Dual selection expression vector system for multiple chain biologies

Abstract

Dual selection expression vector systems are provided in which one vector employs a metabolic selectable marker and the other vector employs an antibiotic resistance selectable marker, compositions and mammalian host cells comprising such expression vector systems, and methods of using the foregoing to express recombinant proteins, including polypeptides comprising three or four unique polypeptide chains.

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 559,778, filed February 29, 2024, which is hereby incorporated by reference in its entirety. Technical Field

[0003] This disclosure provides a dual-selective expression vector system that combines metabolic selectable markers and antibiotic resistance selectable markers, compositions comprising these systems and mammalian host cells (including, but not limited to, Chinese hamster ovary (CHO) cells), and methods for expressing recombinant proteins (e.g., multi-chain biologics, such as recombinant proteins comprising three or four unique polypeptide chains) using the aforementioned substances.

[0004] Submission of sequence list

[0005] The contents of the following sequence list XML are incorporated herein by reference in their entirety: File name: 10589-WO01-SEC_ST26, Creation date: February 27, 2025; Size: 10,676 bytes. Background Technology

[0006] Biologics are used worldwide for a wide range of applications, including therapeutic and diagnostic applications, due to their broad applicability. Mammalian cell lines are the primary expression systems for these biologics, with Chinese hamster ovary (CHO) cells serving as the main cell factory (see Lalonde et al., 2017, J Biotechnol 251:128-140). Especially with the advent of biosimilars, speed to market and cost-effectiveness in biologics manufacturing are now more important than ever.

[0007] The costs associated with manufacturing biologics are very high due to the complexity of their production, which involves multi-step processes including selecting optimal cell lines, mass-culturing cells, and purifying the desired biologic from the cell harvest. Typically, the manufacture of novel antibody forms, such as those with three or four unique antibody chains, is even more complex. While these costs are decreasing due to improvements in various aspects of manufacturing, they can still be prohibitive when new antibody forms are widely adopted as first-line therapies.

[0008] To make biotherapeutic agents more accessible to patients, reducing the commodity cost of the manufacturing process is an attractive proposition. One way to achieve this is to increase the titer associated with the production cell line. Expression vector conformations can help optimize the expression levels of different chains in recombinant proteins, especially for molecules with three or four distinct polypeptide chains, resulting in more balanced chain expression, reduced impurities, and higher product quality.

[0009] Establishing stable host cell lines that generate multi-chain recombinant antibody forms (e.g., CHO cell lines) requires a selection process to integrate heavy and light chains (HC and LC, respectively) from two or more expression vectors (bicistronic or monocistronic) into the genome. The most common selection method for generating CHO cell lines utilizes a single selection approach, which relies primarily on metabolically selectable markers (e.g., dihydrofolate reductase (DHFR) or glutamine synthase (GS)) and inhibitory compounds (e.g., methotrexate (MTX) or methionine sulfoxide (MSX)), regardless of the number of expression vectors. However, this selection approach may not achieve optimized expression levels and chain ratios for complex biologics (e.g., multi-chain recombinant proteins, such as therapeutic molecules with three or four unique polypeptide chains).

[0010] Therefore, there remains a need in the art for expression vector systems that, when transfected into host cell lines, produce recombinant proteins (e.g., molecules with three or four unique polypeptide chains) at high titers while minimizing and / or improving product quality properties, such as reducing product-related impurities (e.g., high molecular weight aggregates). Such expression vector systems would be beneficial for the process development of biopharmaceuticals. Summary of the Invention

[0011] This disclosure relates to a dual-selection strategy for mammalian host cell lines used to express recombinant proteins, including, for example, different antibody-derived forms having three or four unique polypeptide chains. Illustratively, this document provides dual-selection expression vector systems, one vector employing a metabolically selectable marker and the other employing an antibiotic resistance selectable marker, compositions comprising such expression vector systems and mammalian host cells, and methods for expressing recombinant proteins using the aforementioned substances.

[0012] One aspect of this disclosure provides an expression vector system comprising a first expression vector and a second expression vector, wherein:

[0013] The first expression vector contains a first nucleotide sequence encoding a first antibody light chain, a second nucleotide sequence encoding a first antibody heavy chain, an antibody heavy chain fusion, or an Fc chain fusion, and a third nucleotide sequence encoding a metabolically selectable biomarker; and

[0014] The second expression vector contains a fourth nucleotide sequence encoding the light chain of the second antibody, a fifth nucleotide sequence encoding the heavy chain of the second antibody, an antibody heavy chain fusion, or an Fc chain fusion, and a sixth nucleotide sequence encoding an antibiotic resistance selectable marker.

[0015] If the second or fifth nucleotide sequence encodes an Fc chain fusion, then the other nucleotide sequence encodes an antibody heavy chain or an antibody heavy chain fusion.

[0016] In some embodiments, the first expression vector comprises a first nucleotide sequence, a second nucleotide sequence, and a third nucleotide sequence in a 5' to 3' sequence. In some embodiments, the second expression vector comprises a fourth nucleotide sequence, a fifth nucleotide sequence, and a sixth nucleotide sequence in a 5' to 3' sequence. In some embodiments, the first expression vector comprises a first nucleotide sequence, a second nucleotide sequence, and a third nucleotide sequence in a 5' to 3' sequence, and the second expression vector comprises a fourth nucleotide sequence, a fifth nucleotide sequence, and a sixth nucleotide sequence in a 5' to 3' sequence.

[0017] In some embodiments, the metabolic selectable marker is glutamine synthase or dihydrofolate reductase. In some embodiments, the metabolic selectable marker is glutamine synthase. In some embodiments, the metabolic selectable marker is dihydrofolate reductase.

[0018] In some embodiments, the antibiotic resistance selectable marker is a resistance marker against an antibiotic selected from the group consisting of: puromycin, genimycin, hygromycin, blastomycin, and humicin D. In some embodiments, the antibiotic resistance selectable marker is a resistance marker against hygromycin.

[0019] In some embodiments, the metabolic selectable marker is glutamine synthase, and the antibiotic resistance selectable marker is a resistance marker against hygromycin.

[0020] In some embodiments, the first expression vector does not contain a nucleotide sequence encoding an antibiotic resistance selectable marker. In some embodiments, the second expression vector does not contain a nucleotide sequence encoding a metabolic selectable marker. In some embodiments, the first expression vector does not contain a nucleotide sequence encoding an antibiotic resistance selectable marker, and the second expression vector does not contain a nucleotide sequence encoding a metabolic selectable marker.

[0021] In some embodiments, a first promoter is operatively linked to a first nucleotide sequence, a second promoter is operatively linked to a second nucleotide sequence, and a third promoter is operatively linked to a third nucleotide sequence. In some embodiments, a first expression vector comprises, in a 5' to 3' sequence, a first promoter, a first nucleotide sequence, a second promoter, a second nucleotide sequence, a third promoter, and a third nucleotide sequence. In some embodiments, each of the first and second promoters is a CMV-derived promoter (e.g., CMV / GAPDH, CMV / adL, or CMV / EF1a promoter). In some embodiments, each of the first and second promoters is a GAPDH promoter (e.g., CMV / GAPDH promoter). In some embodiments, the third promoter is mPGK or SRα.

[0022] In some embodiments, a fourth promoter is operatively linked to a fourth nucleotide sequence, a fifth promoter is operatively linked to a fifth nucleotide sequence, and a sixth promoter is operatively linked to a sixth nucleotide sequence. In some embodiments, the second expression vector comprises, in a 5' to 3' sequence, a fourth promoter, a fourth nucleotide sequence, a fifth promoter, a fifth nucleotide sequence, a sixth promoter, and a sixth nucleotide sequence. In some embodiments, each of the fourth and fifth promoters is a CMV-derived promoter (e.g., CMV / GAPDH, CMV / adL, or CMV / EF1a promoter). In some embodiments, each of the fourth and fifth promoters is a GAPDH promoter (e.g., a CMV / GAPDH promoter). In some embodiments, the sixth promoter is SV40.

[0023] In some embodiments, a first polyA sequence is operatively linked to a first nucleotide sequence, a second polyA sequence is operatively linked to a second nucleotide sequence, a third polyA sequence is operatively linked to a third nucleotide sequence, a fourth polyA sequence is operatively linked to a fourth nucleotide sequence, a fifth polyA sequence is operatively linked to a fifth nucleotide sequence, and a sixth polyA sequence is operatively linked to a sixth nucleotide sequence. In some embodiments, a first expression vector comprises, in a 5' to 3' order, a first promoter, a first nucleotide sequence, a first polyA sequence, a second promoter, a second nucleotide sequence, a second polyA sequence, a third promoter, a third nucleotide sequence, and a third polyA sequence. In some embodiments, a second expression vector comprises, in a 5' to 3' order, a fourth promoter, a fourth nucleotide sequence, a fourth polyA sequence, a fifth promoter, a fifth nucleotide sequence, a fifth polyA sequence, a sixth promoter, a sixth nucleotide sequence, and a sixth polyA sequence. In some embodiments, each of the first polyA sequence, the second polyA sequence, the third polyA sequence, the fourth polyA sequence, the fifth polyA sequence, and the sixth polyA sequence is independently selected from the group consisting of: rabbit β-globin pA sequence, thymidine kinase pA (TKpA) sequence, and simian virus 40 (SV40) early pA sequence. In some embodiments, each polyA sequence is an SV40 early pA sequence.

[0024] In some embodiments, the expression vector system encodes Fab-heterogeneous Fc-[scFv] Heterologous IgG, asymmetric antibody-cytokine fusion, [VH-VH] Fab]-Heterogeneous Fc, [VH Fab]-Heterogeneous Fc or [Fab] ]-Heterogeneous Fc-[VH VH] molecules.

[0025] Another aspect of this disclosure provides an expression vector system comprising a first expression vector and a second expression vector, wherein:

[0026] The first expression vector comprises, in 5' to 3' order, a first promoter, a first nucleotide sequence encoding the first antibody light chain, a first polyA sequence, a second promoter, a second nucleotide sequence encoding the first antibody heavy chain, an antibody heavy chain fusion, or an Fc chain fusion, and a third nucleotide sequence encoding a metabolically selectable marker; and

[0027] The second expression vector contains a fourth nucleotide sequence encoding the light chain of the second antibody, a fifth nucleotide sequence encoding the heavy chain of the second antibody, an antibody heavy chain fusion, or an Fc chain fusion, and a sixth nucleotide sequence encoding an antibiotic resistance selectable marker.

[0028] If the second or fifth nucleotide sequence encodes an Fc chain fusion, then the other nucleotide sequence encodes an antibody heavy chain or an antibody heavy chain fusion.

[0029] In some embodiments, the third promoter is mPGK or SRα. In some embodiments, the sixth promoter is SV40. In some embodiments, each of the first, second, fourth, and fifth promoters is a CMV-derived promoter (e.g., CMV / GAPDH, CMV / adL, or CMV / EF1a promoter). In some embodiments, each of the first, second, fourth, and fifth promoters is a CMV / GAPDH or CMV / adL promoter. In some embodiments, each of the first, second, fourth, and fifth promoters is a GAPDH promoter (e.g., CMV / GAPDH promoter). In some embodiments, each of the first, second, third, fourth, fifth, and sixth polyA sequences is an early pA sequence of simian virus 40 (SV40).

[0030] In some embodiments, the third promoter is mPGK or SRα; the sixth promoter is SV40; each of the first, second, fourth, and fifth promoters is a CMV / GAPDH or CMV / adL promoter; and each of the first, second, third, fourth, fifth, and sixth polyA sequences is an early pA sequence of simian virus 40 (SV40).

[0031] In some embodiments, the metabolic selectable marker is glutamine synthase, and the antibiotic resistance selectable marker is a resistance marker against hygromycin.

[0032] In some embodiments, the third promoter is mPGK or SRα; the sixth promoter is SV40; each of the first, second, fourth, and fifth promoters is a CMV / GAPDH or CMV / adL promoter; each of the first, second, third, fourth, fifth, and sixth polyA sequences is an early pA sequence of simian virus 40 (SV40); the metabolic selectable marker is glutamine synthase; and the antibiotic resistance selectable marker is a resistance marker against hygromycin.

[0033] In some embodiments, the expression vector system encodes Fab-heterogeneous Fc-[scFv] Heterologous IgG, asymmetric antibody-cytokine fusion, [VH-VH] Fab]-Heterogeneous Fc, [VH Fab]-Heterogeneous Fc or [Fab] ]-Heterogeneous Fc-[VH VH] molecules.

[0034] This disclosure also relates, in another aspect, to compositions comprising the expression vector system described herein. Such compositions can be used in methods for producing recombinant proteins, including, for example, Fab-heterologous Fc-[scFv]. Heterologous IgG, asymmetric antibody-cytokine fusion, [VH-VH] Fab]-Heterogeneous Fc, [VH Fab]-Heterogeneous Fc or [Fab] ]-Heterogeneous Fc-[VH VH] molecules, as described in this article.

[0035] Another aspect of this disclosure relates to mammalian host cells comprising the expression vector systems described herein. For example, this disclosure provides a mammalian host cell comprising a pair of expression vectors, wherein a) a first expression vector comprises a nucleotide sequence encoding a first antibody light chain, a first antibody heavy chain, or an antibody heavy chain fusion, and a metabolically selectable marker; and b) a second expression vector comprises a nucleotide sequence encoding a second antibody light chain, a second antibody heavy chain, or an antibody heavy chain fusion, and an antibiotic resistance selectable marker expressed in the mammalian cell.

[0036] In some embodiments, the metabolically selectable biomarker is selected from the group consisting of glutamine synthase and dihydrofolate reductase. In some embodiments, the metabolically selectable biomarker is glutamine synthase. In some embodiments, the metabolically selectable biomarker is dihydrofolate reductase.

[0037] In some embodiments, the antibiotic resistance selective marker is a resistance marker against an antibiotic selected from the group consisting of: puromycin, genimycin, hygromycin, blastomycin, and hummus-containing mycotoxin D. In some embodiments, the antibiotic resistance selective marker is a resistance marker against puromycin. In some embodiments, the antibiotic resistance selective marker is a resistance marker against genimycin. In some embodiments, the antibiotic resistance selective marker is a resistance marker against hygromycin. In some embodiments, the antibiotic resistance selective marker is a resistance marker against blastomycin. In some embodiments, the antibiotic resistance selective marker is a resistance marker against hummus-containing mycotoxin D.

[0038] In some embodiments, the metabolic selectable marker is glutamine synthase, and the antibiotic resistance marker is a marker of resistance to hygromycin.

[0039] In some embodiments, the metabolic selectable marker is dihydrofolate reductase, and the antibiotic resistance marker is a marker of resistance to puromycin.

[0040] In some embodiments, the first antibody light chain and the second antibody light chain have the same sequence.

[0041] In some embodiments, the first expression vector encodes a first antibody heavy chain, the second expression vector encodes a second antibody heavy chain, and the first antibody heavy chain is different from the second antibody heavy chain.

[0042] In some embodiments, the first expression vector encodes an antibody heavy chain, and the second expression vector encodes an antibody heavy chain fusion.

[0043] In any of the above embodiments, the antibody heavy chain fusion is selected from the group consisting of: antibody heavy chain-scFv, antibody heavy chain-cytokine, and antibody heavy chain-VHH.

[0044] In some embodiments, the promoter is operatively linked to the nucleotide sequences encoding the antibody light chain, antibody heavy chain, or antibody heavy chain fusion, and metabolically selectable markers. In some aspects of this embodiment, the promoter for the metabolically selectable marker is selected from the group consisting of mPGK and SRa, and the promoter for the antibiotic resistance selectable marker is SV40.

[0045] In some embodiments, the metabolically selectable biomarker is glutamine synthase, and the promoter of the metabolically selectable biomarker is mPGK. In some embodiments, the metabolically selectable biomarker is glutamine synthase, and the promoter of the metabolically selectable biomarker is SRa.

[0046] In some embodiments, the promoter of the antibiotic resistance selectable marker is SV40, and the antibiotic resistance selectable marker is a resistance marker against hygromycin.

[0047] In some embodiments, the metabolic selectable marker is glutamine synthase, the promoter of the metabolic selectable marker is mPGK or SRa, the promoter of the antibiotic resistance selectable marker is SV40, and the antibiotic resistance selectable marker is a resistance marker against hygromycin.

[0048] In some embodiments, the polyA sequence is operatively linked to individual nucleotide sequences within the expression vector. In some aspects of this embodiment, the polyA sequences may be identical or different and are selected from the group consisting of: rabbit β-globin pA sequences, thymidine kinase pA (TKpA) sequences, and simian virus 40 (SV40) early pA sequences. In some embodiments, each polyA sequence is a simian virus 40 (SV40) early pA sequence.

[0049] In some embodiments, the mammalian host cell is a Chinese hamster ovary (CHO) cell.

[0050] In some embodiments, CHO cells are dihydrofolate reductase deficient (dhfr-). In some embodiments, CHO cells are dihydrofolate reductase deficient (dhfr-), and the metabolic selectable marker is dihydrofolate reductase.

[0051] In some embodiments, CHO cells are glutamine synthase knockout (GSKO) cells. In some embodiments, CHO cells are glutamine synthase knockout (GSKO) cells, and the metabolic selectable marker is glutamine synthase.

[0052] In one particular aspect, this disclosure relates to mammalian host cells, wherein a) a first expression vector comprises a nucleotide sequence comprising, in a 5' to 3' order, the following elements: 1) a first promoter operably linked to a nucleotide sequence encoding an antibody light chain, followed by a first polyA sequence; 2) a second 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 metabolically selectable marker, followed by a third polyA sequence; and b) a second expression vector comprises a nucleotide sequence comprising, in a 5' to 3' order, the following elements: 1) a first promoter operably linked to a nucleotide sequence encoding an antibody light chain, followed by a first polyA sequence; 2) a second promoter operably linked to a nucleotide sequence encoding an antibody heavy chain-scFv fusion, an antibody heavy chain-VHH fusion, or a heavy chain-cytokine fusion, followed by a second polyA sequence; and 3) The promoter is operatively linked to a nucleotide sequence encoding an antibiotic resistance selectable marker, followed by a third polyA sequence.

[0053] In some embodiments, the first and second promoters of the first expression vector are CMV-derived promoters (e.g., CMV / GAPDH, CMV / adL, or CMV / EF1a promoters). In some embodiments, the first and second promoters of the first expression vector are CMV / GAPDH promoters. In some embodiments, the first and second promoters of the first expression vector are CMV / adL promoters.

[0054] In some embodiments, the first and second promoters of the second expression vector are CMV-derived promoters (e.g., CMV / GAPDH, CMV / adL, or CMV / EF1a promoters). In some embodiments, the first and second promoters of the second expression vector are CMV / GAPDH promoters. In some embodiments, the first and second promoters of the second expression vector are CMV / adL promoters.

[0055] In some embodiments, the first and second promoters of the first expression vector and the first and second promoters of the second expression vector are CMV-derived promoters (e.g., CMV / GAPDH, CMV / adL, or CMV / EF1a promoters). In some embodiments, the first and second promoters of the first expression vector and the first and second promoters of the second expression vector are CMV / GAPDH promoters. In some embodiments, the first and second promoters of the first expression vector and the first and second promoters of the second expression vector are CMV / adL promoters.

[0056] In some embodiments, the mammalian host cell expresses C1 mAb or an antibody-cytokine fusion. In one aspect, the mammalian host cell encodes C1 mAb. In another aspect, the mammalian host cell encodes an antibody-cytokine fusion.

[0057] In some embodiments, the antibody light chain encoded by the first expression vector and the antibody light chain encoded by the second expression vector contain the same amino acid sequence.

[0058] In some embodiments, the metabolically selectable marker is glutamine synthase, the promoter operably linked to the nucleotide sequence encoding the metabolically selectable marker is mPGK or SRa, the promoter operably linked to the nucleotide sequence encoding the antibiotic resistance selectable marker is SV40, and the antibiotic resistance selectable marker is a resistance marker against hygromycin.

[0059] In some embodiments, each polyA sequence is independently selected from the group consisting of: rabbit β-globin pA sequence, thymidine kinase pA (TKpA) sequence, and simian virus 40 (SV40) early pA sequence. In some embodiments, each polyA sequence is a simian virus 40 (SV40) early pA sequence.

[0060] In some embodiments, the mammalian host cell is a Chinese hamster ovary (CHO) cell.

[0061] In some embodiments, CHO cells are dihydrofolate reductase deficient (dhfr-). In some embodiments, CHO cells are dihydrofolate reductase deficient (dhfr-), and the metabolic selectable marker is dihydrofolate reductase.

[0062] In some embodiments, CHO cells are glutamine synthase knockout (GSKO) cells. In some embodiments, CHO cells are glutamine synthase knockout (GSKO) cells, and the metabolic selectable marker is glutamine synthase.

[0063] In another specific aspect, this disclosure relates to mammalian host cells, wherein a) a first expression vector comprises a nucleotide sequence comprising, in a 5' to 3' order, the following elements: 1) a first promoter operably linked to a nucleotide sequence encoding an antibody light chain, followed by a first polyA sequence; 2) a second 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 metabolically selectable marker, followed by a third polyA sequence; and b) a second expression vector comprises a nucleotide sequence comprising, in a 5' to 3' order, the following elements: 1) a first promoter operably linked to a nucleotide sequence encoding an antibody light chain, followed by a first polyA sequence; 2) a second promoter operably linked to a nucleotide sequence encoding a second antibody heavy chain, followed by a second polyA sequence; and 3) A promoter operatively linked to a nucleotide sequence encoding an antibiotic resistance selectable marker, followed by a third polyA sequence, wherein the first antibody heavy chain differs from the second antibody heavy chain.

[0064] In one aspect, mammalian host cells encode heterologous IgG.

[0065] In some embodiments, the antibody light chain encoded by the first expression vector and the antibody light chain encoded by the second expression vector contain the same amino acid sequence. In some embodiments, the antibody light chain encoded by the first expression vector and the antibody light chain encoded by the second expression vector contain different amino acid sequences.

[0066] In some embodiments, the first and second promoters of the first expression vector are CMV-derived promoters (e.g., CMV / GAPDH, CMV / adL, or CMV / EF1a promoters). In some embodiments, the first and second promoters of the first expression vector are CMV / GAPDH promoters. In some embodiments, the first and second promoters of the first expression vector are CMV / adL promoters.

[0067] In some embodiments, the first and second promoters of the second expression vector are CMV-derived promoters (e.g., CMV / GAPDH, CMV / adL, or CMV / EF1a promoters). In some embodiments, the first and second promoters of the second expression vector are CMV / GAPDH promoters. In some embodiments, the first and second promoters of the second expression vector are CMV / adL promoters.

[0068] In some embodiments, the first and second promoters of the first expression vector and the first and second promoters of the second expression vector are CMV-derived promoters (e.g., CMV / GAPDH, CMV / adL, or CMV / EF1a promoters). In some embodiments, the first and second promoters of the first expression vector and the first and second promoters of the second expression vector are CMV / GAPDH promoters. In some embodiments, the first and second promoters of the first expression vector and the first and second promoters of the second expression vector are CMV / adL promoters.

[0069] In some embodiments, the metabolically selectable marker is glutamine synthase, the promoter operably linked to the nucleotide sequence encoding the metabolically selectable marker is mPGK or SRa, the promoter operably linked to the nucleotide sequence encoding the antibiotic resistance selectable marker is SV40, and the antibiotic resistance selectable marker is a resistance marker against hygromycin.

[0070] In some embodiments, each polyA sequence is independently selected from the group consisting of: rabbit β-globin pA sequence, thymidine kinase pA (TKpA) sequence, and simian virus 40 (SV40) early pA sequence. In some embodiments, each polyA sequence is a simian virus 40 (SV40) early pA sequence.

[0071] In some embodiments, the mammalian host cell is a Chinese hamster ovary (CHO) cell. In some aspects of this embodiment, the CHO cell is a dihydrofolate reductase-deficient (dhfr-) or glutamine synthetase knockout (GSKO) cell.

[0072] In some embodiments, CHO cells are dihydrofolate reductase deficient (dhfr-). In some embodiments, CHO cells are dihydrofolate reductase deficient (dhfr-), and the metabolic selectable marker is dihydrofolate reductase.

[0073] In some embodiments, CHO cells are glutamine synthase knockout (GSKO) cells. In some embodiments, CHO cells are glutamine synthase knockout (GSKO) cells, and the metabolic selectable marker is glutamine synthase.

[0074] Another aspect of this disclosure relates to a method for producing a recombinant protein, the method comprising culturing the mammalian host cells described herein in a cell culture medium suitable for metabolic and antibiotic selection, and recovering the recombinant protein. Illustratively, the cell culture medium suitable for metabolic and antibiotic selection may include a cell culture medium lacking components essential for cell survival (components that would otherwise be provided by the expression of metabolic selection markers), and optionally containing a metabolic selector and an antibiotic.

[0075] For example, in some embodiments, the cell culture medium contains metabolic selectors and antibiotics.

[0076] In some embodiments, when the mammalian host cell contains a dual-selective expression vector system and the metabolically selectable marker is glutamine synthase, the metabolic selector is methionine sulfoxide imine. In some embodiments, when the mammalian host cell contains a dual-selective expression system and the metabolically selectable marker is dihydrofolate reductase, the metabolic selector is methotrexate.

[0077] In some embodiments, when the mammalian host cell contains a dual-selection expression system and the antibiotic resistance selectable marker is a resistance marker against puromycin (e.g., the pac gene), the antibiotic is puromycin. In some embodiments, when the mammalian host cell contains a dual-selection expression system and the antibiotic resistance selectable marker is a resistance marker against neomycin (e.g., the neo gene), the antibiotic is neomycin. In some embodiments, when the mammalian host cell contains a dual-selection expression system and the antibiotic resistance selectable marker is a resistance marker against blastcin (e.g., the bsr gene), the antibiotic is blastcin. In some embodiments, when the mammalian host cell contains a dual-selection expression system and the antibiotic resistance selectable marker is a resistance marker against hygromycin (e.g., the ble gene), the antibiotic is hygromycin D. In some embodiments, when the mammalian host cell contains a dual-selection expression system and the antibiotic resistance selectable marker is a resistance marker against hygromycin (e.g., the hph gene), the antibiotic is hygromycin B.

[0078] In some embodiments, when the mammalian host cell contains a dual-selective expression system, and wherein the metabolic selector is glutamine synthase and the antibiotic resistance selector is a resistance marker against hygromycin (e.g., the hph gene), the metabolic selector is methionine sulfoxide imine and the antibiotic is hygromycin B.

[0079] Additionally, in some embodiments, when the mammalian host cell contains a dual-selective expression system and the metabolically selectable marker is glutamine synthase, the cell culture medium does not contain glutamine. In some embodiments, when the mammalian host cell contains a dual-selective expression system and the metabolically selectable marker is glutamine synthase, the cell culture medium contains the metabolic selector methionine sulfoxide imine but does not contain glutamine.

[0080] Additionally, in some embodiments, when the mammalian host cell contains a dual-selective expression system, and wherein the metabolically selectable marker is glutamine synthase and the antibiotic resistance selectable marker is a resistance marker against hygromycin (e.g., the hph gene), the cell culture medium contains the antibiotic hygromycin B but not glutamine. In some embodiments, when the mammalian host cell contains a dual-selective expression system, and wherein the metabolically selectable marker is glutamine synthase and the antibiotic resistance selectable marker is a resistance marker against hygromycin (e.g., the hph gene), the cell culture medium contains the metabolic selector methionine sulfoxide imine and the antibiotic hygromycin B but not glutamine.

[0081] In some embodiments, when the mammalian host cell contains a dual-selective expression system and the metabolically selectable marker is dihydrofolate reductase, the cell culture medium does not contain inosine or thymidine. In some embodiments, when the mammalian host cell contains a dual-selective expression system and the metabolically selectable marker is dihydrofolate reductase, the cell culture medium contains the metabolic selector methotrexate but does not contain inosine or thymidine.

[0082] Illustratively, this disclosure provides a method for generating a recombinant protein (e.g., in antibody form), the method comprising a) culturing mammalian host cells under conditions expressing an antibody chain and a selectable marker, the mammalian host cells comprising 1) a first expression vector encoding a first light chain, a first heavy chain, or a fusion of the first heavy chain, and a metabolic selectable marker; and 2) a second expression vector encoding a second light chain, a second heavy chain, or a fusion of the second heavy chain, and an antibiotic selectable marker; wherein the culture is performed in a culture medium containing a metabolic selector and an antibiotic; and b) recovering the antibody form from the culture.

[0083] In some embodiments, the metabolically selectable marker is glutamine synthase and the metabolic selector is methionine sulfoxide imine, or the metabolically selectable marker is dihydrofolate reductase and the metabolic selector is methotrexate. In some embodiments, the antibiotic resistance marker is the hph gene and the antibiotic is hygromycin B, the antibiotic resistance marker is the pac gene and the antibiotic is puromycin, the antibiotic resistance marker is the neo gene and the antibiotic is neomycin, the antibiotic resistance marker is the bsr gene and the antibiotic is blastomycin, or the antibiotic resistance marker is the ble gene and the antibiotic is fulvicin D.

[0084] In one aspect of the method, a) a first expression vector comprises a nucleotide sequence comprising, in a 5' to 3' order, the following elements: 1) a first promoter operably linked to a nucleotide sequence encoding an antibody light chain, followed by a first polyA sequence; 2) a second 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 metabolically selectable marker, followed by a third polyA sequence; and b) a second expression vector comprises a nucleotide sequence comprising, in a 5' to 3' order, the following elements: 1) a first promoter operably linked to a nucleotide sequence encoding an antibody light chain, followed by a first polyA sequence; 2) a second 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 an antibiotic resistance selectable marker, followed by a third polyA sequence. In one respect, the method encodes C1 mAb.

[0085] In another aspect of the method, a) the first expression vector comprises a nucleotide sequence comprising, in a 5' to 3' order, the following elements: 1) a first promoter operably linked to a nucleotide sequence encoding a first antibody light chain, followed by a first polyA sequence; 2) a second 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 metabolically selectable marker, followed by a third polyA sequence; and b) the second expression vector comprises a nucleotide sequence comprising, in a 5' to 3' order, the following elements: 1) a first promoter operably linked to a nucleotide sequence encoding an antibody light chain, followed by a first polyA sequence; 2) a second promoter operably linked to a nucleotide sequence encoding a second antibody heavy chain, followed by a second polyA sequence; and 3) A promoter operatively linked to a nucleotide sequence encoding an antibiotic resistance selectable marker, followed by a third polyA sequence, wherein the first antibody heavy chain differs from the second heavy chain. In one aspect, the method encodes heterologous IgG.

[0086] On another aspect, a) the first expression vector comprises a nucleotide sequence comprising, in a 5' to 3' order, the following elements: 1) a first promoter operably linked to a nucleotide sequence encoding an antibody light chain, followed by a first polyA sequence; 2) a second 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 metabolically selectable marker, followed by a third polyA sequence; and b) the second expression vector comprises a nucleotide sequence comprising, in a 5' to 3' order, the following elements: 1) a first promoter operably linked to a nucleotide sequence encoding an antibody light chain, followed by a first polyA sequence; 2) a second promoter operably linked to a nucleotide sequence encoding an antibody heavy chain-cytokine fusion, followed by a second polyA sequence; and 3) A promoter, operatively linked to a nucleotide sequence encoding an antibiotic resistance selectable marker, followed by a third polyA sequence, wherein the first antibody heavy chain differs from the second antibody heavy chain. In one aspect, the mammalian host cell encodes an antibody-cytokine fusion.

[0087] In some embodiments of these methods, the mammalian host cell is the Chinese hamster ovary (CHO) cell. In one aspect, the CHO cells are dihydrofolate reductase deficient (dhfr-) or glutamine synthetase knockout (GSKO) cells.

[0088] In some embodiments, CHO cells are dihydrofolate reductase deficient (dhfr-). In some embodiments, CHO cells are dihydrofolate reductase deficient (dhfr-), and the metabolic selectable marker is dihydrofolate reductase.

[0089] In some embodiments, CHO cells are glutamine synthase knockout (GSKO) cells. In some embodiments, CHO cells are glutamine synthase knockout (GSKO) cells, and the metabolic selectable marker is glutamine synthase.

[0090] In some embodiments of any of these methods, the recovered antibody form is purified and formulated into a pharmaceutically acceptable preparation. Attached Figure Description

[0091] Figures 1A-1F Schematic diagrams of several different complex biological forms are provided: Fab-heterologous Fc-[scFv] ]molecular( Figure 1A ); Heterologous IgG molecules ( Figure 1B ); asymmetric antibody-cytokine fusion ( Figure 1C ); [VH-VH Fab-heterologous Fc molecules ( Figure 1D ); [VH Fab-heterologous Fc molecules ( Figure 1E ); and [Fab ]-Heterogeneous Fc-[VH VH]( Figure 1F )molecular.

[0092] Figures 2A-2G A) A schematic diagram of the vector used to evaluate the dual selection strategy in the case of 3-chain C1mAb molecules is provided; B) Recovery time; C) Overall titer (g / L); D) Effective pool titer (g / L); E) SEC-HPLC-HMW%; F) nrCE front peak impurities%; and G) rCE chromatograms: HC-rCE-SDS %, HC-ScFv-rCE-SDS %, and LC-rCE-SDS %. The ideal proportion of perfectly assembled molecules is indicated between the two lines. Titers and product mass were measured on day 10 of fed-batch production. Data are presented as the average of two independent transfections. Error bars represent the standard deviation (SD) of two biological replicates of fed-batch production. GAPDH / CMV refers to the promoter. LC refers to the light chain. HC1 refers to the first heavy chain. HC2-scFV refers to the second heavy chain fused with the scFv sequence. mGS refers to mouse glutamine synthetase. Hygr B refers to hygromycin B.

[0093] Figures 3A-3FProvided are: A) a schematic diagram of the vector used to evaluate the dual selection strategy in the case of 4-chain heterologous IgG molecules; B) recovery time; C) total titer (g / L); D) effective pool titer (g / L); E) SEC-HPLC-HMW%; F) nrCE pre-peak impurities%. Titers and product mass were measured on day 10 of fed-batch production. Data are expressed as the average of two independent transfections. Error bars represent the standard deviation (SD) of two biological replicates of fed-batch production. CMV / GapDH refers to the promoter. LC1 refers to the first light chain. LC2 refers to the second light chain. HC1 refers to the first heavy chain. HC2 refers to the second heavy chain. GS refers to glutamine synthase. Hygro refers to hygromycin B.

[0094] Figures 4A-4G A) A schematic diagram of the vector used to evaluate the dual-selection strategy in the case of antibody-cytokine fusion molecules is provided; B) Recovery time; C) Overall titer (g / L); D) Effective pool titer (g / L); E) SEC-HPLC-HMW%; F) nrCE precursor impurities%; and G) rCE chromatograms: HC-rCE-SDS %, HC-ScFv-rCE-SDS %, and LC-rCE-SDS %. The ideal proportion of perfectly assembled molecules is indicated between the two lines. Titers and product mass were measured on day 10 of fed-batch production. Data are presented as the average of two independent transfections. Error bars represent the standard deviation (SD) of two biological replicates of fed-batch production. LC refers to the light chain. HC1 refers to the first heavy chain. HC2 refers to the second heavy chain. mGS refers to mouse glutamine synthetase. Hyg B refers to hygromycin B. Attachment of cytokine sequences is not shown.

[0095] Figures 5A-5G A) is provided for evaluating [VH-VH] A) Schematic diagram of the dual-selection strategy in the case of Fab-heterologous Fc molecules; B) Recovery time; C) Normalized total titer (normalized relative to single-selection conditions); D) Normalized effective titer (normalized relative to single-selection conditions); E) SEC-HPLC-HMW%; F) nrCE front peak impurities%; and G) rCE chromatograms: HC1-rCE-SDS %, HC2-rCE-SDS %, and LC-rCE-SDS %. Titers and product mass were measured on day 14 of fed-batch production. Data are expressed as the average of two independent transfections and two technical replicates (fed-batch production). LC refers to the light chain. HC1 refers to the first heavy chain. HC2 refers to the second heavy chain. mGS refers to mouse glutamine synthetase. Hyg B refers to hygromycin B.

[0096] Figures 6A-6G A) is provided for evaluating the performance of two [VH] A schematic diagram of the carrier for the dual-selection strategy in the case of Fab-Fc molecules (molecules A and B); B) Recovery time; C) Normalized total titer (normalized relative to single-selection conditions); D) Normalized effective titer (normalized relative to single-selection conditions); E) SEC-HPLC-HMW%; F) nrCE front peak impurities%; and G) rCE chromatograms: HC1-rCE-SDS %, HC2-rCE-SDS %, and LC-rCE-SDS %. Titers and product mass were measured on day 14 of fed-batch production. Data are expressed as the average of two independent transfections and two technical replicates (fed-batch production). LC refers to the light chain. HC1 refers to the first heavy chain. HC2 refers to the second heavy chain. mGS refers to mouse glutamine synthetase. Hyg B refers to hygromycin B.

[0097] Figures 7A-7H A) is provided for evaluating [Fab] ]-Heterogeneous Fc-[VH A) Schematic diagram of the carrier for the dual-selection strategy under the VH molecular condition; B) Recovery time; C) Normalized total titer (normalized relative to single-selection conditions); D) Normalized effective titer (normalized relative to single-selection conditions); E) SEC-HPLC-HMW%; F) nrCE pre-peak impurities%; G) nrCE post-peak impurities%; and H) rCE chromatograms: HC1-rCE-SDS %, HC2-rCE-SDS %, and LC-rCE-SDS %. Titers and product mass were measured on day 14 of fed-batch production. Data are expressed as the average of two independent transfections and two technical replicates (fed-batch production). LC refers to the light chain. HC1 refers to the first heavy chain. HC2 refers to the second heavy chain. mGS refers to mouse glutamine synthetase. Hyg refers to hygromycin B. Detailed Implementation

[0098] This disclosure is based on a vector system design utilizing dual selection in mammalian cells, where one vector utilizes metabolic selection and the other utilizes antibiotic selection. Therefore, this disclosure relates to a mammalian host cell comprising a pair of expression vectors, wherein a) the first expression vector contains a nucleotide sequence encoding a first antibody light chain, a first antibody heavy chain, an antibody heavy chain fusion, or an Fc chain fusion (e.g., a first antibody heavy chain or an antibody heavy chain fusion), and a metabolically selectable marker; and b) the second expression vector contains a nucleotide sequence encoding a second antibody light chain, a second antibody heavy chain, an antibody heavy chain fusion, or an Fc chain fusion (e.g., a second antibody heavy chain or an antibody heavy chain fusion), and an antibiotic resistance selectable marker, wherein the antibiotic resistance selectable marker is expressed in the mammalian host cell. Surprisingly, compared to standard single metabolic selection methods (e.g., using glutamine synthase), this design increases unit productivity and titer while reducing impurities. This dual selection strategy ensures the integration and expression of all antibody chains from both vectors, thus influencing productivity and controlling the chain ratio that improves product quality.

[0099] In the various embodiments described herein, the first and second expression vectors are always different due to different optional biomarkers, but the first and second antibody light chains can be the same (i.e., have the same sequence) or different. The first antibody heavy chain and the second antibody heavy chain can be the same or different. In some embodiments, one expression vector encodes a heavy chain, while the other expression vector encodes a heavy chain fusion, wherein the heavy chain in the heavy chain fusion is linked to, for example, scFv, cytokines, VH / VHH, etc. In other embodiments, one expression vector encodes an antibody heavy chain, while the other expression vector encodes an Fc chain fusion, wherein the heavy chain in the heavy chain fusion is linked to, for example, scFv, cytokines, VH / VHH, etc.

[0100] This strategy is particularly useful for antibody forms with three or four distinct chains expressed on two different vectors. Examples of three- or four-chain antibody forms may have a heavy chain, a heavy chain-scFv fusion, and two light chains (the light chains are identical in the three-chain antibody form or different in the four-chain form). Another example of a four-chain antibody form has two different heavy chains and two different light chains, where each heavy chain-light chain pair is expressed by a different vector. Another example of a three- or four-chain antibody form may have a heavy chain, a heavy chain-cytokine fusion, and two light chains (the light chains are identical in the three-chain antibody form or different in the four-chain form). Yet another example of a three-chain antibody form includes one light chain, one heavy chain, and a heavy chain fusion (where VH is fused to the heavy chain at its N-terminus). Still another example of a three-chain antibody form comprises one light chain, one heavy chain, and an Fc chain fusion (where VH is fused to the N-terminus of Fc). In yet another instance, the triple-chain antibody form comprises a light chain, a heavy chain fusion (where VH is fused to the heavy chain at its C-terminus), and an Fc chain fusion (where VH is fused to the Fc at its C-terminus).

[0101] Certain instances in this application demonstrate the use of this dual-selection strategy to express 3-chain C1mAb (Fab-heterogeneous Fc-[scFv)). [ ]) molecules, 4-chain heterologous IgG molecules, and 3-chain antibody-cytokine fusions. Two plasmids were used to express the antibody chains: sequences encoding the first heavy chain and the first light chain were constructed in a first plasmid using a GS-selectable marker controlled by the Sra promoter, while a second plasmid containing sequences encoding the second heavy chain or heavy chain fusion and the second light chain used a hygromycin B antibiotic-selectable marker controlled by the SV40 promoter. In the 4-chain and 3-chain molecules, the use of dual selection improved productivity by 1.2 to 3.5 times and reduced impurities (precursor peaks) by 3.8 to 4.5 times, respectively. Additional examples illustrate that the dual selection strategy disclosed herein increases certain [VH-VH Fab]-Heterogeneous Fc, [VH Fab]-Heterogeneous Fc and [Fab ]-Heterogeneous Fc-[VH The overall titer and effective titer of VH molecules were reduced, and impurities were decreased.

[0102] By employing the dual expression vector system and mammalian host cells described herein, the production of recombinant proteins can be increased while maintaining or improving product quality. For example, by using the dual expression vectors described herein in mammalian host production cell lines, biopharmaceuticals can be produced in a cheaper and more consistent manner. These inventions are particularly applicable to the commercial production of peptides (e.g., in antibody form) having three or four distinct polypeptide chains.

[0103] The dual expression vectors described herein are used in cell lines (also referred to as “host cells”), preferably mammalian (“mammalian host cells”) grown in cell culture media, to produce recombinant proteins of commercial or scientific significance. Cell lines are typically derived from lineages of primary cultures and can be maintained indefinitely in culture. Genetically engineered cell lines involve transfecting, transforming, or transducing cells with two expression vectors (each containing a nucleotide sequence encoding two antibody chains) to induce the host cells to express an antibody form with a desired chain number. Methods and vectors for genetically engineering cells and / or cell lines to express, for example, target proteins are well known to those skilled in the art; various techniques are described, for example: Current Protocols in Molecular Biology Ausubel et al. (edited., Wiley & Sons, New York, 1988, and quarterly updates); Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Laboratory Press, 1989); Kaufman, RJ, Large Scale Mammalian Cell Culture, 1990, pp. 15–69; and Harlow and Lane Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1990).

[0104] definition

[0105] Although the terminology used herein is standard in the art, definitions of certain terms are provided herein to ensure clarity and definiteness of the meaning of the claims. Units, prefixes, and symbols may be expressed in their International System of Units (SI) accepted forms. The numerical ranges enumerated herein include the numbers defining the ranges and encompass and support every integer within the defined ranges. Unless otherwise indicated, the methods and techniques described herein may be performed according to conventional methods well known in the art and as described in the various general and more specific references cited and discussed throughout this specification.

[0106] As used herein, unless otherwise expressly stated, the term "a / an" means one or more. Furthermore, unless the context requires otherwise, singular terms will include plural and plural terms will include singular. Generally, the nomenclature and techniques used in conjunction with the 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.

[0107] All documents or portions thereof cited in this application, including but not limited to patents, patent applications, articles, books, and monographs, are expressly incorporated herein by reference. The content described in the embodiments of the invention may be combined with other embodiments of the invention.

[0108] This disclosure provides systems and methods for expressing a “target protein” (typically in the form of an antibody). A “target protein” includes naturally occurring proteins, recombinant proteins, and engineered proteins (e.g., proteins that do not exist in nature and have been designed and / or produced by humans). The target protein may, but does not have to be, a protein known or suspected of having therapeutic relevance.

[0109] As used herein, “antibody chain” or “chain” refers to both the antibody light chain and the antibody heavy chain. The terms “antibody heavy chain” and “antibody light chain” have their standard meanings in the art and include, for example, the various antibody heavy and light chains described elsewhere herein (e.g., the heavy and light chains of IgG1, IgG2, IgG3, and IgG4 mAbs). The terms “antibody heavy chain” and “antibody light chain” include standard full-length antibody heavy and light chains.

[0110] As used herein, the terms “antibody heavy chain fusion protein” and “antibody heavy chain fusion protein” refer to a polypeptide containing an antibody heavy chain covalently linked to one or more additional proteins or peptides. For example, an “antibody heavy chain fusion protein” may be an antibody heavy chain covalently linked to cytokines, scFv, VH / VHH, etc. The linking 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 one or more additional proteins at the N-terminus or C-terminus (or both) of the heavy chain. The antibody heavy chain may also be linked to an additional protein sequence, such as scFv, at an internal amino acid residue or between Fab and Fc. In addition to the antibody light chain, the terms “antibody light chain fusion protein” and “antibody light chain fusion protein” have the same meaning as described immediately above for “antibody heavy chain fusion protein.” As used herein, an “antibody fusion protein” refers to an antibody as provided herein, covalently linked (e.g., via the heavy or light chain of an antibody) to one or more additional proteins or peptides. Therefore, antibody fusion proteins contain at least one antibody heavy chain fusion protein or antibody light chain fusion protein as one of the polypeptides in an antibody fusion protein. Most commonly, an antibody fusion protein is a molecule containing two antibody light chains (which may be the same or different), one antibody heavy chain, and an antibody heavy chain fusion protein, such that the additional protein is linked to one of the antibody heavy chains. The term "antibody chain fusion" encompasses both antibody heavy chain fusions and antibody light chain fusions.

[0111] In some embodiments, the antibody heavy chain fusion is a fusion of the heavy chain with a VH, scFv, or cytokine, wherein the VH, scFv, or cytokine is fused to the N-terminus or C-terminus of the antibody heavy chain portion of the antibody heavy chain fusion, or between CH1 and CH2 of the antibody heavy chain portion of the antibody heavy chain fusion. Fusion can be direct or via a linker.

[0112] In some embodiments, the antibody heavy chain fusion compound is a fusion of an antibody heavy chain and a VH. In some embodiments, the antibody heavy chain fusion compound is a fusion of an antibody heavy chain and an scFv. In some embodiments, the antibody heavy chain fusion compound is a fusion of an antibody heavy chain and a cytokine.

[0113] In some embodiments, the antibody heavy chain fusion compound is a direct fusion of the antibody heavy chain and VH. In some embodiments, the antibody heavy chain fusion compound is a direct fusion of the antibody heavy chain and scFv. In some embodiments, the antibody heavy chain fusion compound is a direct fusion of the antibody heavy chain and cytokines.

[0114] In some embodiments, the antibody heavy chain fusion is a fusion of an antibody heavy chain and a vitamin H, wherein the antibody heavy chain fusion includes a linker between the antibody heavy chain and the vitamin H. In some embodiments, the antibody heavy chain fusion is a fusion of an antibody heavy chain and an scFv, wherein the antibody heavy chain fusion includes a linker between the antibody heavy chain and the scFv. In some embodiments, the antibody heavy chain fusion is a fusion of an antibody heavy chain and a cytokine, wherein the antibody heavy chain fusion includes a linker between the antibody heavy chain and the cytokine.

[0115] As used herein, "antibody form" refers to a protein having at least one antibody chain. Antibody forms can have two, three, or four distinct chains, and any or all of these chains may include fusion forms. The references to two-chain, three-chain, or four-chain molecules imply that each chain is unique. Any and all molecules containing antibody chains described herein are considered antibody forms.

[0116] As used herein, the terms “peptide” and “protein” (e.g., as used in the context of a target protein or target peptide) are used interchangeably and refer to polymers of amino acid residues. These terms also apply to amino acid polymers in which one or more amino acid residues are analogs or mimics of the corresponding naturally occurring amino acids, as well as naturally occurring amino acid polymers. These terms may also cover amino acid polymers that have been modified, for example, by adding carbohydrate residues to form glycoproteins or by phosphorylation. Peptides and proteins may be produced by naturally occurring and non-recombinant cells, or by genetically engineered or recombinant cells. Peptides and proteins may comprise molecules having the amino acid sequence of a natural protein, or molecules having one or more amino acids with a natural sequence that have been omitted, added, and / or substituted.

[0117] As used herein, the term "heterologous" in conjunction with nucleic acids means having nucleic acids that are not naturally present in the host cell. This can include mutated sequences, such as sequences different from those naturally occurring. This can include sequences from other species. This can also include sequences located in a different location in the genome than those naturally occurring in the host cell. This generally does not include naturally occurring mutations that may occur in the host cell. Cells that already contain heterologous nucleic acids encoding a target protein, for example through stable integration of an expression cassette, will be considered to contain heterologous nucleic acid sequences. For clarity, CHO cells or derivatives thereof (e.g., DHFR- or GS knockout types) containing nucleic acids encoding antigen-binding proteins will be considered to contain heterologous nucleic acids.

[0118] As used herein, the term "operably ligated" means that the ligated nucleic acid sequences are typically continuous or substantially continuous, and when it is necessary to ligate two protein-coding regions, they are continuous and within the reading frame. However, because enhancers typically function at intervals of several thousand bases from promoters, and intron sequences can have variable lengths, some polynucleotide elements can be operably ligated but not continuously. Two or more nucleic acid sequences can be operably ligated in a manner that produces a nucleic acid molecule capable of guiding the transcription of a given gene and / or the synthesis of a desired protein molecule.

[0119] As used herein, the term "bioreactor" means any container that can be used for the growth of cell cultures. The cell culture of the host cells disclosed herein can be carried out in a bioreactor, and the bioreactor can be selected based on the application of the target protein produced by the cells grown in the bioreactor. Bioreactors can be of any size, as long as they are suitable for cell culture; typically, the size of the bioreactor is appropriate for the volume of the cell culture grown within it. Typically, the bioreactor will be at least 1 liter and can be 2, 5, 10, 50, 100, 200, 250, 500, 1,000, 1,500, 2,000, 2,500, 5,000, 8,000, 10,000, 12,000 liters or larger, or any volume between these values. The internal conditions of the bioreactor, including but not limited to pH and temperature, can be controlled during culture. Those skilled in the art will recognize and be able to select a suitable bioreactor for practicing the methods disclosed herein based on relevant considerations. In one embodiment, a bioreactor of 500 L to 2000 L is used. In one embodiment, a bioreactor of 1000 L to 2000 L is used.

[0120] As used herein, “cell culture” or “culture” means the growth and reproduction of cells outside a multicellular organism or tissue. Suitable culture conditions for mammalian cells are known in the art. See, for example, *Animal Cell Culture: A Practical Approach*, edited by D. Rickwood, Oxford University Press, New York (1992). Mammalian cells can be cultured in suspension or attached to a solid substrate. Fluidized bed bioreactors, hollow fiber bioreactors, roller flasks, shake flasks, or stirred tank bioreactors with or without microcarriers can be used.

[0121] The term "cell culture medium" (also known as "culture medium," "cell culture media," or "tissue culture medium") refers to any nutrient solution used to grow cells (e.g., animal or mammalian cells) and typically provides at least one or more of the following components: energy (usually in the form of carbohydrates, such as glucose); one or more of all essential amino acids, typically twenty basic amino acids plus cysteine; vitamins and / or other organic compounds, typically required in low concentrations; lipids or free fatty acids; and trace elements, such as inorganic compounds or naturally occurring elements, typically required in very low concentrations (usually in the micromolar range).

[0122] Nutrient solutions may optionally be supplemented with additional optional components to optimize cell growth, such as hormones and other growth factors, such as transferrin, epidermal growth factor, insulin-like growth factor, insulin, serum, etc.; salts, such as calcium salts, magnesium salts, and phosphates, and buffers, such as HEPES; nucleosides and bases, such as adenosine, thymidine, hypoxanthine; and protein and tissue hydrolysates, such as hydrolyzed animal or plant proteins (peptones or mixtures of peptones, which may be obtained from animal by-products, purified gelatin, or plant material); antibiotics, such as gentamicin; anti-caking agents; cell protectants or surfactants, such as Pluronic. ® F68 (also known as Lutrol) ® F68 and Kolliphor ® P188); nonionic triblock, consisting of a hydrophobic central chain of polyoxypropylene (poly(propylene oxide)) and two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)) on the flanks; polyamines, such as putrescine, spermidine and spermine (see, for example, International Patent Application Publication No. WO 2008 / 154014) and pyruvate (see, for example, U.S. Patent No. 8,053,238), depending on the needs of the cells to be cultured and / or the desired cell culture parameters.

[0123] Cell culture media include those that are typically used and / or are known to be used in any cell culture process, such as, but not limited to, batch, extended batch, fed-batch, and / or perfusion or continuous cell culture.

[0124] "Basic" (or batch) cell culture medium refers to a cell culture medium that is typically used to initiate cell culture and is sufficiently complete to support cell culture.

[0125] "Feed-batch culture" refers to a form of suspension culture and means a method of culturing cells in which additional components are provided to the culture at one or more points after the start of the culture process. The provided components typically include nutrient supplements that have been depleted for the cells during the culture process. Additionally or alternatively, the additional components may include supplemental components. Fed-batch cultures typically stop at a certain point, and the cells and / or components in the culture medium are harvested and optionally purified.

[0126] “Growth” cell culture medium refers to a cell culture medium that is typically used for cell culture during the exponential growth phase (i.e., the “growth phase”) and is sufficiently complete to support cell culture during this phase. Growth cell culture media may also contain selectants that confer resistance or viability to optional markers incorporated into the host cell line. Such selectants include, but are not limited to, genimycin (G418), neomycin, hygromycin B, puromycin, bleomycin, methionine sulfoxide, methotrexate, glutamine-free cell culture media, glycine-deficient cell culture media, hypoxanthine and thymidine, or thymidine alone.

[0127] "Perfusion" cell culture media are typically used to maintain cell cultures via perfusion or continuous culture methods and are sufficiently complete to support cell culture during the process. Perfusion cell culture medium formulations can be richer or more concentrated than basal cell culture medium formulations to suit the methods used for removing used medium. Perfusion cell culture media can be used during both the growth and production phases.

[0128] “Production” cell culture medium refers to a cell culture medium that is typically used for cell culture during the transition period from the end of exponential growth to the beginning of protein production (i.e., the “transition” and / or “product” phase), and is sufficiently complete to maintain the desired cell density, viability, and / or product titer during this phase.

[0129] Concentrated cell culture media may contain some or all of the nutrients necessary to maintain cell culture; in particular, concentrated media may contain nutrients identified or known to be consumed during the production phase of cell culture. Concentrated media can be based on virtually any cell culture medium formulation. Such concentrated feed media may contain some or all of the components of cell culture media, for example, at approximately 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 20, 30, 50, 100, 200, 400, 600, 800, or even approximately 1000 times their normal amounts.

[0130] The components used to prepare cell culture media can be completely ground into a powdered culture medium formulation; partially ground together with the liquid supplement to be added to the cell culture media as needed; or added to the cell culture in completely liquid form.

[0131] Cell cultures can also be supplemented with separate concentrated feeds containing specific nutrients that may be difficult to formulate in cell cultures or are rapidly depleted within them. Such nutrients can be amino acids such as tyrosine, cysteine, and / or cystine (see, for example, International Patent Application Publication No. WO2012 / 145682). Separate feeding can be initiated before or during the production phase. Separate feeding can be accomplished by adding feed to the cell culture medium in batches on the same day as or different from the concentrated feed medium. Alternatively, separate feed can be infused on the same day as or different from the perfusion medium.

[0132] "Serium-free" refers to cell media that do not contain animal serum, such as fetal bovine serum. Various tissue media (including defined media) are commercially available; for example, any one or a combination of the following cell media can be used: RPMI-1640, RPMI-1641, Dürbeco Modified Eagle Medium (DMEM), Eagle Minimum Essential Medium, F-12K, Ham F12, Iskov Modified Dürbeco Medium, McCoy 5A, Leibovitz L-15, and serum-free media such as EX-CELL. TM Examples of such media include the 300 series (JRH Biosciences, Lenexa, Kansas) and MCDB 302 (Sigma Aldrich Corp., St. Louis, MO). Serum-free forms of these media are also available. Depending on the needs of the cells being cultured and / or the desired cell culture parameters, cell culture media can be supplemented with additional or increased concentrations of components such as amino acids, salts, sugars, vitamins, hormones, growth factors, buffers, antibiotics, lipids, trace elements, etc. Custom-made cell culture media can also be used.

[0133] "Titer" refers to the total amount of a target polypeptide or protein (which may be naturally occurring or recombinant) produced by a cell culture in a given volume of culture medium. Titer can be expressed in milligrams or micrograms per milliliter of culture medium (or other volumetric measure). "Cumulative titer" is the titer produced by the cells during culture and can be determined, for example, by measuring the daily titer and using those values ​​to calculate the cumulative titer.

[0134] As used herein, the term “host cell” should be understood to include cells that have been genetically engineered to express a target polypeptide. Genetic engineering of cells involves transfecting, transforming, or transducing cells with a nucleic acid encoding a recombinant polynucleotide molecule (“target gene”), and / or otherwise altering (e.g., through homologous recombination and gene activation or fusion of recombinant and non-recombinant cells) to induce the host cell to express the desired recombinant polypeptide. Methods and vectors for genetically engineering cells and / or cell lines to express target peptides are well known to those skilled in the art; for example, various techniques are described in Current Protocols in Molecular Biology, edited by Ausubel et al. (Wiley & Sons, New York, 1988, and quarterly updates); Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Laboratory Press, 1989); Kaufman, RJ, Large Scale Mammalian Cell Culture, 1990, pp. 15–69. The term includes progeny of parental cells, regardless of whether the progeny is morphologically or genetically identical to the original parental cell, provided the target gene is present. Cell cultures may contain one or more host cells. “Effective titer” refers to the total titer normalized relative to the percentage of correctly paired products (i.e., the percentage of SEC peak or nrCE peak).

[0135] The “Fc” region, as used in this text, contains the C-cell containing the antibody. H 2 and C H The three-domain structure consists of two heavy-chain segments. These two heavy-chain segments are composed of two or more disulfide bonds and C... H The hydrophobic interactions of the three domains are maintained together.

[0136] As used in this article, "Fc chain fusion" refers to a C chain covalently linked to one or more additional proteins or peptides. H 2 and C H 3. Heavy chain fragments with structural domains. For example, an "Fc chain fusion complex" could be a C-chain fragment covalently linked to cytokines, scFv, VH / VHH, etc. H 2 and C HThe heavy chain fragment has three domains. Linkage can be direct or via peptide linkers (e.g., glycine-serine linkers). In Fc chain fusion proteins, the Fc chain can be linked to one or more additional proteins at the N-terminus or C-terminus (or both) of the heavy chain fragment. The heavy chain fragment can also be linked to additional protein sequences (e.g., scFv) at internal amino acid residues.

[0137] In some embodiments, the Fc chain fusion is a fusion of an Fc chain with a vitamin H, scFv, or cytokine, wherein the vitamin H, scFv, or cytokine is fused to the N-terminus or C-terminus of the Fc chain. In some embodiments, the Fc chain fusion is a fusion of an Fc chain with a vitamin H, scFv, or cytokine, wherein the vitamin H, scFv, or cytokine is fused to the N-terminus of the Fc chain. In some embodiments, the Fc chain fusion is a fusion of an Fc chain with a vitamin H, scFv, or cytokine, wherein the vitamin H, scFv, or cytokine is fused to the C-terminus of the Fc chain. Fusion can be direct or via a linker.

[0138] In some embodiments, the Fc chain fusion is a fusion of the Fc chain and VH. In some embodiments, the Fc chain fusion is a fusion of the Fc chain and scFv. In some embodiments, the Fc chain fusion is a fusion of the Fc chain and cytokines.

[0139] In some embodiments, the Fc chain fusion is a direct fusion of the Fc chain and VH. In some embodiments, the Fc chain fusion is a direct fusion of the Fc chain and scFv. In some embodiments, the Fc chain fusion is a direct fusion of the Fc chain and cytokines.

[0140] In some embodiments, the Fc chain fusion is a fusion of an Fc chain and a VH, wherein the Fc chain fusion includes a linker between the Fc chain and the VH. In some embodiments, the Fc chain fusion is a fusion of an Fc chain and an scFv, wherein the Fc chain fusion includes a linker between the Fc chain and the scFv. In some embodiments, the Fc chain fusion is a fusion of an Fc chain and a cytokine, wherein the Fc chain fusion includes a linker between the Fc chain and the cytokine.

[0141] A "half-antibody" is an immunofunctional immunoglobulin construct comprising a complete heavy chain, a complete light chain, and a second heavy chain Fc region paired with the Fc region of the complete heavy chain. A linker may, but is not necessary, connect the heavy chain Fc region and the second heavy chain Fc region. In a particular embodiment, the half-antibody is the monovalent form of the antigen-binding protein disclosed herein. In other embodiments, a pair of charged residues may be used to associate one Fc region with a second Fc region.

[0142] It should be understood that, regardless of how the embodiments are described herein using the language “comprising,” other similar embodiments are also provided, described in terms of “consisting of” and / or “substantially consisting of”.

[0143] Selectable markers

[0144] To stably transfect mammalian cells, it is known that only a small fraction of cells can integrate foreign DNA into their genome, depending on the expression vector and transfection technique used. To identify and select these integrators, genes encoding selectable markers (expressed in mammalian cells) are typically introduced into host cells via the same expression vector as one or more target genes.

[0145] Selectable marker genes encode proteins essential for the survival and growth of host cells grown in selective media. Typical selectable marker genes encode proteins that (a) confer resistance to antibiotics or other toxins; (b) compensate for cellular nutritional deficiencies; or (c) provide essential nutrients unavailable from complex or limited media through metabolism. The latter two can be considered metabolically selectable markers. The vector system disclosed herein utilizes both an antibiotic resistance selectable marker and a metabolically selectable marker. Both markers are expressed in mammalian host cells. This differs from antibiotic resistance selectable markers expressed in prokaryotes. Therefore, in some embodiments, expression vectors containing antibiotic resistance selectable markers will not have metabolically selectable markers. Unless otherwise stated, antibiotic resistance selectable markers referred to herein are those expressed in mammalian cells.

[0146] Selectable markers of antibiotic resistance expressed in mammalian host cells include kanamycin resistance genes, ampicillin resistance genes, tetracycline resistance genes, hygromycin B resistance genes, puromycin resistance genes, and neomycin resistance genes. Widely used selective markers of antibiotic resistance are the NeoR (neo), BsdR (bsr), HygR (hph), PuroR (pac), and BleoR (ble) genes, which confer resistance to the selective antibiotics G418 / genimycin, blastomycin, hygromycin B, puromycin, and fulvicin D, respectively. See, for example, Guo et al., 2021, J. Biol Chem. [Journal of Biochemistry] 297:100838. Antibiotic resistance selectable markers can be described in this article by gene name or the name of the corresponding antibiotic selector. That is, when referring to antibiotic resistance selectable markers used in expression vectors, hph and resistance markers against hygromycin B, pac and resistance markers against puromycin, etc., can be used interchangeably in this article. The terms antibiotic resistance marker and antibiotic resistance selectable marker can also be used interchangeably in this article.

[0147] In some embodiments disclosed herein, the antibiotic resistance selectable markers used in the expression vector are selected from the NeoR (neo), BsdR (bsr), HygR (hph), PuroR (pac), and BleoR (ble) genes. In some embodiments, the antibiotic resistance selectable marker used in the expression vector is the NeoR (neo) gene. In some embodiments, the antibiotic resistance selectable marker used in the expression vector is the BsdR (bsr) gene. In some embodiments, the antibiotic resistance selectable marker used in the expression vector is the HygR (hph) gene. In some embodiments, the antibiotic resistance selectable marker used in the expression vector is the PuroR (pac) gene. In some embodiments, the antibiotic resistance selectable marker used in the expression vector is the BleoR (ble) gene.

[0148] Specific metabolically selectable biomarkers expressed in mammalian cells include glutamine synthase (GS), dihydrofolate reductase (DHFR), asparaginase (Aspg; see Ha et al. Biotechnol Bioeng. [Biotechnology and Bioengineering] 2023 120:1159-1166), and promoter-free thymidine kinase genes. In some embodiments, the metabolically selectable biomarker used in the expression vector is glutamine synthase. In some embodiments, the metabolically selectable biomarker used in the expression vector is dihydrofolate reductase. In some embodiments, the metabolically selectable biomarker used in the expression vector is asparaginase. In some embodiments, the metabolically selectable biomarker used in the expression vector is promoter-free thymidine kinase.

[0149] The mammalian cell transformants are subjected to selection pressure, where only the transformant is suitable for survival due to the presence of two selectable genes in the two expression vectors. This selection pressure is applied by culturing the transformed cells under conditions of continuously increasing selectant concentrations in the culture medium, resulting in additional stringency and / or amplification of the selectable genes and the DNA encoding the target protein. Consequently, an increased amount of the target polypeptide is synthesized from the amplified DNA.

[0150] In some embodiments, one selectable marker (metabolism) is glutamine synthase or dihydrofolate reductase, and another selectable marker (antibiotic) is HygR. The selector for GS is methionine sulfoxide (MSX). The selector for DHFR is methotrexate (MTX). The selector for HygR is hygromycin B.

[0151] Glutamine synthase (GS) catalyzes glutamine biosynthesis through the condensation of ammonia and glutamate. GS knockout cell lines (GSKO) provide sufficient selection strictness in the absence of MSX or with low MSX concentrations, while coupling 25 mM MSX with GS knockout cell lines results in higher selection efficiency compared to CHOK1SV cell lines with higher MSX concentrations. See Fan et al., 2012, Biotechnol. Bioeng. [Biotechnology and Bioengineering] 109(4):1007-1015. Previous reports have shown that increasing MSX concentration during the seed train phase after clonal selection increases productivity without significantly affecting cell growth, GS and target gene copy number and expression, and maintains product quality properties in multiple GS knockout cell lines. See Tian et al., 2020, Engineering in Life Sciences [Life Sciences Engineering] 20(3-4): 112-125. Adding MSX during pool retrieval / selection can increase stringency and thus influence strand / vector expression.

[0152] In some embodiments, the promoter Sra (also referred to herein as SRα and Srα) or mPGK is operatively linked to a metabolically selectable biomarker, and the SV40 promoter is operatively linked to an antibiotic resistance selectable biomarker. In some embodiments, the promoter Sra is operatively linked to a metabolically selectable biomarker, and the SV40 promoter is operatively linked to an antibiotic resistance selectable biomarker. In some embodiments, the promoter mPGK is operatively linked to a metabolically selectable biomarker, and the SV40 promoter is operatively linked to an antibiotic resistance selectable biomarker.

[0153] In some embodiments, the metabolically selectable marker is glutamine synthase, the mPGK promoter is operatively linked to the metabolically selectable marker, the antibiotic-selectable marker is HygR, and the SV40 promoter is operatively linked to an antibiotic resistance selectable marker.

[0154] In some embodiments, the metabolically selectable marker is glutamine synthase, the promoter SRα is operatively linked to the metabolically selectable marker, the antibiotic-selectable marker is HygR, and the SV40 promoter is operatively linked to an antibiotic resistance selectable marker.

[0155] In some embodiments, the SRa promoter comprises 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical nucleotide sequences to the nucleotide sequence of SEQ ID NO: 2. In some embodiments, the SRa promoter comprises the nucleotide sequence of SEQ ID NO: 2.

[0156] In some embodiments, the mPGK promoter comprises 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical nucleotide sequences to the SEQ ID NO: 3. In some embodiments, the mPGK promoter comprises the nucleotide sequence of SEQ ID NO: 3.

[0157] In some embodiments, the SV40 promoter comprises 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical nucleotide sequences to the nucleotide sequence of SEQ ID NO: 4. In some embodiments, the SV40 promoter comprises the nucleotide sequence of SEQ ID NO: 4.

[0158] expression carrier

[0159] The optional biomarkers disclosed herein will be incorporated into expression vectors, which will typically include expression cassettes containing one or more promoters that are recognized by the host organism and operatively linked to nucleotide sequences encoding antibody chains and optional biomarkers. A promoter is a non-transcriptional sequence (typically within approximately 100 to 1000 bp) located upstream (i.e., 5') of the start codon of a structural gene that controls the transcription of the structural gene. The terms "promoter" and "promoter sequence" are used interchangeably in the embodiments described herein.

[0160] Typical antibodies are Y-shaped molecules with four polypeptide chains (two identical heavy chains and two identical light chains). Such antibodies are preferably expressed by a single vector. However, bispecific antibodies require alternative forms and are typically expressed on two different vectors. (See, for example, Spiess et al., 2015, Mol. Immunol. [Molecular Immunology] 67:95-106; Brinkmann et al., 2017, MAbs [Monoclonal Antibodies] 9:192-212; and Ma et al., 2021, Frontiers in Immunology [Immunology Frontiers] 12:626616).

[0161] The first expression vector for triple- or quadruple-chain antibody formulations typically contains a first promoter driving the expression of a first nucleotide sequence encoding a first antibody chain, a second promoter driving the expression of a second nucleotide sequence encoding a second antibody chain, and a third promoter driving the expression of a sequence encoding a metabolically selectable biomarker. The second expression vector for triple- or quadruple-chain antibody formulations typically contains a fourth promoter driving the expression of a third nucleotide sequence encoding a third antibody chain, a fifth promoter driving the expression of a fifth nucleotide sequence encoding a fourth antibody chain, and a sixth promoter driving the expression of a sequence encoding an antibiotic-selectable biomarker.

[0162] Promoters of particular interest for the nucleotide sequences encoding antibody chains include the human cytomegalovirus IE1 gene promoter enhancer (CMV) (Boshart et al., 1985, Cell 41:521-30, GenBank accession number X03922) and the hamster glyceraldehyde-3-phosphate dehydrogenase promoter and intron (GAPDH) (US Patent No. 10,202,261). Additional sequences can also be combined with promoters to improve expression. One such example is the adenovirus triplet leader sequence (ADL) (see Gingeras et al., 1982, J. Biol. Chem. 257:13475-91, GenBank accession number J01917). All promoters can be different, or any two, three, four, or five promoters can be the same.

[0163] In some embodiments, the first, second, third, fourth, fifth, and sixth promoters are identical. In some embodiments, the first, second, third, fourth, fifth, and sixth promoters are CMV-derived promoters (e.g., CMV / GAPDH, CMV / adL, or CMV / EF1a promoters). In some embodiments, the first, second, third, fourth, fifth, and sixth promoters are CMV / GAPDH promoters. In some embodiments, the first, second, third, fourth, fifth, and sixth promoters are CMV / adL promoters. In some embodiments, the first, second, third, fourth, fifth, and sixth promoters are CMV / EF1a promoters.

[0164] In some embodiments, the first, second, third, fourth, fifth, and sixth promoters are glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoters. In some embodiments, the GAPDH promoters are operatively linked to CMV promoter enhancers; the resulting construct may be referred to as a CMV / GAPDH promoter. In some embodiments, the first, second, third, fourth, fifth, and sixth promoters are CMV / GAPDH promoters. In some embodiments, each CMV / GAPDH promoter contains 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical nucleotide sequences to the nucleotide sequence of SEQ ID NO: 1. In some embodiments, each CMV / GAPDH promoter contains the nucleotide sequence of SEQ ID NO: 1.

[0165] The poly-A tail can follow each gene (i.e., the coding sequence for the first and second antibody chains and the optional marker). PolyA signal sequences are known in the art and include bovine growth hormone (BGH) polyA signal sequences (e.g., Pfarr et al., 1986, DNA, 5(2):115-22; Goodwin and Rottman, 1992, J. Biol. Chem., 267(23):16330-16334), thymidine kinase polyA (TKpA) signal sequences (Cole and Stacy, 1985, Mol Cell Biol., 5(8):2104-13), rabbit β-globin polyA signal sequences (Lanoix et al., 1988; EMBO J., 7(8):2515-22; GenBank accession number MG356850.1) and early polyA signal sequences of simian virus 40 (SV40) (Connelly and Manley, 1988, GenesDev). [Genes and Development], 2(4):440-52; GenBank accession number J02400). In some embodiments, the polyA tail can be selected from the group consisting of: rabbit β-globin pA sequence, thymidine kinase pA (TKpA) sequence, and simian virus 40 (SV40) early pA sequence. Additionally, all polyA tail sequences can be different, or any two, three, four, or five polyA tail sequences can be the same.

[0166] In some embodiments, each polyA sequence is independently selected from rabbit β-globin pA sequence, thymidine kinase pA (TKpA) sequence, and simian virus 40 (SV40) early pA sequence.

[0167] In some embodiments, each polyA sequence is identical.

[0168] In some embodiments, each polyA sequence is a rabbit β-globin pA sequence. In some embodiments, the rabbit β-globin polyA signal sequence comprises 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical nucleotide sequences to the nucleotide sequence of SEQ ID NO: 5. In some embodiments, the rabbit β-globin polyA signal sequence comprises the nucleotide sequence of SEQ ID NO: 5.

[0169] In some embodiments, each polyA sequence is a thymidine kinase pA (TKpA) sequence. In some embodiments, the thymidine kinase polyA (TKpA) signal sequence comprises 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical nucleotide sequences to the nucleotide sequence of SEQ ID NO: 7. In some embodiments, the thymidine kinase polyA (TKpA) signal sequence comprises the nucleotide sequence of SEQ ID NO: 7.

[0170] In some embodiments, each polyA sequence is an early pA sequence of simian virus 40 (SV40), and the SV40 early polyA signal sequence contains 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical nucleotide sequences to the nucleotide sequence of SEQ ID NO: 6. In some embodiments, the SV40 early polyA signal sequence contains the nucleotide sequence of SEQ ID NO: 6.

[0171] For example, in some triple-stranded forms, one expression vector contains coding sequences for one heavy chain and one light chain, while another expression vector contains coding sequences for a heavy chain-scFv fusion or a heavy chain-cytokine fusion and a light chain (3 strands, 1 vector). In this example, the expression vector may contain a metabolically selectable biomarker, while the other expression vector will contain an antibiotic-selectable biomarker. The heavy chain sequences may be the same or different. The light chain sequences may be the same or different. An example of a triple-stranded form is C1mAb(Fab-heterologous Fc-[scFv... ( ), an asymmetric fusion. The first antibody chain in each expression cassette may be a light chain or a heavy chain / heavy chain-scFv fusion. In one aspect, the first antibody chain is a light chain, and the second antibody chain is a heavy chain, a heavy chain-scFv fusion, or a heavy chain-cytokine fusion.

[0172] C1mAb asymmetric fusion (Fab-heterogeneous Fc-[scFv]) The representative scheme of ]) is described in Figure 2A middle.

[0173] For example, in a non-limiting example of the four-chain mode, one expression vector contains coding sequences for a first light chain and a first heavy chain, and a second expression vector contains a second light chain and a second heavy chain. In this example, the two light chains are different, and the two heavy chains are different. This represents a heterologous IgG mAb. In this example, the expression vector may contain a metabolically selectable biomarker, while the other expression vector will contain an antibiotic-selectable biomarker. The first antibody chain in each expression cassette can be either a light chain or a heavy chain.

[0174] A representative regimen for heterologous IgG mAb is described in Figure 3A middle.

[0175] The expression vectors provided herein can provide improved expression, likely due to an improved chain ratio of the expressed peptide. The chain ratio can be measured using techniques well-known in the art.

[0176] It has been found that, for triple-stranded and quadruple-stranded antibody forms, the use of a dual-selection system (one expression vector with a metabolic selection marker and the other with an antibiotic selection marker) surprisingly leads to higher yields of antibody forms.

[0177] These expression vectors can be used to transform host cells and contain additional nucleic acid sequences for plasmid maintenance and for cloning and expressing exogenous nucleotide sequences. Such sequences typically include one or more of the following nucleotide sequences (in addition to one or more promoters, antibody chains, and optional markers mentioned above): one or more enhancer sequences, origin of replication, transcription and translation control sequences, transcription termination sequences, complete intron sequences containing donor and acceptor splicing sites, various pre-sequences / pro-sequences to improve glycosylation or yield, natural or heterologous signal sequences (lead sequences or signal peptides) for polypeptide secretion, ribosome binding sites, polyadenylated sequences, internal ribosome entry sites (IRES) sequences, expression enhancement sequence elements (EASE), triplet leader sequences (TPL) and VA gene RNA from adenovirus 2, and multi-connector regions for inserting multinucleotides encoding the polypeptide to be expressed. Vectors can be constructed from starter vectors (such as commercially available vectors), and additional elements can be obtained separately and ligated into the vector. Methods for obtaining the components are well known to those skilled in the art.

[0178] Vector components can be homologous (i.e., derived from the same species and / or strain as the host cell), heterologous (e.g., derived from a species other than the host cell species or strain), heterozygous (i.e., derived from a combination of flanking sequences from more than one source), synthetic, or natural. The sequences of useful components in these vectors can be obtained using methods well-known in the art, such as those previously identified by mapping and / or by restriction endonucleases. Furthermore, they can be obtained by polymerase chain reaction (PCR) and / or by screening genomic libraries with suitable probes.

[0179] Ribosome binding sites are typically required for the initiation of mRNA translation and are characterized by a Shine-Dalgarno sequence (prokaryotes) or a Kozak sequence (eukaryotes). This element is typically located at the 3' of the promoter and at the 5' of the coding sequence of the polypeptide to be expressed.

[0180] Origin of replication facilitates the amplification of vectors within host cells. These can be included as part of commercially available prokaryotic vectors or chemically synthesized based on known sequences and ligated into vectors. Various viral sources (e.g., SV40, polyomaviruses, adenoviruses, vesicular stomatitis virus (VSV), or papillomaviruses such as HPV or BPV) can be used to clone vectors in mammalian cells.

[0181] Transcriptional and translational control sequences for mammalian host cell expression vectors can be excised from the viral genome. Commonly used enhancer sequences are derived from polyomaviruses, adenovirus 2, simian virus 40 (SV40), and human cytomegalovirus (CMV). For example, the human CMV promoter / enhancer of the immediate early gene 1 can be used. See, for example, Patterson et al., 1994, Applied Microbiol. Biotechnol. [Applied Microbiology and Biotechnology] 40:691-98. DNA sequences derived from the SV40 viral genome, such as SV40-derived, early and late promoters, enhancers, splice sequences, and polyadenylation sites, can be used to provide other genetic elements for the expression of structural gene sequences in mammalian host cells. Early and late viral promoters are particularly useful because they are readily available as fragments from the viral genome and may contain the origin of viral replication (Fiers et al., 1978, Nature 273:113; Kaufman, 1990, Meth. in Enzymol. 185:487-511). Smaller or larger SV40 fragments can also be used, provided they include approximately 250 bp of the sequence extending from the Hind III site to the BglI site located at the SV40 viral origin of replication. For example, enhancer sequences can be inserted into this vector to increase transcription in higher eukaryotes. Enhancers are cis-acting elements of DNA, typically 10–300 bp in length, that act on promoters to increase transcription. Enhancers are relatively independent in orientation and location and have been found at the 5' and 3' positions of transcription units. Several enhancer sequences are known to be derived from mammalian genes (e.g., globin, elastase, albumin, alpha-fetoprotein, and insulin). However, enhancers derived from viruses are typically used. SV40 enhancers, cytomegalovirus early promoter enhancers, polyomavirus enhancers, and adenovirus enhancers known in the art are exemplary enhancing elements for activating eukaryotic promoters. Although enhancers can be located at the 5' or 3' of the coding sequence in a vector, they are typically located at the 5' site of the promoter.

[0182] A sequence encoding an appropriate natural or heterologous signal sequence (lead sequence or signal peptide) can be incorporated into an expression vector to promote the extracellular secretion of the target protein. The choice of signal peptide or leader sequence depends on the type of host cell from which the target protein is to be produced, and the heterologous signal sequence can replace the natural signal sequence. Examples of functional signal peptides in mammalian host cells include: the interleukin-7 signal sequence described in U.S. Patent No. 4,965,195; the interleukin-2 receptor signal sequence described in Cosman et al., 1984, Nature [Nature] 312:768; the interleukin-4 receptor signal peptide described in European Patent No. 0367566; the type I interleukin-1 receptor signal peptide described in U.S. Patent No. 4,968,607; and the type II interleukin-1 receptor signal peptide described in European Patent No. 0460846.

[0183] Additional control sequences that have been shown to improve the expression of heterologous genes from mammalian expression vectors include elements such as expression enhancement sequence elements (EASE) derived from CHO cells (Morris et al., in Animal Cell Technology, pp. 529-534 (1997); U.S. Patent Nos. 6,312,951 B1, 6,027,915 and 6,309,841 B1) and triplet leader sequences (TPL) and VA gene RNA derived from adenovirus 2 (Gingeras et al., 1982, J. Biol. Chem. 257:13475-13491). Virus-derived internal ribosome entry site (IRES) sequences enable efficient translation of bicistronic mRNAs (Oh and Sarnow, 1993, Current Opinion in Genetics and Development 3:295-300; Ramesh et al., 1996, Nucleic Acids Research 24:2697-2700).

[0184] Vectors that are functional in the specific host cell used can be selected (i.e., the vector is compatible with the host cell structure, thereby allowing gene amplification and / or expression to occur). In some embodiments, the vector used employs protein fragment complementation assays using a protein reporter gene (such as dihydrofolate reductase) (see, for example, U.S. Patent No. 6,270,964). Suitable expression vectors are known in the art and are commercially available.

[0185] Table 1 provides non-limiting examples of synthetic nucleotide (DNA) sequences that can be used in certain expression vector components of the expression vectors disclosed herein.

[0186] Table 1. Non-limiting examples of expression vector component sequences

[0187]

[0188]

[0189] Generation of mammalian host cells expressing the target protein

[0190] The expression of target proteins in cells can be achieved transiently or stably using well-known methods (Davis et al., Basic Methods in Molecular Biology, 2nd ed., Appleton & Lange, Norwalk, Connecticut, 1994; Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001).

[0191] Stable integration methods are well known in the art. In short, stable integration is typically achieved by transiently introducing a heteropolynucleotide or a vector encoding a heteropolynucleotide into a host cell, which facilitates the stable integration of the heteropolynucleotide into the cellular genome. Typically, the heteropolynucleotide is flanked by homologous arms, i.e., sequences homologous to upstream and downstream regions of the integration site. Before their introduction into mammalian host cells, circular vectors can be linearized to facilitate integration into the cellular genome. Methods for introducing vectors into cells are well known in the art, including transfection using biological methods (such as viral delivery), chemical methods (such as transfection using cationic polymers, calcium phosphate, cationic lipids, or cationic amino acids), physical methods (such as electroporation or microinjection), or hybrid methods (such as protoplast fusion).

[0192] Stable integration-specific methods utilize recombinase-mediated cassette exchange (RMCE; Bode and Baer, ​​2001, CurrOpin Biotechnol. [Current Biotechnical Perspective] 12:473-80, and Bode et al., 2000, Biol. Chem. [Biochemistry] 381:801-813) for site-specific integration into the genome (also known as “targeted integration”). Site-specific recombinases such as Flp and Cre mediate recombination between two copies of their target sequence, referred to as FRT and loxP, respectively. Using two incompatible target sequences, such as FRT combined with F3 (Schlake and Bode, 1994, Biochemistry [Biochemistry], 33:12746-51) and an inverted recognition target site (Feng et al., 1999, J. Mol. Biol. [Journal of Molecular Biology] 292:779-85), allows the insertion of DNA fragments into predetermined chromosomal sites carrying target sequences of similar conformation. See also European Patent No. EP1781796B1 and European Patent Application Publication No. EP2789691A1.

[0193] RMCE insertion into specific sites in the genome can be mediated by nucleases (e.g., zinc finger proteins (ZFPs), transcription activator-like effector nucleases (TALENs), and clustered regularly spaced short palindromic repeats (CRISPR) / CRISPR-associated protein 9 (Cas9)). These nucleases can be engineered to generate single-strand and double-strand breaks (SSBs / DSBs) in the genome. There are two main and distinct pathways for DSB repair—homologous recombination and non-homologous end joining (NHEJ). Homologous recombination requires the presence of a homologous sequence as a template (e.g., a “donor” containing the RMCE) to guide the cellular repair process, and the repair outcome is error-free and predictable. In the absence of a template (or “donor”) sequence for homologous recombination, cells typically attempt to repair DSBs via the unpredictable and error-prone process of non-homologous end joining (NHEJ).

[0194] Vectors can be any molecule or entity suitable for transferring and / or transporting proteins encoding information to host cells and / or specific locations and / or compartments within host cells (e.g., nucleic acids, plasmids, bacteriophages, transposons, granules, chromosomes, viruses, viral capsids, virions, naked DNA, complex DNA, etc.). Vectors can include viral and nonviral vectors, and non-attachment mammalian vectors. Vectors are commonly referred to as expression vectors, such as recombinant expression vectors and cloning vectors. Vectors can be introduced into host cells to allow the vector itself to replicate and thereby amplify copies of the polynucleotides contained therein. Cloning vectors may contain sequence components that typically include, but are not limited to, origin of replication, promoter sequences, transcription initiation sequences, enhancer sequences, inverted terminal repeat sequences, and optional markers. These elements can be appropriately selected by those skilled in the art.

[0195] After construction, one or more vectors can be inserted into suitable cells for amplification and / or peptide expression. Transformation of the expression vector into selected cells can be accomplished by well-known methods, including transfection, infection, calcium phosphate co-precipitation, electroporation, nuclear transfection, microinjection, DEAE-dextran-mediated transfection, cationic lipid-mediated delivery, liposome-mediated transfection, microbombardment, receptor-mediated gene delivery, and polylysine, histone, chitosan, and peptide-mediated delivery. The chosen method will vary in part depending on the type of host cells used. These methods, and other suitable methods, are well known to those skilled in the art and are described in manuals and other technical publications, such as Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001).

[0196] The term "transformation" refers to a change in the genetic characteristics of a cell. A cell is transformed when it is modified to contain new DNA or RNA. For example, a cell is transformed when new genetic material is introduced into it via transfection, transduction, or other techniques, resulting in genetic modification from its original state. After transfection or transduction, the transformed DNA can either physically integrate into the cell's chromosome and recombine with the cell's DNA, or it can be temporarily maintained as a non-replicating free element, or it can replicate independently as a plasmid. When the transformed DNA replicates with cell division, the cell is considered to have been "stablely transformed."

[0197] The term “transfection” refers to the absorption of foreign or exogenous DNA by cells. Many transfection techniques are well known in the art and are disclosed herein. See, for example, Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, ibid.; Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al., 1981, Gene 13:197.

[0198] The term "transduction" refers to the process by which foreign DNA is introduced into cells via viral vectors. See Jones et al., (1998). Genetics: principles and analysis. Boston: Jones & Bartlett Publ.

[0199] Suitable mammalian host cells can be obtained by transfecting mammalian host cells with a pair of expression vectors as described herein. Therefore, this disclosure relates to mammalian host cells comprising a pair of expression vectors, wherein a) the first expression vector comprises a nucleotide sequence encoding a first antibody light chain, a first antibody heavy chain, or an antibody heavy chain fusion, and a metabolically selectable marker; and b) the second expression vector comprises a nucleotide sequence encoding a second antibody light chain, a second antibody heavy chain, or an antibody heavy chain fusion, and an antibiotic resistance selectable marker, wherein the second expression vector does not contain a metabolically selectable marker.

[0200] cell lines

[0201] In the methods disclosed herein, any mammalian cell line can be used. A variety of mammalian cell lines suitable for growth in cultures are available from the American Type Culture Collection (Manassas, Virginia) and commercial suppliers. Examples of cell lines commonly used in the industry include monkey kidney CVl lines transformed from SV40 (COS-7, ATCC CRL 1651); human embryonic kidney lines (293 cells or subclones used for growth in suspension culture (Graham et al., 1977, J. Gen Virol. [Journal of General Virology] 36:59)); juvenile hamster kidney cells (BHK, ATCC CCL 10); mouse Setolly cells (TM4, Mather, 1980, Biol. Reprod. [Reproductive Biology] 23:243-251); monkey kidney cells (CVl ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); and Buffalo rat hepatocytes (BRL 3A, ATCC CRL). 1442); human lung cells (W138, ATCC CCL 75); human liver cancer cells (Hep G2, HB 8065); mouse mammary tumors (MMT 060562, ATCC CCL51); TRI cells (Mather et al., 1982, Annals NY Acad. Sci. [Annals of the New York Academy of Sciences] 383:44-68); MRC 5 cells or FS4 cells; mammalian myeloma cells, as well as many other cell lines and Chinese hamster ovary (CHO) cells.

[0202] Large-scale production of proteins for commercial applications typically takes place in suspension culture. Therefore, the mammalian host cells used to generate the recombinant mammalian cells described herein can, but need not, be adapted for growth in suspension culture. Several host cells are known to be adapted for growth in suspension culture, including mouse myeloma NS0 cells and CHO cells from the CHO-S, DG44, and DXB11 cell lines. Other suitable cell lines include mouse myeloma SP2 / 0 cells, juvenile hamster kidney BHK-21 cells, and human PER.C6 cells. ® Cells, human embryonic kidney HEK-293 cells, and cell lines derived from or engineered from any cell line disclosed herein.

[0203] CHO cells are widely used for the production of complex recombinant proteins, including CHOK1 cells (ATCC CCL61). Dihydrofolate reductase (DHFR) deficient mutant cell lines (Urlaub et al., 1980, Proc Natl Acad Sci USA [Proceedings of the National Academy of Sciences] 77: 4216-4220) DXB11 and DG-44 are ideal CHO host cell lines because efficient DHFR-selective and amplifiable gene expression systems allow for high-level expression of recombinant proteins in these cells (Kaufman RJ, 1990, Meth Enzymol [Enzyme Methodology] 185:537-566). Also included is the glutamine synthetase (GS) knockout CHOK1SV cell line, selected using methionine sulfoxide imine (MSX) based on glutamine synthetase (GS). Other suitable CHO host cells may include, but are not limited to, the following (ECACC accession numbers are in parentheses): CHO (85050302), CHO (protein-free) (00102307), CHO-K1 (85051005), CHO-K1 / SF (93061607), CHO / DHFR- (94060607), CHO / DHFR-AC-free (05011002), RR-CHOKI (92052129).

[0204] In some embodiments disclosed herein, the mammalian host cell comprising a pair of selection vectors is a CHO cell. In some embodiments, the mammalian host cell is a GS KO CHO cell. In some embodiments, the mammalian host cell is a DHFR-CHO cell.

[0205] Cell culture process

[0206] This disclosure also provides methods for generating recombinant proteins (e.g., in antibody form), comprising a) culturing mammalian host cells under conditions expressing an antibody chain and a selectable biomarker, the mammalian host cells comprising 1) a first expression vector encoding a first light chain, a first heavy chain, or a fusion of the first heavy chain, and a metabolic selectable biomarker; and 2) a second expression vector encoding a second light chain, a second heavy chain, or a fusion of the second heavy chain, and an antibiotic selectable biomarker; wherein the culture is performed in a culture medium containing a metabolic selector and an antibiotic; and b) recovering the recombinant protein (e.g., in antibody form) from the culture.

[0207] The pair of expression vectors used in such a method (i.e., the first expression vector and the second expression vector) can be any expression vector system described herein. Host cells transfected with the vector systems described herein can be used for adherent cultures grown in stirred tank reactors, or suspension cultures (including conventional batch and fed-batch cell cultures, which may but do not necessarily include a rotating filter), perfusion systems (including alternating tangential flow (“ATF”) cultures, acoustic perfusion systems, depth filter perfusion systems, and other systems), hollow fiber bioreactors (HFBs, which in some cases can be used for perfusion processes), and various other cell culture methods (see, for example, Tao et al., 2003, Biotechnol. Bioeng. [Biotechnology and Bioengineering] 82:751-65; Kuystermans and Al-Rubeai, (2011) “Bioreactor Systems for Producing Antibody from Mammalian Cells”). Antibody Expression and Production In [Antibody Expression and Production], Cell Engineering 7:25-52, Al-Rubeai (ed.) Springer; Catapano et al., (2009) “Bioreactor Design and Scale-Up” Cell and Tissue Reaction Engineering: Principles and Practice In [Cellular and Tissue Reaction Engineering: Principles and Practice], Eibl et al. (editors) Springer-Verlag, which is incorporated herein by reference in its entirety.

[0208] During recombinant protein production, a controlled system is desired in which cells grow to a desired density, and then the cells' physiological state transitions to a high-productivity state of growth arrest, where cells use energy and substrates to produce the desired recombinant protein rather than generating more cells. Various methods exist to achieve this goal, including temperature variations and amino acid starvation, as well as the use of cell cycle inhibitors or other molecules that can stop cell growth without inducing cell death.

[0209] The production of recombinant proteins begins with establishing mammalian cell production cultures expressing the protein in culture plates, flasks, tubes, bioreactors, or other suitable containers. For example, in one embodiment, a small production bioreactor, typically 500 L to 2000 L, is used. In another embodiment, a bioreactor of 1000 L to 2000 L is used. The seed cell density used to inoculate the bioreactor can have a positive impact on the level of recombinant protein produced. In some embodiments, at least 0.5 x 10⁻⁶ cells can be inoculated in a serum-free medium. 6 (e.g., like Gundam and over 3.0 x 10) 6 (Number) live cells / mL are inoculated into the bioreactor. In another embodiment, the inoculation amount is 1.0 x 10⁻⁶ cells / mL. 6 live cells / mL.

[0210] The mammalian cells then undergo an exponential growth phase. Cell cultures can be maintained without supplemental feeding until the desired cell density is achieved. Cell cultures can be maintained for up to three days with or without supplemental feeding. Cultures can be inoculated at the desired cell density to begin the production phase without a short growth phase. The transition from the growth phase to the production phase can be initiated by any method known in the art.

[0211] Three methods are typically used in the commercial production of recombinant proteins via mammalian cell culture: batch culture, fed-batch culture, and perfusion culture. Batch culture is a discontinuous method in which cells are grown in a fixed volume of culture medium for a short period of time, followed by complete harvesting. Cultures grown using the batch method experience an increase in cell density until a maximum cell density is reached, after which the viable cell density decreases as culture medium components are consumed and metabolic byproducts such as lactate and ammonia levels accumulate. Harvesting typically occurs when the maximum cell density is achieved (e.g., 5 × 10⁶ cells / year). 6 Cells / mL or higher, depending on the culture medium formulation, cell line, etc. Batch processing is the simplest culture method; however, viable cell density is limited by nutrient availability, and once cells reach maximum density, the culture declines and yields decrease. There is no ability to extend the production phase because the accumulation of waste products and the rapid depletion of nutrients lead to a decline in culture (typically around 3–7 days).

[0212] Fed-batch cultures improve upon the batch process by providing batch or continuous feed to replenish those medium components that have already been consumed. Because fed-batch cultures receive additional nutrients throughout the run, they achieve higher cell densities (>10 to 30 x 10⁻⁶) compared to batch methods. 6The potential for increased product titers (cells / mL, depending on culture medium formulation, cell line, etc.) and feed-batch culture, unlike batch processes, can be generated and maintained in biphasic cultures by manipulating feed strategies and culture medium formulations to differentiate between the cell proliferation phase (growth phase) and the suspension or slow cell growth phase (production phase) to achieve the desired cell density. Therefore, fed-batch culture has the potential to achieve higher product titers compared to batch culture. Typically, batch methods are used during the growth phase and fed-batch methods are used during the production phase, but the feed strategy for fed-batch can be used throughout the entire process. However, unlike batch processes, bioreactor volume is a limiting factor for the amount of feed. Furthermore, as with batch methods, the accumulation of metabolic byproducts will lead to a decline in culture volume, which limits the duration of the production phase, for example, approximately 10 to 21 days. Feed-batch culture is discontinuous, and harvest typically occurs when metabolic byproduct levels or culture viability reach predetermined levels. Compared to non-feeded batch culture, fed-batch culture can produce a larger amount of recombinant protein. See, for example, U.S. Patent No. 5,672,502.

[0213] Perfusion culture is a culture in which cell cultures receive a fresh perfusion supply of medium while the used medium is removed. Perfusion can be continuous, stepwise, intermittent, or any combination of these. The perfusion rate can be less than one working volume per day to multiple working volumes. Cells remain in the culture, and the removed used medium is substantially cell-free or has significantly fewer cells than the culture. Recombinant proteins expressed in the cell culture may also be retained in the culture. Perfusion can be accomplished by a variety of means, including centrifugation, sedimentation, or filtration. See, for example, Voisard et al., 2003, Biotechnology and Bioengineering 82:751-65. An example of a filtration method is alternating tangential flow filtration. Alternating tangential flow is maintained by pumping the medium through a hollow fiber filter module. See, for example, U.S. Patent No. 6,544,424; Furey, 2002, Gen. Eng. News. 22(7):62-63.

[0214] "Perfusion flow rate" is the amount of culture medium that passes through (adds to and removes from) a bioreactor within a given time period, typically expressed as a portion or multiple working volumes. "Working volume" refers to the volume of the bioreactor used for cell culture. In one embodiment, the perfusion flow rate is one working volume per day or less. Perfusion feed media can be formulated to maximize perfusion nutrient concentrations, thereby minimizing the perfusion rate.

[0215] Cell cultures can be supplemented with concentrated feed media containing components (such as nutrients and amino acids) consumed during the cell culture production process.

[0216] Concentrated fed-batch culture media can be based on almost any cell culture medium formulation. Such concentrated fed-batch media can contain most components of cell culture media, for example, approximately 5, 6, 7, 8, 9, 10, 12, 14, 16, 20, 30, 50, 100, 200, 400, 600, 800, or even approximately 1000 times their normal amounts. Concentrated fed-batch culture media are often used in fed-batch culture processes.

[0217] Samples from cell cultures can be monitored and evaluated using any analytical technique known in the art. A variety of parameters, including recombinant proteins and the quality and characteristics of the culture medium, can be monitored throughout the culture period. Samples can be acquired and monitored intermittently at desired frequencies, including continuous monitoring, real-time, or near-real-time.

[0218] Typically, cell cultures (Nx to N-1) preceding the final production culture are used to generate seed cells, which will be used to inoculate the production bioreactor, N-1 culture. Seed cell density can have a positive impact on the level of recombinant protein produced. Product levels tend to increase with increasing seed density. Increased titers are not only associated with higher seed density but may also be influenced by the metabolism and cell cycle state of the cells entering production.

[0219] Seed cells can be produced by any culture method. One such method is perfusion culture using alternating tangential flow filtration. The N-1 bioreactor can be operated using alternating tangential flow filtration to provide high-density cells for seeding the production bioreactor. The N-1 stage can be used to grow cells to a density >90 × 10⁻⁶. 6 Cells / mL. The N-1 bioreactor can be used to generate batch seed cultures or as a rolling seed stock culture, maintaining high seed cell density for inoculating multiple production bioreactors. The duration of the growth phase for production can range from 7 to 14 days and can be designed to maintain cells in exponential growth before inoculating the production bioreactors. The perfusion rate, culture medium formulation, and time are optimized to allow cells to grow and be delivered to the production bioreactors in a state most conducive to optimizing their production. For inoculating production bioreactors, >15 × 10⁶ cells / mL can be achieved. 6 Seed cell density of 100 cells / mL. Higher seed cell density at inoculation can reduce or even eliminate the time required to reach the desired production density.

[0220] In some embodiments, mammalian host cells can be used to generate a high yield of the target protein. High yield or high volumetric productivity corresponds to the cell's ability to produce high levels of the target protein. Using a fed-batch or perfusion culture medium suitable for mammalian host cells and containing amino acids, vitamins, or trace elements, in a culture grown for 10 days under fed-batch or perfusion conditions, a specific yield will depend on the target protein and may be at least 0.05 g / L, at least 0.1 g / L, at least 0.15 g / L, at least 0.2 g / L, at least 0.25 g / L, at least 0.3 g / L, at least 0.35 g / L, at least 0.4 g / L, at least 0.45 g / L, at least 0.5 g / L, at least 0.6 g / L, at least 0.7 g / L, at least 0.8 g / L, at least 0.9 g / L, at least 1 g / L, at least 1.5 g / L, at least 2 g / L, or higher. In specific embodiments, the host cells and methods disclosed herein express the target protein and, when grown under the above-described culture conditions, are capable of producing at least 0.5 g / L, at least 0.6 g / L, at least 0.7 g / L, at least 0.8 g / L, at least 0.9 g / L, at least 1 g / L, at least 1.5 g / L, at least 2 g / L or more, preferably up to about 3 g / L, 4 g / L, 5 g / L or 10 g / L.

[0221] Yield can also be measured based on the unit productivity of a cell line, which is determined by the amount of protein produced per cell per day (expressed as pg / cell / day). Using a fed culture medium suitable for mammalian host cells and containing amino acids, vitamins, or trace elements, in cultures grown for 10 days under fed batch or perfusion conditions, the mammalian host cells disclosed herein are capable of producing at least 1 pg / cell / day, at least 2 pg / cell / day, at least 3 pg / cell / day, at least 4 pg / cell / day, at least 5 pg / cell / day, at least 6 pg / cell / day, at least 7 pg / cell / day, at least 8 pg / cell / day, at least 9 pg / cell / day, at least 10 pg / cell / day, at least 11 pg / cell / day, at least 12 pg / cell / day, at least 13 pg / cell / day, at least 14 pg / cell / day, at least 15 pg / cell / day, at least 20 pg / cell / day, at least 25 pg / cell / day, or more, preferably up to 50 pg / cell / day. In specific embodiments, the mammalian host cells disclosed herein express the target protein and, under the above-described culture conditions, have a unit productivity of at least 10 pg / cell / day, at least 11 pg / cell / day, at least 12 pg / cell / day, at least 13 pg / cell / day, at least 14 pg / cell / day, at least 15 pg / cell / day, at least 20 pg / cell / day, at least 25 pg / cell / day or higher, preferably up to 50 pg / cell / day.

[0222] The mammalian host cells described herein can be used to express the target protein. The expressed protein can be secreted into a culture medium, from which it can be recovered and / or collected. Furthermore, the protein can be purified or partially purified from such a culture or component (e.g., from a culture medium) using known processes and products available from commercial suppliers. The purified protein can then be “formulated” (meaning buffer exchange, sterilization, bulk packaging, and / or packaging for the end user). Suitable formulations for pharmaceutical compositions include those described in Remington's Pharmaceutical Sciences, 18th edition, 1995, Mack Publishing Company, Easton, Pennsylvania.

[0223] In some embodiments, CHO DHFR- cells or CHO GSKO cells can be cultured under methotrexate strict conditions in the case of CHO DHFR- cells or under methionine sulfoxide strict conditions in the case of CHO GSKO cells to facilitate the expression of difficult-to-express chains that pair with stronger GS promoters.

[0224] In some embodiments, CHO DHFR- cells or CHO GSKO cells can be cultured under methotrexate strict conditions in the case of CHO DHFR- cells or under methionine sulfoxide strict conditions in the case of CHO GSKO cells to facilitate the expression of difficult-to-express chains that pair with weaker GS promoters.

[0225] Polynucleotides, peptides, vectors, host cells, immune cells, compositions, etc., according to this disclosure can be prepared using a variety of known techniques.

[0226] Target protein

[0227] Target peptides and proteins (some of which can be produced using the expression vector systems, host cells, and methods described herein) may have scientific or commercial significance, including protein-based therapeutics. Target proteins particularly include secreted proteins, non-secreting proteins, intracellular proteins, or membrane-bound proteins. Target peptides and proteins can be produced using cell culture methods via recombinant animal cell lines and may be referred to as “recombinant proteins.” One or more expressed proteins can be produced intracellularly or secreted into a culture medium from which they can be recovered and / or collected. The terms “isolated protein” or “isolated recombinant protein” refer to a target peptide or protein purified from proteins or peptides or other contaminants that would interfere with its therapeutic, diagnostic, preventative, research, or other uses. Target proteins include proteins that exert therapeutic effects by binding to targets, particularly those listed below (including targets derived from them, associated targets, and modifications thereof).

[0228] Target proteins include "antigen-binding proteins," including, for example, "antibody forms." An "antigen-binding protein" is a protein or polypeptide containing an antigen-binding region or portion that has an affinity for another molecule (antigen) to which it binds. Antigen-binding proteins encompass antibodies, peptides, antibody fragments, antibody derivatives, antibody analogs, fusion proteins (including single-chain variable fragments (scFv), double-chain (bivalent) scFv, and IgG scFv (see, for example, Orcutt et al., 2010, Protein Eng Des2 Sel [Protein Engineering, Design & Selection] 23:221-228), heterologous IgG (see, for example, Liu et al., 2015, J Biol Chem [Journal of Biochemistry] 290:7535-7562), mutant proteins, and XmAbs. ®(Xencor, Inc., Monrovia, California). Examples of antigen-binding proteins include, but are not limited to, human antibodies, humanized antibodies; chimeric antibodies; recombinant antibodies; single-chain antibodies; biantibodies; triantibodies; tetraantibodies; Fab fragments; F(ab')2 fragments; IgD antibodies; IgE antibodies; IgM antibodies; IgG1 antibodies; IgG2 antibodies; IgG3 antibodies; or IgG4 antibodies and fragments thereof. Also included are bispecific T-cell binders (BiTE). ® ) molecules, and bispecific T cell binder molecules with extended durations (such as extended half-life) (e.g., HLE BiTE molecules, heterologous Ig BITE molecules, etc.).

[0229] As used herein, the term “antigen-binding protein” is used in its broadest sense and refers to a protein that contains a portion that binds to an antigen or target, and optionally includes a scaffold or framework portion that allows the antigen-binding portion to adopt a conformation that promotes antigen-binding protein binding to an antigen. Antigen-binding proteins may contain, for example, alternative protein scaffolds or artificial scaffolds with grafted CDRs or CDR derivatives. Such scaffolds include, but are not limited to, antibody-derived scaffolds (containing mutations that introduce, for example, stabilize the three-dimensional structure of the antigen-binding protein) and fully synthetic scaffolds (containing, for example, biocompatible polymers). See, for example, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 53(1):121-129; Roque et al., 2004, Biotechnol. Prog. 20:639-654. In addition, peptide antibody mimics (“PAMs”) and scaffolds based on antibody mimics utilizing fibronectin components as scaffolds may be used.

[0230] Antigen-binding proteins can have structures such as those of naturally occurring immunoglobulins. An immunoglobulin is a tetrameric molecule. In naturally occurring immunoglobulins, each tetramer consists of two pairs of identical polypeptide chains, each pair having a "light chain" (approximately 25 kDa) and a "heavy chain" (approximately 50-70 kDa). The amino-terminal portion of each chain includes a variable region of approximately 100 to 110 or more amino acids, which is primarily responsible for antigen recognition. The carboxyl-terminal portion of each chain defines a constant region primarily responsible for effector function. Human light chains are classified as κ light chains and λ light chains. Heavy chains are classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively.

[0231] Naturally occurring immunoglobulin chains exhibit the same general structure of a relatively conserved framework region (FR) linked by three hypervariable regions (also known as complementarity-determining regions or CDRs). Both the light and heavy chains contain domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the N-terminus to the C-terminus. Each domain can be assigned amino acids according to the definition in Sequences of Proteins of Immunological Interest, 5th Edition, US Dept. of Health and Human Services, PHS, NIH, NIH Publication No. 91-3242, (1991). The CDR can also be redefined according to alternative nomenclature schemes, such as Chothia's nomenclature scheme (see Chothia and Lesk, 1987, J. Mol. Biol. [Journal of Molecular Biology] 196:901-917; Chothia et al., 1989, Nature [Nature] 342:878-883 or Honegger and Pluckthun, 2001, J . Mol. Biol. [Journal of Molecular Biology] 309:657-670).

[0232] In the context of this disclosure, when the dissociation constant (K) D ≤10 -8 When M occurs, the antigen-binding protein is said to "specifically bind" or "selectively bind" to its target antigen. When K occurs... D ≤5×10 -9 At M, the antibody binds to the antigen with "high affinity," while at K... D ≤5×10 -10 When M occurs, the antibody binds to the antigen with "extremely high affinity".

[0233] Unless otherwise stated, the term "antibody" includes any isotype or subclass of glycosylated and non-glycosylated immunoglobulin, or its antigen-binding region that competes with intact antibodies for specific binding. Unless otherwise stated, antibodies include human, humanized, chimeric, multispecific, monoclonal, polyclonal, heterologous IgG, bispecific antibodies, and oligomers. Antibodies include IgG1, IgG2, IgG3, or IgG4.

[0234] Antigen-binding proteins may have one or more binding sites. If more than one binding site is present, these binding sites may be the same as each other or they may be different. For example, naturally occurring human immunoglobulins typically have two identical binding sites, while “bispecific” or “bifunctional” antibodies have two different binding sites. A standard nomenclature for multispecific antibody forms is VERITAS. See Biswas et al., 2023, mAbs [monoclonal antibodies] 15:1-9.

[0235] Antigen-binding fragments or antigen-binding regions include Fab, Fab', F(ab')2, Fv, biantibodies, Fd, dAb, macrobody, single-chain antibody molecules, and single-domain V. H H, complementarity-determining region (CDR) fragments, scFv, biantibodies, triantibodies, tetraantibodies, and polypeptides containing at least a portion of an immunoglobulin sufficient to bind a specific antigen to a target polypeptide.

[0236] Fab fragments are those with V L V H C L and C H 1. A monovalent segment of a structural domain; F(ab')2 segment is a divalent segment having two Fab segments connected by a disulfide bridge in the hinge region; Fd segment has V H and C H 1. Structural domain; the Fv fragment has a V-shaped arm for the antibody. L and V H Structural domain; and the dAb fragment has V H Structural domain, V L structural domain, or V H or V L Antigen-binding fragments of the domain (US Patent Nos. 6,846,634, 6,696,245, US Patent Application Publication Nos. 2005 / 0202512, 2004 / 0202995, 2004 / 0038291, 2004 / 0009507, 2003 / 0039958, Ward et al., 1989, Nature 341:544-546).

[0237] Single-chain antibodies (scFv) are antibodies in which V L and V HRegions are linked via linkers (e.g., synthetic sequences of amino acid residues) to form continuous protein chains, where the linkers are long enough to allow the protein chains to fold back and form monovalent antigen-binding sites (see, for example, Bird et al., 1988, Science 242:423-26 and Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-83, U.S. Patents 7,741,465 and 6,319,494, and Eshhar et al., 1997, Cancer Immunol Immunotherapy 45:131-136). scFv retains the ability of the parent antibody to specifically interact with the target antigen.

[0238] Biantibodies are bivalent antibodies consisting of two polypeptide chains, each containing V proteins linked by a linker. H and V L The linker is too short to allow pairing between two domains on the same chain, thus allowing each domain to pair with a complementary domain on another polypeptide chain (see, for example, Holliger et al., 1993, Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 90:6444-48; and Poljak et al., 1994, Structure 2:1121-23). ​​If the two polypeptide chains of a biantibody are identical, the biantibody produced by pairing them will have two identical antigen-binding sites. Polypeptide chains with different sequences can be used to prepare biantibodies with two different antigen-binding sites. Similarly, triantibodies and tetraantibodies are antibodies that contain three and four polypeptide chains, respectively, forming three and four antigen-binding sites, which may be the same or different.

[0239] For clarity, and as described herein, note that antigen-binding proteins may, but do not have to, be of human origin (e.g., human antibodies), and in some cases will contain non-human proteins, such as rat or mouse proteins, and in other cases antigen-binding proteins may contain hybrids of human and non-human proteins (e.g., humanized antibodies).

[0240] The target protein may include a human antibody. The term "human antibody" includes all antibodies having one or more variable and constant regions derived from a human immunoglobulin sequence. In one embodiment, all variable and constant domains are derived from a human immunoglobulin sequence (a fully human antibody). Such antibodies can be prepared in a variety of ways, including by immunizing mice genetically modified to express antibodies derived from human heavy and / or light chain encoding genes, such as those derived from Xenomouse, with the target antigen.® UltiMab™ or Velocimmune ® The system's mice, or those derived from UniRat ® Rats. Phage-based methods can also be used.

[0241] Alternatively, the target protein may include a humanized antibody. The sequence of a “humanized antibody” differs from that of an antibody derived from a non-human species in that one or more amino acid substitutions, deletions, and / or additions are made such that, when administered to a human subject, the humanized antibody is less likely to induce an immune response and / or induce a less severe immune response compared to a non-human species antibody. In one embodiment, certain amino acid mutations are made in the framework and constant domains of the heavy and / or light chains of a non-human species antibody to produce a humanized antibody. In another embodiment, one or more constant domains from a human antibody are fused to one or more variable domains from a non-human species. Examples of how humanized antibodies can be prepared can be found in U.S. Patent Nos. 6,054,297, 5,886,152, and 5,877,293.

[0242] It also includes modified proteins, such as those chemically modified by non-covalent, covalent, or both covalent and non-covalent bonds. It further includes proteins containing one or more post-translational modifications, which can be prepared by modification through cellular modification systems or by in vitro introduction or other means by enzymatic and / or chemical methods.

[0243] The target protein may also include recombinant fusion proteins, which include, for example, polymerized domains such as leucine zippers, coiled helices, and the Fc portion of immunoglobulins. It also includes proteins containing all or part of the amino acid sequence of the differentiating antigen (called CD proteins) or their ligands, or proteins substantially similar to any of these.

[0244] In some embodiments, the target protein may include proteins that specifically bind to: one or more CD proteins, HER receptor family proteins, cell adhesion molecules, growth factors, nerve growth factor, fibroblast growth factor, transforming growth factor (TGF), insulin-like growth factor, bone-inducing factor, insulin and insulin-related proteins, coagulation and coagulation-related proteins, colony-stimulating factor (CSF), other blood and serum proteins, blood group antigens; receptors, receptor-related proteins, growth hormone, growth hormone receptor, T cell receptors; neurotrophic factors, neurotrophic proteins, relaxin, interferon, interleukin, viral antigens, lipoproteins, integrins, rheumatoid factor, immunotoxins, surface membrane proteins, transport proteins, homing receptors, addressins, regulatory proteins, and immunoadhesins.

[0245] In some embodiments, the target protein binds alone or in any combination to one or more of the following: CD proteins (including, but not limited to, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD25, CD30, CD33, CD34, CD38, CD40, CD70, CD123, CD133, CD138, CD171, and CD174), HER receptor family proteins (including, for example, HER2, HER3, HER4, and EGF receptors), EGFRvIII, cell adhesion molecules (e.g., LFA-1, Mol, p150,95, VLA-4, ICAM-1, VCAM, and αv / β3 integrin), growth factors (including, but not limited to, vascular endothelial growth factor (“VEGF”); VEGFR2, growth hormone, thyroid-stimulating hormone, follicle-stimulating hormone, luteinizing hormone, growth hormone-releasing factor, parathyroid hormone, and Müllerian-inhibiting substances. substance), human macrophage inflammatory protein (MIP-1-α), erythropoietin (EPO), nerve growth factor (such as NGF-β), platelet-derived growth factor (PDGF), fibroblast growth factor (including, for example, aFGF and bFGF), epidermal growth factor (EGF), Cripto, transforming growth factor (TGF) (especially including TGF-α and TGF-β (including TGF-β1, TGF-β2, TGF-β3, TGF-β4 or TGF-β5)), insulin-like growth factor-I and insulin-like growth factor-II (IGF-I and IGF-II), des(1-3)-IGF-I (brain IGF-I) and bone-inducing factor, insulin and insulin-related proteins (including but not limited to insulin, insulin A chain, insulin B chain, proinsulin and insulin-like growth factor binding protein); (coagulation proteins and coagulation-related proteins, especially such as Factor VIII, tissue factor, von Willebrand factor, protein C, α-1-antitrypsin, plasminogen activators (such as urokinase and tissue plasminogen activator (“t-PA”)), bombazine, thrombin, thrombopoietin and thrombopoietin receptors, colony-stimulating factor (CSF) (especially including M-CSF, GM-CSF, and G-CSF), other blood and serum proteins (including but not limited to albumin, IgE, and blood group antigens), receptors and receptor-associated proteins (including, for example, flk2 / flt3 receptors, obesity (OB) receptors, growth hormone receptors, and T-cell receptors); neurotrophic factors, including but not limited to bone-derived neurotrophic factor (BDNF) and neurotrophin-3, neurotrophin-4, neurotrophin-5, or neurotrophin-6 (NT-3, NT-4, NT-5, or NT-6).Relaxin A chain, relaxin B chain and pro-relaxin, interferons (including, for example, interferon α, interferon β and interferon γ), interleukins (ILs) (e.g. IL-1 to IL-10, IL-12, IL-15, IL-17, IL-23, IL-12 / IL-23, IL-2Ra, IL-1-R1, IL-6 receptor, IL-4 receptor and / or IL-13 receptor, IL-13RA2 or IL-17 receptor, IL-1RAP); viral antigens, including but not limited to AIDS envelope virus antigens, lipoproteins, calcitonin, glucagon, atrial natriuretic peptide, pulmonary surfactant. Agents, tumor necrosis factor-α and tumor necrosis factor-β, enkephalin, BCMA, IgKappa, ROR-1, ERBB2, mesothelin, RANTES (activated and regulated normal T cell expression and secretion factors), mouse gonadotropin-related peptide, DNase, FR-α, inhibin and activin, integrin, protein A or D, rheumatoid factor, immunotoxin, bone morphogenetic protein (BMP), superoxide dismutase, surface membrane protein, decay accelerator factor (DAF), AIDS envelope, transport protein, homing receptor, MIC (MIC-a, MIC-B), ULBP 1-6, EPCAM, addressin, regulatory protein, immunoadhesin, antigen-binding protein, growth hormone, CTGF, CTLA4, eotaxin-1, MUC1, CEA, c-MET, Claudin-18, GPC-3, EPHA2, FPA, LMP1, MG7, NY-ESO-1, PSCA, ganglioside GD2, ganglioside GM2, BAFF, OPGL (RANKL), myostatin, Dickkopf-1 (DKK-1), Ang2, NGF, IGF-1 receptor, hepatocyte growth factor (HGF), TRAIL-R2, c-Kit, B7RP-1, PSMA, NKG2D-1, programmed cell death protein 1 and ligand, PD1 and PDL1, mannose receptor / hCGβ, hepatitis C virus, mesothelin dsFv [PE38] conjugate, Legionella pneumophila (lly), IFN γ, interferon-gamma inducible protein 10 (IP10), IFNAR, TALL-1, thymic stromal lymphopoietin (TSLP), proprotein convertase subtilisin / Kexin type 9 (PCSK9), stem cell factor, Flt-3, calcitonin gene-related peptide (CGRP), OX40L, α4β7, platelet-specific (platelet glycoprotein IIb / IIIb (PAC-1)), transforming growth factor β (TFGβ), zona pellucida sperm-binding protein 3 (ZP-3), TWEAK, platelet-derived growth factor receptor α (PDGFRα), sclerostin, and any bioactive fragments or variants of the foregoing.

[0246] Examples of therapeutic proteins include, but are not limited to, abciximab, adalimumab, adelimumab, aflibercept, alemtuzumab, alicurumab, anakinase, acecept, balithimab, belimumab, bevacizumab, biosozumab, bonatumab, brodatumab, mocantozumab, konnatumab, cetuximab, cetuximab, and konnatumab. Dalizumab, denosumab, eculizumab, ezolizumab, efalizumab, epazolizumab, etanercept, evokulumab, galiliximab, genitalumab, gemutuzumab, golimumab, teimozumab, infliximab, ipilimumab, levocurumab, ixazolizumab, imaparumab, motesanib phosphate diphosphate), morotumab-CD3, natecillatab, nimotuzumab, nivolumab, olizumab, olrezumab, olfamumab, olmalizumab, interleukin, palizumab, panitumab, pembrolizumab, pertuzumab, pectizumab, ranituzumab, rituximab, rituximab, romistastatin, romosuzumab, saxagstastatin, tocilizumab, tosimomab, trastuzumab, uterotumab, vedozazumab, vexizumab, voloximab, zalumab, zalumab, and any biosimilars of the foregoing substances.

[0247] The target protein further includes antibodies comprising 1, 2, 3, 4, 5, or 6 complementarity-determining regions (CDRs) of any of the aforementioned antibodies. One or more CDRs may be covalently or non-covalently incorporated into the molecule to make it an antigen-binding protein. The antigen-binding protein may be incorporated into one or more CDRs as part of a larger polypeptide chain, may covalently link one or more CDRs to another polypeptide chain, or may be non-covalently incorporated into one or more CDRs. CDRs allow the antigen-binding protein to bind specifically to a particular target antigen. Variations are also included that include regions identical in amino acid sequence to a reference amino acid sequence of the target protein at 70% or higher, particularly 80% or higher, more particularly 90% or higher, even more particularly 95% or higher, especially 97% or higher, even more particularly 98% or higher, even more particularly 99% or higher. This identity can be determined using a variety of well-known and readily available amino acid sequence analysis software. Preferred software includes those implementing the Smith-Waterman algorithm, which is considered a satisfactory solution to the search and alignment problem. Other algorithms can also be used, especially when speed is a significant consideration. Commonly used programs for DNA, RNA, and peptide alignment and homology matching include FASTA, TFASTA, BLASTN, BLASTP, BLASTX, TBLASTN, PROSRCH, BLAZE, and MPSRCH, the latter being an implementation of the Smith-Waltman algorithm for execution on massively parallel processors manufactured by MasPar.

[0248] Target proteins containing the Fc region (including antigen-binding proteins and Fc chain fusion proteins) form another aspect of this disclosure. Additionally, in the context of this disclosure, a hapten can be a target protein.

[0249] Additional non-limiting exemplary embodiments

[0250] The non-limiting example embodiments disclosed herein also include:

[0251] E1. A mammalian host cell containing a pair of expression vectors, wherein

[0252] a) The first expression vector contains a nucleotide sequence encoding the light chain of a first antibody, the heavy chain of a first antibody, or a fusion of antibody heavy chains, and a metabolically selectable marker; and

[0253] b) The second expression vector contains a nucleotide sequence encoding a second antibody light chain, a second antibody heavy chain, or an antibody heavy chain fusion, as well as an antibiotic resistance selectable marker.

[0254] E2. The mammalian host cell as described in E1, wherein the metabolic selectable marker is selected from the group consisting of glutamine synthase and dihydrofolate reductase.

[0255] E3. Mammalian host cells as described in E1 or E2, wherein the antibiotic resistance marker is a resistance marker against an antibiotic selected from the group consisting of: puromycin, genimycin, hygromycin, blastomycin, and humicin D.

[0256] E4. A mammalian host cell as described in any one of E1-E3, wherein the first antibody light chain and the second antibody light chain are identical.

[0257] E5. A mammalian host cell as described in any one of E1-E4, wherein the first expression vector encodes a first antibody heavy chain and the second expression vector encodes a second antibody heavy chain, and the first antibody heavy chain and the second antibody heavy chain are different.

[0258] E6. A mammalian host cell as described in any one of E1-E4, wherein the first expression vector encodes an antibody heavy chain fusion and the second expression vector encodes an antibody heavy chain.

[0259] E7. A mammalian host cell as described in any one of E1-E4, wherein the first expression vector encodes an antibody heavy chain and the second expression vector encodes an antibody heavy chain fusion.

[0260] E8. The mammalian host cell as described in any one of E1-E7, wherein the antibody heavy chain fusion is selected from the group consisting of: antibody heavy chain-scFv, antibody heavy chain-cytokine, and antibody heavy chain-VHH.

[0261] E9. A mammalian host cell as described in any one of E1-E8, wherein a promoter is operatively linked to nucleotide sequences encoding antibody light chains, antibody heavy chains or antibody heavy chain fusions, and metabolically selectable markers.

[0262] E10. The mammalian host cell of any one of E1-E9, wherein the promoter of the metabolic selectable marker is selected from the group consisting of mPGK and Sra, and the promoter of the antibiotic resistance selectable marker is SV40.

[0263] E11. A mammalian host cell as described in any one of E1-E9, wherein the polyA sequence is operatively linked to each nucleotide sequence.

[0264] E12. The mammalian host cell as described in E11, wherein the polyA sequence is identical or different and is selected from the group consisting of: rabbit β-globin pA sequence, thymidine kinase pA (TKpA) sequence and simian virus 40 (SV40) early pA sequence.

[0265] E13. Mammalian host cells as described in E1, wherein

[0266] a) The first expression vector contains a nucleotide sequence comprising the following elements in a 5' to 3' sequence:

[0267] 1) A first promoter operatively linked to a nucleotide sequence encoding an antibody light chain, followed by a first polyA sequence;

[0268] 2) A second promoter, operatively linked to a nucleotide sequence encoding the antibody heavy chain, followed by a second polyA sequence; and

[0269] 3) A promoter operatively linked to a nucleotide sequence encoding a metabolically selectable marker, followed by a third polyA sequence; and

[0270] b) The second expression vector contains a nucleotide sequence comprising the following elements in a 5' to 3' sequence:

[0271] 1) A first promoter operatively linked to a nucleotide sequence encoding an antibody light chain, followed by a first polyA sequence;

[0272] 2) A second promoter operatively linked to a nucleotide sequence encoding the antibody heavy chain-scFv fusion, followed by a second polyA sequence; and

[0273] 3) A promoter that is operatively linked to a nucleotide sequence encoding an antibiotic resistance selectable marker, followed by a third polyA sequence.

[0274] E14. A mammalian host cell as described in E13, which encodes C1 mAb.

[0275] E15. Mammalian host cells as described in E1, wherein

[0276] a) The first expression vector contains a nucleotide sequence comprising the following elements in a 5' to 3' sequence:

[0277] 1) A first promoter operatively linked to a nucleotide sequence encoding an antibody light chain, followed by a first polyA sequence;

[0278] 2) A second promoter operatively linked to a nucleotide sequence encoding the first antibody heavy chain, followed by a second polyA sequence; and

[0279] 3) A promoter operatively linked to a nucleotide sequence encoding a metabolically selectable marker, followed by a third polyA sequence; and

[0280] b) The second expression vector contains a nucleotide sequence comprising the following elements in a 5' to 3' sequence:

[0281] 1) A first promoter operatively linked to a nucleotide sequence encoding an antibody light chain, followed by a first polyA sequence;

[0282] 2) A second promoter operatively linked to a nucleotide sequence encoding a second antibody heavy chain, followed by a second polyA sequence; and

[0283] 3) A promoter operatively linked to a nucleotide sequence encoding a selectable marker of antibiotic resistance, followed by a third poly-A sequence.

[0284] The first antibody heavy chain and the second antibody heavy chain are different.

[0285] E16. A mammalian host cell as described in E15, which encodes heterologous IgG.

[0286] E17. Mammalian host cells as described in E1, wherein

[0287] a) The first expression vector contains a nucleotide sequence comprising the following elements in a 5' to 3' sequence:

[0288] 1) A first promoter operatively linked to a nucleotide sequence encoding an antibody light chain, followed by a first polyA sequence;

[0289] 2) A second promoter, operatively linked to a nucleotide sequence encoding the antibody heavy chain, followed by a second polyA sequence; and

[0290] 3) A promoter operatively linked to a nucleotide sequence encoding a metabolically selectable marker, followed by a third polyA sequence; and

[0291] b) The second expression vector contains a nucleotide sequence comprising the following elements in a 5' to 3' sequence:

[0292] 4) A first promoter, which is operatively linked to a nucleotide sequence encoding an antibody light chain, followed by a first polyA sequence;

[0293] 5) A second promoter operatively linked to a nucleotide sequence encoding an antibody heavy chain-cytokine fusion protein, followed by a second polyA sequence; and

[0294] 6) A promoter that is operatively linked to a nucleotide sequence encoding an antibiotic resistance selectable marker, followed by a third polyA sequence.

[0295] E18. A mammalian host cell as described in E17, which encodes an antibody-cytokine fusion.

[0296] E19. A mammalian host cell as described in any one of E1-E18, wherein the cell is a Chinese hamster ovary (CHO) cell.

[0297] E20. The mammalian host cell as described in E19, wherein the CHO cell is dihydrofolate reductase deficient (dhfr-) or glutamine synthase knockout (GSKO).

[0298] E21. A method for producing an antibody form, the method comprising:

[0299] a) Culture mammalian host cells under conditions expressing antibody chains and selectable markers, wherein the mammalian host cells contain

[0300] 1) A first expression vector encoding the first light chain, the first heavy chain, or a fusion of the first heavy chain, and a metabolically selectable biomarker; and

[0301] 2) A second expression vector encoding a second light chain, a second heavy chain or a fusion of two heavy chains, and antibiotic-selective biomarkers;

[0302] The culture was carried out in a culture medium containing metabolic selectors and antibiotics; and

[0303] b) Recover the antibody form from the culture.

[0304] E22. The method as described in E21, wherein the metabolic selectable marker is glutamine synthase and the metabolic selector is methionine sulfoxide imine, or the metabolic selectable marker is dihydrofolate reductase and the metabolic selector is methotrexate.

[0305] E23. The method as described in E22, wherein the antibiotic resistance marker is the hph gene and the antibiotic is hygromycin B, the antibiotic resistance marker is the pac gene and the antibiotic is puromycin, the antibiotic resistance marker is the neo gene and the antibiotic is neomycin, the antibiotic resistance marker is the bsr gene and the antibiotic is blastomycin, or the antibiotic resistance marker is the ble gene and the antibiotic is cymoxanil D.

[0306] E24. As described in E21, wherein

[0307] a) The first expression vector contains a nucleotide sequence comprising the following elements in a 5' to 3' sequence:

[0308] 1) A first promoter operatively linked to a nucleotide sequence encoding an antibody light chain, followed by a first polyA sequence;

[0309] 2) A second promoter, operatively linked to a nucleotide sequence encoding the antibody heavy chain, followed by a second polyA sequence; and

[0310] 3) A promoter operatively linked to a nucleotide sequence encoding a metabolically selectable marker, followed by a third polyA sequence; and

[0311] b) The second expression vector contains a nucleotide sequence comprising the following elements in a 5' to 3' sequence:

[0312] 1) A first promoter operatively linked to a nucleotide sequence encoding an antibody light chain, followed by a first polyA sequence;

[0313] 2) A second promoter operatively linked to a nucleotide sequence encoding the antibody heavy chain-scFv fusion, followed by a second polyA sequence; and

[0314] 3) A promoter that is operatively linked to a nucleotide sequence encoding an antibiotic resistance selectable marker, followed by a third polyA sequence.

[0315] E25. The method described in E24 is encoded as C1 mAb.

[0316] E26. As described in E21, wherein

[0317] a) The first expression vector contains a nucleotide sequence comprising the following elements in a 5' to 3' sequence:

[0318] 1) A first promoter operatively linked to a nucleotide sequence encoding an antibody light chain, followed by a first polyA sequence;

[0319] 2) A second promoter operatively linked to a nucleotide sequence encoding the first antibody heavy chain, followed by a second polyA sequence; and

[0320] 3) A promoter operatively linked to a nucleotide sequence encoding a metabolically selectable marker, followed by a third polyA sequence; and

[0321] b) The second expression vector contains a nucleotide sequence comprising the following elements in a 5' to 3' sequence:

[0322] 1) A first promoter operatively linked to a nucleotide sequence encoding an antibody light chain, followed by a first polyA sequence;

[0323] 2) A second promoter operatively linked to a nucleotide sequence encoding a second antibody heavy chain, followed by a second polyA sequence; and

[0324] 3) A promoter operatively linked to a nucleotide sequence encoding a selectable marker of antibiotic resistance, followed by a third poly-A sequence.

[0325] The first antibody heavy chain and the second antibody heavy chain are different.

[0326] E27. As described in E26, which encodes heterologous IgG.

[0327] E28. As described in E21, wherein

[0328] a) The first expression vector contains a nucleotide sequence comprising the following elements in a 5' to 3' sequence:

[0329] 1) A first promoter operatively linked to a nucleotide sequence encoding an antibody light chain, followed by a first polyA sequence;

[0330] 2) A second promoter, operatively linked to a nucleotide sequence encoding the antibody heavy chain, followed by a second polyA sequence; and

[0331] 3) A promoter operatively linked to a nucleotide sequence encoding a metabolically selectable marker, followed by a third polyA sequence; and

[0332] b) The second expression vector contains a nucleotide sequence comprising the following elements in a 5' to 3' sequence:

[0333] 1) A first promoter operatively linked to a nucleotide sequence encoding an antibody light chain, followed by a first polyA sequence;

[0334] 2) A second promoter operatively linked to a nucleotide sequence encoding an antibody heavy chain-cytokine fusion compound, followed by a second polyA sequence; and

[0335] 3) A promoter that is operatively linked to a nucleotide sequence encoding an antibiotic resistance selectable marker, followed by a third polyA sequence.

[0336] E29. As described in E28, which encodes an antibody-cytokine fusion.

[0337] E30. The method of any one of E21-E29, wherein the mammalian host cell is a Chinese hamster ovary (CHO) cell.

[0338] E31. The method as described in E30, wherein the CHO cell is dihydrofolate reductase deficient (dhfr-) or glutamine synthase knockout (GSKO).

[0339] E32. The method of any one of E21-E31, wherein the recovered antibody form is purified and formulated into a pharmaceutically acceptable formulation.

[0340] Further non-limiting exemplary embodiments / features disclosed herein include:

[0341] F1. An expression vector system comprising a first expression vector and a second expression vector, wherein:

[0342] The first expression vector contains a first nucleotide sequence encoding a first antibody light chain, a second nucleotide sequence encoding a first antibody heavy chain, an antibody heavy chain fusion, or an Fc chain fusion, and a third nucleotide sequence encoding a metabolically selectable biomarker; and

[0343] The second expression vector contains a fourth nucleotide sequence encoding the light chain of the second antibody, a fifth nucleotide sequence encoding the heavy chain of the second antibody, an antibody heavy chain fusion, or an Fc chain fusion, and a sixth nucleotide sequence encoding an antibiotic resistance selectable marker.

[0344] If the second or fifth nucleotide sequence encodes an Fc chain fusion, then the other nucleotide sequence encodes an antibody heavy chain or an antibody heavy chain fusion.

[0345] F2. The expression vector system as described in F1, wherein the first expression vector comprises the first nucleotide sequence, the second nucleotide sequence, and the third nucleotide sequence in a 5' to 3' sequence.

[0346] F3. An expression vector system as described in F1 or F2, wherein the second expression vector comprises the fourth nucleotide sequence, the fifth nucleotide sequence, and the sixth nucleotide sequence in a 5' to 3' sequence.

[0347] F4. The expression vector system as described in any one of F1-F3, wherein:

[0348] The first expression vector contains the first nucleotide sequence, the second nucleotide sequence, and the third nucleotide sequence in a 5' to 3' sequence; and

[0349] The second expression vector contains the fourth nucleotide sequence, the fifth nucleotide sequence, and the sixth nucleotide sequence in a 5' to 3' sequence.

[0350] F5. The expression vector system as described in any one of F1-F4, wherein the first expression vector does not contain a nucleotide sequence encoding an antibiotic resistance selectable marker.

[0351] F6. The expression vector system as described in any one of F1-F5, wherein the second expression vector does not contain a nucleotide sequence encoding a metabolically selectable marker.

[0352] F7. The expression vector system as described in any one of F1-F6, wherein the first expression vector does not contain a nucleotide sequence encoding an antibiotic resistance selectable marker, and the second expression vector does not contain a nucleotide sequence encoding a metabolic selectable marker.

[0353] F8. The expression vector system as described in any one of F1-F7, wherein the metabolic marker is either glutamine synthase or dihydrofolate reductase.

[0354] F9. The expression vector system as described in any one of F1-F8, wherein the metabolic selectable marker is glutamine synthase.

[0355] F10. The expression vector system as described in any one of F1-F8, wherein the metabolic selectable marker is dihydrofolate reductase.

[0356] F11. The expression vector system as described in any one of F1-F10, wherein the antibiotic resistance selectable marker is a resistance marker against an antibiotic selected from the group consisting of: puromycin, genimycin, hygromycin, blastomycin, and humicin D.

[0357] F12. The expression vector system as described in any one of F1-F11, wherein the antibiotic resistance selectable marker is a resistance marker against puromycin.

[0358] F13. The expression vector system as described in any one of F1-F11, wherein the antibiotic resistance selectable marker is a resistance marker against genipathomimetic.

[0359] F14. The expression vector system as described in any one of F1-F11, wherein the antibiotic resistance selectable marker is a resistance marker against hygromycin.

[0360] F15. The expression vector system as described in any one of F1-F11, wherein the antibiotic resistance selectable marker is a resistance marker against blast fungicide.

[0361] F16. The expression vector system as described in any one of F1-F11, wherein the antibiotic resistance selectable marker is a resistance marker against fumonisin D.

[0362] F17. The expression vector system as described in any one of F1-F11, wherein the antibiotic resistance marker is optionally a pac gene, hph gene, bsr gene or ble gene.

[0363] F18. An expression vector system as described in any one of F1-F11, F12 or F17, wherein the antibiotic resistance selectable marker is the pac gene.

[0364] F19. An expression vector system as described in any one of F1-F11, F14 or F17, wherein the antibiotic resistance selectable marker is the hph gene.

[0365] F20. An expression vector system as described in any one of F1-F11, F15 or F17, wherein the antibiotic resistance selectable marker is the bsr gene.

[0366] F21. An expression vector system as described in any one of F1-F11, F16 or F17, wherein the antibiotic resistance selectable marker is the ble gene.

[0367] F22. An expression vector system as described in any one of F1-F9, F11 or F14, wherein the metabolic selectable marker is glutamine synthase and the antibiotic resistance selectable marker is a resistance marker against hygromycin.

[0368] F23. An expression vector system as described in any one of F1-F9, F11, F14, F17 or F19, wherein the metabolic selectable marker is glutamine synthase and the antibiotic resistance selectable marker is the hph gene.

[0369] F24. The expression vector system as described in any one of F1-F23, wherein a first promoter is operatively linked to a first nucleotide sequence, a second promoter is operatively linked to a second nucleotide sequence, and a third promoter is operatively linked to a third nucleotide sequence.

[0370] F25. The expression vector system as described in F24, wherein the first expression vector comprises, in a 5' to 3' order, the first promoter, the first nucleotide sequence, the second promoter, the second nucleotide sequence, the third promoter, and the third nucleotide sequence.

[0371] F26. The expression vector system as described in any one of F1-F25, wherein a fourth promoter is operatively linked to the fourth nucleotide sequence, a fifth promoter is operatively linked to the fifth nucleotide sequence, and a sixth promoter is operatively linked to the sixth nucleotide sequence.

[0372] F27. The expression vector system as described in F26, wherein the second expression vector comprises, in a 5' to 3' order, the fourth promoter, the fourth nucleotide sequence, the fifth promoter, the fifth nucleotide sequence, the sixth promoter, and the sixth nucleotide sequence.

[0373] F28. The expression vector system as described in any one of F1-F23, wherein:

[0374] A first promoter is operatively linked to the first nucleotide sequence, a second promoter is operatively linked to the second nucleotide sequence, a third promoter is operatively linked to the third nucleotide sequence, a fourth promoter is operatively linked to the fourth nucleotide sequence, a fifth promoter is operatively linked to the fifth nucleotide sequence, and a sixth promoter is operatively linked to the sixth nucleotide sequence.

[0375] The first expression vector comprises, in a 5' to 3' order, the first promoter, the first nucleotide sequence, the second promoter, the second nucleotide sequence, the third promoter, and the third nucleotide sequence; and

[0376] The second expression vector contains, in 5' to 3' order, the fourth promoter, the fourth nucleotide sequence, the fifth promoter, the fifth nucleotide sequence, the sixth promoter, and the sixth nucleotide sequence.

[0377] F29. An expression vector system as described in any one of F24, F25 or F28, wherein the third promoter is mPGK or SRα.

[0378] F30. An expression vector system as described in any one of F24, F25, F28 or F29, wherein the third promoter is mPGK.

[0379] F31. An expression vector system as described in any one of F24, F25, F28 or F29, wherein the third promoter is SRα.

[0380] F32. The expression vector system as described in any one of F26-F31, wherein the sixth promoter is SV40.

[0381] F33. An expression vector system as described in F26-F29 or F31, wherein the third promoter is mPGK or SRα and the sixth promoter is SV40.

[0382] F34. The expression vector system as described in any one of F1-F23, wherein:

[0383] A first promoter is operatively linked to the first nucleotide sequence, a second promoter is operatively linked to the second nucleotide sequence, a third promoter is operatively linked to the third nucleotide sequence, a fourth promoter is operatively linked to the fourth nucleotide sequence, a fifth promoter is operatively linked to the fifth nucleotide sequence, and a sixth promoter is operatively linked to the sixth nucleotide sequence.

[0384] The first expression vector comprises, in 5' to 3' order, the first promoter, the first nucleotide sequence, the second promoter, the second nucleotide sequence, the third promoter, and the third nucleotide sequence;

[0385] The second expression vector contains, in the order of 5' to 3', the fourth promoter, the fourth nucleotide sequence, the fifth promoter, the fifth nucleotide sequence, the sixth promoter, and the sixth nucleotide sequence;

[0386] The third promoter is either mPGK or SRα; and

[0387] The sixth promoter is SV40.

[0388] F35. The expression vector system as described in any one of F26-F34, wherein each of the first promoter, the second promoter, the fourth promoter and the fifth promoter is a CMV-derived promoter (e.g., CMV / GAPDH, CMV / adL or CMV / EF1a promoter).

[0389] F36. The expression vector system as described in any one of F26-F35, wherein each of the first promoter, the second promoter, the fourth promoter, and the fifth promoter is a CMV / GAPDH or CMV / adL promoter.

[0390] F37. The expression vector system as described in any one of F26-F35, wherein:

[0391] A first promoter is operatively linked to the first nucleotide sequence, a second promoter is operatively linked to the second nucleotide sequence, a third promoter is operatively linked to the third nucleotide sequence, a fourth promoter is operatively linked to the fourth nucleotide sequence, a fifth promoter is operatively linked to the fifth nucleotide sequence, and a sixth promoter is operatively linked to the sixth nucleotide sequence.

[0392] The first expression vector comprises, in 5' to 3' order, the first promoter, the first nucleotide sequence, the second promoter, the second nucleotide sequence, the third promoter, and the third nucleotide sequence;

[0393] The second expression vector contains, in the order of 5' to 3', the fourth promoter, the fourth nucleotide sequence, the fifth promoter, the fifth nucleotide sequence, the sixth promoter, and the sixth nucleotide sequence;

[0394] The third promoter is either mPGK or SRα;

[0395] The sixth promoter is SV40; and

[0396] Each of the first promoter, the second promoter, the fourth promoter, and the fifth promoter is a CMV-derived promoter (e.g., CMV / GAPDH, CMV / adL, or CMV / EF1a promoter).

[0397] F38. The expression vector system as described in any one of F26-F37, wherein:

[0398] A first promoter is operatively linked to the first nucleotide sequence, a second promoter is operatively linked to the second nucleotide sequence, a third promoter is operatively linked to the third nucleotide sequence, a fourth promoter is operatively linked to the fourth nucleotide sequence, a fifth promoter is operatively linked to the fifth nucleotide sequence, and a sixth promoter is operatively linked to the sixth nucleotide sequence.

[0399] The first expression vector comprises, in 5' to 3' order, the first promoter, the first nucleotide sequence, the second promoter, the second nucleotide sequence, the third promoter, and the third nucleotide sequence;

[0400] The second expression vector contains, in the order of 5' to 3', the fourth promoter, the fourth nucleotide sequence, the fifth promoter, the fifth nucleotide sequence, the sixth promoter, and the sixth nucleotide sequence;

[0401] The third promoter is either mPGK or SRα;

[0402] The sixth promoter is SV40; and

[0403] Each of the first promoter, the second promoter, the fourth promoter, and the fifth promoter is a CMV / GAPDH or CMV / adL promoter.

[0404] F39. The expression vector system as described in any one of F1-F38, wherein a first polyA sequence is operatively linked to the first nucleotide sequence, a second polyA sequence is operatively linked to the second nucleotide sequence, a third polyA sequence is operatively linked to the third nucleotide sequence, a fourth polyA sequence is operatively linked to the fourth nucleotide sequence, a fifth polyA sequence is operatively linked to the fifth nucleotide sequence, and a sixth polyA sequence is operatively linked to the sixth nucleotide sequence.

[0405] F40. The expression vector system as described in F39, wherein the first expression vector comprises, in a 5' to 3' order, the first promoter, the first nucleotide sequence, the first polyA sequence, the second promoter, the second nucleotide sequence, the second polyA sequence, the third promoter, the third nucleotide sequence, and the third polyA sequence.

[0406] F41. The expression vector system as described in F39 or F40, wherein the second expression vector comprises, in a 5' to 3' order, the fourth promoter, the fourth nucleotide sequence, the fourth polyA sequence, the fifth promoter, the fifth nucleotide sequence, the fifth polyA sequence, the sixth promoter, the sixth nucleotide sequence, and the sixth polyA sequence.

[0407] F42. The expression vector system as described in any one of F39-F41, wherein:

[0408] The first expression vector comprises, in a 5' to 3' order, the first promoter, the first nucleotide sequence, the first polyA sequence, the second promoter, the second nucleotide sequence, the second polyA sequence, the third promoter, the third nucleotide sequence, and the third polyA sequence; and

[0409] The second expression vector contains, in the order of 5' to 3', the fourth promoter, the fourth nucleotide sequence, the fourth polyA sequence, the fifth promoter, the fifth nucleotide sequence, the fifth polyA sequence, the sixth promoter, the sixth nucleotide sequence, and the sixth polyA sequence.

[0410] F43. The expression vector system as described in any one of F39-F42, wherein each of the first polyA sequence, the second polyA sequence, the third polyA sequence, the fourth polyA sequence, the fifth polyA sequence, and the sixth polyA sequence is independently selected from the group consisting of rabbit β-globin pA, thymidine kinase pA (TKpA) sequence, and simian virus 40 (SV40) early pA sequence.

[0411] F44. The expression vector system as described in any one of F39-F43, wherein each of the first polyA sequence, the second polyA sequence, the third polyA sequence, the fourth polyA sequence, the fifth polyA sequence, and the sixth polyA sequence is a rabbit β-globin pA sequence.

[0412] F45. The expression vector system as described in any one of F39-F43, wherein each of the first polyA sequence, the second polyA sequence, the third polyA sequence, the fourth polyA sequence, the fifth polyA sequence, and the sixth polyA sequence is a thymidine kinase pA (TKpA) sequence.

[0413] F46. The expression vector system as described in any one of F39-F43, wherein each of the first polyA sequence, the second polyA sequence, the third polyA sequence, the fourth polyA sequence, the fifth polyA sequence, and the sixth polyA sequence is an early pA sequence of simian virus 40 (SV40).

[0414] F47. The expression vector system as described in any one of F1-F23, wherein:

[0415] A first promoter and a first polyA sequence are operatively linked to the first nucleotide sequence, a second promoter and a second polyA sequence are operatively linked to the second nucleotide sequence, a third promoter and a third polyA sequence are operatively linked to the third nucleotide sequence, a fourth promoter and a fourth polyA sequence are operatively linked to the fourth nucleotide sequence, a fifth promoter and a fifth polyA sequence are operatively linked to the fifth nucleotide sequence, and a sixth promoter and a sixth polyA sequence are operatively linked to the sixth nucleotide sequence;

[0416] The first expression vector comprises, in a 5' to 3' order, the first promoter, the first nucleotide sequence, the first polyA sequence, the second promoter, the second nucleotide sequence, the second polyA sequence, the third promoter, the third nucleotide sequence, and the third polyA sequence;

[0417] The second expression vector comprises, in the order of 5' to 3', the fourth promoter, the fourth nucleotide sequence, the fourth polyA sequence, the fifth promoter, the fifth nucleotide sequence, the fifth polyA sequence, the sixth promoter, the sixth nucleotide sequence, and the sixth polyA sequence;

[0418] The third promoter is either mPGK or SRα;

[0419] The sixth promoter is SV40;

[0420] Each of the first, second, fourth, and fifth promoters is a CMV / GAPDH or CMV / adL promoter; and

[0421] Each of the first polyA sequence, the second polyA sequence, the third polyA sequence, the fourth polyA sequence, the fifth polyA sequence, and the sixth polyA sequence is an early pA sequence of simian virus 40 (SV40).

[0422] F48. An expression vector system as described in any one of F34, F37, F38 or F47, wherein the metabolic selectable marker is glutamine synthase and the antibiotic resistance selectable marker is a resistance marker against hygromycin.

[0423] F49. An expression vector system as described in any one of F34, F37, F38, F47 or F48, wherein the metabolic selectable marker is glutamine synthase and the antibiotic resistance selectable marker is a resistance marker against hygromycin.

[0424] F50. The expression vector system as described in any one of F1-F49, wherein the first antibody light chain and the second antibody light chain contain the same amino acid sequence.

[0425] F51. The expression vector system as described in any one of F1-F50, wherein the second nucleotide sequence encodes a first antibody heavy chain or antibody heavy chain fusion, and the fifth nucleotide sequence encodes a second antibody heavy chain or antibody heavy chain fusion.

[0426] F52. The expression vector system as described in any one of F1-F51, wherein the first nucleotide sequence encodes a first antibody heavy chain, the fifth nucleotide sequence encodes a second antibody heavy chain, the first antibody heavy chain and the second antibody heavy chain contain different amino acid sequences, and the first antibody light chain and the second antibody light chain contain the same amino acid sequence.

[0427] F53. The expression vector system as described in any one of F1-F51, wherein:

[0428] The first nucleotide sequence encodes a first antibody heavy chain fusion, and the fifth nucleotide sequence encodes a second antibody heavy chain; or

[0429] The first nucleotide sequence encodes the first antibody heavy chain, and the fifth nucleotide sequence encodes the second antibody heavy chain fusion.

[0430] F54. The expression vector system as described in any one of F1-F51 or F53, wherein:

[0431] (a)(i) The first nucleotide sequence encodes a first antibody heavy chain fusion, and the fifth nucleotide sequence encodes a second antibody heavy chain; or (ii) The first nucleotide sequence encodes a first antibody heavy chain, and the fifth nucleotide sequence encodes a second antibody heavy chain fusion; and

[0432] (b) The first antibody light chain and the second antibody light chain contain the same amino acid sequence.

[0433] F55. The expression vector system as described in any one of F1-F50, wherein:

[0434] The first nucleotide sequence encodes a first antibody heavy chain or an antibody heavy chain fusion, and the fifth nucleotide sequence encodes a second Fc chain fusion; or

[0435] The first nucleotide sequence encodes a first Fc chain fusion, and the fifth nucleotide sequence encodes a second antibody heavy chain or an antibody heavy chain fusion.

[0436] F56. The expression vector system as described in any one of F1-F50 or F55, wherein:

[0437] The first nucleotide sequence encodes a first antibody heavy chain fusion, and the fifth nucleotide sequence encodes a second Fc chain fusion; or

[0438] The first nucleotide sequence encodes the first Fc chain fusion, and the fifth nucleotide sequence encodes the second antibody heavy chain fusion.

[0439] F57. The expression vector system as described in any one of F1-F51 or F53-F56, wherein the first antibody heavy chain fusion and / or the second antibody heavy chain fusion are selected from the group consisting of: antibody heavy chain-scFv, antibody heavy chain-cytokine, and antibody heavy chain-VH.

[0440] F58. An expression vector system as described in any one of F1-F50, F55 or F56, wherein the first Fc chain fusion or the second Fc chain fusion is selected from the group consisting of: Fc chain-scFv, Fc chain-cytokine and Fc chain-VH.

[0441] F59. A composition comprising an expression vector system as described in any one of F1-F58.

[0442] F60. A mammalian host cell comprising an expression vector system as described in any one of F1-F58.

[0443] F61. The mammalian host cell as described in F60, wherein the mammalian host cell is a Chinese hamster ovary (CHO) cell.

[0444] F62. A mammalian host cell as described in F60 or F61, wherein the mammalian host cell is a GS KOCHO cell.

[0445] F63. A method for producing a recombinant protein, the method comprising:

[0446] Mammalian host cells, such as any one of F60-F62, are cultured in a cell culture medium suitable for metabolism and antibiotic selection; and

[0447] The recombinant protein was recovered.

[0448] F64. The method as described in F63, wherein the cell culture medium contains antibiotics but does not contain components necessary for cell survival, which would otherwise be provided by the expression of the metabolic selection marker.

[0449] F65. The method described in F63 or F64, wherein the cell culture medium contains antibiotics and metabolic selectors.

[0450] F66. The method of any one of F63-F65, wherein the cell culture medium contains antibiotics and metabolic selectors, but does not contain components necessary for cell survival, which would otherwise be provided by the expression of the metabolic selector.

[0451] F67. The method as described in F65 or F66, wherein the mammalian host cell comprises an expression vector system, wherein the metabolically selectable marker is glutamine synthase, and the metabolic selector is methionine sulfoxide imine.

[0452] F68. The method as described in F65 or F66, wherein the mammalian host cell comprises an expression vector system, wherein the metabolic selectable marker is dihydrofolate reductase, and the metabolic selector is methotrexate.

[0453] F69. The method as described in F65 or F66, wherein the mammalian host cell comprises an expression vector system, wherein the metabolically selectable marker is glutamine synthase, and the antibiotic resistance selectable marker is a resistance marker against hygromycin (e.g., the hph gene), the metabolic selector is methionine sulfoxide imine, and the antibiotic is hygromycin B.

[0454] F70. The method of any one of F63-F66, wherein the mammalian host cell comprises an expression vector system, wherein the metabolic selectable marker is glutamine synthase, and the cell culture medium does not contain glutamine.

[0455] j

[0456] F71. The method as described in F70, wherein the cell culture medium contains methionine sulfoxide imine.

[0457] F72. The method as described in F65 or F66, wherein the mammalian host cell comprises an expression vector system, wherein the metabolically selectable marker is glutamine synthase, and the antibiotic resistance selectable marker is a resistance marker against hygromycin (e.g., the hph gene), the metabolic selector is methionine sulfoxide imine, the antibiotic is hygromycin B, and the cell culture medium does not contain glutamine.

[0458] This invention is not limited in scope to the specific embodiments described herein, which are intended as individual illustrations of various aspects of the invention, and functionally equivalent methods and components are also within the scope of the invention. In fact, various modifications to the invention will become apparent to those skilled in the art from the foregoing description and drawings, in addition to those shown and described herein. Such modifications are intended to fall within the scope of the appended claims.

[0459] Example

[0460] Example 1. A dual-selection strategy to improve the productivity of triple-chain C1mAb antibody forms

[0461] Materials and Methods

[0462] Plasmid generation. The coding sequences for the light and heavy chains were inserted into the pPBGS plasmid backbone using Golden Gate cloning to generate two bicistronic vectors. Briefly, LC and HC were controlled using a CMV-derived promoter with SV40-polyA, followed by an SRα promoter driving mGS-polyA expression and / or an SV40 promoter driving hygromycin B-polyA expression. All fragments were unidirectionally assembled using a combination of overhang sequences to facilitate Golden Gate cloning. Various vector configurations are shown in [Figure / Image / Description / Illustration]. Figure 2A middle.

[0463] Plasmid transfection into GS KO hosts. A proprietary GS KO clone cell host derived from the CHO-K1 parental host was used to generate a stable pool expressing the 3-chain antibody form. Transfection was performed every 3–4 days in shake flasks at 120 rpm, 36°C, and 5% CO2, in proprietary DMEM-F12-based medium at 0.3–0.4 × 10⁻⁶ mg / L. 6 Host cells were passaged at a seeding density of 1 × 10⁶ cells / mL. Twenty-four hours before transfection, host cells were seeded at a density of 1 × 10⁶ cells / mL. 6 10 cells / mL were seeded into the host cells to ensure that the cells were in the exponential growth phase at the time of transfection.

[0464] Stable pools expressing recombinant antibodies were generated using Gene Pulser XCell (BioRad Laboratories; Hercules, CA) according to the manufacturer's protocol. Repeated transfections were performed for each vector conformation. In short, 10 mg of each plasmid was combined with 5 mg of proprietary piggyBac transposase and electroporated to 20 × 10⁻⁶ cells. 6 Transfected cells were collected from host cells. The cells were recovered in 20 mL of growth medium in 50 mL centrifuge tubes at 225 rpm, 36 °C and 5% CO2.

[0465] Selection and recovery. Seventy-two hours after transfection, cells were centrifuged and transferred to selection medium containing 25 µM MSX and 400 mg / ml hygromycin B, glutamine-free. Cells were then thawed every 3–4 days at approximately 1–2 × 10⁻⁶ cells / day. 6 Passage cells at a seeding density of 100 cells / mL until viability exceeds 90%, at which point the seeding density is reduced to 0.3-0.4 × 10⁻⁶ cells / mL. 6 Cells / mL.

[0466] Feed-in batch production. Fully recovered cells are fed into a proprietary basal medium at a rate of 1×10⁻⁶. 6 Cells / mL were seeded for fed-batch production. Proprietary feed was added to the culture on days 3, 6, and 8, and harvesting was completed on day 10. Cell counts and viability were determined using a Vi-Cell BLU cell viability analyzer (Beckman Coulter, Brea, CA). The supernatant was analyzed for 1) titers (protein A-HPLC) and 2) product quality properties, including aggregates, shears, and isotypes, using size exclusion chromatography (SEC-UHPLC) (Waters UPLC H-class series), reductive capillary electrophoresis (rCE-SDS) (Sciex PA 800 Plus pharmaceutical analysis system), and analytical hydrophobic interaction chromatography (HIC-HPLC) (Agilent HPLC 1100 / 1200 series), respectively.

[0467] result

[0468] Figure 2D The results showed that the normalized effective titer using the dual selection method (black) increased by 2.45-fold compared to the optimized carrier configuration (grey) selected using standard single metabolic GS. Furthermore, dual selection reduced impurities measured as an nr-CE peak by 2-fold compared to the optimized carrier configuration. Figure 2F This reduction is due to a very appropriate chain ratio, resulting in perfectly assembled antibodies. Figure 2G ).

[0469] Example 2. A dual-selection strategy to improve heterologous IgG antibody production

[0470] Materials and Methods

[0471] Heterologous IgG mAb plasmid generation. Using Golden Gate cloning, coding sequences for the light and heavy chains were inserted into the pPBGS plasmid backbone to generate two bicistronic vectors. Briefly, LC and HC were controlled using a CMV-derived promoter with SV40-polyA, followed by an SRα or mPGK promoter driving mGS-polyA expression and / or an SV40 promoter driving hygromycin B-polyA expression. All fragments were unidirectionally assembled using a combination of overhang sequences to facilitate Golden Gate cloning. Different vector conformations are shown in [Figure / Image / Description / Illustration]. Figure 3A middle.

[0472] Plasmid transfection into GS KO hosts. A proprietary GS KO clone cell host derived from the CHO-K1 parental host was used to generate a stable pool expressing the 3-chain antibody form. Transfection was performed every 3–4 days in shake flasks at 120 rpm, 36°C, and 5% CO2, in proprietary DMEM-F12-based medium at 0.3–0.4 × 10⁻⁶ mg / L. 6 Host cells were passaged at a seeding density of 1 × 10⁶ cells / mL. Twenty-four hours before transfection, host cells were seeded at a density of 1 × 10⁶ cells / mL. 6 10 cells / mL were seeded into the host cells to ensure that the cells were in the exponential growth phase at the time of transfection.

[0473] Stable pools for expressing recombinant antibodies were generated using Gene Pulser XCell (Bio-Ray Laboratories, Hercules, CA) according to the manufacturer's protocol. Repeat transfections were performed for each vector conformation. In short, 10 mg of each plasmid was combined with 5 mg of the proprietary piggyBac transposase and electroporated to 20 × 10⁻⁶ cells. 6 Transfected cells were collected from host cells. The cells were recovered in 20 mL of growth medium in 50 mL centrifuge tubes at 225 rpm, 36 °C and 5% CO2.

[0474] Selection and recovery. Seventy-two hours after transfection, cells were centrifuged and transferred to selection medium without glutamine and containing 25 µM MSX and 400 µg / ml hygromycin B. Cells were then thawed every 3–4 days at approximately 1–2 × 10⁻⁶ cells / day. 6 Passage cells at a seeding density of 100 cells / mL until viability exceeds 90%, at which point the seeding density is reduced to 0.3-0.4 × 10⁻⁶ cells / mL. 6 Cells / mL.

[0475] Feed-in batch production. Fully recovered cells are fed into a proprietary basal medium at a rate of 1×10⁻⁶. 6Cells / mL were seeded for fed-batch production. Proprietary feed was added to the culture on days 3, 6, and 8, and harvesting was completed on day 10. Cell counts and viability were determined using a Vi-Cell BLU cell viability analyzer (Beckman Coulter, Brea, CA). The supernatant was analyzed for 1) titers (protein A-HPLC) and 2) product quality properties, including aggregates, shears, and isotypes, using size exclusion chromatography (SEC-UHPLC) (Waters UPLC H-class series), reductive capillary electrophoresis (rCE-SDS) (Sciex PA 800 Plus pharmaceutical analysis system), and analytical hydrophobic interaction chromatography (HIC-HPLC) (Agilent HPLC 1100 / 1200 series), respectively.

[0476] result

[0477] Figure 3C and Figure 3D The results showed that, compared with the standard single GS selection (grey), the dual selection method (black) increased the overall titer by 1.2 to 1.4 times, and achieved a higher effective titer. Furthermore, compared with single selection, dual selection produced a comparable %HMW of material via SEC and reduced impurities measured at the nr-CE front by 3.8 times (respectively...). Figure 3E and Figure 3F Overall titer and effective titer are normalized relative to a single GS-selected sample.

[0478] Example 3. A dual-selection strategy to improve antibody-cytokine fusion productivity

[0479] Materials and Methods

[0480] Antibody-cytokine fusion plasmid generation. Using Golden Gate cloning, coding sequences for the light and heavy chains were inserted into the pPBGS plasmid backbone to generate two bicistronic vectors. Briefly, LC and HC were controlled using a CMV-derived promoter with SV40-polyA, followed by an SRα or mPGK promoter driving mGS-polyA expression and / or an SV40 promoter driving hygromycin B-polyA expression. All fragments were unidirectionally assembled using a combination of overhang sequences to facilitate Golden Gate cloning. Different vector conformations are shown in [Figure / Image / Description / Illustration]. Figure 4A middle.

[0481] Plasmid transfection into GS KO hosts. A proprietary GS KO clone cell host derived from the CHO-K1 parental host was used to generate a stable pool expressing the 3-chain antibody form. Transfection was performed every 3–4 days in shake flasks at 120 rpm, 36°C, and 5% CO2, in proprietary DMEM-F12-based medium at 0.3–0.4 × 10⁻⁶ mg / L. 6 Host cells were passaged at a seeding density of 1 × 10⁶ cells / mL. Twenty-four hours before transfection, host cells were seeded at a density of 1 × 10⁶ cells / mL. 6 10 cells / mL were seeded into the host cells to ensure that the cells were in the exponential growth phase at the time of transfection.

[0482] Stable pools for expressing recombinant antibodies were generated using Gene Pulser XCell (Bio-Ray Laboratories, Hercules, CA) according to the manufacturer's protocol. Repeat transfections were performed for each vector conformation. In short, 10 mg of each plasmid was combined with 5 mg of the proprietary piggyBac transposase and electroporated to 20 × 10⁻⁶ cells. 6 Transfected cells were collected from host cells. The cells were recovered in 20 mL of growth medium in 50 mL centrifuge tubes at 225 rpm, 36 °C and 5% CO2.

[0483] Selection and recovery. Seventy-two hours after transfection, cells were centrifuged and transferred to selection medium without glutamine and containing 25 µM MSX and 400 µg / ml hygromycin B. Cells were then thawed every 3–4 days at approximately 1–2 × 10⁻⁶ cells / day. 6 Passage cells at a seeding density of 100 cells / mL until viability exceeds 90%, at which point the seeding density is reduced to 0.3-0.4 × 10⁻⁶ cells / mL. 6 Cells / mL.

[0484] Feed-in batch production. Fully recovered cells are fed into a proprietary basal medium at a rate of 1×10⁻⁶. 6 Cells / mL were seeded for fed-batch production. Proprietary feed was added to the culture on days 3, 6, and 8, and harvesting was completed on day 10. Cell counts and viability were determined using a Vi-Cell BLU cell viability analyzer (Beckman Coulter, Brea, CA). The supernatant was analyzed for 1) titers (protein A-HPLC) and 2) product quality properties, including aggregates, shears, and isotypes, using size exclusion chromatography (SEC-UHPLC) (Waters UPLC H-class series), reductive capillary electrophoresis (rCE-SDS) (Sciex PA 800 Plus pharmaceutical analysis system), and analytical hydrophobic interaction chromatography (HIC-HPLC) (Agilent HPLC 1100 / 1200 series), respectively.

[0485] result

[0486] Figure 4D The results showed that the dual selection method (black) increased the normalized effective titer by 2.2-fold compared to the optimized carrier configuration (grey) using standard single GS selection. Furthermore, dual selection reduced impurities measured at the nr-CE front peak by 1.5-fold compared to the optimized carrier configuration. Figure 4E and Figure 4G ).

[0487] Example 4. Improving [VH-VH] Dual selection strategy for Fab-heterologous Fc molecule productivity

[0488] Materials and Methods

[0489] Plasmid generation. The coding sequences for the light and heavy chains were inserted into the pPBGS plasmid backbone using Golden Gate cloning to generate bicistronic vectors. In short, a CMV-derived promoter controls both the light and heavy chains and carries SV40-polyA, followed by an SRα or mPGK promoter driving mGS-polyA expression and / or an SV40 promoter driving hygromycin B-polyA expression. All fragments were unidirectionally assembled using a combination of overhang sequences to facilitate Golden Gate cloning. Different vector conformations are shown in [Figure / Image / Description]. Figure 5A middle.

[0490] Plasmid transfection into GS KO hosts. A proprietary GS KO clone cell host derived from the CHO-K1 parental host is used to generate cells expressing [VH-VH]. [Fab] - Stabilization pool for heterologous Fc molecules. Sterilization was performed every 3-4 days in shake flasks at 120 rpm, 36°C, and 5% CO2, in a proprietary DMEM-F12-based medium at 0.5-0.6 × 10⁻⁶ mg / L. 6 Host cells were passaged at a seeding density of 1 × 10⁶ cells / mL. Twenty-four hours before transfection, host cells were seeded at a density of 1 × 10⁶ cells / mL. 6 10 cells / mL were seeded into the host cells to ensure that the cells were in the exponential growth phase at the time of transfection.

[0491] Stable pools for expressing recombinant proteins were generated using Gene Pulser XCell (Bio-Ray Laboratories, Hercules, CA) according to the manufacturer's protocol. Repeated transfections were performed for each vector conformation. In short, 10 mg of each plasmid was combined with 5 mg of the proprietary piggyBac transposase and electroporated to 20 × 10⁻⁶ cells. 6 Transfected cells were collected from host cells. The cells were recovered in 20 mL of growth medium in 50 mL centrifuge tubes at 225 rpm, 36 °C and 5% CO2.

[0492] Selection and recovery. Seventy-two hours after transfection, cells were centrifuged and transferred to a glutamine-free selection medium containing 400 µg / mL hygromycin B. Cells were then thawed every 3–4 days at approximately 1–2 × 10⁻⁶ cells / day. 6 Pass the cells at a seeding density of 100 cells / mL until viability exceeds 90%, at which point the seeding density is reduced to 0.5-0.6 × 10⁻⁶ cells / mL. 6 Cells / mL.

[0493] Feed-in batch production. Fully recovered cells were cultured in a proprietary basal medium at 1.3 × 10⁻⁶. 6 Cells / mL were seeded for fed-batch production. Proprietary feed was added to the culture on days 3, 6, 8, 10, and 13, and harvested on day 15. Cell counts and viability were determined using a Vi-Cell BLU cell viability analyzer (Beckman Coulter, Brea, CA). Size exclusion chromatography (SEC-UHPLC) (Waters UPLC H-class series), reductive capillary electrophoresis (rCE-SDS), and non-reductive capillary electrophoresis (nrCE-SDS) (Sciex PA 800 Plus pharmaceutical analysis system) were used to analyze 1) titers (protein A-HPLC) and 2) product quality properties, including aggregates, shears, and isotypes, of the supernatant.

[0494] result

[0495] like Figure 5B As shown, the recovery times for both carrier configurations are essentially the same. However, as Figure 5C and Figure 5D As shown, compared to the single-selection configuration, using the dual-selection support increased the overall titer and effective titer of molecule A by approximately 1.7-fold and 1.9-fold, respectively. Additionally, compared to the single-selection configuration, the dual-selection support configuration reduced the number of impurities measured at the nr-CE front by approximately 2.5-fold. Figure 5F ), while maintaining similar aggregates as measured by SEC-HMW ( Figure 5E The rCE-SDS spectra generated by the two vector configurations are depicted on... Figure 5G middle.

[0496] Example 5. Improving two [VH] Dual selection strategy for Fab-heterologous Fc molecule productivity

[0497] Materials and Methods

[0498] Plasmid generation. The coding sequences for the light and heavy chains of molecules A and B were inserted into the pPBGS plasmid backbone using Golden Gate cloning to generate bicistronic vectors. In short, a CMV-derived promoter controls both the light and heavy chains and carries SV40-polyA, followed by an SRα or mPGK promoter driving mGS-polyA expression and / or an SV40 promoter driving hygromycin B-polyA expression. All fragments were unidirectionally assembled using a combination of overhang sequences to facilitate Golden Gate cloning. Different vector configurations are shown in [Figure / Image / Description / Illustration]. Figure 6A middle.

[0499] Plasmid transfection into the GS KO host. A proprietary GS KO clone cell host derived from the CHO-K1 parental host was used to generate cells expressing two [VH] genera. [Fab] - Stabilization pool for heterologous Fc molecules (molecules A and B). Sterilization was performed every 3-4 days in shake flasks at 120 rpm, 36°C, and 5% CO2, in a proprietary DMEM-F12-based medium at 0.5-0.6 × 10⁻⁶ ppm. 6 Host cells were passaged at a seeding density of 1 × 10⁶ cells / mL. Twenty-four hours before transfection, host cells were seeded at a density of 1 × 10⁶ cells / mL. 6 10 cells / mL were seeded into the host cells to ensure that the cells were in the exponential growth phase at the time of transfection.

[0500] Stable pools for expressing recombinant proteins were generated using Gene Pulser XCell (Bio-Ray Laboratories, Hercules, CA) according to the manufacturer's protocol. Repeated transfections were performed for each vector conformation. In short, 10 mg of each plasmid was combined with 5 mg of the proprietary piggyBac transposase and electroporated to 20 × 10⁻⁶ cells. 6 Transfected cells were collected from host cells. The cells were recovered in 20 mL of growth medium in 50 mL centrifuge tubes at 225 rpm, 36 °C and 5% CO2.

[0501] Selection and recovery. Seventy-two hours after transfection, cells were centrifuged and transferred to a glutamine-free selection medium containing 400 µg / mL hygromycin B. Cells were then thawed every 3–4 days at approximately 1–2 × 10⁻⁶ cells / day. 6 Pass the cells at a seeding density of 100 cells / mL until viability exceeds 90%, at which point the seeding density is reduced to 0.5-0.6 × 10⁻⁶ cells / mL. 6 Cells / mL.

[0502] Feed-in batch production. Fully recovered cells were cultured in a proprietary basal medium at 1.3 × 10⁻⁶. 6Cells / mL were seeded for fed-batch production. Proprietary feed was added to the culture on days 3, 6, 8, 10, and 13, and harvested on day 15. Cell counts and viability were determined using a Vi-Cell BLU cell viability analyzer (Beckman Coulter, Brea, CA). Size exclusion chromatography (SEC-UHPLC) (Waters UPLC H-class series), reductive capillary electrophoresis (rCE-SDS), and non-reductive capillary electrophoresis (nrCE-SDS) (Sciex PA 800 Plus pharmaceutical analysis system) were used to analyze 1) titers (protein A-HPLC) and 2) product quality properties, including aggregates, shears, and isotypes, of the supernatant.

[0503] result

[0504] like Figure 6B As shown, the recovery time for the two carrier configurations is similar for molecule A, but slightly longer (about four days) for molecule B.

[0505] Compared to the single-selective carrier configuration, the dual-selective carrier configuration increased the overall titer and effective titer of molecules A and B by approximately 1.5-fold and 1.3-fold, and 1.7-fold and 3.4-fold, respectively. Figure 6C and Figure 6D As shown. Additionally, for molecule B, the dual-selective support configuration reduced impurities by approximately 1.4-fold or 1.8-fold compared to the single-selective support configuration (via SEC-HMW aggregates, respectively). Figure 6E ) and nr-CE front peak ( Figure 6F (Measurement). In both dual-selective and single-selective support configurations, similar impurity levels were observed in molecule A ( Figure 6E and Figure 6F The rCE-SDS spectra of molecules A and B, generated from two different support configurations, are depicted on... Figure 6G middle.

[0506] Example 6. Improving [Fab] ]-Heterogeneous Fc-[VH VH] Dual-choice strategy for molecular productivity

[0507] Materials and Methods

[0508] Plasmid generation. The coding sequences for the light and heavy chains were inserted into the pPBGS plasmid backbone using Golden Gate cloning to generate bicistronic vectors. In short, a CMV-derived promoter controls both the light and heavy chains and carries SV40-polyA, followed by an SRα or mPGK promoter driving mGS-polyA expression and / or an SV40 promoter driving hygromycin B-polyA expression. All fragments were unidirectionally assembled using a combination of overhang sequences to facilitate Golden Gate cloning. Different vector conformations are shown in [Figure / Image / Description]. Figure 7A middle.

[0509] Plasmid transfection into the GS KO host. A proprietary GS KO clone cell host derived from the CHO-K1 parental host is used to generate expression [Fab]. ]-Heterogeneous Fc-[VH Stabilization pool of VH] molecules. In a shake flask at 120 rpm, 36°C, and 5% CO2, in a proprietary DMEM-F12-based medium, at a concentration of 0.5–0.6 × 10⁻⁶ every 3–4 days. 6 Host cells were passaged at a seeding density of 1 × 10⁶ cells / mL. Twenty-four hours before transfection, host cells were seeded at a density of 1 × 10⁶ cells / mL. 6 10 cells / mL were seeded into the host cells to ensure that the cells were in the exponential growth phase at the time of transfection.

[0510] Stable pools for expressing recombinant proteins were generated using Gene Pulser XCell (Bio-Ray Laboratories, Hercules, CA) according to the manufacturer's protocol. Repeated transfections were performed for each vector conformation. In short, 10 mg of each plasmid was combined with 5 mg of the proprietary piggyBac transposase and electroporated to 20 × 10⁻⁶ cells. 6 Transfected cells were collected from host cells. The cells were recovered in 20 mL of growth medium in 50 mL centrifuge tubes at 225 rpm, 36 °C and 5% CO2.

[0511] Selection and recovery. Seventy-two hours after transfection, cells were centrifuged and transferred to a glutamine-free selection medium containing 400 µg / mL hygromycin B. Cells were then thawed every 3–4 days at approximately 1–2 × 10⁻⁶ cells / day. 6 Pass the cells at a seeding density of 100 cells / mL until viability exceeds 90%, at which point the seeding density is reduced to 0.5-0.6 × 10⁻⁶ cells / mL. 6 Cells / mL.

[0512] Feed-in batch production. Fully recovered cells were cultured in a proprietary basal medium at 1.3 × 10⁻⁶. 6Cells / mL were seeded for fed-batch production. Proprietary feed was added to the culture on days 3, 6, 8, 10, and 13, and harvested on day 15. Cell counts and viability were determined using a Vi-Cell BLU cell viability analyzer (Beckman Coulter, Brea, CA). Size exclusion chromatography (SEC-UHPLC) (Waters UPLC H-class series), reductive capillary electrophoresis (rCE-SDS), and non-reductive capillary electrophoresis (nrCE-SDS) (Sciex PA 800 Plus pharmaceutical analysis system) were used to analyze 1) titers (protein A-HPLC) and 2) product quality properties, including aggregates, shears, and isotypes, of the supernatant.

[0513] result

[0514] like Figure 7B As shown, the recovery times of the dual-selection and single-selection vector configurations are similar. Additionally, compared to the single-selection vector configuration, the dual-selection vector configuration exhibits similar overall titer and effective titer, such as... Figure 7C and Figure 7D As shown. However, compared to the single-selective support configuration, the dual-selective support configuration reduced the number of impurities measured as SEC-HMW aggregates by approximately 1.7-fold and reduced the nr-CE post-peak by approximately 2.2-fold (respectively, respectively). Figure 7E and Figure 7G ), while maintaining a similar nr-CE peak ( Figure 7F The rCE-SDS spectra generated by the two vector configurations are depicted on... Figure 7H middle.

Claims

1. An expression vector system comprising a first expression vector and a second expression vector, wherein: The first expression vector contains a first nucleotide sequence encoding a first antibody light chain, a second nucleotide sequence encoding a first antibody heavy chain, an antibody heavy chain fusion, or an Fc chain fusion, and a third nucleotide sequence encoding a metabolically selectable biomarker; and The second expression vector contains a fourth nucleotide sequence encoding the light chain of the second antibody, a fifth nucleotide sequence encoding the heavy chain of the second antibody, an antibody heavy chain fusion, or an Fc chain fusion, and a sixth nucleotide sequence encoding an antibiotic resistance selectable marker. If the second or fifth nucleotide sequence encodes an Fc chain fusion, then the other nucleotide sequence encodes an antibody heavy chain or an antibody heavy chain fusion.

2. The expression vector system of claim 1, wherein the first expression vector comprises the first nucleotide sequence, the second nucleotide sequence, and the third nucleotide sequence in a 5' to 3' sequence.

3. The expression vector system of claim 1 or claim 2, wherein the second expression vector comprises the fourth nucleotide sequence, the fifth nucleotide sequence, and the sixth nucleotide sequence in a 5' to 3' sequence.

4. The expression vector system of any one of claims 1-3, wherein the first expression vector does not contain a nucleotide sequence encoding an antibiotic resistance selectable marker, and the second expression vector does not contain a nucleotide sequence encoding a metabolic selectable marker.

5. The expression vector system according to any one of claims 1-4, wherein the metabolic selectable marker is glutamine synthase or dihydrofolate reductase.

6. The expression vector system according to any one of claims 1-5, wherein the metabolic selectable marker is glutamine synthase.

7. The expression vector system according to any one of claims 1-6, wherein the antibiotic resistance selectable marker is a resistance marker against an antibiotic selected from the group consisting of: puromycin, genimycin, hygromycin, blastomycin, and humicin D.

8. The expression vector system according to any one of claims 1-7, wherein the antibiotic resistance selectable marker is a resistance marker against hygromycin.

9. The expression vector system of any one of claims 1-8, wherein the metabolic selectable marker is glutamine synthase, and the antibiotic resistance selectable marker is a resistance marker against hygromycin.

10. The expression vector system of any one of claims 1-9, wherein a first promoter is operatively linked to the first nucleotide sequence, a second promoter is operatively linked to the second nucleotide sequence, and a third promoter is operatively linked to the third nucleotide sequence.

11. The expression vector system of claim 10, wherein the first expression vector comprises, in a 5' to 3' order, the first promoter, the first nucleotide sequence, the second promoter, the second nucleotide sequence, the third promoter, and the third nucleotide sequence.

12. The expression vector system of claim 10 or claim 11, wherein the third promoter is mPGK or SRα.

13. The expression vector system of any one of claims 10-12, wherein the third promoter is mPGK.

14. The expression vector system of any one of claims 10-13, wherein the third promoter is SRα.

15. The expression vector system of any one of claims 1-14, wherein a fourth promoter is operatively linked to the fourth nucleotide sequence, a fifth promoter is operatively linked to the fifth nucleotide sequence, and a sixth promoter is operatively linked to the sixth nucleotide sequence.

16. The expression vector system of claim 15, wherein the second expression vector comprises, in a 5' to 3' order, the fourth promoter, the fourth nucleotide sequence, the fifth promoter, the fifth nucleotide sequence, the sixth promoter, and the sixth nucleotide sequence.

17. The expression vector system of claim 15 or claim 16, wherein the sixth promoter is SV40.

18. The expression vector system according to any one of claims 1-15, wherein: A first promoter is operatively linked to the first nucleotide sequence, a second promoter is operatively linked to the second nucleotide sequence, a third promoter is operatively linked to the third nucleotide sequence, a fourth promoter is operatively linked to the fourth nucleotide sequence, a fifth promoter is operatively linked to the fifth nucleotide sequence, and a sixth promoter is operatively linked to the sixth nucleotide sequence. The first expression vector comprises, in 5' to 3' order, the first promoter, the first nucleotide sequence, the second promoter, the second nucleotide sequence, the third promoter, and the third nucleotide sequence; The second expression vector contains, in the order of 5' to 3', the fourth promoter, the fourth nucleotide sequence, the fifth promoter, the fifth nucleotide sequence, the sixth promoter, and the sixth nucleotide sequence; The third promoter is either mPGK or SRα; and The sixth promoter is SV40.

19. The expression vector system of any one of claims 1-18, wherein each of the first promoter, the second promoter, the fourth promoter, and the fifth promoter is a CMV-derived promoter.

20. The expression vector system of any one of claims 1-19, wherein each of the first promoter, the second promoter, the fourth promoter, and the fifth promoter is a CMV / GAPDH or CMV / adL promoter.

21. The expression vector system according to any one of claims 1-20, wherein: A first promoter is operatively linked to the first nucleotide sequence, a second promoter is operatively linked to the second nucleotide sequence, a third promoter is operatively linked to the third nucleotide sequence, a fourth promoter is operatively linked to the fourth nucleotide sequence, a fifth promoter is operatively linked to the fifth nucleotide sequence, and a sixth promoter is operatively linked to the sixth nucleotide sequence. The first expression vector comprises, in 5' to 3' order, the first promoter, the first nucleotide sequence, the second promoter, the second nucleotide sequence, the third promoter, and the third nucleotide sequence; The second expression vector contains, in the order of 5' to 3', the fourth promoter, the fourth nucleotide sequence, the fifth promoter, the fifth nucleotide sequence, the sixth promoter, and the sixth nucleotide sequence; The third promoter is either mPGK or SRα; The sixth promoter is SV40; and Each of the first promoter, the second promoter, the fourth promoter, and the fifth promoter is a CMV / GAPDH or CMV / adL promoter.

22. The expression vector system of any one of claims 1-21, wherein a first polyA sequence is operatively linked to the first nucleotide sequence, a second polyA sequence is operatively linked to the second nucleotide sequence, a third polyA sequence is operatively linked to the third nucleotide sequence, a fourth polyA sequence is operatively linked to the fourth nucleotide sequence, a fifth polyA sequence is operatively linked to the fifth nucleotide sequence, and a sixth polyA sequence is operatively linked to the sixth nucleotide sequence.

23. The expression vector system of claim 22, wherein: The first expression vector comprises, in 5' to 3' order, the first promoter, the first nucleotide sequence, the first polyA sequence, the second promoter, the second nucleotide sequence, the second polyA sequence, the third promoter, the third nucleotide sequence, and the third polyA sequence; The second expression vector contains, in the order of 5' to 3', the fourth promoter, the fourth nucleotide sequence, the fourth polyA sequence, the fifth promoter, the fifth nucleotide sequence, the fifth polyA sequence, the sixth promoter, the sixth nucleotide sequence, and the sixth polyA sequence.

24. The expression vector system of claim 22 or claim 23, wherein each of the first polyA sequence, the second polyA sequence, the third polyA sequence, the fourth polyA sequence, the fifth polyA sequence, and the sixth polyA sequence is independently selected from the group consisting of: rabbit β-globin pA sequence, thymidine kinase pA (TKpA) sequence, and simian virus 40 (SV40) early pA sequence.

25. The expression vector system of any one of claims 22-24, wherein each of the first polyA sequence, the second polyA sequence, the third polyA sequence, the fourth polyA sequence, the fifth polyA sequence, and the sixth polyA sequence is an early pA sequence of simian virus 40 (SV40).

26. The expression vector system according to any one of claims 1-9, wherein: A first promoter and a first polyA sequence are operatively linked to the first nucleotide sequence, a second promoter and a second polyA sequence are operatively linked to the second nucleotide sequence, a third promoter and a third polyA sequence are operatively linked to the third nucleotide sequence, a fourth promoter and a fourth polyA sequence are operatively linked to the fourth nucleotide sequence, a fifth promoter and a fifth polyA sequence are operatively linked to the fifth nucleotide sequence, and a sixth promoter and a sixth polyA sequence are operatively linked to the sixth nucleotide sequence; The first expression vector comprises, in 5' to 3' order, the first promoter, the first nucleotide sequence, the first polyA sequence, the second promoter, the second nucleotide sequence, the second polyA sequence, the third promoter, the third nucleotide sequence, and the third polyA sequence; The second expression vector comprises, in the order of 5' to 3', the fourth promoter, the fourth nucleotide sequence, the fourth polyA sequence, the fifth promoter, the fifth nucleotide sequence, the fifth polyA sequence, the sixth promoter, the sixth nucleotide sequence, and the sixth polyA sequence; The third promoter is either mPGK or SRα; The sixth promoter is SV40; Each of the first, second, fourth, and fifth promoters is a CMV / GAPDH or CMV / adL promoter; and Each of the first polyA sequence, the second polyA sequence, the third polyA sequence, the fourth polyA sequence, the fifth polyA sequence, and the sixth polyA sequence is an early pA sequence of simian virus 40 (SV40).

27. The expression vector system of any one of claims 18, 21 or 26, wherein the metabolic selectable marker is glutamine synthase and the antibiotic resistance selectable marker is a resistance marker against hygromycin.

28. The expression vector system of any one of claims 1-27, wherein the first antibody light chain and the second antibody light chain comprise the same amino acid sequence.

29. The expression vector system of any one of claims 1-28, wherein the second nucleotide sequence encodes a first antibody heavy chain or an antibody heavy chain fusion, and the fifth nucleotide sequence encodes a second antibody heavy chain or an antibody heavy chain fusion.

30. The expression vector system of any one of claims 1-29, wherein the first nucleotide sequence encodes a first antibody heavy chain, the fifth nucleotide sequence encodes a second antibody heavy chain, the first antibody heavy chain and the second antibody heavy chain comprise different amino acid sequences, and the first antibody light chain and the second antibody light chain comprise the same amino acid sequence.

31. The expression vector system according to any one of claims 1-29, wherein: The first nucleotide sequence encodes a first antibody heavy chain fusion, and the fifth nucleotide sequence encodes a second antibody heavy chain; or The first nucleotide sequence encodes the first antibody heavy chain, and the fifth nucleotide sequence encodes the second antibody heavy chain fusion.

32. The expression vector system according to any one of claims 1-29, wherein: (a)(i) The first nucleotide sequence encodes a first antibody heavy chain fusion, and the fifth nucleotide sequence encodes a second antibody heavy chain; or (ii) The first nucleotide sequence encodes a first antibody heavy chain, and the fifth nucleotide sequence encodes a second antibody heavy chain fusion; and (b) The first antibody light chain and the second antibody light chain contain the same amino acid sequence.

33. The expression vector system according to any one of claims 1-28, wherein: The first nucleotide sequence encodes a first antibody heavy chain or an antibody heavy chain fusion, and the fifth nucleotide sequence encodes a second Fc chain fusion. or The first nucleotide sequence encodes a first Fc chain fusion, and the fifth nucleotide sequence encodes a second antibody heavy chain or an antibody heavy chain fusion.

34. The expression vector system according to any one of claims 1-8 or 13, wherein: The first nucleotide sequence encodes a first antibody heavy chain fusion, and the fifth nucleotide sequence encodes a second Fc chain fusion; or The first nucleotide sequence encodes the first Fc chain fusion, and the fifth nucleotide sequence encodes the second antibody heavy chain fusion.

35. The expression vector system according to any one of claims 1-29 or 31-34, wherein the first antibody heavy chain fusion and / or the second antibody heavy chain fusion are selected from the group consisting of: antibody heavy chain-scFv, antibody heavy chain-cytokine, and antibody heavy chain-VH.

36. The expression vector system according to any one of claims 1-28 or 33-35, wherein the first Fc chain fusion or the second Fc chain fusion is selected from the group consisting of: Fc chain-scFv, Fc chain-cytokine, and Fc chain-VH.

37. A composition comprising the expression vector system as described in any one of claims 1-36.

38. A mammalian host cell comprising an expression vector system as described in any one of claims 1-36.

39. The mammalian host cell of claim 38, wherein the mammalian host cell is a Chinese hamster ovary (CHO) cell.

40. A method for producing a recombinant protein, the method comprising: The mammalian host cells as described in claim 38 or claim 39 are cultured in a cell culture medium suitable for metabolism and antibiotic selection; as well as The recombinant protein was recovered.

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