two expression vectors, each expressing a selectable marker, wherein the promoters driving the expression of the selectable marker are different. Background Technology

[0002] Biologics are used worldwide for a variety of applications, such as therapy and diagnostics, due to their wide range of uses. Mammalian cell lines are the primary expression systems for these biologics, with Chinese hamster ovary (CHO) cells being the main cell factory. See Lalonde et al., 2017, J Biotechnol 251:128-140. Especially with the emergence of biosimilars, speed to market and cost-effectiveness are now more important than ever.

[0003] The cost of manufacturing biologics is very high due to the complexity of their production using multi-step processes involving the selection of optimal cell lines, mass culture of cells for production, and purification of the desired biologic from the cell harvest. Typically, manufacturing is even more complex for novel antibody forms, such as those with three or four antibody chains. While these costs are decreasing due to improvements in various aspects of manufacturing, they can still be prohibitive when widely adopted as first-line therapies.

[0004] To make biotherapeutic agents more accessible to patients, reducing the commodity cost of the manufacturing process is an attractive proposition. One way to achieve this is by increasing the titer associated with the production cell line. Proper vector conformation can help optimize the expression levels of different strands in recombinant proteins, particularly for three- or four-stranded molecules, resulting in more balanced strand expression, reduced impurities, and higher product quality.

[0005] Attenuation of the selective markers for glutamine synthetase has been shown to improve product titers. See Sacco et al., 2022, Biotechnol. Bioeng. [Biotechnology and Bioengineering] 119:1712-1727. See also Yang et al., 2019, Bioprocess Biosyst. Eng. [Bioprocess and Biosystems Engineering] 42:799-806.

[0006] There remains a need for vector systems that can produce recombinant proteins, particularly triple- and quadruple-stranded molecules, at high titers when transfected into host cell lines, with minimal impact on growth and productivity. Such vector systems would be beneficial for the process development of biopharmaceuticals. Summary of the Invention

[0007] This disclosure provides a mammalian host cell for expressing an antigen-binding protein having three or four different chains, the mammalian host cell comprising a first expression vector and a second expression vector, wherein: (a) the first expression vector comprises a nucleotide sequence encoding: 1) a first chain and a second chain, wherein the first chain is operatively linked to a first promoter, and the second chain is operatively linked to a second promoter or an IRES sequence or to the first chain via a linker sequence; and 2) a first selectable tag operatively linked to a third promoter; and (b) the second expression vector comprises a nucleotide sequence encoding: 1) The third and fourth chains, wherein the third chain is operatively connected to the fourth promoter, and the fourth chain is operatively connected to the fifth promoter or the IRES sequence or to the third chain via a adapter sequence; and 2) a second optional marker operatively connected to the sixth promoter, wherein the first, second, third, and fourth chains are selected from heavy chains, light chains, antibody-heavy chain fusions, antibody-light chain fusions, ScFv, and ScFv-Fc, wherein two of these chains may be identical; and wherein the sixth promoter is different from the third promoter.

[0008] In some embodiments, the second chain and the fourth chain are operatively connected to the second promoter and the fourth promoter, respectively.

[0009] In some embodiments, the first selectable marker and the second selectable marker are the same. In some aspects, the first selectable marker and the second selectable marker are selected from the group consisting of glutamine synthase and dihydrofolate reductase. In some aspects, the first selectable marker and the second selectable marker are glutamine synthase.

[0010] In some embodiments, the third and sixth promoters are selected from mPGK and Srα.

[0011] In some embodiments, the first, second, fourth, and fifth promoters differ from the third and sixth promoters. In some aspects, the first, second, fourth, and fifth promoters are selected from the group consisting of CMV / GAPDH, CMV / adL, and CMV / EF1α.

[0012] In some embodiments, the first and fourth promoters are identical, and the second and fifth promoters are identical. In some aspects, the first, second, fourth, and fifth promoters are identical. In some aspects, the first and fourth promoters are different from the second and fifth promoters.

[0013] In some embodiments, the stronger of the third and sixth promoters resides on an expression vector with a more difficult-to-express strand. In some aspects, Srα resides on an expression vector with a more difficult-to-express strand, and mPGK resides on an expression vector with a more easily expressed strand.

[0014] In some aspects of the embodiments where the optional marker is glutamine synthase, methionine sulfoxide (MSX) strictness is optimized to favor the expression of more difficult-to-express strands paired with stronger promoters. In some sub-aspects, MSX strictness is less than 75 µM.

[0015] In some embodiments, the stronger of the third and sixth promoters is on an expression vector with a more readily expressible expression chain.

[0016] In some embodiments, the first and third chains are antibody light chains, and the second and fourth chains are antibody heavy chains. In some aspects, the first and second chains are the same antibody light chain. In some aspects, the first and second chains are different antibody light chains.

[0017] In some embodiments, a) the first and third chains are antibody light chains, and b) one of the second or fourth chains is an antibody heavy chain, and the other is an antibody heavy chain fusion. In some aspects, the first and second chains are the same antibody light chain. In some aspects, the first and second chains are different antibody light chains.

[0018] In some embodiments, the antigen-binding protein having three or four chains is selected from the group consisting of heteroIgG or C1mAb.

[0019] In some embodiments, the first expression vector comprises, in 5' to 3' order, a first promoter, a nucleotide sequence encoding a first antibody light chain, a second promoter or IRES, a nucleotide sequence encoding a first heavy chain, a third promoter for Srα, and a selectively labeled nucleotide sequence encoding glutamine synthase; and the second expression vector comprises, in 5' to 3' order, a fourth promoter, a nucleotide sequence encoding a second antibody light chain, a fifth promoter or IRES, a nucleotide sequence encoding a second heavy chain, a sixth promoter for mPGK, and a selectively labeled nucleotide sequence encoding glutamine synthase. In some respects, the first and second antibody light chains are identical. In some respects, the first heavy chain is more difficult to express than the second heavy chain.

[0020] In some embodiments, the first expression vector comprises, in a 5' to 3' order, a first promoter, a nucleotide sequence encoding a first antibody light chain, a second promoter, a nucleotide sequence encoding a first heavy chain, a third promoter for Srα, and a selectively labeled nucleotide sequence encoding glutamine synthase; and the second expression vector comprises, in a 5' to 3' order, a fourth promoter, a nucleotide sequence encoding a second antibody light chain, a fifth promoter, a nucleotide sequence encoding a second heavy chain, a sixth promoter for mPGK, and a selectively labeled nucleotide sequence encoding glutamine synthase. In some respects, the first antibody light chain and the second antibody light chain are identical. In some respects, the first heavy chain is more difficult to express than the second heavy chain.

[0021] In some embodiments, the first expression vector comprises, in a 5' to 3' order, a first promoter, a nucleotide sequence encoding an antibody light chain, a second promoter or IRES, a nucleotide sequence encoding a heavy chain fusion, a third promoter for Srα, and a selectively labeled nucleotide sequence encoding glutamine synthase; and the second expression vector comprises, in a 5' to 3' order, a fourth promoter, a nucleotide sequence encoding an antibody light chain, a fifth promoter or IRES, a nucleotide sequence encoding a heavy chain, a sixth promoter for mPGK, and a selectively labeled nucleotide sequence encoding glutamine synthase. In some respects, the first antibody light chain and the second antibody light chain are identical.

[0022] In some embodiments, the first expression vector comprises, in a 5' to 3' order, a first promoter, a nucleotide sequence encoding an antibody light chain, a second promoter, a nucleotide sequence encoding a heavy chain fusion, a third promoter for Srα, and a selectable label for glutamine synthase; and the second expression vector comprises, in a 5' to 3' order, a fourth promoter, a nucleotide sequence encoding an antibody light chain, a fifth promoter, a nucleotide sequence encoding a heavy chain, a sixth promoter for mPGK, and a nucleotide sequence encoding a selectable label for glutamine synthase. In some respects, the first antibody light chain and the second antibody light chain are identical.

[0023] In some embodiments, the first expression vector and the second expression vector are integrated into the genome.

[0024] In some embodiments, the host cell is a Chinese hamster ovary (CHO) cell. In some respects, CHO cells are deficient in dihydrofolate reductase (DHFR-) or glutamine synthase knockout (GSKO) type.

[0025] This disclosure also provides a method for generating an antigen-binding protein having three or four chains, the method comprising: (a) culturing mammalian host cells as described in any one of the above embodiments, sub-embodiments, aspects and sub-aspects under conditions expressing the antigen-binding protein; and (b) recovering the antigen-binding protein.

[0026] This disclosure also provides a method for generating an antigen-binding protein having three or four chains, the method comprising: (a) culturing mammalian host cells under conditions expressing the antigen-binding protein, and in the case of CHO DHFR- cells under methotrexate strictness or in the case of CHO GSKO cells under methionine sulfoxide imine conditions to facilitate the expression of the poorly expressed chain paired with a stronger GS promoter, the mammalian host cells being Chinese hamster ovary (CHO) cells lacking dihydrofolate reductase (DHFR-) or glutamine synthetase knockout (GSKO); and (b) recovering the antigen-binding protein.

[0027] In some embodiments of these methods, the recovered antigen-binding protein is purified and formulated into a pharmaceutically acceptable preparation.

[0028] This disclosure also provides an antigen-binding protein produced by any mammalian host cell provided herein or by any method provided herein. Attached Figure Description

[0029] Figure 1 A- Figure 1 B describes an example of a mixed promoter expression cassette driving glutamine synthase (GS) expression using 3- or 4-chain molecules. The promoter of the target gene (i.e., the light chain (LC, LC1, and LC2), heavy chain (HC), or heavy chain fusion (HC-mAb)) can be modified according to the target molecule. mPGK and SRα refer to promoters used to drive GS expression. (B) Schematic diagram of 3-chain (C1mAb) and 4-chain (heterologous IgG) molecules requiring vector optimization for evaluation.

[0030] Figure 2 A- Figure 2 F shows a schematic diagram of the expression cassette used in the vector evaluation study of the 3-chain C1 mAb molecule (A). (B) shows that the mPGK / Srα vector combination resulted in an effective titer (normalized relative to %nrCE main peak) of up to approximately 5.3-fold improvement. (C) shows a reduction in %nrCE front peak impurities. (D) shows an increase in %nrCE main peak. (E, F) show an increase in the relative expression of the mAb fusion as measured by rCE.

[0031] Figure 3 A- Figure 3C shows that the vector containing a mixed promoter expression cassette expressing heterologous IgG improved the effective titer (normalized relative to %SEC main peak) by up to 1.2 times on day 10 (A) while having minimal impact on product quality (B, C). Detailed Implementation

[0032] This disclosure is partly based on the unexpected finding that when using dual-vector systems in mammalian host cells, employing different promoters to drive selectively labeled expression can increase the titer of the resulting recombinant protein, particularly for antibody forms with three or four chains. These dual-vector systems can be used in mammalian host cells to generate antibodies with two distinct heavy chains and two distinct light chains, where each heavy-light chain pair is expressed by a different vector. These dual-vector systems can also be used to generate antibody forms with three or four distinct chains.

[0033] Standard antibody production techniques typically employ different promoters to express the heavy and light chains in order to optimize antibody expression. This is often necessary because the heavy and light chains are expressed at different levels. The situation becomes more complex when the antibody structure contains three or four chains. The inventors have unexpectedly discovered that by employing different promoters for the selectable markers on each vector, it is possible to increase the titer of the resulting antibody structure while improving product quality (e.g., reducing aggregation (high molecular weight impurities (HMW)) and reducing degradation (low molecular weight impurities (LMW))).

[0034] By employing the dual-vector system and mammalian host cells described herein, the production of recombinant proteins can be increased while maintaining or improving product quality. Using the dual-vector system described herein in mammalian host production cell lines allows for the production of biopharmaceuticals in a cheaper and more consistent manner. This invention is particularly applicable to the commercial production of antibodies with three or four distinct chains.

[0035] The vector systems described herein are used in cell lines (also referred to as “host cells”), preferably mammalian (“mammalian host cells”) grown in cell culture media, to produce recombinant proteins of commercial or scientific significance. Cell lines are typically derived from lineages of primary cultures and can be maintained indefinitely in culture. Genetically engineered cell lines involve transfecting, transforming, or transducing cells with a dual-vector system (each vector containing a nucleotide sequence encoding two antibody chains) to induce the expression of a target antigen-binding protein in the host cells. Methods and vectors for genetically engineering cells and / or cell lines to express, for example, target proteins are well known to those skilled in the art; for example, various techniques are available in… Current Protocols in Molecular Biology [Modern Methods in Molecular Biology]Ausubel et al., eds. (Wiley & Sons, New York, 1988, and quarterly updates); Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989); Kaufman, RJ., Large Scale Mammalian Cell Culture, 1990, pp. 15–69; and Harlow and Lane Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York (1990).

[0036] definition

[0037] Although the terminology used herein is standard in the art, definitions of certain terms are provided herein to ensure clarity and definiteness of the meaning of the claims. Units, prefixes, and symbols may be expressed in their International System of Units (SI) accepted forms. The numerical ranges enumerated herein include the numbers defining the ranges and include and support every integer within the defined ranges. Unless otherwise indicated, the methods and techniques described herein may be performed according to conventional methods well known in the art and as described in the various general and more specific references cited and discussed throughout this specification.

[0038] As used herein, unless otherwise expressly stated, the terms “a” and “an” mean one or more. Furthermore, unless the context requires otherwise, singular terms will include plural and plural terms will include singular. Generally, the nomenclature and techniques used in conjunction with those described herein for cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization are those well-known and commonly used in the art.

[0039] All documents or portions thereof cited in this application, including but not limited to patents, patent applications, articles, books, and monographs, are expressly incorporated herein by reference. The content described in the embodiments of the invention may be combined with other embodiments of the invention.

[0040] This disclosure provides methods for expressing a “target protein.” A “target protein” includes naturally occurring proteins, recombinant proteins, and engineered proteins (e.g., proteins that do not exist in nature and have been designed and / or produced by humans). The target protein may be, but does not have to be, a protein known or suspected of having therapeutic relevance.

[0041] As used herein, “antibody chain” or “chain” refers to an antibody light chain, antibody heavy chain, antibody light chain fusion, antibody heavy chain fusion, scFv-Fc fusion, VHH fusion, etc. The terms “antibody heavy chain” and “antibody light chain” have their standard meaning in the art and include, for example, the various antibody heavy and light chains described elsewhere herein (e.g., heavy and light chains of IgG1, IgG2, IgG3, and IgG4 mAbs). The terms “antibody heavy chain” and “antibody light chain” include standard full-length heavy and light chains. The terms “antibody heavy chain fusion,” “antibody heavy chain fusion protein,” “heavy chain protein fusion,” and “heavy chain peptide fusion” are used interchangeably herein and refer to a polypeptide containing an antibody heavy chain covalently linked to one or more other proteins or peptides. For example, an “antibody heavy chain fusion” can be an antibody heavy chain covalently linked to a cytokine, VHH, or scFv. The linking can be direct or via a peptide linker (e.g., a glycine-serine linker). In an antibody heavy chain fusion, the antibody heavy chain may be linked to one or more additional proteins at the N-terminus or C-terminus (or both) of the heavy chain. The terms “antibody light chain fusion protein” and “antibody light chain fusion” have the same meaning as described above for “antibody heavy chain fusion,” except that the antibody light chain replaces the antibody heavy chain. As used herein, “antibody fusion” refers to an antibody as provided herein, covalently linked (e.g., via the antibody heavy chain or light chain) to one or more additional proteins or polypeptides. Therefore, an antibody fusion contains at least one antibody heavy chain fusion or antibody light chain fusion as one of the chains of the antibody fusion. Most commonly, an antibody fusion is a molecule containing two antibody light chains, one antibody heavy chain, and one antibody heavy chain fusion, such that an additional protein is linked to one of the antibody heavy chains. An example is a C1mAb with a fusion at the C-terminus of the Fc region.

[0042] As used herein, the terms “peptide” and “protein” (e.g., as used in the context of a target protein or target peptide) are used interchangeably to refer to polymers containing amino acid residues. These terms also apply to amino acid polymers in which one or more amino acid residues are analogs or mimics of the corresponding naturally occurring amino acid, as well as naturally occurring amino acid polymers. These terms may also cover amino acid polymers that have been modified, for example, by adding carbohydrate residues to form glycoproteins or by phosphorylation. Peptides and proteins may be produced by naturally occurring and non-recombinant cells, or by genetically engineered or recombinant cells. Peptides and proteins may comprise molecules having the amino acid sequence of a natural protein, or molecules having one or more amino acids with a natural sequence that have been omitted, added, and / or substituted.

[0043] As used herein, the term "heterologous" in conjunction with nucleic acids means having nucleic acids that are not naturally present in the host cell. This can include mutated sequences, such as sequences different from those naturally occurring. This can include sequences from other species. This can also include sequences located at different sites in the genome than those naturally occurring in the host cell. This generally does not include naturally occurring mutations that may occur in the host cell. Cells that already contain heterologous nucleic acids encoding a target protein, for example through stable integration of an expression cassette, will be considered to contain heterologous nucleic acid sequences. For clarity, CHO cells or derivatives thereof (e.g., DHFR- or GS knockout types) containing nucleic acids encoding antigen-binding proteins will be considered to contain heterologous nucleic acids.

[0044] As used herein, the term "operably ligated" means that the ligated nucleic acid sequences are typically continuous or substantially continuous, and when it is necessary to ligate two protein-coding regions, they are continuous and within the reading frame. However, because enhancers typically function at intervals of several thousand bases from promoters, and intron sequences can have variable lengths, some polynucleotide elements can be operably ligated but are not continuous.

[0045] As used herein, the term "bioreactor" means any container that can be used for the growth of cell cultures. Cell cultures of mammalian cells described herein can be grown in a bioreactor, and the bioreactor can be selected based on the application of the target protein produced by the cells grown in the bioreactor. Bioreactors can be of any size, as long as they are suitable for cell culture; typically, the size of the bioreactor is appropriate for the volume of cell cultures grown within it. Typically, a bioreactor will be at least 1 liter and can be 2, 5, 10, 50, 100, 200, 250, 500, 1,000, 1,500, 2,000, 2,500, 5,000, 8,000, 10,000, 12,000 liters or larger, or any volume between these values. Internal conditions of the bioreactor, including but not limited to pH and temperature, can be controlled during culture. Those skilled in the art will recognize and be able to select a suitable bioreactor for practicing the methods disclosed herein based on relevant considerations.

[0046] As used herein, “cell culture” or “culture” means the growth and reproduction of cells outside a multicellular organism or tissue. Suitable culture conditions for mammalian cells are known in the art. See, for example, *Animal cellculture: A Practical Approach*, edited by D. Rickwood, Oxford University Press, New York (1992). Mammalian cells can be cultured in suspension or attached to a solid substrate. Fluidized bed bioreactors, hollow fiber bioreactors, roller flasks, shake flasks, or stirred tank bioreactors with or without microcarriers can be used. In one embodiment, a bioreactor of 500 L to 2000 L is used. In another embodiment, a bioreactor of 1000 L to 2000 L is used.

[0047] The term "cell culture medium" (also known as "culture medium," "cell culture media," or "tissue culture medium") refers to any nutrient solution used to grow cells (such as animal or mammalian cells) and typically provides at least one or more of the following components: energy (usually in the form of carbohydrates, such as glucose); one or more of all essential amino acids, typically twenty basic amino acids plus cysteine; vitamins and / or other organic compounds, typically required in low concentrations; lipids or free fatty acids; and trace elements, such as inorganic compounds or naturally occurring elements, typically required in very low concentrations (typically in the micromolar range).

[0048] The nutrient solution may optionally be supplemented with additional optional components to optimize cell growth, such as hormones and other growth factors, such as transferrin, epidermal growth factor, serum, etc.; salts, such as calcium salts, magnesium salts, and phosphates, and buffers, such as HEPES; nucleosides and bases, such as adenosine, thymidine, hypoxanthine; and proteins and tissue hydrolysates, such as hydrolyzed animal or plant proteins (peptones or mixtures of peptones, which may be obtained from animal by-products, purified gelatin, or plant material); antibiotics, such as gentamicin; anti-caking agents; and cell protectants or surfactants, such as Pluronic. ® F68 (also known as Lutrol) ® F68 and Kolliphor ® P188; a nonionic triblock consisting of a hydrophobic central chain of polyoxypropylene (poly(propylene oxide)) and two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)) on either side; polyamines, such as putrescine, spermidine, and spermine (see, for example, International Patent Application Publication No. WO 2008 / 154014) and pyruvate (see, for example, U.S. Patent No. 8,053,238), depending on the needs of the cells to be cultured and / or the required cell culture parameters.

[0049] Cell culture media include those that are typically used and / or are known to be used in any cell culture process, such as, but not limited to, batch, extended batch, fed-batch, and / or perfusion or continuous cell culture.

[0050] "Basic" (or batch) cell culture medium refers to a cell culture medium that is typically used to initiate cell culture and is sufficiently complete to support cell culture.

[0051] "Feed-batch culture" refers to a form of suspension culture and means a method of culturing cells in which additional components are provided to the culture at one or more points after the start of the culture process. The provided components typically include nutrient supplements that have been depleted for the cells during the culture process. Alternatively or additionally, the additional components may include supplemental components (e.g., cell cycle inhibitory compounds). Fed-batch cultures are typically stopped at a certain point, and the cells and / or components in the culture medium are collected and optionally purified.

[0052] “Growth” cell culture medium refers to a cell culture medium that is typically used for cell culture during the exponential growth phase (“growth phase”) and is sufficiently complete to support cell culture during this phase. Growth cell culture media may also contain selectants that confer selective marker resistance or viability to host cell lines. Such selectants include, but are not limited to, genimycin (G418), neomycin, hygromycin B, puromycin, bleomycin, methionine sulfoxide, methotrexate, glutamine-free cell culture media, glycine-free cell culture media, hypoxanthine and thymidine, or thymidine alone.

[0053] "Perfusion" cell culture media are typically used to maintain cell cultures via perfusion or continuous culture methods and are sufficiently complete to support cell culture during the process. Perfusion cell culture medium formulations can be richer or more concentrated than basal cell culture medium formulations to suit the methods used for removing used medium. Perfusion cell culture media can be used during both the growth and production phases.

[0054] “Production” cell culture medium refers to a cell culture medium that is typically used for cell culture during the transition period from the end of exponential growth to the beginning of protein production, the “transition” and / or “product” phase, and is sufficiently complete to maintain the desired cell density, viability and / or product titer during that phase.

[0055] Concentrated cell culture media may contain some or all of the nutrients necessary to maintain cell culture; in particular, concentrated media may contain nutrients identified or known to be consumed during the production phase of cell culture. Concentrated media can be based on virtually any cell culture medium formulation. Such concentrated feed media may contain some or all of the components of cell culture media, for example, in normal amounts of about 2X, 3X, 4X, 5X, 6X, 7X, 8X, 9X, 10X, 12X, 14X, 16X, 20X, 30X, 50X, 100X, 200X, 400X, 600X, 800X, or even about 1000X.

[0056] The components used to prepare cell culture media can be completely ground into powdered culture medium formulations; partially ground together with liquid supplements to be added to the cell culture media as needed; or added to the cell culture media in completely liquid form.

[0057] Cell cultures can also be supplemented with separate concentrated feeds containing specific nutrients that may be difficult to formulate in cell cultures or are rapidly depleted in them. Such nutrients can be amino acids such as tyrosine, cysteine, and / or cystine (see, for example, International Patent Application Publication No. WO 2012 / 145682). For example, a concentrated tyrosine solution can be separately fed into cell cultures grown in a tyrosine-containing cell culture medium such that the tyrosine concentration in the cell culture does not exceed 8 mM. In another example, concentrated tyrosine and cysteine ​​solutions are separately fed into cell cultures grown in a cell culture medium lacking tyrosine, cysteine, or cysteine. Separate feeding can be initiated before or during the production phase. Separate feeding can be accomplished by feeding the cell culture medium in batches on the same day or a different day than the concentrated feed medium. Separate feeding can also be performed on the same day or a different day than the perfusion medium.

[0058] "Serium-free" refers to cell media that do not contain animal serum, such as fetal bovine serum. Various tissue media, including defined media, are commercially available. For example, any one or a combination of the following cell media can be used: RPMI-1640, RPMI-1641, Dürbeco Modified Eagle Medium (DMEM), Eagle Minimum Essential Medium, F-12K, Ham F12, Iskov Modified Dürbeco Medium, McCoy 5A, Leibovitz L-15, and serum-free media such as EX-CELL. TM Examples of such media include the 300 series (JRH Biosciences, Lenexa, Kansas) and MCDB 302 (Sigma Aldrich Corp., St. Louis, MO). Serum-free forms of these media are also available. Depending on the needs of the cells being cultured and / or the required cell culture parameters, the cell culture medium can be supplemented with additional or increased concentrations of components such as amino acids, salts, sugars, vitamins, hormones, growth factors, buffers, antibiotics, lipids, and trace elements. Custom-made cell culture media can also be used.

[0059] "Titer" refers to the total amount of a target polypeptide or protein (which may be naturally occurring or recombinant) produced by a cell culture in a given volume of culture medium. Titer can be expressed in milligrams or micrograms per milliliter of culture medium (or other volumetric measure). "Cumulative titer" is the titer produced by the cells during culture and can be determined, for example, by measuring the daily titer and using those values ​​to calculate the cumulative titer.

[0060] As used herein, the term “host cell” should be understood to include cells that have been genetically engineered to express a target polypeptide. Genetic engineering of cell lines involves transfecting, transforming, or transducing cells with a nucleic acid encoding a recombinant polynucleotide molecule (“target gene”), and / or otherwise altering (e.g., through homologous recombination and gene activation or fusion of recombinant and non-recombinant cells) to induce the host cell to express the desired recombinant polypeptide. Methods and vectors for genetically engineering cells and / or cell lines to express target polypeptides are well known to those skilled in the art; for example, various techniques are described in Current Protocols in Molecular Biology. Ausubel et al., eds. (Wiley & Sons, New York, 1988, and quarterly updates); Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Laboratory Press, 1989); Kaufman, RJ, Large Scale Mammalian Cell Culture, 1990, pp. 15–69. This term includes the offspring of the parent cells, regardless of whether the offspring are morphologically or genetically identical to the original parent cells, as long as the target gene is present. Cell cultures may contain one or more host cells.

[0061] It should be understood that, regardless of how the embodiments are described herein using the language “comprising,” other similar embodiments are also provided, described in terms of “consisting of” and / or “substantially consisting of”.

[0062] Optional markers

[0063] To stably transfect mammalian cells, it is known that only a small fraction of cells can integrate foreign DNA into their genome, depending on the expression vector and transfection technique used. To identify and select these integrators, a gene encoding a selectable marker is typically introduced into the host cell into the same expression vector as the target gene.

[0064] Selectable marker genes encode proteins required for the survival and growth of host cells grown in selective media. Typical selectable marker genes encode proteins that: (a) confer resistance to antibiotics or other toxins (e.g., ampicillin, tetracycline, or kanamycin for prokaryotic host cells); (b) compensate for cellular nutritional deficiencies; or (c) provide essential nutrients through metabolism that are not available from complex or limited media. Specific antibiotic resistance selectable markers are kanamycin resistance genes, ampicillin resistance genes, tetracycline resistance genes, and neomycin resistance genes.

[0065] Other selectable gene amplification can be used to amplify genes to be expressed. Amplification is the process by which genes required to produce proteins necessary for growth or cell survival are replicated tandemly within the chromosomes of recombinant cells across successive generations. Examples of suitable selectable markers for mammalian cells include glutamine synthase (GS), dihydrofolate reductase (DHFR), asparaginase (Aspg; see Ha et al. Biotechnol Bioeng. [Biotechnology and Bioengineering] 2023 120:1159-1166), and promoterless thymidine kinase genes.

[0066] Selective pressure is applied to mammalian cell transformants, where only the transformant is viable due to the presence of a selectable gene in the vector. This pressure is exerted by culturing the transformed cells under conditions of continuously increasing selectant concentrations in the culture medium, resulting in further tightening and / or amplification of the selectable gene and the DNA encoding the target protein. Consequently, an increased amount of the target polypeptide is synthesized from the amplified DNA. The selectant for GS is methionine sulfoxide imide (MSX). The selectant for DHFR is methotrexate (MTX).

[0067] Compared to DHFR-based systems, the GS knockout cell line (GSKO) provides sufficient selection strictness in the absence of MSX or with low MSX concentrations, while coupling 25 μM MSX with the GS knockout cell line results in higher selection efficiency compared to the CHOK1SV cell line at higher MSX concentrations (Fan et al., Biotechnol Bioeng. [Biotechnology and Bioengineering], 109(4): 1007-1015 (2012)). Previous reports have shown that increasing the MSX concentration during the seed train stage after clonal selection increases productivity without significantly affecting cell growth, GS and target gene copy number and expression, and maintains product quality properties in multiple GS knockout cell lines (Tian et al., Engineering in Life Sciences [Life Sciences Engineering] 20(3-4): 112-125 (2020)). Chain / vector expression can be influenced by increasing strictness during pool recovery / selection through the addition of MSX. MSX can also be used to support different vectors in the case of mixed GS promoters.

[0068] In some embodiments, the MSX concentration may be optimized for one of the promoters driving GS expression. In specific embodiments, the MSX concentration may be optimized for GS linked to a more difficult-to-express chain.

[0069] In some embodiments, the optional label is glutamine synthase. Glutamine synthase (GS) catalyzes the biosynthesis of glutamine through the condensation of ammonia with glutamate. Mammalian GS enzymes are decamerics consisting of two stacked pentamer rings, with ten active sites located at the junctions of the subunits. Each active site is formed by residues from an N-terminal domain (β-grasp domain, consisting of residues 25-112) from one subunit and residues from a C-terminal domain (catalytic domain, consisting of residues 113-373) from an adjacent subunit.

[0070] In some embodiments, CHO DHFR- cells or CHO GSKO cells can be cultured under methotrexate strict conditions in the case of CHO DHFR- cells or under methionine sulfoxide strict conditions in the case of CHO GSKO cells to facilitate the expression of difficult-to-express chains that pair with stronger GS promoters.

[0071] In some embodiments, CHO DHFR- cells or CHO GSKO cells can be cultured under methotrexate strict conditions in the case of CHO DHFR- cells or under methionine sulfoxide strict conditions in the case of CHO GSKO cells to facilitate the expression of difficult-to-express chains that pair with weaker GS promoters.

[0072] In one embodiment, the MSX strictness is less than 75 µM. In one embodiment, the strictness is less than 50 µM. In one embodiment, the strictness is about 25 µM to 50 µM. In one embodiment, the strictness is about 25 µM to 45 µM. In one embodiment, the strictness is about 25 µM to 40 µM. In one embodiment, the strictness is about 25 µM to 35 µM. In one embodiment, the strictness is about 25 µM to 30 µM. In one embodiment, the strictness is less than or equal to 25 µM. In one embodiment, the strictness is about 5 µM to 25 µM. In one embodiment, the strictness is about 10 µM to 25 µM. In one embodiment, the strictness is about 15 µM to 25 µM. In one embodiment, the strictness is about 20 µM to 25 µM. In one embodiment, the tightness is 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75 µM.

[0073] Hybrid promoters

[0074] Expression and cloning typically involve promoters containing nucleotide sequences that are recognized by the host organism and operatively linked to the target protein. A promoter is a non-transcribed sequence located upstream (i.e., 5') of the start codon (typically within approximately 100 to 1000 bp) of a structural gene that controls transcription. Promoters are generally grouped into one of two categories: inducible promoters and constitutive promoters. Inducible promoters initiate transcription at an increased level in response to changes in culture conditions (such as the presence or absence of nutrients, or temperature changes) of the DNA under their control. Constitutive promoters, on the other hand, consistently transcribe the gene they are operatively linked to, i.e., they have minimal or no control over gene expression. Many promoters recognized by a variety of potential host cells are well-known.

[0075] In the dual-vector system described herein, any suitable combination of promoters can be used, provided that the two promoters are different, suitable for the host cell line, and drive the expression of the linked gene to different degrees. The chain ratio of the expressed peptide can be measured using techniques well known in the art. Such a ratio can provide information about the more difficult and easier-to-express chains. The chain ratio of the expressed peptide can be measured using techniques well known in the art, such as reducing capillary electrophoresis-sodium dodecyl sulfate (rCE-SDS)). Suitable promoters for mammalian host cells are well known and include, but are not limited to, promoters derived from viral genomes, such as polyomaviruses, infectious epitheliomaviruses, adenoviruses (such as adenovirus 2), bovine papillomaviruses, avian sarcomaviruses, cytomegaloviruses, retroviruses, hepatitis B viruses, and simian virus 40 (SV40). Other suitable mammalian promoters include heterologous mammalian promoters, such as heat shock promoters and actin promoters.

[0076] Other promoters of interest include, but are not limited to: the early SV40 promoter (Benoist and Chambon, 1981, Nature 290:304-310); the CMV promoter (Thornsen et al., 1984, Proc. Natl. Acad. USA 81:659-663); promoters contained in the 3' long terminal repeat sequence of Rouss sarcoma virus (Yamamoto et al., 1980, Cell 22:787-797); the herpesvirus thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. USA 78:1444-1445); and promoters and regulatory sequences from the metallothionein gene (Prinster et al., 1982, Nature). 296:39-42); and prokaryotic promoters, such as β-lactamase promoters (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 75:3727-3731); tac promoters (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 80:21-25); human elongation factor 1α promoter (EF-1α) (Kim et al., 1990, Gene [Gene] 91:217-233); SRα promoter (Srα) (Takebe et al., 1988, Mol Cell. Biol. [Molecular Cell Biology] 1; 8(1): 466-72) and mouse mPGK-1 promoter (mPGK) (Adra et al., 1987, Gene [Gene] 60:65-74).

[0077] Promoters of particular interest that encode the nucleotide sequence of the antibody chain include the human cytomegalovirus IE1 gene promoter-enhancer (CMV) (Boshart et al., 1985, Cell 41:521-30, GenBank accession number X03922), the adenovirus triplet leader sequence (adl) (Gingeras et al., 1982, J. Biol. Chem. 257:13475-91, GenBank accession number J01917), and the hamster glyceraldehyde-3-phosphate dehydrogenase promoter and intron (GAPDH) (US Patent No. 10,202,261).

[0078] In some embodiments, the promoter of the antibody chain is selected from combinations of CMV / GAPDH, CMV / adL, and CMV / EF1α. In these promoter combinations, the CMV promoter, along with promoters selected from GAPDH, adL, and EF1α, is operatively linked to the nucleotide sequence encoding the antibody chain, generally making the combination a better promoter than a single promoter. In some embodiments, the CMV promoter is located at the 5' of a promoter selected from GAPDH, adL, and EF1α.

[0079] In some embodiments, the selectable promoters are chosen from mPGK and Srα.

[0080] Promoters in CHO cells have also been explored. See, for example, Tossolini et al., 2022, Plasmid 119-120:102620; Nguyen et al., 2019, Biotechnology. J. 14:e1900125; Romanova et al., 2018, Biotechnology. J. 13:e1700232.

[0081] In the vector system disclosed in this paper, two distinct promoters each drive the expression of selectable labels on two vectors. These promoters have different strengths. Comparisons of promoter strengths can be found in the literature. See, for example, Qin et al., 2010, PloS One [PLOS ONE] 5:e10611. Promoter strength can be predicted based on promoter sequence and comparison with promoter libraries (see, for example, Zhang et al., 2022, ACS Synth Biol. [ACS Synthetic Biology] 11:92-102) or various online tools (see Skrlj et al., 2010, Anal Biochem. [Analytical Biochemistry] 396:83-90; Liang et al., 2021, Anal. Biochem. [Analytical Biochemistry] 630:114335). Srα is known to be a stronger promoter than the mPGK promoter.

[0082] Surprisingly, it has been found that using two different promoters and alternative markers leads to higher antibody yields, regardless of which chain is produced. However, it has been found that the highest yields are observed when the stronger promoter is on the same vector as the more difficult-to-express chain. Therefore, in some embodiments, the stronger promoter is on a vector with the difficult-to-express chain. In other embodiments, the stronger promoter is on a vector with the easier-to-express chain.

[0083] The ease or difficulty of antibody chain expression can be assessed in systems where the vector is identical except for the generated chains. The vector is transfected into the same cell line, whether in the same cells or in different cell pools under the same conditions. As mentioned above, measuring the chain ratio can indicate which chains are more difficult to express and which are easier to express.

[0084] In some embodiments, one vector contains Srα operatively linked to a selectable marker, and another vector contains mPGK operatively linked to a selectable marker. In one aspect of this embodiment, the selectable marker is GS. In one aspect, the Srα-containing vector is on a vector containing a gene encoding a more difficult-to-express strand, while the mPGK is on a vector containing a gene encoding an easier-to-express strand. In another aspect, the Srα-containing vector is on a vector containing a gene encoding an easier-to-express strand, while the mPGK is on a vector containing a gene encoding a more difficult-to-express strand.

[0085] 3- and 4-chain molecules

[0086] Typical antibodies are Y-shaped molecules with four polypeptide chains (two identical heavy chains and two identical light chains). Such antibodies are preferably expressed by a single vector. However, bispecific antibodies require alternative forms and are typically expressed on two different vectors. See, for example, Spiess et al. 2015, Mol. Immunol. [Molecular Immunology] 67:95-106; Brinkmann et al., 2017, MAbs [Monoclonal Antibodies] 9:192-212; and Ma et al., 2021, Frontiers in Immunology [Immunology Frontiers] 12:626616.

[0087] Any form having at least three different chains or that can be expressed on two vectors can be used with the vector system of the present invention. Some embodiments are described below. Other embodiments are described in the cited references. Others can be determined by those skilled in the art.

[0088] The expression vectors provided herein contain at least two antibody chain encoding genes. The two antibody chain encoding genes can be the same or different genes. For example, in some embodiments, the vector or nucleic acid construct may contain two copies of the same antibody light chain gene. Alternatively, in some embodiments, the vector or nucleic acid construct may contain one copy of the antibody light chain gene and one copy of the antibody heavy chain gene, or one copy of the antibody light chain gene from a first antibody and one copy of the antibody light chain gene from a second antibody. Alternatively, in some embodiments, the first expression vector may contain one copy of the first antibody light chain and one copy of the first antibody heavy chain. The second expression may contain one copy of the second antibody light chain and one copy of the second antibody heavy chain. In some embodiments, both the first and second expression vectors contain the same antibody light chain sequence, and the first and second expressions contain different antibody heavy chain sequences. In these embodiments, the two heavy chains can be connected via a knock-in-hole structure or a similar mechanism.

[0089] In some embodiments, the two expression vectors co-produce bispecific antibodies in mammalian host cells. Bispecific antibodies can be classified into five classes (one of which will not be discussed further because it requires chemical conjugation). See Spiess et al., 2015, Mol. Immunol. [Molecular Immunology] 67:95-106.

[0090] Bispecific IgG (BsIgG) is a form that is monovalent for each antigen and is produced using two different heavy chains and two different light chains. BsIgG can also be called heterologous IgG. In this form, typically, the coding sequences for the heavy chain and light chain pairs are on a first vector, and the coding sequences for the different heavy chain and light chain pairs are on a second vector.

[0091] Added IgG is a form in which an additional antigen-binding unit is added to the amino or carboxyl terminus of the light or heavy chain. Examples of added IgG include single-domain antibodies (unpaired V antibodies). L or V H Examples include dual variable domain Ig (DVD-Ig), paired antibody variable domains (e.g., Fv or ScFv), and engineered protein scaffolds. See also LaFleur et al., 2013, mAbs [Monoclonal Antibodies] 5:208-218; Wu et al., 2007, Nat. Biotechnol. [Nature Biotechnology] 25:1290-7. In this form, typically, the coding sequences for the heavy and light chain pairs are present on both the first and second vectors. The coding sequence for an antigen-binding unit (e.g., scFv, VHH domain, or cytokine) can be added to the coding sequence of the heavy chain or the light chain. More than one coding sequence for an antigen-binding unit can be added.

[0092] Bispecific antibody fragments lack the constant antibody domain and can consist of heavy and light chains linked by peptide linkers, scFv fragments, dual-affinity retargeting proteins (DART), and biantibodies (including tetravalent tandem biantibodies). See also Arndt et al., 1999, Blood 94:2562-2568; and Kipriyanov et al., 1999, J. Mol. Bio. 293:41-56. In this form, as an example, two distinct scFv fragments can be expressed on each of two vectors, or in the case of DART, one vector contains the coding sequence for VHA-linker-VLB and the other contains the coding sequence for VHB-linker-VLA.

[0093] Bispecific fusion proteins are antibody fragments linked to other proteins, such as receptors and albumin. In this form, typically, the coding sequences for the heavy and light chain pairs are present in both a first and a second vector. The coding sequence for an antigen-binding unit (e.g., a receptor) can be added to either the coding sequence of the heavy chain or the coding sequence of the light chain. More than one coding sequence for an antigen-binding unit can be added.

[0094] In another example, for expressing bispecific IgG antibodies, it may be desirable to use the following embodiment, wherein a first gene encoding an antibody light chain and a second gene encoding an antibody light chain encode different antibody light chains, and a first gene encoding an antibody heavy chain and a second gene encoding an antibody heavy chain encode different antibody heavy chains. This is useful because a typical bispecific IgG antibody contains four different polypeptide types: i) an antibody light chain of a first antigen-binding moiety, ii) an antibody heavy chain of a first antigen-binding moiety, iii) an antibody light chain of a second antigen-binding moiety, and iv) an antibody heavy chain of a second antigen-binding moiety. Therefore, for bispecific IgG expression, it may be desirable to have a first position in a first vector containing a gene encoding a first antibody light chain, a second position in a first vector containing a gene encoding a second antibody light chain, a first position in a second vector containing a gene encoding a first antibody heavy chain, and a second position in a second vector containing a gene encoding a second antibody heavy chain. Alternatively, for bispecific IgG expression, it may be desirable to have a first position in a first vector containing a gene encoding the light chain of the first antibody, a second position in a first vector containing a gene encoding the heavy chain of the first antibody, a first position in a second vector containing a gene encoding the light chain of the second antibody, and a second position in a second vector containing a gene encoding the heavy chain of the second antibody. As discussed above, additional sequences may be appended to the amino or carboxyl termini of the light or heavy chain.

[0095] Other related embodiments are also provided herein. For example, in some embodiments, the first and second vectors may be used in conjunction with the embodiments provided herein, for example, for expressing bispecific antibodies having a common light chain (see, for example, WO 2021 / 124073). Bispecific antibodies having a common light chain may contain two distinct heavy chains.

[0096] In the embodiments provided herein involving a first vector and a second vector, in some other aspects, the first vector contains a first gene encoding an antibody light chain and a second gene encoding an antibody light chain, and the second vector contains a first gene encoding an antibody heavy chain fusion and a second gene encoding an antibody heavy chain. In some embodiments, the first gene encoding the antibody light chain and the second gene encoding the antibody light chain encode the same antibody light chain (e.g., two copies of the same gene). In some other embodiments, the first gene encoding the antibody light chain and the second gene encoding the antibody light chain encode different antibody light chains (e.g., one copy of each of two different antibody light chain genes).

[0097] In the embodiments provided herein involving a first vector and a second vector, in some other aspects, the first vector contains a first gene encoding an antibody light chain and a second gene encoding an antibody heavy chain, and the second vector contains a first gene encoding an antibody light chain and a second gene encoding an antibody heavy chain. In some embodiments, the first gene encoding the antibody light chain and the second gene encoding the antibody light chain encode the same antibody light chain (e.g., two copies of the same gene). In some other embodiments, the first gene encoding the antibody light chain and the second gene encoding the antibody light chain encode different antibody light chains (e.g., one copy of each of two different antibody light chain genes).

[0098] The choice of whether the first vector contains two copies of the same antibody light chain or one copy of each of two different antibody light chains depends on the specific type of antibody molecule to be manufactured. For example, for an antibody fusion protein involving a standard monospecific IgG mAb covalently linked to another protein via an antibody heavy chain, the molecule typically contains three separate polypeptide types: i) an antibody light chain; ii) an antibody heavy chain; and iii) an antibody heavy chain fusion. In this antibody fusion molecule, there is one chain of each of the antibody heavy chain and the antibody heavy chain fusion, and two copies of the antibody light chain. Since only one type of antibody light chain is present in the molecule, two copies of the same antibody light chain gene are provided in the first vector. In another example, for an antibody fusion protein involving a bispecific IgG mAb covalently linked to another protein via an antibody heavy chain, the molecule typically contains four separate polypeptide types: i) an antibody light chain of a first antigen-binding portion; ii) an antibody heavy chain of a first antigen-binding portion; iii) an antibody light chain of a second antigen-binding portion; and iv) an antibody heavy chain fusion, wherein the antibody heavy chain portion is used for the second antigen-binding portion. This antibody fusion protein molecule contains one copy of each of the antibody heavy chain and the antibody heavy chain fusion body, as well as one copy of each of the antibody light chains. Because the molecule contains two types of antibody light chains, two distinct antibody light chain genes are provided in the first vector.

[0099] In the embodiments provided herein involving two different antibody heavy chains or fusions of antibody heavy chains, optionally, the heavy chains may contain one or more amino acid modifications in a constant region of the heavy chain to promote heterodimer formation between the two different heavy chains. Such modifications are known in the art and include, for example, electrostatic amino acid modifications based on charge and spatial "mortar and pestle" amino acid modifications.

[0100] Vectors can be used to transform host cells and may contain additional nucleic acid sequences that direct and / or control (alongside the host cell) the expression of one or more heterologous coding regions operatively linked to them. Expression constructs may include, but are not limited to, sequences that affect or control transcription, translation, and, in the presence of introns, influence RNA splicing of coding regions operatively linked to them. "Operably linked" means that the components to which this term applies are in a relationship that allows them to perform their inherent functions. For example, the arrangement of control sequences (e.g., promoters) in a vector "operatively linked" to a protein-coding sequence such that normal activity of the control sequence leads to transcription of the protein-coding sequence, resulting in recombinant expression of the encoded protein.

[0101] Vectors that are functional in the specific host cell used can be selected (i.e., the vector is compatible with the host cell structure, thereby allowing gene amplification and / or expression to occur). In some embodiments, the vector used employs protein fragment complementation assays using a protein reporter sequence such as dihydrofolate reductase (see, for example, U.S. Patent No. 6,270,964). Suitable expression vectors are known in the art and are commercially available.

[0102] Typically, vectors used in host cells will contain sequences for plasmid maintenance and for cloning and expressing exogenous nucleotide sequences. Such sequences will typically include one or more of the following nucleotide sequences (in addition to one or more promoters and optional markers mentioned above): one or more enhancer sequences, origin of replication, transcription and translation control sequences, transcription termination sequences, complete intron sequences containing donor and acceptor splicing sites, various pre-sequences / pro-sequences that improve glycosylation or yield, natural or heterologous signal sequences (lead sequences or signal peptides) for polypeptide secretion, ribosome binding sites, polyadenylated sequences, internal ribosome entry sites (IRES) sequences, expression enhancement sequence elements (EASE), triplet leader sequences (TPA) and VA gene RNA from adenovirus 2, and multi-connector regions of multinucleotides encoding the polypeptide to be expressed. Vectors can be constructed from starter vectors (such as commercially available vectors), and other elements can be obtained separately and ligated into the vector. Methods for obtaining the components are well known to those skilled in the art.

[0103] Vector components can be homologous (i.e., derived from the same species and / or strain as the host cell), heterologous (e.g., derived from a species other than the host cell species or strain), heterozygous (i.e., derived from a combination of side sequences from more than one source), synthetic, or natural. The sequences of the useful components in these vectors can be obtained using methods well-known in the art, such as those previously identified by mapping and / or by restriction endonucleases. Alternatively, they can be obtained by polymerase chain reaction (PCR) and / or by screening genomic libraries with suitable probes.

[0104] Ribosome binding sites are typically required for the initiation of mRNA translation and are characterized by a Shine-Dalgarno sequence (prokaryotes) or a Kozak sequence (eukaryotes). This element is typically located at the 3' of the promoter and at the 5' of the coding sequence of the polypeptide to be expressed.

[0105] Origin of replication facilitates the amplification of vectors within host cells. These can be included as part of commercially available prokaryotic vectors or chemically synthesized based on known sequences and ligated into vectors. Various viral sources (e.g., SV40, polyomaviruses, adenoviruses, vesicular stomatitis virus (VSV), or papillomaviruses such as HPV or BPV) can be used to clone vectors in mammalian cells.

[0106] Transcriptional and translational control sequences for mammalian host cell expression vectors can be excised from the viral genome. Commonly used promoter and enhancer sequences are derived from polyomaviruses, adenovirus 2, simian virus 40 (SV40), and human cytomegalovirus (CMV). For example, the human CMV promoter / enhancer of the immediate early gene 1 can be used. See, for example, Patterson et al., 1994, Applied Microbiol. Biotechnol. [Applied Microbiology and Biotechnology] 40:691-98. DNA sequences derived from the SV40 viral genome, such as SV40-derived, early and late promoters, enhancers, splice sequences, and polyadenylation sites, can be used to provide other genetic elements for the expression of structural gene sequences in mammalian host cells. Early and late viral promoters are particularly useful because they are readily available as fragments from the viral genome and can also contain the origin of viral replication (Fiers et al., 1978, Nature 273:113; Kaufman, 1990, Meth.in Enzymol 185:487-511). Smaller or larger SV40 fragments can also be used, provided they include approximately 250 bp of the sequence extending from the Hind III site to the BglI site located at the SV40 viral replication origin.

[0107] Enhancer sequences can be inserted into this vector to increase transcription in higher eukaryotes. Enhancers are cis-acting elements of DNA, typically about 10-300 bp in length, that act on the promoter to increase transcription. Enhancers are relatively independent in orientation and location, and have been found at the 5' and 3' positions of transcription units. Several enhancer sequences are known from mammalian genes (e.g., globulins, elastases, albumins, alpha-fetoproteins, and insulin). However, enhancers derived from viruses are typically used. The SV40 enhancer, cytomegalovirus early promoter enhancer, polyomavirus enhancer, and adenovirus enhancer known in the art are exemplary enhancing elements for activating eukaryotic promoters. Although enhancers can be located at the 5' or 3' of the coding sequence in the vector, they are typically located at the 5' site of the promoter.

[0108] Transcription termination sequences are typically located at the 3′ end of the polypeptide coding region and are used to terminate transcription. In prokaryotic cells, the transcription termination sequence is usually a GC-rich fragment followed by a poly-T sequence. While the sequence can be readily cloned from libraries or even commercially available as part of a vector, it can also be readily synthesized using nucleic acid synthesis methods known to those skilled in the art.

[0109] In some cases, such as when glycosylation is required in eukaryotic host cell expression systems, various pre-sequences can be manipulated to improve glycosylation or yield. For example, the peptidase cleavage site of a specific signal peptide can be altered, or pre-sequences can be added, which can also affect glycosylation. The final protein product may have one or more additional amino acids readily expressible at the -1 position (relative to the first amino acid of the mature protein), which may not be completely removed. For example, the final protein product may have one or two amino acid residues attached to the amino terminus found in the peptidase cleavage site. Alternatively, when the enzyme cleaves in such regions within the mature polypeptide, using some of the enzyme cleavage sites may produce a slightly truncated form of the desired polypeptide.

[0110] A sequence encoding an appropriate natural or heterologous signal sequence (lead sequence or signal peptide) can be incorporated into an expression vector to promote the extracellular secretion of the target protein. The choice of signal peptide or leader sequence depends on the type of host cell from which the target protein is to be produced, and the heterologous signal sequence can replace the natural signal sequence. Examples of functional signal peptides in mammalian host cells include: the interleukin-7 signal sequence described in U.S. Patent No. 4,965,195; the interleukin-2 receptor signal sequence described in Cosman et al., 1984, Nature [Nature] 312:768; the interleukin-4 receptor signal peptide described in European Patent No. 0367566; the type I interleukin-1 receptor signal peptide described in U.S. Patent No. 4,968,607; and the type II interleukin-1 receptor signal peptide described in European Patent No. 0460846.

[0111] Other control sequences that have been shown to improve the expression of heterologous genes from mammalian expression vectors include elements such as expression-enhancing sequence elements (EASE) derived from CHO cells (Morris et al., in Animal Cell Technology, pp. 529-534 (1997); US Patent Nos. 6,312,951 B1, 6,027,915 and 6,309,841 B1) and triplet leader sequences (TPL) and VA gene RNA derived from adenovirus 2 (Gingeras et al., 1982, J. Biol. Chem. 257:13475-13491). Virus-derived internal ribosome entry site (IRES) sequences enable efficient translation of bicistronic mRNAs (Oh and Sarnow, 1993, Current Opinion in Genetics and Development 3:295-300; Ramesh et al., 1996, Nucleic Acids Research 24:2697-2700).

[0112] Target protein

[0113] The target peptides and proteins may have scientific or commercial significance, including protein-based therapeutics. Target proteins particularly include secreted proteins, non-secreted proteins, intracellular proteins, or membrane-bound proteins. Target peptides and proteins can be produced using cell culture methods through recombinant animal cell lines and may be referred to as “recombinant proteins.” One or more expressed proteins can be produced intracellularly or secreted into a culture medium from which they can be recovered and / or collected. The terms “isolated protein” or “isolated recombinant protein” refer to a target peptide or protein purified from proteins or peptides or other contaminants that would interfere with its therapeutic, diagnostic, preventative, research, or other uses. Target proteins include proteins that exert therapeutic effects by binding to targets, particularly those listed below, including targets derived from them, associated targets, and modifications thereof.

[0114] The target protein includes "antigen-binding proteins." An "antigen-binding protein" is a protein or polypeptide that includes an antigen-binding region or region that has an affinity for another molecule (antigen) to which it binds. Antigen-binding proteins encompass antibodies, peptides, antibody fragments, antibody derivatives, antibody analogs, Fc fusion proteins (including single-chain variable fragments (scFv), double-chain (bivalent) scFv, and IgG scFv (see, for example, Orcutt et al., 2010, ProteinEng Des2 Sel [Protein Engineering, Design & Selection] 23:221-228), heterologous IgG (see, for example, Liu et al., 2015, J Biol Chem [Journal of Biochemistry] 290:7535-7562), mutant proteins, and XmAbs. ® (Xencor, Inc., Monrovia, CA). Examples of antigen-binding proteins include human antibodies, humanized antibodies; chimeric antibodies; recombinant antibodies; single-chain antibodies; biantibodies; triantibodies; tetraantibodies; Fab fragments; F(ab')2 fragments; IgD antibodies; IgE antibodies; IgM antibodies; IgG1 antibodies; IgG2 antibodies; IgG3 antibodies; or IgG4 antibodies, and fragments thereof. Also included are bispecific T-cell binding agents (BiTE). ® Bispecific T-cell binding agents with extended durations (e.g., extended half-life) (e.g., HLEBiTE, HeteroIg BITE, etc.)

[0115] As used herein, the term “antigen-binding protein” is used in its broadest sense and refers to a protein that contains a portion that binds to an antigen or target, optionally including a scaffold or framework portion that allows the antigen-binding portion to adopt a conformation that promotes the binding of the antigen-binding protein to the antigen. Antigen-binding proteins may comprise, for example, alternative protein scaffolds or artificial scaffolds with grafted CDRs or CDR derivatives. Such scaffolds include, but are not limited to, antibody-derived scaffolds containing mutations introduced to, for example, stabilize the three-dimensional structure of the antigen-binding protein; and fully synthetic scaffolds containing, for example, biocompatible polymers. See, for example, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 53(1):121-129; Roque et al., 2004, Biotechnol.Prog. 20:639-654. Additionally, peptide antibody mimics (“PAMs”) and scaffolds based on antibody mimics utilizing fibronectin components as scaffolds may be used.

[0116] Antigen-binding proteins can have structures such as those of naturally occurring immunoglobulins. An immunoglobulin is a tetrameric molecule. In naturally occurring immunoglobulins, each tetramer consists of two pairs of identical polypeptide chains, each pair having a "light chain" (approximately 25 kDa) and a "heavy chain" (approximately 50-70 kDa). The amino-terminal portion of each chain includes a variable region of approximately 100 to 110 or more amino acids, which is primarily responsible for antigen recognition. The carboxyl-terminal portion of each chain defines a constant region, primarily responsible for effector functions. Human light chains are classified as κ light chains and λ light chains. Heavy chains are classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively.

[0117] Naturally occurring immunoglobulin chains exhibit the same general structure of a relatively conserved framework region (FR) linked by three hypervariable regions (also known as complementarity-determining regions or CDRs). Both the light and heavy chains contain domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4, from the N-terminus to the C-terminus. Each domain can be assigned amino acids according to the definition in Sequences of Proteins of Immunological Interest, 5th edition, US Dept. of Health and Human Services, PHS, NIH, NIH Publication No. 91-3242, (1991). The CDR can also be redefined according to alternative nomenclature schemes, such as Chothia's nomenclature scheme (see Chothia and Lesk, 1987, J. Mol. Biol. [Journal of Molecular Biology] 196:901-917; Chothia et al., 1989, Nature [Nature] 342:878-883 or Honegger and Pluckthun, 2001, J . Mol. Biol. [Journal of Molecular Biology] 309:657-670).

[0118] In the context of this disclosure, when the dissociation constant (K) D ≤ 10 -8 When M occurs, the antigen-binding protein is said to "specifically bind" or "selectively bind" to its target antigen. When K occurs... D ≤ 5 x 10 -9 At M, the antibody binds to the antigen with "high affinity," while at K... D ≤ 5 x 10 -10 When M occurs, the antibody binds to the antigen with "extremely high affinity".

[0119] Unless otherwise stated, the term "antibody" includes any isotype or subclass of glycosylated and non-glycosylated immunoglobulin, or its antigen-binding region that competes with intact antibodies for specific binding. Additionally, unless otherwise stated, the term "antibody" refers to an intact immunoglobulin that competes with intact antibodies for specific binding.

[0120] Antigen-binding fragments can be produced through recombinant DNA technology or through enzymatic or chemical cleavage of intact antibodies, and can form elements of the target protein. Unless otherwise stated, antibodies include human, humanized, chimeric, multispecific, monoclonal, polyclonal, heterologous IgG, bispecific antibodies, and their oligomers or antigen-binding fragments. Antibodies include IgG1, IgG2, IgG3, or IgG4 types. Also included are proteins with antigen-binding fragments or antigen-binding regions, such as Fab, Fab', F(ab')2, Fv, biantibodies, Fd, dAb, macrobody, single-chain antibody molecules, and single-domain V. H H, complementarity-determining region (CDR) fragments, scFv, biantibodies, triantibodies, tetraantibodies, and polypeptides that contain at least a portion of an immunoglobulin sufficient to bind a specific antigen to a target polypeptide.

[0121] Antigen-binding proteins may have one or more binding sites. If more than one binding site is present, these binding sites may be the same or different from each other. For example, naturally occurring human immunoglobulins typically have two identical binding sites, while "bispecific" or "bifunctional" antibodies have two different binding sites.

[0122] Fab fragments are those with V L V H C L and C H 1. A monovalent segment of a structural domain; F(ab')2 segment is a divalent segment having two Fab segments connected by a disulfide bridge in the hinge region; Fd segment has V H and C H 1. Structural domain; the Fv fragment has a V-shaped arm for the antibody. L and V H Structural domain; and the dAb fragment has V H Structural domain, V L structural domain, or V H or V LAntigen-binding fragments of the domain (US Patent Nos. 6,846,634, 6,696,245, US Patent Application Publication Nos. 2005 / 0202512, 2004 / 0202995, 2004 / 0038291, 2004 / 0009507, 2003 / 0039958, Ward et al., 1989, Nature 341:544-546).

[0123] Single-chain antibodies (scFv) are antibodies in which V L and V H Regions are linked via linkers (e.g., synthetic sequences of amino acid residues) to form continuous protein chains, where the linkers are long enough to allow the protein chains to fold back and form monovalent antigen-binding sites (see, for example, Bird et al., 1988, Science 242:423-26 and Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-83, U.S. Patents 7,741,465 and 6,319,494, and Eshhar et al., 1997, Cancer Immunol Immunotherapy 45:131-136). scFv retains the ability of the parent antibody to specifically interact with the target antigen.

[0124] Biantibodies are bivalent antibodies consisting of two polypeptide chains, each containing V proteins linked by a linker. H and V L The linker is too short to allow pairing between two domains on the same chain, thus allowing each domain to pair with a complementary domain on another polypeptide chain (see, for example, Holliger et al., 1993, Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 90:6444-48; and Poljak et al., 1994, Structure 2:1121-23). ​​If the two polypeptide chains of a biantibody are identical, the biantibody produced by pairing them will have two identical antigen-binding sites. Polypeptide chains with different sequences can be used to prepare biantibodies with two different antigen-binding sites. Similarly, triantibodies and tetraantibodies are antibodies that contain three and four polypeptide chains, respectively, forming three and four antigen-binding sites, which may be the same or different.

[0125] For clarity, and as described herein, note that antigen-binding proteins may, but do not have to, be of human origin (e.g., human antibodies), and in some cases will contain non-human proteins, such as rat or mouse proteins, and in other cases antigen-binding proteins may contain hybrids of human and non-human proteins (e.g., humanized antibodies).

[0126] The target protein may include a human antibody. The term "human antibody" includes all antibodies having one or more variable and constant regions derived from a human immunoglobulin sequence. In one embodiment, all variable and constant domains are derived from a human immunoglobulin sequence (a fully human antibody). Such antibodies can be prepared in a variety of ways, including by immunizing mice genetically modified to express antibodies derived from human heavy and / or light chain encoding genes, such as those derived from Xenomouse, with the target antigen. ® UltiMab™ or Velocimmune ® The system's mice, or those derived from UniRat ® Rats. Phage-based methods can also be used.

[0127] Alternatively, the target protein may include a humanized antibody. The sequence of a “humanized antibody” differs from that of an antibody derived from a non-human species in that one or more amino acid substitutions, deletions, and / or additions are made such that, when administered to a human subject, the humanized antibody is less likely to induce an immune response and / or induce a less severe immune response compared to a non-human species antibody. In one embodiment, certain amino acid mutations are made in the framework and constant domains of the heavy and / or light chains of a non-human species antibody to produce a humanized antibody. In another embodiment, one or more constant domains from a human antibody are fused to one or more variable domains from a non-human species. Examples of how humanized antibodies can be prepared can be found in U.S. Patent Nos. 6,054,297, 5,886,152, and 5,877,293.

[0128] It also includes modified proteins, such as those chemically modified by non-covalent, covalent, or both covalent and non-covalent bonds. It further includes proteins containing one or more post-translational modifications, which can be prepared by modification through cellular modification systems or by in vitro introduction or other means by enzymatic and / or chemical methods.

[0129] In some embodiments, the target protein may include proteins that specifically bind to one or more CD proteins, HER receptor family proteins, cell adhesion molecules, growth factors, nerve growth factors, fibroblast growth factors, transforming growth factors (TGF), insulin-like growth factors, bone-inducing factors, insulin and insulin-related proteins, coagulation and coagulation-related proteins, colony-stimulating factors (CSF), other blood and serum proteins, and blood group antigens; receptors, receptor-related proteins, growth hormone, growth hormone receptors, and T-cell receptors; neurotrophic factors, neurotrophic proteins, relaxin, interferon, interleukin, viral antigens, lipoproteins, integrins, rheumatoid factor, immunotoxins, surface membrane proteins, transport proteins, homing receptors, addressins, regulatory proteins, and immunoadhesins.

[0130] In some embodiments, the target protein may be combined alone or in any combination with one or more of the following proteins: CD proteins (including but not limited to CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD25, CD30, CD33, CD34, CD38, CD40, CD70, CD123, CD133, CD138, CD171, and CD174), HER receptor family proteins (including, for example, HER2, HER3, HER4, and EGF receptors), EGFRvIII, cell adhesion molecules (e.g., LFA-1, Mol, p150,95, VLA-4, ICAM-1, VCAM, and αv / β3 integrin), growth factors (including but not limited to, for example, vascular endothelial growth factor (“VEGF”); VEGFR2, growth hormone, thyroid-stimulating hormone, follicle-stimulating hormone, luteinizing hormone, growth hormone-releasing factor, parathyroid hormone, and Müllerian-inhibiting substances. substance), human macrophage inflammatory protein (MIP-1-α), erythropoietin (EPO), nerve growth factors (such as NGF-β), platelet-derived growth factor (PDGF), fibroblast growth factor (including, for example, aFGF and bFGF), epidermal growth factor (EGF), Cripto, transforming growth factor (TGF) (especially including TGF-α and TGF-β (including TGF-β1, TGF-β2, TGF-β3, TGF-β4 or TGF-β5)), insulin-like growth factor-I and insulin-like growth factor-II (IGF-I and IGF-II), des(1-3)-IGF-I (brain IGF-I) and bone-inducing factor, insulin and insulin-related proteins (including but not limited to insulin, insulin A chain, insulin B chain, proinsulin and insulin-like growth factor binding protein); (coagulation proteins and coagulation-related proteins, especially, such as factor VIII, tissue factor, van Wilbond) Willebrand factor, protein C, α-1-antitrypsin, plasminogen activators (such as urokinase and tissue plasminogen activator (“t-PA”)), bombazine, thrombin, thrombopoietin and thrombopoietin receptor, colony-stimulating factor (CSF) (especially including M-CSF, GM-CSF and G-CSF), other blood and serum proteins (including but not limited to albumin, IgE and blood group antigens), receptors and receptor-associated proteins (including, for example, flk2 / flt3 receptors, obesity (OB) receptors, growth hormone receptors and T-cell receptors); neurotrophic factors, including but not limited to bone-derived neurotrophic factor (BDNF) and neurotrophin-3, neurotrophin-4, neurotrophin-5 or neurotrophin-6 (NT-3, NT-4, NT-5 or NT-6).Relaxin A chain, relaxin B chain and pro-relaxin, interferons (including, for example, interferon α, interferon β and interferon γ), interleukins (ILs) (e.g. IL-1 to IL-10, IL-12, IL-15, IL-17, IL-23, IL-12 / IL-23, IL-2Ra, IL-1-R1, IL-6 receptor, IL-4 receptor and / or IL-13 receptor, IL-13RA2 or IL-17 receptor, IL-1RAP); viral antigens, including but not limited to AIDS envelope virus antigens, lipoproteins, calcitonin, glucagon, atrial natriuretic peptide, pulmonary surfactant. Agents, tumor necrosis factor-α and tumor necrosis factor-β, enkephalin, BCMA, IgKappa, ROR-1, ERBB2, mesothelin, RANTES (activated and regulated normal T cell expression and secretion factors), mouse gonadotropin-related peptide, DNase, FR-α, inhibin and activin, integrin, protein A or D, rheumatoid factor, immunotoxin, bone morphogenetic protein (BMP), superoxide dismutase, surface membrane protein, decay accelerator factor (DAF), AIDS envelope, transport protein, homing receptor, MIC (MIC-a, MIC-B), ULBP 1-6, EPCAM, addressin, regulatory protein, immunoadhesin, antigen-binding protein, growth hormone, CTGF, CTLA4, eotaxin-1, MUC1, CEA, c-MET, Claudin-18, GPC-3, EPHA2, FPA, LMP1, MG7, NY-ESO-1, PSCA, ganglioside GD2, ganglioside GM2, BAFF, OPGL (RANKL), myostatin, Dickkopf-1 (DKK-1), Ang2, NGF, IGF-1 receptor, hepatocyte growth factor (HGF), TRAIL-R2, c-Kit, B7RP-1, PSMA, NKG2D-1, programmed cell death protein 1 and ligand, PD1 and PDL1, mannose receptor / hCGβ, hepatitis C virus, mesothelin dsFv [PE38] conjugate, Legionella pneumophila (lly), IFN γ, interferon-gamma inducible protein 10 (IP10), IFNAR, TALL-1, thymic stromal lymphopoietin (TSLP), proprotein convertase subtilisin / Kexin type 9 (PCSK9), stem cell factor, Flt-3, calcitonin gene-related peptide (CGRP), OX40L, α4β7, platelet-specific (platelet glycoprotein IIb / IIIb (PAC-1), transforming growth factor β (TFGβ), zona pellucida sperm-binding protein 3 (ZP-3), TWEAK, platelet-derived growth factor receptor α (PDGFRα), sclerostin, and any bioactive fragments or variants of the foregoing substances.

[0131] In another embodiment, the target protein includes abciximab, adalimumab, adelimumab, aflibercept, alenmab, alicurumab, anakinase, asceticumab, bailiximab, belimumab, bevacizumab, biotinylate, bonatumab, bentuximab, brodatumab, mocantozumab, konatumab, cetuximab, tertuximab, and konatumab. Dalizumab, denosumab, eculizumab, ezolizumab, efalizumab, epazolizumab, etanercept, evokulumab, galiliximab, genitalumab, gemutuzumab, golimumab, teimozumab, infliximab, ipilimumab, ixazolizumab, levocurumab, lucizumab, mapalimumab, motesanib phosphate diphosphate), morotumab-CD3, natecillatab, nimotuzumab, nivolumab, olizumab, olrezumab, olfamumab, olmalizumab, interleukin, palizumab, panitumab, pembrolizumab, pertuzumab, pectizumab, ranituzumab, rituximab, rituximab, romistastatin, lomoxoluzumab, saxaglastin, tocilizumab, tosimomab, trastuzumab, uterotumab, vedozazumab, vexizumab, voloximab, zalumab, zalumab, and any biosimilars of the foregoing substances.

[0132] The target protein described in this invention encompasses all the foregoing and further includes antibodies containing 1, 2, 3, 4, 5, or 6 complementarity-determining regions (CDRs) of any of the aforementioned antibodies. One or more CDRs can be covalently or non-covalently incorporated into the molecule to make it an antigen-binding protein. The antigen-binding protein can be incorporated into a CDR as part of a larger polypeptide chain, can be covalently linked to another polypeptide chain, or can be non-covalently incorporated into a CDR. The CDR allows the antigen-binding protein to bind specifically to a particular target antigen. Variations are also included that include regions of an amino acid sequence having 70% or higher, particularly 80% or higher, more particularly 90% or higher, even more particularly 95% or higher, especially 97% or higher, even more particularly 98% or higher, even more particularly 99% or higher identity with a reference amino acid sequence of the target protein. This identity can be determined using a variety of well-known and readily available amino acid sequence analysis software. Preferred software includes those implementing the Smith-Waterman algorithm, which is considered a satisfactory solution to the problem of searching and aligning sequences. Other algorithms can also be used, especially when speed is a significant consideration. Commonly used programs for DNA, RNA, and peptide alignment and homology matching include FASTA, TFASTA, BLASTN, BLASTP, BLASTX, TBLASTN, PROSRCH, BLAZE, and MPSRCH, the latter being an implementation of the Smith-Waltman algorithm for execution on massively parallel processors manufactured by MasPar.

[0133] Preferably, the antigen-binding molecule is its antibody fragment, and more preferably one or more single-chain antibody fragments (“scFv”). scFvs are preferred for use in chimeric antigen receptors because they can be engineered to be expressed as part of a single chain. See Krause et al., 1988, J. Exp. Med., 188(4): 619-626; Finney et al., 1998, J Immunol 161: 2791-2797.

[0134] The “Fc” region, as used in this text, contains the C-cell containing the antibody. H 2 and C H The three-domain structure consists of two heavy-chain segments. These two heavy-chain segments are composed of two or more disulfide bonds and C... H The hydrophobic interactions of the three domains maintain their cohesion. Target proteins containing the Fc region (including antigen-binding proteins and Fc fusion proteins) form another aspect of this disclosure.

[0135] A "half-antibody" is an immunofunctional immunoglobulin construct comprising a complete heavy chain, a complete light chain, and a second heavy chain Fc region paired with the Fc region of the complete heavy chain. A linker may, but is not necessary, connect the heavy chain Fc region and the second heavy chain Fc region. In a particular embodiment, the half-antibody is a monovalent form of the antigen-binding protein disclosed herein. In other embodiments, one Fc region may be associated with a second Fc region using paired charged residues. In the context of this disclosure, the half-antibody may be the target protein.

[0136] Production of mammalian host cells expressing the target protein

[0137] The expression of the target protein in cells can be achieved transiently or stably using well-known methods (Davis et al., Basic Methods in Molecular Biology, 2nd ed., Appleton & Lange, Norwalk, Connecticut, 1994; Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001).

[0138] Methods for stable integration are well known in the art. In short, stable integration is typically achieved by transiently introducing a heteropolynucleotide or a vector containing a heteropolynucleotide into a host cell, which facilitates the stable integration of the heteropolynucleotide into the cellular genome. Typically, the heteropolynucleotide is flanked by homologous arms, i.e., sequences homologous to upstream and downstream regions of the integration site. Circular vectors can be linearized to facilitate integration into the cellular genome before being introduced into mammalian host cells. Methods for introducing vectors into cells are well known in the art and include transfection using biological methods (such as viral delivery), chemical methods (such as using cationic polymers, calcium phosphate, cationic lipids, or cationic amino acids), physical methods (such as electroporation or microinjection), or hybrid methods (such as protoplast fusion).

[0139] Stable integration-specific methods utilize recombinase-mediated cassette exchange (RMCE; Bode and Baer, ​​2001, CurrOpin Biotechnol. [Current Biotechnical Perspective] 12:473-80, and Bode et al., 2000, Biol. Chem. [Biochemistry] 381:801-813) for site-specific integration into the genome (also known as “targeted integration”). Site-specific recombinases such as Flp and Cre mediate recombination between two copies of their target sequence, referred to as FRT and loxP, respectively. Using two incompatible target sequences, such as FRT combined with F3 (Schlake and Bode, 1994, Biochemistry [Biochemistry], 33:12746-51) and an inverted recognition target site (Feng et al., 1999, J. Mol. Biol. [Journal of Molecular Biology] 292:779-85), allows the insertion of DNA fragments into predetermined chromosomal sites carrying target sequences of similar conformation. See also European Patent No. EP 1781796 B1 and European Patent Application Publication No. EP 2789691 A1.

[0140] RMCE insertion into specific sites in the genome can be mediated by nucleases (e.g., zinc finger proteins (ZFPs), transcription activator-like effector nucleases (TALENs), and clustered regularly spaced short palindromic repeats (CRISPR) / CRISPR-associated protein 9 (Cas9)). These nucleases can be engineered to generate single-strand and double-strand breaks (SSBs / DSBs) in the genome. There are two main and distinct pathways for DSB repair—homologous recombination and non-homologous end joining (NHEJ). Homologous recombination requires the presence of a homologous sequence as a template (e.g., a “donor” containing the RMCE) to guide the cellular repair process, and the repair outcome is error-free and predictable. In the absence of a template (or “donor”) sequence for homologous recombination, cells typically attempt to repair DSBs via the unpredictable and error-prone process of non-homologous end joining (NHEJ).

[0141] Vectors can be any molecule or entity suitable for transferring and / or transporting protein-coding information to and / or to specific locations and / or compartments within host cells (e.g., nucleic acids, plasmids, bacteriophages, transposons, granules, chromosomes, viruses, viral capsids, virions, naked DNA, complex DNA, etc.). Vectors can include viral and nonviral vectors, and non-attachment mammalian vectors. Vectors are commonly referred to as expression vectors, such as recombinant expression vectors and cloning vectors. Vectors can be introduced into host cells to allow replication of the vector itself, thereby amplifying copies of the polynucleotides contained therein. Cloning vectors may contain sequence components, which typically include, but are not limited to, origin of replication, promoter sequences, transcription initiation sequences, enhancer sequences, and optional markers. These elements can be appropriately selected by those skilled in the art.

[0142] After construction, one or more vectors can be inserted into suitable cells for amplification and / or peptide expression. Transformation of the expression vector into selected cells can be accomplished by well-known methods, including transfection, infection, calcium phosphate co-precipitation, electroporation, nuclear transfection, microinjection, DEAE-dextran-mediated transfection, cationic lipid-mediated delivery, liposome-mediated transfection, microbombardment, receptor-mediated gene delivery, and polylysine, histone, chitosan, and peptide-mediated delivery. This method will vary in some respects depending on the type of host cells used. These methods, and others suitable for use, are well known to those skilled in the art and are described in manuals and other technical publications, such as Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (2001).

[0143] The term "transformation" refers to a change in the genetic characteristics of a cell. A cell is transformed when it is modified to contain new DNA or RNA. For example, a cell is transformed when new genetic material is introduced into it via transfection, transduction, or other techniques, resulting in genetic modification from its original state. After transfection or transduction, the transformed DNA can either physically integrate into the cell's chromosome and recombine with the cell's DNA, or it can be temporarily maintained as a non-replicating free element, or it can replicate independently as a plasmid. When the transformed DNA replicates with cell division, the cell is considered to have been "stablely transformed."

[0144] The term “transfection” refers to the absorption of foreign or exogenous DNA by cells. Many transfection techniques are well known in the art and are disclosed herein. See, for example, Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, ibid.; Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al., 1981, Gene 13:197.

[0145] The term “transduction” refers to the process by which foreign DNA is introduced into cells via viral vectors. See Jones et al. (1998). Genetics: principles and analysis. Boston: Jones & Bartlett Publ.

[0146] cell lines

[0147] In the methods disclosed herein, any mammalian cell line can be used. A variety of mammalian cell lines suitable for growth in culture are available from the American Type Culture Collection (Manassas, Virginia) and commercial suppliers. Examples of cell lines commonly used in the industry include the monkey kidney CVl line transformed from SV40 (COS-7, ATCC CRL 1651); the human embryonic kidney line (293 cells or subclones used for growth in suspension culture (Graham et al., 1977, J. Gen Virol. [Journal of General Virology] 36:59); juvenile hamster kidney cells (BHK, ATCC CCL 10); mouse saturated cells (TM4, Mather, 1980, Biol. Reprod. [Reproductive Biology] 23:243-251); monkey kidney cells (CVl ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); and Buffalo rat hepatocytes (BRL 3A, ATCC CRL). 1442); human lung cells (W138, ATCC CCL 75); human liver cancer cells (Hep G2, HB8065); mouse mammary tumors (MMT 060562, ATCC CCL51); TRI cells (Mather et al., 1982, Annals N.Y Acad. Sci. [Annals of the New York Academy of Sciences] 383:44-68); MRC 5 cells or FS4 cells; mammalian myeloma cells, as well as many other cell lines and Chinese hamster ovary (CHO) cells.

[0148] Large-scale production of proteins for commercial applications typically takes place in suspension culture. Therefore, the mammalian host cells used to generate the recombinant mammalian cells described herein can, but need not, be adapted for growth in suspension culture. Several host cells are known to be adapted for growth in suspension culture, including mouse myeloma NS0 cells and CHO cells from the CHO-S, DG44, and DXB11 cell lines. Other suitable cell lines include mouse myeloma SP2 / 0 cells, juvenile hamster kidney BHK-21 cells, and human PER.C6 cells. ® Cells, human embryonic kidney HEK-293 cells, and cell lines derived from or engineered from any cell line disclosed herein.

[0149] CHO cells are widely used for the production of complex recombinant proteins, including CHOK1 cells (ATCC CCL61). Dihydrofolate reductase (DHFR) deficient mutant cell lines (Urlaub et al., 1980, Proc Natl Acad Sci USA [Proceedings of the National Academy of Sciences] 77: 4216-4220) DXB11 and DG-44 are ideal CHO host cell lines because efficient DHFR-selective and amplifiable gene expression systems allow for high-level expression of recombinant proteins in these cells (Kaufman RJ, 1990, Meth Enzymol [Enzyme Methodology] 185:537-566). Also included is the glutamine synthetase (GS) knockout CHOK1SV cell line, selected using methionine sulfoxide imine (MSX) based on glutamine synthetase (GS). Other suitable CHO host cells may include, but are not limited to, the following (ECACC accession numbers are in parentheses): CHO (85050302), CHO (protein-free) (00102307), CHO-K1 (85051005), CHO-K1 / SF (93061607), CHO / DHFR- (94060607), CHO / DHFR-AC-free (05011002), RR-CHOKI (92052129).

[0150] Cell culture process

[0151] The host cells transfected using the vector systems described herein are suitable for adherent or suspension cultures grown in stirred tank reactors (including conventional batch and fed-batch cell cultures, which may but need not include a rotating filter), perfusion systems (including alternating tangential flow (“ATF”) cultures, acoustic perfusion systems, depth filter perfusion systems, and other systems), hollow fiber bioreactors (HFBs, which in some cases can be used for perfusion processes), and various other cell culture methods (see, for example, Tao et al., 2003, Biotechnol. Bioeng. [Biotechnology and Bioengineering] 82:751-65; Kuystermans and Al-Rubeai, (2011) “Bioreactor Systems for Producing Antibody from Mammalian Cells”). Antibody Expression and Production [ Antibody expression and productionIn Cell Engineering, 7:25-52, Al-Rubeai (ed.) Springer; Catapano et al., (2009) "Bioreactor Design and Scale-Up" Cell and Tissue Reaction Engineering: Principles and Practice [ Cellular and Tissue Reaction Engineering: Principles and Practice In [reference], Eibl et al. (edited) Springer-Verlag, whose full text is incorporated herein by reference.

[0152] Between stages of recombinant protein production, a controlled system is desired in which cells grow to the desired density, and then the cells' physiological state transitions to a high-productivity state of growth arrest, where cells use energy and substrates to produce the desired recombinant protein rather than generating more cells. Various methods exist to achieve this goal, including temperature changes and amino acid starvation, as well as the use of cell cycle inhibitors or other molecules that can stop cell growth without inducing cell death.

[0153] The production of recombinant proteins begins with establishing mammalian cell production cultures expressing the protein in culture plates, flasks, tubes, bioreactors, or other suitable containers. Typically, smaller production bioreactors are used; in one embodiment, the bioreactor is 500 L to 2000 L. In another embodiment, a bioreactor of 1000 L to 2000 L is used. The seed cell density used to inoculate the bioreactor can have a positive impact on the level of recombinant protein produced. In one embodiment, the bioreactor is used in serum-free medium with a density of at least 0.5 × 10⁻⁶ cells / mL. 6 Up to 3.0 × 10 6 1 live cells / mL seeding. In another embodiment, the seeding volume is 1.0 × 10⁶ cells / mL. 6 live cells / mL.

[0154] The mammalian cells then undergo an exponential growth phase. The cell culture can be maintained without supplemental feeding until the desired cell density is reached. In one embodiment, the cell culture is maintained for up to three days with or without supplemental feeding. In another embodiment, the culture can be inoculated at the desired cell density to initiate the production phase without a brief growth phase. In any embodiment herein, the transition from the growth phase to the production phase can also be initiated by any of the foregoing methods.

[0155] Three methods are typically used in the commercial production of recombinant proteins via mammalian cell culture: batch culture, fed-batch culture, and perfusion culture. Batch culture is a discontinuous method in which cells are grown in a fixed volume of culture medium for a short period of time, followed by complete harvesting. Cultures grown using the batch method experience an increase in cell density until reaching maximum cell density, after which the viable cell density decreases as culture medium components are consumed and metabolic byproducts such as lactate and ammonia levels accumulate. Harvesting typically occurs when maximum cell density is reached (e.g., 5 x 10⁻⁶ cells / year). 6 Cells / mL or higher, depending on the culture medium formulation, cell line, etc. Batch processing is the simplest culture method; however, viable cell density is limited by nutrient availability, and once cells reach maximum density, the culture declines and yields decrease. Production phases cannot be extended because the accumulation of waste products and rapid nutrient depletion lead to culture decline (typically around 3-7 days).

[0156] Fed-batch cultures improve upon the batch process by providing feed in clumps or continuous medium to replenish those medium components that have already been consumed. Because fed-batch cultures receive additional nutrients throughout the run, they achieve higher cell densities (>10 to 30 x 10⁻⁶) compared to batch methods. 6 The potential for increased product titers (cells / ml, depending on culture medium formulation, cell line, etc.) and feed-batch culture, unlike batch culture, can be generated and maintained by manipulating feed strategies and culture medium formulations to differentiate between the cell proliferation phase (growth phase) and the suspension or slow cell growth phase (production phase) to achieve the desired cell density. Therefore, fed-batch culture has the potential to achieve higher product titers compared to batch culture. Typically, batch methods are used in the growth phase and fed-batch methods in the production phase, but a fed-batch feed strategy can be used throughout the process. However, unlike batch culture, bioreactor volume is a limiting factor for feed volume. Furthermore, as with batch methods, the accumulation of metabolic byproducts will lead to culture degradation, limiting the duration of the production phase to approximately 10 to 21 days. Feeded-batch culture is discontinuous; harvest typically occurs when metabolic byproduct levels or culture viability reach predetermined levels. Compared to non-feeded batch culture, fed-batch culture can produce significantly larger quantities of recombinant protein. See, for example, U.S. Patent No. 5,672,502.

[0157] The perfusion method offers a potential improvement over batch and fed-batch methods by adding fresh culture medium while simultaneously removing used medium. Typical large-scale commercial cell culture strategies aim to achieve 60–90 (+) x 10⁶ cells / year. 6High cell densities of [number] cells / mL have been achieved, with biomass comprising almost one-third to more than half of the reactor volume. Using perfusion culture, >1 x 10[number] cells / mL have been achieved. 8 Extreme cell densities of [number] cells / mL have been achieved, and even higher densities are predicted. Typical perfusion cultures begin with a batch culture start-up lasting one or two days, followed by continuous, stepwise, and / or intermittent addition of fresh feed medium to the culture. Used medium is removed throughout the growth and production phases of the culture, retaining cells and other high-molecular-weight compounds such as proteins (based on the filtration molecular weight cutoff). Various methods, such as sedimentation, centrifugation, or filtration, can be used to remove used medium while maintaining cell density. Perfusion flow rates ranging from a fraction of one working volume per day to multiple working volumes per day have been reported.

[0158] The advantage of perfusion is that production cultures can be maintained for longer periods than batch or fed-batch methods. However, it requires increased preparation, use, storage, and disposal of the culture medium to support long-term perfusion culture, especially those with high cell densities, and even more nutrients, all of which drive production costs higher compared to batch and fed-batch methods. Furthermore, higher cell densities can cause problems during production, such as maintaining dissolved oxygen levels and increasing gas handling, including supplying more oxygen and removing more carbon dioxide, which leads to more foaming and the need to modify defoaming strategies; and during harvest and downstream processing, the effort required to remove excess cell material can result in product loss, thus negating the benefits of increased titers due to increased cell mass.

[0159] A large-scale cell culture strategy is also provided, which combines fed-batch feeding during the growth phase with continuous perfusion during the subsequent production phase. This method aims to maintain cell cultures at a production phase with a cell volume of less than or equal to 35%.

[0160] In one embodiment, a fed-batch culture with a clump feed is used to maintain the cell culture during the growth phase. Perfusion feed can then be used during the production phase. In one embodiment, perfusion begins when the cells reach the production phase. In another embodiment, perfusion begins on day 3 or approximately day 3 through day 9 of cell culture. In yet another embodiment, perfusion begins on day 5 or approximately day 5 through day 7 of cell culture.

[0161] Using clump feeding during the growth phase allows cells to transition to the production phase, resulting in less dependence on temperature changes as a means of initiating and controlling the production phase; however, a temperature change of approximately 36°C to approximately 31°C can occur between the growth and production phases. In one embodiment, this change is 36°C to 32°C.

[0162] As described in this article, bioreactors can be used in serum-free culture media with a density of at least 0.5 × 10⁻⁶. 6 Up to 3.0 × 10 6 Seed at 1.0 × 10⁶ live cells / mL, for example, 1.0 × 10⁶ cells / mL. 6 live cells / mL.

[0163] Perfusion culture is a culture in which cell cultures receive fresh perfusion feed while the used medium is removed. Perfusion can be continuous, stepwise, intermittent, or any combination of these. The perfusion rate can be less than one working volume to several working volumes per day. Cells remain in the culture, and the removed used medium is substantially cell-free or has significantly fewer cells than the culture. Recombinant proteins expressed in the cell culture may also be retained in the culture. Perfusion can be performed in many ways, including centrifugation, sedimentation, or filtration, see, for example, Voisard et al., 2003, Biotechnology and Bioengineering 82:751-65. An example of a filtration method is alternating tangential flow filtration. Alternating tangential flow is maintained by pumping the medium through a hollow fiber filter module. See, for example, U.S. Patent No. 6,544,424; Furey, 2002, Gen. Eng. News. 22(7):62-63.

[0164] "Perfusion flow rate" is the amount of culture medium that passes through (adds to and removes from) a bioreactor within a given time period, typically expressed as a fraction or multiple of the working volume. "Working volume" refers to the volume of the bioreactor used for cell culture. In one embodiment, the perfusion flow rate is one working volume or less per day. Perfusion feed media can be formulated to maximize the concentration of perfused nutrients, thereby minimizing the perfusion rate.

[0165] Cell cultures can be supplemented with concentrated feed media containing components (such as nutrients and amino acids) consumed during the cell culture production process.

[0166] Concentrated fed-batch culture media can be based on almost any cell culture medium formulation. Such concentrated fed-batch media can contain most components of cell culture media, for example, in normal quantities of approximately 5X, 6X, 7X, 8X, 9X, 10X, 12X, 14X, 16X, 20X, 30X, 50X, 100X, 200X, 400X, 600X, 800X, or even approximately 1000X. Concentrated fed-batch culture media are often used in fed-batch culture processes.

[0167] Samples from cell cultures can be monitored and evaluated using any analytical technique known in the art. A variety of parameters, including recombinant proteins and the quality and characteristics of the culture medium, can be monitored throughout the culture period. Samples can be acquired and monitored intermittently at desired frequencies, including continuous monitoring, real-time or near real-time.

[0168] Typically, cell cultures (Nx to N-1) preceding the final production culture are used to generate seed cells, which will be used to inoculate the production bioreactor, N-1 culture. Seed cell density can have a positive impact on the level of recombinant protein produced. Product levels tend to increase with increasing seed density. Increased titers are not only associated with higher seed density but may also be influenced by the metabolism and cell cycle state of the cells entering production.

[0169] Seed cells can be produced by any culture method. One such method is perfusion culture using alternating tangential flow filtration. The N-1 bioreactor can be operated using alternating tangential flow filtration to provide high-density cells for seeding the production bioreactor. The N-1 stage can be used to grow cells to a density > 90 x 10⁻⁶. 6 Cells / mL. The N-1 bioreactor can be used to generate clump-type seed cultures or as a rolling seed stock culture, which can maintain high seed cell density for inoculating multiple production bioreactors. The duration of the growth phase for production can range from 7 to 14 days and can be designed to maintain cells in exponential growth before inoculating the production bioreactors. The perfusion rate, culture medium formulation, and time are optimized to allow cells to grow and deliver them to the production bioreactors in a state most conducive to optimizing their production. For inoculating production bioreactors, >15 x 10⁻⁶ cells / mL can be achieved. 6 Seed cell density of 100 cells / mL. Higher seed cell density at inoculation can reduce or even eliminate the time required to reach the desired production density.

[0170] In some embodiments, mammalian host cells can be used to generate a high yield of the target protein. High yield, or high volumetric productivity, is the ability of cells to produce high levels of the target protein. Using a fed-batch or perfusion culture medium suitable for mammalian host cells and containing amino acids, vitamins, or trace elements, in a culture grown for 10 days under fed-batch or perfusion conditions, a specific yield will depend on the target protein and may be at least 0.05 g / L, at least 0.1 g / L, at least 0.15 g / L, at least 0.2 g / L, at least 0.25 g / L, at least 0.3 g / L, at least 0.35 g / L, at least 0.4 g / L, at least 0.45 g / L, at least 0.5 g / L, at least 0.6 g / L, at least 0.7 g / L, at least 0.8 g / L, at least 0.9 g / L, at least 1 g / L, at least 1.5 g / L, at least 2 g / L, or higher. In specific embodiments, the host cells and methods disclosed herein express the target protein and, when grown under the above-described culture conditions, are capable of producing at least 0.5 g / L, at least 0.6 g / L, at least 0.7 g / L, at least 0.8 g / L, at least 0.9 g / L, at least 1 g / L, at least 1.5 g / L, at least 2 g / L or more, preferably up to about 3 g / L, 4 g / L, 5 g / L or 10 g / L.

[0171] Yield can also be measured based on the unit productivity of the cell line, which is determined by the amount of protein produced per cell per day (expressed as pg / cell / day). Using a fed culture medium suitable for mammalian host cells and containing amino acids, vitamins, or trace elements, in cultures grown for 10 days under fed batch or perfusion conditions, the mammalian host cells disclosed herein are capable of producing at least 1 pg / cell / day, at least 2 pg / cell / day, at least 3 pg / cell / day, at least 4 pg / cell / day, at least 5 pg / cell / day, at least 6 pg / cell / day, at least 7 pg / cell / day, at least 8 pg / cell / day, at least 9 pg / cell / day, at least 10 pg / cell / day, at least 11 pg / cell / day, at least 12 pg / cell / day, at least 13 pg / cell / day, at least 14 pg / cell / day, at least 15 pg / cell / day, at least 20 pg / cell / day, at least 25 pg / cell / day, or more, preferably up to 50 pg / cell / day. In specific embodiments, the mammalian host cells disclosed herein express the target protein and, under the above-described culture conditions, have a unit productivity of at least 10 pg / cell / day, at least 11 pg / cell / day, at least 12 pg / cell / day, at least 13 pg / cell / day, at least 14 pg / cell / day, at least 15 pg / cell / day, at least 20 pg / cell / day, at least 25 pg / cell / day or higher, preferably up to 50 pg / cell / day.

[0172] The mammalian host cells described herein can be used to express the target protein. The expressed protein can be secreted into a culture medium, from which it can be recovered and / or collected. Furthermore, the protein can be purified or partially purified from such a culture or component (e.g., from a culture medium) using known processes and products available from commercial suppliers. The purified protein can then be “formulated” (meaning buffer exchange, sterilization, batch packaging, and / or packaging for the end user). Suitable formulations for pharmaceutical compositions include those described in Remington's Pharmaceutical Sciences, 18th edition, 1995, Mack Publishing Company, Easton, Pennsylvania.

[0173] The polynucleotides, peptides, vectors, host cells, immune cells, compositions, etc., according to the present invention can be prepared using a variety of known techniques.

[0174] This invention is not limited in scope to the specific embodiments described herein, which are intended as individual illustrations of various aspects of the invention, and functionally equivalent methods and components are also within the scope of the invention. In fact, various modifications to the invention will become apparent to those skilled in the art from the foregoing description and drawings, in addition to those shown and described herein. Such modifications are intended to fall within the scope of the appended claims. Example

[0175] Materials and Methods

[0176] Plasmid transfection and pool recovery

[0177] Using a GenePulser Xcell™ electroporator (Bio-Rad Laboratories, Inc., Hercules, CA), 20 μg of a proprietary plasmid containing the target gene (GOI) and 5 μg of ILTpiggyback transposase (see International Patent Application Publication No. WO 2020123327) were transfected into SAFC™ PF-CHO medium (Ex-Cell) suspended in the medium. ® 325 PF-CHO serum-free medium, catalog number 14340C, Millipore Sigma, St. Louis, Missouri, 20 x 10 6Glutamine synthase knockout (GSKO) in Chinese hamster ovary (CHO) cells. Immediately after transfection, transfected CHO cells were cultured in 20 ml of chemically determined host cell medium. Three days post-transfection, glutamine-negative medium supplemented with 12.5 μM or 25 μM L-methionine sulfoxide (MSX, Millipore Bosigma, St. Louis, Missouri) was used as the selection medium. Transfected cells were passaged every 3 or 4 days until viability reached 85% or higher. Viability and cell density were measured using a Vi-CELL™ BLU cell viability analyzer (Beckman Coulter, Indianapolis, Indiana). Once viability > 85%, the cultures were cultured at 5 x 10⁻⁶ cells / year. 5 Seed at 100 cells / mL, then passaged twice until viability and doubling time stabilize.

[0178] Replenishment in batches

[0179] Under production conditions, the pools were evaluated and sorted using a 15-day fed-batch (FB) cell culture process. N-1 cultures were cultured at 5 x 10⁻⁶ cells / day. 5 Cells / ml were seeded in 10 ml of MSX-free glutamine-negative host medium. After 4 days, the production culture was seeded at 1 x 10⁻⁶ cells / ml. 6 Cells / ml were seeded in 10 ml of MSX-free glutamine-negative medium with a 1:750 Gibco™ anti-caking agent (Thermo Fisher Scientific Inc., Waltham, MA). Production culture could proceed up to day 15 as long as viability was above 50%. On days 3, 6, 8, 10, 13, and 15, the cultures were counted using Vi-CELL™ BLU (Beckman Coulter), and viable cell counts and viability percentages were recorded. On days 3, 6, 8, 10, and 13, BioProfile was used. ® The Flex2 automated cell culture analyzer (Nova Biomedical, Waltham, MA) measured glucose levels in the culture medium. Cultures were fed with 5% v / v proprietary medium supplemented with 0.2% v / v tyrosine-cysteine ​​and glucose on days 3, 6, 8, 10, and 13. Following glucose measurement, glucose levels were adjusted to 12–14 g / L using a 50% glucose stock solution based on glucose consumption on days 3, 6, 8, 10, and 13. On days 10, 13, and 15, 200 μl of harvested cell culture medium (HCCF) was taken for titer measurement to assess productivity.

[0180] On day 15, cell cultures were saturated at 2000 rpm for 10 minutes, and HCCF samples were subjected to ATOLL (protein A-HPLC), size exclusion chromatography (SEC), reductive capillary electrophoresis (rCE), and non-reductive capillary electrophoresis (nrCE) to assess product quality. TIBCO Spotfire was used. ® Software (Cloud Software Group, Inc.) visualized titer and product quality data. Only data from cultures with viability > 60% were used for analysis. The values ​​on the graph represent the average values ​​from two replicate cultures. Fold changes were calculated separately for the 12.5 μM MSX and 25 μM MSX conditions. The fold changes presented on the graph are the average fold changes between the 12.5 μM and 25 μM MSX conditions.

[0181] Example 1

[0182] As part of our vector screening effort to improve the productivity and product quality of 3- and 4-chain molecules (including 3-chain molecules with heavy chains fused to cytokines and heterologous IgG 4-chain molecules) in GSKO hosts, 3- and 4-chain molecules were tested with various combinations of promoters.

[0183] A dual-vector system was employed, using different combinations of mPGK and Srα promoters to drive the expression of glutamine synthase as a selectable marker. In this experiment, a three-chain molecule with a heavy chain fused to a cytokine was generated, wherein the first vector encodes both the light and heavy chains, and the second vector encodes a light and heavy chain-peptide fusion. Figure 1 A schematic diagram of the different carriers used in this experiment is shown.

[0184] The vector was co-transfected into CHO cells and cell cultures as described above. The titer and product quality were determined as described above.

[0185] Compared to pools using either the mPGK or Srα promoter to drive GS expression in two vectors, pools using the GS-selected mPGK promoter in one vector and the Srα promoter in another vector exhibited higher titers and product quality. See also Figure 2 The improvements in titer and product quality were most pronounced when Srα was expressed on a vector containing the expression restriction strand (the more difficult-to-express strand). In the case of 3-strand molecules, compared to the mPGK / mPGK and Srα / Srα pools, the effective titer (nrCE) of the mPGK / Srα pool was increased by up to 5.3-fold, and the product quality was improved by up to 2.6-fold (nrCE% main peak). Figure 2In particular, the mPGK / Srα hybrid vector combination containing Srα on vectors with more difficult-to-express mAb fusion complexes helped increase mAb fusion complex expression by up to 2.4-fold, thereby increasing the ratio of mAb fusion complex to heavy chain, resulting in more balanced chain expression and improved product quality.

[0186] Example 2

[0187] As part of our vector screening effort to improve the productivity and product quality of 3- and 4-chain molecules (including 3-chain molecules with heavy chains fused to cytokines and heterologous IgG 4-chain molecules) in GSKO hosts, 3- and 4-chain molecules were tested with various combinations of promoters.

[0188] A dual-vector system was employed, using different combinations of mPGK and Srα promoters to drive the expression of glutamine synthase as a selectable marker. In this experiment, heterologous IgG was generated, with the first vector encoding both the light and heavy chains, and the second vector encoding a light and heavy chain-protein fusion.

[0189] Using 25 μM MSX during cell growth facilitates the selection of vectors containing Srα-GS and the expression of more difficult-to-express chains. Compared to... mPGK Srα is a stronger promoter, therefore, at higher MSX concentrations of 25 μM, selection via the Srα vector is more effective than selection via... mPGK Vector selection is easier to achieve. By placing the difficult-to-express strand on a less restrictive vector and the easier-to-express strand on a more restrictive vector, strand expression can be balanced.

[0190] For heterologous IgG, it was also observed that co-transfection of vectors with two different GS-selective promoters (mPGK, which is more likely to express LC-HC, and Srα, which is less likely to express LC-HC) resulted in a 1.2-fold increase in titers in the corresponding Srα / Srα vectors, with minimal impact on product quality. Figure 3 ).

[0191] In summary, these results demonstrate that promoters used for selecting label expression can also be used as vector design tools to generate high-quality pools of similar 3- and 4-chain molecules, which can help improve process efficiency and reduce future manufacturing costs.

Claims

1. A mammalian host cell for expressing an antigen binding protein having three or four different chains, the mammalian host cell comprising a first expression vector and a second expression vector, wherein: (a) the first expression vector comprises a nucleotide sequence encoding: 1) a first chain and a second chain, wherein the first chain is operably linked to a first promoter, the second chain is operably linked to a second promoter or an IRES sequence or is linked to the first chain by a linker sequence; and 2) a first selectable marker operably linked to a third promoter; and (b) the second expression vector comprises a nucleotide sequence encoding: 1) a third chain and a fourth chain, wherein the third chain is operably linked to a fourth promoter, the fourth chain is operably linked to a fifth promoter or an IRES sequence or is linked to the third chain by a linker sequence; and 2) a second selectable marker operably linked to a sixth promoter, wherein the first chain, the second chain, the third chain, and the fourth chain are selected from the group consisting of a heavy chain, a light chain, an antibody heavy chain fusion, an antibody light chain fusion, a ScFv, and a ScFv-Fc, wherein two of the chains can be the same; and wherein the sixth promoter is different from the third promoter.

2. The mammalian host cell of claim 1, wherein the second chain and the fourth chain are operably linked to a second promoter and a fourth promoter, respectively.

3. The mammalian host cell of claim 1, wherein the first selectable marker and the second selectable marker are the same.

4. The mammalian host cell of claim 3, wherein the first selectable marker and the second selectable marker are selected from the group consisting of glutamine synthetase and dihydrofolate reductase.

5. The mammalian host cell of claim 4, wherein the first selectable marker and the second selectable marker are glutamine synthetase.

6. The mammalian host cell of claim 1, wherein the third promoter and the sixth promoter are selected from mPGK and Srα.

7. The mammalian host cell of claim 2, wherein the first promoter, the second promoter, the fourth promoter, and the fifth promoter are different from the third promoter and the sixth promoter.

8. The mammalian host cell of claim 7, wherein the first promoter, the second promoter, the fourth promoter, and the fifth promoter are selected from the group consisting of CMV / GAPDH, CMV / adL, and CMV / EF1α.

9. The mammalian host cell of claim 7, wherein the first promoter and the fourth promoter are the same, and the second promoter and the fifth promoter are the same.

10. The mammalian host cell of claim 9, wherein the first promoter, the second promoter, the fourth promoter, and the fifth promoter are the same.

11. The mammalian host cell of claim 9, wherein the first promoter and the fourth promoter are different from the second promoter and the fifth promoter. ​ ​ ​ ​ 12. The mammalian host cell of claim 1, wherein the stronger of the third and sixth promoters is on the expression vector with the more difficult to express strand.

13. The mammalian host cell of claim 12, wherein Srα is on the expression vector with the more difficult to express strand and mPGK is on the expression vector with the more easy to express strand.

14. The mammalian host cell of claim 5, wherein the methionine sulfoximine (MSX) stringency is optimized to favor expression of the more difficult to express strand paired with the stronger promoter.

15. The mammalian host cell of claim 14, wherein the MSX stringency is less than 75 µM.

16. The mammalian host cell of claim 1, wherein the stronger of the third and sixth promoters is on the expression vector with the more easy to express strand.

17. The mammalian host cell of claim 1, wherein the first and third strands are antibody light chains and the second and fourth strands are antibody heavy chains.

18. The mammalian host cell of claim 17, wherein the first and second strands are the same antibody light chain.

19. The mammalian host cell of claim 17, wherein the first and second strands are different antibody light chains.

20. The mammalian host cell of claim 1, wherein a) the first and third strands are antibody light chains and b) one of the second or fourth strands is an antibody heavy chain and the other is an antibody heavy chain fusion.

21. The mammalian host cell of claim 20, wherein the first and second strands are the same antibody light chain.

22. The mammalian host cell of claim 20, wherein the first and second strands are different antibody light chains.

23. The mammalian host cell of claim 1, wherein the antigen binding protein with three or four chains is selected from a hetero-IgG and a C1 mAb.

24. The mammalian host cell of claim 1, wherein the first expression vector comprises, in 5’ to 3’ order, a first promoter, a nucleotide sequence encoding a first antibody light chain, a second promoter or IRES, a nucleotide sequence encoding a first heavy chain, a third promoter that is Srα, and a nucleotide sequence encoding a selectable marker that is glutamine synthetase; and the second expression vector comprises, in 5’ to 3’ order, a fourth promoter, a nucleotide sequence encoding a second antibody light chain, a fifth promoter or IRES, a nucleotide sequence encoding a second heavy chain, a sixth promoter that is mPGK, and a nucleotide sequence encoding a selectable marker that is glutamine synthetase.

25. The mammalian host cell of claim 24, wherein the first and second antibody light chains are the same.

26. The mammalian host cell of claim 24, wherein the first heavy chain is more difficult to express than the second heavy chain.

27. The mammalian host cell of claim 1, wherein the first expression vector comprises, in 5' to 3' order, a first promoter, a nucleotide sequence encoding a first antibody light chain, a second promoter, a nucleotide sequence encoding a first heavy chain, a third promoter that is Srα, and a nucleotide sequence encoding a selectable marker that is glutamine synthetase; and the second expression vector comprises, in 5' to 3' order, a fourth promoter, a nucleotide sequence encoding a second antibody light chain, a fifth promoter, a nucleotide sequence encoding a second heavy chain, a sixth promoter that is mPGK, and a nucleotide sequence encoding a selectable marker that is glutamine synthetase.

28. The mammalian host cell of claim 27, wherein the first antibody light chain and the second antibody light chain are the same.

29. The mammalian host cell of claim 27, wherein the first heavy chain is more difficult to express than the second heavy chain.

30. The mammalian host cell of claim 1, wherein the first expression vector comprises, in 5' to 3' order, a first promoter, a nucleotide sequence encoding an antibody light chain, a second promoter or IRES, a nucleotide sequence encoding a heavy chain fusion, a third promoter that is Srα, and a nucleotide sequence encoding a selectable marker that is glutamine synthetase; and the second expression vector comprises, in 5' to 3' order, a fourth promoter, a nucleotide sequence encoding the antibody light chain, a fifth promoter or IRES, a nucleotide sequence encoding a heavy chain, a sixth promoter that is mPGK, and a nucleotide sequence encoding a selectable marker that is glutamine synthetase.

31. The mammalian host cell of claim 30, wherein the first antibody light chain and the second antibody light chain are the same.

32. The mammalian host cell of claim 1, wherein the first expression vector comprises, in 5' to 3' order, a first promoter, a nucleotide sequence encoding an antibody light chain, a second promoter, a nucleotide sequence encoding a heavy chain fusion, a third promoter that is Srα, and a selectable marker that is glutamine synthetase; and the second expression vector comprises, in 5' to 3' order, a fourth promoter, a nucleotide sequence encoding the antibody light chain, a fifth promoter, a nucleotide sequence encoding a heavy chain, a sixth promoter that is mPGK, and a nucleotide sequence encoding a selectable marker that is glutamine synthetase.

33. The mammalian host cell of claim 32, wherein the first antibody light chain and the second antibody light chain are the same.

34. The mammalian host cell of claim 1, wherein the first expression vector and the second expression vector are integrated in the genome of the host cell.

35. The mammalian host cell of claim 1, wherein the host cell is a Chinese hamster ovary (CHO) cell.

36. The mammalian host cell of claim 35, wherein the CHO cell is deficient in dihydrofolate reductase (DHFR - ) or is glutamine synthetase knock-out (GSKO) type.

37. A method for producing an antigen binding protein having three or four chains, the method comprising a) culturing the mammalian host cell of claim 1 under conditions wherein the antigen binding protein is expressed; and b) recovering the antigen binding protein. ​ ​ 38. The method of claim 37, wherein the recovered antigen-binding protein is purified and formulated into a pharmaceutically acceptable formulation.

39. A method for producing an antigen-binding protein having three or four chains, the method comprising a) culturing the mammalian host cell of claim 36 under conditions wherein the antigen-binding protein is expressed, and under methotrexate stringency in the case of CHO DHFR- cells or under methionine sulfoximine stringency in the case of CHO GSKO cells, to favor expression of a difficult-to-express chain paired with a stronger GS promoter; and b) recovering the antigen-binding protein.

40. The method of claim 39, wherein the recovered antigen-binding protein is purified and formulated into a pharmaceutically acceptable formulation. ​ ​

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