A vector incorporating a combination of promoters that drive the expression of a selected marker.

Abstract

A dual-vector system for the expression of multiple chain molecules and mammalian host cells containing this system are provided, where each vector uses a different promoter that drives the expression of a selected marker. The mammalian host cells can be used to produce the recombinant protein of interest in a mammalian cell culture.

Description

[Technical Field]

[0001] The present invention generally relates to mammalian host cells comprising a vector system for expressing recombinant proteins having molecules comprising three or more chains. Specifically, this vector system comprises two expression vectors, each expressing a different selection marker, and the promoters driving the expression of these selection markers are different. [Background technology]

[0002] Due to their wide range of applications, biological agents are used worldwide in various fields, including therapy and diagnosis. The primary expression system for these biological agents is mammalian cell lines, 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 have become more important than ever.

[0003] The manufacturing costs of biological drugs are high due to the complexity of their production, which involves multi-step processes that include the selection of optimal cell lines, the cultivation of large quantities of producing cells, and the purification of the desired biological drug from the recovered cells. Manufacturing is even more complex, typically for novel antibody modalities, such as those with three or four antibody chains. While these costs have been decreasing through improvements in all aspects of production, they can still be extremely expensive for them to be widely adopted as state-of-the-art therapies.

[0004] To make biological therapies more accessible to patients, the proposal to reduce product costs for the manufacturing process is an attractive one. One way to achieve this objective is to reduce product costs by increasing the titer associated with the producing cell line. Appropriate vector configuration can help optimize the expression levels of various chains in recombinant proteins, particularly with respect to triple-stranded or quadruple-stranded molecules, resulting in more balanced chain expression, reduced impurities, and higher product quality.

[0005] Attenuation of glutamine synthetase selection markers has been shown to improve product titer. See Sacco et al., 2022, Biotechnol. Bioeng. 119:1712-1727. Also see Yang et al., 2019, Bioprocess Biosyst. Eng. 42:799-806. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Lalonde et al.,2017,J Biotechnol 251:128-140 [Non-Patent Document 2] Sacco et al.,2022,Biotechnol.Bioeng.119:1712-1727 [Non-Patent Document 3] Yang et al.,2019,Bioprocess Biosyst.Eng.42:799-806 [Overview of the project] [Problems that the invention aims to solve]

[0007] There still remains a need for a vector system that can produce recombinant proteins (particularly three-chain molecules and four-chain molecules) at high titers while minimizing the impact on growth and productivity when transfected into a host cell line. Such a vector system would be beneficial for process development of biopharmaceuticals. [Means for Solving the Problem]

[0008] The present disclosure provides a mammalian host cell for expressing an antigen-binding protein having 3 or 4 different chains comprising a first expression vector and a second expression vector, wherein: (a) the first expression vector comprises: 1) a nucleotide sequence encoding a first chain and a second chain, wherein the first chain is operably linked to a first promoter, and the second chain is operably linked to a second promoter or an IRES sequence, or is linked to the first chain via a linker sequence; and 2) a nucleotide sequence encoding a first selectable marker operably linked to a third promoter; (b) the second expression vector comprises: 1) a nucleotide sequence encoding a third chain and a fourth chain, wherein the third chain is operably linked to a fourth promoter, and the fourth chain is operably linked to a fifth promoter or an IRES sequence, or is linked to the third chain via a linker sequence; and 2) a nucleotide sequence encoding a second selectable marker operably linked to a sixth promoter; the first, second, third, and fourth chains are selected from a heavy chain, a light chain, an antibody heavy chain fusion, an antibody light chain fusion, an ScFv, and an ScFv-Fc, and two of these chains may be identical; and the sixth promoter is different from the third promoter.

[0009] In certain embodiments, the second chain and the fourth chain are operably linked to the second promoter and the fourth promoter, respectively.

[0010] In certain specific embodiments, the first selectable marker and the second selectable marker are identical. In certain specific aspects, the first selectable marker and the second selectable marker are selected from the group consisting of glutamine synthetase and dihydrofolate reductase. In certain specific aspects, the first selectable marker and the second selectable marker are glutamine synthetase.

[0011] In certain specific embodiments, the third promoter and the sixth promoter are selected from mPGK and Srα.

[0012] In certain specific embodiments, the first, second, fourth, and fifth promoters are different from the third and sixth promoters. In certain specific aspects, the first, second, fourth, and fifth promoters are selected from the group consisting of CMV / GAPDH, CMV / adL, and CMV / EF1α.

[0013] In certain specific embodiments, the first and fourth promoters are identical, and the second and fifth promoters are identical. In certain specific aspects, the first, second, fourth, and fifth promoters are identical. In certain specific aspects, the first and fourth promoters are different from the second and fifth promoters.

[0014] In certain specific embodiments, the stronger promoter among the third promoter and the sixth promoter is present on an expression vector carrying a chain that is more difficult to express. In certain specific aspects, Srα is present on an expression vector carrying a chain that is more difficult to express, and mPGK is present on an expression vector carrying a chain that is easier to express.

[0015] In certain specific aspects of embodiments where the selectable marker is glutamine synthetase, methionine sulfoximine (MSX) stringency is optimized to favor expression of the more difficult-to-express chain that is paired with a stronger promoter. In certain specific sub-aspects, the MSX stringency is less than 75 μM.

[0016] In certain embodiments, the stronger of the third and sixth promoters is located on an expression vector having a chain that is more readily expressed.

[0017] In a particular embodiment, the first and third chains are antibody light chains, and the second and fourth chains are antibody heavy chains. In a particular embodiment, the first and second chains are identical antibody light chains. In a particular embodiment, the first and second chains are different antibody light chains.

[0018] In a particular embodiment, 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 a particular embodiment, the first and second chains are identical antibody light chains. In a particular embodiment, the first and second chains are different antibody light chains.

[0019] In a particular embodiment, the antigen-binding protein having three or four chains is selected from the group consisting of heteroIgG and C1mAb.

[0020] In certain embodiments, the first expression vector comprises, in 5' to 3' order, a first promoter, a nucleotide sequence encoding the first antibody light chain, a second promoter or IRES, a nucleotide sequence encoding the first heavy chain, a third promoter which is Srα, and a nucleotide sequence encoding a select marker which is glutamine synthetase; and the second expression vector comprises, in 5' to 3' order, a fourth promoter, a nucleotide sequence encoding the second antibody light chain, a fifth promoter or IRES, a nucleotide sequence encoding the second heavy chain, a sixth promoter which is mPGK, and a nucleotide sequence encoding a select marker which is glutamine synthetase. In certain embodiments, the first and second antibody light chains are identical. In certain embodiments, the first heavy chain is more difficult to express than the second heavy chain.

[0021] In certain embodiments, the first expression vector comprises, in 5' to 3' order, a first promoter, a nucleotide sequence encoding the first antibody light chain, a second promoter, a nucleotide sequence encoding the first heavy chain, a third promoter which is Srα, and a nucleotide sequence encoding a select marker which is glutamine synthetase; and the second expression vector comprises, in 5' to 3' order, a fourth promoter, a nucleotide sequence encoding the second antibody light chain, a fifth promoter, a nucleotide sequence encoding the second heavy chain, a sixth promoter which is mPGK, and a nucleotide sequence encoding a select marker which is glutamine synthetase. In certain embodiments, the first and second antibody light chains are identical. In certain embodiments, the first heavy chain is more difficult to express than the second heavy chain.

[0022] In a particular embodiment, the first expression vector comprises, in 5' to 3' order, a first promoter, a nucleotide sequence encoding the antibody light chain, a second promoter or IRES, a nucleotide sequence encoding the heavy chain fusion, a third promoter which is Srα, and a nucleotide sequence encoding a select marker which 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 the heavy chain, a sixth promoter which is mPGK, and a nucleotide sequence encoding a select marker which is glutamine synthetase. In a particular embodiment, the first antibody light chain and the second antibody light chain are identical.

[0023] In a particular embodiment, the first expression vector comprises, in 5' to 3' order, a first promoter, a nucleotide sequence encoding the antibody light chain, a second promoter, a nucleotide sequence encoding the heavy chain fusion, a third promoter which is Srα, and a select marker which 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 the heavy chain, a sixth promoter which is mPGK, and a nucleotide sequence encoding the select marker which is glutamine synthetase. In a particular embodiment, the first antibody light chain and the second antibody light chain are identical.

[0024] In certain embodiments, the first and second expression vectors are integrated into the genome.

[0025] In certain embodiments, the host cells are Chinese hamster ovary (CHO) cells. In certain aspects, the CHO cells are either dihydrofolate reductase-deficient (DHFR-) or glutamine synthetase knockout (GSKO).

[0026] The disclosure also provides a method for producing an antigen-binding protein having three or four chains, comprising: a) culturing mammalian host cells of any of the embodiments, subembodiments, aspects, and sub-existences described above under conditions for expressing the antigen-binding protein; and b) recovering the antigen-binding protein.

[0027] The disclosure also provides a method for producing an antigen-binding protein having three or four chains, comprising (a) culturing mammalian host cells, which are Chinese hamster ovary (CHO) cells lacking dihydrofolate reductase (DHFR-) or glutamine synthetase knockout (GSKO), under conditions for expressing the antigen-binding protein, and under methotrexate stringency in the case of CHO DHFR- cells, or under methionine sulfoxamine in the case of CHO GSKO cells, to favor the expression of a less expressible chain paired with a stronger GS promoter; and (b) recovering the antigen-binding protein.

[0028] In certain embodiments of these methods, the recovered antigen-binding protein is purified and formulated into a pharmaceutically acceptable formulation.

[0029] This disclosure also provides antigen-binding proteins produced by any of the mammalian host cells provided herein or by any of the methods provided herein. [Brief explanation of the drawing]

[0030] [Figure 1] A) Examples of mixed promoter expression cassettes driving glutamine synthetase (GS) selection for triple-stranded or quadruple-stranded molecules are shown. The promoter of the gene of interest (i.e., light chain (LC, LC1, and LC2), heavy chain (HC), or heavy chain fusion (HC-mAb)) may be modified depending on the molecule of interest. mPGK and SRα refer to the promoters used to drive GS expression. (B) Schematic diagrams of evaluated triple-stranded (C1mAb) and quadruple-stranded (heteroIgG) molecules requiring vector optimization. [Figure 2-1] (A) A schematic diagram of the expression cassette used in the vector evaluation test of the triple-stranded C1mAb molecule is shown. [Figure 2-2](B) shows an improvement of up to approximately 5.3 times in the yield of the mPGK / Srα vector combination in the effective titer normalized to the nrCE main peak%, (C) shows a reduction in the nrCE pre-peak impurity%, (D) shows an increase in the nrCE main peak%, and (E, F) shows an increase in the relative expression of the mAb-fusion when measured by rCE. [Figure 3] This study demonstrates that a vector containing a mixed promoter expression cassette expressing heteroIgG improves the day 10 effective titer, normalized to the SEC main peak%, by up to 1.2 times (A) while minimizing the impact on product quality (B, C). [Modes for carrying out the invention]

[0031] This disclosure is partly based on the surprising discovery that when two vector systems are utilized in mammalian host cells, the titer of the recombinant protein produced can be increased, particularly with respect to antibody modalities having three or four chains, by using different promoters that drive the expression of a selection marker. These two vector systems can be utilized in mammalian host cells to produce antibodies having two different heavy chains and two different light chains, where each heavy-light chain pair is expressed from a different vector. These two vector systems can also be used to produce antibody modalities having three or four distinct chains.

[0032] In standard antibody production techniques, different promoters are often used for the expression of heavy and light chains to optimize antibody expression. This is often necessary because the heavy and light chains are expressed at different levels. This situation becomes even more complex when the antibody structure contains three or four chains. Surprisingly, we have found that by using different promoters for the selection markers on each vector, the titer of the resulting antibody structure can be increased along with improved product quality (e.g., reduction of aggregation (high molecular weight impurities (HMW)) and degradation (reduction of low molecular weight impurities (LMW))).

[0033] By utilizing the two-vector system and mammalian host cells described herein, the production of recombinant proteins can be increased while maintaining or improving product quality. Using the two-vector system described herein in mammalian host-producing cell lines allows for the more cost-effective and consistent production of biopharmaceuticals. The present invention is particularly useful in the commercial production of antibodies having three or four distinct chains.

[0034] The vector systems described herein are used in cell lines (also referred to as “host cells”) grown in cell culture media for the production of recombinant proteins for commercial or scientific purposes, preferably in mammals (“mammalian host cells”). The cell lines are typically derived from strains resulting from primary cultures that can be maintained in culture for an unlimited period of time. Genetic manipulation of cell lines involves transfecting, transforming, or transducing cells with a two-vector system, each vector containing nucleotide sequences encoding two antibody chains, to cause the host cells to express an antigen-binding protein of interest. For example, methods and vectors for genetically engineering cells and / or cell lines to express a target protein are well known to those skilled in the art, and various techniques are described below, for example: Current Protocols in Molecular Biology, Ausubel et al., eds. (Wiley & Sons, New York, 1988 and updated four times a year); Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring 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, Cold Spring Harbor, NY (1990).

[0035] definition The terminology used in this application is standard in the art; however, this specification provides definitions of certain terms to ensure clarity and unambiguity in the sense of the claims. Units, prefixes, and symbols may be expressed in the form recognized by the SI (International System of Units). Numerical ranges listed herein include the number defining the range, each integer within the defined range, and supporting it. Unless otherwise indicated, the methods and techniques described herein are generally carried out in accordance with the prior art and as described in the various general and more specific references cited and discussed throughout this specification.

[0036] As used herein, the terms “a” and “an” mean one or more unless otherwise specifically indicated. Furthermore, unless otherwise required by context, singular terms shall include plural forms and plural terms shall include singular forms. In general, the nomenclature and techniques used in relation to cell and tissue culture, molecular biology, immunology, microbiology, genetics, and the chemistry and hybridization of proteins and nucleic acids described herein are well known and commonly used in the art.

[0037] All documents or parts of documents cited herein, including but not limited to patents, patent applications, papers, books, and academic articles, are expressly incorporated herein by reference. Those described in the embodiments of the present invention may be combined with other embodiments of the present invention.

[0038] This disclosure provides a method for expressing a “protein of interest.” “Protein of interest” includes naturally occurring proteins, recombinant proteins, and engineered proteins (e.g., proteins that are not naturally occurring and are designed and / or produced by humans). The protein of interest may, but does not have to be, a protein known or thought to be therapeutically relevant.

[0039] 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 meanings in the art and include, for example, the various antibody heavy and light chains described elsewhere in this specification (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 synonymously herein and refer to polypeptides containing an antibody heavy chain covalently linked to one or more additional proteins or peptides. For example, an “antibody heavy chain fusion” may be an antibody heavy chain covalently linked to a cytokine, VHH, or scFv. This linkage may be direct or mediated via a peptide linker (e.g., a glycine-serine linker). In an antibody heavy chain fusion, the antibody heavy chain may be linked to an additional protein at its N-terminus or C-terminus (or both). The terms “antibody light chain fusion protein” and “antibody light chain fusion” have the same meaning as described above with respect to “antibody heavy chain fusion,” except that the antibody light chain replaces the antibody heavy chain. As used herein, “antibody fusion” refers to an antibody provided herein that is covalently linked (e.g., via the antibody's heavy or light chain) to one or more additional proteins or polypeptides. Thus, an antibody fusion includes at least an antibody heavy chain fusion or an antibody light chain fusion as one of the chains of the antibody fusion. Most commonly, an antibody fusion is a molecule comprising two antibody light chains, one antibody heavy chain, and one antibody heavy chain fusion, such that the additional protein is linked to one of the antibody's heavy chains. An example is a C1mAb fused to the C-terminus of the Fc region.

[0040] As used herein, the terms “polypeptide” and “protein” (for example, as used in the context of the protein or polypeptide of interest) are used synonymously to refer to polymers of amino acid residues. These terms also apply to amino acid polymers and naturally occurring amino acid polymers, as well as naturally occurring amino acid polymers, in which one or more amino acid residues are analogs or mimetic to the corresponding naturally occurring amino acids. These terms may also include amino acid polymers modified, for example, by the addition of carbohydrate residues to form glycoproteins, or by phosphorylation. Polypeptides and proteins may be produced by naturally occurring non-recombinant cells, or they may be produced by genetically modified or recombinant cells. Polypeptides and proteins may include molecules having the amino acid sequence of a natural protein, or molecules having the deletion, addition, and / or substitution of one or more amino acids of the natural sequence.

[0041] As used herein, the term “heterogeneous” in relation to nucleic acids means having nucleic acids that are not naturally present in the host cell. This term may include mutant sequences (e.g., sequences different from naturally occurring sequences). This term may include sequences of other species origin. This term may also include having sequences at genomic locations different from those naturally occurring in the host cell. In general, this term does not include spontaneous mutations that may occur in the host cell. For example, a cell that already contains heterogeneous nucleic acids encoding a target protein by stably incorporating an expression cassette will be considered to contain heterogeneous nucleic acid sequences. For clarity, CHO cells or their derivatives (e.g., DHFR- or GS knockout) that have nucleic acids encoding an antigen-binding protein will be considered to contain heterogeneous nucleic acids.

[0042] As used herein, the term “operably linked” means that the linked nucleic acid sequences are typically contiguous or substantially contiguous, and, where necessary, link two protein-coding regions that are contiguous within a reading frame. However, since enhancers generally function when separated from promoters by several kilobases, and intron sequences can be of variable length, some polynucleotide elements can be operafully linked but not contiguous.

[0043] As used herein, the term “bioreactor” means any container useful for growing cell cultures. Mammalian cell cultures disclosed herein may be grown in a bioreactor, which may be selected based on the intended use of the protein produced by the cells growing within it. A bioreactor can be of any size, as long as it is useful for cell culture; typically, a bioreactor is of an appropriate size for the volume of cell culture grown within it. Typically, a bioreactor is at least 1 liter and may 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 more, or any volume in between. Internal conditions of the bioreactor, including but not limited to pH and temperature, may be controlled during the culture period. Those skilled in the art will be able to recognize and select a bioreactor suitable for use when carrying out the methods disclosed herein, based on relevant considerations.

[0044] As used herein, “cell culture” or “culture” means the proliferation and reproduction of cells outside of a multicellular organism or tissue. Suitable culture conditions for mammalian cells are known in the art. See, for example, Animal cell culture: A Practical Approach, D. Rickwood, ed., Oxford University Press, New York (1992). Mammalian cells can be cultured in suspension or attached to a solid culture medium. Fluidized bed bioreactors, hollow fiber bioreactors, roller bottles, shaking flasks, or agitated tank bioreactors may be used, with or without microcarriers. In one embodiment, a 500 L to 2000 L bioreactor is used. In another embodiment, a 1000 L to 2000 L bioreactor is used.

[0045] The term “cell culture medium” (also called “culture medium,” “cell culture media,” or “tissue culture medium”) refers to any nutrient solution used for growing cells, such as animal or mammalian cells, and generally provides at least one or more components derived from: energy sources (usually in the form of carbohydrates such as glucose); all essential amino acids, and generally one or more of the 20 basic amino acids and 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, usually in the micromolar concentration range.

[0046] The nutrient solution may contain additional optional components to optimize cell proliferation, depending on the requirements of the cells being cultured and / or the desired cell culture parameters, such as hormones and other growth factors, e.g., transferrin, epidermal growth factor, serum; salts, e.g., calcium, magnesium, and phosphates, as well as buffers, e.g., HEPES; nucleosides and bases, e.g., adenosine, thymidine, hypoxanthine; and proteins and tissue hydrolysates, e.g., hydrolyzed animal or plant proteins (peptone or peptone mixtures which can be obtained from animal by-products, purified gelatin, or plant materials); antibiotics, e.g., gentamicin; and anticaking agents. Cytoprotective agents or surfactants, such as Pluronic® F68 (also known as Lutrol® F68 and Kolliphor® P188): a nonionic triblock copolymer consisting of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) sandwiched between two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)); polyamines, such as putrescine, spermidine, and spermine (see, for example, International Publication No. 2008 / 154014), and pirubate (see, for example, U.S. Patent No. 8,053,238), may be optionally supplemented.

[0047] Examples of cell culture media include, but are not limited to, those typically used in any cell culture process such as batch culture, extended batch culture, fed-batch culture, and / or perfusion culture or continuous culture, and / or those known for use in such processes.

[0048] "Basic" (or batch) cell culture medium typically refers to a cell culture medium that is used to initiate a cell culture and is complete enough to support the cell culture.

[0049] "Fed-batch culture" refers to a form of suspension culture in which additional components are supplied to the culture medium at the start of the culture process or at some point thereafter. The supplied components typically include cellular nutritional supplements that have been depleted during the culture process. In addition, or / or, the additional components may include supplement components (e.g., cell cycle inhibitors). Fed-batch culture is typically stopped at some point, and the cells and / or components in the medium are harvested and optionally purified.

[0050] "Proliferation" cell culture media typically refer to cell culture media used for cell culture during the "proliferation phase," which is a period of exponential growth, and which are sufficiently complete to support cell culture during this phase. Proliferation cell culture media may also include selective agents that confer resistance or survival to selective markers incorporated into the host cell line. Such selective agents include, but are not limited to, genethecin (G418), neomycin, hygromycin B, puromycin, zeosin, methionine sulfamine, methotrexate, glutamine-free cell culture media, glycine, hypoxanthine, and thymidine-free or thymidine-free cell culture media.

[0051] "Perfused" cell culture media typically refers to cell culture media used for cell culture that are maintained by perfusion or continuous culture methods and are sufficiently complete to support cell culture during this process. Perfused cell culture medium formulations may be more enriched or more concentrated than basal cell culture medium formulations to accommodate the method used to remove used medium. Perfused cell culture media may be used in both the growth and production phases.

[0052] "Productive" cell culture media typically refers to cell culture media used during the "transition" and / or "productive" phases of cell culture, which occur when exponential growth ends and is replaced by protein production, and which are sufficiently complete to maintain the desired cell density, viability, and / or productive titer during this phase.

[0053] Concentrated cell culture media may contain some or all of the nutrients necessary to maintain cell culture, and in particular, concentrated media may contain nutrients that have been confirmed or are known to be consumed during the production phase of cell culture. Concentrated media may be based on almost any cell culture medium formulation. Such concentrated feed media may contain some or all of the components of cell culture media in amounts, for example, about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 20, 30, 50, 100, 200, 400, 600, 800, or about 1000 times their normal amounts.

[0054] The components used to prepare cell culture media can be completely pulverized to form a powdered medium formulation, partially pulverized together with liquid supplements added to the cell culture medium as needed, or added to the cell culture medium in a completely liquid form.

[0055] Cell cultures may also be supplemented with individual concentrated feeds for certain nutrients that are difficult to formulate or that may be rapidly depleted in the cell culture medium. Such nutrients may be amino acids such as tyrosine, cysteine, and / or cystine (see, for example, International Publication No. 2012 / 145682). For example, a concentrated tyrosine solution may be individually fed to cell cultures grown in a tyrosine-containing cell culture medium, such that the concentration of tyrosine in the cell culture medium does not exceed 8 mM. In another example, concentrated solutions of tyrosine and cystine may be individually fed to cell cultures grown in a cell culture medium lacking tyrosine, cystine, or cysteine. Individual feeds may be started before or at the beginning of the production phase. Individual feeds may be added to the cell culture medium on the same day or a different day as the concentrated feed medium. Individual feeds may also be perfused on the same day or a different day as the perfusion medium.

[0056] "Serum-free" applies to cell culture media that do not contain animal serum, such as fetal bovine serum. Various tissue culture media, including defined culture media, are commercially available, and for example, one or a combination of the following cell culture media may be used: among many, RPMI-1640 medium, RPMI-1641 medium, Dulbecco's modified Eagle medium (DMEM), Minimum Essential Medium Eagle, F-12K medium, Ham F12 medium, Iskov modified Dulbecco's medium, McCoy's 5A medium, Leibowitz L-15 medium, and serum-free media such as EX-CELL® 300 series (JRH Biosciences, Lenexa, Kansas), MCDB 302 (Sigma Aldrich Corp., St. Louis, MO). Serum-free versions of such culture media are also available. Cell culture media may be supplemented with additional components or increased concentrations of components such as amino acids, salts, sugars, vitamins, hormones, growth factors, buffers, antibiotics, lipids, and trace elements, depending on the requirements of the cells being cultured and / or the desired cell culture parameters. Customized cell culture media may also be used.

[0057] "Titer" refers to the total amount of the target polypeptide or protein (which may be naturally occurring or a recombinant protein) produced by cell culture in a given volume of culture medium. Titer can be expressed in milligrams or micrograms per milliliter (or other volume indicator) of culture medium. "Cumulative titer" refers to the titer produced by cells during the culture process and can be determined, for example, by measuring the titer daily and using those values ​​to calculate the cumulative titer.

[0058] As used herein, the term “host cell” is understood to include cells that have been genetically engineered to express the polypeptide of interest. Genetic engineering of cells includes transfecting, transforming, or transducing a host cell with a nucleic acid encoding a recombinant polynucleotide molecule (the “gene of interest”), and / or modifying it in other ways (e.g., by homologous recombination and gene activation, or by fusion of recombinant and non-recombinant cells) to cause the host cell to express the recombinant polypeptide of interest. Methods and vectors for genetically engineering cells and / or cell lines to express the polypeptide of interest are well known to those skilled in the art, and various techniques are exemplified, for example, in Current Protocols in Molecular Biology. Ausubel et al., eds. (Wiley & Sons, New York, 1988 and updated quarterly); 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 offspring of a parent cell, regardless of whether they are morphologically or genetically identical to the original parent cell, as long as the gene of interest is present. A cell culture may contain one or more host cells.

[0059] Wherever an embodiment is described herein with the word “comprising,” it will be understood that other similar embodiments are also provided, which are described using the terms “consisting of” and / or “essentially consisting of.”

[0060] Selection Marker Regarding the stable transfection of mammalian cells, it is known that, depending on the expression vector and transfection technique used, only a small fraction of the cells can incorporate foreign DNA into their genome. To identify and select these incorporates, a gene encoding a selection marker is typically introduced into the host cell using the same expression vector as the target gene.

[0061] Selection marker genes encode proteins necessary for the survival and proliferation of host cells growing in a selective culture medium. Typical selection marker genes encode proteins that (a) confer resistance to antibiotics or other toxins (e.g., ampicillin, tetracycline, or kanamycin) to prokaryotic host cells, (b) compensate for cellular nutritional deficiencies, or (c) supply essential nutrients that cannot be obtained from the combined or limited medium through metabolism. Specific antibiotic resistance selection markers include kanamycin resistance genes, ampicillin resistance genes, tetracycline resistance genes, and neomycin resistance genes.

[0062] Other selectable genes can be used to amplify the expressed genes. Amplification is the process by which genes required for the production of proteins crucial for proliferation or cell survival are repeated in tandem within the chromosomes of recombinant cells over generations. Examples of suitable selective markers for mammalian cells include glutamine synthetase (GS), dihydrofolate reductase (DHFR), asparaginase (Aspg; Ha et al. Biotechnol Bioeng. 2023 120:1159-1166), and the promoter resthymidine kinase gene.

[0063] Mammalian cell transformants are placed under selective pressure, ensuring that only that transformant is adapted to survive through the selectable gene present in the vector. Selective pressure is applied by culturing the transformed cells under conditions of continuously increasing the concentration of the selective agent in the culture medium, thereby resulting in further stringency and / or amplification of both the selectable gene and the DNA encoding the target protein. As a result, a large amount of the target polypeptide is synthesized from the amplified DNA. The selective agent for GS is methionine sulfoxamine (MSX). The selective agent for DHFR is methotrexate (MTX).

[0064] Compared to DHFR-based systems, GS knockout cell lines (GSKOs) provide sufficient selective stringency without MSX or at low MSX concentrations, however, 25 μM MSX with GS knockout cell lines resulted in higher selectivity compared to CHOK1SV cell lines at higher MSX concentrations (Fan, et al., Biotechnol Bioeng., 109(4):1007-1015 (2012)). Previous reports have shown that increasing MSX concentration during the seed train stage after clonal selection increases productivity in multiple GS knockout cell lines without significantly affecting cell proliferation, GS and target gene copy number, and expression, while maintaining product quality attributes (Tian et al., Engineering in Life Sciences 20(3-4):112-125 (2020)). Chain / vector expression may be affected by increasing stringency during pool recovery / selection by adding MSX. MSX can also be used to favor different vectors in the case of mixed GS promoters.

[0065] In certain embodiments, the MSX concentration may be optimized for one of the promoters driving GS expression. In certain embodiments, the MSX concentration is optimized for GS ligated to a more difficult-to-express chain.

[0066] In certain embodiments, the selection marker is glutamine synthetase. Glutamine synthetase (GS) catalyzes glutamine biosynthesis by condensation of ammonia and glutamate. The mammalian GS enzyme is a decamer composed of two stacked pentameric rings, each having 10 active sites located at the subunit junctions. Each active site is formed by residues from the N-terminal domain of one subunit (the β-grasp domain consisting of residues 25-112) and residues from the C-terminal domain of the adjacent subunit (the catalytic domain consisting of residues 113-373).

[0067] In certain embodiments, CHO DHFR- cells or CHO GSKO cells may be cultured under conditions that express antibody chains, either under methotrexate stringing in the case of CHO DHFR- cells or under methionine sulfoxamine stringing in the case of CHO GSKO cells, to favor the expression of a difficult-to-express chain paired with a stronger GS promoter.

[0068] In certain embodiments, CHO DHFR- cells or CHO GSKO cells may be cultured under conditions that favor the expression of antibody chains that are difficult to express in conjunction with a weaker GS promoter, such as under methotrexate stringing in the case of CHO DHFR- cells, or under methionine sulfoxamine stringing in the case of CHO GSKO cells.

[0069] In one embodiment, the MSX stringency is less than 75 μM. In one embodiment, this stringency is less than 50 μM. In one embodiment, this stringency is approximately 25 μM to 50 μM. In one embodiment, this stringency is approximately 25 μM to 45 μM. In one embodiment, this stringency is approximately 25 μM to 40 μM. In one embodiment, this stringency is approximately 25 μM to 35 μM. In one embodiment, this stringency is approximately 25 μM to 30 μM. In one embodiment, this stringency is approximately 25 μM or less. In one embodiment, this stringency is approximately 5 μM to 25 μM. In one embodiment, this stringency is approximately 10 μM to 25 μM. In one embodiment, this stringency is approximately 15 μM to 25 μM. In one embodiment, this stringency is approximately 20 μM to 25 μM. In one embodiment, this stringency 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.

[0070] Mixed promoter Expression and cloning typically involve a promoter that is recognized by the host organism and operably ligated to a nucleotide sequence encoding the protein of interest. The promoter is a non-transcriptional sequence located upstream (i.e., 5') of the start codon of a structural gene (generally within approximately 100–1000 bp) and controls the transcription of that structural gene. Traditionally, promoters have been classified into two classes: inductive promoters and constitutive promoters. Inductive promoters, under their control, initiate an increase in transcription levels from DNA in response to any change in culture conditions, such as the presence or absence of nutrients or changes in temperature. Constitutive promoters, on the other hand, transcribe the gene to which they operably ligate uniformly (i.e., with little or no regulation of gene expression). Numerous promoters recognized by various potential host cells are well known.

[0071] In the two-vector systems described herein, any suitable combination of promoters may be used, provided that the two promoters are different, appropriate for the host cell line, and drive the expression of the bound gene to different degrees. The chain ratio of the expressed polypeptide can be measured using techniques well known in the art. Such ratios may provide information about which chains are more difficult to express and which are easier to express. The chain ratio of the expressed polypeptide can be measured using techniques well known in the art, such as reduced capillary electrophoresis-sodium dodecyl sulfate (rCE-SDS). Promoteri suitable for use with mammalian host cells are well known and include, but are not limited to, those derived from the genomes of viruses such as polyomaviruses, fowlpox virus, adenoviruses (e.g., adenovirus type 2), bovine papillomavirus, aerosarcoma virus, cytomegalovirus, retroviruses, hepatitis B virus, and Simian virus 40 (SV40). Other suitable mammalian promoters include heteromammalian promoters, such as heat shock promoters and actin promoters.

[0072] Further promoters of note include, but are not limited to, the following: 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); the promoter included in the long terminal repeat at the 3' end of Rous sarcoma virus (Yamamoto et al., 1980, Cell 22:787-797); the herpesthymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. USA 78:1444-1445); promoters and regulatory sequences derived from the metallothionein gene (Prinster et al., 1982, Nature 296:39-42) and prokaryotic promoters such as the beta-lactamase promoter (Villa-Kamaroff et al.) al., 1978, Proc. Natl. Acad. Sci. USA 75:3727-3731); tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25), human elongation factor 1α promoter (EF-1α) (Kim et al., 1990, Gene 91:217-233), SRα promoter (Srα) (Takebe et al., 1988, Mol Cell. Biol. Jan; 8(1):466-72, and mouse mPGK-1 promoter (mPGK) (Adra et al., 1987, Gene 60:65-74).

[0073] Promoter of particular interest to nucleotide sequences encoding antibody chains include the human cytomegalovirus IE1 gene promoter-enhancer (CMV) (Boshart et al., 1985, Cell 41:521-30, GenBank Accession No. X03922), the adenovirus tripartite leader (adl) (Gingeras et al., 1982, J. Biol. Chem. 257:13475-91, GenBank Accession No. J01917), and the hamster glyceraldehyde-3-phosphate dehydrogenase promoter and intron (GAPDH) (U.S. Patent No. 10,202,261).

[0074] In certain embodiments, the antibody chain promoter is selected from the combinations CMV / GAPDH, CMV / adL, and CMV / EF1α. In these promoter combinations, both the CMV promoter and the promoter selected from GAPDH, adL, and EF1α are typically operably ligated to the nucleotide sequence encoding the antibody chain, such that this combination is a better promoter than the individual promoters. In certain embodiments, the CMV promoter is located at the 5' end of the promoter selected from GAPDH, adL, and EF1α.

[0075] In a particular embodiment, the promoter of the selection marker is selected from mPGK and Srα.

[0076] Promoters in CHO cells have also been investigated. 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.

[0077] In the vector systems disclosed herein, two different promoters drive the expression of two different select markers on two different 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 5:e10611. Promoter strengths can be predicted based on their sequences and comparison with promoter libraries (see, for example, Zhang et Al., 2022, ACS Synth Biol. 11:92-102) or various web tools (see Skrlj et Al., 2010, Anal Biochem. 396:83-90; Liang et al., 2021, Anal. Biochem. 630:114335). Srα is known to be a stronger promoter than the mPGK promoter.

[0078] Surprisingly, it has been found that using two different promoters with a selection marker results in higher antibody production regardless of which chain is being expressed. However, it has been found that the highest production is observed when the stronger promoter is present on the same vector as the more difficult-to-express chain. Therefore, in certain embodiments, the stronger promoter is present on the vector with the difficult-to-express chain. In other embodiments, the stronger promoter is present on the vector with the easier-to-express chain.

[0079] The ease of antibody chain expression can be evaluated in a system where the vector is identical except for the chain produced. The vector is transfected into the same cell line, regardless of whether it is in the same cells or in different cell pools under the same conditions. As mentioned above, by measuring the chain ratio, information can be obtained about which chains are more difficult to express and which are more easily expressed.

[0080] In a particular embodiment, one vector contains Srα operably linked to a selection marker, and the other vector contains mPGK operably linked to a selection marker. In one aspect of this embodiment, the selection marker is GS. In one aspect, the vector containing Srα is a vector containing a gene encoding a strand that is more difficult to express, and the mPGK is on a vector containing a gene encoding a strand that is more easily expressed. In another aspect, the vector containing Srα is a vector containing a gene encoding a strand that is more easily expressed, and the mPGK is on a vector containing a gene encoding a strand that is more difficult to express.

[0081] 3- and 4-chain molecules Typical antibodies are Y-shaped molecules with four polypeptide chains (two identical heavy chains and two identical light chains). Such antibodies are preferably expressed from a single vector. However, bispecific antibodies require the use of alternative forms and are usually expressed from two different vectors. See, for example, Spiess et al., 2015, MolImmunol. 67:95-106; Brinkmann et al., 2017, MAbs 9:192-212; and Ma et al., 2021, Frontiers in Immunology 12:626616.

[0082] Any form having at least three distinct strands or being expressible on two vectors may be used with the vector system of the present invention. Certain embodiments are described below. Other embodiments are described in the references cited. Others may be determined by those skilled in the art.

[0083] The expression vectors provided herein include genes encoding at least two antibody chains. The genes encoding the two antibody chains may be the same gene or different genes. For example, in some embodiments, the vector or nucleic acid construct may include two copies of the same antibody light chain gene. Alternatively, in some embodiments, the vector or nucleic acid construct may include 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 include one copy of the first antibody light chain and one copy of the first antibody heavy chain. The second expression may include 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 include the same antibody light chain sequence, while the first and second expression vectors include different antibody heavy chain sequences. In these embodiments, the two heavy chains may be linked via a knob-in-hole or similar mechanism.

[0084] In certain embodiments, two expression vectors jointly produce bispecific antibodies in mammalian host cells. These bispecific antibodies can be classified into five categories (one of which requires chemical coupling and is therefore not further considered). See Spiess, et al., 2015, Mol.Immunol.67:95-106.

[0085] Bispecific IgG (BsIgG) is a form produced using two different heavy chains and two different light chains that are monovalent for each antigen. BsIgG may also be called heteroIgG. In this form, typically, the coding sequences for one pair of heavy chains and light chains are located on a first vector, and the coding sequences for another pair of heavy chains and light chains are located on a second vector.

[0086] The IgG to be added is in the form of an additional antigen-binding unit being added to either the amino or carboxyl terminus of either the light or heavy chain. An example of the IgG to be added is a single-domain antibody (unpaired V). L or V H Examples include, for instance, a bivariable domain Ig (DVD-Ig), a paired antibody-variable domain (e.g., Fv or ScFv), and an engineered protein scaffold. See also LaFleur et al., 2013, mAbs 5:208-218; Wu et al., 2007, Nat. Biotechnol. 25:1290-7. In this form, typically, the coding sequences for the heavy and light chain pairs are present in both the first and second vectors. The coding sequence for an antigen-binding unit (e.g., scFv, VHH domain, or cytokine) may be added to either the heavy chain coding sequence or the light chain coding sequence. Multiple coding sequences for antigen-binding units may be added.

[0087] Bispecific antibody fragments lack an antibody constant domain and may consist of heavy and light chains linked by diabodies such as peptide linkers, scFv fragments, biaffinity retargeting proteins (DARTs), and tetravalent tandem diabodies. See also Arndt et al., 1999, Blood 94:2562-2568; and Kipriyanov et al., 1999, J.Mol.Bio.293:41-56. In this form, for example, two different scFv fragments may be expressed in each of two vectors, or in the case of DARTs, one vector may contain the coding sequence VHA-linker-VLB and the other vector may contain the coding sequence VHB-linker-VLA.

[0088] A bispecific fusion protein is an antibody fragment linked to a receptor and another protein, such as albumin. In this form, typically, the coding sequences of a heavy chain and a light chain are present in the first and second vectors. The coding sequence of an antigen-binding unit (e.g., a receptor) may be added to either the coding sequence of the heavy chain or the coding sequence of the light chain. Multiple coding sequences relating to the antigen-binding unit may be added.

[0089] In another example, to express a bispecific IgG antibody, it may be desirable to use an embodiment in which 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 beneficial because a typical bispecific IgG antibody contains the following four different polypeptide types: i) an antibody light chain relating to a first antigen-binding moiety, ii) an antibody heavy chain relating to a first antigen-binding moiety, iii) an antibody light chain relating to a second antigen-binding moiety, and iv) an antibody heavy chain relating to a second antigen-binding moiety. Therefore, for bispecific IgG expression, it may be desirable to have a first position in a first vector containing the gene encoding the first antibody light chain, a second position in a first vector containing the gene encoding the second antibody light chain, a first position in a second vector containing the gene encoding the first antibody heavy chain, and a second position in a second vector containing the gene encoding the second antibody heavy chain. Alternatively, for bispecific IgG expression, it may be desirable to have a first position in the first vector containing the gene encoding the first antibody light chain, a second position in the first vector containing the gene encoding the first antibody heavy chain, a first position in the second vector containing the gene encoding the second antibody light chain, and a second position in the second vector containing the gene encoding the second antibody heavy chain. As described above, additional sequences may be added to the amino or carboxyl terminus of either the light chain or either heavy chain.

[0090] Other relevant embodiments are also provided herein. For example, in some embodiments, the first and second vectors may be used together with the embodiments provided herein for, for example, the expression of a bispecific antibody having a common light chain (see, for example, International Publication No. 2021 / 124073). A bispecific antibody having a common light chain may comprise two different heavy chains.

[0091] In embodiments provided herein, involving a first vector and a second vector, in some other embodiments, the first vector comprises a first gene encoding an antibody light chain and a second gene encoding an antibody light chain, and the second vector comprises 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 each of two different antibody light chain genes).

[0092] In embodiments provided herein, involving a first vector and a second vector, in some other embodiments, the first vector comprises a first gene encoding an antibody light chain and a second gene encoding an antibody heavy chain, and the second vector comprises a first gene encoding an antibody light chain and a second gene encoding an antibody heavy chain. In some embodiments, the first gene encoding an antibody light chain and the second gene encoding an 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 an antibody light chain and the second gene encoding an antibody light chain encode different antibody light chains (e.g., one copy each of two different antibody light chain genes).

[0093] The choice in the first vector—whether to have two copies of the same antibody light chain or one copy each of two different antibody light chains—depends on the specific type of antibody molecule being produced. For example, in the case of an antibody fusion protein containing a standard monospecificity IgG mAb covalently linked to another protein via an antibody heavy chain, this molecule typically contains three distinct polypeptide types: i) antibody light chain; ii) antibody heavy chain; and iii) antibody heavy chain fusion. In this antibody fusion molecule, there are one chain each of the antibody heavy chain and antibody heavy chain fusion, as well as two copies of the antibody light chain. Since this molecule contains only one type of antibody light chain, two copies of the same antibody light chain gene are provided in the first vector. In another example, consider an antibody fusion protein containing a bispecific IgG mAb covalently linked to another protein via an antibody heavy chain. This molecule typically contains four distinct polypeptide types: i) an antibody light chain relating to a first antigen-binding moiety; ii) an antibody heavy chain relating to a first antigen-binding moiety; iii) an antibody light chain relating to a second antigen-binding moiety; and iv) an antibody heavy chain fusion in which the antibody heavy chain portion relates to a second antigen-binding moiety. This antibody fusion protein molecule contains one copy each of the antibody heavy chain and antibody heavy chain fusion, as well as one copy each of the antibody light chains. Since this molecule contains two types of antibody light chains, two different antibody light chain genes are provided in the first vector.

[0094] In embodiments provided herein, involving two different antibody heavy chains, or an antibody heavy chain and an antibody heavy chain fusion, the heavy chains may optionally include one or more amino acid modifications in the constant region of the heavy chains to facilitate heterodimerization between the two different heavy chains. Such modifications are known in the art and include, for example, charge-based electrostatic amino acid modifications and stereo-based "knob-into-hole" amino acid modifications.

[0095] A vector is useful for transforming host cells and may contain additional nucleic acid sequences that induce and / or regulate the expression of one or more heterogeneous coding regions operably ligated to this nucleic acid sequence (in cooperation with the host cell). An expression construct may contain, but is not limited to, sequences that affect or control transcription, translation, and, where introns are present, sequences that affect RNA splicing of coding regions operably ligated to these introns. "Operatably ligated" means that the components to which this term applies are in a relationship that allows them to perform their intrinsic function. For example, a regulatory sequence (e.g., a promoter) in a vector "operably ligated" to a protein coding sequence is positioned so that the normal activity of this regulatory sequence leads to transcription of the protein coding sequence, resulting in recombinant expression of the encoded protein.

[0096] Vectors can be selected to function within the specific host cells in which they are used (i.e., the vector is compatible with the host's cellular mechanisms and allows for gene amplification and / or expression). In some embodiments, vectors are used in protein fragment complementation assays that utilize protein reporters 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.

[0097] Typically, vectors used in host cells include sequences for maintaining plasmids and sequences for cloning and expressing exogenous nucleotide sequences. Such sequences typically include one or more of the following nucleotide sequences (in addition to the promoter and selection markers mentioned above): one or more enhancer sequences, origins of replication, transcription and translation regulatory sequences, transcription termination sequences, complete intron sequences including donor and acceptor splice sites, various pre-sequences or pro-sequences for glycosylation or yield improvement, native or heterologous signal sequences (leader sequences or signal peptides) for polypeptide secretion, ribosome binding sites, polyadenylation sequences, internal ribosome entry sites (IRES) sequences, expression enhancement sequence elements (EASE), three-part leader (TPA) and VA gene RNA derived from adenovirus type 2, and polylinker regions for inserting polynucleotides encoding the polypeptide to be expressed. Vectors can be constructed from starting vectors, such as commercially available vectors, and additional elements can be obtained individually and ligated into the vector. Methods used to obtain each of the components are well known to those skilled in the art.

[0098] Vector components can be homogeneous (i.e., derived from the same species and / or strain as the host cell), heterogeneous (e.g., derived from a species other than the host cell species or host cell line), hybrid (i.e., a combination of adjacent sequences derived from two or more sources), synthetic, or native. Sequences of components useful in a vector can be obtained by methods known in the art, such as those previously identified by mapping and / or restriction endonucleases. Furthermore, they can be obtained by polymerase chain reaction (PCR) and / or by screening a genomic library with a suitable probe.

[0099] The ribosome binding site is typically required for mRNA translation initiation and is characterized by a Shine-Dalgano sequence (prokaryotes) or a Kozak sequence (eukaryotes). This element is typically located at the 3' end of the promoter and the 5' end of the coding sequence of the polypeptide to be expressed.

[0100] Origins of replication assist in the amplification of vectors in host cells. These origins may be included as part of commercially available prokaryotic vectors, or they may be chemically synthesized based on known sequences and ligated into vectors. Various viral origins (e.g., SV40, polyomas, adenoviruses, varicella-stomatitis virus (VSV), or papillomaviruses such as HPV or BPV) are useful for cloning vectors in mammalian cells.

[0101] Transcriptional and translational regulatory sequences for expression vectors in mammalian host cells can be excised from viral genomes. Commonly used promoter and enhancer sequences are derived from polyomaviruses, adenovirus type 2, Simianvirus 40 (SV40), and human cytomegalovirus (CMV). For example, the human CMV promoter / enhancer of early gene 1 can be used. See, for example, Patterson et al., 1994, Applied Microbiol. Biotechnol. 40:691-98. DNA sequences derived from the SV40 viral genome, e.g., the SV40 origin, early and late promoters, enhancers, splices, and polyadenylation sites, can be used to provide other genetic elements for expressing structural gene sequences in mammalian host cells. Both early and late viral promoters are readily obtainable as fragments from the viral genome and may also contain the viral replication origin, making them particularly useful (Fiers et al., 1978, Nature 273:113; Kaufman, 1990, Meth. in Enzymol. 185:487-511). Smaller or larger SV40 fragments can also be used if they contain a sequence of approximately 250 bp extending from the Hind III site towards the BglI site located at the SV40 viral replication origin.

[0102] Enhancer sequences can be inserted into vectors to increase transcription by higher eukaryotes. Enhancers are cis-acting elements of DNA, typically about 10–300 bp long, that act on promoters to increase transcription. Enhancers are relatively independent of direction and position and can be found at both the 5' and 3' ends of the transcription unit. Several enhancer sequences are known to be available from mammalian genes (e.g., globin, elastase, albumin, alpha-fetoprotein, and insulin). However, viral enhancers are typically used. The SV40 enhancer, cytomegalovirus initial promoter enhancer, polyoma enhancer, and adenovirus enhancer, known in this technique, are exemplary enhancing elements for eukaryotic promoter activation. Enhancers can be located at either the 5' or 3' end of the coding sequence in the vector, but are typically located at the 5' end from the promoter.

[0103] Transcription termination sequences are typically located 3' to the end of the polypeptide coding region and play a role in terminating transcription. In prokaryotic cells, the transcription termination sequence is usually a GC-rich fragment followed by a poly-T sequence. While this sequence can be readily cloned from libraries or commercially purchased as part of a vector, it can also be readily synthesized using methods for nucleic acid synthesis known to those skilled in the art.

[0104] In some cases where glycosylation is desired in eukaryotic host cell expression systems, various pre-sequences or pro-sequences can be manipulated to improve glycosylation or yield. For example, the peptidase cleavage site of a particular signal peptide may be modified, or a pro-sequence may be added, which can also affect glycosylation. The final protein product may have one or more additional amino acids associated with expression at position -1 (relative to the first amino acid of the mature protein), but these amino acids do not have to be completely removed. For example, the final protein product may have one or two amino acid residues found at the peptidase cleavage site, attached to the amino terminus. Alternatively, using several enzymatic cleavage sites may result in a slightly shortened form of the desired polypeptide when cleaved enzymatically in such a region within the mature polypeptide.

[0105] Sequences encoding appropriate native or heterologous signal sequences (leader sequences or signal peptides) can be incorporated into expression vectors to promote extracellular secretion of the target protein. The choice of signal peptide or leader depends on the type of host cell intended to produce the target protein, and heterologous signal sequences may replace native signal sequences. Examples of signal peptides that function in mammalian host cells include: the signal sequence for interleukin-7 described in U.S. Patent No. 4,965,195; the signal sequence for the interleukin-2 receptor described in Cosman et al., 1984, 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.

[0106] Further regulatory sequences that have been shown to improve the expression of heterologous genes from mammalian expression vectors include: expression-enhancing sequence elements (EASE) derived from CHO cells (Morris et al., Animal Cell Technology, pp. 529-534 (1997); U.S. Patent Nos. 6,312,951B1, 6,027,915, and 6,309,841B1), and elements such as the three-part leader (TPL) and VA gene RNA derived from adenovirus type 2 (Gingeras et al., 1982, J. Biol. Chem. 257: 13475-13491). The viral internal ribosome entry site (IRES) sequence enables efficient translation of bicistronic mRNA (Oh and Sarnow, 1993, Current Opinion in Genetics and Development 3:295-300; Ramesh et al., 1996, Nucleic Acids Research 24:2697-2700).

[0107] Target protein The polypeptides and proteins of interest may be for scientific or commercial purposes, including protein-based therapeutics. Among the many types of proteins of interest are secreted proteins, non-secreted proteins, intracellular proteins, or membrane-bound proteins. The polypeptides and proteins of interest may be produced by recombinant animal cell lines using cell culture methods and may be referred to as “recombinant proteins.” The expressed proteins may be produced intracellularly, secreted into the culture medium, and recovered and / or collected from there. The terms “isolated protein” or “isolated recombinant protein” refer to the polypeptide or protein of interest that has been isolated and purified from a protein or polypeptide or other contaminants that would inhibit therapeutic, diagnostic, preventive, research, or other uses. Among the proteins of interest are proteins that exert a therapeutic effect by binding to a target (in particular, targets listed below, e.g., targets derived from these, targets associated with these, and their modifications).

[0108] The target protein is an "antigen-binding protein." An antigen-binding protein refers to a protein or polypeptide that contains an antigen-binding region or part that has affinity for another molecule (antigen) to which it binds. Antigen-binding proteins include antibodies, peptide bodies, antibody fragments, antibody derivatives, antibody analogs, Fc fusion proteins (e.g., single-chain variable fragments (scFv), double-chain (bivalent) scFv, and IgGscFv (see, for example, Orcutt et al., 2010, Protein Eng Des2 Sel 23:221-228)), heteroIgG (see, for example, Liu et al., 2015, J Biol Chem 290:7535-7562), mutains, and XmAb® (Xencor, Inc., Monrovia, CA). Examples of antigen-binding proteins include human antibodies, humanized antibodies, chimeric antibodies, recombinant antibodies, single-chain antibodies, diabodies, triabodies, tetrabodies, Fab fragments, F(ab')2 fragments, IgD antibodies, IgE antibodies, IgM antibodies, IgG1 antibodies, IgG2 antibodies, IgG3 antibodies, or IgG4 antibodies, and their fragments. Similarly, bispecific T cell engagers (BiTE®), bispecific T cell engagers with extended half-lives, such as HLE BiTE and HeteroIg BITE, are also mentioned.

[0109] As used herein, the term “antigen-binding protein” is used in its broadest sense and refers to a protein comprising a moiety that binds to an antigen or target, and a scaffold or framework moiety that optionally allows the antigen-binding moiety to adopt a three-dimensional structure that promotes the binding of the antigen-binding protein to the antigen. Antigen-binding proteins may include, for example, alternative or artificial protein scaffolds having grafted CDRs or CDR derivatives. Such scaffolds include, but are not limited to, fully synthetic scaffolds containing biocompatible polymers, as well as, for example, antibody-derived scaffolds containing mutations introduced to stabilize the three-dimensional structure of the antigen-binding protein. 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. In addition, peptide antibody mimes ("PAMs"), as well as antibody mimeograph-based scaffolds using fibronectin components as scaffolds, may also be used.

[0110] Antigen-binding proteins can have structures similar to naturally occurring immunoglobulins. "Immunoglobulins" are tetrameric molecules. In naturally occurring immunoglobulins, each tetramer consists of two identical pairs of polypeptide chains, each having one "light" chain (approximately 25 kDa) and one "heavy" chain (approximately 50-70 kDa). The amino-terminus of each chain contains a variable region consisting of approximately 100-110 or more amino acids, primarily involved in antigen recognition. The carboxyl-terminus of each chain defines a constant region primarily involved in effector function. Human light chains are classified into kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and the antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively.

[0111] Naturally occurring immunoglobulin chains exhibit the same general structure of relatively conserved framework regions (FR) linked by three hypervariable regions, also called complementarity determining regions or CDRs. From the N-terminus to the C-terminus, both light chains and heavy chains comprise domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The assignment of amino acids to each domain is described by Kabat et al. in Sequences of Proteins of Immunological Interest, 5 th Ed., US Dept. of Health and Human Services, PHS, NIH, NIH Publication no. 91-3242, (1991), and can be performed in accordance with the definition thereof. If desired, CDRs can also be redefined according to an alternative nomenclature scheme such as that of Chothia (see Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917; Chothia et al., 1989, Nature 342:878-883, or Honegger and Pluckthun, 2001, J. Mol. Biol. 309:657-670).

[0112] In the context of the present disclosure, an antigen-binding protein has a dissociation constant (K D ) of 10 -8 M or less, it is said to "specifically bind" or "selectively bind" to its target antigen. An antibody specifically binds to an antigen with "high affinity" when its K D is 5×10 -9 M or less, and specifically binds to an antigen with "very high affinity" when its K D is 5×10 -10 M or less.

[0113] The term "antibody," unless otherwise specified, includes both glycosylated and nonglycosylated immunoglobulins of any isotype or subclass, or references to their antigen-binding region that competes with intact antibodies for specific binding. In addition, the term "antibody," unless otherwise specified, refers to intact immunoglobulins that compete with intact antibodies for specific binding.

[0114] The antigen-binding region can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies, and can form elements of the target protein. Unless otherwise specified, antibodies include human, humanized, chimeric, multispecific, monoclonal, polyclonal, heteroIgG, bispecific, and their oligomers or antigen-binding fragments. Antibodies include IgG1, IgG2, IgG3, or IgG4 types. Similarly, proteins having antigen-binding fragments or regions, such as Fab, Fab', F(ab')2, Fv, diabody, Fd, dAb, maxibody, single-chain antibody molecules, and single-domain V. H The polypeptide comprises H, a complementarity-determining region (CDR) fragment, scFv, a diabody, a triabody, a tetrabody, and at least a portion of immunoglobulin sufficient to confer specific antigen binding to the target polypeptide.

[0115] Antigen-binding proteins may have one or more binding sites. If multiple binding sites exist, they may be identical or distinct from one another. For example, naturally occurring human immunoglobulins typically have two identical binding sites, while "bispecific" or "bifunctional" antibodies have two distinct binding sites.

[0116] Fab fragment is V L , V H , C L , and C HThe monovalent fragment having one domain, the F(ab')2 fragment is a bivalent fragment having two Fab fragments linked by disulfide bridges in the hinge region, and the Fd fragment is V H and C H It has one domain, and the Fv fragment is the V of the single arm of the antibody. L and V H It has a domain, and the dAb fragment is V H Domain, V L Domain, or V H Or V L It has an antigen-binding fragment in the domain (U.S. Patent Nos. 6,846,634, 6,696,245, U.S. Patent Publication Nos. 2005 / 0202512, 2004 / 0202995, 2004 / 0038291, 2004 / 0009507, 2003 / 0039958, Ward et al., 1989, Nature 341:544-546).

[0117] Single-chain antibodies (scFv) are V L and V H An antibody is one in which regions are linked via a linker (e.g., a synthetic sequence of amino acid residues) to form a continuous protein chain, and this linker is long enough to allow the protein chain to fold itself and form a monovalent antigen-binding site (see, e.g., Bird et al., 1988, Science 242:423-26 and Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-83), U.S. Patent Nos. 7,741,465 and 6,319,494, and Eshhar et al., 1997, Cancer Immunol Immunotherapy 45:131-136. scFv retains the ability of the parental antibody to specifically interact with the target antigen.

[0118] The diabody is a bivalent antibody containing two polypeptide chains, each polypeptide chain linked by a linker that is too short to allow pairing between the two domains on the same chain. H and V L The diabody contains domains, which allow each domain to pair with a complementary domain on another polypeptide chain (see, for example, Holliger et al., 1993, Proc. Natl. Acad. Sci. USA 90:6444-48; and Poljak et al., 1994, Structure 2:1121-23). ​​If the two polypeptide chains of a diabody are identical, the resulting diabody will have two identical antigen-binding sites. Diabodies with two different antigen-binding sites can be constructed using polypeptide chains with different sequences. Similarly, triabodies and tetrabodies are antibodies containing three and four polypeptide chains, respectively, and forming three and four antigen-binding sites, respectively, where the three and four antigen-binding sites may be identical or different.

[0119] For clarity, and as described herein, it should be noted that antigen-binding proteins may, but do not have to be, of human origin (e.g., human antibodies), and may, in some cases, include non-human proteins, such as rat or mouse proteins, and in other cases, antigen-binding proteins may include hybrids of human and non-human proteins (e.g., humanized antibodies).

[0120] The target protein may include human antibodies. 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 (fully human antibody). Such antibodies can be prepared by various methods, including immunization with the target antigen of genetically modified mice that express antibodies derived from genes encoding human heavy and / or light chains, such as mice from the Xenomouse®, UltiMab®, or Velocimmune® lines, or rats from the UniRat® line. Phage-based methods may also be used.

[0121] Alternatively, the target protein may include a humanized antibody. A "humanized antibody" has a sequence different from that of an antibody derived from a non-human species due to the substitution, deletion, and / or addition of one or more amino acids. Therefore, when administered to a human subject, a humanized antibody is less likely to induce an immune response and / or induces a less severe immune response compared to a non-human antibody. In one embodiment, a humanized antibody is produced by mutating certain amino acids within the framework domain and constant domain of the heavy and / or light chain of a non-human antibody. In another embodiment, a constant domain derived from a human antibody is fused to a variable domain of a non-human species. Examples of methods for producing humanized antibodies can be found in U.S. Patent No. 6,054,297, No. 5,886,152, and No. 5,877,293.

[0122] Similarly included are modified proteins, such as those chemically modified by non-covalent, covalent, or both covalent and non-covalent bonds. Similarly included are proteins further comprising one or more post-translational modifications that can be generated by cell modification systems, or modifications that are introduced ex vivo by enzymatic and / or chemical methods or otherwise.

[0123] In some embodiments, the protein of interest may include one or more CD proteins, HER receptor family proteins, cell adhesion molecules, growth factors, nerve growth factor, fibroblast growth factor, transforming growth factor (TGF), insulin-like growth factor, bone-inducing factors, insulin and insulin-related proteins, coagulation and coagulation-related proteins, colony-stimulating factor (CSF), other blood and serum proteins, blood group antigens; receptors, receptor-related proteins, growth hormone, growth hormone receptor, T cell receptor; neurotrophic factors, neurotrophins, relaxin, interferon, interleukin, viral antigens, lipoproteins, integrins, rheumatoid factor, immunotoxins, surface membrane proteins, transport proteins, homing receptors, adresins, regulatory proteins, and proteins that specifically bind to immunoadhesins.

[0124] In some embodiments, the target protein binds to one or more of the following, either alone or in any combination: CD proteins, e.g., 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, e.g., HER2, HER3, HER4, and EGF receptor, EGFRvIII; cell adhesion molecules, e.g., LFA-1, Mo l, p150, 95, VLA-4, ICAM-1, VCAM, and alpha-v / beta-3 integrin, growth factors, e.g., vascular endothelial growth factor ("VEGF"); VEGFR2, growth hormone, thyroid-stimulating hormone, follicle-stimulating hormone, luteinizing hormone, growth hormone-releasing factor, parathyroid hormone, Müllerian duct inhibitor, human macrophage inflammatory protein (MIP-1-alpha), erythropoietin (EPO), nerve growth factor, e.g., NGF-beta, platelet-derived growth factor (PDGF), fibroblast growth factor, e.g., aFGF and bFGF, epidermal growth factor (EGF), Cripto, transforming growth factor (TGF), for example, TGF-α and TGF-β, for example, TGF-β1, TGF-β2, TGF-β3, TGF-β4, or TGF-β5, insulin-like growth factor-I and-II (IGF-I and IGF-II), des(1-3)-IGF-I (brain IGF-I), and bone induction factors, insulin and insulin-related proteins, for example, insulin, insulin A chain, insulin B chain, proinsulin, and insulin-like proteins, for example, but not limited to these. Long factor-binding proteins; coagulation and coagulation-related proteins, such as, among many others, factor VIII, tissue factor, von Willebrand factor, protein C, alpha-1-antitrypsin, plasminogen activators, such as urokinase and tissue plasminogen activator ("t-PA"), bombadin, thrombin, thrombopoietin, and thrombopoietin receptor, colony-stimulating factors (CSFs), such as, among many others, M-CSF, GM-CSF, and G-CSF listed below, and other blood and serum proteins, such as, but not limited to,Albumin, IgE, and blood group antigens, receptors, and receptor-related proteins, e.g., flk2 / flt3 receptor, obesity (OB) receptor, growth hormone receptor, and T cell receptor; neurotrophic factors, e.g., but not limited to bone-derived neurotrophic factor (BDNF), and neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6); relaxin A chain, relaxin B chain, and prorelaxin; interferons, e.g., interferon-alpha, -beta, and -gamma; interleukin (IL), e.g., I L-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 to IL-13RA2, or IL-17 receptor IL-1RAP; viral antigens, e.g., but not limited to AIDS enveloped virus antigens, lipoproteins, calcitonin, glucagon, atrial natriuretic factor, pulmonary surfactants, tumor necrosis factor-alpha and beta, enkephalinase, BCMA, Ig kappa, ROR-1, ERBB2, mesotherapy Phosphorus, RANTES (regulated by activation, expressed and secreted by normal T cells), mouse gonadotropin-related peptide, DNase, FR-alpha, inhibin, and activin, integrin, protein A or D, rheumatoid factor, immunotoxin, bone morphogenetic protein (BMP), superoxide dismutase, surface membrane protein, disintegration factor (DAF), AIDS envelope, transport protein, homing receptor, MIC (MIC-A, MIC-B), ULBP1-6, EPCAM, adresin, regulatory protein, immunoadhesin, antigen binding Proteins, somatropin, 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 ligands,PD1 and PDL1, mannose receptor / hCGβ, hepatitis C virus, mesoserine dsFv[PE38] conjugate, Legionella pneumophila (LLY), IFN gamma, interferon gamma-inducible protein 10 (IP10), IFNAR, TALL-1, thymic stromal lymphocyte neoplastic factor (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 beta (TGF) (IGRB), zona pellucida sperm-binding protein 3 (ZP-3), TWEAK, platelet-derived growth factor receptor alpha (PDGFRα), sclerostin, and any of the aforementioned biologically active fragments or variants.

[0125] In another embodiment, the target protein may include: absiximab, adalimumab, adecatumumab, aflibercept, alemtuzumab, alirocumab, anakinra, atacicept, basiliximab, belimumab, bevacizumab, biosozumab, blinatumomab, brentuximab vedotin, brodalumab, cantuzumab meltansine, canakinumab, cetuximab, certolizumab pegol, conatumumab, daclizumab, denosumab, eculizumab, edrecolomab, efalizumab, epratuzumab, etanercept, evolocumab, galiximab, ganitumamab, gemtuzumab, golimumab, ibritumomab tiuxetan, i Nfliximab, ipilimumab, ixekizumab, reldelimumab, lumiliximab, mapatumumab, motesanib diphosphate, muromonab-CD3, natalizumab, nesiritide, nimotuzumab, nivolumab, ocrelizumab, ofatumumab, omalizumab, oprelbequin, palivizumab, panitumumab, pembrolizumab Pertuzumab, paxerizumab, ranibizumab, rilotumumab, rituximab, romiplostim, romosozumab, salglamostim, tocilizumab, tositumomab, trastuzumab, ustekinumab, vedolizumab, vizilizumab, borosiximab, zanorimumab, saltumumab, and any of the aforementioned biosimilars.

[0126] The target protein according to the present invention encompasses all of the foregoing and further comprises an antibody containing one, two, three, four, five, or six complementarity-determining regions (CDRs) of any of the aforementioned antibodies. One or more CDRs may be incorporated into a molecule covalently or non-covalently to form an antigen-binding protein. The antigen-binding protein may incorporate the CDR as part of a larger polypeptide chain, which may be covalently linked to another polypeptide chain, or it may be incorporated non-covalently. This CDR makes it possible to specifically bind the antigen-binding protein to a particular target antigen. Similarly included are variants containing regions whose amino acid sequence is identical to that of the reference amino acid sequence of the target protein by 70% or more, particularly 80% or more, more particularly 90% or more, even more particularly 95% or more, particularly 97% or more, even more particularly 98% or more, and even more particularly 99% or more. This identity can be determined using various well-known and readily available amino acid sequence analysis software. Preferred software includes implementations of the Smith-Waterman algorithm, which are considered satisfactory solutions for sequence searching and alignment problems. Other algorithms may also be used, especially when speed is a critical requirement. In this regard, commonly used programs for DNA, RNA, and polypeptide alignment and homology matching include FASTA, TFASTA, BLASTN, BLASTP, BLASTX, TBLASTN, PROSRCH, BLAZE, and MPSRCH, the latter being an implementation of the Smith-Waterman algorithm that runs on a MasPar massively parallel processor.

[0127] Preferably, the antigen-binding molecule is its antibody fragment, more preferably one or more single-chain antibody fragments ("scFv"). Since scFv can be engineered to be expressed as part of a single chain, it is preferable to use it in a chimeric antigen receptor. See Krause et al., 1988, J. Exp. Med., 188(4); 619-626; Finney et al., 1998, J Immunol 161; 2791-2797.

[0128] The "Fc" region is the C region of the antibody when this term is used herein. H 2 domains and C H It contains two heavy chain fragments, each containing three domains. The two heavy chain fragments have two or more disulfide bonds and C H The three domains are bound by hydrophobic interactions. The target protein, which includes an antigen-binding protein and an Fc fusion protein, and which includes an Fc region, constitutes another aspect of this disclosure.

[0129] A "hemibody" is an immunologically functional 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 required, be used to conjugate the heavy chain Fc region to the second heavy chain Fc region. In certain embodiments, the hemibody is an antigen-binding protein in the monovalent form disclosed herein. In other embodiments, a pair of charged residues may be used to conjugate one Fc region to the second Fc region. The hemibody may be the protein of interest in the context of this disclosure.

[0130] Creation of mammalian host cells that express the target protein. The expression of a target protein in cells can be achieved by either transient or stable expression, using well-known methods (Davis et al., Basic Methods in Molecular Biology, 2 nd ., Appleton & Lange, Norwalk, Conn., 1994; Sambrook et al., Molecular Cloning; A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001).

[0131] Methods for stable integration are well known in the art. Briefly, stable integration is generally achieved by transiently introducing heterologous polynucleotides or vectors containing heterologous polynucleotides into host cells, thereby promoting the stable integration of the heterologous polynucleotides into the cellular genome. Typically, heterologous polynucleotides are adjacent to homologous arms (i.e., homologous sequences in the upstream and downstream regions relative to the integration site). Before introducing them into mammalian host cells, circular vectors may be linearized to facilitate integration into the cellular genome. Methods for introducing vectors into cells are well known in the art and include biological methods such as viral delivery; chemical methods such as the use of cationic polymers, calcium phosphate, cationic lipids, or cationic amino acids; physical methods such as electroporation or microinjection; or transfection by mixed methods such as protoplast fusion.

[0132] A specific method for stable integration uses recombinase-mediated cassette exchange (RMCE; Bode and Baer, ​​2001, Curr Opin Biotechnol. 12:473-80, and Bode et al., 2000, Biol. Chem. 381:801-813) for site-specific integration (also called "targeted integration") in the genome. Site-specific recombinases such as Flp and Cre mediate recombination between two copies of their target sequences, called FRT and loxP, respectively. By using two incompatible target sequences (e.g., FRT combined with F3) (Schlake and Bode, 1994, Biochemistry, 33:12746-51) and inversion recognition target sites (Feng et al., 1999, J. Mol. Biol. 292:779-85), DNA segments can be inserted into a given chromosomal locus containing the target sequence in a similar configuration. See also European Patent No. 1781796B1 and European Patent Application Publication No. 2789691A1.

[0133] The insertion of RMCEs into specific sites in the genome can be mediated by nucleases (e.g., zinc finger proteins (ZFPs), activator-like effector nucleases (TALENs), clustered and regularly arranged short palindromic sequence repeats (CRISPR) / CRISPR-related protein 9 (Cas9)) that can be manipulated to create single-strand and double-strand breaks (SSBs / DSBs) in the genome. There are two main and distinct pathways for repairing DSBs: homologous recombination and non-homologous end joining (NHEJ). Homologous recombination requires the presence of a homologous sequence (e.g., a “donor” containing an RMCE) as a template to guide the cellular repair process, and the repair outcome is error-free and predictable. If a template (or “donor”) sequence for homologous recombination is not present, the cell typically attempts to repair the DSB via the unpredictable and error-prone process of non-homologous end joining (NHEJ).

[0134] A vector can be any molecule or entity (e.g., nucleic acids, plasmids, bacteriophages, transposons, cosmids, chromosomes, viruses, viral capsids, virions, naked DNA, complexed DNA, etc.) suitable for use in transposing and / or transporting information-coding proteins to host cells and / or specific locations and / or compartments within host cells. Examples of vectors include viral vectors and non-viral vectors, and non-episomal mammalian vectors. Vectors are often referred to as expression vectors (e.g., recombinant expression vectors and cloning vectors). A vector can be introduced into a host cell to enable replication of the vector itself, thereby amplifying copies of the polynucleotides it contains. Cloning vectors may, but are not limited to, include sequence components such as an origin of replication, promoter sequence, transcription start sequence, enhancer sequence, and selection marker. These elements may be selected as needed by those skilled in the art.

[0135] After construction, one or more vectors can be inserted into cells suitable for amplification and / or polypeptide expression. Transformation of selected cells with expression vectors can be achieved by well-known methods such as transfection, infection, co-precipitation with calcium phosphate, electroporation, nucleofection, microinjection, DEAE-dextran-mediated transfection, cationic lipid-mediated delivery, liposome-mediated transfection, microparticle guns, receptor-mediated gene delivery, and delivery mediated by polylysine, histones, chitosan, and peptides. The method selected depends to some extent on the type of host cell used. These methods and other suitable methods are well-known to those skilled in the art and are described in manuals and other technical publications, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001).

[0136] The term "transformation" refers to a change in the genetic characteristics of a cell. A cell is transformed if it has been modified to contain new DNA or RNA. For example, a cell is transformed if it has been genetically modified from its native state by introducing new genetic material through transfection, transduction, or other techniques. Following transfection or transduction, the transformed DNA may be incorporated into the cell's DNA by physically integrating it into the cell's chromosomes, or it may be temporarily maintained as an episomal element without replication, or it may be replicated independently as a plasmid. If the transformed DNA is replicated by cell division, the cell is considered "stablely transformed."

[0137] The term "transfection" refers to the uptake of exogenous or foreign 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, op. cit.; Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; and Chu et al., 1981, Gene 13:197.

[0138] The term "transduction" refers to the process by which foreign DNA is introduced into cells via a viral vector. See Jones et al., (1998). Genetics: principles and analysis. Boston: Jones & Bartlett Publ.

[0139] cell line The methods disclosed herein may use any mammalian cell line. A wide variety of mammalian cell lines suitable for growth in culture are available from the American Type Culture Collection (Manassas, Va.) and distributors. Examples of cell lines commonly used in this industry include: SV40-transformed monkey kidney CV1 cell line (COS-7, ATCC CRL 1651); human fetal kidney cell line (293 or 293 cells subcloned for growth in suspension culture (Graham et al., 1977, J. Gen Virol. 36:59)); baby hamster kidney cells (BHK, ATCC CCL 10); mouse Sertoli cells (TM4, Mather, 1980, Biol. Reprod. 23:243-251); monkey kidney cells (CV1 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); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocellular carcinoma cells (Hep G2, HB 8065); mouse mammary tumor cells (MMT 060562, ATCC CCL51); TRI cells (Mather et al., 1982, Annals NY Acad. Sci. 383:44-68); MRC5 cells or FS4 cells; mammalian myeloma cells, and many other cell lines, as well as Chinese hamster ovary (CHO) cells.

[0140] Large-scale production of proteins for commercial use is typically carried out in suspension culture. Therefore, mammalian host cells used to produce the recombinant mammalian cells described herein may, but do not need to, be adapted for growth in suspension culture. Various host cells adapted for growth in suspension culture are known, including mouse myeloma NS0 cells, as well as CHO cells derived from the CHO-S, DG44, and DXB11 cell lines. Other suitable cell lines include mouse myeloma SP2 / 0 cells, baby hamster kidney BHK-21 cells, human PER.C6® cells, human fetal kidney HEK-293 cells, and cell lines derived from or manipulated from any of the cell lines disclosed herein.

[0141] CHO cells are widely used to produce complex recombinant proteins, including CHOK1 cells (ATCC CCL61). Dihydrofolate reductase (DHFR)-deficient mutant cell lines (Urlaub et al., 1980, Proc Natl Acad Sci USA 77:4216-4220), DXB11, and DG-44 are desirable CHO host cell lines because high levels of recombinant protein expression are possible in these cells through efficient DHFR-selectable and amplified gene expression systems (Kaufman RJ, 1990, Meth Enzymol 185:537-566). Similarly, glutamine synthase (GS) knockout CHOK1SV cell lines utilize glutamine synthase (GS)-based methionine sulfoximine (MSX) selection. Other suitable CHO host cells include, but are not limited to, the following (ECACC accession numbers in parentheses): CHO (85050302), CHO (protein-free) (00102307), CHO-K1 (85051005), CHO-K1 / SF (93061607), CHO / DHFR- (94060607), CHO / DHFR-AC-free (05011002), and RR-CHOKI (92052129).

[0142] Cell culture process Host cells transfected with the vector systems described herein for adherent or suspension cultures grown in various other cell culture methods, including agitated tank reactors (which may or may not include spin filters, including conventional batch and fed-batch cell cultures), perfusion systems (including alternating tangential flow ("ATF") cultures, acoustic perfusion systems, depth filter perfusion systems, and other systems), hollow fiber bioreactors (HFB, which may optionally be used in the perfusion process), and various other cell culture methods (e.g., Tao et al., 2003, Biotechnol. Bioeng. 82:751-65; Kuystermans & Al-Rubeai, (2011) “Bioreactor Systems for Producing Antibody from Mammalian Cells” in Antibody Expression and Production, Cell Engineering 7:25-52, Al-Rubeai(ed) Springer; Catapano et al., (2009) “Bioreactor Design and See "Scale-Up" in Cell and Tissue Reaction Engineering; Principles and Practice, Eibl et al. (eds.) Springer-Verlag.

[0143] It is desirable to have a controlled system that allows cells to grow to a desired density while producing recombinant proteins, and then switches the physiological state of the cells to a high-productivity state where growth is stopped, allowing the cells to use energy and culture medium to produce the target recombinant protein instead of creating more cells. Various methods exist to achieve this objective, including temperature changes and amino acid deficiencies, as well as the use of cell cycle inhibitors or other molecules that can stop cell growth without causing cell death.

[0144] Recombinant protein production begins with establishing a mammalian cell production culture of protein-expressing cells in a culture plate, flask, tube, bioreactor, or other suitable container. Typically, smaller production bioreactors are used; in one embodiment, the bioreactor is 500 L to 2000 L. In another embodiment, a 1000 L to 2000 L bioreactor is used. The seed cell density used for seeding the bioreactor can have a positive effect on the level of recombinant protein produced. In one embodiment, at least 0.5 × 10⁶ cells are used in serum-free culture medium. 6 The following and 3.0 × 10 6 Seedling is performed in the bioreactor at a rate of more than 100 viable cells / mL. In another embodiment, seeding is performed at a rate of 1.0 × 10⁶ 6 This is the number of viable cells / mL.

[0145] Next, the mammalian cells undergo an exponential growth phase. The cell culture may be maintained without additional feeding until a desired cell density is achieved. In one embodiment, the cell culture is maintained for up to three days, with or without additional feeding. In another embodiment, the culture may be seeded at a desired cell density to initiate the production phase without a short growth phase. In any of the embodiments herein, the transition from the growth phase to the production phase may also be initiated by any of the methods described above.

[0146] To produce recombinant proteins through mammalian cell culture, three methods are typically used commercially: 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 increase in cell density until they reach maximum cell density, after which the viable cell density decreases as the culture medium components are consumed and metabolic by-products (such as lactate and ammonia) accumulate. Harvesting is typically performed when the maximum cell density is reached (e.g., 5 × 10⁶ depending on the culture medium formulation, cell line, etc.). 6(More than 100 cells / mL). The batch process is the simplest culture method, but the viable cell density is limited by nutrient availability, and the culture decreases and production declines once the cell density reaches its maximum. The production phase cannot be extended (typically around 3-7 days) because the culture rapidly decreases due to the accumulation of waste products and depletion of nutrients.

[0147] Fed-batch culture improves upon the batch process by providing a bolus or continuous medium feed to replenish consumed media components. Because fed-batch culture receives additional nutrients throughout its operation, it results in a higher cell density (10-30 × 10⁶ depending on the medium formulation, cell line, etc.) compared to the batch method. 6 It is possible to achieve higher cell density (over 100 cells / ml) and increased productivity. Unlike batch processes, by manipulating the feed method and medium formulation, a two-phase culture can be generated and maintained, and the cell proliferation phase (growth phase) to achieve the desired cell density can be distinguished from the cell growth arrest or stagnation phase (production phase). Therefore, compared to batch culture, fed-batch culture may achieve a higher productivity. Typically, the batch method is used during the growth phase and the fed-batch method is used during the production phase, but a fed-batch culture feed strategy can be used throughout the entire process. However, unlike the batch process, the volume of the bioreactor becomes a limiting factor, and the amount of feed is limited. Also, similar to the batch method, the culture decreases due to the accumulation of metabolic byproducts, which limits the duration of the production phase to approximately 10-21 days. Fed-batch culture is discontinuous, and harvesting is typically performed when the level of metabolic byproducts or the viability of the culture reaches a predetermined level. Compared to batch culture without feeding, fed-batch culture can produce a larger amount of recombinant protein. For example, see U.S. Patent No. 5,672,502.

[0148] Perfusion methods offer potential improvements compared to batch and feed-add methods by adding fresh medium and simultaneously removing used medium. In typical large-scale commercial cell culture systems, biomass accounts for approximately one-third to more than one-half of the reactor volume, resulting in 60-90(+) × 10⁻¹⁰ cells. 6 We aim to achieve a high cell density of 1 × 10⁶ cells / mL through perfusion culture. 8 Very high cell densities of over 100 cells / mL have been achieved, and even higher densities are predicted. Typical perfusion culture begins with initiating a batch culture lasting one or two days, and then, throughout the growth and production phases of the culture, fresh feed medium is continuously, stepwise, and / or intermittently added to the culture while retaining cells and further high molecular weight compounds, such as proteins (based on the molecular weight cutoff of the filter), and simultaneously removing the used medium. Various methods such as sedimentation, centrifugation, or filtration can be used to remove the used medium while maintaining cell density. Perfusion flow rates ranging from a small working volume per day to many multiple working volumes per day have been reported.

[0149] The advantage of the perfusion process is that it allows for the maintenance of productive cultures for longer periods compared to batch or fed-batch cultures. However, supporting long-term perfusion culture, especially at high cell densities, requires the preparation, use, storage, and disposal of more culture medium, as well as more nutrients, all of which further increase production costs compared to batch and fed-batch methods. In addition, high cell densities can lead to problems during production, such as maintaining dissolved oxygen levels, and problems associated with increased gas treatment, including supplying more oxygen and removing more carbon dioxide. This can result in more foaming, necessitating changes to the defoaming method. Similarly, during recovery and downstream processes, the effort required to remove excess cellular material can lead to product loss, which can negate the advantage of increased titer due to increased cell volume.

[0150] Also offered is a large-scale cell culture method that combines fed-boil feeding during the growth phase with continuous perfusion during the production phase. This method is intended for the production phase, maintaining the cell culture at a filled cell volume of 35% or less.

[0151] In one embodiment, fed-batch culture with a bolus feed is used to maintain cell culture during the proliferation phase. Perfusion feed may then be used during the production phase. In one embodiment, perfusion is started when the cells reach the production phase. In another embodiment, perfusion is started approximately 3 to 9 days into the cell culture. In yet another embodiment, perfusion is started approximately 5 to 7 days into the cell culture.

[0152] During the proliferation phase, using a bolus feed allows cells to transition to the production phase, resulting in reduced reliance on temperature changes as a means of initiating and controlling the production phase, although a temperature change of approximately 36°C to 31°C can occur between the proliferation and production phases. In one embodiment, this change is from 36°C to 32°C.

[0153] As described herein, at least 0.5 × 10⁶ in serum-free culture medium 6 From 3.0 × 10⁶ viable cells / mL 6 More than 100 viable cells / mL, for example, 1.0 × 10⁶ 6 The cells can be seeded into the bioreactor at a rate of 100 viable cells / mL.

[0154] In perfusion culture, fresh perfusion feed medium is supplied to the cell culture while used medium is removed simultaneously. Perfusion can be continuous, stepwise, intermittent, or a combination of any or all of these. The perfusion rate can range from less than a working volume per day to a large working volume. Cells are retained in the culture, and the removed used medium contains virtually no cells, or only a very small number of cells compared to the culture. Recombinant proteins expressed by the cell culture may also be retained in the culture. Perfusion can be achieved by many means, including centrifugation, sedimentation, or filtration. See, for example, Voisard et al., 2003, Biotechnology and Bioengineering 82:751-65. One 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. For example, see U.S. Patent No. 6,544,424; Furey, 2002, Gen.Eng.News.22(7):62-63.

[0155] "Perfusion flow rate" refers to the amount of culture medium that passes through (is added to and removed from) the bioreactor, and is typically expressed as a fraction of a working volume within a given time. "Working volume" refers to the amount of bioreactor volume used for cell culture. In one embodiment, the perfusion flow rate is less than or equal to one working volume per day. The perfusion feed medium may be formulated to maximize the perfusion nutrient concentration and minimize the perfusion rate.

[0156] Cell cultures can be supplemented with concentrated feed medium containing nutrients and components such as amino acids that are consumed during the production phase of the cell culture.

[0157] Concentrated feed media can be based on almost any cell culture medium formulation. Such concentrated feed media may contain most of the components of the cell culture medium at, for example, about 5, 6, 7, 8, 9, 10, 12, 14, 16, 20, 30, 50, 100, 200, 400, 600, 800, or about 1000 times the normal amounts. Concentrated feed media are often used in fed-batch culture processes.

[0158] Samples from cell cultures can be monitored and evaluated using any analytical technique known in the art. Various parameters, including the quality and characteristics of recombinant proteins and culture media, can be monitored during the culture period. Samples can be intermittently collected and monitored at any desired frequency, including continuous monitoring, real-time or near real-time.

[0159] Typically, cell cultures preceding the final production culture (Nx~N-1) are used to generate seed cells, N-1 cultures, which are used to seed into the production bioreactor. Seed cell density can positively influence the level of recombinant protein produced. Production levels tend to increase as seed density increases. The increase in titer is not only linked to higher seed density, but can also affect the metabolic and cell cycle state of the cells fed into production.

[0160] Seed cells can be prepared by any culture method. One such method is perfusion culture using alternating tangential flow filtration. Alternating tangential flow filtration can be used to operate an N-1 bioreactor and provide a high density of cells for seeding into a production bioreactor. Using the N-1 step, cells can be prepared to >90 × 10⁶ 6Cells can be grown to densities exceeding 15 × 10⁶ cells / mL. The N-1 bioreactor can be used to generate bolus seed cultures or as a rotating seed stock culture that may be maintained for seeding at high seed cell densities into multiple production bioreactors. The product growth phase can range from 7 to 14 days and may be designed to maintain cells in an exponential growth state before seeding in the production bioreactor. Perfusion rate, medium composition, and timing are optimized to deliver cells to the production bioreactor in a state that best facilitates production optimization. Seed cell density of 15 × 10⁶ 6 By achieving a cell density exceeding 100 cells / mL, seeding into the production bioreactor can be accomplished. Increasing the seed cell density at the time of seeding can shorten or reduce the time required to reach the desired production density.

[0161] In certain embodiments, mammalian host cells can be used to produce high-yield target proteins. High-yield or high-volume productivity refers to the ability of cells to produce high levels of the target protein. The specific yield depends 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 more, in a 10-day culture grown in a feed medium suitable for mammalian host cells and under fed-add or perfusion conditions containing amino acids, vitamins, or trace elements. In certain embodiments, the host cells and methods of the present disclosure, when expressing the protein of interest and growing under the culture conditions described above, 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.

[0162] Yield can also be measured in terms of the specific productivity of the cell line, and is determined based on the amount of protein produced by one cell per day (expressed as pg / cell / day). The mammalian host cells of this disclosure 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 a culture at day 10 grown using a feed medium suitable for mammalian host cells and under fed-add or perfusion conditions containing amino acids, vitamins, or trace elements. In certain embodiments, the mammalian host cells of the Disclosure express the protein of interest and have a specific 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 more, preferably up to 50 pg / cell / day, under the culture conditions described above.

[0163] The desired protein may be expressed using mammalian host cells as described herein. The expressed protein may be secreted into the culture medium, from which it may be recovered and / or collected. Furthermore, the protein may be purified or partially purified from such culture or components (e.g., from the culture medium) using known processes and products available from distributors. The purified protein may then be “formulated,” meaning buffer exchange, sterilization, bulk packaging, and / or packaging for the end user. Suitable formulations for pharmaceutical compositions are described in Remington's Pharmaceutical Sciences, 18th ed. 1995, Mack Publishing Company, Easton, Pa.

[0164] Various known techniques can be used to produce polynucleotides, polypeptides, vectors, host cells, immune cells, compositions, etc., according to the present invention.

[0165] The present invention is intended as a single description of individual aspects of the invention and is not limited in scope by the specific embodiments described herein; functionally equivalent methods and components are within the scope of the invention. In fact, in addition to those shown and described herein, various modifications of the invention will be apparent to those skilled in the art from the above and the accompanying drawings. Such modifications are intended to be included within the scope of the appended claims. [Examples]

[0166] Materials and methods Plasmid transfection and pool recovery 20 μg of a dedicated plasmid containing the target gene (GOI) and 5 μg of ILT piggyback transposase (see International Publication No. 2020123327) were suspended in SAFC® PF-CHO medium (Ex-Cell® 325 PF-CHO serum-free medium, catalog number 14340C, Millipore Sigma, St. Louis, MO) using a GenePulser Xcell® electroporator (Bio-Rad Laboratories, Inc., Hercules, CA) and 20 × 10⁻¹⁶ of the sample. 6 Glutamine synthetase knockout (GSKO) Chinese hamster ovary (CHO) cells were transfected. Immediately after transfection, the transfected CHO cells were cultured in 20 ml of chemically defined host cell medium. Three days after transfection, chemically defined glutamine-negative medium supplemented with 12.5 μM or 25 μM L-methionine sulfoximine (MSX, Millipore Sigma, St. Louis, MO) was used as the selective medium. The transfected pool was passaged every 3 or 4 days until the viability reached 85% or higher. Viability and cell density were measured using a Vi-CELL® BLU cell viability analyzer (Beckman Coulter, Indianapolis, IN). Once the viability exceeded 85%, the culture was further passaged for 5 × 10⁶ cells over two more passages until the viability and doubling time stabilized. 5 Cells were seeded at a rate of 1 / mL.

[0167] Ryuka The pool was evaluated and ranked under production conditions using a 15-day fed-batch (FB) cell culture process. N-1 cultures were placed in 10 ml of glutamine-negative host medium without MSX, with 5 × 10⁶ cells per 10 ml. 5The cells were seeded at a rate of 100 cells / ml. After 4 days, the produced culture was added to 10 ml of glutamine-negative culture medium without MSX and Gibco® Anti-Clumping Agent (Thermo Fisher Scientific Inc., Waltham, MA) at a ratio of 1:750. 6 Cells were seeded at a rate of 100 cells / ml. Production culture was carried out until day 15, as long as the viability was above 50%. The culture was counted on days 3, 6, 8, 10, 13, and 15 using Vi-CELL® BLU (Beckman Coulter Inc.), and the viable cell count and viability were recorded. Glucose levels in the culture medium were measured on days 3, 6, 8, 10, and 13 using BioProfile® Flex2 Automated Cell Culture Analyzer (Nova Biomedical, Waltham, MA). On days 3, 6, 8, 10, and 13, the production culture was supplied with a 5v / v% dedicated medium supplemented with 0.2v / v% tyrosine-cystine and glucose. After glucose measurement, on days 3, 6, 8, 10, and 13, glucose levels were adjusted to 12-14 g / L with a 50% glucose stock solution according to glucose consumption. 200 μl of the collected cell culture medium (HCCF) was subjected to titer measurement on days 10, 13, and 15 to evaluate productivity.

[0168] On day 15, the cell cultures were centrifuged at 2000 rpm for 10 minutes, and the HCCF was subjected to ATOLL (protein A-HPLC), size exclusion chromatography (SEC), reductive capillary electrophoresis (rCE), and non-reductive capillary electrophoresis (nrCE) to evaluate product quality. Titer and product quality data were visualized using TIBCO Spotfire® software (Cloud Software Group, Inc.). Only data from cultures with a viability of over 60% were used for analysis. The values ​​on the graph represent the average of two repeated cultures. The magnification change was calculated for both the 12.5 μM MSX condition and the 25 μM MSX condition. The magnification change shown in the graph is the average magnification change between the 12.5 μM and 25 μM MSX conditions.

[0169] Example 1 As part of our vector screening efforts to improve the productivity and product quality of triple-stranded and quadruple-stranded molecules (e.g., triple-stranded molecules with heavy chains fused to cytokines, and hetero-IgG quadruple-stranded molecules) in the GSKO host, we tested various combinations of promoters.

[0170] We utilized a two-vector system to drive the expression of glutamine synthetase as a selection marker, using various combinations of the mPGK promoter and the Srα promoter. In this experiment, we generated a triple-chain molecule with a heavy chain fused to the cytokine, where the first vector encoded the light and heavy chains, and the second vector encoded the light and heavy chain-peptide fusion. Figure 1 shows a schematic of the various vectors used in this experiment.

[0171] The vector was co-transfected into CHO cells, and the cells were cultured as described above. The titer and product quality were determined as described above.

[0172] Pools using an mPGK promoter for the GS selection marker on one vector and a Srα promoter for the other vector showed higher titer and product quality compared to pools using either the mPGK or Srα promoter to drive GS expression in both vectors. See Figure 2. The improvement in titer and product quality was most evident when Srα was present on a vector containing an expression-restricting chain (a chain that is more difficult to express). For triple-stranded molecules, compared to the mPGK / mPGK and Srα / Srα pools, the mPGK / Srα pool showed up to a 5.3-fold increase in effective titer (nrCE) and up to a 2.6-fold improvement in product quality (nrCE main peak %) (Figure 2). In particular, the mPGK / Srα combination, a mixed vector containing Srα on a vector with a more difficult-to-express mAb fusion, helped to increase the expression of the mAb fusion by up to 2.4-fold, resulting in an increased mAb fusion-to-heavy chain ratio, more balanced chain expression, and improved product quality.

[0173] Example 2 As part of our vector screening efforts to improve the productivity and product quality of triple-stranded and quadruple-stranded molecules (e.g., triple-stranded molecules with heavy chains fused to cytokines, and hetero-IgG quadruple-stranded molecules) in the GSKO host, we tested various combinations of promoters.

[0174] We utilized a two-vector system to drive the expression of glutamine synthetase as a select marker, using various combinations of the mPGK promoter and the Srα promoter. In this experiment, hetero-IgG was generated, where the first vector encoded the light and heavy chains, and the second vector encoded the light and heavy chain-protein fusion.

[0175] To select vectors containing Srα-GS and to favor the expression of more difficult-to-express chains, 25 μM MSX was used during cell proliferation. Srα is a stronger promoter compared to mPGK, and therefore, selection with a Srα vector will be easier to overcome than selection with an mPGK vector at a higher 25 μM MSX concentration. Chain expression can be balanced by placing difficult-to-express chains in less stringent vectors and easier-to-express chains in more stringent selection.

[0176] Regarding heteroIgG, we observed that co-transfection of vectors with two different GS-selective promoters (mPGK for LC-HC, which is easier to express, and Srα for LC-HC, which is more difficult to express) resulted in a 1.2-fold improvement in titer in the corresponding Srα / Srα vector while minimizing the impact on product quality (Figure 3).

[0177] Overall, these results suggest that promoters for selective marker expression can also be used as vector design levers to create a high-quality pool for similar triple-stranded and quadruple-stranded molecules, potentially improving process efficiency and reducing future manufacturing costs.

Claims

1. Mammalian host cells for expressing an antigen-binding protein having three or four different chains, including a first expression vector and a second expression vector, (a) The first expression vector comprises: 1) a nucleotide sequence encoding a first strand and a second strand, wherein the first strand is operably linked to a first promoter, and the second strand is operably linked to a second promoter or an IRES sequence, or linked to the first strand via a linker sequence; and 2) a nucleotide sequence encoding a first selection marker operably linked to a third promoter. (b) The second expression vector comprises: 1) a nucleotide sequence encoding a third and a fourth chain, wherein the third chain is operably linked to a fourth promoter, and the fourth chain is operably linked to a fifth promoter or an IRES sequence, or linked to the third chain via a linker sequence; and 2) a nucleotide sequence encoding a second select marker operably linked to a sixth promoter. The first, second, third, and fourth chains are selected from the group consisting of heavy chains, light chains, antibody heavy chain fusions, antibody light chain fusions, ScFv, and ScFv-Fc, and two of the chains may be identical. The sixth promoter is different from the third promoter. Mammalian host cells.

2. The mammalian host cell according to claim 1, wherein the second and fourth strands are operably connected to the second and fourth promoters, respectively.

3. The mammalian host cell according to claim 1, wherein the first selection marker and the second selection marker are the same.

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

5. The mammalian host cell according to claim 4, wherein the first and second selection markers are glutamine synthetases.

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

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

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

9. The mammalian host cell according to claim 7, wherein the first and fourth promoters are identical, and the second and fifth promoters are identical.

10. The mammalian host cell according to claim 9, wherein the first, second, fourth, and fifth promoters are identical.

11. The mammalian host cell according to claim 9, wherein the first and fourth promoters are different from the second and fifth promoters.

12. The mammalian host cell according to claim 1, wherein the more potent promoter among the third promoter and the sixth promoter is present on the expression vector having a chain that is less expressible.

13. The mammalian host cell according to claim 12, wherein Srα is present on the expression vector having the chain that is more difficult to express, and mPGK is present on the expression vector having the chain that is more easily expressed.

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

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

16. The mammalian host cell according to claim 1, wherein the more potent promoter among the third promoter and the sixth promoter is present on the expression vector having the chain that facilitates expression.

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

18. The mammalian host cell according to claim 17, wherein the first and second chains are identical antibody light chains.

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

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

21. The mammalian host cell according to claim 20, wherein the first and second chains are identical antibody light chains.

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

23. The mammalian host cell according to claim 1, wherein the antigen-binding protein having three or four chains is selected from heteroIgG and C1mAb.

24. The first expression vector comprises, in order from 5' to 3', 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 which is Srα, and a nucleotide sequence encoding a select marker which is glutamine synthetase. The second expression vector comprises, in order from 5' to 3', a fourth promoter, a nucleotide sequence encoding the second antibody light chain, a fifth promoter or IRES, a nucleotide sequence encoding the second heavy chain, a sixth promoter which is mPGK, and a nucleotide sequence encoding a selection marker which is glutamine synthetase. A mammalian host cell according to claim 1.

25. The mammalian host cell according to claim 24, wherein the first antibody light chain and the second antibody light chain are the same.

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

27. The mammalian host cell according to claim 1, wherein the first expression vector comprises, in order from 5' to 3', 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 which is Srα, and a nucleotide sequence encoding a select marker which is glutamine synthetase; and the second expression vector comprises, in order from 5' to 3', 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 which is mPGK, and a nucleotide sequence encoding a select marker which is glutamine synthetase.

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

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

30. The mammalian host cell according to claim 1, wherein the first expression vector comprises, in order from 5' to 3', 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 which is Srα, and a nucleotide sequence encoding a select marker which is glutamine synthetase; and the second expression vector comprises, in order from 5' to 3', 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 which is mPGK, and a nucleotide sequence encoding a select marker which is glutamine synthetase.

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

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

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

34. The mammalian host cell according to claim 1, wherein the first and second expression vectors are incorporated into the genome of the host cell.

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

36. The aforementioned CHO cells lack dihydrofolate reductase (DHFR). － ) or glutamine synthetase knockout (GSKO) according to claim 35.

37. A method for producing an antigen-binding protein having three or four chains, a) culturing the mammalian host cells described in claim 1 under conditions for expressing the antigen-binding protein; and b) Recovering the antigen-binding protein. A method that includes this.

38. The method according to claim 37, comprising purifying the recovered antigen-binding protein and formulating it into a pharmaceutically acceptable formulation.

39. A method for producing an antigen-binding protein having three or four chains, a) Culturing the mammalian host cells according to claim 36 under conditions that express the antigen-binding protein, and under methotrexate stringing in the case of CHO DHFR- cells, or under methionine sulfoxamine stringing in the case of CHO GSKO cells, in such a manner that favors the expression of a chain that is difficult to express and pairs with a stronger GS promoter; and b) Recovering the antigen-binding protein. A method that includes this.

40. The method according to claim 39, comprising purifying the recovered antigen-binding protein and formulating it into a pharmaceutically acceptable formulation.

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