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WO2025155774A2 - Vector engineering strategies to enhance volumetric productivity and decrease impurity formation across a diverse set of modalities - Google Patents

Vector engineering strategies to enhance volumetric productivity and decrease impurity formation across a diverse set of modalities

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Publication number
WO2025155774A2
WO2025155774A2 PCT/US2025/011954 US2025011954W WO2025155774A2 WO 2025155774 A2 WO2025155774 A2 WO 2025155774A2 US 2025011954 W US2025011954 W US 2025011954W WO 2025155774 A2 WO2025155774 A2 WO 2025155774A2
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Prior art keywords
antibody
heavy chain
expression
nucleotide sequence
fusion
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French (fr)
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WO2025155774A3 (en
Inventor
Kristine M. DARIS
Noelia BLANCO TALAVAN
Natalia GOMEZ
Marissa Mock
Fuyi CHEN
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Amgen Inc
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Amgen Inc
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Publication of WO2025155774A2 publication Critical patent/WO2025155774A2/en
Publication of WO2025155774A3 publication Critical patent/WO2025155774A3/en
Pending legal-status Critical Current
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
    • C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/40—Systems of functionally co-operating vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/42—Vector systems having a special element relevant for transcription being an intron or intervening sequence for splicing and/or stability of RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/50—Vector systems having a special element relevant for transcription regulating RNA stability, not being an intron, e.g. poly A signal

Definitions

  • the present disclosure provides expression cassettes comprising a polynucleotide sequence which comprises the following elements in 5’ to 3’ order: 1) a first copy of a GAPDH promoter operably linked to a nucleotide sequence encoding a first antibody chain or antibody chain fusion followed by a first polyA signal sequence; 2) a second copy of the GAPDH promoter operably linked to a nucleotide sequence encoding a second antibody chain or antibody chain fusion followed by a second polyA signal sequence; and 3) a promoter operably linked to a nucleotide sequence encoding a selectable marker followed by a third polyA signal sequence, wherein, when the first antibody chain or antibody chain fusion is an antibody light chain, the second antibody chain or antibody chain fusion is an antibody heavy chain or antibody heavy chain fusion, wherein, when the first antibody chain or antibody chain fusion is an antibody heavy chain or antibody heavy chain fusion, the second antibody chain or antibody chain fusion is an antibody light chain, and wherein the antibody heavy chain
  • the first antibody chain or antibody chain fusion is an antibody light chain
  • the second antibody chain or antibody chain fusion is an antibody heavy chain or antibody heavy chain fusion.
  • the antibody heavy chain fusion is a heavy chain-scFv.
  • the antibody heavy chain fusion is a heavy chain-cytokine.
  • the antibody heavy chain fusion is a heavy chain VH.
  • the first antibody chain or antibody chain fusion is an antibody heavy chain or antibody heavy chain fusion
  • the second antibody chain or antibody chain fusion is an antibody light chain.
  • the antibody heavy chain fusion is a heavy chain-scFv.
  • the antibody heavy chain fusion is a heavy chain-cytokine.
  • the antibody heavy chain fusion is a heavy chain VH.
  • the first GAPDH promoter is operably linked to a nucleotide sequence encoding an antibody light chain and the second GAPDH promoter is operably linked to an antibody heavy chain or antibody heavy chain fusion. In certain embodiments, the first GAPDH promoter is operably linked to a nucleotide sequence encoding an antibody light chain and the second GAPDH promoter is operably linked to an antibody heavy chain. In certain embodiments, the first GAPDH promoter is operably linked to a nucleotide sequence encoding an antibody light chain and the second GAPDH promoter is operably linked to an antibody heavy chain fusion.
  • the antibody heavy chain fusion is a heavy chain-scFv.
  • the antibody heavy chain fusion is a direct fusion of a heavy chain with a VH. In some embodiments, the antibody heavy chain fusion is a direct fusion of a heavy chain with a scFv. In some embodiments, the antibody heavy chain fusion is a direct fusion of a heavy chain with a cytokine.
  • the antibody heavy chain fusion is a fusion of a heavy chain with a VH, wherein the fusion comprises a linker between the heavy chain and the VH.
  • the antibody heavy chain fusion is a fusion of a heavy chain with a scFv, wherein the fusion comprises a linker between the heavy chain and the scFv.
  • the antibody heavy chain fusion is a fusion of a heavy chain with a cytokine, wherein the fusion comprises a linker between the heavy chain and the cytokine.
  • the antibody heavy chain fusion comprises a VH, scFv, or cytokine fused to the C-terminus of the heavy chain portion of the heavy chain fusion. In some embodiments, the antibody heavy chain fusion comprises a VH fused to the C-terminus of the heavy chain portion of the heavy chain fusion. In some embodiments, the antibody heavy chain fusion comprises a scFv fused to the C-terminus of the heavy chain portion of the heavy chain fusion. In some embodiments, the antibody heavy chain fusion comprises a cytokine fused to the C-terminus of the heavy chain portion of the heavy chain fusion.
  • the antibody heavy chain fusion comprises a VH, scFv, or cytokine fused between the CHI and CH2 of the heavy chain portion of the heavy chain fusion. In some embodiments, the antibody heavy chain fusion comprises a VH fused between the CHI and CH2 of the heavy chain portion of the heavy chain fusion. In some embodiments, the antibody heavy chain fusion comprises a scFv fused between the CHI and CH2 of the heavy chain portion of the heavy chain fusion. In some embodiments, the antibody heavy chain fusion comprises a cytokine fused between the CHI and CH2 of the heavy chain portion of the heavy chain fusion.
  • the selectable marker is selected from the group consisting of glutamine synthetase and dihydrofolate reductase. In certain embodiments, the selectable marker is glutamine synthetase. In certain embodiments, the selectable marker is dihydrofolate reductase.
  • the promoter for the chains of the antibody modality is a combination of the CMV promoter enhancer and GAPDH (CMV/GAPDH).
  • CMV/GAPDH is referred to as a promoter.
  • a representative CMV/GAPDH promoter is provided in SEQ ID NO: 1.
  • both the CMV promoter enhancer and GAPDH promoter are operably linked to the nucleotide sequence encoding the antibody chain, such that the combination may be a better promoter than GAPDH alone.
  • the CMV promoter is 5’ of the GAPDH.
  • the CMV/GAPDH promoter comprises a nucleotide sequence of SEQ ID NO: 1.
  • the promoter operably linked to the nucleotide sequence encoding the selectable marker is selected from the group consisting of mPGK, SRoc, and SV40 promoters. In some embodiments, the promoter operably linked to the nucleotide sequence encoding the selectable marker is a mPGK promoter. In some embodiments, the promoter operably linked to the nucleotide sequence encoding the selectable marker is a SRoc promoter. In some embodiments, the promoter operably linked to the nucleotide sequence encoding the selectable marker is a SV40 promoter.
  • the selectable marker on the first expression vector is glutamine synthetase. In some embodiments, the selectable marker on the second expression vector is glutamine synthetase. In some embodiments, the selectable marker on the first expression vector and the selectable marker on the second expression vector is glutamine synthetase.
  • the promoter operably linked to a nucleotide sequence encoding a selectable marker on the first expression vector is mPGK
  • the promoter operably linked to a nucleotide sequence encoding a selectable marker on the second expression vector is mPGK
  • the selectable marker on the first expression vector and the selectable marker on the second expression vector is glutamine synthetase
  • each of the first, second, and third polyA signal sequences on the first expression vector is a simian virus 40 (SV40) early polyA signal sequence.
  • each of the first, second, and third polyA signal sequences on the second expression vector is a simian virus 40 (SV40) early polyA signal sequence.
  • each of the first, second, and third polyA signal sequences on the first expression vector is a simian virus 40 (SV40) early polyA signal sequence
  • each of the first, second, and third polyA signal sequences on the second expression vector is a simian virus 40 (SV40) early polyA signal sequence.
  • the nucleotide sequence encoding the first antibody heavy chain may precede the nucleotide sequence encoding the first antibody light chain (in 5’ to 3’ order). Additionally, in alternative embodiments, on the second expression vector, the nucleotide sequence encoding the second antibody heavy chain may precede the nucleotide sequence encoding the second antibody light chain (in 5’ to 3’ order).
  • the present disclosure also provides methods for producing an antibody modality comprising culturing any of the mammalian host cells described above or herein under conditions in which the antibody chains are expressed, and recovering the antibody modality from the culture.
  • the recovered antibody modality is purified and formulated in a pharmaceutically acceptable formulation.
  • a “pharmaceutically acceptable formulation” refers to a formulation that is generally safe, non-toxic, and neither biologically nor otherwise undesirable for use in a subject.
  • FIG. 1 is an example of plasmid map for recombinant expression of an antibody molecule having one light chain and one heavy chain.
  • FIGs. 2A-2B show Pool Titer in g/L (A) and Cell Specific Productivity (qp) in pg/cell/day (B) from each light chain (LC), heavy chain (heavy chain fusion) (HC) and glutamine synthetase (GS) promoter and MSX combination. Titer was measured on day 10 of a fed-batch production. Data is normalized and presented as mean values for two independent transfections. Error bars represent the standard deviation (SD) of two technical replicates for fed-batch production. Data is shaded based on MSX concentrations (0 pM, 12.5 pM and 25 pM).
  • FIGs. 17A-17B show Pool Titer in g/L (A) and Cell Specific Productivity in pg/cell/day (B) from each light chain (LC)/heavy chain (HC) promoter combination and glutamine synthetase (GS) promoter and MSX combination for a B2Hmab and a C2Hmab. Titer was measured on day 10 of a fed-batch production. Data is normalized and presented as mean values for two independent transfections. Error bars represent the standard deviation (SD) of three technical replicates for fed- batch production.
  • SD standard deviation
  • FIGs. 18A-18C show Product quality (A) % main Peak in SEC, (B) % main peak in nonreduced capillary electrophoresis (nrMCE), and (C) B: rCE-Clipping; in cell culture of CHO cell pools from the different vector configurations for a B2Hmab and a C2Hmab. Data is normalized and presented as mean values for two independent transfections.
  • antibody modality refers to a protein having at least one antibody chain.
  • An antibody modality can have two, three, or four unique chains, wherein any or all of the chains can include fusions.
  • Reference to two, three, or four chain molecules implies that each chain is unique. Any and all of the antibody chain containing molecules described herein are considered antibody modalities.
  • polypeptide and protein are used interchangeably herein to refer to a polymer of amino acid residues.
  • the terms also apply to amino acid polymers in which one or more amino acid residues is an analog or mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
  • the terms can also encompass amino acid polymers that have been modified, e.g., by the addition of carbohydrate residues to form glycoproteins, or phosphorylated.
  • the internal conditions of the bioreactor including, but not limited to pH and temperature, can be controlled during the culturing period.
  • suitable bioreactors for use in practicing the methods disclosed herein based on the relevant considerations.
  • the nutrient solution may optionally be supplemented with additional optional components to optimize growth of cells, such as hormones and other growth factors, e.g., transferrin, epidermal growth factor, insulin, insulin-like growth factor, serum, and the like; salts, e.g., calcium, magnesium and phosphate, and buffers, e.g., HEPES; nucleosides, and bases, e.g., adenosine, thymidine, hypoxanthine; and protein and tissue hydrolysates, e.g., hydrolyzed animal or plant protein (peptone or peptone mixtures, which can be obtained from animal byproducts, purified gelatin or plant material); antibiotics, e.g., gentamycin; anti-clumping agents; cell protectants or surfactants such as Pluronic®F68 (also referred to as Lutrol® F68 and Kolliphor® P188; nonionic triblock composed of a central hydrophobic chain of polyoxy
  • a “base” (or batch) cell culture medium refers to a cell culture medium that is typically used to initiate a cell culture and is sufficiently complete to support the cell culture.
  • a “growth” cell culture medium refers to a cell culture medium that is typically used in cell cultures during a period of exponential growth, a “growth phase,” and is sufficiently complete to support the cell culture during this phase.
  • a growth cell culture medium may also contain selection agents that confer resistance or survival to selectable markers incorporated into the host cell line. Such selection agents include, but are not limited to, geneticin (G418), neomycin, hygromycin B, puromycin, zeocin, methionine sulfoximine, methotrexate, glutamine-free cell culture medium, cell culture medium lacking glycine, hypoxanthine and thymidine, or thymidine alone.
  • a “perfusion” cell culture medium refers to a cell culture medium that is typically used in cell cultures that are maintained by perfusion or continuous culture methods and is sufficiently complete to support the cell culture during this process.
  • Perfusion cell culture medium formulations may be richer or more concentrated than base cell culture medium formulations to accommodate the method used to remove the spent medium.
  • Perfusion cell culture medium can be used during both the growth and production phases.
  • a “production” cell culture medium refers to a cell culture medium that is typically used in cell cultures during the transition when exponential growth is ending and protein production takes over, “transition” and/or “product” phases, and is sufficiently complete to maintain a desired cell density, viability and/or product titer during this phase.
  • Cell cultures can also be supplemented with independent concentrated feeds of particular nutrients which may be difficult to formulate or are quickly depleted in cell cultures.
  • Such nutrients may be amino acids such as tyrosine, cysteine and/or cystine (see e.g., International Patent Application Publication No. WO2012/145682).
  • the independent feeds can begin prior to or at the start of the production phase.
  • the independent feeds can be accomplished by fed batch to the cell culture medium on the same or different days as the concentrated feed medium.
  • the independent feeds can also be perfused on the same or different days as the perfused medium.
  • tissue culture media that does not contain animal sera, such as fetal bovine serum.
  • tissue culture media including defined culture media, are commercially available, for example, any one or a combination of the following cell culture media can be used: RPMI-1640 Medium, RPMI-1641 Medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium Eagle, F-12K Medium, Ham's F12 Medium, Iscove's Modified Dulbecco's Medium, McCoy's 5 A Medium, Leibovitz's L-15 Medium, and serum -free media such as EX-CELLTM 300 Series (JRH Biosciences, Lenexa, Kansas), MCDB 302 (Sigma Aldrich Corp., St.
  • Titer means the total amount of a polypeptide or protein of interest (which may be a naturally occurring or recombinant protein of interest) produced by a cell culture in a given amount of medium volume. Titer can be expressed in units of milligrams or micrograms of polypeptide or protein per milliliter (or other measure of volume) of medium. “Cumulative titer” is the titer produced by the cells during the course of the culture, and can be determined, for example, by measuring daily titers and using those values to calculate the cumulative titer.
  • the term “host cell” is understood to include a cell that has been genetically engineered to express a polypeptide of interest. Genetically engineering a cell involves transfecting, transforming, or transducing the cell with a nucleic acid encoding a recombinant polynucleotide molecule (a “gene of interest”), and/or otherwise altering (e.g., by homologous recombination and gene activation or fusion of a recombinant cell with a non-recombinant cell) so as to cause the host cell to express a desired recombinant polypeptide.
  • a recombinant polynucleotide molecule e.g., a recombinant polynucleotide molecule
  • Expression and cloning will typically include expression cassettes that contain one or more promoters that are recognized by the host organism and are operably linked to the nucleotide sequence encoding a protein of interest. Promoters are untranscribed sequences located upstream (i.e., 5') to the start codon of a structural gene (generally within about 100 to 1000 bp) that control transcription of the structural gene.
  • An expression cassette for an antibody modality will generally contain a first promoter driving expression of a first nucleotide sequence encoding a first antibody chain or antibody chain fusion, a second promoter driving expression of a second nucleotide sequence encoding a second antibody chain or antibody chain fusion, and a promoter driving expression of a coding sequence encoding a selectable marker.
  • a polyA tail can follow each gene (i.e., the coding sequences encoding the first and second antibody or antibody fusion chains and the selectable marker).
  • the first promoter and the second promoter are both glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoters.
  • the GAPDH promoter is operably linked to the CMV promoter enhancer; the resulting construct referred to as a CMV/GADPH promoter.
  • a typical antibody is a Y-shaped molecule having four polypeptide chains - two identical heavy chains and two identical light chains. Such an antibody is preferably expressed from a single vector.
  • bispecific antibodies require the use of alternative formats and can be expressed on a single vector or two different vectors depending on the number of chains being produced. See, e.g., Spiess et al., 2015, Mol. Immunol. 67:95-106; Brinkmann et al., 2017, MAbs 9: 192-212; and Ma et al., 2021, Frontiers in Immunology 12:626616.
  • a single expression cassette is used, i.e., 2 chains, 1 vector.
  • the expression cassette is useful for the expression of a monoclonal antibody (having one heavy chain sequence and one light chain sequence) or a symmetrical fusion (for example, having a heavy chain sequence fused to a scFv or VH and a light chain sequence), such as a B2 mab (Fab- scFv-Fc) or C2 mab (IgG-scFv) mAb, B2Hmab (Fab-VH-Fc), C2Hmab (IgG-VH) or (IgG-cytokine) mAb (2 chains, 1 vector).
  • a monoclonal antibody having one heavy chain sequence and one light chain sequence
  • a symmetrical fusion for example, having a heavy chain sequence fused to a scFv or VH and a light chain sequence
  • a B2 mab Fab- scFv-Fc
  • C2 mab IgG-scFv
  • B2Hmab Fab-
  • FIG. 20A A representative scheme for a mAb is depicted in FIG. 20A.
  • FIG. 20B A representative scheme for a B2 mab or C2 mAb is depicted in FIG. 20B.
  • the first antibody chain is each expression cassette can be either the light chain or the heavy chain/heavy chain-scFv fusion.
  • the first antibody chain is the light chain and the second antibody chain is either a heavy chain or a heavy chain-scFv fusion.
  • Another example is a ClmAb (Fab-heteroFc-[cytokine], asymmetric fusion).
  • FIG. 20C A representative scheme for a ClmAb, asymmetric fusion is depicted in FIG. 20C.
  • FIG. 20D A representative scheme for a heteroIgG mAb is depicted in FIG. 20D.
  • Promoters of particular interest for 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) and hamster glyceraldehyde-3-phosphate dehydrogenase promoter and intron (GAPDH) (U.S. Patent No. 10,202,261). Additional sequences can also be combined with promoters to improve expression.
  • CMV human cytomegalovirus IE1 gene promoter enhancer
  • GPDH hamster glyceraldehyde-3-phosphate dehydrogenase promoter and intron
  • Additional sequences can also be combined with promoters to improve expression.
  • ADL adenovirus tripartite leader
  • the promoter for the chains of the antibody modality is the combination of the CMV promoter enhancer and GAPDH (CMV/GAPDH).
  • CMV/GAPDH CMV promoter enhancer and GAPDH
  • both the CMV promoter enhancer and GAPDH promoter are operably linked to the nucleotide sequence encoding the antibody chain, such that the combination may be a better promoter than the GAPDH promoter alone.
  • the CMV promoter is 5 ’ of the GAPDH.
  • CMV/GAPDH is referred to as a promoter.
  • a representative CMV/GAPDH promoter is provided in SEQ ID NO: 1.
  • each GAPDH promoter of an expression cassette is a CMV/GAPDH promoter.
  • each CMV/GAPDH promoter of an expression cassette comprises a nucleotide sequence that is 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 1.
  • each CMV/GAPDH promoter comprises the nucleotide sequence of SEQ ID NO: 1.
  • each GAPDH promoter of an expression cassette in a pair of expression vectors is a CMV/GAPDH promoter.
  • each CMV/GAPDH promoter of an expression cassette in a pair of expression vectors comprises a nucleotide sequence that is 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 1.
  • each CMV/GAPDH promoter comprises the nucleotide sequence of SEQ ID NO: 1.
  • a polyA signal sequence can follow each gene (i.e., the coding sequences encoding the antibody or antibody fusion chains and the selectable marker).
  • PolyA signal sequences are known in the art and include a bovine growth hormone (BGH) polyA signal sequence (e.g., Regen et al., 1986, DNA, 5(2): 115-22; Goodwin and Rottman, 1992, J. Biol. Chem., 267(23): 16330-16334), athymidine kinase polyA (TKpA) signal sequence (Cole and Stacy, 1985, Mol Cell Biol., 5(8):2104- 13), a rabbit beta-globin polyA signal sequence (Lanoix et al., 1988; EMBO J. 7(8):2515-22; GenBank Accession No.
  • BGH bovine growth hormone
  • TKpA athymidine kinase polyA
  • each of the first, second, and third polyA signal sequences on each expression cassette is a bovine growth hormone (BGH) polyA signal sequence.
  • BGH bovine growth hormone
  • each of the first, second, and third polyA signal sequences on each expression cassette is a simian virus 40 (SV40) early polyA signal sequence.
  • SV40 simian virus 40
  • the BGH polyA signal sequence comprises a nucleotide sequence that is 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 5. In some embodiments, the BGH polyA signal sequence comprises the nucleotide sequence of SEQ ID NO: 5.
  • the rabbit beta-globin polyA signal sequence comprises a nucleotide sequence that is 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 6. In some embodiments, the rabbit beta-globin polyA signal sequence comprises the nucleotide sequence of SEQ ID NO: 6.
  • the SV40 early polyA signal sequence comprises a nucleotide sequence that is 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 7. In some embodiments, the SV40 early polyA signal sequence comprises the nucleotide sequence of SEQ ID NO: 7.
  • the thymidine kinase polyA (TKpA) signal sequence comprises a nucleotide sequence that is 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 8. In some embodiments, the thymidine kinase polyA (TKpA) signal sequence comprises the nucleotide sequence of SEQ ID NO: 8.
  • selectable genes may be used to amplify the gene that will be expressed. Amplification is the process wherein genes that are required for production of a protein critical for growth or cell survival are reiterated in tandem within the chromosomes of successive generations of recombinant cells.
  • suitable selectable markers for mammalian cells include, but are not limited to, glutamine synthetase (GS), dihydrofolate reductase (DHFR), asparaginase (Aspg; see Ha et al. Biotechnol Bioeng. 2023 120: 1159-1166), and promoterless thymidine kinase genes.
  • Mammalian cell transformants are placed under selection pressure wherein only the transformants are uniquely adapted to survive by virtue of the selectable gene present in the vector.
  • Selection pressure is imposed by culturing the transformed cells under conditions in which the concentration of selection agent in the medium is successively increased, thereby leading to additional stringency and/or amplification of both the selectable gene and the DNA that encodes a protein of interest.
  • increased quantities of a polypeptide of interest are synthesized from the amplified DNA.
  • the selection agent for GS is methionine sulfoximine (MSX).
  • the selection agent for DHFR is methotrexate (MTX).
  • GS knockout cell lines provide sufficient selection stringency without MSX or with low MSX concentrations, while 25 pM MSX coupled with the GS- knockout cell line led to higher selection efficiency compared with CHOK1SV cell lines at higher MSX concentrations (Fan, et al., Biotechnol Bioeng., 109(4): 1007-1015 (2012)).
  • a previous report showed that increasing the MSX concentration in the seed train stage after clone selection increased productivity without significant impacts on cell growth, GS, and target gene copy numbers and expression, and maintained product quality attributes in multiple GS knockout cell lines (Tian et al., Engineering in Life Sciences 20(3-4): 112-125 (2020)).
  • Chain/vector expression can be influenced by increasing stringency during pool recovery/selection by adding MSX.
  • the MSX concentration can be optimized for one of the promoters driving GS expression.
  • the MSX concentration is optimized for the GS linked to the more difficult to express chain.
  • the selectable marker in an expression cassette is glutamine synthetase.
  • Glutamine synthetase (GS) catalyzes glutamine biosynthesis by the condensation of ammonia with glutamate.
  • the selectable marker in each expression cassette is glutamine synthetase.
  • the promoter SRoc is operably linked to the selectable marker.
  • the promoter mPGK is operably linked to the selectable marker.
  • Other suitable promoters can be selected from those well known in the art.
  • the present disclosure provides expression cassettes comprising a polynucleotide sequence which comprises the following elements in 5’ to 3’ order: a first GAPDH promoter operably linked to a nucleotide sequence encoding a first antibody chain or antibody chain fusion followed by a first polyA signal sequence; a second GAPDH promoter operably linked to a nucleotide sequence encoding a second antibody chain or antibody chain fusion followed by a second polyA signal sequence; and a promoter operably linked to a nucleotide sequence encoding a selectable marker followed by a third polyA signal sequence.
  • Such expression cassettes may be part of expression vectors or pairs of expression vectors in which each expression vector comprises an expression cassette having the aforementioned configuration.
  • the selectable marker is glutamine synthetase.
  • the promoter operably linked to the nucleotide sequence encoding the selectable marker is a SRoc promoter, a mPGK promoter, or a SV40 promoter.
  • the selectable marker is glutamine synthetase
  • the promoter operably linked to the nucleotide sequence encoding the selectable marker is a SRoc promoter, a mPGK promoter, or a SV40 promoter.
  • the promoter operably linked to the nucleotide sequence encoding the selectable marker is a SRoc promoter.
  • the selectable marker is glutamine synthetase
  • the promoter operably linked to the nucleotide sequence encoding the selectable marker is a SRoc promoter.
  • the promoter operably linked to the nucleotide sequence encoding the selectable marker is a mPGK promoter.
  • the selectable marker is glutamine synthetase, and the promoter operably linked to the nucleotide sequence encoding the selectable marker is a mPGK promoter.
  • the promoter operably linked to the nucleotide sequence encoding the selectable marker is a SV40 promoter.
  • the selectable marker is glutamine synthetase, and the promoter operably linked to the nucleotide sequence encoding the selectable marker is a SV40 promoter.
  • the SRoc promoter comprises a nucleotide sequence that is 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 2. In some embodiments, the SRoc promoter comprises the nucleotide sequence of SEQ ID NO: 2.
  • the mPGK promoter comprises a nucleotide sequence that is 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 3. In some embodiments, the mPGK promoter comprises the nucleotide sequence of SEQ ID NO: 3.
  • the SV40 promoter comprises a nucleotide sequence that is 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 4. In some embodiments, the SV40 promoter comprises the nucleotide sequence of SEQ ID NO: 4. ADDITIONAL COMPONENTS OF EXPRESSION VECTORS
  • expression cassetes described herein are employed in expression vectors. Such expression vectors are useful for transformation of a host cell and can contain additional nucleic acid sequences for plasmid maintenance and for cloning and expression of exogenous nucleotide sequences.
  • sequences will typically include one or more of the following nucleotide sequences (in addition to the promoter(s)/enhancer fragment(s), antibody chains, selectable marker(s), and other sequences (e.g., polyadenylation sequences) described above): one or more enhancer sequences, an origin of replication, transcriptional and translational control sequences, a transcriptional termination sequence, a complete intron sequence containing a donor and acceptor splice site, various pre- or prosequences to improve glycosylation or yield, a native or heterologous signal sequence (leader sequence or signal peptide) for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, internal ribosome entry site (IRES) sequences, an expression augmenting sequence element (EASE), tripartite leader (TPL) and VA gene RNAs from Adenovirus 2, and a polylinker region for inserting the polynucleotide encoding the polypeptide to be expressed.
  • Vector components may be homologous (i.e., from the same species and/or strain as the host cell), heterologous (e.g., from a species other than the host cell species or strain), hybrid (i.e., a combination of flanking sequences from more than one source), synthetic or native.
  • sequences of components useful in the vectors may be obtained by methods well known in the art, such as those previously identified by mapping and/or by restriction endonuclease. In addition, they can be obtained by polymerase chain reaction (PCR) and/or by screening a genomic library with suitable probes.
  • a ribosome-binding site is usually necessary fortranslation initiation of mRNA and is characterized by a Shine-Dalgamo sequence (prokaryotes) or a Kozak sequence (eukaryotes).
  • the element is typically located 3' to the promoter and 5' to the coding sequence of the polypeptide to be expressed.
  • An origin of replication aids in the amplification of the vector in a host cell. They may be included as part of commercially available prokaryotic vectors and may also be chemically synthesized based on a known sequence and ligated into the vector.
  • Various viral origins e.g., SV40, polyoma, adenovirus, vesicular stomatitis virus (VSV), or papillomaviruses such as HPV or BPV
  • SV40 polyoma
  • adenovirus vesicular stomatitis virus
  • papillomaviruses such as HPV or BPV
  • Transcriptional and translational control sequences for mammalian host cell expression vectors can be excised from viral genomes.
  • Commonly used enhancer sequences are derived from polyoma virus, adenovirus 2, simian virus 40 (SV40), and human cytomegalovirus (CMV).
  • SV40 simian virus 40
  • CMV human cytomegalovirus
  • the human CMV promoter/enhancer of immediate early gene 1 may be used. See e.g. Paterson et al., 1994, Applied Microbiol. Biotechnol. 40:691-98.
  • DNA sequences derived from the SV40 viral genome for example, SV40 origin, early and late promoter, enhancer, splice, and polyadenylation sites can be used to provide other genetic elements for expression of a structural gene sequence in a mammalian host cell.
  • Viral early and late promoters are particularly useful because both are easily obtained from a viral genome as a fragment, which can also contain a viral origin of replication (Fiers et al., 1978, Nature 273: 113; Kaufman, 1990, Meth, in Enzymol. 185:487-511).
  • Smaller or larger SV40 fragments can also be used, provided the approximately 250 bp sequence extending from the Hind III site toward the Bgll site located in the SV40 viral origin of replication site is included.
  • an enhancer sequence may be inserted into the vector to increase transcription by higher eukaryotes. Enhancers are cis-acting elements of DNA, usually about 10-300 bp in length, that act on the promoter to increase transcription. Enhancers are relatively orientation and position independent, having been found at positions both 5' and 3' to the transcription unit. Several enhancer sequences available from mammalian genes are known (e.g., globin, elastase, albumin, alpha-feto-protein and insulin).
  • an enhancer from a virus is used.
  • the SV40 enhancer, the cytomegalovirus early promoter enhancer, the polyoma enhancer, and adenovirus enhancers known in the art are exemplary enhancing elements for the activation of eukaryotic promoters. While an enhancer may be positioned in the vector either 5' or 3' to a coding sequence, it is typically located at a site 5' from the promoter.
  • the final protein product may have, in the -1 position (relative to the first amino acid of the mature protein), one or more additional amino acids incident to expression, which may not have been totally removed.
  • the final protein product may have one or two amino acid residues found in the peptidase cleavage site, atached to the aminoterminus.
  • use of some enzyme cleavage sites may result in a slightly truncated form of the desired polypeptide if the enzyme cuts at such area within the mature polypeptide.
  • a sequence encoding an appropriate native or heterologous signal sequence can be incorporated into an expression vector, to promote extracellular secretion of the protein of interest.
  • the choice of signal peptide or leader depends on the type of host cells in which the protein of interest to be produced, and a heterologous signal sequence can replace the native signal sequence.
  • Examples of signal peptides that are functional in mammalian host cells include, but are not limited to, the following: the signal sequence for interleukin-7 described in U.S. Patent No. 4,965,195; the signal sequence for interleukin-2 receptor described in Cosman et al., 1984, Nature 312:768; the interleukin-4 receptor signal peptide described in EP Patent No. 0367 566; the type I interleukin-1 receptor signal peptide described in U.S. Pat. No. 4,968,607; the type II interleukin-1 receptor signal peptide described in EP Patent No. 0 460 846.
  • Additional control sequences shown to improve expression of heterologous genes from mammalian expression vectors include, but are not limited to, such elements as the expression augmenting sequence element (EASE) derived from CHO cells (Morris et al., in Animal Cell Technology, pp. 529-534 (1997); U.S. Patent Nos. 6,312,951 Bl, 6,027,915, and 6,309,841 Bl) and the tripartite leader (TPL) and VA gene RNAs from Adenovirus 2 (Gingeras et al., 1982, J. Biol. Chem. 257: 13475-13491).
  • EASE expression augmenting sequence element
  • TPL tripartite leader
  • VA gene RNAs from Adenovirus 2
  • IVS internal ribosome entry site
  • TABLE A provides non-limiting example synthetic nucleotide (DNA) sequences for certain expression cassette/vector components that may be used in expression cassettes and vectors of the present disclosure.
  • isolated protein or “isolated recombinant protein” refers to a polypeptide or protein of interest, that is purified away from proteins or polypeptides or other contaminants that would interfere with its therapeutic, diagnostic, prophylactic, research, or other use.
  • Proteins of interest include proteins that exert a therapeutic effect by binding a target, particularly a target among those listed below, including targets derived therefrom, targets related thereto, and modifications thereof.
  • Proteins of interest include “antigen-binding proteins,” in particular “antibody modalities.”
  • Antigen-binding protein refers to proteins or polypeptides that comprise an antigen-binding region or antigen-binding portion that has affinity for another molecule to which it binds (antigen).
  • Antigenbinding proteins encompass antibodies, peptibodies, antibody fragments, antibody derivatives, antibody analogs, fusion proteins (including single-chain variable fragments (scFvs), double-chain (divalent) scFvs, and IgGscFv (see, e.g., Orcutt et al., 2010, Protein Eng Des2 Sei 23:221-228), hetero-IgG (see, e.g., Liu et al., 2015, J Biol Chem 290:7535-7562), muteins, and XmAb® (Xencor, Inc., Monrovia, CA).
  • antigen binding proteins include, but are not limited to, a human antibody, a humanized antibody, a chimeric antibody, a recombinant antibody, a single chain antibody, a diabody, a triabody, a tetrabody, a Fab fragment, a F(ab’)2 fragment, an IgD antibody, an IgE antibody, an IgM antibody, an IgGl antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody, and fragments thereof.
  • bispecific T cell engager Bispecific T cell engager (BiTE®) molecules, bispecific T cell engagers having extensions, such as half-life extensions, for example HLE BiTE molecules, Heterolg BITE molecules, and others.
  • the term “antigen binding protein” is used in its broadest sense and means a protein comprising a portion that binds to an antigen or target and, optionally, a scaffold or framework portion that allows the antigen binding portion to adopt a conformation that promotes binding of the antigen binding protein to the antigen.
  • the antigen binding protein can comprise, for example, an alternative protein scaffold or artificial scaffold with grafted CDRs or CDR derivatives.
  • Such scaffolds include, but are not limited to, antibody-derived scaffolds comprising mutations introduced to, for example, stabilize the three-dimensional structure of the antigen binding protein as well as wholly synthetic scaffolds comprising, for example, a biocompatible polymer.
  • PAMs peptide antibody mimetics
  • An antigen binding protein can have, for example, the structure of a naturally occurring immunoglobulin.
  • An “immunoglobulin” is a tetrameric molecule. In a naturally occurring immunoglobulin, each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa).
  • the amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition, i.e., the VL and VH domains.
  • the carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function.
  • Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody’s isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
  • Naturally occurring immunoglobulin chains exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs. From N-terminus to C-terminus, both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain can be done in accordance with the definitions of Kabat et al.
  • antibody includes reference to both glycosylated and non-glycosylated immunoglobulins of any isotype or subclass or to an antigen-binding region thereof that competes with the intact antibody for specific binding, unless otherwise specified. Unless otherwise specified, antibodies include human, humanized, chimeric, multi-specific, monoclonal, polyclonal, heteroIgG, bispecific, and oligomers. Antibodies include the IgGl-, IgG2-, IgG3-, or lgG4-type.
  • An antigen binding protein can have one or more binding sites. If there is more than one binding site, the binding sites can be identical to one another or can be different. For example, a naturally occurring human immunoglobulin typically has two identical binding sites, while a “bispecific” or “bifunctional” antibody has two different binding sites.
  • VERITAS One standard nomenclature for multispecific antibody modalities is VERITAS. See Biswas et al., 2023, mAbs 15: 1-9.
  • An antigen binding fragment or region include Fab, Fab', F(ab')2, Fv, diabodies, Fd, dAb, maxibodies, single chain antibody molecules, single domain VHH, complementarity determining region (CDR) fragments, scFv, diabodies, triabodies, tetrabodies, and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to a target polypeptide.
  • CDR complementarity determining region
  • a VH domain is the heavy chain variable domain.
  • the variable region gives the antibody its ability to bind to antigens.
  • a VH domain includes the variable regions of UniAbsTM, which are called UniDabsTM. See, e.g., Clarke et al., 2018, Front Immunol. 9:3037.
  • a Fab fragment is a monovalent fragment having the VL, VH, CL and CHI domains; a F(ab’)2 fragment is a bivalent fragment having two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment has the VH and CHI domains; an Fv fragment has the VL and VH domains of a single arm of an antibody; and a dAb fragment has a VH domain, a VL domain, or an antigen-binding fragment of a VH or VL domain (U.S. Pat. Nos. 6,846,634, 6,696,245, U.S. Patent Application Publication Nos.
  • a single-chain antibody is an antibody in which a VL and a VH region are joined via a linker (e.g., a synthetic sequence of amino acid residues) to form a continuous protein chain wherein the linker is long enough to allow the protein chain to fold back on 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.
  • a linker e.g., a synthetic sequence of amino acid residues
  • an antigen binding protein can, but need not, be of human origin (e.g., a human antibody), and in some cases will comprise a non-human protein, for example, a rat or murine protein, and in other cases an antigen binding protein can comprise a hybrid of human and non-human proteins (e.g., a humanized antibody).
  • a protein of interest can comprise a human antibody.
  • human antibody includes all antibodies that have one or more variable and constant regions derived from human immunoglobulin sequences. In one embodiment, all of the variable and constant domains are derived from human immunoglobulin sequences (a fully human antibody).
  • Such antibodies can be prepared in a variety of ways, including through the immunization with an antigen of interest of a mouse that is genetically modified to express antibodies derived from human heavy and/or light chain-encoding genes, such as a mouse derived from a Xenomouse®, UltiMabTM, or Velocimmune® system, or a rat derived from UniRat®. Phage-based approaches can also be employed.
  • a protein of interest can comprise a humanized antibody.
  • a “humanized antibody” has a sequence that differs from the sequence of an antibody derived from a non-human species by one or more amino acid substitutions, deletions, and/or additions, such that the humanized antibody is less likely to induce an immune response, and/or induces a less severe immune response, as compared to the non-human species antibody, when it is administered to a human subject.
  • certain amino acids in the framework and constant domains of the heavy and/or light chains of the non-human species antibody are mutated to produce the humanized antibody.
  • the constant domain(s) from a human antibody are fused to the variable domain(s) of a non-human species. Examples of how to make humanized antibodies can be found in U.S. Pat. Nos. 6,054,297, 5,886,152 and 5,877,293.
  • modified proteins such as are proteins modified chemically by a non- covalent bond, covalent bond, or both a covalent and non-covalent bond. Also included are proteins further comprising one or more post-translational modifications, which may be made by cellular modification systems or modifications introduced ex vivo by enzymatic and/or chemical methods or introduced in other ways.
  • Proteins of interest may also include recombinant fusion proteins comprising, for example, a multimerization domain, such as a leucine zipper, a coiled coil, an Fc portion of an immunoglobulin, and the like. Also included are proteins comprising all or part of the amino acid sequences of differentiation antigens (referred to as CD proteins) or their ligands or proteins substantially similar to either of these.
  • a multimerization domain such as a leucine zipper, a coiled coil, an Fc portion of an immunoglobulin, and the like.
  • CD proteins proteins comprising all or part of the amino acid sequences of differentiation antigens
  • proteins of interest may include proteins that bind specifically to one or more CD proteins, HER receptor family proteins, cell adhesion molecules, growth factors, nerve growth factors, fibroblast growth factors, transforming growth factors (TGF), insulin-like growth factors, osteoinductive factors, insulin and insulin-related proteins, coagulation and coagulation- related proteins, colony stimulating factors (CSFs), other blood and serum proteins, blood group antigens, receptors, receptor-associated proteins, growth hormones, growth hormone receptors, T-cell receptors; neurotrophic factors, neurotrophins, relaxins, interferons, interleukins, viral antigens, lipoproteins, integrins, rheumatoid factors, immunotoxins, surface membrane proteins, transport proteins, homing receptors, addressins, regulatory proteins, and immunoadhesins.
  • CD proteins HER receptor family proteins
  • CSFs colony stimulating factors
  • proteins of interest bind to one of more of the following, alone or in any combination: CD proteins including but not limited to CD3, CD4, CD5, CD7, CD8, CD 19, CD20, CD22, CD25, CD30, CD33, CD34, CD38, CD40, CD70, CD123, CD133, CD138, CD171, and CD 174, HER receptor family proteins, including, for instance, HER2, HER3, HER4, and the EGF receptor, EGFRvIII, cell adhesion molecules, for example, LFA-1, Mol, pl50,95, VLA-4, ICAM-1, VCAM, and alpha v/beta 3 integrin, growth factors, including but not limited to, for example, vascular endothelial growth factor (“VEGF”); VEGFR2, growth hormone, thyroid stimulating hormone, follicle stimulating hormone, luteinizing hormone, growth hormone releasing factor, parathyroid hormone, mullerian-inhibiting substance, human macrophage inflammatory protein (MIP- 1-alpha),
  • VEGF vascular end
  • proteins of interest include abciximab, adalimumab, adecatumumab, aflibercept, alemtuzumab, alirocumab, anakinra, atacicept, basiliximab, belimumab, bevacizumab, biosozumab, blinatumomab, brentuximab vedotin, brodalumab, cantuzumab mertansine, canakinumab, cetuximab, certolizumab pegol, conatumumab, daclizumab, denosumab, eculizumab, edrecolomab, efalizumab, epratuzumab, etanercept, evolocumab, galiximab, ganitumab, gemtuzumab, golimumab, ibritumo
  • the antigen binding molecule may be an antibody fragment thereof, including one or more single chain antibody fragment (“scFv”).
  • scFvs are preferred for use in chimeric antigen receptors because they can be engineered to be expressed as part of a single chain. See Krause et al., 1988, J. Exp. Med., 188(4): 619-626; Finney et al., 1998, J Immunol 161: 2791-2797.
  • An “Fc” region comprises two heavy chain fragments comprising the CH2 and CH3 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains. Proteins of interest comprising an Fc region, including antigen binding proteins and Fc fusion proteins, form another aspect of the instant disclosure.
  • heterologous polynucleotide or a vector containing the heterologous polynucleotide into the host cell, which facilitates the stable integration of said heterologous polynucleotide into the cell genome.
  • the heterologous polynucleotide is flanked by homology arms, i.e., sequences homologous to the region upstream and downstream to the integration site.
  • circular vectors may be linearized to facilitate integration into the cell genome.
  • Methods for the introduction of vectors into cells include transfection with biological methods, such as viral delivery, with chemical methods, such as using cationic polymers, calcium phosphate, cationic lipids or cationic amino acids; with physical methods, such as electroporation or microinjection; or with mixed approaches, such as protoplast fusion.
  • biological methods such as viral delivery
  • chemical methods such as using cationic polymers, calcium phosphate, cationic lipids or cationic amino acids
  • physical methods such as electroporation or microinjection
  • mixed approaches such as protoplast fusion.
  • a specific method of 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 in the genome (also termed “targeted integration”).
  • Sitespecific recombinases such as Flp and Cre mediate recombination between two copies of their target sequence termed FRT and loxP, respectively.
  • Insertion of RMCE into a specific site in the genome can be mediated by nucleases (e.g., zinc finger protein (ZFP), transcription activator-like effector nuclease (TALEN), clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)) that can be engineered to create single- and double-stranded breaks (SSBs/DSBs) in the genome.
  • ZFP zinc finger protein
  • TALEN transcription activator-like effector nuclease
  • CRISPR clustered regularly interspaced short palindromic repeats
  • Cas9 CRISPR-associated protein 9
  • Homologous recombination requires the presence of a homologous sequence as a template (e.g., "donor” containing RMCE) to guide the cellular repair process and the results of the repair are error-free and predictable.
  • a template or "donor”
  • the cell In the absence of a template (or "donor") sequence for homologous recombination, the cell typically attempts to repair the DSB via the unpredictable and error-prone process of non-homologous end-joining (NHEJ).
  • NHEJ non-homologous end-joining
  • a vector may be any molecule or entity (e.g., nucleic acid, plasmid, bacteriophage, transposon, cosmid, chromosome, virus, virus capsid, virion, naked DNA, complexed DNA and the like) suitable for use to transfer and/or transport protein encoding information into a host cell and/or to a specific location and/or compartment within a host cell.
  • Vectors can include viral and non-viral vectors, non-episomal mammalian vectors. Vectors are often referred to as expression vectors, for example, recombinant expression vectors and cloning vectors.
  • expression cassettes that may be incorporated into expression vectors useful for expressing various proteins of interest, including certain antibody modalities discussed above.
  • Expression vectors according to the present disclosure may be introduced into a host cell to allow replication of the vector itself and thereby amplify the copies of the polynucleotide contained therein.
  • cloning vectors may contain sequence components generally include, without limitation, an origin of replication, promoter sequences, transcription initiation sequences, enhancer sequences, and selectable markers. These elements may be selected as appropriate by a person of ordinary skill in the art.
  • one or more vectors may be inserted into a suitable cell for amplification and/or polypeptide expression.
  • the transformation of an expression vector into a selected cell may be accomplished by well-known methods including transfection, infection, calcium phosphate co-precipitation, electroporation, nucleofection, microinjection, DEAE-dextran mediated transfection, cationic lipids mediated delivery, liposome mediated transfection, microprojectile bombardment, receptor-mediated gene delivery, delivery mediated by polylysine, histone, chitosan, and peptides.
  • the method selected will in part be a function of the type of host cell to be used.
  • transformation refers to a change in a cell's genetic characteristics, and a cell has been transformed when it has been modified to contain new DNA or RNA.
  • a cell is transformed where it is genetically modified from its native state by introducing new genetic material via transfection, transduction, or other techniques.
  • the transforming DNA can recombine with that of the cell by physically integrating into a chromosome of the cell or can be maintained transiently as an episomal element without being replicated, or can replicate independently as a plasmid.
  • a cell is considered to have been “stably transformed” when the transforming DNA is replicated with the division of the cell.
  • transfection refers to the uptake of foreign or exogenous DNA by a cell.
  • a number of transfection techniques are well known in the art and are disclosed herein. See, e.g., Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, supra; Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al., 1981, Gene 13: 197.
  • transduction refers to the process whereby foreign DNA is introduced into a cell via viral vector. See Jones et al., (1998). Genetics: principles and analysis. Boston: Jones & Bartlett Publ.
  • any mammalian cell line can be used, with CHO cell lines being one preferred example of a cell line that may be used in combination with expression vectors of the present disclosure.
  • a wide variety of mammalian cell lines suitable for growth in culture are available from the American Type Culture Collection (Manassas, Va.) and commercial vendors.
  • Non- limiting examples of cell lines commonly used in the industry include monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture (Graham et al, 1977, J. Gen Virol.
  • 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 VOD-76, ATCC CRL- 1587
  • human cervical carcinoma 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 hepatoma cells Hep G2, HB 8065
  • mouse mammary tumor MMT 060562, ATCC CCL51
  • TRI cells Mather et al., 1982, Annals N.Y Acad. Sci. 383:44-68
  • MRC 5 cells or FS4 cells mammalian myeloma cells, and a number of other cell lines and Chinese hamster ovary (CHO) cells.
  • mammalian host cells used to generate the recombinant mammalian cells described herein can, but need not be, adapted to growth in suspension culture.
  • a variety of host cells adapted to growth in suspension culture are known, including mouse myeloma NS0 cells and CHO cells from CHO-S, DG44, and DXB11 cell lines.
  • Other suitable cell lines include, but are not limited to, mouse myeloma SP2/0 cells, baby hamster kidney BHK-21 cells, human PER.C6® cells, human embryonic kidney HEK-293 cells, and cell lines derived or engineered from any of the cell lines disclosed herein.
  • CHO cells are widely used to produce complex recombinant proteins, including CHOK1 cells (ATCC CCL61).
  • the 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 the efficient DHFR selectable and amplifiable gene expression system allows high level recombinant protein expression in these cells (Kaufman R. J., 1990, Meth Enzymol 185:537-566).
  • GS glutamine synthase
  • MSX methionine sulfoximine
  • suitable CHO host cells could include, but are not limited to, the following (ECACC accession numbers in brackets): CHO (85050302), CHO (PROTEIN FREE) (00102307), CHO-K1 (85051005), CHO-K1/SF (93061607), CHO/DHFR-(94060607), CHO/DHFR-AC-free (05011002), RR-CHOKI (92052129).
  • Host cells transfected with the vector systems described herein may be cultured in adherent culture or suspension cultures grown in stirred tank reactors (including traditional batch and fed-batch cell cultures, which may but need not comprise a spin fdter), perfusion systems (including alternating tangential flow (“ATF”) cultures, acoustic perfusion systems, depth fdter perfusion systems, and other systems), and hollow fiber bioreactors (HFB, which in some cases may be employed in perfusion processes), as well as various other cell culture methods (see, e.g., Tao et al., 2003, Biotechnol. Bioeng.
  • ATF alternating tangential flow
  • HFB hollow fiber bioreactors
  • the bioreactor is inoculated with at least 0.5 xl0 6 ,1.0 x 10 6 , 2.0 xlO 6 3.0 xlO 6 , 5.0 xlO 6 or 10 x 10 6 viable cells/mL in a serum-free culture medium.
  • the mammalian cells then undergo an exponential growth phase.
  • the cell culture can be maintained without supplemental feeding until a desired cell density is achieved.
  • the cell culture is maintained for up to three days with or without supplemental feeding.
  • the culture can be inoculated at a desired cell density to begin the production phase without a brief growth phase.
  • the switch from the growth phase to production phase can also be initiated by any of the aforementioned methods.
  • Batch culture is a discontinuous method where cells are grown in a fixed volume of culture media for a short period of time followed by a full harvest. Cultures grown using the batch method experience an increase in cell density until a maximum cell density is reached, followed by a decline in viable cell density as the media components are consumed and levels of metabolic by-products (such as lactate and ammonia) accumulate. Harvest typically occurs at the point when the maximum cell density is achieved (e.g., 5xl0 6 cells/mL or greater, depending on media formulation, cell line, etc.).
  • the batch process is the simplest culture method; however, viable cell density is limited by the nutrient availability and once the cells are at maximum density, the culture declines and production decreases. There is no ability to extend a production phase because the accumulation of waste products and nutrient depletion rapidly lead to culture decline (typically around 3 to 7 days).
  • a batch method is used during the growth phase and a fed-batch method used during the production phase, but a fed-batch feeding strategy can be used throughout the entire process.
  • bioreactor volume is a limiting factor which limits the amount of feed.
  • metabolic by-product accumulation will lead to culture decline, which limits the duration of the production phase, about 10 to 21 days.
  • Fed-batch cultures are discontinuous, and harvest typically occurs when metabolic byproduct levels or culture viability reach predetermined levels. When compared to a batch culture, in which no feeding occurs, a fed batch culture can produce greater amounts of recombinant protein. See e.g., U.S. Patent No. 5,672,502.
  • Perfusion culture is one in which the cell culture receives fresh perfusion feed medium while simultaneously removing spent medium.
  • Perfusion can be continuous, stepwise, intermittent, or a combination of any or all of any of these. Perfusion rates can be less than a working volume to many working volumes per day.
  • the cells are retained in the culture and the spent medium that is removed is substantially free of cells or has significantly fewer cells than the culture. Recombinant proteins expressed by the cell culture can also be retained in the culture.
  • Perfusion can be accomplished by a number of means including centrifugation, sedimentation, or filtration, See e.g. Voisard et al., 2003, Biotechnology and Bioengineering 82:751-65.
  • An example of a filtration method is alternating tangential flow filtration. Alternating tangential flow is maintained by pumping medium through hollow-fiber filter modules. See e.g. US Patent No. 6,544,424; Furey, 2002, Gen. Eng. News. 22 (7):62-63.
  • Perfusion flow rate is the amount of media that is passed through (added and removed) from a bioreactor, typically expressed as some portion or multiple of the working volume, in a given time.
  • Working volume refers to the amount of bioreactor volume used for cell culture.
  • the perfusion flow rate is one working volume or less per day.
  • Perfusion feed medium can be formulated to maximize perfusion nutrient concentration to minimize perfusion rate.
  • Perfusion methods offer potential improvement over the batch and fed-batch methods by adding fresh media and simultaneously removing spent media. Typical large scale commercial cell culture strategies strive to reach high cell densities, 60 - 90(+) x 10 6 cells/mL where almost a third to over one-half of the reactor volume is biomass.
  • An advantage of the perfusion process is that the production culture can be maintained for longer periods than batch or fed-batch culture methods. However, increased media preparation, use, storage and disposal are necessary to support a long-term perfusion culture, particularly those with high cell densities, which also need even more nutrients, and all of this drives the production costs even higher, compared to batch and fed batch methods.
  • a large-scale cell culture strategy that combines fed batch feeding during the growth phase with continuous perfusion during the production phase may be used to express proteins of interest. Such a method may target a production phase in which the cell culture is maintained at a packed cell volume of less than or equal to 35%.
  • a recombinant antigen-binding protein e.g., a protein derived from an antibody which comprises two chains, one based on antibody heavy chain and one based on antibody light chain
  • a recombinant antigen-binding protein e.g., a protein derived from an antibody which comprises two chains, one based on antibody heavy chain and one based on antibody light chain
  • GS glutamine synthetase
  • selection of high-producing cell lines is based on controlling the balance between the expression level of GS and the concentration of its specific inhibitor, L-methionine sulfoximine (MSX).
  • each vector in the plurality of vectors comprises a promoter having a unique transcriptional strength driving the expression of the heavy chain and/or light chain and/or a promoter having a unique transcriptional strength driving the expression of the glutamine synthetase gene.
  • An expression cassette comprising a polynucleotide sequence which comprises the following elements in 5 ’ to 3 ’ order: a) a first GAPDH promoter operably linked to a nucleotide sequence encoding a first antibody chain or antibody chain fusion followed by a first polyA sequence; b) a second GAPDH promoter operably linked to a nucleotide sequence encoding a second antibody chain antibody chain fusion followed by a second polyA sequence; and c) a promoter operably linked to a nucleotide sequence encoding a selectable marker followed by a third polyA sequence; wherein when the first antibody chain or antibody chain fusion is an antibody light chain, the second antibody chain or antibody chain fusion is an antibody heavy chain or antibody heavy chain fusion, wherein when the first antibody chain or antibody chain fusion is an antibody heavy chain or heavy chain fusion, the second antibody chain or antibody chain fusion is an antibody light chain, and wherein the heavy chain fusion is selected from the group consisting of a heavy
  • E7 An expression vector which comprises the expression cassette of any of E1-E6.
  • the first expression vector comprises an expression cassette comprising a polynucleotide sequence which comprises the following elements in 5’ to 3’ order: a) a first copy of a GAPDH promoter operably linked to a nucleotide sequence encoding an antibody light chain followed by a first polyA sequence; b) a second copy of the GAPDH promoter operably linked to a nucleotide sequence encoding an antibody heavy chain followed by a second polyA sequence; and c) a promoter operably linked to a nucleotide sequence encoding a selectable marker followed by a third polyA sequence; and
  • the second expression vector comprises an expression cassette comprising a polynucleotide sequence which comprises the following elements in 5’ to 3’ order: a) a first copy of a GAPDH promoter operably linked to a nucleotide sequence encoding a second antibody light chain followed by a first polyA sequence; b) a second copy of the GAPDH promoter operably linked to a nucleotide sequence encoding a second antibody heavy chain followed by a second polyA sequence; and c) a promoter operably linked to a nucleotide sequence encoding a selectable marker followed by a third polyA sequence.
  • E14 A mammalian host cell comprising the expression vector of any of E7-E9.
  • E 15 A mammalian host cell comprising the pair of expression vectors of any of E 10-E 13.
  • E16 The mammalian host cell of E14 or E15 which is selected from the group consisting of CHO.
  • El 7 The mammalian host cell of El 6, wherein the CHO cell is a dhfir- or GSKO.
  • El 8. Method for producing an antibody modality comprising culturing the mammalian host cell of any of E14-E17 under conditions in which the antibody chains are expressed, and recovering the antibody modality from the culture.
  • the expression cassette of Fl wherein the first antibody chain or antibody chain fusion is an antibody light chain, and the second antibody chain or antibody chain fusion is an antibody heavy chain fusion.
  • F5. The expression cassette of Fl, wherein the first antibody chain or antibody chain fusion is an antibody heavy chain fusion, and the second antibody chain or antibody chain fusion is an antibody light chain.
  • F6 The expression cassette of any one of Fl, F3, or F5, wherein the antibody heavy chain fusion is a direct fusion of a heavy chain with a VH, a direct fusion of a heavy chain with a scFv, or a direct fusion of a heavy chain with a cytokine.
  • F8 The expression cassette of any one of Fl, F3, F5, or F6, wherein the antibody heavy chain fusion comprises a VH, scFv, or cytokine fused to the N-terminus of the heavy chain portion of the antibody heavy chain fusion.
  • F9 The expression cassete of any one of Fl, F3, F5, or F6, wherein the antibody heavy chain fusion comprises a VH, scFv, or cytokine fused between the CHI and CH2 of the heavy chain portion of the antibody heavy chain fusion.
  • F10 The expression cassete of any one of Fl, F3, or F5, wherein the antibody heavy chain fusion comprises a linker between the heavy chain and the VH, scFv, or cytokine.
  • Fl 1 The expression cassete of any one of Fl, F3, or F5-F10, wherein the antibody heavy chain fusion is a heavy chain-scFv fusion.
  • F12 The expression cassete of any one of Fl, F3, or F5-F10, wherein the antibody heavy chain fusion is a heavy chain-cytokine fusion.
  • F13 The expression cassete of any one of Fl, F3, or F5-F10, wherein the antibody heavy chain fusion is a heavy chain VH fusion.
  • Fl 6 The expression cassete of any one of Fl -Fl 4, wherein the selectable marker is dihydrofolate reductase.
  • Fl 7 The expression cassete of any one of Fl -Fl 6, wherein the GAPDH promoter is a
  • Fl 8 The expression cassete of Fl 7, wherein the CMV promoter enhancer is 5’ of the GAPDH promoter.
  • Fl 9 The expression cassete of F17 or F18, wherein the GAPDH promoter comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 1.
  • F20 The expression cassete of any one of F17-F19, wherein the GAPDH promoter comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO: 1.
  • F21 The expression cassete of any one of F17-F20, wherein the GAPDH promoter comprises the nucleotide sequence of SEQ ID NO: 1.
  • F22 The expression cassete of any one of F1-F21, wherein the promoter operably linked to the nucleotide sequence encoding the selectable marker is selected from the group consisting of mPGK, SRa, and SV40 promoters.
  • F23 The expression cassete of any one of F 1-F22, wherein the promoter operably linked to the nucleotide sequence encoding the selectable marker is a mPGK promoter.
  • F24 The expression cassete of any one of F 1-F22, wherein the promoter operably linked to the nucleotide sequence encoding the selectable marker is a SRa promoter.
  • F25 The expression cassete of any one of F 1-F22, wherein the promoter operably linked to the nucleotide sequence encoding the selectable marker is a SV40 promoter.
  • each of the first, second, and third polyA signal sequences is independently selected from the group consisting of a bovine growth hormone (BGH) polyA signal sequence, a thymidine kinase polyA (TKpA) signal sequence, a rabbit beta-globin polyA signal sequence, and a simian virus 40 (SV40) early polyA signal sequence.
  • BGH bovine growth hormone
  • TKpA thymidine kinase polyA
  • SV40 simian virus 40
  • F27 The expression cassete of any one of F1-F26, wherein each of the first, second, and third polyA signal sequences is the same.
  • F28 The expression cassete of any one of F1-F27, wherein each of the first, second, and third polyA signal sequences is a simian virus 40 (SV40) early polyA signal sequence.
  • SV40 simian virus 40
  • F29 The expression cassete of Fl, wherein the expression cassete encodes an antibody light chain and an antibody heavy chain, the GAPDH promoter is a CMV/GAPDH promoter, the selectable marker is glutamine synthetase, the promoter operably linked to a nucleotide sequence encoding the selectable marker is mPGK, and each of the first, second, and third polyA signal sequences is a simian virus 40 (SV40) early polyA signal sequence.
  • SV40 simian virus 40
  • the expression cassete of Fl wherein the expression cassete encodes an antibody light chain and an antibody heavy chain fusion, the GAPDH promoter is a CMV/GAPDH promoter, the selectable marker is glutamine synthetase, the promoter operably linked to a nucleotide sequence encoding the selectable marker is SRa, and each of the first, second, and third polyA signal sequences is a simian virus 40 (SV40) early polyA signal sequence.
  • SV40 simian virus 40
  • F31 The expression cassete of F30, wherein the antibody heavy chain fusion is a direct fusion of a heavy chain with a VH, a direct fusion of a heavy chain with a scFv, or a direct fusion of a heavy chain with a cytokine.
  • F32 The expression cassete of F30 or F31, wherein the antibody heavy chain fusion comprises a VH, scFv, or cytokine fused to the C-terminus of the heavy chain portion of the antibody heavy chain fusion.
  • F33 The expression cassete of F30 or F31, wherein the antibody heavy chain fusion comprises a VH, scFv, or cytokine fused to the N-terminus of the heavy chain portion of the antibody heavy chain fusion.
  • F34 The expression cassete of F30 or F31, wherein the antibody heavy chain fusion comprises a VH, scFv, or cytokine fused between the CHI and CH2 of the heavy chain portion of the antibody heavy chain fusion.
  • F35 The expression cassete of F30, wherein the antibody heavy chain fusion comprises a linker between the heavy chain and the VH, scFv, or cytokine.
  • F36 The expression cassete of any one of F30-F35, wherein the antibody heavy chain fusion is a heavy chain-scFv fusion.
  • F37 The expression cassete of any one of F30-F35, wherein the antibody heavy chain fusion is a heavy chain-cytokine fusion.
  • F38 The expression cassete of any one of F30-F35, wherein the antibody heavy chain fusion is a heavy chain VH fusion.
  • F42 The expression vector of F39 or F41, encoding a B2/C2 mAb.
  • the first expression vector comprises an expression cassette comprising a polynucleotide sequence which comprises the following elements in 5’ to 3’ order: a) a first copy of a GAPDH promoter operably linked to a nucleotide sequence encoding an antibody light chain followed by a first polyA signal sequence; b) a second copy of the GAPDH promoter operably linked to a nucleotide sequence encoding an antibody heavy chain followed by a second polyA signal sequence; and c) a promoter operably linked to a nucleotide sequence encoding a selectable marker followed by a third polyA signal sequence; and
  • the first expression vector comprises an expression cassette comprising a polynucleotide sequence which comprises the following elements in 5’ to 3’ order: a) a first copy of a GAPDH promoter operably linked to a nucleotide sequence encoding an antibody heavy chain followed by a first polyA signal sequence; b) a second copy of the GAPDH promoter operably linked to a nucleotide sequence encoding an antibody light chain followed by a second polyA signal sequence; and c) a promoter operably linked to a nucleotide sequence encoding a selectable marker followed by a third polyA signal sequence; and 2) the second expression vector comprises an expression cassette comprising a polynucleotide sequence which comprises the following elements in 5’ to 3’ order: a) a first copy of a GAPDH promoter operably linked to a nucleotide sequence encoding an antibody light chain followed by a first polyA signal sequence; b) a second copy of the GAPDH promoter operably linked
  • F45 A pair of expression vectors, wherein:
  • the second expression vector comprises an expression cassette comprising a polynucleotide sequence which comprises the following elements in 5’ to 3’ order: a) a first copy of a GAPDH promoter operably linked to a nucleotide sequence encoding an antibody heavy chain fusion followed by a first polyA signal sequence; b) a second copy of the GAPDH promoter operably linked to a nucleotide sequence encoding an antibody light chain followed by a second polyA signal sequence; and c) a promoter operably linked to a nucleotide sequence encoding a selectable marker followed by a third polyA signal sequence.
  • F46 A pair of expression vectors, wherein:
  • the first expression vector comprises an expression cassette comprising a polynucleotide sequence which comprises the following elements in 5’ to 3’ order: a) a first copy of a GAPDH promoter operably linked to a nucleotide sequence encoding an antibody heavy chain followed by a first polyA signal sequence; b) a second copy of the GAPDH promoter operably linked to a nucleotide sequence encoding an antibody light chain followed by a second polyA signal sequence; and c) a promoter operably linked to a nucleotide sequence encoding a selectable marker followed by a third polyA signal sequence; and
  • the second expression vector comprises an expression cassette comprising a polynucleotide sequence which comprises the following elements in 5 ’ to 3 ’ order a) a first copy of a GAPDH promoter operably linked to a nucleotide sequence encoding an antibody heavy chain fusion followed by a first polyA signal sequence; b) a second copy of the GAPDH promoter operably linked to a nucleotide sequence encoding an antibody light chain followed by a second polyA signal sequence; and c) a promoter operably linked to a nucleotide sequence encoding a selectable marker followed by a third polyA signal sequence.
  • F47 The pair of expression vectors of any one of F43-F46, wherein the antibody light chain of the first expression vector is the same as the antibody light chain of the second expression vector.
  • F48 The pair of expression vectors of any one of F43-F47, wherein the antibody heavy chain of the first expression vector is the same as the antibody heavy chain portion of the antibody heavy chain fusion of the second expression vector.
  • F49 The pair of expression vectors of any one of F43-F48, wherein the antibody light chain of the first expression vector is the same as the antibody light chain of the second expression vector, and the antibody heavy chain of the first expression vector is the same as the antibody heavy chain portion of the antibody heavy chain fusion of the second expression vector.
  • F50 The pair of expression vectors of any one of F43-F46, wherein the antibody light chain of the first expression vector is different from the antibody light chain of the second expression vector.
  • F51 The pair of expression vectors of any one of F43-F46 or F50, wherein the antibody heavy chain of the first expression vector is different from the antibody heavy chain portion of the antibody heavy chain fusion of the second expression vector.
  • F52 The pair of expression vectors of any one of F43-F46, F50, or F51, wherein the antibody light chain of the first expression vector is different from the antibody light chain of the second expression vector, and the antibody heavy chain of the first expression vector is different from the antibody heavy chain portion of the antibody heavy chain fusion of the second expression vector.
  • F53 The pair of expression vectors of any one of F43-F52, wherein the antibody heavy chain fusion is a heavy chain-scFv fusion.
  • F54 The pair of expression vectors of F43, which encodes a Cl mAb.
  • F55 The pair of expression vectors of any one of F43-F54, wherein the promoter operably linked to the nucleotide sequence encoding the selectable marker on each of the first expression vector and the second expression vector is independently selected from the group consisting of mPGK, SRa, and SV40 promoters.
  • F56 The pair of expression vectors of any one of F43-F55, wherein the promoter operably linked to a nucleotide sequence encoding a selectable marker on the first expression vector is SRa.
  • F57 The pair of expression vectors of any one of F43-F56, wherein the promoter operably linked to a nucleotide sequence encoding a selectable marker on the second expression vector is SRa.
  • F58 The pair of expression vectors of any one of F43-F57, wherein the promoter operably linked to a nucleotide sequence encoding a selectable marker on the first expression vector is SRa, and the promoter operably linked to a nucleotide sequence encoding a selectable marker on the second expression vector is SRa.
  • F59 The pair of expression vectors of any one of F43-F58, wherein the selectable marker on the first expression vector and the selectable marker on the second expression vector is glutamine synthetase.
  • F60 The pair of expression vectors of any one of F43-F59, wherein each of the first, second, and third polyA signal sequences on the first expression vector and the second expression vector is independently selected from the group consisting of a bovine growth hormone (BGH) polyA signal sequence, a thymidine kinase polyA (TKpA) signal sequence, a rabbit beta-globin polyA signal sequence, and a simian virus 40 (SV40) early polyA signal sequence.
  • BGH bovine growth hormone
  • TKpA thymidine kinase polyA
  • SV40 simian virus 40
  • F62 The pair of expression vectors of any one of F43-F61, wherein each of the first, second, and third polyA signal sequences on the second expression vector is a simian virus 40 (SV40) early polyA signal sequence.
  • SV40 simian virus 40
  • F63 The pair of expression vectors of any one of F43-F62, wherein each of the first, second, and third polyA signal sequences on the first expression vector is a simian virus 40 (SV40) early polyA signal sequence, and each of the first, second, and third polyA signal sequences on the second expression vector is a simian virus 40 (SV40) early polyA signal sequence.
  • SV40 simian virus 40
  • each of the first, second, and third polyA signal sequences on the first expression vector and the second expression vector is a simian virus 40 (SV40) early polyA signal sequence
  • the promoter operably linked to a nucleotide sequence encoding a selectable marker on the first expression vector is SRa
  • the promoter operably linked to a nucleotide sequence encoding a selectable marker on the second expression vector is SRa
  • the selectable marker on the first expression vector and the selectable marker on the second expression vector is glutamine synthetase.
  • F65 A pair of expression vectors, wherein:
  • the first expression vector comprises an expression cassette comprising a polynucleotide sequence which comprises the following elements in 5’ to 3’ order: a) a first copy of a GAPDH promoter operably linked to a nucleotide sequence encoding a first antibody light chain followed by a first polyA signal sequence; b) a second copy of the GAPDH promoter operably linked to a nucleotide sequence encoding a first antibody heavy chain followed by a second polyA signal sequence; and c) a promoter operably linked to a nucleotide sequence encoding a selectable marker followed by a third polyA signal sequence; and
  • the second expression vector comprises an expression cassette comprising a polynucleotide sequence which comprises the following elements in 5’ to 3’ order: a) a first copy of a GAPDH promoter operably linked to a nucleotide sequence encoding a second antibody light chain followed by a first polyA signal sequence; b) a second copy of the GAPDH promoter operably linked to a nucleotide sequence encoding a second antibody heavy chain followed by a second polyA signal sequence; and c) a promoter operably linked to a nucleotide sequence encoding a selectable marker followed by a third polyA signal sequence.
  • F66 A pair of expression vectors, wherein:
  • the first expression vector comprises an expression cassette comprising a polynucleotide sequence which comprises the following elements in 5’ to 3’ order: a) a first copy of a GAPDH promoter operably linked to a nucleotide sequence encoding a first antibody heavy chain followed by a first polyA signal sequence; b) a second copy of the GAPDH promoter operably linked to a nucleotide sequence encoding a first antibody light chain followed by a second polyA signal sequence; and c) a promoter operably linked to a nucleotide sequence encoding a selectable marker followed by a third polyA signal sequence; and
  • the second expression vector comprises an expression cassette comprising a polynucleotide sequence which comprises the following elements in 5’ to 3’ order: a) a first copy of a GAPDH promoter operably linked to a nucleotide sequence encoding a second antibody light chain followed by a first polyA signal sequence; b) a second copy of the GAPDH promoter operably linked to a nucleotide sequence encoding a second antibody heavy chain followed by a second polyA signal sequence; and c) a promoter operably linked to a nucleotide sequence encoding a selectable marker followed by a third polyA signal sequence.
  • F67 A pair of expression vectors, wherein:
  • the first expression vector comprises an expression cassette comprising a polynucleotide sequence which comprises the following elements in 5’ to 3’ order: a) a first copy of a GAPDH promoter operably linked to a nucleotide sequence encoding a first antibody light chain followed by a first polyA signal sequence; b) a second copy of the GAPDH promoter operably linked to a nucleotide sequence encoding a first antibody heavy chain followed by a second polyA signal sequence; and c) a promoter operably linked to a nucleotide sequence encoding a selectable marker followed by a third polyA signal sequence; and
  • the second expression vector comprises an expression cassette comprising a polynucleotide sequence which comprises the following elements in 5’ to 3’ order: a) a first copy of a GAPDH promoter operably linked to a nucleotide sequence encoding a second antibody heavy chain followed by a first polyA signal sequence; b) a second copy of the GAPDH promoter operably linked to a nucleotide sequence encoding a second antibody light chain followed by a second polyA signal sequence; and c) a promoter operably linked to a nucleotide sequence encoding a selectable marker followed by a third polyA signal sequence.
  • F68 A pair of expression vectors, wherein:
  • the first expression vector comprises an expression cassette comprising a polynucleotide sequence which comprises the following elements in 5’ to 3’ order: a) a first copy of a GAPDH promoter operably linked to a nucleotide sequence encoding a first antibody heavy chain followed by a first polyA signal sequence; b) a second copy of the GAPDH promoter operably linked to a nucleotide sequence encoding a first antibody light chain followed by a second polyA signal sequence; and c) a promoter operably linked to a nucleotide sequence encoding a selectable marker followed by a third polyA signal sequence; and
  • the second expression vector comprises an expression cassette comprising a polynucleotide sequence which comprises the following elements in 5’ to 3’ order: a) a first copy of a GAPDH promoter operably linked to a nucleotide sequence encoding a second antibody heavy chain followed by a first polyA signal sequence; b) a second copy of the GAPDH promoter operably linked to a nucleotide sequence encoding a second antibody light chain followed by a second polyA signal sequence; and c) a promoter operably linked to a nucleotide sequence encoding a selectable marker followed by a third polyA signal sequence.
  • F69 The pair of expression vectors of any one of F65-F68, which encodes a heteroIgG.
  • F70 The pair of expression vectors of any one of F65-F69, wherein the promoter operably linked to the nucleotide sequence encoding the selectable marker on each of the first expression vector and the second expression vector is independently selected from the group consisting of mPGK, SRa, and SV40 promoters.
  • F71 The pair of expression vectors of any one of F65-F70, wherein the promoter operably linked to a nucleotide sequence encoding a selectable marker on the first expression vector is mPGK.
  • F72 The pair of expression vectors of any one of F65-F71, wherein the promoter operably linked to a nucleotide sequence encoding a selectable marker on the second expression vector is mPGK.
  • F73 The pair of expression vectors of any one of F65-F72, wherein the promoter operably linked to a nucleotide sequence encoding a selectable marker on the first expression vector is mPGK, and the promoter operably linked to a nucleotide sequence encoding a selectable marker on the second expression vector is mPGK.
  • F74 The pair of expression vectors of any one of F65-F73, wherein the selectable marker on the first expression vector and the selectable marker on the second expression vector is glutamine synthetase.
  • F75 The pair of expression vectors of any one of F65-F74, wherein each of the first, second, and third polyA signal sequences on the first expression vector and the second expression vector is independently selected from the group consisting of a bovine growth hormone (BGH) polyA signal sequence, a thymidine kinase polyA (TKpA) signal sequence, a rabbit beta-globin polyA signal sequence, and a simian virus 40 (SV40) early polyA signal sequence.
  • BGH bovine growth hormone
  • TKpA thymidine kinase polyA
  • SV40 simian virus 40
  • F77 The pair of expression vectors of any one of F65-F76, wherein each of the first, second, and third polyA signal sequences on the second expression vector is a simian virus 40 (SV40) early polyA signal sequence.
  • SV40 simian virus 40
  • F78 The pair of expression vectors of any one of F65-F77, wherein each of the first, second, and third polyA signal sequences on the first expression vector is a simian virus 40 (SV40) early polyA signal sequence, and each of the first, second, and third polyA signal sequences on the second expression vector is a simian virus 40 (SV40) early polyA signal sequence.
  • SV40 simian virus 40
  • each of the first, second, and third polyA signal sequences on the first expression vector and the second expression vector is a simian virus 40 (SV40) early polyA signal sequence
  • the promoter operably linked to a nucleotide sequence encoding a selectable marker on the first expression vector is mPGK
  • the promoter operably linked to a nucleotide sequence encoding a selectable marker on the second expression vector is mPGK
  • the selectable marker on the first expression vector and the selectable marker on the second expression vector is glutamine synthetase.
  • F80 The pair of expression vectors of any one of F43-F79, wherein the GAPDH promoter on the first expression vector is a CMV/GAPDH promoter.
  • F81 The pair of expression vectors of any one of F43-F80, wherein the GAPDH promoter on the second expression vector is a CMV/GAPDH promoter.
  • F82 The pair of expression vectors of any one of F43-F81, wherein the GAPDH promoter on the first expression vector is a CMV/GAPDH promoter, and the GAPDH promoter on the second expression vector is a CMV/GAPDH promoter.
  • F83 The pair of expression vectors of any one of F80-F82, wherein the CMV promoter enhancer is 5 ’ of the GAPDH promoter.
  • F84 The pair of expression vectors of any one of F80-F83, wherein the GAPDH promoter on the first expression vector and/or the second expression vector comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 1.
  • F85 The pair of expression vectors of any one of F80-F84, wherein the GAPDH promoter on the first expression vector and/or the second expression vector comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO: 1.
  • F86 The pair of expression vectors of any one of F80-F85, wherein the GAPDH promoter on the first expression vector and/or the second expression vector comprises the nucleotide sequence of SEQ ID NO: 1.
  • F87 A mammalian host cell comprising an expression vector of any one of F39-F32.
  • F88 A mammalian host cell comprising a pair of expression vectors of any one of F43-F86.
  • F89 The mammalian host cell of F87 or F88, which is a Chinese hamster ovary (CHO) cell.
  • F90 The mammalian host cell of F89, wherein the CHO cell is a dhfr- CHO cell or a GSKO CHO cell.
  • F91 The mammalian host cell of F89, wherein the CHO cell is a GSKO CHO cell.
  • a method for producing an antibody modality comprising culturing a mammalian host cell of any of any one of F87-F91 under conditions in which the antibody modality is produced, and recovering the antibody modality from the culture.
  • F93 The method of F92, wherein the recovered antibody modality is purified and formulated in a pharmaceutically acceptable formulation.
  • Plasmid Generation The coding sequences of the LCs and HCs (or HC-fusions) were inserted in the pPBGS plasmid backbone using Golden Gate cloning to generate a tri-cistronic vector. Briefly, the CMV/ADL, CMV/GAPDH promoters/enhancer fragments were used to control the LCs and HCs (or HC-fusions) with SV40-Poly A fragments following the SRa or mPGK promoters driving the expression of mGS-polyA in that order (TABLE 1). All the fragments were unidirectionally assembled using combinations of overhang sequences to facilitate Golden Gate cloning. MSX was used at 0, 12.5, 25, and 50 pM.
  • GS KO knockout clonal cell host, derived from the CHO-K1 parental host, was used for generating stable pools expressing a number of different antibody modalities as listed in TABLE 1.
  • Host cells were passaged at a seeding density of 0.4-0.3 x 10 6 cells/mL every 3-4 days in a proprietary DMEM-F 12-based media in shake flasks at 120 rpm, 36°C, and 5% CO2. Twenty-four hours before transfection, the host cells were seeded at 1 x 10 6 cells/mL to ensure the cells would be in exponential growth phase at transfection.
  • the cells were spun down and transferred into selection media without glutamine or growth factor and with 0, 12.5, 25, and 50 pM of MSX.
  • the cells were passaged at seeding densities around 1-2 x 10 6 cells/mL every 3-4 days until viability reached over 90%, when the seeding density was reduced to 0.4-0.3 x 10 6 cells/mL.
  • Results in FIGs. 2A-2B show higher titers (A) and cell specific productivity (B) obtained using the CMV/GADPH promoter for light chain (HC), heavy chain (LC) in combination with SRa or mPGK promoters for glutamine synthetase (GS) at different MSX levels.
  • FIGs. 3A-3C show similar Viability (A), Viable Dell Density (VCD) (B) and Integrated Viable Cell Density (VCD) (C) profiles for all vector configurations during fed-batch production. Color corresponds to the MSX concentration levels.
  • B2 mAh Plasmid Generation The coding sequences of a HC-scFv and LC were inserted in the pPBGS plasmid backbone using Golden Gate cloning to generate a tri-cistronic vector. Briefly, CMV/ADL, CMV/GAPDH, or CMW/EF 1 promoters/enhancer fragments were used to control the HC-scFv and LC with SV40-Poly A fragments following the SRa, mPGK, or SV40 promoters driving the expression of mGS-polyA in that order (TABLE 2). All the fragments were uni -directionally assembled using combinations of overhang sequences to facilitate Golden Gate cloning. MSX was used at 0, 12.5, 25, and 50 pM.
  • Supernatants were analyzed for 1) titer (protein A-HPLC) and 2) product quality atributes including aggregates, clips, and isoforms using size-exclusion chromatography (SEC-UHPLC) (Waters UPLC H-class series), reduced capillary electrophoresis (rCE-SDS) (Sciex PA 800 Plus Pharmaceutical Analysis System), and analytical hydrophobic interaction chromatography (HIC-HPLC) (Agilent HPLC 1100/1200 series), respectively.
  • SEC-UHPLC size-exclusion chromatography
  • rCE-SDS reduced capillary electrophoresis
  • HIC-HPLC analytical hydrophobic interaction chromatography
  • Results in FIGs. 4A-4B show higher titers (A) and cell specific productivity (B) obtained using the CMV/GADPH promoter for light chain (HC), heavy chain (LC) in combination with SRa or mPGK promoters for glutamine synthetase (GS) in 12.5 pM, 25 pM, and 50 pM and 0 pM and 12.5 pM MSX, respectively.
  • FIGs. 5A-5C show aggregates (SEC HMW) (A), clips (rCE low-molecular weight and middle-molecular weight species, LMW+MMW) (B), and isoforms (HIC-HPLC- postpeaks) (C). Impurities were comparable among the vector configurations with the highest titer: CMV/GADPH with SRa/mPGK promoters combinations in 25 pM and 12.5 pM MSX, respectively.
  • Viability A
  • Viable Dell Density B
  • Integrated Viable Cell Density C
  • CHO Chinese Hamster Ovary
  • HC and LC heavy and light chains
  • MSX L-methionine sulfoximine
  • This Example describes a further vector optimization by using promoters with different strengths to modulate the expression levels of the HC, LC, and GS genes in combination with various MSX concentrations to improve productivity of bispecific antibodies in CHO cells.
  • the vector engineering strategy described herein can be expanded to improve and optimize productivity for other recombinant proteins expressed in stable pools in general.
  • Stable pools expressing 2+2 ScFv molecule A or B were generated using a Gene Pulser XCell (BioRad Laboratories; Hercules, CA) following the manufacturer’s protocol. Duplicate transfections were performed for each of the vector configurations. Briefly, 20 pg of each plasmid in combination with 5 pg of a proprietary piggybac transposase were electroporated into 20 x 10 6 host cells. The transfected cells were recovered in 20 mL of growth media in 50 mL spin tubes at 225 rpm, 36°C, and 5% CO 2 .
  • Supernatants were analyzed for 1) titer (protein A-HPLC) and 2) product quality atributes including aggregates, clips, and isoforms using size-exclusion chromatography (SEC-UHPLC) (Waters UPLC H-class series), reduced capillary electrophoresis (rCE-SDS) (Sciex PA 800 Plus Pharmaceutical Analysis System), and analytical hydrophobic interaction chromatography (HIC-HPLC) (Agilent HPLC 1100/1200 series), respectively.
  • SEC-UHPLC size-exclusion chromatography
  • rCE-SDS reduced capillary electrophoresis
  • HIC-HPLC analytical hydrophobic interaction chromatography
  • Results in FIGs. 7A-7B show higher titers (A) and cell specific productivity (B) for 2+2 ScFv molecule A obtained using the CMV/GADPH promoter for light chain (HC), heavy chain (LC) in combination with SRa or mPGK promoters for glutamine synthetase (GS) in 12.5 pM, 25 pM, and 50 pM and 0 pM and 12.5 pM MSX, respectively.
  • FIGs. 10A-10C show aggregates (SEC HMW) (A), clips (rCE low-molecular weight and middle-molecular weight species, LMW+MMW) (B), and isoforms (HIC-HPLC- post-peaks) (C) for 2+2 ScFv molecule B.
  • Impurities were comparable among the vector configurations with the highest titer: CMV/GADPH with SRa/mPGK promoters combinations in 25 pM and 12.5 pM MSX, respectively.
  • FIGs. 11A-11C show similar Viability (A), Viable Dell Density (VCD) (B), and Integrated Viable Cell Density (VCD) (C) profiles for all vector configurations with the same MSX concentration for 2+2 ScFv molecule A during fed-batch production except for CMV/GADPH + mPGK + 25 pM MSX and CMV/GADPH + SV40 +2 5pM MSX.
  • CHO Chinese Hamster Ovary
  • HC and LC heavy and light chains
  • MSX L-methionine sulfoximine
  • This Example describes a further vector optimization by using promoters with different strengths to modulate the expression levels of the HC, LC, and GS genes in combination with various MSX concentrations to improve productivity of monoclonal antibodies in CHO cells.
  • GS KO knockout clonal cell host, derived from the CHO-K1 parental host, was used for generating stable pools expressing MAbl or MAb2.
  • Host cells were passaged at a seeding density of 0.3 -0.4 x 10 6 cells/mL every 3-4 days in a proprietary DMEM-F 12-based media in shake flasks at 130 rpm, 36°C and 5% CO2. Twenty-four hours before transfection, the host cells were seeded at 1 x 10 6 cells/mL to ensure the cells would be in exponential growth phase at transfection.
  • Stable pools expressing MAbl or MAb2 were generated using a Lipofectamine LTX (Gibco, Billings, MT) and Opti-MEM I Reduced Serum Media (Gibco, Billings, MT) following the manufacturer’s protocol. Single transfection was performed for each of the vector configurations.
  • each bicistronic plasmid in combination with 2 pg of a proprietary piggybac transposase were added into 4 x 10 6 host cells.
  • 1 pg of each plasmid encoding LC or HC was used.
  • the transfected cells were recovered in 4 mb of growth media in a 6- well plate at 225 rpm, 36°C and 5% CO2.
  • Fc-containing proteins in cell culture medium were purified using ProA affinity capture ( 1 ml HiTrap MabSelect SuRe column, GE Life Sciences), eluted with 100 mM sodium acetate, pH 3.6 followed immediately by buffer exchange into 10 mM sodium acetate, 150 mM NaCl, pH 5.2 using a 5 ml HiTrap Desalting column (GE Life Sciences) as described previously (Gong et al., 2021, MAbs 13(1): 1870058). ProA yield was calculated by measuring the absorbance at 280 nm (A280).
  • Results in FIGs. 13A-13B show higher titers and proA yields obtained using the CMV/GADPH promoter for light chain (HC) and heavy chain (LC) in a bicistronic vectors in combination with SRa, mPGK, SV40, or miniSV40 promoters for glutamine synthetase (GS) in 10 pM MSX.
  • FIG. 14 shows % main peak from SEC for all vector configurations evaluated. Impurities (100% - % main peak) were comparable among all the vector configurations.
  • Results in FIG. 15 shows highest titer obtained using the CMV/GADPH promoter for light chain (HC), heavy chain (LC) in combination with mPGK promoter for glutamine synthetase (GS) in 25 pM MSX.
  • FIGs. 16A-16B show % main peak from SEC (A), and % main peak from a non-reduced capillary electrophoresis assay (nrMCE) (B). Impurities were comparable among the top titer vector configurations evaluated (low titer configurations were not tested for product quality).
  • This Example describes vector optimization using promoters with different strengths to modulate the expression levels of the HC-UniDab, LC, and GS genes in combination with different MSX concentrations to improve productivity of bispecific antibodies in CHO cells.
  • the vector engineering strategy described herein can be expanded to improve and optimize productivity for other recombinant proteins expressed in stable pools in general.
  • B2- and C2-Hmah Plasmid Generation The coding sequences of HC-UniDab and LC were inserted into PBGS plasmid backbone using Golden Gate cloning.
  • the CMV/adL, CMV/GapdH promoters/enhancer fragments were used to control HC -Unidab and LC with SV40-polyA fragments followed by SRa or mpGK promoters driving expression of mGS-PolyA (TABLE 6). All fragments were uni-directionally assembled using a combination of overhang sequences to facilitate Golden Gate cloning.
  • MSX was used at 18.75 or 37.5 uM MSX concentration depending on the GS promoter.
  • a GS KO (knockout) clonal cell host derived from the CHO-K1 parental host, was used for generating stable pools expressing the B2- and C2- Hmabs.
  • Host cells were passaged at a seeding density of 0.4-0.3 x 106 cells/mL every 3-4 days in a proprietary DMEM-F 12-based media in shake flasks at 120 rpm, 36°C and 5% CO2. Twenty-four hours before transfection, the host cells were seeded at 1 x 10 6 cells/mL to ensure the cells would be in exponential growth phase at transfection.
  • Supernatants were analyzed for 1) titer (protein A-HPLC) and 2) product quality attributes including aggregates, clips, and isoforms using size-exclusion chromatography (SEC-UHPLC) (Waters UPLC H-class series), reduced and non-reduced capillary electrophoresis (rCE- and nrCE-SDS) (Sciex PA 800 Plus Pharmaceutical Analysis System).
  • SEC-UHPLC Size-exclusion chromatography
  • rCE- and nrCE-SDS reduced and non-reduced capillary electrophoresis
  • FIG. 17A Higher titers (FIG. 17A) and cell specific productivity (FIG. 17B) was observed for both B2- Hmab and C2-Hmab molecules using CMV/GAPDH for light chain (LC), heavy chain (HC -Unidab) in combination with SRa or mPGK promoters for glutamine synthetase (GS) in 37.5 pM MSX and 18.75 pM MSX, respectively.
  • Titers (FIG. 17A) are normalized to CMV/adL, SRa-GS pools.
  • Impurities are comparable across the different vector configurations for aggregates (FIG. 18A), partial species (FIG. 18B), and clips (FIG. 18C).

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Abstract

Disclosed herein are expression cassettes, expression vectors, dual vector systems, and mammalian host cells, including but not limited to Chinese hamster ovary (CHO) cells, comprising the foregoing, for the expression of certain antibody modalities, wherein each expression vector employs a promoter set optimized for the antibody modality. Also disclosed herein are methods of producing certain antibody modalities using these mammalian host cells.

Description

VECTOR ENGINEERING STRATEGIES TO ENHANCE VOLUMETRIC PRODUCTIVITY AND
DECREASE IMPURITY FORMATION ACROSS A DIVERSE SET OF MODALITIES
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority to U.S. Provisional Patent Application
No. 63/623,063, filed January 19, 2024, which is hereby incorporated by reference in its entirety.
FIELD
[0002] The present disclosure provides expression cassettes, expression vectors, and dual vector systems for use in mammalian host cells, including but not limited to Chinese hamster ovary (CHO) cells, for the expression of antibody modalities.
SUBMISSION OF SEQUENCE LISTING
[0003] The content of the following Sequence Listing XML is incorporated herein by reference in its entirety: file name: 10614-W001-SEC, date created: January 13, 2025; size: 11,843 bytes.
BACKGROUND
[0004] Due to their broad utility, biologies are used worldwide in a variety of applications, such as therapeutics and diagnostics. Mammalian cell lines are the predominant expression systems for biologies, with Chinese hamster ovary (CHO) cells being the predominant cellular factory. (See Lalonde etal., 2017, J Biotechnol 251: 128-140.) Particularly with the advent of biosimilars, speed-to- market and cost-efficiency are now more important than ever before.
[0005] The costs of manufacturing biologies are high due to their complex production strategies, which involve multi-step processes involving the selection of optimal cell lines, culturing production cells in large quantities, and purification of the desired biologic from the cell harvest. Manufacturing is typically even more complex for new antibody modalities, such as those having three or four unique antibody chains. While these costs are decreasing due to improvements in all facets of production, costs can still be prohibitive in their widespread adoption as front-line therapies.
[0006] In order to make biological therapeutics more accessible to patients, decreasing the cost of goods for the manufacturing process is an attractive proposition. One way to achieve this objective is to reduce the cost of goods by increasing the titers associated with production cell lines.
[0007] There still exists a need for expression cassettes and vector systems, which, when transfected into host cell lines, produce recombinant proteins at high titers, with minimal impact on product quality attributes. Novel vector configurations can help to optimize expression levels of different chains in recombinant proteins, such as, for example, for two-, three-, or four-chain molecules, resulting in more balanced chain expression, reduced impurities, and higher product quality. Expression cassettes and vector systems employing such novel vector configurations would benefit the process development of biologies. SUMMARY
[0008] The present disclosure provides expression cassettes comprising a polynucleotide sequence which comprises the following elements in 5’ to 3’ order: 1) a first copy of a GAPDH promoter operably linked to a nucleotide sequence encoding a first antibody chain or antibody chain fusion followed by a first polyA signal sequence; 2) a second copy of the GAPDH promoter operably linked to a nucleotide sequence encoding a second antibody chain or antibody chain fusion followed by a second polyA signal sequence; and 3) a promoter operably linked to a nucleotide sequence encoding a selectable marker followed by a third polyA signal sequence, wherein, when the first antibody chain or antibody chain fusion is an antibody light chain, the second antibody chain or antibody chain fusion is an antibody heavy chain or antibody heavy chain fusion, wherein, when the first antibody chain or antibody chain fusion is an antibody heavy chain or antibody heavy chain fusion, the second antibody chain or antibody chain fusion is an antibody light chain, and wherein the antibody heavy chain fusion is selected from the group consisting of a heavy chain-scFv, a heavy chain-cytokine, and a heavy chain VH. In some embodiments, such an expression cassette is capable being expressed in a mammalian cell, such as, for example, a Chinese hamster ovary (CHO) cell.
[0009] In some embodiments, the first antibody chain or antibody chain fusion is an antibody light chain, and the second antibody chain or antibody chain fusion is an antibody heavy chain or antibody heavy chain fusion. In some embodiments, the antibody heavy chain fusion is a heavy chain-scFv. In some embodiments, the antibody heavy chain fusion is a heavy chain-cytokine. In some embodiments, the antibody heavy chain fusion is a heavy chain VH.
[0010] In some embodiments, the first antibody chain or antibody chain fusion is an antibody heavy chain or antibody heavy chain fusion, and the second antibody chain or antibody chain fusion is an antibody light chain. In some embodiments, the antibody heavy chain fusion is a heavy chain-scFv. In some embodiments, the antibody heavy chain fusion is a heavy chain-cytokine. In some embodiments, the antibody heavy chain fusion is a heavy chain VH.
[0011] In certain embodiments, the first GAPDH promoter is operably linked to a nucleotide sequence encoding an antibody light chain and the second GAPDH promoter is operably linked to an antibody heavy chain or antibody heavy chain fusion. In certain embodiments, the first GAPDH promoter is operably linked to a nucleotide sequence encoding an antibody light chain and the second GAPDH promoter is operably linked to an antibody heavy chain. In certain embodiments, the first GAPDH promoter is operably linked to a nucleotide sequence encoding an antibody light chain and the second GAPDH promoter is operably linked to an antibody heavy chain fusion.
[0012] In certain embodiments, the first GAPDH promoter is operably linked to a nucleotide sequence encoding an antibody heavy chain or antibody heavy chain fusion and the second GAPDH promoter is operably linked to an antibody light chain. In certain embodiments, the first GAPDH promoter is operably linked to a nucleotide sequence encoding an antibody heavy chain and the second GAPDH promoter is operably linked to an antibody light chain. In certain embodiments, the first GAPDH promoter is operably linked to a nucleotide sequence encoding an antibody heavy chain fusion and the second GAPDH promoter is operably linked to an antibody light chain.
[0013] In some embodiments, the antibody heavy chain fusion is a heavy chain-scFv.
[0014] In some embodiments, the antibody heavy chain fusion is a heavy chain-cytokine.
[0015] In some embodiments, the antibody heavy chain fusion is a heavy chain VH.
[0016] In some embodiments, the antibody heavy chain fusion is a direct fusion of a heavy chain with a VH. In some embodiments, the antibody heavy chain fusion is a direct fusion of a heavy chain with a scFv. In some embodiments, the antibody heavy chain fusion is a direct fusion of a heavy chain with a cytokine.
[0017] In some embodiments, the antibody heavy chain fusion is a fusion of a heavy chain with a VH, wherein the fusion comprises a linker between the heavy chain and the VH. In some embodiments, the antibody heavy chain fusion is a fusion of a heavy chain with a scFv, wherein the fusion comprises a linker between the heavy chain and the scFv. In some embodiments, the antibody heavy chain fusion is a fusion of a heavy chain with a cytokine, wherein the fusion comprises a linker between the heavy chain and the cytokine.
[0018] In some embodiments, the antibody heavy chain fusion comprises a VH, scFv, or cytokine fused to the C-terminus of the heavy chain portion of the heavy chain fusion. In some embodiments, the antibody heavy chain fusion comprises a VH fused to the C-terminus of the heavy chain portion of the heavy chain fusion. In some embodiments, the antibody heavy chain fusion comprises a scFv fused to the C-terminus of the heavy chain portion of the heavy chain fusion. In some embodiments, the antibody heavy chain fusion comprises a cytokine fused to the C-terminus of the heavy chain portion of the heavy chain fusion.
[0019] In some embodiments, the antibody heavy chain fusion comprises a VH, scFv, or cytokine fused to the N-terminus of the heavy chain portion of the heavy chain fusion. In some embodiments, the antibody heavy chain fusion comprises a VH fused to the N-terminus of the heavy chain portion of the heavy chain fusion. In some embodiments, the antibody heavy chain fusion comprises a scFv fused to the N-terminus of the heavy chain portion of the heavy chain fusion. In some embodiments, the antibody heavy chain fusion comprises a cytokine fused to the N-terminus of the heavy chain portion of the heavy chain fusion.
[0020] In some embodiments, the antibody heavy chain fusion comprises a VH, scFv, or cytokine fused between the CHI and CH2 of the heavy chain portion of the heavy chain fusion. In some embodiments, the antibody heavy chain fusion comprises a VH fused between the CHI and CH2 of the heavy chain portion of the heavy chain fusion. In some embodiments, the antibody heavy chain fusion comprises a scFv fused between the CHI and CH2 of the heavy chain portion of the heavy chain fusion. In some embodiments, the antibody heavy chain fusion comprises a cytokine fused between the CHI and CH2 of the heavy chain portion of the heavy chain fusion.
[0021] In certain embodiments, the selectable marker is selected from the group consisting of glutamine synthetase and dihydrofolate reductase. In certain embodiments, the selectable marker is glutamine synthetase. In certain embodiments, the selectable marker is dihydrofolate reductase.
[0022] In some embodiments described herein, the promoter for the chains of the antibody modality is a combination of the CMV promoter enhancer and GAPDH (CMV/GAPDH). Collectively, CMV/GAPDH is referred to as a promoter. A representative CMV/GAPDH promoter is provided in SEQ ID NO: 1. In this combination, both the CMV promoter enhancer and GAPDH promoter are operably linked to the nucleotide sequence encoding the antibody chain, such that the combination may be a better promoter than GAPDH alone.
[0023] In certain embodiments, the CMV promoter is 5’ of the GAPDH.
[0024] In certain embodiments, the CMV/GAPDH promoter comprises a nucleotide sequence of SEQ ID NO: 1.
[0025] In certain embodiments, the promoter operably linked to the nucleotide sequence encoding the selectable marker is selected from the group consisting of mPGK, SRoc, and SV40 promoters. In some embodiments, the promoter operably linked to the nucleotide sequence encoding the selectable marker is a mPGK promoter. In some embodiments, the promoter operably linked to the nucleotide sequence encoding the selectable marker is a SRoc promoter. In some embodiments, the promoter operably linked to the nucleotide sequence encoding the selectable marker is a SV40 promoter.
[0026] In certain embodiments, an expression cassette is provided as above, wherein the first, second, and third polyA signal sequences are the same or different and are selected from the group consisting of a thymidine kinase pA (TKpA) signal sequence, a rabbit beta-globin pA, and a simian virus 40 (SV40) early pA signal sequence.
[0027] In some embodiments, the first, second, and third polyA signal sequences are the same.
[0028] In some embodiments, at least one of the first, second, and third polyA signal sequences is different.
[0029] In some embodiments, the first, second, and third polyA signal sequences are independently selected from the group consisting of a bovine growth hormone (BGH) polyA signal sequence, a thymidine kinase polyA (TKpA) signal sequence, a rabbit beta-globin polyA signal sequence, and a simian virus 40 (SV40) early polyA signal sequence.
[0030] In some embodiments, each of the first, second, and third polyA signal sequences is a bovine growth hormone (BGH) polyA signal sequence.
[0031] In some embodiments, each of the first, second, and third polyA signal sequences is a thymidine kinase polyA (TKpA) signal sequence. [0032] In some embodiments, each of the first, second, and third polyA signal sequences is a rabbit beta-globin polyA signal sequence.
[0033] In some embodiments, each of the first, second, and third polyA signal sequences is a simian virus 40 (SV40) early polyA signal sequence.
[0034] In some embodiments, the expression cassette comprises a polynucleotide sequence which comprises the following elements in 5’ to 3’ order: a) a first GAPDH promoter operably linked to a nucleotide sequence encoding an antibody light chain followed by a first polyA signal sequence; b) a second GAPDH promoter operably linked to a nucleotide sequence encoding an antibody heavy chain followed by a second polyA signal sequence; and c) a promoter operably linked to a nucleotide sequence encoding a selectable marker followed by a third polyA signal sequence.
[0035] In some embodiments, the first GAPDH promoter is a CMV/GAPDH promoter. In some embodiments, the first GAPDH promoter comprises a nucleotide sequence that is 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the first GAPDH promoter comprises the nucleotide sequence of SEQ ID NO: 1. [0036] In some embodiments, the second GAPDH promoter is a CMV/GAPDH promoter. In some embodiments, the second GAPDH promoter comprises a nucleotide sequence that is 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the second GAPDH promoter comprises the nucleotide sequence of SEQ ID NO: 1. [0037] In some embodiments, each of the first GAPDH promoter and the second GAPDH promoter is a CMV/GAPDH promoter. In some embodiments, each of the first GAPDH promoter and the second GAPDH promoter comprises a nucleotide sequence that is 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 1. In some embodiments, each of the first GAPDH promoter and the second GAPDH promoter comprises the nucleotide sequence of SEQ ID NO: 1.
[0038] In some embodiments, the promoter operably linked to a nucleotide sequence encoding the selectable marker is mPGK.
[0039] In some embodiments, the selectable marker is glutamine synthetase.
[0040] In some embodiments, the selectable marker is glutamine synthetase and the promoter operably linked to a nucleotide sequence encoding the selectable marker is mPGK.
[0041] In some embodiments, each of the first, second, and third polyA signal sequences is a simian virus 40 (SV40) early polyA signal sequence.
[0042] In some embodiments, each of the first GAPDH promoter and the second GAPDH promoter is a CMV/GAPDH promoter, the selectable marker is glutamine synthetase, the promoter operably linked to a nucleotide sequence encoding the selectable marker is mPGK, and each of the first, second, and third polyA signal sequences is a simian virus 40 (SV40) early polyA signal sequence. [0043] In some embodiments, the expression cassette comprises a polynucleotide sequence which comprises the following elements in 5’ to 3’ order: d) a first GAPDH promoter operably linked to a nucleotide sequence encoding an antibody heavy chain followed by a first polyA signal sequence; e) a second GAPDH promoter operably linked to a nucleotide sequence encoding an antibody light chain followed by a second polyA signal sequence; and f) a promoter operably linked to a nucleotide sequence encoding a selectable marker followed by a third polyA signal sequence.
[0044] In some embodiments, the first GAPDH promoter is a CMV/GAPDH promoter. In some embodiments, the first GAPDH promoter comprises a nucleotide sequence that is 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the first GAPDH promoter comprises the nucleotide sequence of SEQ ID NO: 1. [0045] In some embodiments, the second GAPDH promoter is a CMV/GAPDH promoter. In some embodiments, the second GAPDH promoter comprises a nucleotide sequence that is 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the second GAPDH promoter comprises the nucleotide sequence of SEQ ID NO: 1. [0046] In some embodiments, each of the first GAPDH promoter and the second GAPDH promoter is a CMV/GAPDH promoter. In some embodiments, each of the first GAPDH promoter and the second GAPDH promoter comprises a nucleotide sequence that is 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 1. In some embodiments, each of the first GAPDH promoter and the second GAPDH promoter comprises the nucleotide sequence of SEQ ID NO: 1.
[0047] In some embodiments, the promoter operably linked to a nucleotide sequence encoding the selectable marker is mPGK.
[0048] In some embodiments, the selectable marker is glutamine synthetase.
[0049] In some embodiments, the selectable marker is glutamine synthetase and the promoter operably linked to a nucleotide sequence encoding the selectable marker is mPGK.
[0050] In some embodiments, each of the first, second, and third polyA signal sequences is a simian virus 40 (SV40) early polyA signal sequence.
[0051] In some embodiments, each of the first GAPDH promoter and the second GAPDH promoter is a CMV/GAPDH promoter, the selectable marker is glutamine synthetase, the promoter operably linked to a nucleotide sequence encoding the selectable marker is mPGK, and each of the first, second, and third polyA signal sequences is a simian virus 40 (SV40) early polyA signal sequence. [0052] In some embodiments, the expression cassette comprises a polynucleotide sequence which comprises the following elements in 5’ to 3’ order: g) a first GAPDH promoter operably linked to a nucleotide sequence encoding an antibody light chain followed by a first polyA signal sequence; h) a second GAPDH promoter operably linked to a nucleotide sequence encoding an antibody heavy chain fusion followed by a second polyA signal sequence; and i) a promoter operably linked to a nucleotide sequence encoding a selectable marker followed by a third polyA signal sequence.
[0053] In some embodiments, the antibody heavy chain fusion is a HC-scFv.
[0054] In some embodiments, the antibody heavy chain fusion is a direct fusion of a heavy chain with a VH.
[0055] In some embodiments, the antibody heavy chain fusion is a fusion of a heavy chain with a VH, wherein the fusion comprises a linker between the heavy chain and the VH.
[0056] In some embodiments, the antibody heavy chain fusion comprises a VH fused to the C- terminus of the heavy chain portion of the heavy chain fusion.
[0057] In some embodiments, the antibody heavy chain fusion comprises a VH fused to the N- terminus of the heavy chain portion of the heavy chain fusion.
[0058] In some embodiments, the antibody heavy chain fusion comprises a VH fused between the CHI and CH2 of the heavy chain portion of the heavy chain fusion.
[0059] In some embodiments, the first GAPDH promoter is a CMV/GAPDH promoter. In some embodiments, the first GAPDH promoter comprises a nucleotide sequence that is 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the first GAPDH promoter comprises the nucleotide sequence of SEQ ID NO: 1. [0060] In some embodiments, the second GAPDH promoter is a CMV/GAPDH promoter. In some embodiments, the second GAPDH promoter comprises a nucleotide sequence that is 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the second GAPDH promoter comprises the nucleotide sequence of SEQ ID NO: 1. [0061] In some embodiments, each of the first GAPDH promoter and the second GAPDH promoter is a CMV/GAPDH promoter. In some embodiments, each of the first GAPDH promoter and the second GAPDH promoter comprises a nucleotide sequence that is 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 1. In some embodiments, each of the first GAPDH promoter and the second GAPDH promoter comprises the nucleotide sequence of SEQ ID NO: 1.
[0062] In some embodiments, the promoter operably linked to a nucleotide sequence encoding the selectable marker is SRa.
[0063] In some embodiments, the selectable marker is glutamine synthetase. [0064] In some embodiments, the selectable marker is glutamine synthetase and the promoter operably linked to a nucleotide sequence encoding the selectable marker is SRa.
[0065] In some embodiments, each of the first, second, and third polyA signal sequences is a simian virus 40 (SV40) early polyA signal sequence.
[0066] In some embodiments, each of the first GAPDH promoter and the second GAPDH promoter is a CMV/GAPDH promoter, the selectable marker is glutamine synthetase, the promoter operably linked to a nucleotide sequence encoding the selectable marker is SRa, and each of the first, second, and third polyA signal sequences is a simian virus 40 (SV40) early polyA signal sequence.
[0067] In some embodiments, the antibody heavy chain fusion is a HC-scFv, each of the first GAPDH promoter and the second GAPDH promoter is a CMV/GAPDH promoter, the selectable marker is glutamine synthetase, the promoter operably linked to a nucleotide sequence encoding the selectable marker is SRa, and each of the first, second, and third polyA signal sequences is a simian virus 40 (SV40) early polyA signal sequence.
[0068] In some embodiments, the expression cassette comprises a polynucleotide sequence which comprises the following elements in 5’ to 3’ order: j) a first GAPDH promoter operably linked to a nucleotide sequence encoding an antibody heavy chain fusion followed by a first polyA signal sequence; k) a second GAPDH promoter operably linked to a nucleotide sequence encoding an antibody light chain followed by a second polyA signal sequence; and l) a promoter operably linked to a nucleotide sequence encoding a selectable marker followed by a third polyA signal sequence.
[0069] In some embodiments, the antibody heavy chain fusion is a HC-scFv.
[0070] In some embodiments, the antibody heavy chain fusion is a direct fusion of a heavy chain with a VH.
[0071] In some embodiments, the antibody heavy chain fusion is a fusion of a heavy chain with a VH, wherein the fusion comprises a linker between the heavy chain and the VH.
[0072] In some embodiments, the antibody heavy chain fusion comprises a VH fused to the C- terminus of the heavy chain portion of the heavy chain fusion.
[0073] In some embodiments, the antibody heavy chain fusion comprises a VH fused to the N- terminus of the heavy chain portion of the heavy chain fusion.
[0074] In some embodiments, the antibody heavy chain fusion comprises a VH fused between the CHI and CH2 of the heavy chain portion of the heavy chain fusion.
[0075] In some embodiments, the first GAPDH promoter is a CMV/GAPDH promoter. In some embodiments, the first GAPDH promoter comprises a nucleotide sequence that is 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the first GAPDH promoter comprises the nucleotide sequence of SEQ ID NO: 1. [0076] In some embodiments, the second GAPDH promoter is a CMV/GAPDH promoter. In some embodiments, the second GAPDH promoter comprises a nucleotide sequence that is 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the second GAPDH promoter comprises the nucleotide sequence of SEQ ID NO: 1. [0077] In some embodiments, each of the first GAPDH promoter and the second GAPDH promoter is a CMV/GAPDH promoter. In some embodiments, each of the first GAPDH promoter and the second GAPDH promoter comprises a nucleotide sequence that is 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 1. In some embodiments, each of the first GAPDH promoter and the second GAPDH promoter comprises the nucleotide sequence of SEQ ID NO: 1.
[0078] In some embodiments, the promoter operably linked to a nucleotide sequence encoding the selectable marker is SRa.
[0079] In some embodiments, the selectable marker is glutamine synthetase.
[0080] In some embodiments, the selectable marker is glutamine synthetase and the promoter operably linked to a nucleotide sequence encoding the selectable marker is SRa.
[0081] In some embodiments, each of the first, second, and third polyA signal sequences is a simian virus 40 (SV40) early polyA signal sequence.
[0082] In some embodiments, each of the first GAPDH promoter and the second GAPDH promoter is a CMV/GAPDH promoter, the selectable marker is glutamine synthetase, the promoter operably linked to a nucleotide sequence encoding the selectable marker is SRa, and each of the first, second, and third polyA signal sequences is a simian virus 40 (SV40) early polyA signal sequence.
[0083] In some embodiments, the antibody heavy chain fusion is a HC-scFv, each of the first GAPDH promoter and the second GAPDH promoter is a CMV/GAPDH promoter, the selectable marker is glutamine synthetase, the promoter operably linked to a nucleotide sequence encoding the selectable marker is SRa, and each of the first, second, and third polyA signal sequences is a simian virus 40 (SV40) early polyA signal sequence.
[0084] The present disclosure also provides expression vectors which comprise any of the expression cassettes disclosed above or anywhere herein.
[0085] In certain embodiments, the polynucleotide sequence encodes a monoclonal antibody.
[0086] In certain embodiments, the polynucleotide sequence encodes a B2mab, C2mAb, B2Hmab, or C2Hmab.
[0087] The present disclosure also provides pairs of expression vectors. In one embodiment, the pair has a first expression vector which comprises an expression cassette comprising a polynucleotide sequence which comprises the following elements in 5’ to 3’ order: 1) a first copy of a GAPDH promoter operably linked to a nucleotide sequence encoding an antibody light chain followed by a first polyA sequence; 2) a second copy of the GAPDH promoter operably linked to a nucleotide sequence encoding an antibody heavy chain followed by a second polyA sequence; and 3) a promoter operably linked to a nucleotide sequence encoding a selectable marker followed by a third polyA sequence; and a second expression vector which comprises an expression cassette comprising a polynucleotide sequence which comprises the following elements in 5’ to 3’ order: 1) a first copy of a GAPDH promoter operably linked to a nucleotide sequence encoding an antibody light chain followed by a first polyA sequence; 2) a second copy of the GAPDH promoter operably linked to a nucleotide sequence encoding an antibody heavy chain-scFv fusion followed by a second polyA sequence; and 3) a promoter operably linked to a nucleotide sequence encoding a selectable marker followed by a third polyA sequence. In one aspect of this embodiment, the pair of vectors encodes a Cl mAb.
[0088] In some embodiments, the antibody light chain of the first expression vector is the same as the antibody light chain of the second expression vector.
[0089] In some embodiments, the antibody heavy chain of the first expression vector is the same as the antibody heavy chain portion of the antibody heavy chain-scFv fusion of the second expression vector.
[0090] In some embodiments, the antibody light chain of the first expression vector is the same as the antibody light chain of the second expression vector, and the antibody heavy chain of the first expression vector is the same as the antibody heavy chain portion of the antibody heavy chain-scFv fusion of the second expression vector.
[0091] In some embodiments, the antibody light chain of the first expression vector is different from the antibody light chain of the second expression vector.
[0092] In some embodiments, the antibody heavy chain of the first expression vector is different from the antibody heavy chain portion of the antibody heavy chain-scFv fusion of the second expression vector.
[0093] In some embodiments, the antibody light chain of the first expression vector is different from the antibody light chain of the second expression vector, and the antibody heavy chain of the first expression vector is different from the antibody heavy chain portion of the antibody heavy chain-scFv fusion of the second expression vector.
[0094] In some embodiments, each copy of the GAPDH promoter on the first expression vector is a CMV/GAPDH promoter. In some embodiments, each copy of the GAPDH promoter on the first expression vector comprises a nucleotide sequence that is 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 1 . In some embodiments, each copy of the GAPDH promoter on the first expression vector comprises the nucleotide sequence of SEQ ID NO: 1. [0095] In some embodiments, each copy of the GAPDH promoter on the second expression vector is a CMV/GAPDH promoter. In some embodiments, each copy of the GAPDH promoter on the second expression vector comprises a nucleotide sequence that is 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 1 . In some embodiments, each copy of the GAPDH promoter on the second expression vector comprises the nucleotide sequence of SEQ ID NO: 1.
[0096] In some embodiments, each copy of the GAPDH promoter on the first expression vector and each copy of the GAPDH promoter on the second expression vector is a CMV/GAPDH promoter. In some embodiments, each copy of the GAPDH promoter on the first expression vector and each copy of the GAPDH promoter on the second expression vector comprises a nucleotide sequence that is 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 1. In some embodiments, each copy of the GAPDH promoter on the first expression vector and each copy of the GAPDH promoter on the second expression vector comprises the nucleotide sequence of SEQ ID NO: 1.
[0097] In some embodiments, the promoter operably linked to a nucleotide sequence encoding a selectable marker on the first expression vector is SRa. In some embodiments, the promoter operably linked to a nucleotide sequence encoding a selectable marker on the second expression vector is SRa. In some embodiments, the promoter operably linked to a nucleotide sequence encoding a selectable marker on the first expression vector is SRa, and the promoter operably linked to a nucleotide sequence encoding a selectable marker on the second expression vector is SRa.
[0098] In some embodiments, the selectable marker on the first expression vector is glutamine synthetase. In some embodiments, the selectable marker on the second expression vector is glutamine synthetase. In some embodiments, the selectable marker on the first expression vector and the selectable marker on the second expression vector is glutamine synthetase.
[0099] In some embodiments, the promoter operably linked to a nucleotide sequence encoding a selectable marker on the first expression vector is SRa, the promoter operably linked to a nucleotide sequence encoding a selectable marker on the second expression vector is SRa, and the selectable marker on the first expression vector and the selectable marker on the second expression vector is glutamine synthetase.
[0100] In some embodiments, each of the first, second, and third polyA signal sequences on the first expression vector is a simian virus 40 (SV40) early polyA signal sequence. In some embodiments, each of the first, second, and third polyA signal sequences on the second expression vector is a simian virus 40 (SV40) early polyA signal sequence. In some embodiments, each of the first, second, and third polyA signal sequences on the first expression vector is a simian virus 40 (SV40) early polyA signal sequence, and each of the first, second, and third polyA signal sequences on the second expression vector is a simian virus 40 (SV40) early polyA signal sequence.
[0101] In some embodiments, each of the first, second, and third polyA signal sequences on the first expression vector and the second expression vector is a simian virus 40 (SV40) early polyA signal sequence, the promoter operably linked to a nucleotide sequence encoding a selectable marker on the first expression vector is SRa, the promoter operably linked to a nucleotide sequence encoding a selectable marker on the second expression vector is SRa, and the selectable marker on the first expression vector and the selectable marker on the second expression vector is glutamine synthetase. [0102] In some embodiments, each copy of the GAPDH promoter on the first expression vector and each copy of the GAPDH promoter on the second expression vector is a CMV/GAPDH promoter, each of the first, second, and third polyA signal sequences on the first expression vector and the second expression vector is a simian virus 40 (SV40) early polyA signal sequence, the promoter operably linked to a nucleotide sequence encoding a selectable marker on the first expression vector is SRa, the promoter operably linked to a nucleotide sequence encoding a selectable marker on the second expression vector is SRa, and the selectable marker on the first expression vector and the selectable marker on the second expression vector is glutamine synthetase.
[0103] In alternative embodiments, on the first expression vector, the nucleotide sequence encoding the antibody heavy chain may precede the nucleotide sequence encoding the antibody light chain (in 5’ to 3’ order). Additionally, in alternative embodiments, on the second expression vector, the nucleotide sequence encoding the antibody heavy chain-scFv fusion may precede the nucleotide sequence encoding the antibody light chain (in 5’ to 3’ order). Furthermore, in alternative embodiments, on the first expression vector, the nucleotide sequence encoding the antibody heavy chain may precede the nucleotide sequence encoding the antibody light chain, and, on the second expression vector, the nucleotide sequence encoding the antibody heavy chain-scFv fusion may precede the nucleotide sequence encoding the antibody light chain (all in 5’ to 3’ order).
[0104] In another embodiment, the pair has a first expression vector which comprises an expression cassette comprising a polynucleotide sequence which comprises the following elements in 5 ’ to 3 ’ order: 1) a first copy of a GAPDH promoter operably linked to a nucleotide sequence encoding a first antibody light chain followed by a first polyA sequence; 2) a second copy of the GAPDH promoter operably linked to a nucleotide sequence encoding a first antibody heavy chain followed by a second polyA sequence; and 3) a promoter operably linked to a nucleotide sequence encoding a selectable marker followed by a third polyA sequence; and the second expression vector which comprises an expression cassette comprising a polynucleotide sequence which comprises the following elements in 5’ to 3’ order: 1) a first copy of a GAPDH promoter operably linked to a nucleotide sequence encoding a second antibody light chain followed by a first polyA sequence; 2) a second copy of the GAPDH promoter operably linked to a nucleotide sequence encoding a second antibody heavy chain followed by a second polyA sequence; and 3) a promoter operably linked to a nucleotide sequence encoding a selectable marker followed by a third polyA sequence. In certain aspects of this embodiment, there are two unique light chains and two unique heavy chains giving a four-chain antibody (i.e., the first antibody light chain and the second antibody light chain are different, and the first antibody heavy chain and the second antibody heavy chain are different). In one aspect of this embodiment, the pair of vector encodes a heteroIgG.
[0105] In some embodiments, each copy of the GAPDH promoter on the first expression vector is a CMV/GAPDH promoter. In some embodiments, each copy of the GAPDH promoter on the first expression vector comprises a nucleotide sequence that is 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 1 . In some embodiments, each copy of the GAPDH promoter on the first expression vector comprises the nucleotide sequence of SEQ ID NO: 1. [0106] In some embodiments, each copy of the GAPDH promoter on the second expression vector is a CMV/GAPDH promoter. In some embodiments, each copy of the GAPDH promoter on the second expression vector comprises a nucleotide sequence that is 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 1 . In some embodiments, each copy of the GAPDH promoter on the second expression vector comprises the nucleotide sequence of SEQ ID NO: 1.
[0107] In some embodiments, each copy of the GAPDH promoter on the first expression vector and each copy of the GAPDH promoter on the second expression vector is a CMV/GAPDH promoter. In some embodiments, each copy of the GAPDH promoter on the first expression vector and each copy of the GAPDH promoter on the second expression vector comprises a nucleotide sequence that is 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 1. In some embodiments, each copy of the GAPDH promoter on the first expression vector and each copy of the GAPDH promoter on the second expression vector comprises the nucleotide sequence of SEQ ID NO: 1.
[0108] In some embodiments, the promoter operably linked to a nucleotide sequence encoding a selectable marker on the first expression vector is mPGK. In some embodiments, the promoter operably linked to a nucleotide sequence encoding a selectable marker on the second expression vector is mPGK. In some embodiments, the promoter operably linked to a nucleotide sequence encoding a selectable marker on the first expression vector is mPGK, and the promoter operably linked to a nucleotide sequence encoding a selectable marker on the second expression vector is mPGK.
[0109] In some embodiments, the selectable marker on the first expression vector is glutamine synthetase. In some embodiments, the selectable marker on the second expression vector is glutamine synthetase. In some embodiments, the selectable marker on the first expression vector and the selectable marker on the second expression vector is glutamine synthetase.
[0110] In some embodiments, the promoter operably linked to a nucleotide sequence encoding a selectable marker on the first expression vector is mPGK, the promoter operably linked to a nucleotide sequence encoding a selectable marker on the second expression vector is mPGK, and the selectable marker on the first expression vector and the selectable marker on the second expression vector is glutamine synthetase.
[0111] In some embodiments, each of the first, second, and third polyA signal sequences on the first expression vector is a simian virus 40 (SV40) early polyA signal sequence. In some embodiments, each of the first, second, and third polyA signal sequences on the second expression vector is a simian virus 40 (SV40) early polyA signal sequence. In some embodiments, each of the first, second, and third polyA signal sequences on the first expression vector is a simian virus 40 (SV40) early polyA signal sequence, and each of the first, second, and third polyA signal sequences on the second expression vector is a simian virus 40 (SV40) early polyA signal sequence.
[0112] In some embodiments, each of the first, second, and third polyA signal sequences on the first expression vector and the second expression vector is a simian virus 40 (SV40) early polyA signal sequence, the promoter operably linked to a nucleotide sequence encoding a selectable marker on the first expression vector is mPGK, the promoter operably linked to a nucleotide sequence encoding a selectable marker on the second expression vector is mPGK, and the selectable marker on the first expression vector and the selectable marker on the second expression vector is glutamine synthetase. [0113] In some embodiments, each copy of the GAPDH promoter on the first expression vector and each copy of the GAPDH promoter on the second expression vector is a CMV/GAPDH promoter, each of the first, second, and third polyA signal sequences on the first expression vector and the second expression vector is a simian virus 40 (SV40) early polyA signal sequence, the promoter operably linked to a nucleotide sequence encoding a selectable marker on the first expression vector is mPGK, the promoter operably linked to a nucleotide sequence encoding a selectable marker on the second expression vector is mPGK, and the selectable marker on the first expression vector and the selectable marker on the second expression vector is glutamine synthetase.
[0114] In some embodiments, the first antibody light chain and the second antibody light chain are different, the first antibody heavy chain and the second antibody heavy chain are different, each copy of the GAPDH promoter on the first expression vector and each copy of the GAPDH promoter on the second expression vector is a CMV/GAPDH promoter, each of the first, second, and third polyA signal sequences on the first expression vector and the second expression vector is a simian virus 40 (SV40) early polyA signal sequence, the promoter operably linked to a nucleotide sequence encoding a selectable marker on the first expression vector is mPGK, the promoter operably linked to a nucleotide sequence encoding a selectable marker on the second expression vector is mPGK, and the selectable marker on the first expression vector and the selectable marker on the second expression vector is glutamine synthetase.
[0115] In alternative embodiments, on the first expression vector, the nucleotide sequence encoding the first antibody heavy chain may precede the nucleotide sequence encoding the first antibody light chain (in 5’ to 3’ order). Additionally, in alternative embodiments, on the second expression vector, the nucleotide sequence encoding the second antibody heavy chain may precede the nucleotide sequence encoding the second antibody light chain (in 5’ to 3’ order). Furthermore, in alternative embodiments, on the first expression vector, the nucleotide sequence encoding the first antibody heavy chain may precede the nucleotide sequence encoding the first antibody light chain, and, on the second expression vector, the nucleotide sequence encoding the second antibody heavy chain may precede the nucleotide sequence encoding the second antibody light chain (all in 5’ to 3’ order). [0116] The present disclosure also provides a mammalian host cell comprising any of the expression vectors described above or any of the pairs of expression vectors described above. In one embodiment, the mammalian host cell is a Chinese Hamster Ovary (CHO) cell. In one aspect of this embodiment, the CHO cell is a dihydrofolate reductase deficient (dhfr-) CHO cell or a glutamine synthetase knockout (GSKO) CHO cell.
[0117] The present disclosure also provides methods for producing an antibody modality comprising culturing any of the mammalian host cells described above or herein under conditions in which the antibody chains are expressed, and recovering the antibody modality from the culture. In one embodiment, the recovered antibody modality is purified and formulated in a pharmaceutically acceptable formulation. In some embodiments, a “pharmaceutically acceptable formulation” refers to a formulation that is generally safe, non-toxic, and neither biologically nor otherwise undesirable for use in a subject.
[0118] Further aspects and advantages will be apparent to those of ordinary skill in the art from a review of the following detailed description. The description hereafter includes specific cases, embodiments, and examples with the understanding that the disclosure is illustrative and is not intended to limit the embodiments of the present disclosure to the specific cases, embodiments, and examples described herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0119] FIG. 1 is an example of plasmid map for recombinant expression of an antibody molecule having one light chain and one heavy chain.
[0120] FIGs. 2A-2B show Pool Titer in g/L (A) and Cell Specific Productivity (qp) in pg/cell/day (B) from each light chain (LC), heavy chain (heavy chain fusion) (HC) and glutamine synthetase (GS) promoter and MSX combination. Titer was measured on day 10 of a fed-batch production. Data is normalized and presented as mean values for two independent transfections. Error bars represent the standard deviation (SD) of two technical replicates for fed-batch production. Data is shaded based on MSX concentrations (0 pM, 12.5 pM and 25 pM).
[0121] FIGs. 3A-3C show Viability (A), Viable Dell Density (VCD) (B) and Integrated Viable Cell Density (VCD) (C) in cell culture of CHO cell pools from the different vector configurations. Data is presented as mean values for two independent transfections. Error bars represent the standard deviation (SD) of two technical replicates for fed-batch production. Data is shaded based on MSX concentrations (0 pM, 12.5 pM and 25 pM).
[0122] FIGs. 4A-4B show the pool Titer in g/L (A) and Cell Specific Productivity (qp) in pg/cell/day (B) from each light chain (LC), heavy chain (HC) and glutamine synthetase (GS) promoter and MSX combination. Titer was measured on day 10 of a fed-batch production. Data is normalized and presented as mean values for two independent transfections. Error bars represent the standard deviation (SD) of three technical replicates for fed-batch production. Data is shaded based on light and heavy chain promoters (CMV/ADL, CMV/GAPDH and CMV/EF1 + CMV/GAPDH), GS promoters (SRa, mPGK and SV40) and MSX concentrations (0 pM, 12.5 pM, 25 pM and 50 pM). [0123] FIGs. 5A-5C show the product quality in cell culture of CHO cell pools from the different vector configurations: A: SEC-HMW (%); B: rCE-Clipping; C: HIC-HPLC. Data was measured on day 10 of a fed-batch production. Data is presented as mean values for two independent transfections. Error bars represent the standard deviation (SD) of three technical replicates for fed-batch production. Data is shaded based on light and heavy chain promoters (CMV/ADL, CMV/GAPDH and CMV/EF1 + CMV/GAPDH), GS promoters (SRa, mPGK and SV40) and MSX concentrations (0 pM, 12.5 pM, 25 pM and 50 pM).
[0124] FIGs. 6A-6C show the Viability (A), Viable Dell Density (VCD) (B) and Integrated Viable Cell Density (VCD) (C) in cell culture of CHO cell pools from the different vector configurations. Data is presented as mean values for two independent transfections. Error bars represent the standard deviation (SD) of three technical replicates for fed-batch production. Data is shaded based on MSX concentrations (0 pM, 12.5 pM, 25 pM and 50 pM).
[0125] FIGs. 7A-7B show Pool Titer in g/L (A) and Cell Specific Productivity in pg/cell/day (B) from each light chain (LC), heavy chain (HC) and glutamine synthetase (GS) promoter and MSX combination for 2+2 ScFv molecule A. Titer was measured on day 10 of a fed-batch production. Data is normalized and presented as mean values for two independent transfections. Error bars represent the standard deviation (SD) of three technical replicates for fed-batch production. Data is shaded based on light and heavy chain promoters (CMV/ADL and CMV/GAPDH), GS promoters (SRa, mPGK and SV40) and MSX concentrations (0 pM, 12.5 pM, 25 pM and 50 pM).
[0126] FIGs. 8A-8B show Pool Titer in g/L (A) and Cell Specific Productivity in pg/cell/day (B) from each light chain (LC), heavy chain (HC) and glutamine synthetase (GS) promoter and MSX combination for 2+2 ScFv molecule B. Titer was measured on day 10 of a fed-batch production. Data is normalized and presented as mean values for two independent transfections. Error bars represent the standard deviation (SD) of three technical replicates for fed-batch production. Data is shaded based on light and heavy chain promoters (CMV/ADL and CMV/GAPDH), GS promoters (SRa, mPGK and SV40) and MSX concentrations (0 pM, 12.5 pM and 25 pM). [0127] FIGs. 9A-9C show the product quality in cell culture of CHO cell pools from the different vector configurations for a 2+2 ScFv molecule A: A: SEC-HMW (%); B: rCE-Clipping; C: HIC- HPLC. Data was measured on Day 10 of a fed-batch production. Data is presented as mean values for two independent transfections. Error bars represent the standard deviation (SD) of three technical replicates. Data is shaded based on light and heavy chain promoters (CMV/ADL and CMV/GAPDH), GS promoters (SRa, mPGK and SV40) and MSX concentrations (OpM, 12.5pM, 25pM and 50pM). [0128] FIGs. 10A-10C show the product quality in cell culture of CHO cell pools from the different vector configurations for 2+2 ScFv molecule B: A: SEC-HMW (%); B: rCE-Clipping; C: HIC-HPLC. Data was measured on Day 10 of a fed-batch production. Data is presented as mean values for two independent transfections. Error bars represent the standard deviation (SD) of three technical replicates. Data is shaded based on light and heavy chain promoters (CMV/ADL and CMV/GAPDH), GS promoters (SRa, mPGK and SV40) and MSX concentrations (OpM, 12.5pM and 25pM).
[0129] FIGs. 11A-11C show Viability (A), Viable Dell Density (VCD) (B) and Integrated Viable Cell Density (VCD) (C) for 2+2 ScFv molecule A in cell culture of CHO cell pools from the different vector configurations. Data is presented as mean values for two independent transfections. Error bars represent the standard deviation (SD) of three technical replicates for fed-batch production. Data is shaded based on MSX concentrations (0 pM, 12.5 pM, 25 pM and 50 pM).
[0130] FIGs. 12A-12C show Viability (A), Viable Dell Density (VCD) (B) and Integrated Viable Cell Density (VCD) (C) for 2+2 ScFv molecule B in cell culture of CHO cell pools from the different vector configurations. Data is presented as mean values for two independent transfections. Error bars represent the standard deviation (SD) of three technical replicates for fed-batch production. Data is shaded based on MSX concentrations (0 pM, 12.5 pM and 25 pM).
[0131] FIGs. 13A-13B show normalized titers (A) and normalized Protein A (proA) yields (B) in mg/L from each light chain (LC), heavy chain (HC) and glutamine synthetase (GS) promoter combinations in two different GS hosts (Hl and H2) in either monocistronic or bicistronic configurations. Titer was measured on day 10 of a fed-batch production. ProA yields were calculated by measuring protein concentrations after purification.
[0132] FIG. 14 shows Product quality (% main Peak in SEC) in cell culture of CHO cell pools from the different vector configurations.
[0133] FIG. 15 shows Protein A recovery in g/L from each light chain (LC), heavy chain (HC) and glutamine synthetase (GS) promoter. Titer was measured on day 7 of a batch production.
[0134] FIGs. 16A-16B show Product quality (A) % main Peak in SEC, and (B) %main peak in nonreduced capillary electrophoresis (nrMCE) in cell culture of CHO cell pools from the different vector configurations.
[0135] FIGs. 17A-17B show Pool Titer in g/L (A) and Cell Specific Productivity in pg/cell/day (B) from each light chain (LC)/heavy chain (HC) promoter combination and glutamine synthetase (GS) promoter and MSX combination for a B2Hmab and a C2Hmab. Titer was measured on day 10 of a fed-batch production. Data is normalized and presented as mean values for two independent transfections. Error bars represent the standard deviation (SD) of three technical replicates for fed- batch production. Data is shaded based on light and heavy chain promoters (CMV/ADL and CMV/GAPDH), GS promoters (SRa and mPGK) and MSX concentrations (18.75 pM and 37.5 pM). [0136] FIGs. 18A-18C show Product quality (A) % main Peak in SEC, (B) % main peak in nonreduced capillary electrophoresis (nrMCE), and (C) B: rCE-Clipping; in cell culture of CHO cell pools from the different vector configurations for a B2Hmab and a C2Hmab. Data is normalized and presented as mean values for two independent transfections. Error bars represent the standard deviation (SD) of three technical replicates for fed-batch production. Data is shaded based on light and heavy chain promoters (CMV/ADL and CMV/GAPDH), GS promoters (SRa and mPGK) and MSX concentrations (18.75 pM and 37.5 pM).
[0137] FIGs. 19A-19C show Viability (A), Viable Dell Density (VCD) (B) and Integrated Viable Cell Density (VCD) (C) for a B2Hmab and a C2Hmab in cell culture of CHO cell pools from the different vector configurations. Data is normalized and presented as mean values for two independent transfections. Data is shaded based on MSX concentrations (18.75 pM and 37.5 pM).
[0138] FIG. 20A is a representative scheme for an expression cassette for a mAb. [0139] FIG. 20B is a representative scheme for an expression cassette for a B2/C2 mAb. [0140] FIG. 20C is a representative scheme for a pair of expression cassettes for a ClmAb, asymmetric fusion.
[0141] FIG. 20D is a representative scheme for a pair of expression cassettes for a heteroIgG mAb.
DETAILED DESCRIPTION
[0142] The present disclosure is based, in part, on the discovery that certain combinations of promoters in expression cassettes used to drive expression of different antibody chains can increase the titer of the antibody being produced, including standard antibodies (2 chain), multi-specific antibodies with 2 chains, and antibody modalities with 3- or 4 chains, such as modalities in which one or two chains are modified with scFv, cytokines, VH, etc. Expression cassettes having specific arrangements of promoters and antibody chains and a selectable marker can be integrated into expression vectors for expression in mammalian host cells to produce antibodies. This is also true for antibody modalities having three- or four-chains expressed on two different vectors. Two different expression cassettes can be employed on two different expression vectors in order to produce antibody modalities having three- or four- chains. A three- or four-chain antibody modality can have a heavy chain, a heavy chain-scFv fusion, or a heavy chain-VH fusion and two light chains (the light chains being the same in the case of a 3-chain antibody modality or different in the case of a 4-chain modality). Another example of a four-chain antibody modality is a modality comprising two different heavy chains and two different light chains, where each heavy chain - light chain pair is expressed from a different vector.
[0143] Standard antibody production techniques often employ different promoters for the expression of the heavy chain and the light chain to optimize expression of the antibody. This is often necessary because the heavy and light chain are expressed at different levels. The situation becomes even more complicated when the antibody structures contain three or four chains. The inventors have surprisingly found that, by employing specific expression cassettes for different antibody modalities, the titers of the resulting antibody structures can be increased with improved product quality, such as, for example, reduced aggregation, clipping, or presence of non-desired isoforms. Additionally, by using the expression cassettes and expression vectors described herein in mammalian host production cell lines, biopharmaceuticals may be produced in a less expensive and more consistent manner. The expression cassettes, expression vectors, and mammalian host cells may find particular utility in the commercial production of standard antibodies and antibody modalities having two, three, or four unique chains.
[0144] The expression cassettes and expression vectors described herein are employed in cell lines (also referred to as “host cells”), preferably mammalian (“mammalian host cells”), grown in cell culture media to produce a recombinant protein of commercial or scientific interest. Cell lines are typically derived from a lineage arising from a primary culture that can be maintained in culture for an unlimited time. Genetically engineering the cell line involves transfecting, transforming or transducing the cells with one or two expression vectors where each vector contains nucleotide sequences encoding two antibody chains so as to cause the host cell to express an antibody modality having the desired number of chains. Methods and vectors for genetically engineering cells and/or cell lines to express, for example, a protein of interest, are well known to those of skill in the art; for example, various techniques are illustrated in Current Protocols in Molecular Biology. Ausubel et al., eds. (Wiley & Sons, New York, 1988, and quarterly updates); Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Laboratory Press, 1989); Kaufman, R.J., 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, N.Y. (1990).
DEFINITIONS
[0145] While the terminology used in this application is standard within the art, definitions of certain terms are provided herein to assure clarity and definiteness in the meaning of the claims. Units, prefixes, and symbols may be denoted in their SI (International System of Units) accepted form. Numeric ranges recited herein are inclusive of the numbers defining the range and include and are supportive of each integer within the defined range. The methods and techniques described herein are generally performed according to conventional methods well-known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated.
[0146] As used herein, the terms “a” and “an” mean one or more unless specifically indicated otherwise. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art.
[0147] All documents, or portions of documents, cited in this application, including but not limited to patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference. What is described in an embodiment of the invention can be combined with other embodiments of the invention.
[0148] The present disclosure provides tools for expressing a “protein of interest,” generally an antibody modality. A “protein of interest” includes naturally occurring proteins, recombinant proteins, and engineered proteins (e.g., proteins that do not occur in nature and which have been designed and/or created by humans). A protein of interest can, but need not be, a protein that is known or suspected to be therapeutically relevant.
[0149] As used herein, an “antibody chain” or “chain” refers to antibody light chains and antibody heavy chains. The terms “antibody heavy chain” and “antibody light chain” have the standard meaning in the art and include, for example, the various antibody heavy and light chains described elsewhere herein (e.g., heavy and light chains of IgGl, IgG2, IgG3, and IgG4 mAbs). The terms “antibody heavy chain” and “antibody light chain” include standard full-length antibody heavy chains and light chains.
[0150] As used herein, the terms “antibody heavy chain fusion” and “antibody heavy chain fusion protein” refer to a polypeptide that contains an antibody heavy chain covalently linked to one or more additional proteins or peptides. For example, an “antibody heavy chain fusion protein” can be an antibody heavy chain covalently linked to a cytokine, scFv, VH, and the like. The linkage may be direct, or via a peptide linker (e.g., a glycine-serine linker). In an antibody heavy chain fusion protein, the antibody heavy chain may be linked to additional protein(s) at the N- terminus or the C-terminus of the heavy chain (or both locations). The antibody heavy chain may also be linked to additional protein sequences, such as an scFv, at an internal amino acid residue or be between the Fab and the Fc. The terms “antibody light chain fusion protein” and “antibody light chain fusion” have the same meaning as described immediately above for “antibody heavy chain fusion protein”, except for an antibody light chain. As used herein, an “antibody fusion protein” refers to an antibody as provided herein which is covalently linked to one or more additional proteins or polypeptides (e.g., via a heavy chain or light chain of the antibody). Thus, an antibody fusion protein contains at least an antibody heavy chain fusion protein or an antibody light chain fusion protein as one of the polypeptides of the antibody fusion protein. Most commonly, an antibody fusion protein is a molecule that contains two antibody light chains, one antibody heavy chain, and one antibody heavy chain fusion protein, such that the additional protein is linked to one of the heavy chains of the antibody. An “antibody chain fusion” encompasses both antibody heavy chain fusions and antibody light chain fusions.
[0151] As used herein, “antibody modality” refers to a protein having at least one antibody chain. An antibody modality can have two, three, or four unique chains, wherein any or all of the chains can include fusions. Reference to two, three, or four chain molecules implies that each chain is unique. Any and all of the antibody chain containing molecules described herein are considered antibody modalities.
[0152] As used herein, the terms “polypeptide” and “protein” (e.g., as used in the context of a protein of interest or a polypeptide of interest) are used interchangeably herein to refer to a polymer of amino acid residues. The terms also apply to amino acid polymers in which one or more amino acid residues is an analog or mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The terms can also encompass amino acid polymers that have been modified, e.g., by the addition of carbohydrate residues to form glycoproteins, or phosphorylated. Polypeptides and proteins can be produced by a naturally-occurring and non-recombinant cell, or polypeptides and proteins can be produced by a genetically-engineered or recombinant cell. Polypeptides and proteins can comprise molecules having the amino acid sequence of a native protein, or molecules having deletions from, additions to, and/or substitutions of one or more amino acids of the native sequence.
[0153] As used herein, the term “heterologous” used in connection with a nucleic acid means having a nucleic acid not naturally occurring within a host cell. This can include mutated sequences, e.g., sequences differing from the naturally occurring sequence. This can include sequences from other species. This can also include having a sequence at a different position in the genome than that naturally occurring in the host cell. This generally does not include natural mutations that may occur in a host cell. A cell already containing a heterologous nucleic acid encoding a protein of interest, for example, by stable integration of an expression cassette, would be considered to contain a heterologous nucleic acid sequence. For clarity, a CHO cell or a derivative thereof (e.g., a DHFR- or GS knockout) having a nucleic acid encoding an antigen binding protein would be considered to have a heterologous nucleic acid.
[0154] As used herein, the term “operably linked” refers to that the nucleic acid sequences being linked are typically contiguous, or substantially contiguous, and, where necessary to join two protein coding regions, contiguous and in reading frame. However, since enhancers generally function when separated from the promoter by several kilobases and intronic sequences may be of variable lengths, some polynucleotide elements may be operably linked but not contiguous. Two or more nucleic acid sequences may be operably linked in such a manner that a nucleic acid molecule capable of directing the transcription of a given gene and/or the synthesis of a desired protein molecule is produced.
[0155] As used herein, the term “bioreactor” means any vessel useful for the growth of a cell culture. The cell cultures of the instant disclosure can be grown in a bioreactor, which can be selected based on the application of a protein of interest that is produced by cells growing in the bioreactor. A bioreactor can be of any size so long as it is useful for the culturing of cells; typically, a bioreactor is sized appropriate to the volume of cell culture being grown inside of it. Typically, a bioreactor will be at least 1 liter and may be 2, 5, 10, 50, 100, 200, 250, 500, 1,000, 1500, 2000, 2,500, 5,000, 8,000, 10,000, 12,000 liters or more, or any volume in between. The internal conditions of the bioreactor, including, but not limited to pH and temperature, can be controlled during the culturing period. Those of ordinary skill in the art will be aware of, and will be able to select, suitable bioreactors for use in practicing the methods disclosed herein based on the relevant considerations.
[0156] As used herein, “cell culture” or “culture” is meant to refer to the growth and propagation of cells outside of a multicellular organism or tissue. Suitable culture conditions for mammalian cells are known in the art. See e.g. Animal cell culture: A Practical Approach, D. Rickwood, ed., Oxford University Press, New York (1992). Mammalian cells may be cultured in suspension or while attached to a solid substrate. Fluidized bed bioreactors, hollow fiber bioreactors, roller bottles, shake flasks, or stirred tank bioreactors, with or without microcarriers, can be used. 500L to 2000L bioreactors can be used as well as WOOL to 2000L bioreactors.
[0157] The term “cell culture medium” (also called “culture medium,” “cell culture media,” or “tissue culture media”) refers to any nutrient solution used for growing cells, e.g., animal or mammalian cells, and which generally provides at least one or more components from the following: an energy source (usually in the form of a carbohydrate such as glucose); one or more of all essential amino acids, and generally the twenty basic amino acids, plus cysteine; vitamins and/or other organic compounds typically required at low concentrations; lipids or free fatty acids; and trace elements, e.g., inorganic compounds or naturally occurring elements that are typically required at very low concentrations, usually in the micromolar range.
[0158] The nutrient solution may optionally be supplemented with additional optional components to optimize growth of cells, such as hormones and other growth factors, e.g., transferrin, epidermal growth factor, insulin, insulin-like growth factor, serum, and the like; salts, e.g., calcium, magnesium and phosphate, and buffers, e.g., HEPES; nucleosides, and bases, e.g., adenosine, thymidine, hypoxanthine; and protein and tissue hydrolysates, e.g., hydrolyzed animal or plant protein (peptone or peptone mixtures, which can be obtained from animal byproducts, purified gelatin or plant material); antibiotics, e.g., gentamycin; anti-clumping agents; cell protectants or surfactants such as Pluronic®F68 (also referred to as Lutrol® F68 and Kolliphor® P188; nonionic triblock composed of a central hydrophobic chain of polyoxypropylene (polypropylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (polyethylene oxide)); polyamines, e.g., putrescine, spermidine and spermine (see e.g., International Patent Application Publication No. WO 2008/154014) and pyruvate (see e.g. U.S. Pat. No. 8,053,238) depending on the requirements of the cells to be cultured and/or the desired cell culture parameters.
[0159] Cell culture media include those that are typically employed in and/or are known for use with any cell culture process, such as, but not limited to, batch, extended batch, fed-batch, and/or perfusion or continuous culturing of cells.
[0160] A “base” (or batch) cell culture medium refers to a cell culture medium that is typically used to initiate a cell culture and is sufficiently complete to support the cell culture.
[0161] A “fed-batch culture” refers to a form of suspension culture and means a method of culturing cells in which additional components are provided to the culture at a time or times subsequent to the beginning of the culture process. The provided components typically comprise nutritional supplements for the cells which have been depleted during the culturing process. Additionally, or alternatively, the additional components may include supplementary components (e.g., a cell-cycle inhibitory compound). A fed-batch culture is typically stopped at some point and the cells and/or components in the medium are harvested and optionally purified.
[0162] A “growth” cell culture medium refers to a cell culture medium that is typically used in cell cultures during a period of exponential growth, a “growth phase,” and is sufficiently complete to support the cell culture during this phase. A growth cell culture medium may also contain selection agents that confer resistance or survival to selectable markers incorporated into the host cell line. Such selection agents include, but are not limited to, geneticin (G418), neomycin, hygromycin B, puromycin, zeocin, methionine sulfoximine, methotrexate, glutamine-free cell culture medium, cell culture medium lacking glycine, hypoxanthine and thymidine, or thymidine alone.
[0163] A “perfusion” cell culture medium refers to a cell culture medium that is typically used in cell cultures that are maintained by perfusion or continuous culture methods and is sufficiently complete to support the cell culture during this process. Perfusion cell culture medium formulations may be richer or more concentrated than base cell culture medium formulations to accommodate the method used to remove the spent medium. Perfusion cell culture medium can be used during both the growth and production phases.
[0164] A “production” cell culture medium refers to a cell culture medium that is typically used in cell cultures during the transition when exponential growth is ending and protein production takes over, “transition” and/or “product” phases, and is sufficiently complete to maintain a desired cell density, viability and/or product titer during this phase.
[0165] Concentrated cell culture medium can contain some or all of the nutrients necessary to maintain the cell culture; in particular, concentrated medium can contain nutrients identified as or known to be consumed during the course of the production phase of the cell culture. Concentrated medium may be based on just about any cell culture media formulation. Such a concentrated feed medium can contain some or all the components of the cell culture medium at, for example, about 2X, 3X, 4X, 5X, 6X, 7X, 8X, 9X, 10X, 12X, 14X, 16X, 20X, 30X, 50X, lOOx, 200X, 400X, 600X, 800X, or even about 1000X of their normal amount.
[0166] The components used to prepare cell culture medium may be completely milled into a powder medium formulation; partially milled with liquid supplements added to the cell culture medium as needed; or added in a completely liquid form to the cell culture.
[0167] Cell cultures can also be supplemented with independent concentrated feeds of particular nutrients which may be difficult to formulate or are quickly depleted in cell cultures. Such nutrients may be amino acids such as tyrosine, cysteine and/or cystine (see e.g., International Patent Application Publication No. WO2012/145682). The independent feeds can begin prior to or at the start of the production phase. The independent feeds can be accomplished by fed batch to the cell culture medium on the same or different days as the concentrated feed medium. The independent feeds can also be perfused on the same or different days as the perfused medium.
[0168] “Serum -free” applies to a cell culture medium that does not contain animal sera, such as fetal bovine serum. Various tissue culture media, including defined culture media, are commercially available, for example, any one or a combination of the following cell culture media can be used: RPMI-1640 Medium, RPMI-1641 Medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium Eagle, F-12K Medium, Ham's F12 Medium, Iscove's Modified Dulbecco's Medium, McCoy's 5 A Medium, Leibovitz's 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), among others. Serum -free versions of such culture media are also available. Cell culture media may be supplemented with additional or increased concentrations of components such as amino acids, salts, sugars, vitamins, hormones, growth factors, buffers, antibiotics, lipids, trace elements and the like, depending on the requirements of the cells to be cultured and/or the desired cell culture parameters. Customized cell culture media can also be used.
[0169] “Titer” means the total amount of a polypeptide or protein of interest (which may be a naturally occurring or recombinant protein of interest) produced by a cell culture in a given amount of medium volume. Titer can be expressed in units of milligrams or micrograms of polypeptide or protein per milliliter (or other measure of volume) of medium. “Cumulative titer” is the titer produced by the cells during the course of the culture, and can be determined, for example, by measuring daily titers and using those values to calculate the cumulative titer.
[0170] As used herein, the term “host cell” is understood to include a cell that has been genetically engineered to express a polypeptide of interest. Genetically engineering a cell involves transfecting, transforming, or transducing the cell with a nucleic acid encoding a recombinant polynucleotide molecule (a “gene of interest”), and/or otherwise altering (e.g., by homologous recombination and gene activation or fusion of a recombinant cell with a non-recombinant cell) so as to cause the host cell to express a desired recombinant polypeptide. Methods and vectors for genetically engineering cells and/or cell lines to express a polypeptide of interest are well-known to those of skill in the art; for example, various techniques are illustrated in Current Protocols in Molecular Biology. Ausubel et al., eds. (Wiley & Sons, New York, 1988, and quarterly updates); Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Laboratory Press, 1989); Kaufman, R.J., Large Scale Mammalian Cell Culture, 1990, pp. 15-69. The term includes the progeny of the parent cell, whether or not the progeny is identical in morphology or in genetic makeup to the original parent cell, so long as the gene of interest is present. A cell culture can comprise one or more host cells.
[0171] It is understood that wherever embodiments are described herein with the language “comprising,” otherwise analogous embodiments described in terms of “consisting of’ and/or “consisting essentially of’ are also provided.
EXPRESSION CASSETTES
[0172] Expression and cloning will typically include expression cassettes that contain one or more promoters that are recognized by the host organism and are operably linked to the nucleotide sequence encoding a protein of interest. Promoters are untranscribed sequences located upstream (i.e., 5') to the start codon of a structural gene (generally within about 100 to 1000 bp) that control transcription of the structural gene.
[0173] An expression cassette for an antibody modality will generally contain a first promoter driving expression of a first nucleotide sequence encoding a first antibody chain or antibody chain fusion, a second promoter driving expression of a second nucleotide sequence encoding a second antibody chain or antibody chain fusion, and a promoter driving expression of a coding sequence encoding a selectable marker. A polyA tail can follow each gene (i.e., the coding sequences encoding the first and second antibody or antibody fusion chains and the selectable marker). In embodiments disclosed herein, the first promoter and the second promoter are both glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoters. In certain embodiments, the GAPDH promoter is operably linked to the CMV promoter enhancer; the resulting construct referred to as a CMV/GADPH promoter.
[0174] A typical antibody is a Y-shaped molecule having four polypeptide chains - two identical heavy chains and two identical light chains. Such an antibody is preferably expressed from a single vector. However, bispecific antibodies require the use of alternative formats and can be expressed on a single vector or two different vectors depending on the number of chains being produced. See, e.g., Spiess et al., 2015, Mol. Immunol. 67:95-106; Brinkmann et al., 2017, MAbs 9: 192-212; and Ma et al., 2021, Frontiers in Immunology 12:626616. [0175] In one embodiment, a single expression cassette is used, i.e., 2 chains, 1 vector. In certain aspects, the expression cassette is useful for the expression of a monoclonal antibody (having one heavy chain sequence and one light chain sequence) or a symmetrical fusion (for example, having a heavy chain sequence fused to a scFv or VH and a light chain sequence), such as a B2 mab (Fab- scFv-Fc) or C2 mab (IgG-scFv) mAb, B2Hmab (Fab-VH-Fc), C2Hmab (IgG-VH) or (IgG-cytokine) mAb (2 chains, 1 vector). The first antibody chain can be either the light chain or the heavy chain/heavy chain-scFv fusion/heavy chain-VH fusion. In one aspect, the first antibody chain is the light chain and the second antibody chain is either a heavy chain, a heavy chain-scFv fusion, or a heavy chain-VH fusion.
[0176] A representative scheme for a mAb is depicted in FIG. 20A.
[0177] A representative scheme for a B2 mab or C2 mAb is depicted in FIG. 20B.
[0178] In other embodiments, two expression cassettes are used. In certain aspects, the two expression cassettes are useful for the expression of a three-chain or four-chain antibody modality. For example, for a three-chain modality, one expression cassette contains coding sequences for one heavy chain and one light chain and the other expression cassette contains coding sequences for a heavy chain-scFv fusion and a light chain (3 chains, 2 vectors). The heavy chain sequences can be the same or different. The light chain sequences can be the same or different. An example of three-chain modality is a ClmAb (Fab-heteroFc-[scFv*], asymmetric fusion). The first antibody chain is each expression cassette can be either the light chain or the heavy chain/heavy chain-scFv fusion. In one aspect, the first antibody chain is the light chain and the second antibody chain is either a heavy chain or a heavy chain-scFv fusion. Another example is a ClmAb (Fab-heteroFc-[cytokine], asymmetric fusion).
[0179] A representative scheme for a ClmAb, asymmetric fusion is depicted in FIG. 20C.
[0180] For example, for a four-chain modality, one expression cassette contains coding sequences for a first light chain and a first heavy chain and the second expression cassette contains a second heavy chain and a second light chain. This represents a heteroIgG mAb. The first antibody chain in each expression cassette can be either the light chain or the heavy chain.
[0181] In another aspect, the first antibody chain is the light chain, and the second antibody chain is either a heavy chain, a heavy chain-scFv fusion, or heavy chain-VH fusion.
[0182] A representative scheme for a heteroIgG mAb is depicted in FIG. 20D.
[0183] Promoters of particular interest for 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) and hamster glyceraldehyde-3-phosphate dehydrogenase promoter and intron (GAPDH) (U.S. Patent No. 10,202,261). Additional sequences can also be combined with promoters to improve expression. One such example is the adenovirus tripartite leader (ADL). (See Gingeras et al., 1982, J. Biol. Chem. 257: 13475-91, GenBank Accession No. JO 1917) . [0184] It has been found that using CMV/GAPDH as the promoter driving expression of all of the antibody chains for certain modalities surprisingly results in higher production of the antibody modality, as illustrated in the Examples of this application. In embodiments described herein, the promoter for the chains of the antibody modality is the combination of the CMV promoter enhancer and GAPDH (CMV/GAPDH). In this combination, both the CMV promoter enhancer and GAPDH promoter are operably linked to the nucleotide sequence encoding the antibody chain, such that the combination may be a better promoter than the GAPDH promoter alone. In certain embodiments, the CMV promoter is 5 ’ of the GAPDH. Collectively, CMV/GAPDH is referred to as a promoter. A representative CMV/GAPDH promoter is provided in SEQ ID NO: 1.
[0185] In some embodiments, each GAPDH promoter of an expression cassette is a CMV/GAPDH promoter. In some embodiments, each CMV/GAPDH promoter of an expression cassette comprises a nucleotide sequence that is 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 1. In some embodiments, each CMV/GAPDH promoter comprises the nucleotide sequence of SEQ ID NO: 1.
[0186] In some embodiments, each GAPDH promoter of an expression cassette in a pair of expression vectors is a CMV/GAPDH promoter. In some embodiments, each CMV/GAPDH promoter of an expression cassette in a pair of expression vectors comprises a nucleotide sequence that is 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 1. In some embodiments, each CMV/GAPDH promoter comprises the nucleotide sequence of SEQ ID NO: 1. [0187] In the expression cassettes described herein, a polyA signal sequence can follow each gene (i.e., the coding sequences encoding the antibody or antibody fusion chains and the selectable marker). PolyA signal sequences are known in the art and include a bovine growth hormone (BGH) polyA signal sequence (e.g., Pfarr et al., 1986, DNA, 5(2): 115-22; Goodwin and Rottman, 1992, J. Biol. Chem., 267(23): 16330-16334), athymidine kinase polyA (TKpA) signal sequence (Cole and Stacy, 1985, Mol Cell Biol., 5(8):2104- 13), a rabbit beta-globin polyA signal sequence (Lanoix et al., 1988; EMBO J. 7(8):2515-22; GenBank Accession No. MG356850.1), and a simian virus 40 (SV40) early polyA signal sequence (Connelly and Manley, 1988, Genes Dev., 2(4):440-52; GenBank Accession No. J02400). In some embodiments, the first, second, and third polyA signal sequences on each expression cassette are independently selected from the group consisting of a bovine growth hormone (BGH) polyA signal sequence, a thymidine kinase polyA (TKpA) signal sequence, a rabbit beta-globin polyA signal sequence, and a simian virus 40 (SV40) early polyA signal sequence.
[0188] In some embodiments, each of the first, second, and third polyA signal sequences on each expression cassette is a bovine growth hormone (BGH) polyA signal sequence.
[0189] In some embodiments, each of the first, second, and third polyA signal sequences on each expression cassette is a thymidine kinase polyA (TKpA) signal sequence. [0190] In some embodiments, each of the first, second, and third polyA signal sequences on each expression cassette is a rabbit beta-globin polyA signal sequence.
[0191] In some embodiments, each of the first, second, and third polyA signal sequences on each expression cassette is a simian virus 40 (SV40) early polyA signal sequence.
[0192] In some embodiments, the BGH polyA signal sequence comprises a nucleotide sequence that is 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 5. In some embodiments, the BGH polyA signal sequence comprises the nucleotide sequence of SEQ ID NO: 5.
[0193] In some embodiments, the rabbit beta-globin polyA signal sequence comprises a nucleotide sequence that is 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 6. In some embodiments, the rabbit beta-globin polyA signal sequence comprises the nucleotide sequence of SEQ ID NO: 6.
[0194] In some embodiments, the SV40 early polyA signal sequence comprises a nucleotide sequence that is 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 7. In some embodiments, the SV40 early polyA signal sequence comprises the nucleotide sequence of SEQ ID NO: 7.
[0195] In some embodiments, the thymidine kinase polyA (TKpA) signal sequence comprises a nucleotide sequence that is 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 8. In some embodiments, the thymidine kinase polyA (TKpA) signal sequence comprises the nucleotide sequence of SEQ ID NO: 8.
[0196] The expression cassettes provided herein may provide improved expression, possibly due to improved chain ratios of the expressed polypeptides. Chain ratios can be measured using techniques well-known in the art.
SELECTABLE MARKERS
[0197] For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker is generally introduced into the host cells in the same expression vector as the gene(s) of interest.
[0198] A selectable marker gene encodes a protein necessary for the survival and growth of a host cell grown 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 for prokaryotic host cells; (b) complement auxotrophic deficiencies of the cell; or (c) supply critical nutrients not available from complex or defined media through metabolism. Specific antibiotic- resistance selectable markers are the kanamycin resistance gene, the ampicillin resistance gene, the tetracycline resistance gene, and the neomycin resistance gene.
[0199] Other selectable genes may be used to amplify the gene that will be expressed. Amplification is the process wherein genes that are required for production of a protein critical for growth or cell survival are reiterated in tandem within the chromosomes of successive generations of recombinant cells. Examples of suitable selectable markers for mammalian cells include, but are not limited to, glutamine synthetase (GS), dihydrofolate reductase (DHFR), asparaginase (Aspg; see Ha et al. Biotechnol Bioeng. 2023 120: 1159-1166), and promoterless thymidine kinase genes.
[0200] Mammalian cell transformants are placed under selection pressure wherein only the transformants are uniquely adapted to survive by virtue of the selectable gene present in the vector. Selection pressure is imposed by culturing the transformed cells under conditions in which the concentration of selection agent in the medium is successively increased, thereby leading to additional stringency and/or amplification of both the selectable gene and the DNA that encodes a protein of interest. As a result, increased quantities of a polypeptide of interest are synthesized from the amplified DNA. The selection agent for GS is methionine sulfoximine (MSX). The selection agent for DHFR is methotrexate (MTX).
[0201] Relative to DHFR-based systems, GS knockout cell lines (GSKO) provide sufficient selection stringency without MSX or with low MSX concentrations, while 25 pM MSX coupled with the GS- knockout cell line led to higher selection efficiency compared with CHOK1SV cell lines at higher MSX concentrations (Fan, et al., Biotechnol Bioeng., 109(4): 1007-1015 (2012)). A previous report showed that increasing the MSX concentration in the seed train stage after clone selection increased productivity without significant impacts on cell growth, GS, and target gene copy numbers and expression, and maintained product quality attributes in multiple GS knockout cell lines (Tian et al., Engineering in Life Sciences 20(3-4): 112-125 (2020)). Chain/vector expression can be influenced by increasing stringency during pool recovery/selection by adding MSX.
[0202] In certain embodiments, the MSX concentration can be optimized for one of the promoters driving GS expression. In a specific embodiment, the MSX concentration is optimized for the GS linked to the more difficult to express chain.
[0203] In certain embodiments, the selectable marker in an expression cassette is glutamine synthetase. Glutamine synthetase (GS) catalyzes glutamine biosynthesis by the condensation of ammonia with glutamate. In some embodiments, the selectable marker in each expression cassette is glutamine synthetase.
[0204] In certain embodiments, the promoter SRoc is operably linked to the selectable marker. In other embodiments, the promoter mPGK is operably linked to the selectable marker. Other suitable promoters can be selected from those well known in the art. [0205] The present disclosure provides expression cassettes comprising a polynucleotide sequence which comprises the following elements in 5’ to 3’ order: a first GAPDH promoter operably linked to a nucleotide sequence encoding a first antibody chain or antibody chain fusion followed by a first polyA signal sequence; a second GAPDH promoter operably linked to a nucleotide sequence encoding a second antibody chain or antibody chain fusion followed by a second polyA signal sequence; and a promoter operably linked to a nucleotide sequence encoding a selectable marker followed by a third polyA signal sequence. Such expression cassettes may be part of expression vectors or pairs of expression vectors in which each expression vector comprises an expression cassette having the aforementioned configuration.
[0206] In some embodiments, the selectable marker is glutamine synthetase.
[0207] In some embodiments, the promoter operably linked to the nucleotide sequence encoding the selectable marker is a SRoc promoter, a mPGK promoter, or a SV40 promoter. In some embodiments, the selectable marker is glutamine synthetase, and the promoter operably linked to the nucleotide sequence encoding the selectable marker is a SRoc promoter, a mPGK promoter, or a SV40 promoter. [0208] In some embodiments, the promoter operably linked to the nucleotide sequence encoding the selectable marker is a SRoc promoter. In some embodiments, the selectable marker is glutamine synthetase, and the promoter operably linked to the nucleotide sequence encoding the selectable marker is a SRoc promoter.
[0209] In some embodiments, the promoter operably linked to the nucleotide sequence encoding the selectable marker is a mPGK promoter. In some embodiments, the selectable marker is glutamine synthetase, and the promoter operably linked to the nucleotide sequence encoding the selectable marker is a mPGK promoter.
[0210] In some embodiments, the promoter operably linked to the nucleotide sequence encoding the selectable marker is a SV40 promoter. In some embodiments, the selectable marker is glutamine synthetase, and the promoter operably linked to the nucleotide sequence encoding the selectable marker is a SV40 promoter.
[0211] In some embodiments, the SRoc promoter comprises a nucleotide sequence that is 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 2. In some embodiments, the SRoc promoter comprises the nucleotide sequence of SEQ ID NO: 2.
[0212] In some embodiments, the mPGK promoter comprises a nucleotide sequence that is 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 3. In some embodiments, the mPGK promoter comprises the nucleotide sequence of SEQ ID NO: 3.
[0213] In some embodiments, the SV40 promoter comprises a nucleotide sequence that is 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 4. In some embodiments, the SV40 promoter comprises the nucleotide sequence of SEQ ID NO: 4. ADDITIONAL COMPONENTS OF EXPRESSION VECTORS
[0214] The expression cassetes described herein are employed in expression vectors. Such expression vectors are useful for transformation of a host cell and can contain additional nucleic acid sequences for plasmid maintenance and for cloning and expression of exogenous nucleotide sequences. Such sequences will typically include one or more of the following nucleotide sequences (in addition to the promoter(s)/enhancer fragment(s), antibody chains, selectable marker(s), and other sequences (e.g., polyadenylation sequences) described above): one or more enhancer sequences, an origin of replication, transcriptional and translational control sequences, a transcriptional termination sequence, a complete intron sequence containing a donor and acceptor splice site, various pre- or prosequences to improve glycosylation or yield, a native or heterologous signal sequence (leader sequence or signal peptide) for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, internal ribosome entry site (IRES) sequences, an expression augmenting sequence element (EASE), tripartite leader (TPL) and VA gene RNAs from Adenovirus 2, and a polylinker region for inserting the polynucleotide encoding the polypeptide to be expressed. Vectors may be constructed from a starting vector such as a commercially available vector, additional elements may be individually obtained and ligated into the vector. Methods used for obtaining each of the components are well known to one skilled in the art.
[0215] Vector components may be homologous (i.e., from the same species and/or strain as the host cell), heterologous (e.g., from a species other than the host cell species or strain), hybrid (i.e., a combination of flanking sequences from more than one source), synthetic or native. The sequences of components useful in the vectors may be obtained by methods well known in the art, such as those previously identified by mapping and/or by restriction endonuclease. In addition, they can be obtained by polymerase chain reaction (PCR) and/or by screening a genomic library with suitable probes. [0216] A ribosome-binding site is usually necessary fortranslation initiation of mRNA and is characterized by a Shine-Dalgamo sequence (prokaryotes) or a Kozak sequence (eukaryotes). The element is typically located 3' to the promoter and 5' to the coding sequence of the polypeptide to be expressed.
[0217] An origin of replication aids in the amplification of the vector in a host cell. They may be included as part of commercially available prokaryotic vectors and may also be chemically synthesized based on a known sequence and ligated into the vector. Various viral origins (e.g., SV40, polyoma, adenovirus, vesicular stomatitis virus (VSV), or papillomaviruses such as HPV or BPV) are useful for cloning vectors in mammalian cells.
[0218] Transcriptional and translational control sequences for mammalian host cell expression vectors can be excised from viral genomes. Commonly used enhancer sequences are derived from polyoma virus, adenovirus 2, simian virus 40 (SV40), and human cytomegalovirus (CMV). For example, the human CMV promoter/enhancer of immediate early gene 1 may be used. See e.g. Paterson et al., 1994, Applied Microbiol. Biotechnol. 40:691-98. DNA sequences derived from the SV40 viral genome, for example, SV40 origin, early and late promoter, enhancer, splice, and polyadenylation sites can be used to provide other genetic elements for expression of a structural gene sequence in a mammalian host cell. Viral early and late promoters are particularly useful because both are easily obtained from a viral genome as a fragment, which can also contain a viral origin of replication (Fiers et al., 1978, Nature 273: 113; Kaufman, 1990, Meth, in Enzymol. 185:487-511). Smaller or larger SV40 fragments can also be used, provided the approximately 250 bp sequence extending from the Hind III site toward the Bgll site located in the SV40 viral origin of replication site is included. For examples, an enhancer sequence may be inserted into the vector to increase transcription by higher eukaryotes. Enhancers are cis-acting elements of DNA, usually about 10-300 bp in length, that act on the promoter to increase transcription. Enhancers are relatively orientation and position independent, having been found at positions both 5' and 3' to the transcription unit. Several enhancer sequences available from mammalian genes are known (e.g., globin, elastase, albumin, alpha-feto-protein and insulin). Typically, however, an enhancer from a virus is used. The SV40 enhancer, the cytomegalovirus early promoter enhancer, the polyoma enhancer, and adenovirus enhancers known in the art are exemplary enhancing elements for the activation of eukaryotic promoters. While an enhancer may be positioned in the vector either 5' or 3' to a coding sequence, it is typically located at a site 5' from the promoter.
[0219] In some cases, such as where glycosylation is desired in a eukaryotic host cell expression system, one may manipulate the various pre- or pro-sequences to improve glycosylation or yield. For example, one may alter the peptidase cleavage site of a particular signal peptide, or add prosequences, which also may affect glycosylation. The final protein product may have, in the -1 position (relative to the first amino acid of the mature protein), one or more additional amino acids incident to expression, which may not have been totally removed. For example, the final protein product may have one or two amino acid residues found in the peptidase cleavage site, atached to the aminoterminus. Alternatively, use of some enzyme cleavage sites may result in a slightly truncated form of the desired polypeptide if the enzyme cuts at such area within the mature polypeptide.
[0220] A sequence encoding an appropriate native or heterologous signal sequence (leader sequence or signal peptide) can be incorporated into an expression vector, to promote extracellular secretion of the protein of interest. The choice of signal peptide or leader depends on the type of host cells in which the protein of interest to be produced, and a heterologous signal sequence can replace the native signal sequence. Examples of signal peptides that are functional in mammalian host cells include, but are not limited to, the following: the signal sequence for interleukin-7 described in U.S. Patent No. 4,965,195; the signal sequence for interleukin-2 receptor described in Cosman et al., 1984, Nature 312:768; the interleukin-4 receptor signal peptide described in EP Patent No. 0367 566; the type I interleukin-1 receptor signal peptide described in U.S. Pat. No. 4,968,607; the type II interleukin-1 receptor signal peptide described in EP Patent No. 0 460 846.
[0221] Additional control sequences shown to improve expression of heterologous genes from mammalian expression vectors include, but are not limited to, such elements as the expression augmenting sequence element (EASE) derived from CHO cells (Morris et al., in Animal Cell Technology, pp. 529-534 (1997); U.S. Patent Nos. 6,312,951 Bl, 6,027,915, and 6,309,841 Bl) and the tripartite leader (TPL) and VA gene RNAs from Adenovirus 2 (Gingeras et al., 1982, J. Biol. Chem. 257: 13475-13491). The internal ribosome entry site (IRES) sequences of viral origin allows bicistronic mRNAs to be translated efficiently (Oh and Sarnow, 1993, Current Opinion in Genetics and Development 3:295-300; Ramesh et al., 1996, Nucleic Acids Research 24:2697-2700).
[0222] Vectors may be selected to be functional in the particular host cell employed (i.e., the vector is compatible with the host cell machinery, permitting amplification and/or expression of the gene can occur). In some embodiments, vectors are used that employ protein-fragment complementation assays using protein reporters, such as dihydrofolate reductase (see, for example, U.S. Pat. No. 6,270,964). Suitable expression vectors are known in the art and are also commercially available.
[0223] TABLE A provides non-limiting example synthetic nucleotide (DNA) sequences for certain expression cassette/vector components that may be used in expression cassettes and vectors of the present disclosure.
TABLE A. Non-Limiting Example Expression Cassette/Vector Component Sequences
PROTEINS OF INTEREST
[0224] The present disclosure provides expression cassettes and expression systems useful for expressing certain proteins of interest in host cells (e.g., mammalian host cells, e.g., CHO cells). Polypeptides and proteins of interest can be of scientific or commercial interest, including proteinbased therapeutics. Proteins of interest include, among other things, secreted proteins, non-secreted proteins, intracellular proteins, or membrane-bound proteins. Polypeptides and proteins of interest can be produced by recombinant animal cell lines using cell culture methods and may be referred to as “recombinant proteins.” The expressed protein(s) may be produced intracellularly or secreted into the culture medium from which it can be recovered and/or collected. The term “isolated protein” or “isolated recombinant protein” refers to a polypeptide or protein of interest, that is purified away from proteins or polypeptides or other contaminants that would interfere with its therapeutic, diagnostic, prophylactic, research, or other use. Proteins of interest include proteins that exert a therapeutic effect by binding a target, particularly a target among those listed below, including targets derived therefrom, targets related thereto, and modifications thereof.
[0225] Proteins of interest include “antigen-binding proteins,” in particular “antibody modalities.” Antigen-binding protein refers to proteins or polypeptides that comprise an antigen-binding region or antigen-binding portion that has affinity for another molecule to which it binds (antigen). Antigenbinding proteins encompass antibodies, peptibodies, antibody fragments, antibody derivatives, antibody analogs, fusion proteins (including single-chain variable fragments (scFvs), double-chain (divalent) scFvs, and IgGscFv (see, e.g., Orcutt et al., 2010, Protein Eng Des2 Sei 23:221-228), hetero-IgG (see, e.g., Liu et al., 2015, J Biol Chem 290:7535-7562), muteins, and XmAb® (Xencor, Inc., Monrovia, CA). Examples of antigen binding proteins include, but are not limited to, a human antibody, a humanized antibody, a chimeric antibody, a recombinant antibody, a single chain antibody, a diabody, a triabody, a tetrabody, a Fab fragment, a F(ab’)2 fragment, an IgD antibody, an IgE antibody, an IgM antibody, an IgGl antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody, and fragments thereof. Also included are bispecific T cell engager (BiTE®) molecules, bispecific T cell engagers having extensions, such as half-life extensions, for example HLE BiTE molecules, Heterolg BITE molecules, and others.
[0226] As used herein, the term “antigen binding protein” is used in its broadest sense and means a protein comprising a portion that binds to an antigen or target and, optionally, a scaffold or framework portion that allows the antigen binding portion to adopt a conformation that promotes binding of the antigen binding protein to the antigen. The antigen binding protein can comprise, for example, an alternative protein scaffold or artificial scaffold with grafted CDRs or CDR derivatives. Such scaffolds include, but are not limited to, antibody-derived scaffolds comprising mutations introduced to, for example, stabilize the three-dimensional structure of the antigen binding protein as well as wholly synthetic scaffolds comprising, for example, a biocompatible polymer. See, e.g., Komdorfer 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 mimetics (“PAMs”) can be used, as well as scaffolds based on antibody mimetics utilizing fibronectin components as a scaffold.
[0227] An antigen binding protein can have, for example, the structure of a naturally occurring immunoglobulin. An “immunoglobulin” is a tetrameric molecule. In a naturally occurring immunoglobulin, each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition, i.e., the VL and VH domains. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody’s isotype as IgM, IgD, IgG, IgA, and IgE, respectively. [0228] Naturally occurring immunoglobulin chains exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs. From N-terminus to C-terminus, both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain can be done in accordance with the definitions of Kabat et al. in Sequences of Proteins of Immunological Interest, 5th Ed., US Dept, of Health and Human Services, PHS, NIH, NIH Publication no. 91-3242, (1991). As desired, the 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).
[0229] In the context of the instant disclosure, an antigen binding protein is said to “specifically bind” or “selectively bind” its target antigen when the dissociation constant (KD) is <10-8 M. The antibody specifically binds antigen with “high affinity” when the KD is <5x 10'9 M, and with “very high affinity” when the KD is <5x 10 10 M.
[0230] The term “antibody” includes reference to both glycosylated and non-glycosylated immunoglobulins of any isotype or subclass or to an antigen-binding region thereof that competes with the intact antibody for specific binding, unless otherwise specified. Unless otherwise specified, antibodies include human, humanized, chimeric, multi-specific, monoclonal, polyclonal, heteroIgG, bispecific, and oligomers. Antibodies include the IgGl-, IgG2-, IgG3-, or lgG4-type.
[0231] An antigen binding protein can have one or more binding sites. If there is more than one binding site, the binding sites can be identical to one another or can be different. For example, a naturally occurring human immunoglobulin typically has two identical binding sites, while a “bispecific” or “bifunctional” antibody has two different binding sites. One standard nomenclature for multispecific antibody modalities is VERITAS. See Biswas et al., 2023, mAbs 15: 1-9.
[0232] An antigen binding fragment or region include Fab, Fab', F(ab')2, Fv, diabodies, Fd, dAb, maxibodies, single chain antibody molecules, single domain VHH, complementarity determining region (CDR) fragments, scFv, diabodies, triabodies, tetrabodies, and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to a target polypeptide.
[0233] A VH domain is the heavy chain variable domain. The variable region gives the antibody its ability to bind to antigens. A VH domain includes the variable regions of UniAbs™, which are called UniDabs™. See, e.g., Clarke et al., 2018, Front Immunol. 9:3037.
[0234] A Fab fragment is a monovalent fragment having the VL, VH, CL and CHI domains; a F(ab’)2 fragment is a bivalent fragment having two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment has the VH and CHI domains; an Fv fragment has the VL and VH domains of a single arm of an antibody; and a dAb fragment has a VH domain, a VL domain, or an antigen-binding fragment of a VH or VL domain (U.S. Pat. Nos. 6,846,634, 6,696,245, U.S. Patent Application Publication Nos. 2005/0202512, 2004/0202995, 2004/0038291, 2004/0009507, 2003/0039958, Ward etal., 1989, Nature 341:544-546). [0235] A single-chain antibody (scFv) is an antibody in which a VL and a VH region are joined via a linker (e.g., a synthetic sequence of amino acid residues) to form a continuous protein chain wherein the linker is long enough to allow the protein chain to fold back on 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 as well as Eshhar et al., 1997, Cancer Immunol Immunotherapy 45: 131-136. An scFv retains the parent antibody's ability to specifically interact with target antigen.
[0236] For purposes of clarity, and as described herein, it is noted that an antigen binding protein can, but need not, be of human origin (e.g., a human antibody), and in some cases will comprise a non-human protein, for example, a rat or murine protein, and in other cases an antigen binding protein can comprise a hybrid of human and non-human proteins (e.g., a humanized antibody).
[0237] A protein of interest can comprise a human antibody. The term “human antibody” includes all antibodies that have one or more variable and constant regions derived from human immunoglobulin sequences. In one embodiment, all of the variable and constant domains are derived from human immunoglobulin sequences (a fully human antibody). Such antibodies can be prepared in a variety of ways, including through the immunization with an antigen of interest of a mouse that is genetically modified to express antibodies derived from human heavy and/or light chain-encoding genes, such as a mouse derived from a Xenomouse®, UltiMab™, or Velocimmune® system, or a rat derived from UniRat®. Phage-based approaches can also be employed.
[0238] Alternatively, a protein of interest can comprise a humanized antibody. A “humanized antibody” has a sequence that differs from the sequence of an antibody derived from a non-human species by one or more amino acid substitutions, deletions, and/or additions, such that the humanized antibody is less likely to induce an immune response, and/or induces a less severe immune response, as compared to the non-human species antibody, when it is administered to a human subject. In one embodiment, certain amino acids in the framework and constant domains of the heavy and/or light chains of the non-human species antibody are mutated to produce the humanized antibody. In another embodiment, the constant domain(s) from a human antibody are fused to the variable domain(s) of a non-human species. Examples of how to make humanized antibodies can be found in U.S. Pat. Nos. 6,054,297, 5,886,152 and 5,877,293.
[0239] Also included are modified proteins, such as are proteins modified chemically by a non- covalent bond, covalent bond, or both a covalent and non-covalent bond. Also included are proteins further comprising one or more post-translational modifications, which may be made by cellular modification systems or modifications introduced ex vivo by enzymatic and/or chemical methods or introduced in other ways.
[0240] Proteins of interest may also include recombinant fusion proteins comprising, for example, a multimerization domain, such as a leucine zipper, a coiled coil, an Fc portion of an immunoglobulin, and the like. Also included are proteins comprising all or part of the amino acid sequences of differentiation antigens (referred to as CD proteins) or their ligands or proteins substantially similar to either of these.
[0241] In some embodiments, proteins of interest may include proteins that bind specifically to one or more CD proteins, HER receptor family proteins, cell adhesion molecules, growth factors, nerve growth factors, fibroblast growth factors, transforming growth factors (TGF), insulin-like growth factors, osteoinductive factors, insulin and insulin-related proteins, coagulation and coagulation- related proteins, colony stimulating factors (CSFs), other blood and serum proteins, blood group antigens, receptors, receptor-associated proteins, growth hormones, growth hormone receptors, T-cell receptors; neurotrophic factors, neurotrophins, relaxins, interferons, interleukins, viral antigens, lipoproteins, integrins, rheumatoid factors, immunotoxins, surface membrane proteins, transport proteins, homing receptors, addressins, regulatory proteins, and immunoadhesins.
[0242] In some embodiments, proteins of interest bind to one of more of the following, alone or in any combination: CD proteins including but not limited to CD3, CD4, CD5, CD7, CD8, CD 19, CD20, CD22, CD25, CD30, CD33, CD34, CD38, CD40, CD70, CD123, CD133, CD138, CD171, and CD 174, HER receptor family proteins, including, for instance, HER2, HER3, HER4, and the EGF receptor, EGFRvIII, cell adhesion molecules, for example, LFA-1, Mol, pl50,95, VLA-4, ICAM-1, VCAM, and alpha v/beta 3 integrin, growth factors, including but not limited to, for example, vascular endothelial growth factor (“VEGF”); VEGFR2, growth hormone, thyroid stimulating hormone, follicle stimulating hormone, luteinizing hormone, growth hormone releasing factor, parathyroid hormone, mullerian-inhibiting substance, human macrophage inflammatory protein (MIP- 1-alpha), erythropoietin (EPO), nerve growth factor, such as NGF-beta, platelet-derived growth factor (PDGF), fibroblast growth factors, including, for instance, aFGF and bFGF, epidermal growth factor (EGF), Cripto, transforming growth factors (TGF), including, among others, TGF-a and TGF-J3, including TGF-J31, TGF-J32, TGF-J33, TGF-J34, or TGF-J35, insulin-like growth factors-I and -II (IGF-I and IGF-II), des(l-3)-IGF-I (brain IGF-I), and osteoinductive factors, insulins and insulin-related proteins, including but not limited to insulin, insulin A-chain, insulin B-chain, proinsulin, and insulinlike growth factor binding proteins; (coagulation and coagulation-related proteins, such as, among others, factor VIII, tissue factor, von Willebrand factor, protein C, alpha- 1 -antitrypsin, plasminogen activators, such as urokinase and tissue plasminogen activator (“t-PA”), bombazine, thrombin, thrombopoietin, and thrombopoietin receptor, colony stimulating factors (CSFs), including the following, among others, M-CSF, GM-CSF, and G-CSF, other blood and serum proteins, including but not limited to albumin, IgE, and blood group antigens, receptors and receptor-associated proteins, including, for example, flk2/flt3 receptor, obesity (OB) receptor, growth hormone receptors, and T- cell receptors; neurotrophic factors, including 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, including for example, interferon-alpha, -beta, and -gamma, interleukins (ILs), e g., IL-1 to IL-10, IL-12, IL-15, IL-17, IL-23, IL-12/IL-23, IL-2Ra, IL1-R1, IL-6 receptor, IL-4 receptor and/or IL-13 to the receptor, IL-13RA2, or IL-17 receptor, IL-1RAP; viral antigens, including but not limited to, an AIDS envelope viral antigen, lipoproteins, calcitonin, glucagon, atrial natriuretic factor, lung surfactant, tumor necrosis factor-alpha and -beta, enkephalinase, BCMA, IgKappa, ROR-1, ERBB2, mesothelin, RANTES (regulated on activation normally T-cell expressed and secreted), mouse gonadotropin-associated peptide, DNase, FR-alpha, inhibin, and activin, integrin, protein A or D, rheumatoid factors, immunotoxins, bone morphogenetic protein (BMP), superoxide dismutase, surface membrane proteins, decay accelerating factor (DAF), AIDS envelope, transport proteins, homing receptors, MIC (MIC-a, MIC-B), ULBP 1-6, EPCAM, addressins, regulatory proteins, immunoadhesins, 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 ligand, PD1 and PDL1, mannose receptor/hCGp, hepatitis-C virus, mesothelin dsFv[PE38] conjugate, Legionella pneumophila (lly), IFN gamma, interferon gamma induced protein 10 (IP 10), IFNAR, TALL-1, thymic stromal lymphopoietin (TSLP), proprotein convertase subtilisin/Kexin Type 9 (PCSK9), stem cell factors, Flt- 3, calcitonin gene-related peptide (CGRP), OX40L, a4p7, platelet specific (platelet glycoprotein Ilb/IIIb (PAC-1), transforming growth factor beta (TFGP), Zona pellucida sperm-binding protein 3 (ZP-3), TWEAK, platelet derived growth factor receptor alpha (PDGFRa), sclerostin, and biologically active fragments or variants of any of the foregoing.
[0243] In another embodiment, proteins of interest include abciximab, adalimumab, adecatumumab, aflibercept, alemtuzumab, alirocumab, anakinra, atacicept, basiliximab, belimumab, bevacizumab, biosozumab, blinatumomab, brentuximab vedotin, brodalumab, cantuzumab mertansine, canakinumab, cetuximab, certolizumab pegol, conatumumab, daclizumab, denosumab, eculizumab, edrecolomab, efalizumab, epratuzumab, etanercept, evolocumab, galiximab, ganitumab, gemtuzumab, golimumab, ibritumomab tiuxetan, infliximab, ipilimumab, lerdelimumab, lumiliximab, Ixdkizumab, mapatumumab, motesanib diphosphate, muromonab-CD3, natalizumab, nesiritide, nimotuzumab, nivolumab, ocrelizumab, ofatumumab, omalizumab, oprelvekin, palivizumab, panitumumab, pembrolizumab, pertuzumab, pexelizumab, ranibizumab, rilotumumab, rituximab, romiplostim, romosozumab, sargamostim, tocilizumab, tositumomab, trastuzumab, ustekinumab, vedolizumab, visilizumab, volociximab, zanolimumab, zalutumumab, and biosimilars of any of the foregoing. [0244] Proteins of interest according to the invention encompass all of the foregoing and further include antibodies comprising 1, 2, 3, 4, 5, or 6 of the complementarity determining regions (CDRs) of any of the aforementioned antibodies. One or more CDRs can be incorporated into a molecule either covalently or noncovalently to make it an antigen binding protein. An antigen binding protein can incorporate the CDR(s) as part of a larger polypeptide chain, can covalently link the CDR(s) to another polypeptide chain, or can incorporate the CDR(s) noncovalently. The CDRs permit the antigen binding protein to specifically bind to a particular antigen of interest. Also included are variants that comprise a region that is 70% or more, especially 80% or more, more especially 90% or more, yet more especially 95% or more, particularly 97% or more, more particularly 98% or more, yet more particularly 99% or more identical in amino acid sequence to a reference amino acid sequence of a protein of interest. Identity in this regard can be determined using a variety of well-known and readily available amino acid sequence analysis software. Preferred software includes those that implement the Smith-Waterman algorithms, considered a satisfactory solution to the problem of searching and aligning sequences. Other algorithms also may be employed, particularly where speed is an important consideration. Commonly employed programs for alignment and homology matching of DNAs, RNAs, and polypeptides that can be used in this regard include FASTA, TFASTA, BLASTN, BLASTP, BLASTX, TBLASTN, PROSRCH, BLAZE, and MPSRCH, the latter being an implementation of the Smith-Waterman algorithm for execution on massively parallel processors made by MasPar.
[0245] The antigen binding molecule may be an antibody fragment thereof, including one or more single chain antibody fragment (“scFv”). scFvs are preferred for use in chimeric antigen receptors because they can be engineered to be expressed as part of a single chain. See Krause et al., 1988, J. Exp. Med., 188(4): 619-626; Finney et al., 1998, J Immunol 161: 2791-2797.
[0246] An “Fc” region, as the term is used herein, comprises two heavy chain fragments comprising the CH2 and CH3 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains. Proteins of interest comprising an Fc region, including antigen binding proteins and Fc fusion proteins, form another aspect of the instant disclosure.
GENERATION OF MAMMALIAN HOST CELLS EXPRESSING A PROTEIN OF INTEREST [0247] Expression of a protein of interest in a cell can be achieved by well-known methods, either transiently or by stable expression (Davis et al., Basic Methods in Molecular Biology, 2nd ed., Appleton & Lange, Norwalk, Conn., 1994; Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001). [0248] Methods for stable integration are well known in the art. Briefly, stable integration is commonly achieved by transiently introducing a heterologous polynucleotide or a vector containing the heterologous polynucleotide into the host cell, which facilitates the stable integration of said heterologous polynucleotide into the cell genome. Typically, the heterologous polynucleotide is flanked by homology arms, i.e., sequences homologous to the region upstream and downstream to the integration site. Before their introduction into the mammalian host cell, circular vectors may be linearized to facilitate integration into the cell genome. Methods for the introduction of vectors into cells are well known in the art and include transfection with biological methods, such as viral delivery, with chemical methods, such as using cationic polymers, calcium phosphate, cationic lipids or cationic amino acids; with physical methods, such as electroporation or microinjection; or with mixed approaches, such as protoplast fusion.
[0249] A specific method of 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 in the genome (also termed “targeted integration”). Sitespecific recombinases such as Flp and Cre mediate recombination between two copies of their target sequence termed FRT and loxP, respectively. The use of two incompatible target sequences, for example FRT in combination with F3 (Schlake and Bode, 1994, Biochemistry, 33: 12746-51) as well as inverted recognition target sites (Feng et al., 1999, J. Mol. Biol. 292:779-85) allows the insertion of DNA segments into a predefined chromosomal locus carrying target sequences in a similar configuration. See also EP Patent No. EP1781796B1 and EP Patent Application Publication No. EP2789691A1.
[0250] Insertion of RMCE into a specific site in the genome can be mediated by nucleases (e.g., zinc finger protein (ZFP), transcription activator-like effector nuclease (TALEN), clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)) that can be engineered to create single- and double-stranded breaks (SSBs/DSBs) in the genome. There are two major and distinct pathways to repair DSBs — homologous recombination and non-homologous endjoining (NHEJ). Homologous recombination requires the presence of a homologous sequence as a template (e.g., "donor” containing RMCE) to guide the cellular repair process and the results of the repair are error-free and predictable. In the absence of a template (or "donor") sequence for homologous recombination, the cell typically attempts to repair the DSB via the unpredictable and error-prone process of non-homologous end-joining (NHEJ).
[0251] A vector may be any molecule or entity (e.g., nucleic acid, plasmid, bacteriophage, transposon, cosmid, chromosome, virus, virus capsid, virion, naked DNA, complexed DNA and the like) suitable for use to transfer and/or transport protein encoding information into a host cell and/or to a specific location and/or compartment within a host cell. Vectors can include viral and non-viral vectors, non-episomal mammalian vectors. Vectors are often referred to as expression vectors, for example, recombinant expression vectors and cloning vectors. The present disclosure provides specific examples of expression cassettes that may be incorporated into expression vectors useful for expressing various proteins of interest, including certain antibody modalities discussed above. Expression vectors according to the present disclosure may be introduced into a host cell to allow replication of the vector itself and thereby amplify the copies of the polynucleotide contained therein. As described above, cloning vectors may contain sequence components generally include, without limitation, an origin of replication, promoter sequences, transcription initiation sequences, enhancer sequences, and selectable markers. These elements may be selected as appropriate by a person of ordinary skill in the art.
[0252] Following construction, one or more vectors may be inserted into a suitable cell for amplification and/or polypeptide expression. The transformation of an expression vector into a selected cell may be accomplished by well-known methods including transfection, infection, calcium phosphate co-precipitation, electroporation, nucleofection, microinjection, DEAE-dextran mediated transfection, cationic lipids mediated delivery, liposome mediated transfection, microprojectile bombardment, receptor-mediated gene delivery, delivery mediated by polylysine, histone, chitosan, and peptides. The method selected will in part be a function of the type of host cell to be used. These methods and other suitable methods are well known to the skilled artisan and are set forth in manuals and other technical publications, for example, in Sambrook et al.. Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001).
[0253] The term “transformation” refers to a change in a cell's genetic characteristics, and a cell has been transformed when it has been modified to contain new DNA or RNA. For example, a cell is transformed where it is genetically modified from its native state by introducing new genetic material via transfection, transduction, or other techniques. Following transfection or transduction, the transforming DNA can recombine with that of the cell by physically integrating into a chromosome of the cell or can be maintained transiently as an episomal element without being replicated, or can replicate independently as a plasmid. A cell is considered to have been “stably transformed” when the transforming DNA is replicated with the division of the cell.
[0254] The term “transfection” refers to the uptake of foreign or exogenous DNA by a cell. A number of transfection techniques are well known in the art and are disclosed herein. See, e.g., Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, supra; Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al., 1981, Gene 13: 197.
[0255] The term “transduction” refers to the process whereby foreign DNA is introduced into a cell via viral vector. See Jones et al., (1998). Genetics: principles and analysis. Boston: Jones & Bartlett Publ.
CELL LINES
[0256] In the methods disclosed herein, any mammalian cell line can be used, with CHO cell lines being one preferred example of a cell line that may be used in combination with expression vectors of the present disclosure. A wide variety of mammalian cell lines suitable for growth in culture are available from the American Type Culture Collection (Manassas, Va.) and commercial vendors. Non- limiting examples of cell lines commonly used in the industry include monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney 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 carcinoma 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 hepatoma cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., 1982, Annals N.Y Acad. Sci. 383:44-68); MRC 5 cells or FS4 cells; mammalian myeloma cells, and a number of other cell lines and Chinese hamster ovary (CHO) cells.
[0257] Large-scale production of proteins for commercial applications is typically carried out in suspension culture. Therefore, mammalian host cells used to generate the recombinant mammalian cells described herein can, but need not be, adapted to growth in suspension culture. A variety of host cells adapted to growth in suspension culture are known, including mouse myeloma NS0 cells and CHO cells from CHO-S, DG44, and DXB11 cell lines. Other suitable cell lines include, but are not limited to, mouse myeloma SP2/0 cells, baby hamster kidney BHK-21 cells, human PER.C6® cells, human embryonic kidney HEK-293 cells, and cell lines derived or engineered from any of the cell lines disclosed herein.
[0258] CHO cells are widely used to produce complex recombinant proteins, including CHOK1 cells (ATCC CCL61). The 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 the efficient DHFR selectable and amplifiable gene expression system allows high level recombinant protein expression in these cells (Kaufman R. J., 1990, Meth Enzymol 185:537-566). Also included are the glutamine synthase (GS)-knockout CHOK1SV cell lines, making use of glutamine synthetase (GS)-based methionine sulfoximine (MSX) selection. Other suitable CHO host cells could include, but are not limited to, the following (ECACC accession numbers in brackets): CHO (85050302), CHO (PROTEIN FREE) (00102307), CHO-K1 (85051005), CHO-K1/SF (93061607), CHO/DHFR-(94060607), CHO/DHFR-AC-free (05011002), RR-CHOKI (92052129).
CELL CULTURE PROCESSES
[0259] Host cells transfected with the vector systems described herein may be cultured in adherent culture or suspension cultures grown in stirred tank reactors (including traditional batch and fed-batch cell cultures, which may but need not comprise a spin fdter), perfusion systems (including alternating tangential flow (“ATF”) cultures, acoustic perfusion systems, depth fdter perfusion systems, and other systems), and hollow fiber bioreactors (HFB, which in some cases may be employed in perfusion processes), as well as various other cell culture methods (see, 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 Scale-Up” in Cell and Tissue Reaction Engineering: Principles and Practice. Eibl et al. (eds) Springer- Verlag, incorporated herein by reference in their entireties).
[0260] During recombinant protein production, it is desirable to have a controlled system where cells are grown to a desired density and then the physiological state of the cells is switched to a growth- arrested, high productivity state where the cells use energy and substrates to produce the recombinant protein of interest instead of making more cells. Various methods for accomplishing this goal exist, and include temperature shifts and amino acid starvation, as well as use of a cell-cycle inhibitor or other molecule that can arrest cell growth without causing cell death.
[0261] The production of a recombinant protein begins with establishing a mammalian cell production culture of cells that express the protein, in a culture plate, flask, tube, bioreactor or other suitable vessel. Suitable bioreactors volumes include, but are not limited to, 500L, WOOL, 2000L, 5000L, 10000L, up to 20000L. The seed cell density used to inoculate the bioreactor can have a positive impact on the level of recombinant protein produced. In one embodiment, the bioreactor is inoculated with at least 0.5 xl06,1.0 x 106, 2.0 xlO6 3.0 xlO6, 5.0 xlO6 or 10 x 106 viable cells/mL in a serum-free culture medium.
[0262] The mammalian cells then undergo an exponential growth phase. The cell culture can be maintained without supplemental feeding until a desired cell density is achieved. In one embodiment, the cell culture is maintained for up to three days with or without supplemental feeding. In another embodiment, the culture can be inoculated at a desired cell density to begin the production phase without a brief growth phase. In any of the embodiments herein, the switch from the growth phase to production phase can also be initiated by any of the aforementioned methods.
[0263] Three methods are typically used in commercial processes for the production of recombinant proteins by mammalian cell culture: batch culture, fed-batch culture, and perfusion culture. Batch culture is a discontinuous method where cells are grown in a fixed volume of culture media for a short period of time followed by a full harvest. Cultures grown using the batch method experience an increase in cell density until a maximum cell density is reached, followed by a decline in viable cell density as the media components are consumed and levels of metabolic by-products (such as lactate and ammonia) accumulate. Harvest typically occurs at the point when the maximum cell density is achieved (e.g., 5xl06 cells/mL or greater, depending on media formulation, cell line, etc.). The batch process is the simplest culture method; however, viable cell density is limited by the nutrient availability and once the cells are at maximum density, the culture declines and production decreases. There is no ability to extend a production phase because the accumulation of waste products and nutrient depletion rapidly lead to culture decline (typically around 3 to 7 days).
[0264] Fed-batch culture improves on the batch process by providing bolus or continuous media feeds to replenish those media components that have been consumed. Since fed-batch cultures receive additional nutrients throughout the run, they have the potential to achieve higher cell densities (>10 to 30xl06 cells/ml, depending on media formulation, cell line, etc.) and increased product titers, when compared to the batch method. Unlike the batch process, a biphasic culture can be created and sustained by manipulating feeding strategies and media formulations to distinguish the period of cell proliferation to achieve a desired cell density (the growth phase) from the period of suspended or slow cell growth (the production phase). As such, fed batch cultures have the potential to achieve higher product titers compared to batch cultures. Typically, a batch method is used during the growth phase and a fed-batch method used during the production phase, but a fed-batch feeding strategy can be used throughout the entire process. However, unlike the batch process, bioreactor volume is a limiting factor which limits the amount of feed. Also, as with the batch method, metabolic by-product accumulation will lead to culture decline, which limits the duration of the production phase, about 10 to 21 days. Fed-batch cultures are discontinuous, and harvest typically occurs when metabolic byproduct levels or culture viability reach predetermined levels. When compared to a batch culture, in which no feeding occurs, a fed batch culture can produce greater amounts of recombinant protein. See e.g., U.S. Patent No. 5,672,502.
[0265] Perfusion culture is one in which the cell culture receives fresh perfusion feed medium while simultaneously removing spent medium. Perfusion can be continuous, stepwise, intermittent, or a combination of any or all of any of these. Perfusion rates can be less than a working volume to many working volumes per day. The cells are retained in the culture and the spent medium that is removed is substantially free of cells or has significantly fewer cells than the culture. Recombinant proteins expressed by the cell culture can also be retained in the culture. Perfusion can be accomplished by a number of means including centrifugation, sedimentation, or filtration, See e.g. Voisard et al., 2003, Biotechnology and Bioengineering 82:751-65. An example of a filtration method is alternating tangential flow filtration. Alternating tangential flow is maintained by pumping medium through hollow-fiber filter modules. See e.g. US Patent No. 6,544,424; Furey, 2002, Gen. Eng. News. 22 (7):62-63.
[0266] “Perfusion flow rate” is the amount of media that is passed through (added and removed) from a bioreactor, typically expressed as some portion or multiple of the working volume, in a given time. “Working volume” refers to the amount of bioreactor volume used for cell culture. In one embodiment, the perfusion flow rate is one working volume or less per day. Perfusion feed medium can be formulated to maximize perfusion nutrient concentration to minimize perfusion rate. [0267] Perfusion methods offer potential improvement over the batch and fed-batch methods by adding fresh media and simultaneously removing spent media. Typical large scale commercial cell culture strategies strive to reach high cell densities, 60 - 90(+) x 106 cells/mL where almost a third to over one-half of the reactor volume is biomass. With perfusion culture, extreme cell densities of >1 x 108 cells/mL have been achieved and even higher densities are predicted. Typical perfusion cultures begin with a batch culture start-up lasting for a day or two followed by continuous, step-wise and/or intermittent addition of fresh feed media to the culture and simultaneous removal of spent media with the retention of cells and additional high molecular weight compounds such as proteins (based on the fdter molecular weight cutoff) throughout the growth and production phases of the culture. Various methods, such as sedimentation, centrifugation, or filtration, can be used to remove spent media, while maintaining cell density. Perfusion flow rates of a fraction of a working volume per day up to many multiple working volumes per day have been reported.
[0268] An advantage of the perfusion process is that the production culture can be maintained for longer periods than batch or fed-batch culture methods. However, increased media preparation, use, storage and disposal are necessary to support a long-term perfusion culture, particularly those with high cell densities, which also need even more nutrients, and all of this drives the production costs even higher, compared to batch and fed batch methods. In addition, higher cell densities can cause problems during production, such as maintaining dissolved oxygen levels and problems with increased gassing, including supplying more oxygen and removing more carbon dioxide, which would result in more foaming and the need for alterations to antifoam strategies; as well as during harvest and downstream processing where the efforts required to remove the excessive cell material can result in loss of product, negating the benefit of increased titer due to increased cell mass.
[0269] A large-scale cell culture strategy that combines fed batch feeding during the growth phase with continuous perfusion during the production phase may be used to express proteins of interest. Such a method may target a production phase in which the cell culture is maintained at a packed cell volume of less than or equal to 35%.
[0270] In one embodiment, a fed-batch culture with bolus feeds is used to maintain a cell culture during the growth phase. Perfusion feeding can then be used during a production phase. In one embodiment, perfusion begins when the cells have reached a production phase. In another embodiment, perfusion begins on or about day 3 to on or about day 9 of the cell culture. In another embodiment, perfusion begins on or about day 5 to on or about day 7 of the cell culture.
[0271] Using bolus feeding during the growth phase allows the cells to transition into the production phase, resulting in less dependence on a temperature shift as a means of initiating and controlling the production phase, however a temperature shift of about 36°C to about 31°C can take place between the growth phase and production phase. In one embodiment, the shift is from 36°C to 32°C. [0272] In some embodiments, the bioreactor can be inoculated with at least 0.5 xlO6 up to and beyond 3.0 xlO6 viable cells/mL in a serum-free culture medium, for example, l.OxlO6 viable cells/mL.
[0273] Cell cultures can be supplemented with concentrated feed medium containing components, such as nutrients and amino acids, which are consumed during the course of the production phase of the cell culture.
[0274] Concentrated feed medium may be based on just about any cell culture media formulation. Such a concentrated feed medium can contain most of the components of the cell culture medium at, for example, about 5X, 6X, 7X, 8X, 9X, 10X, 12X, 14X, 16X, 20X, 30X, 50X, lOOx, 200X, 400X, 600X, 800X, or even about 1000X of their normal amount. Concentrated feed media are often used in fed batch culture processes.
[0275] Samples from the cell culture can be monitored and evaluated using any of the analytical techniques known in the art. A variety of parameters including recombinant protein and medium quality and characteristics can be monitored for the duration of the culture. Samples can be taken and monitored intermittently at a desirable frequency, including continuous monitoring, real time or near real time.
[0276] Typically, the cell cultures that precede the final production culture (N-x to N-l) are used to generate the seed cells that will be used to inoculate the production bioreactor, the N-l culture. The seed cell density can have a positive impact on the level of recombinant protein produced. Product levels tend to increase with increasing seed density. Improvement in titer is tied not only to higher seed density but is likely to be influenced by the metabolic and cell cycle state of the cells that are placed into production.
[0277] Seed cells can be produced by any culture method. One such method is a perfusion culture using alternating tangential flow filtration. An N-l bioreactor can be run using alternating tangential flow filtration to provide cells at high density to inoculate a production bioreactor. The N-l stage may be used to grow cells to densities of >90 x 106 cells/mL. The N-l bioreactor can be used to generate bolus seed cultures or can be used as a rolling seed stock culture that could be maintained to seed multiple production bioreactors at high seed cell density. The duration of the growth stage of production can range from 7 to 14 days and can be designed so as to maintain cells in exponential growth prior to inoculation of the production bioreactor. Perfusion rates, medium formulation and timing are optimized to grow cells and deliver them to the production bioreactor in a state that is most conducive to optimizing their production. Seed cell densities of >15 x 106 cells/mL can be achieved for seeding production bioreactors. Higher seed cell densities at inoculation can decrease or even eliminate the time needed to reach a desired production density.
[0278] In certain embodiments, the mammalian host cells can be used to generate high yield of a protein of interest. High yield, or high volumetric productivity, to the ability of cells to produce high levels of a protein of interest. The particular yield will depend on the protein of interest and can 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 fed batch or perfusion conditions, using a feed medium suitable for the mammalian host cell and containing amino acids, vitamins, or trace elements. In specific embodiments, the host cells and methods of the present disclosure express a protein of interest and 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 when grown under the culture conditions described above.
[0279] Yield can also be measured in terms of the specific productivity of a cell line, determined based on the amount of protein produced per cell per day (expressed as pg/cell/day). Mammalian host cells used with expression vector systems of the present 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, at least 50 pg/cell/day, at least 75 pg/cell/day or up to 100 pg/cell/day in a 10-day culture grown in fed batch or perfusion conditions, using a feed medium suitable for the mammalian host cell and containing amino acids, vitamins, or trace elements. In specific embodiments, mammalian host cells used with expression vector systems of the present disclosure express an 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. [0280] The mammalian host cells described herein can be used to express a protein of interest. The expressed protein may be secreted into the culture medium from which they can be recovered and/or collected. In addition, the proteins can be purified, or partially purified, from such culture or component (e.g., from culture medium) using known processes and products available from commercial vendors. The purified proteins can then be “formulated,” meaning buffer exchanged, sterilized, bulk-packaged, and/or packaged for a final user. Suitable formulations for pharmaceutical compositions (i.e., pharmaceutically acceptable formulations) include those described in Remington’s Pharmaceutical Sciences, 18th ed. 1995, Mack Publishing Company, Easton, PA.
[0281] In certain embodiments, a CHO DHFR- cell or a CHO GSKO cell can be cultured under conditions to express antibody chains under methotrexate stringency in the case of a CHO DHFR- cell or methionine sulfoximine stringency in the case of a CHO GSKO cell to favor expression of the difficult-to-express chain paired with a stronger GS promoter. [0282] In certain embodiments, a CHO DHFR- cell or a CHO GSKO cell can be cultured under conditions to express antibody chains under methotrexate stringency in the case of a CHO DHFR- cell or methionine sulfoximine stringency in the case of a CHO GSKO cell to favor expression of the difficult-to-express chain paired with a weaker GS promoter.
[0283] A variety of known techniques can be utilized in making the polynucleotides, polypeptides, vectors, host cells, and the like according to the present disclosure.
SCREENING METHODS
[0284] Typically, to establish a cell line producing a recombinant antigen-binding protein (e.g., a protein derived from an antibody which comprises two chains, one based on antibody heavy chain and one based on antibody light chain), it is often necessary to integrate the heavy and light chain coding sequences from a single or separate vector into the genome followed by a stringency and efficiency metabolic selection to find high-producing cell lines. In a glutamine synthetase (GS)-CHO expression system, selection of high-producing cell lines is based on controlling the balance between the expression level of GS and the concentration of its specific inhibitor, L-methionine sulfoximine (MSX). As disclosed and exemplified in Example 2, one method of expressing an antigen-binding protein disclosed herein is to optimize the expression of the heavy chain and/or light chain using promoters with different strengths under different MSX concentrations. As GS acts both as a selectable marker and a means to achieve gene amplification, the optimal selection stringency for each promoter was determined using various MSX concentrations. Results from these experiments established the preferred vector design plus selection stringency for approximately seven different biologic modalities (including multispecific proteins). As such, provided herein are vector design strategies for efficient expression of antigen-binding proteins, including the biologic modalities described in Example 2.
[0285] Accordingly, disclosed herein is a method for optimizing expression of an antigen-binding protein, wherein said antigen-binding protein comprises a heavy chain and a light chain, comprising: expressing a plurality of vectors, each in a population of host cells and under a plurality of different concentrations of methionine sulfoximine (MSX), wherein each vector in the plurality of vectors comprises: (i) a promoter having a unique transcriptional strength; (ii) a glutamine synthetase gene operably linked to the promoter; and (iii) a sequence encoding said heavy chain and/or said light chain operably linked to the promoter; and measuring productivity of the antigen-binding protein from each combination of promoter and MSX concentration.
[0286] In some embodiments, the promoter drives the expression of the heavy chain and/or light chain.
[0287] In some embodiments, the promoter is a CMV/ADL promoter. [0288] In some embodiments, the promoter is a GAPDH promoter. In some embodiments, the promoter is a CMV/GAPDH promoter.
[0289] In some embodiments, the promoter is a CMV/EFla promoter.
[0290] In some embodiments, a first vector in the plurality of vectors comprises a GAPDH promoter and a second vector in the plurality of vectors comprises a CMV/ADL promoter. In some embodiments, a first vector in the plurality of vectors comprises a CMV/GAPDH promoter and a second vector in the plurality of vectors comprises a CMV/ADL promoter.
[0291] In some embodiments, the promoter drives the expression of the glutamine synthetase gene. In some embodiments, the promoter is a SRa promoter. In some embodiments, the promoter is a mPGK promoter.
[0292] In some embodiments, each vector in the plurality of vectors comprises a promoter having a unique transcriptional strength driving the expression of the heavy chain and/or light chain and/or a promoter having a unique transcriptional strength driving the expression of the glutamine synthetase gene.
[0293] In some embodiments, the plurality of different MSX concentrations comprises 0 pM. In some embodiments, the plurality of different MSX concentrations comprises 12.5 pM. In some embodiments, the plurality of different MSX concentrations comprises 25 pM In some embodiments, the plurality of different MSX concentrations comprises 12.5 pM and 25 pM. In some embodiments, the plurality of different MSX concentrations comprises 0 pM, 12.5 pM, and 25 pM.
[0294] The present invention is not to be limited in scope by the specific embodiments described herein that are intended as single illustrations of individual aspects of the invention, and functionally equivalent methods and components are within the scope of the invention. Indeed, various modifications of the invention, in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.
ADDITIONAL NON-LIMITING EXAMPLE EMBODIMENTS
[0295] Non-limiting example embodiments of the present disclosure also include:
El . An expression cassette comprising a polynucleotide sequence which comprises the following elements in 5 ’ to 3 ’ order: a) a first GAPDH promoter operably linked to a nucleotide sequence encoding a first antibody chain or antibody chain fusion followed by a first polyA sequence; b) a second GAPDH promoter operably linked to a nucleotide sequence encoding a second antibody chain antibody chain fusion followed by a second polyA sequence; and c) a promoter operably linked to a nucleotide sequence encoding a selectable marker followed by a third polyA sequence; wherein when the first antibody chain or antibody chain fusion is an antibody light chain, the second antibody chain or antibody chain fusion is an antibody heavy chain or antibody heavy chain fusion, wherein when the first antibody chain or antibody chain fusion is an antibody heavy chain or heavy chain fusion, the second antibody chain or antibody chain fusion is an antibody light chain, and wherein the heavy chain fusion is selected from the group consisting of a heavy chain-scFv, a heavy chain-cytokine, and a heavy chain vHH.
E2. The expression cassette of El, wherein the first GAPDH promoter is operably linked to a nucleotide sequence encoding an antibody light chain and the second GAPDH promoter is operably linked to a heavy chain or heavy chain fusion.
E3. The expression cassette of El or E2, wherein the selectable marker is selected from the group consisting of glutamine synthetase and dihydrofolate reductase.
E4. The expression cassette of any one of E1-E3, wherein the GAPDH promoter comprises a nucleotide sequence of SEQ ID NO: 1.
E5. The expression cassette of any of E 1 -E4, wherein the promoter operably linked to the nucleotide sequence encoding the selectable marker is selected from the group consisting of mPGK Sra, and SV40.
E6. The expression cassette of any of E1-E5, wherein the first, second and third polyA sequences are the same or different and are selected from the group consisting of a thymidine kinase pA (TKpA) sequence, rabbit beta-globin pA and a simian virus 40 (SV40) early pA sequence.
E7. An expression vector which comprises the expression cassette of any of E1-E6.
E8. The expression vector of E7, wherein the polynucleotide sequence encodes a monoclonal antibody.
E9. The expression vector of E7, wherein the polynucleotide sequence encodes a B2/C2 mAb. E10. A pair of expression vectors, wherein
1) the first expression vector comprises an expression cassette comprising a polynucleotide sequence which comprises the following elements in 5’ to 3’ order: a) a first copy of a GAPDH promoter operably linked to a nucleotide sequence encoding an antibody light chain followed by a first polyA sequence; b) a second copy of the GAPDH promoter operably linked to a nucleotide sequence encoding an antibody heavy chain followed by a second polyA sequence; and c) a promoter operably linked to a nucleotide sequence encoding a selectable marker followed by a third polyA sequence; and
2) the second expression vector comprises an expression cassette comprising a polynucleotide sequence which comprises the following elements in 5 ’ to 3 ’ order a) a first copy of a GAPDH promoter operably linked to a nucleotide sequence encoding an antibody light chain followed by a first polyA sequence; b) a second copy of the GAPDH promoter operably linked to a nucleotide sequence encoding an antibody heavy chain-scFv fusion followed by a second polyA sequence; and c) a promoter operably linked to a nucleotide sequence encoding a selectable marker followed by a third polyA sequence.
El l. The pair of vectors of E 10, which encodes a C 1 mAb .
E12. A pair of expression vectors, wherein
1) The first expression vector comprises an expression cassette comprising a polynucleotide sequence which comprises the following elements in 5’ to 3’ order: a) a first copy of a GAPDH promoter operably linked to a nucleotide sequence encoding a first antibody light chain followed by a first polyA sequence; b) a second copy of the GAPDH promoter operably linked to a nucleotide sequence encoding a first antibody heavy chain followed by a second polyA sequence; and c) a promoter operably linked to a nucleotide sequence encoding a selectable marker followed by a third polyA sequence; and
2) The second expression vector comprises an expression cassette comprising a polynucleotide sequence which comprises the following elements in 5’ to 3’ order: a) a first copy of a GAPDH promoter operably linked to a nucleotide sequence encoding a second antibody light chain followed by a first polyA sequence; b) a second copy of the GAPDH promoter operably linked to a nucleotide sequence encoding a second antibody heavy chain followed by a second polyA sequence; and c) a promoter operably linked to a nucleotide sequence encoding a selectable marker followed by a third polyA sequence.
E 13. The pair of vectors of E 11 , which encodes a heteroIgG.
E14. A mammalian host cell comprising the expression vector of any of E7-E9.
E 15. A mammalian host cell comprising the pair of expression vectors of any of E 10-E 13.
E16. The mammalian host cell of E14 or E15 which is selected from the group consisting of CHO.
El 7. The mammalian host cell of El 6, wherein the CHO cell is a dhfir- or GSKO.
El 8. Method for producing an antibody modality comprising culturing the mammalian host cell of any of E14-E17 under conditions in which the antibody chains are expressed, and recovering the antibody modality from the culture.
E19. The method of E18, wherein the recovered antibody modality is purified and formulated in a pharmaceutically acceptable formulation.
[0296] Further non-limiting example embodiments/features of the present disclosure include:
Fl . An expression cassette comprising a polynucleotide sequence which comprises the following elements in 5 ’ to 3 ’ order: a) a first copy of a GAPDH promoter operably linked to a nucleotide sequence encoding a first antibody chain or antibody chain fusion followed by a first polyA signal sequence; b) a second copy of the GAPDH promoter operably linked to a nucleotide sequence encoding a second antibody chain or antibody chain fusion followed by a second polyA signal sequence; and c) a promoter operably linked to a nucleotide sequence encoding a selectable marker followed by a third polyA signal sequence, wherein, when the first antibody chain or antibody chain fusion is an antibody light chain, the second antibody chain or antibody chain fusion is an antibody heavy chain or antibody heavy chain fusion, wherein, when the first antibody chain or antibody chain fusion is an antibody heavy chain or antibody heavy chain fusion, the second antibody chain or antibody chain fusion is an antibody light chain, and wherein the antibody heavy chain fusion is selected from the group consisting of a heavy chain-scFv fusion, a heavy chain-cytokine fusion, and a heavy chain VH fusion.
F2. The expression cassette of Fl, wherein the first antibody chain or antibody chain fusion is an antibody light chain, and the second antibody chain or antibody chain fusion is an antibody heavy chain.
F3. The expression cassette of Fl, wherein the first antibody chain or antibody chain fusion is an antibody light chain, and the second antibody chain or antibody chain fusion is an antibody heavy chain fusion.
F4. The expression cassette of Fl, wherein the first antibody chain or antibody chain fusion is an antibody heavy chain, and the second antibody chain or antibody chain fusion is an antibody light chain.
F5. The expression cassette of Fl, wherein the first antibody chain or antibody chain fusion is an antibody heavy chain fusion, and the second antibody chain or antibody chain fusion is an antibody light chain.
F6. The expression cassette of any one of Fl, F3, or F5, wherein the antibody heavy chain fusion is a direct fusion of a heavy chain with a VH, a direct fusion of a heavy chain with a scFv, or a direct fusion of a heavy chain with a cytokine.
F7. The expression cassette of any one of Fl, F3, F5, or F6, wherein the antibody heavy chain fusion comprises a VH, scFv, or cytokine fused to the C-terminus of the heavy chain portion of the antibody heavy chain fusion.
F8. The expression cassette of any one of Fl, F3, F5, or F6, wherein the antibody heavy chain fusion comprises a VH, scFv, or cytokine fused to the N-terminus of the heavy chain portion of the antibody heavy chain fusion. F9. The expression cassete of any one of Fl, F3, F5, or F6, wherein the antibody heavy chain fusion comprises a VH, scFv, or cytokine fused between the CHI and CH2 of the heavy chain portion of the antibody heavy chain fusion.
F10. The expression cassete of any one of Fl, F3, or F5, wherein the antibody heavy chain fusion comprises a linker between the heavy chain and the VH, scFv, or cytokine.
Fl 1. The expression cassete of any one of Fl, F3, or F5-F10, wherein the antibody heavy chain fusion is a heavy chain-scFv fusion.
F12. The expression cassete of any one of Fl, F3, or F5-F10, wherein the antibody heavy chain fusion is a heavy chain-cytokine fusion.
F13. The expression cassete of any one of Fl, F3, or F5-F10, wherein the antibody heavy chain fusion is a heavy chain VH fusion.
F14. The expression cassete of any one of Fl-Fl 3, wherein the selectable marker is glutamine synthetase or dihydrofolate reductase.
F15. The expression cassete of any one of Fl -Fl 4, wherein the selectable marker is glutamine synthetase.
Fl 6. The expression cassete of any one of Fl -Fl 4, wherein the selectable marker is dihydrofolate reductase.
Fl 7. The expression cassete of any one of Fl -Fl 6, wherein the GAPDH promoter is a
CMV/GAPDH promoter.
Fl 8. The expression cassete of Fl 7, wherein the CMV promoter enhancer is 5’ of the GAPDH promoter.
Fl 9. The expression cassete of F17 or F18, wherein the GAPDH promoter comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 1. F20. The expression cassete of any one of F17-F19, wherein the GAPDH promoter comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO: 1.
F21. The expression cassete of any one of F17-F20, wherein the GAPDH promoter comprises the nucleotide sequence of SEQ ID NO: 1.
F22. The expression cassete of any one of F1-F21, wherein the promoter operably linked to the nucleotide sequence encoding the selectable marker is selected from the group consisting of mPGK, SRa, and SV40 promoters.
F23. The expression cassete of any one of F 1-F22, wherein the promoter operably linked to the nucleotide sequence encoding the selectable marker is a mPGK promoter.
F24. The expression cassete of any one of F 1-F22, wherein the promoter operably linked to the nucleotide sequence encoding the selectable marker is a SRa promoter.
F25. The expression cassete of any one of F 1-F22, wherein the promoter operably linked to the nucleotide sequence encoding the selectable marker is a SV40 promoter.
F26. The expression cassete of any one of F1-F25, wherein each of the first, second, and third polyA signal sequences is independently selected from the group consisting of a bovine growth hormone (BGH) polyA signal sequence, a thymidine kinase polyA (TKpA) signal sequence, a rabbit beta-globin polyA signal sequence, and a simian virus 40 (SV40) early polyA signal sequence.
F27. The expression cassete of any one of F1-F26, wherein each of the first, second, and third polyA signal sequences is the same.
F28. The expression cassete of any one of F1-F27, wherein each of the first, second, and third polyA signal sequences is a simian virus 40 (SV40) early polyA signal sequence.
F29. The expression cassete of Fl, wherein the expression cassete encodes an antibody light chain and an antibody heavy chain, the GAPDH promoter is a CMV/GAPDH promoter, the selectable marker is glutamine synthetase, the promoter operably linked to a nucleotide sequence encoding the selectable marker is mPGK, and each of the first, second, and third polyA signal sequences is a simian virus 40 (SV40) early polyA signal sequence. F30. The expression cassete of Fl, wherein the expression cassete encodes an antibody light chain and an antibody heavy chain fusion, the GAPDH promoter is a CMV/GAPDH promoter, the selectable marker is glutamine synthetase, the promoter operably linked to a nucleotide sequence encoding the selectable marker is SRa, and each of the first, second, and third polyA signal sequences is a simian virus 40 (SV40) early polyA signal sequence.
F31. The expression cassete of F30, wherein the antibody heavy chain fusion is a direct fusion of a heavy chain with a VH, a direct fusion of a heavy chain with a scFv, or a direct fusion of a heavy chain with a cytokine.
F32. The expression cassete of F30 or F31, wherein the antibody heavy chain fusion comprises a VH, scFv, or cytokine fused to the C-terminus of the heavy chain portion of the antibody heavy chain fusion.
F33. The expression cassete of F30 or F31, wherein the antibody heavy chain fusion comprises a VH, scFv, or cytokine fused to the N-terminus of the heavy chain portion of the antibody heavy chain fusion.
F34. The expression cassete of F30 or F31, wherein the antibody heavy chain fusion comprises a VH, scFv, or cytokine fused between the CHI and CH2 of the heavy chain portion of the antibody heavy chain fusion.
F35. The expression cassete of F30, wherein the antibody heavy chain fusion comprises a linker between the heavy chain and the VH, scFv, or cytokine.
F36. The expression cassete of any one of F30-F35, wherein the antibody heavy chain fusion is a heavy chain-scFv fusion.
F37. The expression cassete of any one of F30-F35, wherein the antibody heavy chain fusion is a heavy chain-cytokine fusion.
F38. The expression cassete of any one of F30-F35, wherein the antibody heavy chain fusion is a heavy chain VH fusion.
F39. An expression vector which comprises an expression cassete of any one of F1-F38. F40. The expression vector of F39, encoding a monoclonal antibody.
F41. The expression vector of F39, encoding a B2/C2 mAh, C2mAb, B2Hmab, or C2Hmab.
F42. The expression vector of F39 or F41, encoding a B2/C2 mAb.
F43. A pair of expression vectors, wherein:
1) the first expression vector comprises an expression cassette comprising a polynucleotide sequence which comprises the following elements in 5’ to 3’ order: a) a first copy of a GAPDH promoter operably linked to a nucleotide sequence encoding an antibody light chain followed by a first polyA signal sequence; b) a second copy of the GAPDH promoter operably linked to a nucleotide sequence encoding an antibody heavy chain followed by a second polyA signal sequence; and c) a promoter operably linked to a nucleotide sequence encoding a selectable marker followed by a third polyA signal sequence; and
2) the second expression vector comprises an expression cassette comprising a polynucleotide sequence which comprises the following elements in 5’ to 3’ order: a) a first copy of a GAPDH promoter operably linked to a nucleotide sequence encoding an antibody light chain followed by a first polyA signal sequence; b) a second copy of the GAPDH promoter operably linked to a nucleotide sequence encoding an antibody heavy chain fusion followed by a second polyA signal sequence; and c) a promoter operably linked to a nucleotide sequence encoding a selectable marker followed by a third polyA signal sequence.
F44. A pair of expression vectors, wherein:
1) the first expression vector comprises an expression cassette comprising a polynucleotide sequence which comprises the following elements in 5’ to 3’ order: a) a first copy of a GAPDH promoter operably linked to a nucleotide sequence encoding an antibody heavy chain followed by a first polyA signal sequence; b) a second copy of the GAPDH promoter operably linked to a nucleotide sequence encoding an antibody light chain followed by a second polyA signal sequence; and c) a promoter operably linked to a nucleotide sequence encoding a selectable marker followed by a third polyA signal sequence; and 2) the second expression vector comprises an expression cassette comprising a polynucleotide sequence which comprises the following elements in 5’ to 3’ order: a) a first copy of a GAPDH promoter operably linked to a nucleotide sequence encoding an antibody light chain followed by a first polyA signal sequence; b) a second copy of the GAPDH promoter operably linked to a nucleotide sequence encoding an antibody heavy chain fusion followed by a second polyA signal sequence; and c) a promoter operably linked to a nucleotide sequence encoding a selectable marker followed by a third polyA signal sequence.
F45. A pair of expression vectors, wherein:
1) the first expression vector comprises an expression cassette comprising a polynucleotide sequence which comprises the following elements in 5’ to 3’ order: a) a first copy of a GAPDH promoter operably linked to a nucleotide sequence encoding an antibody light chain followed by a first polyA signal sequence; b) a second copy of the GAPDH promoter operably linked to a nucleotide sequence encoding an antibody heavy chain followed by a second polyA signal sequence; and c) a promoter operably linked to a nucleotide sequence encoding a selectable marker followed by a third polyA signal sequence; and
2) the second expression vector comprises an expression cassette comprising a polynucleotide sequence which comprises the following elements in 5’ to 3’ order: a) a first copy of a GAPDH promoter operably linked to a nucleotide sequence encoding an antibody heavy chain fusion followed by a first polyA signal sequence; b) a second copy of the GAPDH promoter operably linked to a nucleotide sequence encoding an antibody light chain followed by a second polyA signal sequence; and c) a promoter operably linked to a nucleotide sequence encoding a selectable marker followed by a third polyA signal sequence.
F46. A pair of expression vectors, wherein:
1) the first expression vector comprises an expression cassette comprising a polynucleotide sequence which comprises the following elements in 5’ to 3’ order: a) a first copy of a GAPDH promoter operably linked to a nucleotide sequence encoding an antibody heavy chain followed by a first polyA signal sequence; b) a second copy of the GAPDH promoter operably linked to a nucleotide sequence encoding an antibody light chain followed by a second polyA signal sequence; and c) a promoter operably linked to a nucleotide sequence encoding a selectable marker followed by a third polyA signal sequence; and
2) the second expression vector comprises an expression cassette comprising a polynucleotide sequence which comprises the following elements in 5 ’ to 3 ’ order a) a first copy of a GAPDH promoter operably linked to a nucleotide sequence encoding an antibody heavy chain fusion followed by a first polyA signal sequence; b) a second copy of the GAPDH promoter operably linked to a nucleotide sequence encoding an antibody light chain followed by a second polyA signal sequence; and c) a promoter operably linked to a nucleotide sequence encoding a selectable marker followed by a third polyA signal sequence.
F47. The pair of expression vectors of any one of F43-F46, wherein the antibody light chain of the first expression vector is the same as the antibody light chain of the second expression vector.
F48. The pair of expression vectors of any one of F43-F47, wherein the antibody heavy chain of the first expression vector is the same as the antibody heavy chain portion of the antibody heavy chain fusion of the second expression vector.
F49. The pair of expression vectors of any one of F43-F48, wherein the antibody light chain of the first expression vector is the same as the antibody light chain of the second expression vector, and the antibody heavy chain of the first expression vector is the same as the antibody heavy chain portion of the antibody heavy chain fusion of the second expression vector.
F50. The pair of expression vectors of any one of F43-F46, wherein the antibody light chain of the first expression vector is different from the antibody light chain of the second expression vector.
F51. The pair of expression vectors of any one of F43-F46 or F50, wherein the antibody heavy chain of the first expression vector is different from the antibody heavy chain portion of the antibody heavy chain fusion of the second expression vector. F52. The pair of expression vectors of any one of F43-F46, F50, or F51, wherein the antibody light chain of the first expression vector is different from the antibody light chain of the second expression vector, and the antibody heavy chain of the first expression vector is different from the antibody heavy chain portion of the antibody heavy chain fusion of the second expression vector.
F53. The pair of expression vectors of any one of F43-F52, wherein the antibody heavy chain fusion is a heavy chain-scFv fusion.
F54. The pair of expression vectors of F43, which encodes a Cl mAb.
F55. The pair of expression vectors of any one of F43-F54, wherein the promoter operably linked to the nucleotide sequence encoding the selectable marker on each of the first expression vector and the second expression vector is independently selected from the group consisting of mPGK, SRa, and SV40 promoters.
F56. The pair of expression vectors of any one of F43-F55, wherein the promoter operably linked to a nucleotide sequence encoding a selectable marker on the first expression vector is SRa.
F57. The pair of expression vectors of any one of F43-F56, wherein the promoter operably linked to a nucleotide sequence encoding a selectable marker on the second expression vector is SRa.
F58. The pair of expression vectors of any one of F43-F57, wherein the promoter operably linked to a nucleotide sequence encoding a selectable marker on the first expression vector is SRa, and the promoter operably linked to a nucleotide sequence encoding a selectable marker on the second expression vector is SRa.
F59. The pair of expression vectors of any one of F43-F58, wherein the selectable marker on the first expression vector and the selectable marker on the second expression vector is glutamine synthetase.
F60. The pair of expression vectors of any one of F43-F59, wherein each of the first, second, and third polyA signal sequences on the first expression vector and the second expression vector is independently selected from the group consisting of a bovine growth hormone (BGH) polyA signal sequence, a thymidine kinase polyA (TKpA) signal sequence, a rabbit beta-globin polyA signal sequence, and a simian virus 40 (SV40) early polyA signal sequence. F61. The pair of expression vectors of any one of F43-F60, wherein each of the first, second, and third polyA signal sequences on the first expression vector is a simian virus 40 (SV40) early polyA signal sequence.
F62. The pair of expression vectors of any one of F43-F61, wherein each of the first, second, and third polyA signal sequences on the second expression vector is a simian virus 40 (SV40) early polyA signal sequence.
F63. The pair of expression vectors of any one of F43-F62, wherein each of the first, second, and third polyA signal sequences on the first expression vector is a simian virus 40 (SV40) early polyA signal sequence, and each of the first, second, and third polyA signal sequences on the second expression vector is a simian virus 40 (SV40) early polyA signal sequence.
F64. The pair of expression vectors of any one of F43-F63, wherein each of the first, second, and third polyA signal sequences on the first expression vector and the second expression vector is a simian virus 40 (SV40) early polyA signal sequence, the promoter operably linked to a nucleotide sequence encoding a selectable marker on the first expression vector is SRa, the promoter operably linked to a nucleotide sequence encoding a selectable marker on the second expression vector is SRa, and the selectable marker on the first expression vector and the selectable marker on the second expression vector is glutamine synthetase.
F65. A pair of expression vectors, wherein:
1) the first expression vector comprises an expression cassette comprising a polynucleotide sequence which comprises the following elements in 5’ to 3’ order: a) a first copy of a GAPDH promoter operably linked to a nucleotide sequence encoding a first antibody light chain followed by a first polyA signal sequence; b) a second copy of the GAPDH promoter operably linked to a nucleotide sequence encoding a first antibody heavy chain followed by a second polyA signal sequence; and c) a promoter operably linked to a nucleotide sequence encoding a selectable marker followed by a third polyA signal sequence; and
2) the second expression vector comprises an expression cassette comprising a polynucleotide sequence which comprises the following elements in 5’ to 3’ order: a) a first copy of a GAPDH promoter operably linked to a nucleotide sequence encoding a second antibody light chain followed by a first polyA signal sequence; b) a second copy of the GAPDH promoter operably linked to a nucleotide sequence encoding a second antibody heavy chain followed by a second polyA signal sequence; and c) a promoter operably linked to a nucleotide sequence encoding a selectable marker followed by a third polyA signal sequence.
F66. A pair of expression vectors, wherein:
1) the first expression vector comprises an expression cassette comprising a polynucleotide sequence which comprises the following elements in 5’ to 3’ order: a) a first copy of a GAPDH promoter operably linked to a nucleotide sequence encoding a first antibody heavy chain followed by a first polyA signal sequence; b) a second copy of the GAPDH promoter operably linked to a nucleotide sequence encoding a first antibody light chain followed by a second polyA signal sequence; and c) a promoter operably linked to a nucleotide sequence encoding a selectable marker followed by a third polyA signal sequence; and
2) the second expression vector comprises an expression cassette comprising a polynucleotide sequence which comprises the following elements in 5’ to 3’ order: a) a first copy of a GAPDH promoter operably linked to a nucleotide sequence encoding a second antibody light chain followed by a first polyA signal sequence; b) a second copy of the GAPDH promoter operably linked to a nucleotide sequence encoding a second antibody heavy chain followed by a second polyA signal sequence; and c) a promoter operably linked to a nucleotide sequence encoding a selectable marker followed by a third polyA signal sequence.
F67. A pair of expression vectors, wherein:
1) the first expression vector comprises an expression cassette comprising a polynucleotide sequence which comprises the following elements in 5’ to 3’ order: a) a first copy of a GAPDH promoter operably linked to a nucleotide sequence encoding a first antibody light chain followed by a first polyA signal sequence; b) a second copy of the GAPDH promoter operably linked to a nucleotide sequence encoding a first antibody heavy chain followed by a second polyA signal sequence; and c) a promoter operably linked to a nucleotide sequence encoding a selectable marker followed by a third polyA signal sequence; and
2) the second expression vector comprises an expression cassette comprising a polynucleotide sequence which comprises the following elements in 5’ to 3’ order: a) a first copy of a GAPDH promoter operably linked to a nucleotide sequence encoding a second antibody heavy chain followed by a first polyA signal sequence; b) a second copy of the GAPDH promoter operably linked to a nucleotide sequence encoding a second antibody light chain followed by a second polyA signal sequence; and c) a promoter operably linked to a nucleotide sequence encoding a selectable marker followed by a third polyA signal sequence.
F68. A pair of expression vectors, wherein:
1) the first expression vector comprises an expression cassette comprising a polynucleotide sequence which comprises the following elements in 5’ to 3’ order: a) a first copy of a GAPDH promoter operably linked to a nucleotide sequence encoding a first antibody heavy chain followed by a first polyA signal sequence; b) a second copy of the GAPDH promoter operably linked to a nucleotide sequence encoding a first antibody light chain followed by a second polyA signal sequence; and c) a promoter operably linked to a nucleotide sequence encoding a selectable marker followed by a third polyA signal sequence; and
2) the second expression vector comprises an expression cassette comprising a polynucleotide sequence which comprises the following elements in 5’ to 3’ order: a) a first copy of a GAPDH promoter operably linked to a nucleotide sequence encoding a second antibody heavy chain followed by a first polyA signal sequence; b) a second copy of the GAPDH promoter operably linked to a nucleotide sequence encoding a second antibody light chain followed by a second polyA signal sequence; and c) a promoter operably linked to a nucleotide sequence encoding a selectable marker followed by a third polyA signal sequence.
F69. The pair of expression vectors of any one of F65-F68, which encodes a heteroIgG.
F70. The pair of expression vectors of any one of F65-F69, wherein the promoter operably linked to the nucleotide sequence encoding the selectable marker on each of the first expression vector and the second expression vector is independently selected from the group consisting of mPGK, SRa, and SV40 promoters.
F71. The pair of expression vectors of any one of F65-F70, wherein the promoter operably linked to a nucleotide sequence encoding a selectable marker on the first expression vector is mPGK.
F72. The pair of expression vectors of any one of F65-F71, wherein the promoter operably linked to a nucleotide sequence encoding a selectable marker on the second expression vector is mPGK.
F73. The pair of expression vectors of any one of F65-F72, wherein the promoter operably linked to a nucleotide sequence encoding a selectable marker on the first expression vector is mPGK, and the promoter operably linked to a nucleotide sequence encoding a selectable marker on the second expression vector is mPGK.
F74. The pair of expression vectors of any one of F65-F73, wherein the selectable marker on the first expression vector and the selectable marker on the second expression vector is glutamine synthetase.
F75. The pair of expression vectors of any one of F65-F74, wherein each of the first, second, and third polyA signal sequences on the first expression vector and the second expression vector is independently selected from the group consisting of a bovine growth hormone (BGH) polyA signal sequence, a thymidine kinase polyA (TKpA) signal sequence, a rabbit beta-globin polyA signal sequence, and a simian virus 40 (SV40) early polyA signal sequence. F76. The pair of expression vectors of any one of F65-F75, wherein each of the first, second, and third polyA signal sequences on the first expression vector is a simian virus 40 (SV40) early polyA signal sequence.
F77. The pair of expression vectors of any one of F65-F76, wherein each of the first, second, and third polyA signal sequences on the second expression vector is a simian virus 40 (SV40) early polyA signal sequence.
F78. The pair of expression vectors of any one of F65-F77, wherein each of the first, second, and third polyA signal sequences on the first expression vector is a simian virus 40 (SV40) early polyA signal sequence, and each of the first, second, and third polyA signal sequences on the second expression vector is a simian virus 40 (SV40) early polyA signal sequence.
F79. The pair of expression vectors of any one of F65-F78, wherein each of the first, second, and third polyA signal sequences on the first expression vector and the second expression vector is a simian virus 40 (SV40) early polyA signal sequence, the promoter operably linked to a nucleotide sequence encoding a selectable marker on the first expression vector is mPGK, the promoter operably linked to a nucleotide sequence encoding a selectable marker on the second expression vector is mPGK, and the selectable marker on the first expression vector and the selectable marker on the second expression vector is glutamine synthetase.
F80. The pair of expression vectors of any one of F43-F79, wherein the GAPDH promoter on the first expression vector is a CMV/GAPDH promoter.
F81. The pair of expression vectors of any one of F43-F80, wherein the GAPDH promoter on the second expression vector is a CMV/GAPDH promoter.
F82. The pair of expression vectors of any one of F43-F81, wherein the GAPDH promoter on the first expression vector is a CMV/GAPDH promoter, and the GAPDH promoter on the second expression vector is a CMV/GAPDH promoter.
F83. The pair of expression vectors of any one of F80-F82, wherein the CMV promoter enhancer is 5 ’ of the GAPDH promoter. F84. The pair of expression vectors of any one of F80-F83, wherein the GAPDH promoter on the first expression vector and/or the second expression vector comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 1.
F85. The pair of expression vectors of any one of F80-F84, wherein the GAPDH promoter on the first expression vector and/or the second expression vector comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO: 1.
F86. The pair of expression vectors of any one of F80-F85, wherein the GAPDH promoter on the first expression vector and/or the second expression vector comprises the nucleotide sequence of SEQ ID NO: 1.
F87. A mammalian host cell comprising an expression vector of any one of F39-F32.
F88. A mammalian host cell comprising a pair of expression vectors of any one of F43-F86.
F89. The mammalian host cell of F87 or F88, which is a Chinese hamster ovary (CHO) cell.
F90. The mammalian host cell of F89, wherein the CHO cell is a dhfr- CHO cell or a GSKO CHO cell.
F91. The mammalian host cell of F89, wherein the CHO cell is a GSKO CHO cell.
F92. A method for producing an antibody modality comprising culturing a mammalian host cell of any of any one of F87-F91 under conditions in which the antibody modality is produced, and recovering the antibody modality from the culture.
F93. The method of F92, wherein the recovered antibody modality is purified and formulated in a pharmaceutically acceptable formulation.
EXAMPLES
EXAMPLE 1. Vector Engineering Strategy to Improve Productivity for Various Antibody Modalities Summary
[0297] Establishing stable Chinese Hamster Ovary (CHO) cell lines producing therapeutic recombinant antibodies involves integration of the heavy and light chains (HC and LC, respectively) from an expression vector(s) into the genome through a selection process. In the glutamine synthetase knockout (GS KO) expression system, selection of stable pools can be controlled by balancing the expression level of the exogenous GS gene and the concentration of its specific inhibitor, L- methionine sulfoximine (MSX).
[0298] This Example describes a further vector optimization by using promoters with different strengths to modulate the expression levels of the HC, LC, and GS genes in combination with various MSX concentrations to improve productivity of different therapeutic modalities in CHO cells. The vector engineering strategy described herein can be expanded to improve and optimize productivity for other recombinant proteins expressed in stable pools in general.
Materials and Methods
[0299] Plasmid Generation. The coding sequences of the LCs and HCs (or HC-fusions) were inserted in the pPBGS plasmid backbone using Golden Gate cloning to generate a tri-cistronic vector. Briefly, the CMV/ADL, CMV/GAPDH promoters/enhancer fragments were used to control the LCs and HCs (or HC-fusions) with SV40-Poly A fragments following the SRa or mPGK promoters driving the expression of mGS-polyA in that order (TABLE 1). All the fragments were unidirectionally assembled using combinations of overhang sequences to facilitate Golden Gate cloning. MSX was used at 0, 12.5, 25, and 50 pM.
TABLE 1. Different Vector Configurations
[0300] Transfection of Plasmids into GS KO Host. A GS KO (knockout) clonal cell host, derived from the CHO-K1 parental host, was used for generating stable pools expressing a number of different antibody modalities as listed in TABLE 1. Host cells were passaged at a seeding density of 0.4-0.3 x 106 cells/mL every 3-4 days in a proprietary DMEM-F 12-based media in shake flasks at 120 rpm, 36°C, and 5% CO2. Twenty-four hours before transfection, the host cells were seeded at 1 x 106 cells/mL to ensure the cells would be in exponential growth phase at transfection.
[0301] Stable pools expressing the antibodies were generated using a Gene Pulser XCell (BioRad Laboratories; Hercules, CA) following the manufacturer’s protocol. Duplicate transfections were performed for each of the vector configurations. Briefly, 20 pg of each plasmid in combination with 5 pg of a proprietary piggybac transposase were electroporated into 20 x 106 host cells. The transfected cells were recovered in 20 mL of growth media in 50 mL spin tubes at 225 rpm, 36°C and 5% CO2. [0302] Selection and Recovery. Seventy-two hours post transfection, the cells were spun down and transferred into selection media without glutamine or growth factor and with 0, 12.5, 25, and 50 pM of MSX. The cells were passaged at seeding densities around 1-2 x 106 cells/mL every 3-4 days until viability reached over 90%, when the seeding density was reduced to 0.4-0.3 x 106 cells/mL.
[0303] Fed-batch Production. Fully recovered cells were inoculated for fed-batch production at 1 x 106 cells/mL in a proprietary basal media. The cultures were supplemented with proprietary feeds on days 3, 6, and 8 and harvested on day 10. Cell count and viability were determined using a Vi-Cell BLU cell viability analyzer (Beckman Coulter, Brea, CA). Supernatants were analyzed for titer (protein A-HPLC).
Results
[0304] Results in FIGs. 2A-2B show higher titers (A) and cell specific productivity (B) obtained using the CMV/GADPH promoter for light chain (HC), heavy chain (LC) in combination with SRa or mPGK promoters for glutamine synthetase (GS) at different MSX levels.
[0305] Additionally, FIGs. 3A-3C show similar Viability (A), Viable Dell Density (VCD) (B) and Integrated Viable Cell Density (VCD) (C) profiles for all vector configurations during fed-batch production. Color corresponds to the MSX concentration levels.
EXAMPLE 2. Vector Engineering Strategy to Improve Productivity for a B2 mAb Bispecific Molecule
Summary
[0306] Establishing stable Chinese Hamster Ovary (CHO) cell lines producing therapeutic recombinant antibodies and molecules derived from antibodies involves integration of the heavy and light chains (HC and LC, respectively) from an expression vector(s) into the genome through a selection process. In the glutamine synthetase knockout (GS KO) expression system, selection of stable pools can be controlled by balancing the expression level of the exogenous GS gene and the concentration of its specific inhibitor, L-methionine sulfoximine (MSX). [0307] This Example describes a further vector optimization by using promoters with different strengths to modulate the expression levels of the HC, LC, and GS genes in combination with various MSX concentrations to improve productivity of bispecific antibodies in CHO cells. The vector engineering strategy described herein can be expanded to improve and optimize productivity for other recombinant proteins expressed in stable pools in general.
Materials and Methods
[0308] B2 mAh Plasmid Generation. The coding sequences of a HC-scFv and LC were inserted in the pPBGS plasmid backbone using Golden Gate cloning to generate a tri-cistronic vector. Briefly, CMV/ADL, CMV/GAPDH, or CMW/EF 1 promoters/enhancer fragments were used to control the HC-scFv and LC with SV40-Poly A fragments following the SRa, mPGK, or SV40 promoters driving the expression of mGS-polyA in that order (TABLE 2). All the fragments were uni -directionally assembled using combinations of overhang sequences to facilitate Golden Gate cloning. MSX was used at 0, 12.5, 25, and 50 pM.
TABLE 2. Different Vector Configurations
[0309] Transfection of Plasmids into GS KO Host. A GS KO (knockout) clonal cell host, derived from the CHO-K1 parental host, was used for generating stable pools expressing the B2 mAb. Host cells were passaged at a seeding density of 0.4-0.3 x 106 cells/mL every 3-4 days in a proprietary DMEM-F 12-based media in shake flasks at 120 rpm, 36°C, and 5% CO2. Twenty-four hours before transfection, the host cells were seeded at 1 x 106 cells/mL to ensure the cells would be in exponential growth phase at transfection.
[0310] Stable pools expressing the B2 mAb were generated using a Gene Pulser XCell (BioRad Laboratories; Hercules, CA) following the manufacturer’s protocol. Duplicate transfections were performed for each of the vector configurations. Briefly, 20 pg of each plasmid in combination with 5 pg of a proprietary piggybac transposase were electroporated into 20 x 106 host cells. The transfected cells were recovered in 20 mb of growth media in 50 mb spin tubes at 225 rpm, 36°C, and 5% CO2. [0311] Selection and Recovery. Seventy-two hours post transfection, the cells were spun down and transferred into selection media without glutamine and with 0, 12.5, 25, and 50 pM of MSX. The cells were passaged at seeding densities around 1-2 x 106 cells/mL every 3-4 days until viability reached over 90%, when the seeding density was reduced to 0.4-0.3 x 106 cells/mL.
[0312] Fed-batch Production. Fully recovered cells were inoculated for fed-batch production at 1 x 106 cells/mL in a proprietary basal media. The cultures were supplemented with proprietary feeds on days 3, 6, and 8 and harvested on day 10. Cell count and viability were determined using a Vi-Cell BLU cell viability analyzer (Beckman Coulter, Brea, CA). Supernatants were analyzed for 1) titer (protein A-HPLC) and 2) product quality atributes including aggregates, clips, and isoforms using size-exclusion chromatography (SEC-UHPLC) (Waters UPLC H-class series), reduced capillary electrophoresis (rCE-SDS) (Sciex PA 800 Plus Pharmaceutical Analysis System), and analytical hydrophobic interaction chromatography (HIC-HPLC) (Agilent HPLC 1100/1200 series), respectively.
Results
[0313] Results in FIGs. 4A-4B show higher titers (A) and cell specific productivity (B) obtained using the CMV/GADPH promoter for light chain (HC), heavy chain (LC) in combination with SRa or mPGK promoters for glutamine synthetase (GS) in 12.5 pM, 25 pM, and 50 pM and 0 pM and 12.5 pM MSX, respectively.
[0314] Additionally, FIGs. 5A-5C show aggregates (SEC HMW) (A), clips (rCE low-molecular weight and middle-molecular weight species, LMW+MMW) (B), and isoforms (HIC-HPLC- postpeaks) (C). Impurities were comparable among the vector configurations with the highest titer: CMV/GADPH with SRa/mPGK promoters combinations in 25 pM and 12.5 pM MSX, respectively. [0315] Finally, FIGs. 6A-6C show similar Viability (A), Viable Dell Density (VCD) (B), and Integrated Viable Cell Density (VCD) (C) profiles for all vector configurations with the same MSX concentration during fed-batch production except for CMV/GADPH + SV40 + 50pM MSX.
EXAMPLE 3. Vector Engineering Strategy to Improve Productivity for a ScFv 2 + 2 A and B Bispecific Molecules
Summary
[0316] Establishing stable Chinese Hamster Ovary (CHO) cell lines producing therapeutic recombinant antibodies and molecules derived from antibodies involves integration of the heavy and light chains (HC and LC, respectively) from an expression vector(s) into the genome through a selection process. In the glutamine synthetase knockout (GS KO) expression system, selection of stable pools can be controlled by balancing the expression level of the exogenous GS gene and the concentration of its specific inhibitor, L-methionine sulfoximine (MSX).
[0317] This Example describes a further vector optimization by using promoters with different strengths to modulate the expression levels of the HC, LC, and GS genes in combination with various MSX concentrations to improve productivity of bispecific antibodies in CHO cells. The vector engineering strategy described herein can be expanded to improve and optimize productivity for other recombinant proteins expressed in stable pools in general.
Materials and Methods
[0318] ScFv 2+2 Plasmid Generation. The coding sequences of the LCs and HCs were inserted in the pPBGS plasmid backbone using Golden Gate cloning to generate a tri-cistronic vector. Briefly, the CMV/ADL or CMV/GAPDH promoters/enhancer fragments were used to control the LCs and HCs with SV40-Poly A fragments following the SRa, mPGK, or SV40 promoters driving the expression of mGS-polyA in that order (TABLE 3). All the fragments were uni -directionally assembled using combinations of overhang sequences to facilitate Golden Gate cloning. MSX was used at 0, 12.5, 25, and 50 pM.
TABLE 3. Different Vector Configurations [0319] Transfection of Plasmids into GS KO Host. A GS KO (knockout) clonal cell host, derived from the CHO-K1 parental host, was used for generating stable pools expressing either 2+2 ScFv molecule A or B. Host cells were passaged at a seeding density of 0.4-0.3 x 106 cells/mL every 3-4 days in a proprietary DMEM-F 12-based media in shake flasks at 120 rpm, 36°C, and 5% CO2. Twenty-four hours before transfection, the host cells were seeded at 1 x 106 cells/mL to ensure the cells would be in exponential growth phase at transfection.
[0320] Stable pools expressing 2+2 ScFv molecule A or B were generated using a Gene Pulser XCell (BioRad Laboratories; Hercules, CA) following the manufacturer’s protocol. Duplicate transfections were performed for each of the vector configurations. Briefly, 20 pg of each plasmid in combination with 5 pg of a proprietary piggybac transposase were electroporated into 20 x 106 host cells. The transfected cells were recovered in 20 mL of growth media in 50 mL spin tubes at 225 rpm, 36°C, and 5% CO2.
[0321] Selection and Recovery. Seventy-two hours post transfection, the cells were spun down and transferred into selection media without glutamine and with 0, 12.5, 25, and 50 pM of MSX. The cells were passaged at seeding densities around 1-2 x 106 cells/mL every 3-4 days until viability reached over 90%, when the seeding density was reduced to 0.4-0.3 x 106 cells/mL.
[0322] Fed-batch Production. Fully recovered cells were inoculated for fed-batch production at 1 x 106 cells/mL in a proprietary basal media. The cultures were supplemented with proprietary feeds on days 3, 6, and 8 and harvested on day 10. Cell count and viability were determined using a Vi-Cell BLU cell viability analyzer (Beckman Coulter, Brea, CA). Supernatants were analyzed for 1) titer (protein A-HPLC) and 2) product quality atributes including aggregates, clips, and isoforms using size-exclusion chromatography (SEC-UHPLC) (Waters UPLC H-class series), reduced capillary electrophoresis (rCE-SDS) (Sciex PA 800 Plus Pharmaceutical Analysis System), and analytical hydrophobic interaction chromatography (HIC-HPLC) (Agilent HPLC 1100/1200 series), respectively.
Results
[0323] Results in FIGs. 7A-7B show higher titers (A) and cell specific productivity (B) for 2+2 ScFv molecule A obtained using the CMV/GADPH promoter for light chain (HC), heavy chain (LC) in combination with SRa or mPGK promoters for glutamine synthetase (GS) in 12.5 pM, 25 pM, and 50 pM and 0 pM and 12.5 pM MSX, respectively.
[0324] Results in FIGs. 8A-8B show higher titers (A) and cell specific productivity (B) for 2+2 ScFv molecule B obtained using the CMV/GADPH promoter for light chain (HC), heavy chain (LC) in combination with SRa or mPGK promoters for glutamine synthetase (GS) in 12.5 pM, 25 pM, and 50 pM and 0 pM and 12.5 pM MSX, respectively. [0325] FIGs. 9A-9C show aggregates (SEC HMW) (A), clips (rCE low-molecular weight and middle-molecular weight species, LMW+MMW) (B), and isoforms (HIC-HPLC- post-peaks) (C) for 2+2 ScFv molecule A. Impurities were comparable among the vector configurations with the highest titer: CMV/GADPH with SRa/mPGK promoters combinations in 25 pM and 12.5 pM MSX, respectively.
[0326] FIGs. 10A-10C show aggregates (SEC HMW) (A), clips (rCE low-molecular weight and middle-molecular weight species, LMW+MMW) (B), and isoforms (HIC-HPLC- post-peaks) (C) for 2+2 ScFv molecule B. Impurities were comparable among the vector configurations with the highest titer: CMV/GADPH with SRa/mPGK promoters combinations in 25 pM and 12.5 pM MSX, respectively.
[0327] FIGs. 11A-11C show similar Viability (A), Viable Dell Density (VCD) (B), and Integrated Viable Cell Density (VCD) (C) profiles for all vector configurations with the same MSX concentration for 2+2 ScFv molecule A during fed-batch production except for CMV/GADPH + mPGK + 25 pM MSX and CMV/GADPH + SV40 +2 5pM MSX.
[0328] FIGs. 12A-12C shows similar Viability (A), Viable Dell Density (VCD) (B), and Integrated Viable Cell Density (VCD) (C) profiles for all vector configurations for 2+2 ScFv molecule B with the same MSX concentration during fed-batch production except for CMV/GADPH + mPGK + 25 pM MSX and CMV/GADPH + Sra + 25 pM MSX.
EXAMPLE 4. Vector Engineering Strategy to Improve Productivity for MAbs Summary
[0329] Establishing stable Chinese Hamster Ovary (CHO) cell lines producing therapeutic recombinant antibodies and molecules derived from antibodies involves integration of the heavy and light chains (HC and LC, respectively) from an expression vector(s) into the genome through a selection process. In the glutamine synthetase knockout (GS KO) expression system, selection of stable pools can be controlled by balancing the expression level of the exogenous GS gene and the concentration of its specific inhibitor, L-methionine sulfoximine (MSX).
[0330] This Example describes a further vector optimization by using promoters with different strengths to modulate the expression levels of the HC, LC, and GS genes in combination with various MSX concentrations to improve productivity of monoclonal antibodies in CHO cells.
Materials and Methods
[0331] MAh Plasmid Generation. The coding sequences of the LCs and HCs for MAbl and MAb2 were inserted in the pBMV plasmid backbone using Golden Gate cloning to generate a tri- or bi- cistronic vector. Briefly, the CMV/ADL, CMV/GAPDH, or CMV/EF1 promoters/enhancer fragments were used to control the LCs and HCs with SV40-Poly A fragments following the SRa, mPGK, SV40, or miniSV40 promoters driving the expression of mGS-polyA in that order (TABLEs 4 and 5). All the fragments were uni-directionally assembled using combinations of overhang sequences to facilitate Golden Gate cloning. MSX was used at 10 (MAbl) or 25 pM (MAb2).
TABLE 4. Monocistronic and Bicistronic Vector Configurations for MAbl
TABLE 5. Different Vector Configurations for MAb2
[0332] Transfection ofMAbl andMAb2 Plasmids into GS KO Host. A GS KO (knockout) clonal cell host, derived from the CHO-K1 parental host, was used for generating stable pools expressing MAbl or MAb2. Host cells were passaged at a seeding density of 0.3 -0.4 x 106 cells/mL every 3-4 days in a proprietary DMEM-F 12-based media in shake flasks at 130 rpm, 36°C and 5% CO2. Twenty-four hours before transfection, the host cells were seeded at 1 x 106 cells/mL to ensure the cells would be in exponential growth phase at transfection.
[0333] Stable pools expressing MAbl or MAb2 were generated using a Lipofectamine LTX (Gibco, Billings, MT) and Opti-MEM I Reduced Serum Media (Gibco, Billings, MT) following the manufacturer’s protocol. Single transfection was performed for each of the vector configurations.
Briefly, 2 pg of each bicistronic plasmid in combination with 2 pg of a proprietary piggybac transposase were added into 4 x 106 host cells. For the monicistronic plasmids, 1 pg of each plasmid encoding LC or HC was used. The transfected cells were recovered in 4 mb of growth media in a 6- well plate at 225 rpm, 36°C and 5% CO2.
[0334] Selection and Recovery. Forty-eight to seventy-two hours post transfection, the cells were spun down and transferred into selection media without glutamine and with 0, 12.5, 25 and 50 pM of MSX. The cells were passaged at seeding densities around 0.5-0.8 x 106 cells/mL every 3-4 days until viability reached over 90%, when the seeding density was reduced to 0.4-0.3 x 106 cells/mL.
[0335] Fed-batch Production. Recovered cells were used to seed 4-mL fed-batch production cultures or 40-mL fed-batch production cultures in 50-mL conical TPP TubeSpin® bioreactor tubes in 24-well culture blocks at 1 x 106 cells/mL, which were harvested after 10 days. During production, viable cell density and viability were monitored using a Vi-Cell® counter (Beckman Coulter, Brea, CA) and media was exchanged at days 3, 6, and 8. At day 10, viable cell density and viability were measured and the conditioned media from these batch productions were used to determine titer by ForteBio OCTET® Red equipped with Protein A biosensors. After harvesting, Fc-containing proteins in cell culture medium were purified using ProA affinity capture ( 1 ml HiTrap MabSelect SuRe column, GE Life Sciences), eluted with 100 mM sodium acetate, pH 3.6 followed immediately by buffer exchange into 10 mM sodium acetate, 150 mM NaCl, pH 5.2 using a 5 ml HiTrap Desalting column (GE Life Sciences) as described previously (Gong et al., 2021, MAbs 13(1): 1870058). ProA yield was calculated by measuring the absorbance at 280 nm (A280).
Results for MAbl
[0336] Results in FIGs. 13A-13B show higher titers and proA yields obtained using the CMV/GADPH promoter for light chain (HC) and heavy chain (LC) in a bicistronic vectors in combination with SRa, mPGK, SV40, or miniSV40 promoters for glutamine synthetase (GS) in 10 pM MSX.
[0337] Additionally, FIG. 14 shows % main peak from SEC for all vector configurations evaluated. Impurities (100% - % main peak) were comparable among all the vector configurations.
Results for MAb2
[0338] Results in FIG. 15 shows highest titer obtained using the CMV/GADPH promoter for light chain (HC), heavy chain (LC) in combination with mPGK promoter for glutamine synthetase (GS) in 25 pM MSX.
[0339] Additionally, FIGs. 16A-16B show % main peak from SEC (A), and % main peak from a non-reduced capillary electrophoresis assay (nrMCE) (B). Impurities were comparable among the top titer vector configurations evaluated (low titer configurations were not tested for product quality).
EXAMPLE 5. Vector Engineering Strategy to Improve Productivity of UniDab-Containing B2- and C2-Hmabs
Summary
[0340] Establishing stable Chinese Hamster Ovary (CHO) cell lines producing therapeutic recombinant antibodies and molecules derived from antibodies involves integration of the heavy and light chains (HC and LC, respectively) from an expression vector(s) into the genome through a selection process. In the glutamine synthetase knockout (GS KO) expression system, selection of stable pools can be controlled by balancing the expression level of the exogenous GS gene and the concentration of its specific inhibitor, L-methionine sulfoximine (MSX).
[0341] This Example describes vector optimization using promoters with different strengths to modulate the expression levels of the HC-UniDab, LC, and GS genes in combination with different MSX concentrations to improve productivity of bispecific antibodies in CHO cells. The vector engineering strategy described herein can be expanded to improve and optimize productivity for other recombinant proteins expressed in stable pools in general.
Materials and Methods
[0342] B2- and C2-Hmah Plasmid Generation. The coding sequences of HC-UniDab and LC were inserted into PBGS plasmid backbone using Golden Gate cloning. The CMV/adL, CMV/GapdH promoters/enhancer fragments were used to control HC -Unidab and LC with SV40-polyA fragments followed by SRa or mpGK promoters driving expression of mGS-PolyA (TABLE 6). All fragments were uni-directionally assembled using a combination of overhang sequences to facilitate Golden Gate cloning. MSX was used at 18.75 or 37.5 uM MSX concentration depending on the GS promoter.
TABLE 6. Different Vector Configurations
[0343] Transfection of Plasmids into GS KO Host. A GS KO (knockout) clonal cell host, derived from the CHO-K1 parental host, was used for generating stable pools expressing the B2- and C2- Hmabs. Host cells were passaged at a seeding density of 0.4-0.3 x 106 cells/mL every 3-4 days in a proprietary DMEM-F 12-based media in shake flasks at 120 rpm, 36°C and 5% CO2. Twenty-four hours before transfection, the host cells were seeded at 1 x 106 cells/mL to ensure the cells would be in exponential growth phase at transfection.
[0344] Stable pools expressing the B2- and C2-HmAb were generated using a Gene Pulser XCell (BioRad Laboratories; Hercules, CA) following the manufacturer’s protocol. Quadruplicate transfections were performed for each of the vector configurations. Briefly, 20 pg of each plasmid in combination with 5 pg of a proprietary piggybac transposase were electroporated into 20 x 106 host cells. The transfected cells were recovered in 20 mL of growth media in 50 mL spin tubes at 225 rpm, 36°C and 5% CO2.
[0345] Selection and Recovery. Seventy-two hours post transfection, the cells were spun down and transferred into selection media without glutamine and with 18.75 or 37.5 pM MSX. The cells were passaged at seeding densities around 1-2 x 106 cells/mL every 3-4 days until viability reached over 90%, when the seeding density was reduced to 0.4-0.3 x 106 cells/mL.
[0346] Fed-batch Production. Fully recovered cells were inoculated for fed-batch production at 1 x 106 cells/mL in a proprietary basal media. The cultures were supplemented with proprietary feeds on days 3, 6, 8, 10, and 13 and harvested on day 15. Cell count and viability were determined using a Vi- Cell BLU cell viability analyzer (Beckman Coulter, Brea, CA). Supernatants were analyzed for 1) titer (protein A-HPLC) and 2) product quality attributes including aggregates, clips, and isoforms using size-exclusion chromatography (SEC-UHPLC) (Waters UPLC H-class series), reduced and non-reduced capillary electrophoresis (rCE- and nrCE-SDS) (Sciex PA 800 Plus Pharmaceutical Analysis System).
Results
[0347] Higher titers (FIG. 17A) and cell specific productivity (FIG. 17B) was observed for both B2- Hmab and C2-Hmab molecules using CMV/GAPDH for light chain (LC), heavy chain (HC -Unidab) in combination with SRa or mPGK promoters for glutamine synthetase (GS) in 37.5 pM MSX and 18.75 pM MSX, respectively. Titers (FIG. 17A) are normalized to CMV/adL, SRa-GS pools.
[0348] Impurities are comparable across the different vector configurations for aggregates (FIG. 18A), partial species (FIG. 18B), and clips (FIG. 18C).
[0349] Pools exhibited similar viability (FIG. 19A), viable cell density (FIG. 19B), and integrated viable cell density (FIG. 19C) over the course of the fed batch culture.

Claims

What is claimed is:
1. An expression cassette comprising a polynucleotide sequence which comprises the following elements in 5 ’ to 3 ’ order: a) a first copy of a GAPDH promoter operably linked to a nucleotide sequence encoding a first antibody chain or antibody chain fusion followed by a first polyA signal sequence; b) a second copy of the GAPDH promoter operably linked to a nucleotide sequence encoding a second antibody chain or antibody chain fusion followed by a second polyA signal sequence; and c) a promoter operably linked to a nucleotide sequence encoding a selectable marker followed by a third polyA signal sequence, wherein, when the first antibody chain or antibody chain fusion is an antibody light chain, the second antibody chain or antibody chain fusion is an antibody heavy chain or antibody heavy chain fusion, wherein, when the first antibody chain or antibody chain fusion is an antibody heavy chain or antibody heavy chain fusion, the second antibody chain or antibody chain fusion is an antibody light chain, and wherein the antibody heavy chain fusion is selected from the group consisting of a heavy chain-scFv fusion, a heavy chain-cytokine fusion, and a heavy chain VH fusion.
2. The expression cassette of claim 1, wherein the first antibody chain or antibody chain fusion is an antibody light chain, and the second antibody chain or antibody chain fusion is an antibody heavy chain.
3. The expression cassette of claim 1, wherein the first antibody chain or antibody chain fusion is an antibody light chain, and the second antibody chain or antibody chain fusion is an antibody heavy chain fusion.
4. The expression cassette of claim 1, wherein the first antibody chain or antibody chain fusion is an antibody heavy chain, and the second antibody chain or antibody chain fusion is an antibody light chain.
5. The expression cassette of claim 1, wherein the first antibody chain or antibody chain fusion is an antibody heavy chain fusion, and the second antibody chain or antibody chain fusion is an antibody light chain.
6. The expression cassete of any one of claims 1, 3, or 5, wherein the antibody heavy chain fusion is a heavy chain-scFv fusion.
7. The expression cassete of any one of claims 1, 3, or 5, wherein the antibody heavy chain fusion is a heavy chain VH fusion.
8. The expression cassete of any one of claims 1-7, wherein the selectable marker is glutamine synthetase or dihydrofolate reductase.
9. The expression cassete of any one of claims 1-8, wherein the selectable marker is glutamine synthetase.
10. The expression cassete of any one of claims 1-9, wherein the GAPDH promoter is a CMV/GAPDH promoter.
11. The expression cassete of claim 10, wherein the CMV promoter enhancer is 5’ of the GAPDH promoter.
12. The expression cassete of any one of claims 1-11, wherein the GAPDH promoter comprises the nucleotide sequence of SEQ ID NO: 1.
13. The expression cassete of any one of claims 1-12, wherein the promoter operably linked to the nucleotide sequence encoding the selectable marker is selected from the group consisting of mPGK, SRa, and SV40 promoters.
14. The expression cassete of any one of claims 1-13, wherein each of the first, second, and third polyA signal sequences is independently selected from the group consisting of a bovine growth hormone (BGH) polyA signal sequence, a thymidine kinase polyA (TKpA) signal sequence, a rabbit beta-globin polyA signal sequence, and a simian virus 40 (SV40) early polyA signal sequence.
15. The expression cassete of any one of claims 1-14, wherein each of the first, second, and third polyA signal sequences is a simian virus 40 (SV40) early polyA signal sequence.
16. The expression cassete of claim 1, wherein the expression cassete encodes an antibody light chain and an antibody heavy chain, the GAPDH promoter is a CMV/GAPDH promoter, the selectable marker is glutamine synthetase, the promoter operably linked to a nucleotide sequence encoding the selectable marker is mPGK, and each of the first, second, and third polyA signal sequences is a simian virus 40 (SV40) early polyA signal sequence.
17. The expression cassette of claim 1, wherein the expression cassette encodes an antibody light chain and an antibody heavy chain fusion, the GAPDH promoter is a CMV/GAPDH promoter, the selectable marker is glutamine synthetase, the promoter operably linked to a nucleotide sequence encoding the selectable marker is SRa, and each of the first, second, and third polyA signal sequences is a simian virus 40 (SV40) early polyA signal sequence.
18. The expression cassette of claim 17, wherein the antibody heavy chain fusion is a heavy chain-scFv fusion.
19. The expression cassette of claim 17, wherein the antibody heavy chain fusion is a heavy chain VH fusion.
20. An expression vector which comprises an expression cassette of any one of claims 1-19.
21. A pair of expression vectors, wherein:
1) the first expression vector comprises an expression cassette comprising a polynucleotide sequence which comprises the following elements in 5’ to 3’ order: a) a first copy of a GAPDH promoter operably linked to a nucleotide sequence encoding an antibody light chain followed by a first polyA signal sequence; b) a second copy of the GAPDH promoter operably linked to a nucleotide sequence encoding an antibody heavy chain followed by a second polyA signal sequence; and c) a promoter operably linked to a nucleotide sequence encoding a selectable marker followed by a third polyA signal sequence; and
2) the second expression vector comprises an expression cassette comprising a polynucleotide sequence which comprises the following elements in 5’ to 3’ order: a) a first copy of a GAPDH promoter operably linked to a nucleotide sequence encoding an antibody light chain followed by a first polyA signal sequence; b) a second copy of the GAPDH promoter operably linked to a nucleotide sequence encoding an antibody heavy chain fusion followed by a second polyA signal sequence; and c) a promoter operably linked to a nucleotide sequence encoding a selectable marker followed by a third polyA signal sequence.
22. The pair of expression vectors of claim 21, wherein the antibody light chain of the first expression vector is the same as the antibody light chain of the second expression vector, and the antibody heavy chain of the first expression vector is the same as the antibody heavy chain portion of the antibody heavy chain fusion of the second expression vector.
23. The pair of expression vectors of claim 21, wherein the antibody light chain of the first expression vector is different from the antibody light chain of the second expression vector, and the antibody heavy chain of the first expression vector is different from the antibody heavy chain portion of the antibody heavy chain fusion of the second expression vector.
24. The pair of expression vectors of any one of claims 21-23, wherein the antibody heavy chain fusion is a heavy chain-scFv fusion.
25. The pair of expression vectors of any one of claims 21-24, wherein each of the first, second, and third polyA signal sequences on the first expression vector and the second expression vector is a simian virus 40 (SV40) early polyA signal sequence, the promoter operably linked to a nucleotide sequence encoding a selectable marker on the first expression vector is SRa, the promoter operably linked to a nucleotide sequence encoding a selectable marker on the second expression vector is SRa, and the selectable marker on the first expression vector and the selectable marker on the second expression vector is glutamine synthetase.
26. A pair of expression vectors, wherein:
1) the first expression vector comprises an expression cassette comprising a polynucleotide sequence which comprises the following elements in 5’ to 3’ order: a) a first copy of a GAPDH promoter operably linked to a nucleotide sequence encoding a first antibody light chain followed by a first polyA signal sequence; b) a second copy of the GAPDH promoter operably linked to a nucleotide sequence encoding a first antibody heavy chain followed by a second polyA signal sequence; and c) a promoter operably linked to a nucleotide sequence encoding a selectable marker followed by a third polyA signal sequence; and
2) the second expression vector comprises an expression cassette comprising a polynucleotide sequence which comprises the following elements in 5’ to 3’ order: a) a first copy of a GAPDH promoter operably linked to a nucleotide sequence encoding a second antibody light chain followed by a first polyA signal sequence; b) a second copy of the GAPDH promoter operably linked to a nucleotide sequence encoding a second antibody heavy chain followed by a second polyA signal sequence; and c) a promoter operably linked to a nucleotide sequence encoding a selectable marker followed by a third polyA signal sequence.
27. The pair of expression vectors of claim 26, which encodes a heteroIgG.
28. The pair of expression vectors of claim 26 or claim 27, wherein each of the first, second, and third polyA signal sequences on the first expression vector and the second expression vector is a simian virus 40 (SV40) early polyA signal sequence, the promoter operably linked to a nucleotide sequence encoding a selectable marker on the first expression vector is mPGK, the promoter operably linked to a nucleotide sequence encoding a selectable marker on the second expression vector is mPGK, and the selectable marker on the first expression vector and the selectable marker on the second expression vector is glutamine synthetase.
29. The pair of expression vectors of any one of claims 21-28, wherein the GAPDH promoter on the first expression vector is a CMV/GAPDH promoter, and the GAPDH promoter on the second expression vector is a CMV/GAPDH promoter.
30. The pair of expression vectors of claim 29, wherein the CMV promoter enhancer is 5’ of the GAPDH promoter.
31. The pair of expression vectors of any one of claims 21-30, wherein the GAPDH promoter on the first expression vector and/or the second expression vector comprises the nucleotide sequence of SEQ ID NO: 1.
32. A mammalian host cell comprising an expression vector of claim 20 or a pair of expression vectors of any one of claims 21-31.
33. The mammalian host cell of claim 32, which is a Chinese hamster ovary (CHO) cell.
34. The mammalian host cell of claim 33, wherein the CHO cell is a glutamine synthetase (GS) knockout CHO cell.
35. A method for producing an antibody modality comprising culturing a mammalian host cell of any of any one of claims 32-34 under conditions in which the antibody modality is produced, and recovering the antibody modality from the culture.
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