Carrier engineering strategies for improving volumetric productivity and reducing impurity formation in multiple forms

Abstract

This article discloses expression cassettes, expression vectors, dual-vector systems, and mammalian host cells comprising the aforementioned, including but not limited to Chinese hamster ovary (CHO) cells, wherein each expression vector employs a promoter set optimized for that antibody form. This article also discloses methods for generating certain antibody forms using these mammalian host cells.

Description

Cross-references to related applications

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 623,063, filed January 19, 2024, which is hereby incorporated by reference in its entirety. Technical Field

[0002] This disclosure provides expression cassettes, expression vectors, and dual-vector systems for expressing antibody forms in mammalian host cells, including but not limited to Chinese hamster ovary (CHO) cells. Submit sequence list

[0003] The contents of the following sequence list XML are incorporated herein by reference in their entirety: File name: 10614-WO01-SEC, Creation date: January 13, 2025; Size: 11,843 bytes. Background Technology

[0004] Biologics are used worldwide in a variety of applications, such as therapy and diagnostics, due to their broad utility. Mammalian cell lines are the primary expression systems for biologics, with Chinese hamster ovary (CHO) cells being the main cell factory (see Lalonde et al., 2017, J Biotechnol [Biotechnology Journal] 251:128-140). In particular, with the advent of biosimilars, speed to market and cost-effectiveness are now more important than ever.

[0005] The manufacturing strategy for biologics is complex, involving multiple steps such as selecting optimal cell lines, mass-culturing cells, and purifying the desired biologic from the cell harvest, resulting in high manufacturing costs. 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 widely adopted as first-line therapies.

[0006] To make biological therapeutics more accessible to patients, reducing the commodity cost of the manufacturing process is an attractive proposition. One way to achieve this is by increasing the titers associated with the production cell lines.

[0007] There remains a need for expression cassettes and vector systems that produce recombinant proteins at high titers upon transfection into host cell lines while minimizing the impact on product quality properties. Novel vector conformations can help optimize the expression levels of different strands in recombinant proteins (e.g., double-, triple-, or quadruple-stranded molecules), resulting in more balanced strand expression, reduced impurities, and higher product quality. Expression cassettes and vector systems employing such novel vector conformations will benefit the process development of biopharmaceuticals. Summary of the Invention

[0008] This disclosure provides expression cassettes comprising a polynucleotide sequence comprising, in a 5' to 3' order, the following elements: 1) a first copy of a GAPDH promoter operatively linked to a nucleotide sequence encoding a first antibody chain or antibody chain fusion, followed by a first polyA signal sequence; 2) a second copy of a GAPDH promoter operatively linked to a nucleotide sequence encoding a second antibody chain or antibody chain fusion, followed by a second polyA signal sequence; and 3) a promoter operatively linked to a nucleotide sequence encoding a selectable marker, followed by a third polyA signal sequence, wherein when the first antibody chain or antibody chain fusion is an antibody light chain, the second antibody chain or antibody chain fusion is an antibody heavy chain or antibody heavy chain fusion, wherein when the first antibody chain or antibody chain fusion is an antibody heavy chain or antibody heavy chain fusion, the second antibody chain or antibody chain fusion is an antibody light chain, and wherein the antibody heavy chain fusion is selected from the group consisting of: heavy chain-scFv, heavy chain-cytokine, and heavy chain VH. In some embodiments, such expression cassettes can be expressed in mammalian cells, such as Chinese hamster ovary (CHO) cells.

[0009] In some embodiments, the first antibody chain or antibody chain fusion is an antibody light chain, and the second antibody chain or antibody chain fusion is an antibody heavy chain or antibody heavy chain fusion. In some embodiments, the antibody heavy chain fusion is a heavy chain-scFv. In some embodiments, the antibody heavy chain fusion is a heavy chain-cytokine. In some embodiments, the antibody heavy chain fusion is a heavy chain VH.

[0010] In some embodiments, the first antibody chain or antibody chain fusion is an antibody heavy chain or antibody heavy chain fusion, and the second antibody chain or antibody chain fusion is an antibody light chain. In some embodiments, the antibody heavy chain fusion is a heavy chain-scFv. In some embodiments, the antibody heavy chain fusion is a heavy chain-cytokine. In some embodiments, the antibody heavy chain fusion is a heavy chain VH.

[0011] In some embodiments, a first GAPDH promoter is operatively linked to a nucleotide sequence encoding an antibody light chain, and a second GAPDH promoter is operatively linked to an antibody heavy chain or an antibody heavy chain fusion.

[0012] In some embodiments, a first GAPDH promoter is operatively linked to a nucleotide sequence encoding an antibody heavy chain or an antibody heavy chain fusion, and a second GAPDH promoter is operatively linked to an antibody light chain. In some embodiments, a first GAPDH promoter is operatively linked to a nucleotide sequence encoding an antibody heavy chain, and a second GAPDH promoter is operatively linked to an antibody light chain. In some embodiments, a first GAPDH promoter is operatively linked to a nucleotide sequence encoding an antibody heavy chain fusion, and a second GAPDH promoter is operatively linked to an antibody light chain.

[0013] In some embodiments, the antibody heavy chain fusion is a heavy chain-scFv.

[0014] In some embodiments, the antibody heavy chain fusion is a heavy chain-cytokine.

[0015] In some embodiments, the antibody heavy chain fusion is a heavy chain VH.

[0016] In some embodiments, the antibody heavy chain fusion compound is a direct fusion of the heavy chain and VH. In some embodiments, the antibody heavy chain fusion compound is a direct fusion of the heavy chain and scFv. In some embodiments, the antibody heavy chain fusion compound is a direct fusion of the heavy chain and cytokines.

[0017] In some embodiments, the antibody heavy chain fusion is a fusion of a heavy chain and a vitamin H, wherein the fusion includes a linker between the heavy chain and the vitamin H. In some embodiments, the antibody heavy chain fusion is a fusion of a heavy chain and an scFv, wherein the fusion includes a linker between the heavy chain and the scFv. In some embodiments, the antibody heavy chain fusion is a fusion of a heavy chain and a cytokine, wherein the fusion includes a linker between the heavy chain and the cytokine.

[0018] In some embodiments, the antibody heavy chain fusion polymer comprises a VH, scFv, or cytokine fused to the C-terminus of the heavy chain portion of the heavy chain fusion polymer. In some embodiments, the antibody heavy chain fusion polymer comprises a VH fused to the C-terminus of the heavy chain portion of the heavy chain fusion polymer. In some embodiments, the antibody heavy chain fusion polymer comprises an scFv fused to the C-terminus of the heavy chain portion of the heavy chain fusion polymer. In some embodiments, the antibody heavy chain fusion polymer comprises a cytokine fused to the C-terminus of the heavy chain portion of the heavy chain fusion polymer.

[0019] In some embodiments, the antibody heavy chain fusion polymer comprises a VH, scFv, or cytokine fused to the N-terminus of the heavy chain portion of the heavy chain fusion polymer. In some embodiments, the antibody heavy chain fusion polymer comprises a VH fused to the N-terminus of the heavy chain portion of the heavy chain fusion polymer. In some embodiments, the antibody heavy chain fusion polymer comprises an scFv fused to the N-terminus of the heavy chain portion of the heavy chain fusion polymer. In some embodiments, the antibody heavy chain fusion polymer comprises a cytokine fused to the N-terminus of the heavy chain portion of the heavy chain fusion polymer.

[0020] In some embodiments, the antibody heavy chain fusion composite includes a VH, scFv, or cytokine fused between CH1 and CH2 of the heavy chain portion of the heavy chain fusion composite. In some embodiments, the antibody heavy chain fusion composite includes a VH fused between CH1 and CH2 of the heavy chain portion of the heavy chain fusion composite. In some embodiments, the antibody heavy chain fusion composite includes an scFv fused between CH1 and CH2 of the heavy chain portion of the heavy chain fusion composite. In some embodiments, the antibody heavy chain fusion composite includes a cytokine fused between CH1 and CH2 of the heavy chain portion of the heavy chain fusion composite.

[0021] In some embodiments, the biomarker may be selected from the group consisting of glutamine synthase and dihydrofolate reductase. In some embodiments, the biomarker may be glutamine synthase. In some embodiments, the biomarker may be dihydrofolate reductase.

[0022] In some embodiments described herein, the promoter for the chain in antibody form is a combination of a CMV promoter enhancer and GAPDH (CMV / GAPDH). Generally, CMV / GAPDH is referred to as the promoter. A representative CMV / GAPDH promoter is provided in SEQ ID NO:1. In this combination, both the CMV promoter enhancer and the GAPDH promoter are operatively linked to the nucleotide sequence encoding the antibody chain, making this combination a better promoter than GAPDH alone.

[0023] In some embodiments, the CMV promoter is at the 5' of the GAPDH.

[0024] In some embodiments, the CMV / GAPDH promoter contains the nucleotide sequence of SEQ ID NO: 1.

[0025] In some embodiments, the promoter operatively linked to the nucleotide sequence encoding the selectable marker is selected from the group consisting of mPGK, SRα, and SV40 promoters. In some embodiments, the promoter operatively linked to the nucleotide sequence encoding the selectable marker is the mPGK promoter. In some embodiments, the promoter operatively linked to the nucleotide sequence encoding the selectable marker is the SRα promoter. In some embodiments, the promoter operatively linked to the nucleotide sequence encoding the selectable marker is the SV40 promoter.

[0026] In some embodiments, the expression cassette is provided as described above, wherein the first polyA signal sequence, the second polyA signal sequence, and the third polyA signal sequence are the same or different, and are selected from the group consisting of: thymidine kinase pA (TKpA) signal sequence, rabbit β-globin pA, and simian virus 40 (SV40) early pA signal sequence.

[0027] In some embodiments, the first polyA signal sequence, the second polyA signal sequence, and the third polyA signal sequence are the same.

[0028] In some embodiments, at least one of the first polyA signal sequence, the second polyA signal sequence, and the third polyA signal sequence is different.

[0029] In some embodiments, the first polyA signal sequence, the second polyA signal sequence, and the third polyA signal sequence are independently selected from the group consisting of bovine growth hormone (BGH) polyA signal sequence, thymidine kinase polyA (TKpA) signal sequence, rabbit β-globin polyA signal sequence, and simian virus 40 (SV40) early polyA signal sequence.

[0030] In some embodiments, each of the first polyA signal sequence, the second polyA signal sequence, and the third polyA signal sequence is a bovine growth hormone (BGH) polyA signal sequence.

[0031] In some embodiments, each of the first polyA signal sequence, the second polyA signal sequence, and the third polyA signal sequence is a thymidine kinase polyA (TKpA) signal sequence.

[0032] In some embodiments, each of the first polyA signal sequence, the second polyA signal sequence, and the third polyA signal sequence is a rabbit β-globin polyA signal sequence.

[0033] In some embodiments, each of the first polyA signal sequence, the second polyA signal sequence, and the third polyA signal sequence is an early polyA signal sequence of simian virus 40 (SV40).

[0034] In some embodiments, the expression cassette comprises a polynucleotide sequence containing the following elements in a 5' to 3' order:

[0035] a) A first GAPDH promoter operatively linked to a nucleotide sequence encoding an antibody light chain, followed by a first polyA signal sequence;

[0036] b) A second GAPDH promoter operatively linked to a nucleotide sequence encoding the antibody heavy chain, followed by a second poly-A signal sequence; and

[0037] c) A promoter operatively linked to a nucleotide sequence encoding an optional marker, followed by a third poly-A signal sequence.

[0038] In some embodiments, the first GAPDH promoter is a CMV / GAPDH promoter. In some embodiments, the first GAPDH promoter comprises 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical nucleotide sequences to the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the first GAPDH promoter comprises the nucleotide sequence of SEQ ID NO: 1.

[0039] In some embodiments, the second GAPDH promoter is a CMV / GAPDH promoter. In some embodiments, the second GAPDH promoter comprises 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical nucleotide sequences to the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the second GAPDH promoter comprises the nucleotide sequence of SEQ ID NO: 1.

[0040] In some embodiments, each of the first GAPDH promoter and the second GAPDH promoter is a CMV / GAPDH promoter. In some embodiments, each of the first GAPDH promoter and the second GAPDH promoter comprises 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical nucleotide sequences to the nucleotide sequence of SEQ ID NO: 1. In some embodiments, each of the first GAPDH promoter and the second GAPDH promoter comprises the nucleotide sequence of SEQ ID NO: 1.

[0041] In some embodiments, the promoter operatively linked to the nucleotide sequence encoding an optional marker is mPGK.

[0042] In some embodiments, the marker may be glutamine synthase.

[0043] In some embodiments, the selectable marker is glutamine synthase, and the promoter operatively linked to the nucleotide sequence encoding the selectable marker is mPGK.

[0044] In some embodiments, each of the first polyA signal sequence, the second polyA signal sequence, and the third polyA signal sequence is an early polyA signal sequence of simian virus 40 (SV40).

[0045] In some embodiments, each of the first GAPDH promoter and the second GAPDH promoter is a CMV / GAPDH promoter, the selectable marker is glutamine synthase, the promoter operatively linked to the nucleotide sequence encoding the selectable marker is mPGK, and each of the first polyA signal sequence, the second polyA signal sequence, and the third polyA signal sequence is an early polyA signal sequence of simian virus 40 (SV40).

[0046] In some embodiments, the expression cassette comprises a polynucleotide sequence containing the following elements in a 5' to 3' order:

[0047] d) A first GAPDH promoter operatively linked to a nucleotide sequence encoding an antibody heavy chain, followed by a first polyA signal sequence;

[0048] e) A second GAPDH promoter operatively linked to a nucleotide sequence encoding an antibody light chain, followed by a second polyA signal sequence; and

[0049] f) A promoter operatively linked to a nucleotide sequence encoding an optional marker, followed by a third polyA signal sequence.

[0050] In some embodiments, the first GAPDH promoter is a CMV / GAPDH promoter. In some embodiments, the first GAPDH promoter comprises 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical nucleotide sequences to the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the first GAPDH promoter comprises the nucleotide sequence of SEQ ID NO: 1.

[0051] In some embodiments, the second GAPDH promoter is a CMV / GAPDH promoter. In some embodiments, the second GAPDH promoter comprises 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical nucleotide sequences to the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the second GAPDH promoter comprises the nucleotide sequence of SEQ ID NO: 1.

[0052] In some embodiments, each of the first GAPDH promoter and the second GAPDH promoter is a CMV / GAPDH promoter. In some embodiments, each of the first GAPDH promoter and the second GAPDH promoter comprises 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical nucleotide sequences to the nucleotide sequence of SEQ ID NO: 1. In some embodiments, each of the first GAPDH promoter and the second GAPDH promoter comprises the nucleotide sequence of SEQ ID NO: 1.

[0053] In some embodiments, the promoter operatively linked to the nucleotide sequence encoding an optional marker is mPGK.

[0054] In some embodiments, the marker may be glutamine synthase.

[0055] In some embodiments, the selectable marker is glutamine synthase, and the promoter operatively linked to the nucleotide sequence encoding the selectable marker is mPGK.

[0056] In some embodiments, each of the first polyA signal sequence, the second polyA signal sequence, and the third polyA signal sequence is an early polyA signal sequence of simian virus 40 (SV40).

[0057] In some embodiments, each of the first GAPDH promoter and the second GAPDH promoter is a CMV / GAPDH promoter, the selectable marker is glutamine synthase, the promoter operatively linked to the nucleotide sequence encoding the selectable marker is mPGK, and each of the first polyA signal sequence, the second polyA signal sequence, and the third polyA signal sequence is an early polyA signal sequence of simian virus 40 (SV40).

[0058] In some embodiments, the expression cassette comprises a polynucleotide sequence containing the following elements in a 5' to 3' order:

[0059] g) A first GAPDH promoter operatively linked to a nucleotide sequence encoding an antibody light chain, followed by a first polyA signal sequence;

[0060] h) A second GAPDH promoter operatively linked to a nucleotide sequence encoding an antibody heavy chain fusion protein, followed by a second poly-A signal sequence; and

[0061] i) A promoter that is operatively linked to a nucleotide sequence encoding an optional marker, followed by a third poly-A signal sequence.

[0062] In some embodiments, the antibody heavy chain fusion is HC-scFv.

[0063] In some embodiments, the antibody heavy chain fusion is a direct fusion of the heavy chain and VH.

[0064] In some embodiments, the antibody heavy chain fusion is a fusion of the heavy chain and VH, wherein the fusion includes a linker between the heavy chain and VH.

[0065] In some embodiments, the antibody heavy chain fusion composite includes a VH at the C-terminus of the heavy chain portion fused to the heavy chain fusion composite.

[0066] In some embodiments, the antibody heavy chain fusion composite includes a VH at the N-terminus of the heavy chain portion fused to the heavy chain fusion composite.

[0067] In some embodiments, the antibody heavy chain fusion complex includes a VH fused between CH1 and CH2 of the heavy chain portion of the heavy chain fusion complex.

[0068] In some embodiments, the first GAPDH promoter is a CMV / GAPDH promoter. In some embodiments, the first GAPDH promoter comprises 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical nucleotide sequences to the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the first GAPDH promoter comprises the nucleotide sequence of SEQ ID NO: 1.

[0069] In some embodiments, the second GAPDH promoter is a CMV / GAPDH promoter. In some embodiments, the second GAPDH promoter comprises 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical nucleotide sequences to the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the second GAPDH promoter comprises the nucleotide sequence of SEQ ID NO: 1.

[0070] In some embodiments, each of the first GAPDH promoter and the second GAPDH promoter is a CMV / GAPDH promoter. In some embodiments, each of the first GAPDH promoter and the second GAPDH promoter comprises 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical nucleotide sequences to the nucleotide sequence of SEQ ID NO: 1. In some embodiments, each of the first GAPDH promoter and the second GAPDH promoter comprises the nucleotide sequence of SEQ ID NO: 1.

[0071] In some embodiments, the promoter operatively linked to the nucleotide sequence encoding the selectable marker is SRα.

[0072] In some embodiments, the marker may be glutamine synthase.

[0073] In some embodiments, the selectable marker is glutamine synthase, and the promoter operably linked to the nucleotide sequence encoding the selectable marker is SRα.

[0074] In some embodiments, each of the first polyA signal sequence, the second polyA signal sequence, and the third polyA signal sequence is an early polyA signal sequence of simian virus 40 (SV40).

[0075] In some embodiments, each of the first GAPDH promoter and the second GAPDH promoter is a CMV / GAPDH promoter, the selectable marker is glutamine synthase, the promoter operatively linked to the nucleotide sequence encoding the selectable marker is SRα, and each of the first polyA signal sequence, the second polyA signal sequence and the third polyA signal sequence is an early polyA signal sequence of simian virus 40 (SV40).

[0076] In some embodiments, the antibody heavy chain fusion is HC-scFv, each of the first and second GAPDH promoters is a CMV / GAPDH promoter, the selectable marker is glutamine synthase, the promoter operatively linked to the nucleotide sequence encoding the selectable marker is SRα, and each of the first, second, and third polyA signal sequences is an early polyA signal sequence of simian virus 40 (SV40).

[0077] In some embodiments, the expression cassette comprises a polynucleotide sequence containing the following elements in a 5' to 3' order:

[0078] j) A first GAPDH promoter operatively linked to a nucleotide sequence encoding an antibody heavy chain fusion, followed by a first polyA signal sequence;

[0079] k) A second GAPDH promoter, operatively linked to a nucleotide sequence encoding an antibody light chain, followed by a second polyA signal sequence; and

[0080] l) A promoter that is operatively linked to a nucleotide sequence encoding a selectable marker, followed by a third polyA signal sequence.

[0081] In some embodiments, the antibody heavy chain fusion is HC-scFv.

[0082] In some embodiments, the antibody heavy chain fusion is a direct fusion of the heavy chain and VH.

[0083] In some embodiments, the antibody heavy chain fusion is a fusion of the heavy chain and VH, wherein the fusion includes a linker between the heavy chain and VH.

[0084] In some embodiments, the antibody heavy chain fusion composite includes a VH at the C-terminus of the heavy chain portion fused to the heavy chain fusion composite.

[0085] In some embodiments, the antibody heavy chain fusion composite includes a VH at the N-terminus of the heavy chain portion fused to the heavy chain fusion composite.

[0086] In some embodiments, the antibody heavy chain fusion complex includes a VH fused between CH1 and CH2 of the heavy chain portion of the heavy chain fusion complex.

[0087] In some embodiments, the first GAPDH promoter is a CMV / GAPDH promoter. In some embodiments, the first GAPDH promoter comprises 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical nucleotide sequences to the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the first GAPDH promoter comprises the nucleotide sequence of SEQ ID NO: 1.

[0088] In some embodiments, the second GAPDH promoter is a CMV / GAPDH promoter. In some embodiments, the second GAPDH promoter comprises 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical nucleotide sequences to the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the second GAPDH promoter comprises the nucleotide sequence of SEQ ID NO: 1.

[0089] In some embodiments, each of the first GAPDH promoter and the second GAPDH promoter is a CMV / GAPDH promoter. In some embodiments, each of the first GAPDH promoter and the second GAPDH promoter comprises 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical nucleotide sequences to the nucleotide sequence of SEQ ID NO: 1. In some embodiments, each of the first GAPDH promoter and the second GAPDH promoter comprises the nucleotide sequence of SEQ ID NO: 1.

[0090] In some embodiments, the promoter operatively linked to the nucleotide sequence encoding the selectable marker is SRα.

[0091] In some embodiments, the marker may be glutamine synthase.

[0092] In some embodiments, the selectable marker is glutamine synthase, and the promoter operably linked to the nucleotide sequence encoding the selectable marker is SRα.

[0093] In some embodiments, each of the first polyA signal sequence, the second polyA signal sequence, and the third polyA signal sequence is an early polyA signal sequence of simian virus 40 (SV40).

[0094] In some embodiments, each of the first GAPDH promoter and the second GAPDH promoter is a CMV / GAPDH promoter, the selectable marker is glutamine synthase, the promoter operatively linked to the nucleotide sequence encoding the selectable marker is SRα, and each of the first polyA signal sequence, the second polyA signal sequence and the third polyA signal sequence is an early polyA signal sequence of simian virus 40 (SV40).

[0095] In some embodiments, the antibody heavy chain fusion is HC-scFv, each of the first and second GAPDH promoters is a CMV / GAPDH promoter, the selectable marker is glutamine synthase, the promoter operatively linked to the nucleotide sequence encoding the selectable marker is SRα, and each of the first, second, and third polyA signal sequences is an early polyA signal sequence of simian virus 40 (SV40).

[0096] This disclosure also provides expression carriers that contain any expression boxes disclosed above or anywhere herein.

[0097] In some embodiments, the polynucleotide sequence encodes a monoclonal antibody.

[0098] In some embodiments, the polynucleotide sequence encodes B2mab, C2mAb, B2Hmab, or C2Hmab.

[0099] This disclosure also provides pairs of expression carriers. In one embodiment, the pair has a first expression vector comprising an expression cassette containing a polynucleotide sequence comprising, in a 5' to 3' order, the following elements: 1) a first copy of a GAPDH promoter operably linked to a nucleotide sequence encoding an antibody light chain, followed by a first polyA sequence; 2) a second copy of the GAPDH promoter operably linked to a nucleotide sequence encoding an antibody heavy chain, followed by a second polyA sequence; and 3) a promoter operably linked to a nucleotide sequence encoding a selectable marker, followed by a third polyA sequence; and a second expression vector comprising an expression cassette containing a polynucleotide sequence comprising, in a 5' to 3' order, the following elements: 1) a first copy of a GAPDH promoter operably linked to a nucleotide sequence encoding an antibody light chain, followed by a first polyA sequence; 2) a second copy of the GAPDH promoter operably linked to a nucleotide sequence encoding an antibody heavy chain-scFv fusion, followed by a second polyA sequence; and 3) a first copy of a promoter operably linked to a first polyA sequence; 2) a second copy of the GAPDH promoter operably linked to a nucleotide sequence encoding an antibody heavy chain-scFv fusion, followed by a second polyA sequence; and 3) a second ... copy of a promoter; 2) a second copy of a promoter operably linked to a nucleotide sequence encoding an antibody heavy chain-scFv fusion, followed by a second polyA sequence; and 3) a second copy of a promoter operably linked to a first copy of a promoter; 2) a second copy of a promoter operably linked to a first copy of a promoter; 3) a second copy of a promoter; 4) a second copy of a promoter; 5) a second copy of a promoter; 6) a second A promoter, operatively linked to a nucleotide sequence encoding an optional marker, followed by a third polyA sequence. In one aspect of this embodiment, the vector pairs encode C1 mAb.

[0100] In some embodiments, the antibody light chain of the first expression vector is the same as the antibody light chain of the second expression vector.

[0101] In some embodiments, the antibody heavy chain of the first expression vector is identical to the antibody heavy chain portion of the antibody heavy chain-scFv fusion of the second expression vector.

[0102] In some embodiments, the antibody light chain of the first expression vector is the same as the antibody light chain of the second expression vector, and the antibody heavy chain of the first expression vector is the same as the antibody heavy chain portion of the antibody heavy chain-scFv fusion of the second expression vector.

[0103] In some embodiments, the antibody light chain of the first expression vector is different from the antibody light chain of the second expression vector.

[0104] In some embodiments, the antibody heavy chain of the first expression vector is different from the antibody heavy chain portion of the antibody heavy chain-scFv fusion of the second expression vector.

[0105] In some embodiments, the antibody light chain of the first expression vector is different from the antibody light chain of the second expression vector, and the antibody heavy chain of the first expression vector is different from the antibody heavy chain portion of the antibody heavy chain-scFv fusion of the second expression vector.

[0106] In some embodiments, each copy of the GAPDH promoter on the first expression vector is a CMV / GAPDH promoter. In some embodiments, each copy of the GAPDH promoter on the first expression vector 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 copy of the GAPDH promoter on the first expression vector contains the nucleotide sequence of SEQ ID NO: 1.

[0107] In some embodiments, each copy of the GAPDH promoter on the second expression vector is a CMV / GAPDH promoter. In some embodiments, each copy of the GAPDH promoter on the second expression vector 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 copy of the GAPDH promoter on the second expression vector contains the nucleotide sequence of SEQ ID NO: 1.

[0108] In some embodiments, each copy of the GAPDH promoter on the first expression vector and each copy of the GAPDH promoter on the second expression vector are both CMV / GAPDH promoters. In some embodiments, each copy of the GAPDH promoter on the first expression vector and each copy of the GAPDH promoter on the second expression vector contain 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical nucleotide sequences to the nucleotide sequence of SEQ ID NO: 1. In some embodiments, each copy of the GAPDH promoter on the first expression vector and each copy of the GAPDH promoter on the second expression vector contain the nucleotide sequence of SEQ ID NO: 1.

[0109] In some embodiments, the promoter operably linked to the nucleotide sequence encoding the selectable marker on the first expression vector is SRα. In some embodiments, the promoter operably linked to the nucleotide sequence encoding the selectable marker on the second expression vector is SRα. In some embodiments, the promoter operably linked to the nucleotide sequence encoding the selectable marker on the first expression vector is SRα, and the promoter operably linked to the nucleotide sequence encoding the selectable marker on the second expression vector is SRα.

[0110] In some embodiments, the selectable biomarker on the first expression vector is glutamine synthase. In some embodiments, the selectable biomarker on the second expression vector is glutamine synthase. In some embodiments, the selectable biomarker on the first expression vector and the selectable biomarker on the second expression vector are both glutamine synthase.

[0111] In some embodiments, the promoter operably linked to the nucleotide sequence encoding the selectable marker on the first expression vector is SRα, the promoter operably linked to the nucleotide sequence encoding the selectable marker on the second expression vector is SRα, and the selectable marker on the first expression vector and the selectable marker on the second expression vector are glutamine synthase.

[0112] In some embodiments, each of the first, second, and third polyA signal sequences on the first expression vector is an early polyA signal sequence of simian virus 40 (SV40). In some embodiments, each of the first, second, and third polyA signal sequences on the second expression vector is an early polyA signal sequence of simian virus 40 (SV40). In some embodiments, each of the first, second, and third polyA signal sequences on the first expression vector is an early polyA signal sequence of simian virus 40 (SV40), and each of the first, second, and third polyA signal sequences on the second expression vector is an early polyA signal sequence of simian virus 40 (SV40).

[0113] In some embodiments, each of the first polyA signal sequence, the second polyA signal sequence, and the third polyA signal sequence on the first expression vector and the second expression vector is an early polyA signal sequence of simian virus 40 (SV40), the promoter on the first expression vector operably linked to the nucleotide sequence encoding the selectable marker is SRα, the promoter on the second expression vector operably linked to the nucleotide sequence encoding the selectable marker is SRα, and the selectable marker on the first expression vector and the selectable marker on the second expression vector are glutamine synthase.

[0114] In some embodiments, each copy of the GAPDH promoter on the first expression vector and each copy of the GAPDH promoter on the second expression vector are CMV / GAPDH promoters, each of the first polyA signal sequence, the second polyA signal sequence and the third polyA signal sequence on the first expression vector and the second expression vector is an early polyA signal sequence of simian virus 40 (SV40), the promoter on the first expression vector operably linked to the nucleotide sequence encoding the selectable marker is SRα, the promoter on the second expression vector operably linked to the nucleotide sequence encoding the selectable marker is SRα, and the selectable marker on the first expression vector and the selectable marker on the second expression vector are glutamine synthase.

[0115] In alternative embodiments, on the first expression vector, the nucleotide sequence encoding the antibody heavy chain may precede the nucleotide sequence encoding the antibody light chain (in a 5' to 3' order). Additionally, in alternative embodiments, on the second expression vector, the nucleotide sequence encoding the antibody heavy chain-scFv fusion may precede the nucleotide sequence encoding the antibody light chain (in a 5' to 3' order). Furthermore, in alternative embodiments, on the first expression vector, the nucleotide sequence encoding the antibody heavy chain may precede the nucleotide sequence encoding the antibody light chain, and on the second expression vector, the nucleotide sequence encoding the antibody heavy chain-scFv fusion may precede the nucleotide sequence encoding the antibody light chain (both in a 5' to 3' order).

[0116] In another embodiment, the pair has a first expression vector comprising an expression cassette containing a polynucleotide sequence comprising, in a 5' to 3' order, the following elements: 1) a first copy of a GAPDH promoter operably linked to a nucleotide sequence encoding a first antibody light chain, followed by a first polyA sequence; 2) a second copy of the GAPDH promoter operably linked to a nucleotide sequence encoding a first antibody heavy chain, followed by a second polyA sequence; and 3) a promoter operably linked to a nucleotide sequence encoding a selectable marker, followed by a third polyA sequence; and a second expression vector comprising an expression cassette containing a polynucleotide sequence comprising, in a 5' to 3' order, the following elements: 1) a first copy of a GAPDH promoter operably linked to a nucleotide sequence encoding a second antibody light chain, followed by a first polyA sequence; 2) a second copy of the GAPDH promoter operably linked to a nucleotide sequence encoding a second antibody heavy chain, followed by a second polyA sequence; and 3 ... A promoter, operatively linked to a nucleotide sequence encoding a selectable marker, followed by a third polyA sequence. In some aspects of this embodiment, there are two distinct light chains and two distinct heavy chains that generate a four-chain antibody (i.e., the first antibody light chain and the second antibody light chain are different, and the first antibody heavy chain and the second antibody heavy chain are different). In one aspect of this embodiment, the vector pairs encode heterologous IgG.

[0117] In some embodiments, each copy of the GAPDH promoter on the first expression vector is a CMV / GAPDH promoter. In some embodiments, each copy of the GAPDH promoter on the first expression vector 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 copy of the GAPDH promoter on the first expression vector contains the nucleotide sequence of SEQ ID NO: 1.

[0118] In some embodiments, each copy of the GAPDH promoter on the second expression vector is a CMV / GAPDH promoter. In some embodiments, each copy of the GAPDH promoter on the second expression vector 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 copy of the GAPDH promoter on the second expression vector contains the nucleotide sequence of SEQ ID NO: 1.

[0119] In some embodiments, each copy of the GAPDH promoter on the first expression vector and each copy of the GAPDH promoter on the second expression vector are both CMV / GAPDH promoters. In some embodiments, each copy of the GAPDH promoter on the first expression vector and each copy of the GAPDH promoter on the second expression vector contain 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical nucleotide sequences to the nucleotide sequence of SEQ ID NO: 1. In some embodiments, each copy of the GAPDH promoter on the first expression vector and each copy of the GAPDH promoter on the second expression vector contain the nucleotide sequence of SEQ ID NO: 1.

[0120] In some embodiments, the promoter operably linked to the nucleotide sequence encoding the selectable marker on the first expression vector is mPGK. In some embodiments, the promoter operably linked to the nucleotide sequence encoding the selectable marker on the second expression vector is mPGK. In some embodiments, the promoter operably linked to the nucleotide sequence encoding the selectable marker on the first expression vector is mPGK, and the promoter operably linked to the nucleotide sequence encoding the selectable marker on the second expression vector is mPGK.

[0121] In some embodiments, the selectable biomarker on the first expression vector is glutamine synthase. In some embodiments, the selectable biomarker on the second expression vector is glutamine synthase. In some embodiments, the selectable biomarker on the first expression vector and the selectable biomarker on the second expression vector are both glutamine synthase.

[0122] In some embodiments, the promoter operably linked to the nucleotide sequence encoding the selectable marker on the first expression vector is mPGK, the promoter operably linked to the nucleotide sequence encoding the selectable marker on the second expression vector is mPGK, and the selectable marker on the first expression vector and the selectable marker on the second expression vector are glutamine synthase.

[0123] In some embodiments, each of the first, second, and third polyA signal sequences on the first expression vector is an early polyA signal sequence of simian virus 40 (SV40). In some embodiments, each of the first, second, and third polyA signal sequences on the second expression vector is an early polyA signal sequence of simian virus 40 (SV40). In some embodiments, each of the first, second, and third polyA signal sequences on the first expression vector is an early polyA signal sequence of simian virus 40 (SV40), and each of the first, second, and third polyA signal sequences on the second expression vector is an early polyA signal sequence of simian virus 40 (SV40).

[0124] In some embodiments, each of the first polyA signal sequence, the second polyA signal sequence, and the third polyA signal sequence on the first expression vector and the second expression vector is an early polyA signal sequence of simian virus 40 (SV40), the promoter on the first expression vector operably linked to the nucleotide sequence encoding the selectable marker is mPGK, the promoter on the second expression vector operably linked to the nucleotide sequence encoding the selectable marker is mPGK, and the selectable marker on the first expression vector and the selectable marker on the second expression vector are glutamine synthase.

[0125] In some embodiments, each copy of the GAPDH promoter on the first expression vector and each copy of the GAPDH promoter on the second expression vector are CMV / GAPDH promoters, each of the first polyA signal sequence, the second polyA signal sequence and the third polyA signal sequence on the first expression vector and the second expression vector is an early polyA signal sequence of simian virus 40 (SV40), the promoter on the first expression vector operably linked to the nucleotide sequence encoding the selectable marker is mPGK, the promoter on the second expression vector operably linked to the nucleotide sequence encoding the selectable marker is mPGK, and the selectable marker on the first expression vector and the selectable marker on the second expression vector are glutamine synthase.

[0126] In some embodiments, the first antibody light chain and the second antibody light chain are different, the first antibody heavy chain and the second antibody heavy chain are different, each copy of the GAPDH promoter on the first expression vector and each copy of the GAPDH promoter on the second expression vector are CMV / GAPDH promoters, each of the first polyA signal sequence, the second polyA signal sequence and the third polyA signal sequence on the first expression vector and the second expression vector is an early polyA signal sequence of simian virus 40 (SV40), the promoter on the first expression vector operably linked to the nucleotide sequence encoding the selectable marker is mPGK, the promoter on the second expression vector operably linked to the nucleotide sequence encoding the selectable marker is mPGK, and the selectable marker on the first expression vector and the selectable marker on the second expression vector are glutamine synthase.

[0127] In alternative embodiments, on the first expression vector, the nucleotide sequence encoding the first antibody heavy chain may precede the nucleotide sequence encoding the first antibody light chain (in a 5' to 3' order). Additionally, in alternative embodiments, on the second expression vector, the nucleotide sequence encoding the second antibody heavy chain may precede the nucleotide sequence encoding the second antibody light chain (in a 5' to 3' order). Furthermore, in alternative embodiments, on the first expression vector, the nucleotide sequence encoding the first antibody heavy chain may precede the nucleotide sequence encoding the first antibody light chain, and on the second expression vector, the nucleotide sequence encoding the second antibody heavy chain may precede the nucleotide sequence encoding the second antibody light chain (both in a 5' to 3' order).

[0128] This disclosure also provides a mammalian host cell comprising any of the above-described expression vectors or pairs of the above-described expression vectors. In one embodiment, the mammalian host cell is a Chinese hamster ovary (CHO) cell. In one aspect of this embodiment, the CHO cell is a dihydrofolate reductase-deficient (dhfr-) CHO cell or a glutamine synthetase knockout (GSKO) CHO cell.

[0129] This disclosure also provides methods for generating antibody forms, which include culturing any mammalian host cells described above or herein under conditions expressing an antibody chain, and recovering the antibody form from the culture. In one embodiment, the recovered antibody form is purified and formulated into a pharmaceutically acceptable formulation. In some embodiments, a "pharmaceutically acceptable formulation" means a formulation that is generally safe, non-toxic, and not biologically or otherwise undesirable for use in a subject.

[0130] Further aspects and advantages will become apparent to those skilled in the art upon reading the following detailed description. The description below includes specific situations, embodiments, and examples, and it should be understood that this disclosure is illustrative and not intended to limit the embodiments described herein to the specific situations, embodiments, and examples. Attached Figure Description

[0131] Figure 1 This is an example of a plasmid map used for recombinant expression of antibody molecules having one light chain and one heavy chain.

[0132] Figure 2A-2B Pool titers (in g / L) (A) and cell productivity (qp) (in pg / cell / day) for each light chain (LC), heavy chain (heavy chain fusion) (HC), and glutamine synthase (GS) promoter and MSX combination are shown (B). Titers were measured on day 10 of fed-batch production. Data were normalized and presented as the mean of two independent transfections. Error bars represent the standard deviation (SD) of two technical replicates of fed-batch production. Data were stained according to MSX concentrations (0 µM, 12.5 µM, and 25 µM).

[0133] Figures 3A-3C Viability (A), viable cell density (VCD) (B), and integrated viable cell density (VCD) (C) in CHO cell cultures with different carrier configurations are shown. Data are presented as the average of two independent transfections. Error bars represent the standard deviation (SD) of two technical replicates in fed-batch production. Data are stained according to MSX concentrations (0 µM, 12.5 µM, and 25 µM).

[0134] Figures 4A-4B Pool titers (in g / L) and cell productivity (qp) (in pg / cell / day) for each light chain (LC), heavy chain (HC), and glutamine synthase (GS) promoter and MSX combination are shown (A). Titers were measured on day 10 of fed-batch production. Data were normalized and expressed as the mean of two independent transfections. Error bars represent the standard deviation (SD) of three technical replicates of fed-batch production. Data were stained according to light and heavy chain promoters (CMV / ADL, CMV / GAPDH, and CMV / EF1+ CMV / GAPDH), GS promoters (SRα, mPGK, and SV40), and MSX concentrations (0 µM, 12.5 µM, 25 µM, and 50 µM).

[0135] Figures 5A-5CProduct mass in cell cultures from CHO cell pools with different vector configurations is shown: A: SEC-HMW (%); B: rCE-Clipping; C: HIC-HPLC. Data were measured on day 10 of fed-batch production. Data are presented as the mean of two independent transfections. Error bars represent the standard deviation (SD) of three technical replicates of fed-batch production. Data are stained according to light and heavy chain promoters (CMV / ADL, CMV / GAPDH, and CMV / EF1+CMV / GAPDH), GS promoters (SRα, mPGK, and SV40), and MSX concentrations (0 µM, 12.5 µM, 25 µM, and 50 µM).

[0136] Figures 6A-6C Viability (A), viable cell density (VCD) (B), and total viable cell density (VCD) (C) in CHO cell pools with different carrier configurations are shown. Data are presented as the mean of two independent transfections. Error bars represent the standard deviation (SD) of three technical replicates in fed-batch production. Data are stained according to MSX concentrations (0 µM, 12.5 µM, 25 µM, and 50 µM).

[0137] Figures 7A-7B Pool titers (in g / L) and cell productivity (in pg / cell / day) for each light chain (LC), heavy chain (HC), and glutamine synthase (GS) promoter and MSX combination of 2+2 ScFv molecule A are shown (A). Titers were measured on day 10 of fed-batch production. Data were normalized and expressed as the mean of two independent transfections. Error bars represent the standard deviation (SD) of three technical replicates of fed-batch production. Data were stained according to light and heavy chain promoters (CMV / ADL and CMV / GAPDH), GS promoters (SRα, mPGK, and SV40), and MSX concentrations (0 µM, 12.5 µM, 25 µM, and 50 µM).

[0138] Figures 8A-8B Pool titers (in g / L) and cell productivity (in pg / cell / day) for each light chain (LC), heavy chain (HC), and glutamine synthase (GS) promoter and MSX combination of 2+2 ScFv molecule B are shown (A). Titers were measured on day 10 of fed-batch production. Data were normalized and expressed as the mean of two independent transfections. Error bars represent the standard deviation (SD) of three technical replicates of fed-batch production. Data were stained according to light and heavy chain promoters (CMV / ADL and CMV / GAPDH), GS promoters (SRα, mPGK, and SV40), and MSX concentrations (0 µM, 12.5 µM, and 25 µM).

[0139] Figures 9A-9C Product masses in cell cultures from CHO cell pools with different vector configurations of 2+2 ScFv molecule A are shown: A: SEC-HMW (%); B: rCE-shear; C: HIC-HPLC. Data were measured on day 10 of fed-batch production. Data are presented as the mean of two independent transfections. Error bars represent the standard deviation (SD) of three technical replicates. Data were stained according to light and heavy chain promoters (CMV / ADL and CMV / GAPDH), GS promoters (SRα, mPGK, and SV40), and MSX concentrations (0 µM, 12.5 µM, 25 µM, and 50 µM).

[0140] Figures 10A-10C Product mass in cell cultures of CHO cell pools with different vector configurations of 2+2 ScFv molecule B is shown: A: SEC-HMW (%); B: rCE-shear; C: HIC-HPLC. Data were measured on day 10 of fed-batch production. Data are presented as the mean of two independent transfections. Error bars represent the standard deviation (SD) of three technical replicates. Data are stained according to light and heavy chain promoters (CMV / ADL and CMV / GAPDH), GS promoters (SRα, mPGK, and SV40), and MSX concentrations (0 µM, 12.5 µM, and 25 µM).

[0141] Figure 11A-11C The activity (A), viable cell density (VCD) (B), and total viable cell density (VCD) (C) of 2+2 ScFv molecules A in cell cultures from CHO cell pools with different carrier configurations are shown. Data are presented as the mean of two independent transfections. Error bars represent the standard deviation (SD) of three technical replicates in fed-batch production. Data are stained according to MSX concentrations (0 µM, 12.5 µM, 25 µM, and 50 µM).

[0142] Figures 12A-12C The viability (A), viable cell density (VCD) (B), and total viable cell density (VCD) (C) of 2+2 ScFv molecules B in cell cultures from CHO cell pools with different carrier configurations are shown. Data are presented as the mean of two independent transfections. Error bars represent the standard deviation (SD) of three technical replicates in fed-batch production. Data are stained according to MSX concentrations (0 µM, 12.5 µM, and 25 µM).

[0143] Figures 13A-13BNormalized titers (A) and normalized protein A (proA) yields (B) (in mg / L) for each light chain (LC), heavy chain (HC), and glutamine synthase (GS) promoter combination in two different GS hosts (H1 and H2) with monocistronic or bicistronic conformations. Titers were measured on day 10 of fed-batch production. ProA yield was calculated by measuring the concentration of purified protein.

[0144] Figure 14 The product quality (% of the main peak in SEC) in cell cultures of CHO cell pools with different carrier configurations is shown.

[0145] Figure 15 Protein A recovery (in g / L) is shown for each light chain (LC), heavy chain (HC), and glutamine synthase (GS) promoter. Titers were measured on day 7 of batch production.

[0146] Figures 16A-16B The product mass in cell cultures of CHO cell pools with different carrier configurations is shown as (A) the percentage of the main peak in SEC and (B) the main peak in non-reducing capillary electrophoresis (nrMCE).

[0147] Figures 17A-17B Pool titers (in g / L) (A) and cell productivity (in pg / cell / day) (B) for each light chain (LC) / heavy chain (HC) promoter combination and glutamine synthase (GS) promoter combination of B2Hmab and C2Hmab are shown. Titers were measured on day 10 of fed-batch production. Data were normalized and expressed as the mean of two independent transfections. Error bars represent the standard deviation (SD) of three technical replicates of fed-batch production. Data were stained according to light and heavy chain promoters (CMV / ADL and CMV / GAPDH), GS promoters (SRα and mPGK), and MSX concentrations (18.75 µM and 37.5 µM).

[0148] Figures 18A-18C Product masses in cell cultures from CHO cell pools with different vector configurations of B2Hmab and C2Hmab are shown as (A) % of the main peak in SEC, (B) % of the main peak in non-reducing capillary electrophoresis (nrMCE), and (C) B:rCE-shear. Data were normalized and expressed as the mean of two independent transfections. Error bars represent the standard deviation (SD) of three technical replicates in fed-batch production. Data were stained according to light and heavy chain promoters (CMV / ADL and CMV / GAPDH), GS promoters (SRα and mPGK), and MSX concentrations (18.75 µM and 37.5 µM).

[0149] Figures 19A-19CViability (A), viable cell density (VCD) (B), and total viable cell density (VCD) (C) of B2Hmab and C2Hmab in cell cultures from CHO cell pools with different vector configurations are shown. Data were normalized and expressed as the average of two independent transfections. Data were stained according to MSX concentrations (18.75 µM and 37.5 µM).

[0150] Figure 20A This is a representative scheme for mAb expression boxes.

[0151] Figure 20B This is a representative scheme for the expression cassette of B2 / C2 mAb.

[0152] Figure 20C It is a representative scheme of expression box pair for C1mAb asymmetric fusion.

[0153] Figure 20D This is a representative scheme for the expression cassette pair of heterologous IgG mAb. Detailed Implementation

[0154] This disclosure is based in part on the finding that certain combinations of promoters in expression cassettes used to drive the expression of different antibody chains can increase the titer of the produced antibody. These antibodies include standard antibodies (2 chains), multispecific antibodies with 2 chains, and antibody forms with 3 or 4 chains, such as those where one or two chains are modified with scFv, cytokines, VH, etc. Expression cassettes with specific arrangements of promoters and antibody chains, as well as selectable markers, can be integrated into expression vectors to produce antibodies in mammalian host cells. The same applies to antibody forms with three or four chains expressed on two different vectors. Two different expression cassettes can be used on two different expression vectors to produce antibody forms with three or four chains. The three- or four-chain antibody forms can have a heavy chain, a heavy chain-scFv fusion, or a heavy chain-VH fusion, and two light chains (the light chains are the same in the case of the 3-chain antibody form or different in the case of the 4-chain form). Another example of a four-chain antibody is a form containing two different heavy chains and two different light chains, where each heavy-light chain pair is expressed by a different vector.

[0155] Standard antibody production techniques typically employ different promoters to express the heavy and light chains to optimize antibody expression. This is often necessary because the heavy and light chains are expressed at different levels. The situation becomes more complex when the antibody structure contains three or four chains. The inventors have unexpectedly discovered that by employing specific expression cassettes for different antibody forms, the titer of the resulting antibody structure can increase with improvements in product quality (e.g., reductions in aggregation, cleavage, or the presence of undesirable isoforms). Furthermore, by using the expression cassettes and expression vectors described herein in mammalian host production cell lines, biopharmaceuticals can be produced in a cheaper and more consistent manner. Expression cassettes, expression vectors, and mammalian host cells may have specific utilities in the commercial production of standard antibodies and antibody forms with two, three, or four unique chains.

[0156] The expression cassettes and expression vectors described herein are used in cell lines (also referred to as “host cells”) (preferably mammalian cells (“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 one or two expression vectors (each containing a nucleotide sequence encoding two antibody chains) to induce the host cells to express an antibody form with the 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, New York (1990).

[0157] definition

[0158] 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.

[0159] As used herein, unless otherwise explicitly 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 those described herein for cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization are those well-known and commonly used in the art.

[0160] 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.

[0161] This disclosure provides tools for expressing a “target protein” (typically in antibody form). 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.

[0162] 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.

[0163] 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, 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, antibody fusion proteins are molecules containing two antibody light chains, 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.

[0164] 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.

[0165] 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.

[0166] 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.

[0167] 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.

[0168] As used herein, the term "bioreactor" means any container that can be used for the growth of cell cultures. The cell cultures disclosed herein can be grown in a bioreactor, and the bioreactor can be selected based on the application of the target protein produced by the cells grown in the bioreactor. Bioreactors can be of any size, as long as they are suitable for cell culture; typically, the size of the bioreactor is appropriate for the volume of the cell cultures 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.

[0169] As used herein, “cell culture” or “culture” means the growth and proliferation 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. Bioreactors ranging from 500L to 2000L and from 1000L to 2000L can be used.

[0170] 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).

[0171] 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, insulin-like growth factor, serum, etc.; salts, such as calcium salts, magnesium salts, and phosphates, and buffers, such as HEPES; nucleosides and bases, such as adenosine, thymidine, and 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; and 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.

[0172] 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.

[0173] "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.

[0174] "Feed-batch culture" refers to a form of suspension culture and means a method in which cultured cells are provided with additional components at one or more points after the start of the culture process. The provided components typically include nutrient supplements that have been depleted for the cells during the culture process. Alternatively, the additional components may include supplemental components (e.g., cell cycle inhibitory compounds). Fed-batch cultures typically stop at a certain point, and the cells and / or components in the culture medium are harvested and optionally purified.

[0175] “Growth” cell culture medium refers to a cell culture medium that is typically used for cell culture during the exponential growth phase (“growth phase”) and is sufficiently complete to support cell culture during this phase. Growth cell culture media may also contain selectants that confer 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.

[0176] "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.

[0177] “Production” cell culture medium refers to a cell culture medium that is typically used for cell culture during the transition period from the end of exponential growth to the beginning of protein production, the “transition” and / or “product” phase, and is sufficiently complete to maintain the desired cell density, viability and / or product titer during that phase.

[0178] Concentrated cell culture media may contain some or all of the nutrients necessary to maintain cell culture; in particular, concentrated media may contain nutrients identified or known to be consumed during the production phase of cell culture. Concentrated media can be based on virtually any cell culture medium formulation. Such concentrated feed media may contain some or all of the components of cell culture media, for example, in normal amounts of about 2X, 3X, 4X, 5X, 6X, 7X, 8X, 9X, 10X, 12X, 14X, 16X, 20X, 30X, 50X, 100X, 200X, 400X, 600X, 800X, or even about 1000X.

[0179] 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.

[0180] Cell cultures can also be supplemented with separate concentrated feeds containing specific nutrients that may be difficult to formulate in cell cultures or are rapidly depleted in them. Such nutrients can be amino acids such as tyrosine, cysteine, and / or cystine (see, for example, International Patent Application Publication No. WO 2012 / 145682). Separate feeding can be initiated before or during the production phase. Separate feeding can be accomplished by adding the 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.

[0181] "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. TMExamples 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, and trace elements. Custom-made cell culture media are also available.

[0182] "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.

[0183] 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 the offspring of the parent cell, regardless of whether the offspring are morphologically or genetically identical to the original parent cell, provided the target gene is present. Cell cultures may contain one or more host cells.

[0184] 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”.

[0185] Expression Box

[0186] Expression and cloning typically involve expression cassettes containing one or more promoters that are recognized by the host organism and operatively linked to a nucleotide sequence encoding a target protein. Promoters are non-transcribed sequences (typically within approximately 100 to 1000 bp) located upstream (i.e., 5') of the start codon of a structural gene that control the transcription of the structural gene.

[0187] Expression cassettes for antibody form typically contain a first promoter driving the expression of a first nucleotide sequence encoding a first antibody chain or antibody chain fusion, a second promoter driving the expression of a second nucleotide sequence encoding a second antibody chain or antibody chain fusion, and a promoter driving the expression of a coding sequence encoding an optional biomarker. A poly-A tail may follow each gene (i.e., the coding sequences encoding the first and second antibody or antibody fusion chains and the optional biomarker). In the embodiments disclosed herein, both the first and second promoters are glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoters. In some embodiments, the GAPDH promoter is operatively linked to a CMV promoter enhancer; the resulting construct is referred to as the CMV / GADPH promoter.

[0188] 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 can be expressed on a single vector or two different vectors, depending on the number of chains produced. 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 [Frontiers in Immunology] 12:626616.

[0189] In one embodiment, a single expression cassette is used, i.e., two chains and one vector. In some aspects, the expression cassette can be used to express monoclonal antibodies (having a heavy chain sequence and a light chain sequence) or symmetrical fusions (e.g., having a heavy chain sequence fused to scFv or VH and a light chain sequence), such as B2 mab (Fab-scFv-Fc) or C2 mab (IgG-scFv) mAb, B2Hmab (Fab-VH-Fc), C2Hmab (IgG-VH), or (IgG-cytokine) mAb (two chains, one vector). The first antibody chain can be a light chain or a heavy chain / heavy chain-scFv fusion / heavy chain-VH fusion. On one hand, 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-VH fusion.

[0190] A representative scheme of mAb is described in Figure 20A middle.

[0191] Representative schemes for B2 mAb or C2 mAb are described in Figure 20B middle.

[0192] In other embodiments, two expression cassettes are used. In some aspects, the two expression cassettes can be used to express tri- or quadruple-chain antibody forms. For example, for a tri-chain form, one expression cassette contains coding sequences for one heavy chain and one light chain, and the other expression cassette contains coding sequences for the heavy chain-scFv fusion and the light chain (3 chains, 2 vectors). The heavy chain sequences can be the same or different. The light chain sequences can be the same or different. An example of a tri-chain form is C1mAb(Fab-heterologous Fc-[scFv... [Asymmetric fusion]. The first antibody chain in each expression cassette can be a light chain or a heavy chain / heavy chain-scFv fusion. On one hand, the first antibody chain is a light chain, and the second antibody chain is a heavy chain or a heavy chain-scFv fusion. Another example is C1mAb (Fab-heterologous Fc-[cytokine], asymmetric fusion).

[0193] A representative scheme of C1mAb asymmetric fusion is described in Figure 20C middle.

[0194] For example, in a four-chain configuration, one expression cassette contains coding sequences for a first light chain and a first heavy chain, and a second expression cassette contains a second heavy chain and a second light chain. This represents a heterologous IgG mAb. The first antibody chain in each expression cassette can be either a light chain or a heavy chain.

[0195] On the other hand, 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-VH fusion.

[0196] A representative regimen for heterologous IgG mAb is described in Figure 20D middle.

[0197] 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).

[0198] It has been found that using CMV / GAPDH as a promoter to drive the expression of all antibody chains in certain forms unexpectedly results in higher yields of antibody forms, as described in the examples of this application. In the embodiments described herein, the promoter for the antibody-form chain is a combination of a CMV promoter enhancer and a GAPDH (CMV / GAPDH). In this combination, both the CMV promoter enhancer and the GAPDH promoter are operatively linked to the nucleotide sequence encoding the antibody chain, making this combination a better promoter than a GAPDH promoter alone. In some embodiments, the CMV promoter is at the 5' of the GAPDH. Generally, CMV / GAPDH is referred to as the promoter. Representative CMV / GAPDH promoters are provided in SEQ ID NO:1.

[0199] In some embodiments, each GAPDH promoter in the expression cassette is a CMV / GAPDH promoter. In some embodiments, each CMV / GAPDH promoter in the expression cassette 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.

[0200] In some embodiments, each GAPDH promoter in the expression cassette of the expression vector pair is a CMV / GAPDH promoter. In some embodiments, each CMV / GAPDH promoter in the expression cassette of the expression vector pair 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.

[0201] In the expression cassette described herein, the polyA signal sequence may follow each gene (i.e., the coding sequence for antibody or antibody fusion chain and optional markers). 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, Genes Dev.). [Genes and Development], 2(4):440-52; GenBank accession number J02400). In some embodiments, the first polyA signal sequence, the second polyA signal sequence, and the third polyA signal sequence on each expression cassette are independently selected from the group consisting of: bovine growth hormone (BGH) polyA signal sequence, thymidine kinase polyA (TKpA) signal sequence, rabbit β-globin polyA signal sequence, and simian virus 40 (SV40) early polyA signal sequence.

[0202] In some embodiments, each of the first polyA signal sequence, the second polyA signal sequence, and the third polyA signal sequence on each expression cassette is a bovine growth hormone (BGH) polyA signal sequence.

[0203] In some embodiments, each of the first polyA signal sequence, the second polyA signal sequence, and the third polyA signal sequence on each expression cassette is a thymidine kinase polyA (TKpA) signal sequence.

[0204] In some embodiments, each of the first polyA signal sequence, the second polyA signal sequence, and the third polyA signal sequence on each expression cassette is a rabbit β-globin polyA signal sequence.

[0205] In some embodiments, each of the first polyA signal sequence, the second polyA signal sequence, and the third polyA signal sequence on each expression cassette is an early polyA signal sequence of simian virus 40 (SV40).

[0206] In some embodiments, the BGH 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 BGH polyA signal sequence comprises the nucleotide sequence of SEQ ID NO: 5.

[0207] 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: 6. In some embodiments, the rabbit β-globin polyA signal sequence comprises the nucleotide sequence of SEQ ID NO: 6.

[0208] In some embodiments, the SV40 early poly-A 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 SV40 early poly-A signal sequence comprises the nucleotide sequence of SEQ ID NO: 7.

[0209] 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: 8. In some embodiments, the thymidine kinase polyA (TKpA) signal sequence comprises the nucleotide sequence of SEQ ID NO: 8.

[0210] The expression cassettes provided herein can provide improved expression, possibly due to an improved chain ratio of the expressed peptide. The chain ratio can be measured using techniques well known in the art.

[0211] Selectable markers

[0212] To stably transfect mammalian cells, it is known that only a small fraction of cells can integrate foreign DNA into their genome, depending on the expression vector and transfection technique used. To identify and select these integrators, a gene encoding a selectable marker is typically introduced into a host cell within an expression vector identical to one or more target genes.

[0213] Selectable biomarker genes encode proteins required for the survival and growth of host cells grown in selective media. Typical selectable biomarker genes encode proteins that: (a) confer resistance to antibiotics or other toxins (e.g., ampicillin, tetracycline, or kanamycin for prokaryotic host cells); (b) compensate for cellular nutritional deficiencies; or (c) provide essential nutrients via metabolism that are not available from complex or limited media. Specific antibiotic resistance selectable biomarkers include kanamycin resistance genes, ampicillin resistance genes, tetracycline resistance genes, and neomycin resistance genes.

[0214] Other alternative gene amplification can be used to amplify the gene to be expressed. Amplification is the process by which the gene required to produce proteins necessary for growth or cell survival is replicated tandemly within the chromosome of consecutive generations of recombinant cells. Examples of suitable alternative markers for mammalian cells include, but are not limited to, glutamine synthase (GS), dihydrofolate reductase (DHFR), asparaginase (Aspg; see Ha et al. Biotechnol Bioeng. [Biotechnology and Bioengineering] 2023120:1159-1166), and promoterless thymidine kinase genes.

[0215] Selective pressure is applied to mammalian cell transformants, where only the transformant is viable due to the presence of a selectable gene in the vector. This pressure is exerted by culturing the transformed cells under conditions of progressively increasing selectant concentrations in the culture medium, resulting in additional stringency and / or amplification of the selectable gene and the DNA encoding the target protein. Consequently, an increased amount of the target polypeptide is synthesized from the amplified DNA. The selectant for GS is methionine sulfoxide imide (MSX). The selectant for DHFR is methotrexate (MTX).

[0216] Compared to DHFR-based systems, the GS knockout cell line (GSKO) provides sufficient selection strictness in the absence of MSX or with low MSX concentrations, while coupling 25 μM MSX with the GS knockout cell line results in higher selection efficiency compared to the CHOK1SV cell line at higher MSX concentrations (Fan et al., Biotechnol Bioeng. [Biotechnology and Bioengineering], 109(4): 1007-1015 (2012)). Previous reports have shown that increasing the MSX concentration during the seedtrain stage after clonal selection increases productivity without significantly affecting cell growth, GS and target gene copy number and expression, and maintains product quality properties in multiple GS knockout cell lines (Tian et al., Engineering in LifeSciences [Life Science Engineering] 20(3-4): 112-125 (2020)). Chain / vector expression can be affected by increasing strictness during pool recovery / selection through the addition of MSX.

[0217] In some embodiments, the MSX concentration may be optimized for one of the promoters driving GS expression. In specific embodiments, the MSX concentration may be optimized for GS linked to a more difficult-to-express chain.

[0218] In some embodiments, the optional marker in the expression cassette is glutamine synthase. Glutamine synthase (GS) catalyzes the biosynthesis of glutamine through the condensation of ammonia and glutamate. In some embodiments, the optional marker in each expression cassette is glutamine synthase.

[0219] In some embodiments, the promoter SRα is operatively connected to a selectable marker. In other embodiments, the promoter mPGK is operatively connected to a selectable marker. Other suitable promoters may be selected from those well known in the art.

[0220] This disclosure provides expression cassettes comprising a polynucleotide sequence comprising, in a 5' to 3' order, the following elements: a first GAPDH promoter operatively linked to a nucleotide sequence encoding a first antibody chain or antibody chain fusion, followed by a first polyA signal sequence; a second GAPDH promoter operatively linked to a nucleotide sequence encoding a second antibody chain or antibody chain fusion, followed by a second polyA signal sequence; and a promoter operatively linked to a nucleotide sequence encoding a selectable marker, followed by a third polyA signal sequence. Such expression cassettes may be part of an expression vector or a pair of expression vectors, wherein each expression vector comprises an expression cassette having the aforementioned configuration.

[0221] In some embodiments, the marker may be glutamine synthase.

[0222] In some embodiments, the promoter operatively linked to the nucleotide sequence encoding the selectable marker is an SRα promoter, an mPGK promoter, or an SV40 promoter. In some embodiments, the selectable marker is glutamine synthase, and the promoter operatively linked to the nucleotide sequence encoding the selectable marker is an SRα promoter, an mPGK promoter, or an SV40 promoter.

[0223] In some embodiments, the promoter operatively linked to the nucleotide sequence encoding the selectable marker is an SRα promoter. In some embodiments, the selectable marker is glutamine synthase, and the promoter operatively linked to the nucleotide sequence encoding the selectable marker is an SRα promoter.

[0224] In some embodiments, the promoter operatively linked to the nucleotide sequence encoding the selectable marker is an mPGK promoter. In some embodiments, the selectable marker is glutamine synthase, and the promoter operatively linked to the nucleotide sequence encoding the selectable marker is an mPGK promoter.

[0225] In some embodiments, the promoter operatively linked to the nucleotide sequence encoding the selectable marker is the SV40 promoter. In some embodiments, the selectable marker is glutamine synthase, and the promoter operatively linked to the nucleotide sequence encoding the selectable marker is the SV40 promoter.

[0226] In some embodiments, the SRα promoter comprises 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical nucleotide sequences to the SEQ ID NO: 2. In some embodiments, the SRα promoter comprises the nucleotide sequence of SEQ ID NO: 2.

[0227] 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.

[0228] 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.

[0229] Additional components of the expression vector

[0230] The expression cassettes described herein are used in expression vectors. Such 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 promoter / enhancer fragments, antibody chains, one or more optional markers, and other sequences (e.g., polyadenylated sequences)): one or more enhancer sequences, origin of replication, transcription and translation control sequences, transcription termination sequences, complete intron sequences containing donor and acceptor splicing sites, various pre-sequences / pro-sequences that improve glycosylation or yield, natural or heterologous signal sequences (lead sequences or signal peptides) for polypeptide secretion, ribosome binding sites, polyadenylated sequences, internal ribosome entry sites (IRES) sequences, expression enhancement sequence elements (EASE), triplet leader sequences (TPL) and VA gene RNA from adenovirus 2, and multi-connector regions of multinucleotides encoding the polypeptide to be expressed. Vectors can be constructed from starter vectors (such as commercially available vectors), and additional elements can be obtained separately and ligated into the vector. The methods used to obtain the components are well known to those skilled in the art.

[0231] Vector components can be homologous (i.e., from the same species and / or strain as the host cell), heterologous (e.g., from a species or strain different from the host cell), heterozygous (i.e., a combination of side 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.

[0232] 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.

[0233] 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.

[0234] 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.

[0235] In some cases, such as when glycosylation is desired in eukaryotic host cell expression systems, various pre-sequences can be manipulated to improve glycosylation or yield. For example, the peptidase cleavage site of a specific signal peptide can be altered, or pre-sequences can be added, which can also affect glycosylation. The final protein product may have one or more easily expressed additional amino acids at the -1 position (relative to the first amino acid of the mature protein), which may not be completely removed. For example, the final protein product may have one or two amino acid residues attached to the amino terminus found in the peptidase cleavage site. Alternatively, when the enzyme cleaves in such regions within the mature polypeptide, using some of the enzyme cleavage sites may produce a slightly truncated form of the desired polypeptide.

[0236] 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 signal peptides that are functional in mammalian host cells include, but are not limited to, the following: 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. 0367 566; 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. 0460 846.

[0237] Additional control sequences that have been shown to improve the expression of heterologous genes from mammalian expression vectors include, but are not limited to, elements such as expression-enhancing 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).

[0238] Vectors that are functional in the specific host cell used can be selected (i.e., the vector is compatible with the host cell structure, thereby allowing gene amplification and / or expression to occur). In some embodiments, the vector used employs protein fragment complementation assays using a protein reporter sequence such as dihydrofolate reductase (see, for example, U.S. Patent No. 6,270,964). Suitable expression vectors are known in the art and are commercially available.

[0239] Table A provides non-limiting examples of synthetic nucleotide (DNA) sequences that can be used in certain expression cassette / vector components of the expression cassettes and vectors disclosed herein.

[0240] Table A. Non-limiting examples of expression cassette / vector component sequences

[0241]

[0242] Target protein

[0243] This disclosure provides expression cassettes and systems for expressing certain target proteins in host cells (e.g., mammalian host cells, such as CHO cells). The target peptides and proteins may have scientific or commercial significance, including protein-based therapeutics. Target proteins particularly include secreted proteins, non-secreted proteins, intracellular proteins, or membrane-bound proteins. Target peptides and proteins can be produced using cell culture methods via recombinant animal cell lines and may be referred to as “recombinant proteins.” One or more expressed proteins may 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).

[0244] Target proteins include "antigen-binding proteins," particularly "antibody forms." An "antigen-binding protein" is a protein or polypeptide containing an antigen-binding region or moiety 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, bispecific T cell binders with extended durations (such as extended half-life) (e.g., HLE BiTE molecules, heterologous Ig BITE molecules, etc.).

[0245] 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 comprise, for example, alternative protein scaffolds or artificial scaffolds with grafted CDRs or CDR derivatives. Such scaffolds include, but are not limited to, antibody-derived scaffolds containing mutations introduced to, for example, stabilize the three-dimensional structure of the antigen-binding protein; and fully synthetic scaffolds containing, for example, biocompatible polymers. See, for example, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 53(1):121-129; Roque et al., 2004, Biotechnol. Prog. 20:639-654. Furthermore, peptide antibody mimics (“PAMs”) and scaffolds based on antibody mimics utilizing fibronectin components as scaffolds may be used.

[0246] 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 primarily responsible for antigen recognition, namely the VL and VH domains. 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.

[0247] 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).

[0248] 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".

[0249] 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 types.

[0250] 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.

[0251] 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.

[0252] The VH domain is a variable domain of the heavy chain. The variable region endows the antibody with the ability to bind to the antigen. The VH domain includes the variable region of UniAbs™, referred to as UniDabs™. See, for example, Clarke et al., 2018, Front Immunol. [Immunology Frontiers] 9:3037.

[0253] 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 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).

[0254] 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.

[0255] 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).

[0256] 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.

[0257] 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.

[0258] 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.

[0259] 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.

[0260] 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 receptor; 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.

[0261] 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 factor VIII, tissue factor, van Wilbond) Willebrand factor, protein C, α-1-antitrypsin, plasminogen activators (such as urokinase and tissue plasminogen activator (“t-PA”)), bombazine, thrombin, thrombopoietin and thrombopoietin receptor, colony-stimulating factor (CSF) (especially including M-CSF, GM-CSF and G-CSF), other blood and serum proteins (including but not limited to albumin, IgE and blood group antigens), receptors and receptor-related 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.

[0262] In another embodiment, the target protein includes abciximab, adalimumab, adelimumab, aflibercept, alenmab, alicurumab, anakinase, asceticumab, bailiximab, belimumab, bevacizumab, biotinylate, bonatumab, bentuximab, brodatumab, mocantozumab, konatumab, cetuximab, tertuximab, konatumab, and dalizumab. Antibiotics, denosumab, eculimab, eculoxetine, efalizumab, epazolizumab, etanercept, evokulumab, galiliximab, genitalumab, gemutuzumab, golimumab, teimomab, infliximab, ipilimumab, levofloxacin, levofloxacin, levofloxacin (lxdkizumab), mapamumumab, motesanib phosphate diphosphate), morotumab-CD3, natecillatab, nimotuzumab, nivolumab, olizumab, olrezumab, olfamumab, olmalizumab, interleukin, palizumab, panitumab, pembrolizumab, pertuzumab, pectizumab, ranituzumab, rituximab, rituximab, romistastatin, lomoxoluzumab, saxaglastin, tocilizumab, tosimomab, trastuzumab, uterotumab, vedozazumab, vexizumab, voloximab, zalumab, zalumab, and any biosimilars of the foregoing substances.

[0263] The target protein according to the invention encompasses all the foregoing and further includes antibodies containing 1, 2, 3, 4, 5, or 6 complementarity-determining regions (CDRs) of any of the aforementioned antibodies. One or more CDRs can be covalently or non-covalently incorporated into the molecule to make it an antigen-binding protein. The antigen-binding protein can be incorporated into one or more CDRs as part of a larger polypeptide chain, can be covalently linked to one or more CDRs to another polypeptide chain, or can 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 problem of searching and aligning sequences. Other algorithms can also be used, especially when speed is a significant consideration. Commonly used programs for DNA, RNA, and peptide alignment and homology matching include FASTA, TFASTA, BLASTN, BLASTP, BLASTX, TBLASTN, PROSRCH, BLAZE, and MPSRCH, the latter being an implementation of the Smith-Waltman algorithm for execution on massively parallel processors manufactured by MasPar.

[0264] Antigen-binding molecules can be their antibody fragments, including one or more single-chain antibody fragments (“scFv”). scFvs are preferred for use in chimeric antigen receptors because they can be engineered to be expressed as part of a single chain. See Krause et al., 1988, J. Exp. Med., 188(4): 619-626; Finney et al., 1998, J Immunol 161: 2791-2797.

[0265] The “Fc” region, as used in this text, contains the C-cell containing the antibody. H 2 and C H The three-domain structure consists of two heavy-chain segments. These two heavy-chain segments are composed of two or more disulfide bonds and C... H The hydrophobic interactions of the three domains maintain their cohesion. Target proteins containing the Fc region (including antigen-binding proteins and Fc fusion proteins) form another aspect of this disclosure.

[0266] Generation of mammalian host cells expressing the target protein

[0267] 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).

[0268] Stable integration methods are well known in the art. In short, stable integration is typically achieved by transiently introducing a heteropolynucleotide or a vector containing a heteropolynucleotide into a host cell, which facilitates the stable integration of the heteropolynucleotide into the cellular genome. Typically, the heteropolynucleotide is flanked by homologous arms, i.e., sequences homologous to upstream and downstream regions of the integration site. Circular vectors can be linearized to facilitate integration into the cellular genome before being introduced into mammalian host cells. Methods for introducing vectors into cells are well known in the art, 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).

[0269] Stable integration-specific methods utilize recombinase-mediated cassette exchange (RMCE; Bode and Baer, ​​2001, CurrOpin Biotechnol. [Current Biotechnical Perspective] 12:473-80, and Bode et al., 2000, Biol. Chem. [Biochemistry] 381:801-813) for site-specific integration into the genome (also known as “targeted integration”). Site-specific recombinases such as Flp and Cre mediate recombination between two copies of their target sequence, referred to as FRT and loxP, respectively. Using two incompatible target sequences, such as FRT combined with F3 (Schlake and Bode, 1994, Biochemistry [Biochemistry], 33:12746-51) and an inverted recognition target site (Feng et al., 1999, J. Mol. Biol. [Journal of Molecular Biology] 292:779-85), allows the insertion of DNA fragments into predetermined chromosomal sites carrying target sequences of similar conformation. See also European Patent No. EP 1781796 B1 and European Patent Application Publication No. EP 2789691 A1.

[0270] 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).

[0271] 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. This disclosure provides specific examples of expression cassettes that can be incorporated into expression vectors that can be used to express a variety of target proteins, including certain antibody forms discussed above. Expression vectors according to this disclosure can be introduced into host cells to allow replication of the vector itself and thereby amplify copies of the polynucleotides contained therein. As mentioned above, cloning vectors may contain sequence components that generally include, but are not limited to, origin of replication, promoter sequences, transcription initiation sequences, enhancer sequences, and optional markers. These elements can be appropriately selected by those skilled in the art.

[0272] 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, New York (2001).

[0273] 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."

[0274] 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.

[0275] 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.

[0276] cell lines

[0277] In the methods disclosed herein, any mammalian cell line can be used, with the CHO cell line being a preferred example of a cell line that can be used in combination with the expression vectors disclosed herein. A variety of mammalian cell lines suitable for growth in cultures are available from the American Type Culture Collection (Manassas, Virginia) and commercial suppliers. Non-limiting examples of industrially used cell lines include the monkey kidney CVl line transformed from SV40 (COS-7, ATCC CRL 1651); the human embryonic kidney line (293 cells or subclones used for growth in suspension culture (Graham et al., 1977, J. Gen Virol. [Journal of General Virology] 36:59)); juvenile hamster kidney cells (BHK, ATCC CCL 10); mouse Setori 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 N.Y Acad. Sci. [Annals of the New York Academy of Sciences] 383:44-68); MRC 5 cells or FS4 cells; mammalian myeloma cells, as well as many other cell lines and Chinese hamster ovary (CHO) cells.

[0278] 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, but are not limited to, 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 of the cell lines disclosed herein.

[0279] 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 CHOK1SV cell line, a glutamine synthetase (GS) knockout GS 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).

[0280] Cell culture process

[0281] Host cells transfected using the vector systems described herein can be cultured in adherent or suspension cultures grown in stirred tank reactors (including conventional batch and fed-batch cell cultures, which may but need not include a rotating filter), perfusion systems (including alternating tangential flow (“ATF”) cultures, acoustic perfusion systems, depth filter perfusion systems, and other systems), and hollow fiber bioreactors (HFBs, which in some cases can be used for perfusion processes), as well as various other cell culture methods (see, for example, Tao et al., 2003, Biotechnol. Bioeng. [Biotechnology and Bioengineering] 82:751-65; Kuystermans and Al-Rubeai, (2011) “Bioreactor Systems for Producing Antibody from Mammalian Cells”). Antibody Expression and Production [ Antibody expression and productionIn Cell Engineering, 7:25-52, Al-Rubeai (ed.) Springer; Catapano et al., (2009) "Bioreactor Design and Scale-Up" Cell and Tissue Reaction Engineering: Principles and Practice [ Cellular and Tissue Reaction Engineering: Principles With practice [In Eibl et al. (edited) Springer-Verlag, which is incorporated herein by reference in its entirety].

[0282] 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.

[0283] 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. Suitable bioreactor volumes include, but are not limited to, 500 L, 1000 L, 2000 L, 5000 L, 10000 L, and up to 20000 L. The seed cell density used to inoculate the bioreactor can have a positive impact on the level of recombinant protein produced. In one embodiment, the bioreactor is used in serum-free medium with at least 0.5 × 10⁶ cells / mL. 6 1.0 × 10 6 2.0 × 10 6 3.0 × 10 6 5.0 × 10 6 Or 10 × 10 6 Inoculation with 1 live cell / mL.

[0284] The mammalian cells then undergo an exponential growth phase. The cell culture can be maintained without supplemental feeding until the desired cell density is achieved. In one embodiment, the cell culture is maintained for up to three days with or without supplemental feeding. In another embodiment, the culture can be inoculated at the desired cell density to initiate the production phase without a short growth phase. In any embodiment herein, the transition from the growth phase to the production phase can also be initiated by any of the foregoing methods.

[0285] 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, as the accumulation of waste and depletion of nutrients rapidly lead to culture decline (typically around 3–7 days).

[0286] 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 are able to achieve higher cell densities (>10 to 30 × 10⁻⁶) compared to batch methods. 6 The potential for increased product titers (cells / ml, depending on culture medium formulation, cell line, etc.) and feed-batch culture, unlike batch culture, can be generated and maintained by manipulating feed strategies and culture medium formulations to differentiate between the cell proliferation phase (growth phase) and the suspension or slow cell growth phase (production phase) to achieve the desired cell density. Therefore, fed-batch culture has the potential to achieve higher product titers compared to batch culture. Typically, batch methods are used in the growth phase and fed-batch methods in the production phase, but a fed-batch feed strategy can be used throughout the process. However, unlike batch culture, bioreactor volume is a limiting factor for feed volume. Furthermore, as with batch methods, the accumulation of metabolic byproducts will lead to culture degradation, limiting the duration of the production phase to approximately 10 to 21 days. Feeded-batch culture is discontinuous, 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 significantly larger quantities of recombinant protein. See, for example, U.S. Patent No. 5,672,502.

[0287] 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 performed in many ways, including centrifugation, sedimentation, or filtration, see, for example, Voisard et al., 2003, Biotechnology and Bioengineering 82:751-65. An example of a filtration method is alternating tangential flow filtration. Alternating tangential flow is maintained by pumping the medium through a hollow fiber filter module. See, for example, U.S. Patent No. 6,544,424; Furey, 2002, Gen. Eng. News 22(7):62-63.

[0288] "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.

[0289] The perfusion method offers a potential improvement over batch and fed-batch methods by adding fresh culture medium while simultaneously removing used medium. Typical large-scale commercial cell culture strategies aim to achieve 60–90 (+) × 10⁶ cells / years. 6 High cell densities of 100 cells / mL have been achieved, with biomass comprising almost one-third to more than half of the reactor volume. Using perfusion culture, >1 × 10⁻⁶ cells / mL has been achieved. 8 Extreme cell densities of [number] cells / mL have been achieved, and even higher densities are predicted. Typical perfusion cultures begin with a batch culture start-up lasting one or two days, followed by continuous, stepwise, and / or intermittent addition of fresh feed medium to the culture. Used medium is removed throughout the growth and production phases of the culture, retaining cells and additional high-molecular-weight compounds such as proteins (based on the filtration molecular weight cutoff). Various methods, such as sedimentation, centrifugation, or filtration, can be used to remove used medium while maintaining cell density. Perfusion flow rates ranging from a portion of one working volume per day to multiple working volumes per day have been reported.

[0290] The advantage of perfusion is that production cultures can be maintained for longer periods than batch or fed-batch methods. However, it requires increased preparation, use, storage, and disposal of the culture medium to support long-term perfusion culture, especially those with high cell densities, and even more nutrients, all of which drive production costs higher compared to batch and fed-batch methods. Furthermore, higher cell densities can cause problems during production, such as maintaining dissolved oxygen levels and increasing gas handling, including supplying more oxygen and removing more carbon dioxide, which leads to more foaming and the need to modify defoaming strategies; and problems during harvesting and downstream processing, where the effort required to remove excess cell material can result in product loss, negating the benefits of increased titers due to increased cell mass.

[0291] A large-scale cell culture strategy that combines batch feeding during the growth phase with continuous perfusion during the production phase can be used to express target proteins. This approach can be used to maintain cell cultures at a production stage with a cell volume of less than or equal to 35%.

[0292] In one embodiment, the cell culture is maintained during the growth phase using a fed-batch culture system. Perfusion feeding can then be used during the production phase. In one embodiment, perfusion begins when the cells reach the production phase. In another embodiment, perfusion begins on day 3 or approximately day 3 through day 9 of cell culture. In yet another embodiment, perfusion begins on day 5 or approximately day 5 through day 7 of cell culture.

[0293] Using batch feeding during the growth phase allows cells to transition to the production phase, resulting in less dependence on temperature changes as a means of initiating and controlling the production phase; however, a temperature change of approximately 36°C to approximately 31°C can occur between the growth and production phases. In one embodiment, this change is 36°C to 32°C.

[0294] In some embodiments, the bioreactor can be used in a serum-free culture medium of at least 0.5 × 10⁻⁶. 6 Up to 3.0 × 10 6 Seed at 1.0 × 10⁶ live cells / mL, for example, 1.0 × 10� 6 live cells / mL.

[0295] Cell cultures can be supplemented with concentrated feed media containing components (such as nutrients and amino acids) consumed during the cell culture production process.

[0296] Concentrated fed-batch culture media can be based on almost any cell culture medium formulation. Such concentrated fed-batch media can contain most components of cell culture media, for example, in normal quantities of approximately 5X, 6X, 7X, 8X, 9X, 10X, 12X, 14X, 16X, 20X, 30X, 50X, 100X, 200X, 400X, 600X, 800X, or even approximately 1000X. Concentrated fed-batch culture media are often used in fed-batch culture processes.

[0297] 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.

[0298] 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.

[0299] 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 cell inoculation for 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.

[0300] In some embodiments, mammalian host cells can be used to generate a high yield of the target protein. High yield, or high volumetric productivity, is the ability of cells to produce high levels of the target protein. Using a fed-batch or perfusion culture medium suitable for mammalian host cells and containing amino acids, vitamins, or trace elements, in a culture grown for 10 days under fed-batch or perfusion conditions, a specific yield will depend on the target protein and may be at least 0.05 g / L, at least 0.1 g / L, at least 0.15 g / L, at least 0.2 g / L, at least 0.25 g / L, at least 0.3 g / L, at least 0.35 g / L, at least 0.4 g / L, at least 0.45 g / L, at least 0.5 g / L, at least 0.6 g / L, at least 0.7 g / L, at least 0.8 g / L, at least 0.9 g / L, at least 1 g / L, at least 1.5 g / L, at least 2 g / L, or higher. In specific embodiments, the host cells and methods disclosed herein express the target protein and, when grown under the above-described culture conditions, are capable of producing at least 0.5 g / L, at least 0.6 g / L, at least 0.7 g / L, at least 0.8 g / L, at least 0.9 g / L, at least 1 g / L, at least 1.5 g / L, at least 2 g / L or more, preferably up to about 3 g / L, 4 g / L, 5 g / L or 10 g / L.

[0301] 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, and grown for 10 days under fed-batch or perfusion conditions, mammalian host cells used with the expression vector system disclosed herein are able to produce at least 1 pg / cell / day, at least 2 pg / cell / day, at least 3 pg / cell / day, at least 4 pg / cell / day, at least 5 pg / cell / day, at least 6 pg / cell / day, at least 7 pg / cell / day, at least 8 pg / cell / day, at least 9 pg / cell / day, at least 10 pg / cell / day, at least 11 pg / cell / day, at least 12 pg / cell / day, at least 13 pg / cell / day, at least 14 pg / cell / day, at least 15 pg / cell / day, at least 20 pg / cell / day, at least 25 pg / cell / day, at least 50 pg / cell / day, at least 75 pg / cell / day, or up to 100 pg / cell / day. In a particular embodiment, mammalian host cells used with the expression vector system disclosed herein express the target protein and, under the culture conditions described above, 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.

[0302] 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 (i.e., pharmaceutically acceptable formulations) for use in pharmaceutical compositions include those described in Remington's Pharmaceutical Sciences, 18th edition, 1995, Mack Publishing Company, Easton, Pennsylvania.

[0303] 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.

[0304] 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.

[0305] Polynucleotides, peptides, vectors, host cells, etc., according to this disclosure can be prepared using a variety of known techniques.

[0306] Filtering methods

[0307] Typically, to establish cell lines that produce recombinant antigen-binding proteins (e.g., proteins derived from antibodies comprising two chains, one based on the antibody heavy chain and the other on the antibody light chain), it is usually necessary to integrate the heavy and light chain coding sequences from a single or separate vector into the genome, followed by rigorous and efficient metabolic selection to identify high-yielding cell lines. In glutamine synthetase (GS)-CHO expression systems, the selection of high-yielding cell lines is based on a balance between controlling the expression level of GS and the concentration of its specific inhibitor, L-methionine sulfoxide (MSX). As disclosed and illustrated in Example 2, one approach to expressing the antigen-binding proteins disclosed herein is to optimize the expression of the heavy and / or light chains using promoters of varying strengths at different MSX concentrations. Since GS acts as both a selectable marker and a means of gene amplification, different MSX concentrations are used to determine the optimal selectivity for each promoter. The results of these experiments established preferred vector designs with selectivity for approximately seven different biological forms, including multispecific proteins. Therefore, this paper provides vector design strategies for the efficient expression of antigen-binding proteins, including the biological forms described in Example 2.

[0308] Therefore, this paper discloses a method for optimizing the expression of an antigen-binding protein, wherein the antigen-binding protein comprises a heavy chain and a light chain, the method comprising:

[0309] Multiple vectors are expressed, each in a host cell population and at various concentrations of methionine sulfoxide (MSX), wherein each of the multiple vectors comprises: (i) a promoter with unique transcriptional strength; (ii) a glutamine synthase gene operably linked to the promoter; and (iii) a sequence operably linked to the promoter encoding the heavy chain and / or the light chain; and

[0310] The production rate of antigen-binding proteins was measured for each combination of promoter and MSX concentration.

[0311] In some embodiments, the promoter drives the expression of the heavy chain and / or the light chain.

[0312] In some embodiments, the promoter is a CMV / ADL promoter.

[0313] In some embodiments, the promoter is a GAPDH promoter. In some embodiments, the promoter is a CMV / GAPDH promoter.

[0314] In some embodiments, the promoter is the CMV / EF1a promoter.

[0315] In some embodiments, a first vector among a plurality of vectors contains a GAPDH promoter, and a second vector among a plurality of vectors contains a CMV / ADL promoter.

[0316] In some embodiments, the promoter drives the expression of the glutamine synthase gene. In some embodiments, the promoter is the SRα promoter. In some embodiments, the promoter is the mPGK promoter.

[0317] In some embodiments, each of the plurality of vectors contains a promoter with unique transcriptional strength driving heavy chain and / or light chain expression and / or a promoter with unique transcriptional strength driving glutamine synthase gene expression.

[0318] In some embodiments, multiple different MSX concentrations include 0 µM. In some embodiments, multiple different MSX concentrations include 12.5 µM. In some embodiments, multiple different MSX concentrations include 25 µM. In some embodiments, multiple different MSX concentrations include 12.5 µM and 25 µM. In some embodiments, multiple different MSX concentrations include 0 µM, 12.5 µM, and 25 µM.

[0319] 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.

[0320] Additional non-limiting exemplary embodiments

[0321] The non-limiting example embodiments disclosed herein also include:

[0322] E1. An expression cassette comprising a polynucleotide sequence containing the following elements in a 5' to 3' sequence:

[0323] a) A first GAPDH promoter operatively linked to a nucleotide sequence encoding a first antibody chain or antibody chain fusion, followed by a first polyA sequence;

[0324] b) A second GAPDH promoter operatively linked to a nucleotide sequence encoding a second antibody chain or antibody chain fusion, followed by a second polyA sequence; and

[0325] c) A promoter operatively linked to a nucleotide sequence encoding an optional marker, followed by a third polyA sequence;

[0326] Where the first antibody chain or antibody chain fusion is an antibody light chain, the second antibody chain or antibody chain fusion is an antibody heavy chain or antibody heavy chain fusion.

[0327] Where the first antibody chain or antibody chain fusion is an antibody heavy chain or heavy chain fusion, the second antibody chain or antibody chain fusion is an antibody light chain, and

[0328] The heavy chain fusion compound is selected from the group consisting of: heavy chain-scFv, heavy chain-cytokine, and heavy chain vHH.

[0329] E2. The expression cassette as described in E1, wherein the first GAPDH promoter is operatively linked to a nucleotide sequence encoding an antibody light chain, and the second GAPDH promoter is operatively linked to a heavy chain or a heavy chain fusion.

[0330] E3. An expression cassette as described in E1 or E2, wherein the selectable marker is selected from the group consisting of glutamine synthase and dihydrofolate reductase.

[0331] E4. The expression cassette as described in any one of E1-E3, wherein the GAPDH promoter comprises the nucleotide sequence of SEQ ID NO: 1.

[0332] E5. The expression cassette as described in any one of E1-E4, wherein the promoter operatively linked to the nucleotide sequence encoding the selectable marker is selected from the group consisting of mPGK, Sra, and SV40.

[0333] E6. The expression cassette as described in any one of E1-E5, wherein the first polyA sequence, the second polyA sequence, and the third polyA sequence are the same or different, and are selected from the group consisting of: thymidine kinase pA (TKpA) sequence, rabbit β-globin pA, and simian virus 40 (SV40) early pA sequence.

[0334] E7. An expression vector comprising an expression cassette as described in any one of E1-E6.

[0335] E8. An expression vector as described in E7, wherein the polynucleotide sequence encodes a monoclonal antibody.

[0336] E9. An expression vector as described in E7, wherein the polynucleotide sequence encodes B2 / C2 mAb.

[0337] E10. An expression vector pair, wherein

[0338] 1) The first expression vector contains an expression cassette containing a polynucleotide sequence comprising the following elements in a 5' to 3' sequence:

[0339] a) The first copy of the GAPDH promoter, which is operatively linked to a nucleotide sequence encoding an antibody light chain, followed by a first polyA sequence;

[0340] b) A second copy of the GAPDH promoter, operatively linked to a nucleotide sequence encoding the antibody heavy chain, followed by a second polyA sequence; and

[0341] c) A promoter operatively linked to a nucleotide sequence encoding a selectable marker, followed by a third polyA sequence; and

[0342] 2) The second expression vector contains an expression cassette containing a polynucleotide sequence comprising the following elements in a 5' to 3' sequence:

[0343] a) The first copy of the GAPDH promoter, which is operatively linked to a nucleotide sequence encoding an antibody light chain, followed by a first polyA sequence;

[0344] b) A second copy of the GAPDH promoter, operatively linked to a nucleotide sequence encoding the antibody heavy chain-scFv fusion, followed by a second polyA sequence; and

[0345] c) A promoter operatively linked to a nucleotide sequence encoding an optional marker, followed by a third polyA sequence.

[0346] E11. The carrier pair as described in E10, which encodes C1 mAb.

[0347] E12. An expression vector pair, wherein

[0348] 1) The first expression vector contains an expression cassette containing a polynucleotide sequence comprising the following elements in a 5' to 3' sequence:

[0349] a) A first copy of the GAPDH promoter, which is operatively linked to a nucleotide sequence encoding a first antibody light chain, followed by a first polyA sequence;

[0350] b) A second copy of the GAPDH promoter, operatively linked to a nucleotide sequence encoding a first antibody heavy chain, followed by a second polyA sequence; and

[0351] c) A promoter operatively linked to a nucleotide sequence encoding a selectable marker, followed by a third polyA sequence; and

[0352] 2) The second expression vector contains an expression cassette containing a polynucleotide sequence comprising the following elements in a 5' to 3' sequence:

[0353] a) A first copy of the GAPDH promoter, which is operatively linked to a nucleotide sequence encoding a second antibody light chain, followed by a first polyA sequence;

[0354] b) A second copy of the GAPDH promoter, operatively linked to a nucleotide sequence encoding a second antibody heavy chain, followed by a second polyA sequence; and

[0355] c) A promoter operatively linked to a nucleotide sequence encoding an optional marker, followed by a third polyA sequence.

[0356] E13. The vector pair as described in E11, which encodes heterologous IgG.

[0357] E14. A mammalian host cell comprising an expression vector as described in any one of E7-E9.

[0358] E15. A mammalian host cell comprising an expression vector pair as described in any one of E10-E13.

[0359] E16. Mammalian host cells as described in E14 or E15, selected from the group consisting of CHO.

[0360] E17. A mammalian host cell as described in E16, wherein the CHO cell is dhfr- or GSKO.

[0361] E18. A method for producing an antibody form, the method comprising culturing mammalian host cells as described in any one of E14-E17 under conditions expressing an antibody chain, and recovering the antibody form from the culture.

[0362] E19. The method as described in E18, wherein the recovered antibody is purified and formulated into a pharmaceutically acceptable preparation.

[0363] Further non-limiting exemplary embodiments / features disclosed herein include:

[0364] F1. An expression cassette comprising a polynucleotide sequence containing the following elements in a 5' to 3' sequence:

[0365] a) A first copy of the GAPDH promoter, operatively linked to a nucleotide sequence encoding a first antibody chain or antibody chain fusion, followed by a first polyA signal sequence;

[0366] b) A second copy of the GAPDH promoter, operatively linked to a nucleotide sequence encoding a second antibody chain or antibody chain fusion, followed by a second polyA signal sequence; and

[0367] c) A promoter operatively linked to a nucleotide sequence encoding a selectable marker, followed by a third poly-A signal sequence.

[0368] Where the first antibody chain or antibody chain fusion is an antibody light chain, the second antibody chain or antibody chain fusion is an antibody heavy chain or antibody heavy chain fusion.

[0369] Where the first antibody chain or antibody chain fusion is an antibody heavy chain or antibody heavy chain fusion, the second antibody chain or antibody chain fusion is an antibody light chain, and

[0370] The antibody heavy chain fusion is selected from the following groups: heavy chain-scFv fusion, heavy chain-cytokine fusion, and heavy chain VH fusion.

[0371] F2. An expression cassette as described in F1, wherein the first antibody chain or antibody chain fusion is an antibody light chain and the second antibody chain or antibody chain fusion is an antibody heavy chain.

[0372] F3. The expression cassette as described in F1, wherein the first antibody chain or antibody chain fusion is an antibody light chain and the second antibody chain or antibody chain fusion is an antibody heavy chain fusion.

[0373] F4. An expression cassette as described in F1, wherein the first antibody chain or antibody chain fusion is an antibody heavy chain and the second antibody chain or antibody chain fusion is an antibody light chain.

[0374] F5. The expression cassette as described in F1, wherein the first antibody chain or antibody chain fusion is an antibody heavy chain fusion, and the second antibody chain or antibody chain fusion is an antibody light chain.

[0375] F6. An expression cassette as described in any one of F1, F3 or F5, wherein the antibody heavy chain fusion is a direct fusion of the heavy chain with VH, a direct fusion of the heavy chain with scFv, or a direct fusion of the heavy chain with a cytokine.

[0376] F7. An expression cassette as described in any one of F1, F3, F5, or F6, wherein the antibody heavy chain fusion comprises a VH, scFv, or cytokine fused to the C-terminus of the heavy chain portion of the antibody heavy chain fusion.

[0377] F8. An expression cassette as described in any one of F1, F3, F5, or F6, wherein the antibody heavy chain fusion comprises a VH, scFv, or cytokine fused to the N-terminus of the heavy chain portion of the antibody heavy chain fusion.

[0378] F9. An expression cassette as described in any one of F1, F3, F5, or F6, wherein the antibody heavy chain fusion comprises a VH, scFv, or cytokine fused between CH1 and CH2 of the heavy chain portion of the antibody heavy chain fusion.

[0379] F10. An expression cassette as described in any one of F1, F3, or F5, wherein the antibody heavy chain fusion comprises a linker between the heavy chain and the VH, scFv, or cytokine.

[0380] F11. An expression cassette as described in any one of F1, F3, or F5-F10, wherein the antibody heavy chain fusion is a heavy chain-scFv fusion.

[0381] F12. An expression cassette as described in any one of F1, F3, or F5-F10, wherein the antibody heavy chain fusion is a heavy chain-cytokine fusion.

[0382] F13. An expression cassette as described in any one of F1, F3, or F5-F10, wherein the antibody heavy chain fusion is a heavy chain VH fusion.

[0383] F14. An expression cassette as described in any one of F1-F13, wherein the optional marker is glutamine synthase or dihydrofolate reductase.

[0384] F15. An expression cassette as described in any one of F1-F14, wherein the optional marker is glutamine synthase.

[0385] F16. An expression cassette as described in any one of F1-F14, wherein the optional marker is dihydrofolate reductase.

[0386] F17. The expression box as described in any one of F1-F16, wherein the GAPDH promoter is a CMV / GAPDH promoter.

[0387] F18. The expression box as described in F17, wherein the CMV promoter enhancer is located at the 5' of the GAPDH promoter.

[0388] F19. An expression cassette as described in F17 or F18, wherein the GAPDH promoter contains at least 90% identical nucleotide sequences to the nucleotide sequence of SEQ ID NO: 1.

[0389] F20. The expression cassette as described in any one of F17-F19, wherein the GAPDH promoter comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO: 1.

[0390] F21. An expression cassette as described in any one of F17-F20, wherein the GAPDH promoter comprises the nucleotide sequence of SEQ ID NO: 1.

[0391] F22. The expression cassette as described in any one of F1-F21, wherein the promoter operatively linked to the nucleotide sequence encoding the selectable marker is selected from the group consisting of mPGK, SRα, and SV40 promoters.

[0392] F23. An expression cassette as described in any one of F1-F22, wherein the promoter operatively linked to the nucleotide sequence encoding an optional marker is an mPGK promoter.

[0393] F24. An expression cassette as described in any one of F1-F22, wherein the promoter operatively linked to the nucleotide sequence encoding an optional marker is an SRα promoter.

[0394] F25. An expression cassette as described in any one of F1-F22, wherein the promoter operatively linked to the nucleotide sequence encoding an optional marker is an SV40 promoter.

[0395] F26. The expression cassette as described in any one of F1-F25, wherein each of the first polyA signal sequence, the second polyA signal sequence, and the third polyA signal sequence is independently selected from the group consisting of: bovine growth hormone (BGH) polyA signal sequence, thymidine kinase polyA (TKpA) signal sequence, rabbit β-globin polyA signal sequence, and simian virus 40 (SV40) early polyA signal sequence.

[0396] F27. The expression box as described in any one of F1-F26, wherein each of the first polyA signal sequence, the second polyA signal sequence, and the third polyA signal sequence is identical.

[0397] F28. An expression cassette as described in any one of F1-F27, wherein each of the first polyA signal sequence, the second polyA signal sequence, and the third polyA signal sequence is an early polyA signal sequence of simian virus 40 (SV40).

[0398] F29. An expression cassette as described in F1, wherein the expression cassette encodes an antibody light chain and an antibody heavy chain, the GAPDH promoter is a CMV / GAPDH promoter, the selectable marker is glutamine synthase, the promoter operatively linked to the nucleotide sequence encoding the selectable marker is mPGK, and each of the first polyA signal sequence, the second polyA signal sequence, and the third polyA signal sequence is an early polyA signal sequence of simian virus 40 (SV40).

[0399] F30. An expression cassette as described in F1, wherein the expression cassette encodes an antibody light chain and antibody heavy chain fusion, the GAPDH promoter is a CMV / GAPDH promoter, the selectable marker is glutamine synthase, the promoter operatively linked to the nucleotide sequence encoding the selectable marker is SRα, and each of the first polyA signal sequence, the second polyA signal sequence, and the third polyA signal sequence is an early polyA signal sequence of simian virus 40 (SV40).

[0400] F31. The expression cassette as described in F30, wherein the antibody heavy chain fusion is a direct fusion of the heavy chain with VH, a direct fusion of the heavy chain with scFv, or a direct fusion of the heavy chain with a cytokine.

[0401] F32. An expression cassette as described in F30 or F31, wherein the antibody heavy chain fusion comprises VH, scFv, or a cytokine fused to the C-terminus of the heavy chain portion of the antibody heavy chain fusion.

[0402] F33. An expression cassette as described in F30 or F31, wherein the antibody heavy chain fusion comprises a VH, scFv, or cytokine fused to the N-terminus of the heavy chain portion of the antibody heavy chain fusion.

[0403] F34. An expression cassette as described in F30 or F31, wherein the antibody heavy chain fusion contains a VH, scFv, or cytokine fused between CH1 and CH2 of the heavy chain portion of the antibody heavy chain fusion.

[0404] F35. An expression cassette as described in F30, wherein the antibody heavy chain fusion contains a linker between the heavy chain and the VH, scFv, or cytokine.

[0405] F36. The expression cassette as described in any one of F30-F35, wherein the antibody heavy chain fusion is a heavy chain-scFv fusion.

[0406] F37. The expression cassette as described in any one of F30-F35, wherein the antibody heavy chain fusion is a heavy chain-cytokine fusion.

[0407] F38. The expression cassette as described in any one of F30-F35, wherein the antibody heavy chain fusion is a heavy chain VH fusion.

[0408] F39. An expression vector comprising an expression cassette as described in any one of F1-F38.

[0409] F40. An expression vector as described in F39, which encodes a monoclonal antibody.

[0410] F41. An expression vector as described in F39, which encodes B2 / C2 mAb, C2mAb, B2Hmab, or C2Hmab.

[0411] F42. An expression vector as described in F39 or F41, which encodes B2 / C2 mAb.

[0412] F43. An expression carrier pair, wherein:

[0413] 1) The first expression vector contains an expression cassette containing a polynucleotide sequence comprising the following elements in a 5' to 3' sequence:

[0414] a) The first copy of the GAPDH promoter, which is operatively linked to a nucleotide sequence encoding an antibody light chain, followed by a first polyA signal sequence;

[0415] b) A second copy of the GAPDH promoter, operatively linked to a nucleotide sequence encoding the antibody heavy chain, followed by a second polyA signal sequence; and

[0416] c) A promoter operatively linked to a nucleotide sequence encoding a selectable marker, followed by a third poly-A signal sequence; and

[0417] 2) The second expression vector contains an expression cassette containing a polynucleotide sequence comprising the following elements in a 5' to 3' sequence:

[0418] a) The first copy of the GAPDH promoter, which is operatively linked to a nucleotide sequence encoding an antibody light chain, followed by a first polyA signal sequence;

[0419] b) A second copy of the GAPDH promoter, operatively linked to a nucleotide sequence encoding an antibody heavy chain fusion protein, followed by a second polyA signal sequence; and

[0420] c) A promoter operatively linked to a nucleotide sequence encoding an optional marker, followed by a third poly-A signal sequence.

[0421] F44. A pair of expression carriers, wherein:

[0422] 1) The first expression vector contains an expression cassette containing a polynucleotide sequence comprising the following elements in a 5' to 3' sequence:

[0423] a) The first copy of the GAPDH promoter, which is operatively linked to a nucleotide sequence encoding the antibody heavy chain, followed by the first polyA signal sequence;

[0424] b) A second copy of the GAPDH promoter, operatively linked to a nucleotide sequence encoding an antibody light chain, followed by a second polyA signal sequence; and

[0425] c) A promoter operatively linked to a nucleotide sequence encoding a selectable marker, followed by a third poly-A signal sequence; and

[0426] 2) The second expression vector contains an expression cassette containing a polynucleotide sequence comprising the following elements in a 5' to 3' sequence:

[0427] a) The first copy of the GAPDH promoter, which is operatively linked to a nucleotide sequence encoding an antibody light chain, followed by a first polyA signal sequence;

[0428] b) A second copy of the GAPDH promoter, operatively linked to a nucleotide sequence encoding an antibody heavy chain fusion protein, followed by a second polyA signal sequence; and

[0429] c) A promoter operatively linked to a nucleotide sequence encoding an optional marker, followed by a third poly-A signal sequence.

[0430] F45. A pair of expression vectors, wherein:

[0431] 1) The first expression vector contains an expression cassette containing a polynucleotide sequence comprising the following elements in a 5' to 3' sequence:

[0432] a) The first copy of the GAPDH promoter, which is operatively linked to a nucleotide sequence encoding an antibody light chain, followed by a first polyA signal sequence;

[0433] b) A second copy of the GAPDH promoter, operatively linked to a nucleotide sequence encoding the antibody heavy chain, followed by a second polyA signal sequence; and

[0434] c) A promoter operatively linked to a nucleotide sequence encoding a selectable marker, followed by a third poly-A signal sequence; and

[0435] 2) The second expression vector contains an expression cassette containing a polynucleotide sequence comprising the following elements in a 5' to 3' sequence:

[0436] a) The first copy of the GAPDH promoter, which is operatively linked to a nucleotide sequence encoding an antibody heavy chain fusion, followed by a first polyA signal sequence;

[0437] b) A second copy of the GAPDH promoter, operatively linked to a nucleotide sequence encoding an antibody light chain, followed by a second polyA signal sequence; and

[0438] c) A promoter operatively linked to a nucleotide sequence encoding an optional marker, followed by a third poly-A signal sequence.

[0439] F46. An expression carrier pair, wherein:

[0440] 1) The first expression vector contains an expression cassette containing a polynucleotide sequence comprising the following elements in a 5' to 3' sequence:

[0441] a) The first copy of the GAPDH promoter, which is operatively linked to a nucleotide sequence encoding the antibody heavy chain, followed by the first polyA signal sequence;

[0442] b) A second copy of the GAPDH promoter, operatively linked to a nucleotide sequence encoding an antibody light chain, followed by a second polyA signal sequence; and

[0443] c) A promoter operatively linked to a nucleotide sequence encoding a selectable marker, followed by a third poly-A signal sequence; and

[0444] 2) The second expression vector contains an expression cassette containing a polynucleotide sequence comprising the following elements in a 5' to 3' sequence:

[0445] a) The first copy of the GAPDH promoter, which is operatively linked to a nucleotide sequence encoding an antibody heavy chain fusion, followed by a first polyA signal sequence;

[0446] b) A second copy of the GAPDH promoter, operatively linked to a nucleotide sequence encoding an antibody light chain, followed by a second polyA signal sequence; and

[0447] c) A promoter operatively linked to a nucleotide sequence encoding an optional marker, followed by a third poly-A signal sequence.

[0448] F47. The expression vector pair as described in any one of F43-F46, wherein the antibody light chain of the first expression vector is identical to the antibody light chain of the second expression vector.

[0449] F48. The expression vector pair as described in any one of F43-F47, wherein the antibody heavy chain of the first expression vector is identical to the antibody heavy chain portion of the antibody heavy chain fusion of the second expression vector.

[0450] F49. The expression vector pair as described in any one of F43-F48, wherein the antibody light chain of the first expression vector is identical to the antibody light chain of the second expression vector, and the antibody heavy chain of the first expression vector is identical to the antibody heavy chain portion of the antibody heavy chain fusion of the second expression vector.

[0451] F50. An expression vector pair as described in any one of F43-F46, wherein the antibody light chain of the first expression vector is different from the antibody light chain of the second expression vector.

[0452] F51. An expression vector pair as described in any one of F43-F46 or F50, wherein the antibody heavy chain of the first expression vector is different from the antibody heavy chain portion of the antibody heavy chain fusion of the second expression vector.

[0453] F52. An expression vector pair as described in any one of F43-F46, F50, or F51, wherein the antibody light chain of the first expression vector is different from the antibody light chain of the second expression vector, and the antibody heavy chain of the first expression vector is different from the antibody heavy chain portion of the antibody heavy chain fusion of the second expression vector.

[0454] F53. The expression vector pair as described in any one of F43-F52, wherein the antibody heavy chain fusion is a heavy chain-scFv fusion.

[0455] F54. The expression vector pair as described in F43, which encodes C1 mAb.

[0456] F55. An expression vector pair as described in any one of F43-F54, wherein the promoter operatively linked to the nucleotide sequence encoding the selectable marker on each of the first and second expression vectors is independently selected from the group consisting of mPGK, SRα, and SV40 promoters.

[0457] F56. An expression vector pair as described in any one of F43-F55, wherein the promoter operably linked to the nucleotide sequence encoding an optional marker on the first expression vector is SRα.

[0458] F57. An expression vector pair as described in any one of F43-F56, wherein the promoter operably linked to the nucleotide sequence encoding an optional marker on the second expression vector is SRα.

[0459] F58. The expression vector pair as described in any one of F43-F57, wherein the promoter operably linked to the nucleotide sequence encoding the selectable marker on the first expression vector is SRα, and the promoter operably linked to the nucleotide sequence encoding the selectable marker on the second expression vector is SRα.

[0460] F59. The expression vector pair as described in any one of F43-F58, wherein the selectable marker on the first expression vector and the selectable marker on the second expression vector are glutamine synthase.

[0461] F60. The expression vector pair as described in any one of F43-F59, wherein each of the first polyA signal sequence, the second polyA signal sequence, and the third polyA signal sequence on the first and second expression vectors is independently selected from the group consisting of: bovine growth hormone (BGH) polyA signal sequence, thymidine kinase polyA (TKpA) signal sequence, rabbit β-globin polyA signal sequence, and simian virus 40 (SV40) early polyA signal sequence.

[0462] F61. The expression vector pair as described in any one of F43-F60, wherein each of the first polyA signal sequence, the second polyA signal sequence and the third polyA signal sequence on the first expression vector is an early polyA signal sequence of simian virus 40 (SV40).

[0463] F62. The expression vector pair as described in any one of F43-F61, wherein each of the first polyA signal sequence, the second polyA signal sequence and the third polyA signal sequence on the second expression vector is an early polyA signal sequence of simian virus 40 (SV40).

[0464] F63. The expression vector pair as described in any one of F43-F62, wherein each of the first polyA signal sequence, the second polyA signal sequence and the third polyA signal sequence on the first expression vector is an early polyA signal sequence of simian virus 40 (SV40), and each of the first polyA signal sequence, the second polyA signal sequence and the third polyA signal sequence on the second expression vector is an early polyA signal sequence of simian virus 40 (SV40).

[0465] F64. The expression vector pair as described in any one of F43-F63, wherein each of the first polyA signal sequence, the second polyA signal sequence, and the third polyA signal sequence on the first and second expression vectors is an early polyA signal sequence of simian virus 40 (SV40), the promoter on the first expression vector operably linked to a nucleotide sequence encoding a selectable marker is SRα, the promoter on the second expression vector operably linked to a nucleotide sequence encoding a selectable marker is SRα, and the selectable marker on the first and second expression vectors is glutamine synthase.

[0466] F65. A pair of expression vectors, wherein:

[0467] 1) The first expression vector contains an expression cassette containing a polynucleotide sequence comprising the following elements in a 5' to 3' sequence:

[0468] a) A first copy of the GAPDH promoter, which is operatively linked to a nucleotide sequence encoding a first antibody light chain, followed by a first polyA signal sequence;

[0469] b) A second copy of the GAPDH promoter, operatively linked to a nucleotide sequence encoding a first antibody heavy chain, followed by a second polyA signal sequence; and

[0470] c) A promoter operatively linked to a nucleotide sequence encoding a selectable marker, followed by a third poly-A signal sequence; and

[0471] 2) The second expression vector contains an expression cassette containing a polynucleotide sequence comprising the following elements in a 5' to 3' sequence:

[0472] a) A first copy of the GAPDH promoter, which is operatively linked to a nucleotide sequence encoding a second antibody light chain, followed by a first polyA signal sequence;

[0473] b) A second copy of the GAPDH promoter, operatively linked to a nucleotide sequence encoding a second antibody heavy chain, followed by a second polyA signal sequence; and

[0474] c) A promoter operatively linked to a nucleotide sequence encoding an optional marker, followed by a third poly-A signal sequence.

[0475] F66. A pair of expression vectors, wherein:

[0476] 1) The first expression vector contains an expression cassette containing a polynucleotide sequence comprising the following elements in a 5' to 3' sequence:

[0477] a) A first copy of the GAPDH promoter, which is operatively linked to a nucleotide sequence encoding a first antibody heavy chain, followed by a first polyA signal sequence;

[0478] b) A second copy of the GAPDH promoter, operatively linked to a nucleotide sequence encoding a first antibody light chain, followed by a second polyA signal sequence; and

[0479] c) A promoter operatively linked to a nucleotide sequence encoding a selectable marker, followed by a third poly-A signal sequence; and

[0480] 2) The second expression vector contains an expression cassette containing a polynucleotide sequence comprising the following elements in a 5' to 3' sequence:

[0481] a) A first copy of the GAPDH promoter, which is operatively linked to a nucleotide sequence encoding a second antibody light chain, followed by a first polyA signal sequence;

[0482] b) A second copy of the GAPDH promoter, operatively linked to a nucleotide sequence encoding a second antibody heavy chain, followed by a second polyA signal sequence; and

[0483] c) A promoter operatively linked to a nucleotide sequence encoding an optional marker, followed by a third poly-A signal sequence.

[0484] F67. An expression vector pair, wherein:

[0485] 1) The first expression vector contains an expression cassette containing a polynucleotide sequence comprising the following elements in a 5' to 3' sequence:

[0486] a) A first copy of the GAPDH promoter, which is operatively linked to a nucleotide sequence encoding a first antibody light chain, followed by a first polyA signal sequence;

[0487] b) A second copy of the GAPDH promoter, operatively linked to a nucleotide sequence encoding a first antibody heavy chain, followed by a second polyA signal sequence; and

[0488] c) A promoter operatively linked to a nucleotide sequence encoding a selectable marker, followed by a third poly-A signal sequence; and

[0489] 2) The second expression vector contains an expression cassette containing a polynucleotide sequence comprising the following elements in a 5' to 3' sequence:

[0490] a) A first copy of the GAPDH promoter, which is operatively linked to a nucleotide sequence encoding a second antibody heavy chain, followed by a first polyA signal sequence;

[0491] b) A second copy of the GAPDH promoter, operatively linked to a nucleotide sequence encoding a second antibody light chain, followed by a second polyA signal sequence; and

[0492] c) A promoter operatively linked to a nucleotide sequence encoding an optional marker, followed by a third poly-A signal sequence.

[0493] F68. A pair of expression vectors, wherein:

[0494] 1) The first expression vector contains an expression cassette containing a polynucleotide sequence comprising the following elements in a 5' to 3' sequence:

[0495] a) A first copy of the GAPDH promoter, which is operatively linked to a nucleotide sequence encoding a first antibody heavy chain, followed by a first polyA signal sequence;

[0496] b) A second copy of the GAPDH promoter, operatively linked to a nucleotide sequence encoding a first antibody light chain, followed by a second polyA signal sequence; and

[0497] c) A promoter operatively linked to a nucleotide sequence encoding a selectable marker, followed by a third poly-A signal sequence; and

[0498] 2) The second expression vector contains an expression cassette containing a polynucleotide sequence comprising the following elements in a 5' to 3' sequence:

[0499] a) A first copy of the GAPDH promoter, which is operatively linked to a nucleotide sequence encoding a second antibody heavy chain, followed by a first polyA signal sequence;

[0500] b) A second copy of the GAPDH promoter, operatively linked to a nucleotide sequence encoding a second antibody light chain, followed by a second polyA signal sequence; and

[0501] c) A promoter operatively linked to a nucleotide sequence encoding an optional marker, followed by a third poly-A signal sequence.

[0502] F69. The expression vector pair as described in any one of F65-F68, which encodes heterologous IgG.

[0503] F70. An expression vector pair as described in any one of F65-F69, wherein the promoter operatively linked to the nucleotide sequence encoding the selectable marker on each of the first and second expression vectors is independently selected from the group consisting of mPGK, SRα, and SV40 promoters.

[0504] F71. An expression vector pair as described in any one of F65-F70, wherein the promoter operatively linked to the nucleotide sequence encoding an optional marker on the first expression vector is mPGK.

[0505] F72. An expression vector pair as described in any one of F65-F71, wherein the promoter operatively linked to the nucleotide sequence encoding an optional marker on the second expression vector is mPGK.

[0506] F73. The expression vector pair as described in any one of F65-F72, wherein the promoter operably linked to the nucleotide sequence encoding the selectable marker on the first expression vector is mPGK, and the promoter operably linked to the nucleotide sequence encoding the selectable marker on the second expression vector is mPGK.

[0507] F74. The expression vector pair as described in any one of F65-F73, wherein the selectable marker on the first expression vector and the selectable marker on the second expression vector are glutamine synthase.

[0508] F75. The expression vector pair as described in any one of F65-F74, wherein each of the first polyA signal sequence, the second polyA signal sequence, and the third polyA signal sequence on the first and second expression vectors is independently selected from the group consisting of: bovine growth hormone (BGH) polyA signal sequence, thymidine kinase polyA (TKpA) signal sequence, rabbit β-globin polyA signal sequence, and simian virus 40 (SV40) early polyA signal sequence.

[0509] F76. The expression vector pair as described in any one of F65-F75, wherein each of the first polyA signal sequence, the second polyA signal sequence and the third polyA signal sequence on the first expression vector is an early polyA signal sequence of simian virus 40 (SV40).

[0510] F77. The expression vector pair as described in any one of F65-F76, wherein each of the first polyA signal sequence, the second polyA signal sequence and the third polyA signal sequence on the second expression vector is an early polyA signal sequence of simian virus 40 (SV40).

[0511] F78. The expression vector pair as described in any one of F65-F77, wherein each of the first polyA signal sequence, the second polyA signal sequence and the third polyA signal sequence on the first expression vector is an early polyA signal sequence of simian virus 40 (SV40), and each of the first polyA signal sequence, the second polyA signal sequence and the third polyA signal sequence on the second expression vector is an early polyA signal sequence of simian virus 40 (SV40).

[0512] F79. The expression vector pair as described in any one of F65-F78, wherein each of the first polyA signal sequence, the second polyA signal sequence, and the third polyA signal sequence on the first and second expression vectors is an early polyA signal sequence of simian virus 40 (SV40), the promoter on the first expression vector operably linked to a nucleotide sequence encoding an optional marker is mPGK, the promoter on the second expression vector operably linked to a nucleotide sequence encoding an optional marker is mPGK, and the optional marker on the first and second expression vectors is glutamine synthase.

[0513] F80. An expression vector pair as described in any one of F43-F79, wherein the GAPDH promoter on the first expression vector is a CMV / GAPDH promoter.

[0514] F81. The expression vector pair as described in any one of F43-F80, wherein the GAPDH promoter on the second expression vector is a CMV / GAPDH promoter.

[0515] F82. The expression vector pair as described in any one of F43-F81, wherein the GAPDH promoter on the first expression vector is a CMV / GAPDH promoter, and the GAPDH promoter on the second expression vector is a CMV / GAPDH promoter.

[0516] F83. The expression vector pair as described in any one of F80-F82, wherein the CMV promoter enhancer is located at the 5' of the GAPDH promoter.

[0517] F84. The expression vector pair as described in any one of F80-F83, wherein the GAPDH promoter on the first expression vector and / or the second expression vector contains a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 1.

[0518] F85. The expression vector pair as described in any one of F80-F84, wherein the GAPDH promoter on the first expression vector and / or the second expression vector contains a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO: 1.

[0519] F86. The expression vector pair as described in any one of F80-F85, wherein the GAPDH promoter on the first expression vector and / or the second expression vector comprises the nucleotide sequence of SEQ ID NO: 1.

[0520] F87. A mammalian host cell comprising an expression vector as described in any one of F39-F32.

[0521] F88. A mammalian host cell comprising an expression vector pair as described in any one of F43-F86.

[0522] F89. Mammalian host cells as described in F87 or F88, specifically Chinese hamster ovary (CHO) cells.

[0523] F90. A mammalian host cell as described in F89, wherein the CHO cell is a dhfr-CHO cell or a GSKO CHO cell.

[0524] F91. A mammalian host cell as described in F89, wherein the CHO cell is a GSKO CHO cell.

[0525] F92. A method for producing an antibody form, the method comprising culturing mammalian host cells, as described in any one of F87-F91, under conditions for producing the antibody form, and recovering the antibody form from the culture.

[0526] F93. The method as described in F92, wherein the recovered antibody is purified and formulated into a pharmaceutically acceptable preparation. Example

[0527] Example 1. Vector engineering strategies for improving the productivity of various antibody forms

[0528] Overview

[0529] Establishing stable Chinese hamster ovary (CHO) cell lines that generate therapeutic recombinant antibodies involves integrating heavy and light chains (HC and LC, respectively) from one or more expression vectors into the genome via a selection process. In glutamine synthase knockout (GS KO) expression systems, the selection of the stable pool can be controlled by balancing the expression level of the exogenous GS gene with the concentration of its specific inhibitor, L-methionine sulfoxide (MSX).

[0530] This example describes further vector optimization to improve the productivity of different therapeutic forms in CHO cells by using promoters with varying strengths to modulate the expression levels of the HC, LC, and GS genes in combination with different MSX concentrations. The vector engineering strategies described herein can be extended to improve and optimize the productivity of other recombinant proteins typically expressed in stable pools.

[0531] Materials and Methods

[0532] Plasmid generation. The coding sequences of LC and HC (or HC-fusion) were inserted into the pPBGS plasmid backbone using Golden Gate cloning to generate tricistronic vectors. Briefly, LC and HC (or HC-fusion) with the SV40-polyA fragment were controlled using CMV / ADL and CMV / GAPDH promoter / enhancer fragments, subsequently driving the SRα or mPGK promoter (in order) for mGS-polyA expression (Table 1). All fragments were unidirectionally assembled using combinations of overhang sequences to facilitate Golden Gate cloning. MSX was used at 0, 12.5, 25, and 50 µM.

[0533] Table 1. Different carrier configurations

[0534]

[0535] Plasmid transfection into GS KO hosts. GS KO (knockout) clone cell hosts derived from the CHO-K1 parental host were used to generate stable pools expressing many different antibody forms as listed in Table 1. 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 0.4–0.3 × 10⁻⁶ ppm. 6Host 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.

[0536] Stable pools of expression antibodies were generated using Gene Pulser XCell (BioRad Laboratories; Hercules, CA) according to the manufacturer's protocol. Repeat transfection was performed for each vector conformation. Briefly, 20 μg of each plasmid was combined with 5 μg of proprietary piggybac transposase and electroporated to 20 × 10⁻⁶ cells / mL. 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.

[0537] Selection and recovery. Seventy-two hours after transfection, cells were centrifuged and transferred to selection medium containing 0, 12.5, 25, and 50 µM MSX, which was free of glutamine or growth factors. Cells were then rotated every 3–4 days at approximately 1–2 × 10⁶ 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 0.4–0.3 × 10⁻⁶ cells / mL. 6 Cells / mL.

[0538] Feed-in batch production. Fully recovered cells are incubated in a proprietary basal medium at a rate of 1 × 10⁻⁶. 6 Cells were seeded at 100 cells / mL for fed-batch production. Proprietary feed was added to the culture on days 3, 6, and 8, and the culture was harvested on day 10. Cell counts and viability were determined using a Vi-Cell BLU cell viability analyzer (Beckman Coulter, Brea, CA). The titer of the supernatant was analyzed (protein A-HPLC).

[0539] result

[0540] Figure 2A-2B The results showed that at different MSX levels, higher titers (A) and cell unit productivity (B) were obtained by using a combination of the CMV / GADPH promoter for light chain (HC) and heavy chain (LC) with the SRα or mPGK promoter for glutamine synthase (GS).

[0541] in addition, Figures 3A-3C This displays an overview of similar viability (A), live cell density (VCD) (B), and total live cell density (VCD) (C) for all carrier configurations during fed-batch production. Colors correspond to MSX concentration levels.

[0542] Example 2. Carrier engineering strategies for improving the productivity of B2 mAb bispecific molecules

[0543] Overview

[0544] Establishing stable Chinese hamster ovary (CHO) cell lines that generate therapeutic recombinant antibodies and antibody-derived molecules involves integrating heavy and light chains (HC and LC, respectively) from one or more expression vectors into the genome via a selection process. In glutamine synthase knockout (GS KO) expression systems, the selection of the stable pool can be controlled by balancing the expression level of the exogenous GS gene with the concentration of its specific inhibitor, L-methionine sulfoxide (MSX).

[0545] This example describes further vector optimization to improve the production of bispecific antibodies in CHO cells by using promoters with varying strengths to modulate the expression levels of the HC, LC, and GS genes in combination with different MSX concentrations. The vector engineering strategies described herein can be extended to improve and optimize the production of other recombinant proteins typically expressed in stable pools.

[0546] Materials and Methods

[0547] B2 mAb plasmid generation. The coding sequences of HC-scFv and LC were inserted into the pPBGS plasmid backbone using Golden Gate cloning to generate tricistronic vectors. Briefly, HC-scFv and LC with the SV40-polyA fragment were controlled using CMV / ADL, CMV / GAPDH, or CMW / EF1 promoter / enhancer fragments, ultimately driving SRα, mPGK, or SV40 promoters (in order) for mGS-polyA expression (Table 2). All fragments were unidirectionally assembled using combinations of overhang sequences to promote Golden Gate cloning. MSX was used at 0, 12.5, 25, and 50 µM.

[0548] Table 2. Different carrier configurations

[0549]

[0550] Plasmid transfection into GS KO hosts. GS KO (knockout) clone cell hosts derived from the CHO-K1 parental host were used to generate a stable pool expressing B2 mAb. 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 0.4–0.3 × 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. 610 cells / mL were seeded into the host cells to ensure that the cells were in the exponential growth phase at the time of transfection.

[0551] Stable pools expressing B2 mAb were generated using Gene Pulser XCell (Bio-Ray Laboratories, Hercules, CA) according to the manufacturer's protocol. Repeat transfection was performed for each vector conformation. Briefly, 20 μg of each plasmid was combined with 5 μg 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.

[0552] Selection and recovery. Seventy-two hours after transfection, cells were centrifuged and transferred to selection medium containing 0, 12.5, 25, and 50 µM MSX, which was glutamine-free. Cells were then cultured every 3–4 days at approximately 1–2 × 10⁶ 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 0.4–0.3 × 10⁻⁶ cells / mL. 6 Cells / mL.

[0553] Feed-in batch production. Fully recovered cells are incubated in 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 pharmaceutical analysis system), and analytical hydrophobic interaction chromatography (HIC-HPLC) (Agilent HPLC 1100 / 1200 series), respectively.

[0554] result

[0555] Figures 4A-4B The results showed that at 12.5 µM, 25 µM, and 50 µM, and at 0 µM and 12.5 µM MSX, respectively, the combination of the CMV / GADPH promoter for light chain (HC) and heavy chain (LC) with the SRα or mPGK promoter for glutamine synthase (GS) yielded higher titers (A) and cell unit productivity (B).

[0556] in addition, Figures 5A-5C The aggregates (SEC HMW) (A), shear products (low and medium molecular weight rCE species, LMW+MMW) (B), and isotypes (HIC-HPLC post-peak) (C) are shown. The impurities are comparable in the following carrier configurations with the highest titers: the combination of CMV / GADPH and the SRα / mPGK promoter (at 25 µM and 12.5 µM MSX, respectively).

[0557] final, Figures 6A-6C The table shows the similar viability (A), viable cell density (VCD) (B), and total viable cell density (VCD) (C) of all vector configurations at the same MSX concentration during fed-batch production, except for CMV / GADPH + SV40 + 50 µM MSX.

[0558] Example 3. Carrier engineering strategies for improving the productivity of ScFv2+2 A and B bispecific molecules.

[0559] Overview

[0560] Establishing stable Chinese hamster ovary (CHO) cell lines that generate therapeutic recombinant antibodies and antibody-derived molecules involves integrating heavy and light chains (HC and LC, respectively) from one or more expression vectors into the genome via a selection process. In glutamine synthase knockout (GS KO) expression systems, the selection of the stable pool can be controlled by balancing the expression level of the exogenous GS gene with the concentration of its specific inhibitor, L-methionine sulfoxide (MSX).

[0561] This example describes further vector optimization to improve the production of bispecific antibodies in CHO cells by using promoters with varying strengths to modulate the expression levels of the HC, LC, and GS genes in combination with different MSX concentrations. The vector engineering strategies described herein can be extended to improve and optimize the production of other recombinant proteins typically expressed in stable pools.

[0562] Materials and Methods

[0563] ScFv 2+2 plasmid generation. The coding sequences of LC and HC were inserted into the pPBGS plasmid backbone using Golden Gate cloning to generate tricistronic vectors. Briefly, LC and HC with the SV40-polyA fragment were controlled using CMV / ADL or CMV / GAPDH promoter / enhancer fragments, subsequently driving SRα, mPGK, or SV40 promoters (in order) for mGS-polyA expression (Table 3). All fragments were unidirectionally assembled using combinations of overhang sequences to promote Golden Gate cloning. MSX was used at 0, 12.5, 25, and 50 µM.

[0564] Table 3. Different carrier configurations

[0565]

[0566] Plasmid transfection into GS KO hosts. GS KO (knockout) clone cell hosts derived from the CHO-K1 parental host were used to generate stable pools expressing 2+2 ScFv molecules A or B. 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 0.4–0.3 × 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.

[0567] Stable pools expressing 2+2 ScFv molecules A or B were generated using Gene Pulser XCell (Bio-Ray Laboratories, Hercules, CA) according to the manufacturer's protocol. Repeat transfection was performed for each vector conformation. Briefly, 20 μg of each plasmid was combined with 5 μg 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.

[0568] Selection and recovery. Seventy-two hours after transfection, cells were centrifuged and transferred to selection medium containing 0, 12.5, 25, and 50 µM MSX, which was glutamine-free. Cells were then cultured every 3–4 days at approximately 1–2 × 10⁶ 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 0.4–0.3 × 10⁻⁶ cells / mL. 6 Cells / mL.

[0569] Feed-in batch production. Fully recovered cells are incubated in 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 pharmaceutical analysis system), and analytical hydrophobic interaction chromatography (HIC-HPLC) (Agilent HPLC 1100 / 1200 series), respectively.

[0570] result

[0571] Figures 7A-7B The results showed that the combination of the CMV / GADPH promoter for light chain (HC) and heavy chain (LC) with the SRα or mPGK promoter for glutamine synthase (GS) resulted in higher titers (A) and higher cellular unit productivity (B) of 2+2 ScFv molecules at 12.5 µM, 25 µM and 50 µM and 0 µM and 12.5 µM MSX, respectively.

[0572] Figures 8A-8B The results showed that the combination of the CMV / GADPH promoter for light chain (HC) and heavy chain (LC) with the SRα or mPGK promoter for glutamine synthase (GS) at 12.5 µM, 25 µM and 50 µM and 0 µM and 12.5 µM MSX, respectively, resulted in higher titers (A) and cellular unit productivity (B) of 2+2 ScFv molecules.

[0573] Figures 9A-9C The aggregates (SEC HMW) (A), cleavage products (low and medium molecular weight rCEs, LMW+MMW) (B), and isoforms (post-HIC-HPLC peaks) of 2+2 ScFv molecule A are shown. The impurities are comparable in the following carrier configurations with the highest titers: the combination of CMV / GADPH and the SRα / mPGK promoter (at 25 µM and 12.5 µM MSX, respectively).

[0574] Figures 10A-10CThe aggregates (SEC HMW) (A), cleavage products (low and medium molecular weight rCEs, LMW+MMW) (B), and isoforms (post-HIC-HPLC peaks) of 2+2 ScFv molecule B are shown. The impurities are comparable in the following carrier configurations with the highest titers: the combination of CMV / GADPH and the SRα / mPGK promoter (at 25 µM and 12.5 µM MSX, respectively).

[0575] Figure 11A-11C The table shows the similar viability (A), viable cell density (VCD) (B), and total viable cell density (VCD) (C) of all carrier configurations of 2+2 ScFv molecule A at the same MSX concentration during fed-batch production, except for CMV / GADPH + mPGK + 25µM MSX and CMV / GADPH + SV40 + 2 5µM MSX.

[0576] Figures 12A-12C The table shows the similar viability (A), viable cell density (VCD) (B), and total viable cell density (VCD) (C) of all carrier configurations of 2+2 ScFv molecule B at the same MSX concentration during fed-batch production, except for CMV / GADPH + mPGK + 25µM MSX and CMV / GADPH + Srα + 25µM MSX.

[0577] Example 4. Carrier engineering strategies for improving MAb productivity

[0578] Overview

[0579] Establishing stable Chinese hamster ovary (CHO) cell lines that generate therapeutic recombinant antibodies and antibody-derived molecules involves integrating heavy and light chains (HC and LC, respectively) from one or more expression vectors into the genome via a selection process. In glutamine synthase knockout (GS KO) expression systems, the selection of the stable pool can be controlled by balancing the expression level of the exogenous GS gene with the concentration of its specific inhibitor, L-methionine sulfoxide (MSX).

[0580] This example describes further vector optimization to improve monoclonal antibody production in CHO cells by using promoters with different strengths to regulate the expression levels of the HC, LC, and GS genes in combination with different MSX concentrations.

[0581] Materials and Methods

[0582] MAb plasmid generation. The coding sequences for LC and HC of MAb1 and MAb2 were inserted into the pBMV plasmid backbone using Golden Gate cloning to generate tricistronic or bicistronic vectors. Briefly, LC and HC with the SV40-polyA fragment were controlled using CMV / ADL, CMV / GAPDH, or CMV / EF1 promoter / enhancer fragments, subsequently driving the SRα, mPGK, SV40, or miniSV40 promoters (in order) expressing mGS-polyA (Tables 4 and 5). All fragments were unidirectionally assembled using a combination of overhang sequences to facilitate Golden Gate cloning. MSX was used at 10 (MAb1) or 25 µM (MAb2).

[0583] Table 4. Monocistronic and biscistronic carrier configurations of MAb1

[0584]

[0585] Table 5. Different carrier configurations of MAb2

[0586]

[0587] Transfection of MAb1 and MAb2 plasmids into GS KO hosts. GS KO (knockout) clone cell hosts derived from the CHO-K1 parental host were used to generate stable pools expressing MAb1 or MAb2. Transfection was performed every 3–4 days in shake flasks at 130 rpm, 36°C, and 5% CO2, in proprietary DMEM-F12-based medium at 0.3–0.4 × 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.

[0588] Stable pools expressing MAb1 or MAb2 were generated according to the manufacturer's protocol using Lipofectamine LTX (Gibco, Billings, Montana) and Opti-MEM I serum-depleted medium (Gibco, Billings, Montana). Single transfection was performed for each vector conformation. Briefly, 2 μg of each bicistronic plasmid was added in combination with 2 μg of proprietary piggybac transposase to a concentration of 4 × 10⁻⁶. 6 Transfected cells were collected in 1 host cell. For monocistronic plasmids, 1 μg of each plasmid encoding LC or HC was used. Transfected cells were recovered in 4 mL of growth medium in 6-well plates at 225 rpm, 36°C, and 5% CO2.

[0589] Selection and recovery. Forty-eight to seventy-two hours after transfection, cells were centrifuged and transferred to selection medium containing 0, 12.5, 25, and 50 µM MSX, glutamine-free. Cells were then cultured every 3–4 days at approximately 0.5–0.8 × 10⁶ 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 0.4–0.3 × 10⁻⁶ cells / mL. 6 Cells / mL.

[0590] Feed-in batch production. The recovered cells are used for production at 1 × 10⁻⁶ batches. 6 Cells / mL were seeded in 24-well culture blocks and produced in 4-mL fed-batch production cultures in 50-mL conical TPP TubeSpin® bioreactor tubes, or in 40-mL fed-batch production cultures, harvested after 10 days. During production, Vi-Cell was used. ® A counter (Beckman Coulter, Brea, California) was used to monitor viable cell density and viability, with the culture medium changed on days 3, 6, and 8. On day 10, viable cell density and viability were measured using a ForteBio OCTET equipped with a protein A biosensor. ® Red, titers were determined using conditioned media from these batches. Following harvest, Fc-containing proteins in the cell culture medium were purified using ProA affinity capture (1 ml HiTrap MabSelect SuRe column, GE Life Sciences), eluted with 100 mM sodium acetate (pH 3.6), and immediately buffer-exchanged to 10 mM sodium acetate, 150 mM NaCl (pH 5.2) (using a 5 ml HiTrap desalting column, GE Life Sciences), as previously described (Gong et al., 2021, MAbs [Monoclonal Antibodies] 13(1): 1870058). ProA yield was calculated by measuring absorbance at 280 nm (A280).

[0591] Results of MAb1

[0592] Figures 13A-13B The results show that higher titers and proA yields were obtained by using a combination of the CMV / GADPH promoter for light chain (HC) and heavy chain (LC) in a bicistronic vector with the SRα, mPGK, SV40, or miniSV40 promoter for glutamine synthase (GS) at 10 µM MSX.

[0593] in addition, Figure 14The percentage of the main peak (100% - main peak) of the SEC is shown for all evaluated vector configurations. Impurities (100% - main peak) are comparable across all vector configurations.

[0594] Results of MAb2

[0595] Figure 15 The results showed the highest titers obtained at 25 µM MSX using a combination of the CMV / GADPH promoter for light chain (HC) and heavy chain (LC) and the mPGK promoter for glutamine synthase (GS).

[0596] in addition, Figures 16A-16B The percentage of the main peak from SEC (A) and the percentage of the main peak from non-reducing capillary electrophoresis (nrMCE) are shown (B). Impurities were comparable in the highest titer carrier configurations evaluated (product quality for lower titer configurations was not tested).

[0597] Example 5. Carrier engineering strategies for improving the productivity of B2- and C2-hmab containing UniDab

[0598] Overview

[0599] Establishing stable Chinese hamster ovary (CHO) cell lines that generate therapeutic recombinant antibodies and antibody-derived molecules involves integrating heavy and light chains (HC and LC, respectively) from one or more expression vectors into the genome via a selection process. In glutamine synthase knockout (GS KO) expression systems, the selection of the stable pool can be controlled by balancing the expression level of the exogenous GS gene with the concentration of its specific inhibitor, L-methionine sulfoxide (MSX).

[0600] This example describes vector optimization using promoters of varying strengths to modulate the expression levels of the HC-UniDab, LC, and GS genes in combinations of different MSX concentrations to improve the production of bispecific antibodies in CHO cells. The vector engineering strategies described herein can be extended to improve and optimize the production of other recombinant proteins typically expressed in stable pools.

[0601] Materials and Methods

[0602] B2- and C2-Hmab plasmids were generated. The coding sequences for HC-UniDab and LC were inserted into the PBGS plasmid backbone using Golden Gate cloning. HC-Unidab and LC with the SV40-polyA fragment were controlled using the CMV / adL and CMV / GapdH promoter / enhancer fragments, subsequently driving the SRα or mpGK promoter for mGS-polyA expression (Table 6). All fragments were unidirectionally assembled using combinations of overhang sequences to promote Golden Gate cloning. MSX was used at a concentration of 18.75 or 37.5 uMMSX, depending on the GS promoter.

[0603] Table 6. Different carrier configurations

[0604]

[0605] Transfection of plasmids into GS KO hosts. GS KO (knockout) clone cell hosts derived from the CHO-K1 parental host were used to generate stable pools expressing B2-Hmab and C2-Hmab. Host cells were passaged every 3–4 days in shake flasks at 120 rpm, 36°C, and 5% CO2 in proprietary DMEM-F12-based medium at a seeding density of 0.4–0.3 × 10⁶ cells / mL. Twenty-four hours prior to transfection, the cells were passaged at a seeding 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.

[0606] Stable pools expressing B2- and C2-HmAb were generated using Gene Pulser XCell (Bio-Rad Laboratories, Hercules, CA) according to the manufacturer's protocol. Four transfections were performed for each vector conformation. Briefly, 20 μg of each plasmid was combined with 5 μg 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 a 50 mL centrifuge tube at 225 rpm, 36°C and 5% CO2.

[0607] Selection and recovery. Seventy-two hours after transfection, cells were centrifuged and transferred to selection medium without glutamine and containing 18.75 or 37.5 µM MSX. Cells were then rotated every 3–4 days at approximately 1–2 × 10⁶ 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 0.4–0.3 × 10⁻⁶ cells / mL. 6 Cells / mL.

[0608] Feed-in batch production. Fully recovered cells are incubated in 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, 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), reducing and non-reducing capillary electrophoresis (rCE- and 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.

[0609] result

[0610] Higher titers of B2-Hmab and C2-Hmab molecules were observed at 37.5 µM MSX and 18.75 µM MSX, respectively, using a combination of CMV / GAPDH for the light chain (LC) and heavy chain (HC-Unidab) with an SRα or mPGK promoter for glutamine synthase (GS). Figure 17A ) and cell unit productivity ( Figure 17B ). The titer ( Figure 17A (This is relative to CMV / adL and SRa-GS pool normalization.)

[0611] Aggregates ( Figure 18A ), some types ( Figure 18B ) and shears ( Figure 18C The impurities in different carrier configurations are comparable.

[0612] During the fed-batch culture process, the tanks exhibited similar viability ( Figure 19A ), live cell density ( Figure 19B ) and total viable cell density ( Figure 19C ).

Claims

1. An expression cassette comprising a polynucleotide sequence containing the following elements in a 5' to 3' sequence: a) A first copy of the GAPDH promoter, operatively linked to a nucleotide sequence encoding a first antibody chain or antibody chain fusion, followed by a first polyA signal sequence; b) A second copy of the GAPDH promoter, operatively linked to a nucleotide sequence encoding a second antibody chain or antibody chain fusion, followed by a second polyA signal sequence; and c) A promoter operatively linked to a nucleotide sequence encoding a selectable marker, followed by a third poly-A signal sequence. Where the first antibody chain or antibody chain fusion is an antibody light chain, the second antibody chain or antibody chain fusion is an antibody heavy chain or antibody heavy chain fusion. Where the first antibody chain or antibody chain fusion is an antibody heavy chain or antibody heavy chain fusion, the second antibody chain or antibody chain fusion is an antibody light chain, and The antibody heavy chain fusion is selected from the following groups: heavy chain-scFv fusion, heavy chain-cytokine fusion, and heavy chain VH fusion.

2. The expression cassette of claim 1, wherein the first antibody chain or antibody chain fusion is an antibody light chain, and the second antibody chain or antibody chain fusion is an antibody heavy chain.

3. The expression cassette of claim 1, wherein the first antibody chain or antibody chain fusion is an antibody light chain, and the second antibody chain or antibody chain fusion is an antibody heavy chain fusion.

4. The expression cassette of claim 1, wherein the first antibody chain or antibody chain fusion is an antibody heavy chain, and the second antibody chain or antibody chain fusion is an antibody light chain.

5. The expression cassette of claim 1, wherein the first antibody chain or antibody chain fusion is an antibody heavy chain fusion, and the second antibody chain or antibody chain fusion is an antibody light chain.

6. The expression cassette according to any one of claims 1, 3, or 5, wherein the antibody heavy chain fusion is a heavy chain-scFv fusion.

7. The expression cassette according to any one of claims 1, 3, or 5, wherein the antibody heavy chain fusion is a heavy chain VH fusion.

8. The expression cassette according to any one of claims 1-7, wherein the optional marker is glutamine synthase or dihydrofolate reductase.

9. The expression cassette according to any one of claims 1-8, wherein the optional marker is glutamine synthase.

10. The expression cassette according to any one of claims 1-9, wherein the GAPDH promoter is a CMV / GAPDH promoter.

11. The expression box of claim 10, wherein the CMV promoter enhancer is at the 5' of the GAPDH promoter.

12. The expression cassette according to any one of claims 1-11, wherein the GAPDH promoter comprises the nucleotide sequence of SEQ ID NO:

1.

13. The expression cassette of any one of claims 1-12, wherein the promoter operatively linked to the nucleotide sequence encoding the selectable marker is selected from the group consisting of mPGK, SRα, and SV40 promoters.

14. The expression cassette according to any one of claims 1-13, wherein each of the first polyA signal sequence, the second polyA signal sequence, and the third polyA signal sequence is independently selected from the group consisting of: bovine growth hormone (BGH) polyA signal sequence, thymidine kinase polyA (TKpA) signal sequence, rabbit β-globin polyA signal sequence, and simian virus 40 (SV40) early polyA signal sequence.

15. The expression cassette of any one of claims 1-14, wherein each of the first polyA signal sequence, the second polyA signal sequence, and the third polyA signal sequence is an early polyA signal sequence of simian virus 40 (SV40).

16. The expression cassette of claim 1, wherein the expression cassette encodes an antibody light chain and an antibody heavy chain, the GAPDH promoter is a CMV / GAPDH promoter, the selectable marker is glutamine synthase, the promoter operatively linked to the nucleotide sequence encoding the selectable marker is mPGK, and each of the first polyA signal sequence, the second polyA signal sequence, and the third polyA signal sequence is an early polyA signal sequence of simian virus 40 (SV40).

17. The expression cassette of claim 1, wherein the expression cassette encodes a fusion of an antibody light chain and an antibody heavy chain, the GAPDH promoter is a CMV / GAPDH promoter, the selectable marker is glutamine synthase, the promoter operatively linked to the nucleotide sequence encoding the selectable marker is SRα, and each of the first polyA signal sequence, the second polyA signal sequence, and the third polyA signal sequence is an early polyA signal sequence of simian virus 40 (SV40).

18. The expression cassette of claim 17, wherein the antibody heavy chain fusion is a heavy chain-scFv fusion.

19. The expression cassette of claim 17, wherein the antibody heavy chain fusion is a heavy chain VH fusion.

20. An expression vector comprising an expression cassette as claimed in any one of claims 1-19.

21. A pair of expression carriers, wherein: 1) The first expression vector contains an expression cassette containing a polynucleotide sequence comprising the following elements in a 5' to 3' sequence: a) The first copy of the GAPDH promoter, which is operatively linked to a nucleotide sequence encoding an antibody light chain, followed by a first polyA signal sequence; b) A second copy of the GAPDH promoter, operatively linked to a nucleotide sequence encoding the antibody heavy chain, followed by a second polyA signal sequence; and c) A promoter operatively linked to a nucleotide sequence encoding a selectable marker, followed by a third poly-A signal sequence; and 2) The second expression vector contains an expression cassette containing a polynucleotide sequence comprising the following elements in a 5' to 3' sequence: a) The first copy of the GAPDH promoter, which is operatively linked to a nucleotide sequence encoding an antibody light chain, followed by a first polyA signal sequence; b) A second copy of the GAPDH promoter, operatively linked to a nucleotide sequence encoding an antibody heavy chain fusion protein, followed by a second polyA signal sequence; and c) A promoter operatively linked to a nucleotide sequence encoding an optional marker, followed by a third poly-A signal sequence.

22. The expression vector pair of claim 21, wherein the antibody light chain of the first expression vector is identical to the antibody light chain of the second expression vector, and the antibody heavy chain of the first expression vector is identical to the antibody heavy chain portion of the antibody heavy chain fusion of the second expression vector.

23. The expression vector pair of claim 21, wherein the antibody light chain of the first expression vector is different from the antibody light chain of the second expression vector, and the antibody heavy chain of the first expression vector is different from the antibody heavy chain portion of the antibody heavy chain fusion of the second expression vector.

24. The expression vector pair according to any one of claims 21-23, wherein the antibody heavy chain fusion is a heavy chain-scFv fusion.

25. The expression vector pair according to any one of claims 21-24, wherein each of the first polyA signal sequence, the second polyA signal sequence, and the third polyA signal sequence on the first expression vector and the second expression vector is an early polyA signal sequence of simian virus 40 (SV40), the promoter on the first expression vector operably linked to a nucleotide sequence encoding a selectable marker is SRα, the promoter on the second expression vector operably linked to a nucleotide sequence encoding a selectable marker is SRα, and the selectable marker on the first expression vector and the selectable marker on the second expression vector are glutamine synthases.

26. A pair of expression carriers, wherein: 1) The first expression vector contains an expression cassette containing a polynucleotide sequence comprising the following elements in a 5' to 3' sequence: a) A first copy of the GAPDH promoter, which is operatively linked to a nucleotide sequence encoding a first antibody light chain, followed by a first polyA signal sequence; b) A second copy of the GAPDH promoter, operatively linked to a nucleotide sequence encoding a first antibody heavy chain, followed by a second polyA signal sequence; and c) A promoter operatively linked to a nucleotide sequence encoding a selectable marker, followed by a third poly-A signal sequence; and 2) The second expression vector contains an expression cassette containing a polynucleotide sequence comprising the following elements in a 5' to 3' sequence: a) A first copy of the GAPDH promoter, which is operatively linked to a nucleotide sequence encoding a second antibody light chain, followed by a first polyA signal sequence; b) A second copy of the GAPDH promoter, operatively linked to a nucleotide sequence encoding a second antibody heavy chain, followed by a second polyA signal sequence; and c) A promoter operatively linked to a nucleotide sequence encoding an optional marker, followed by a third poly-A signal sequence.

27. The expression vector pair as described in claim 26, wherein the expression vector pairs encode heterologous IgG.

28. The expression vector pair of claim 26 or claim 27, wherein each of the first polyA signal sequence, the second polyA signal sequence, and the third polyA signal sequence on the first and second expression vectors is an early polyA signal sequence of simian virus 40 (SV40), the promoter on the first expression vector operably linked to a nucleotide sequence encoding a selectable marker is mPGK, the promoter on the second expression vector operably linked to a nucleotide sequence encoding a selectable marker is mPGK, and the selectable marker on the first and second expression vectors is glutamine synthase.

29. The expression vector pair according to any one of claims 21-28, wherein the GAPDH promoter on the first expression vector is a CMV / GAPDH promoter, and the GAPDH promoter on the second expression vector is a CMV / GAPDH promoter.

30. The expression vector pair of claim 29, wherein the CMV promoter enhancer is located at the 5' of the GAPDH promoter.

31. The expression vector pair according to any one of claims 21-30, wherein the GAPDH promoter on the first expression vector and / or the second expression vector comprises the nucleotide sequence of SEQ ID NO:

1.

32. A mammalian host cell comprising the expression vector as described in claim 20 or a pair of expression vectors as described in any one of claims 21-31.

33. The mammalian host cell as described in claim 32, wherein it is a Chinese hamster ovary (CHO) cell.

34. The mammalian host cell of claim 33, wherein the CHO cell is a glutamine synthase (GS) knockout CHO cell.

35. A method for producing an antibody form, the method comprising culturing a mammalian host cell as described in any one of claims 32-34 under conditions for producing the antibody form, and recovering the antibody form from the culture.

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