Expression vector system with two light chain copies

Abstract

This article discloses an expression system for generating multispecific antibodies with only one Fab or with two identical Fabs, a host cell containing such an expression system, and a method for generating multispecific antibodies using such an expression system, including but not limited to Chinese hamster ovary (CHO) cells.

Description

Cross-reference to related applications

[0001] This application claims the priority benefit of U.S. Provisional Patent Application No. 63 / 623,121, filed January 19, 2024, which is hereby incorporated by reference in its entirety. Technical Field

[0002] This disclosure provides an expression system for generating multispecific antibodies having only one Fab or having two identical Fabs, a host cell containing such an expression system, and a method for generating multispecific antibodies using such an expression system, the 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: 10837-WO01-SEC, Creation date: January 13, 2025; Size: 11,756 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 production strategies for biologics are 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. The production of novel antibody forms, such as multispecific antibodies with three unique antibody chains, is even more complex. Although these costs are decreasing due to improvements in various aspects of production, they can still be prohibitive when widely adopted as first-line therapies.

[0006] To make biotherapeutic agents more accessible to patients, reducing the commodity cost of the manufacturing process is an attractive proposition. One way to achieve this is by increasing the titer associated with the production cell line. Proper vector conformation can help optimize the expression levels of different strands in recombinant proteins, particularly for three-stranded molecules, resulting in more balanced strand expression, reduced impurities, and higher product quality.

[0007] U.S. Patent No. 11,396,557 describes the expression of bispecific antibodies with two different Fabs expressed by two vectors having a common light chain and a heavy chain with different heavy chain variable regions.

[0008] However, there remains a need for expression vector systems that, when transfected into host cell lines, produce recombinant proteins at high titers with minimal or potential improvement in product quality properties, and that include expression vector systems for recombinant proteins with the same Fab region or only one Fab region. Such expression vector systems would be beneficial for the process development of biopharmaceuticals. Summary of the Invention

[0009] This disclosure provides an expression system comprising: 1) a first expression vector comprising a polynucleotide sequence comprising, in a 5' to 3' order, the following elements: a) a first promoter operably linked to a nucleotide sequence encoding a light chain, followed by a polyadenylation (polyA) signal sequence; b) a second promoter operably linked to a nucleotide sequence encoding a heavy chain, a heavy chain fusion, or an Fc fusion, followed by a second polyA signal sequence; and c) a third promoter operably linked to a nucleotide sequence encoding a selectable marker, followed by a third polyA signal sequence; and 2) a second expression vector comprising a polynucleotide sequence comprising, in a 5' to 3' order, the following elements: a) a first promoter operably linked to a nucleotide sequence encoding an identical copy of a light chain, followed by a polyA signal sequence; b) a second promoter operably linked to a nucleotide sequence encoding a heavy chain, a heavy chain fusion, or an Fc fusion, followed by a second polyA signal sequence; and c) A third promoter, operatively linked to a nucleotide sequence encoding an selectable marker, followed by a third polyA signal sequence, wherein when expressed in a host cell, the expression system produces a multispecific antibody having only one Fab or having two identical Fabs.

[0010] In some embodiments, a multispecific antibody has only one Fab.

[0011] In some embodiments, the multispecific antibody has two identical Fabs.

[0012] In some embodiments, the multispecific antibody is a trispecific antibody. For example, in some embodiments, the trispecific antibody has 1) a first heavy chain fusion body having a VH or cytokine fused to the N-terminus or C-terminus of the heavy chain portion of the first heavy chain fusion body; and 2) a second heavy chain fusion body having an scFv fused to the N-terminus of the heavy chain portion of the second heavy chain fusion body.

[0013] In some embodiments, the trispecific antibody has 1) a first heavy chain fusion body having a VH fused to the N-terminus or C-terminus of the heavy chain portion of the first heavy chain fusion body; and 2) a second heavy chain fusion body having an scFv fused to the N-terminus of the heavy chain portion of the second heavy chain fusion body. In some embodiments, the trispecific antibody has 1) a first heavy chain fusion body having a VH fused to the N-terminus of the heavy chain portion of the first heavy chain fusion body; and 2) a second heavy chain fusion body having an scFv fused to the N-terminus of the heavy chain portion of the second heavy chain fusion body. In some embodiments, the trispecific antibody has 1) a first heavy chain fusion body having a VH fused to the C-terminus of the heavy chain portion of the first heavy chain fusion body; and 2) a second heavy chain fusion body having an scFv fused to the N-terminus of the heavy chain portion of the second heavy chain fusion body.

[0014] In some embodiments, the trispecific antibody comprises 1) a first heavy chain fusion body having a cytokine fused to the N-terminus or C-terminus of the heavy chain portion of the first heavy chain fusion body; and 2) a second heavy chain fusion body having an scFv fused to the N-terminus of the heavy chain portion of the second heavy chain fusion body. In some embodiments, the trispecific antibody comprises 1) a first heavy chain fusion body having a cytokine fused to the N-terminus of the heavy chain portion of the first heavy chain fusion body; and 2) a second heavy chain fusion body having an scFv fused to the N-terminus of the heavy chain portion of the second heavy chain fusion body. In some embodiments, the trispecific antibody comprises 1) a first heavy chain fusion body having a cytokine fused to the C-terminus of the heavy chain portion of the first heavy chain fusion body; and 2) a second heavy chain fusion body having an scFv fused to the N-terminus of the heavy chain portion of the second heavy chain fusion body.

[0015] In some embodiments, the first expression vector is a mammalian expression vector. In some embodiments, the second expression vector is a mammalian expression vector. In some embodiments, both the first and second expression vectors are mammalian expression vectors.

[0016] In some embodiments, each heavy chain fusion or Fc fusion is independently a fusion of a heavy chain or Fc with VH, scFv, or a cytokine, wherein the VH, scFv, or cytokine is fused to the N-terminus or C-terminus of the heavy chain portion of the heavy chain fusion or the Fc portion of the Fc fusion, or between CH1 and CH2 of the heavy chain portion of the heavy chain fusion. Fusion can be direct or via a linker.

[0017] In some embodiments, each heavy chain fusion compound is a fusion of a heavy chain and a VH. In some embodiments, each heavy chain fusion compound is a fusion of a heavy chain and an scFv. In some embodiments, each heavy chain fusion compound is a fusion of a heavy chain and a cytokine.

[0018] In some embodiments, each heavy chain fusion compound is a direct fusion of the heavy chain and VH. In some embodiments, each heavy chain fusion compound is a direct fusion of the heavy chain and scFv. In some embodiments, each heavy chain fusion compound is a direct fusion of the heavy chain and cytokines.

[0019] In some embodiments, each 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, each 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, each 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.

[0020] In some embodiments, each Fc fusion is a fusion of Fc and VH. In some embodiments, each Fc fusion is a fusion of Fc and scFv. In some embodiments, each Fc fusion is a fusion of Fc and cytokines.

[0021] In some embodiments, each Fc fusion is a direct fusion of Fc and VH. In some embodiments, each Fc fusion is a direct fusion of Fc and scFv. In some embodiments, each Fc fusion is a direct fusion of Fc and cytokines.

[0022] In some embodiments, each Fc fusion is a fusion of Fc and VH, wherein the fusion includes a linker between Fc and VH. In some embodiments, each Fc fusion is a fusion of Fc and scFv, wherein the fusion includes a linker between Fc and scFv. In some embodiments, each Fc fusion is a fusion of Fc and a cytokine, wherein the fusion includes a linker between Fc and the cytokine.

[0023] In some embodiments, the selectable markers of the first expression vector and the selectable markers of the second expression vector are the same. In some embodiments, the selectable markers of the first expression vector and the selectable markers of the second expression vector are different.

[0024] In some embodiments, the selectable biomarker for the first expression vector is glutamine synthase or dihydrofolate reductase. In some embodiments, the selectable biomarker for the second expression vector is glutamine synthase or dihydrofolate reductase. In some aspects of these embodiments, the selectable biomarker for both the first and second expression vectors is glutamine synthase.

[0025] In some embodiments, the first polyA signal sequence, the second polyA signal sequence, and the third polyA signal sequence on each expression vector are identical.

[0026] In some embodiments, at least one of the first polyA signal sequence, the second polyA signal sequence, and the third polyA signal sequence on each expression vector is different.

[0027] In some embodiments, the first polyA signal sequence, the second polyA signal sequence, and the third polyA signal sequence on each expression vector 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.

[0028] In some embodiments, a first expression vector encodes a heavy chain, and a second expression vector encodes a heavy chain fusion polymer having a VH, scFv, or cytokine fused to the C-terminus of the heavy chain portion of the heavy chain fusion polymer. In some embodiments, a first expression vector encodes a heavy chain, and a second expression vector encodes a heavy chain fusion polymer having a VH fused to the C-terminus of the heavy chain portion of the heavy chain fusion polymer. In some embodiments, a first expression vector encodes a heavy chain, and a second expression vector encodes a heavy chain fusion polymer having an scFv fused to the C-terminus of the heavy chain portion of the heavy chain fusion polymer. In some embodiments, a first expression vector encodes a heavy chain, and a second expression vector encodes a heavy chain fusion polymer having a cytokine fused to the C-terminus of the heavy chain portion of the heavy chain fusion polymer.

[0029] In some embodiments, a first expression vector encodes a heavy chain, and a second expression vector encodes an Fc fusion having an scFv fused to the N-terminus of the Fc portion of the Fc fusion.

[0030] In some embodiments, a first expression vector encodes a heavy chain fusion polymer having a VH or cytokine fused to the C-terminus of the heavy chain portion of the heavy chain fusion polymer, and a second expression vector encodes a heavy chain fusion polymer having an scFv fused to the N-terminus of the heavy chain portion of the heavy chain fusion polymer. In some embodiments, a first expression vector encodes a heavy chain fusion polymer having a VH fused to the C-terminus of the heavy chain portion of the heavy chain fusion polymer, and a second expression vector encodes a heavy chain fusion polymer having an scFv fused to the N-terminus of the heavy chain portion of the heavy chain fusion polymer. In some embodiments, a first expression vector encodes a heavy chain fusion polymer having a cytokine fused to the C-terminus of the heavy chain portion of the heavy chain fusion polymer, and a second expression vector encodes a heavy chain fusion polymer having an scFv fused to the N-terminus of the heavy chain portion of the heavy chain fusion polymer.

[0031] This disclosure also provides a mammalian host cell comprising any of the expression systems described herein. In some embodiments, the mammalian host cell is a Chinese hamster ovary (CHO) cell. In some aspects of this embodiment, the CHO cell is a dihydrofolate reductase-deficient (DHFR-) or glutamine synthetase knockout (GSKO) CHO cell. In some embodiments, the CHO cell is a dihydrofolate reductase-deficient (DHFR-) CHO cell. In some embodiments, the CHO cell is a glutamine synthetase knockout (GSKO) CHO cell.

[0032] Additionally, this disclosure provides a method for generating multispecific antibodies having only one Fab or having two identical Fabs, wherein the method comprises: introducing the expression system described herein into mammalian host cells; and culturing the mammalian host cells to generate the multispecific antibody. In some embodiments, the mammalian host cells are Chinese hamster ovary (CHO) cells. In some embodiments, the CHO cells are dihydrofolate reductase-deficient (DHFR-) CHO cells or glutamine synthetase knockout (GSKO) CHO cells. In some embodiments, the CHO cells are dihydrofolate reductase-deficient (DHFR-) CHO cells. In some embodiments, the CHO cells are glutamine synthetase knockout (GSKO) CHO cells.

[0033] This disclosure further provides a method for preparing a multispecific antibody having only one Fab or having two identical Fabs, wherein the method comprises the steps of: a) introducing two different expression vectors into a host cell, wherein a first expression vector encodes 1) a light chain and 2) a heavy chain, a heavy chain fusion, or an Fc-fusion; and a second expression vector encodes 1) the same light chain and 2) a heavy chain, a heavy chain fusion, or an Fc-fusion; and b) culturing the host cell in a mammalian cell culture under conditions in which the multispecific antibody is expressed in the expression vector, wherein the multispecific antibody has only one Fab or has two identical Fabs.

[0034] In some embodiments, each heavy chain fusion polymer or Fc fusion polymer is independently a fusion polymer with scFv, VH, or a cytokine. In some embodiments, each heavy chain fusion polymer or Fc fusion polymer is independently a fusion polymer with scFv. In some embodiments, each heavy chain fusion polymer or Fc fusion polymer is independently a fusion polymer with VH. In some embodiments, each heavy chain fusion polymer or Fc fusion polymer is independently a fusion polymer with a cytokine.

[0035] In some embodiments, each heavy chain fusion compound is a fusion of a heavy chain and a VH. In some embodiments, each heavy chain fusion compound is a fusion of a heavy chain and an scFv. In some embodiments, each heavy chain fusion compound is a fusion of a heavy chain and a cytokine.

[0036] In some embodiments, each heavy chain fusion compound is a direct fusion of the heavy chain and VH. In some embodiments, each heavy chain fusion compound is a direct fusion of the heavy chain and scFv. In some embodiments, each heavy chain fusion compound is a direct fusion of the heavy chain and cytokines.

[0037] In some embodiments, each 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, each 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, each 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.

[0038] In some embodiments, each Fc fusion is a fusion of Fc and VH. In some embodiments, each Fc fusion is a fusion of Fc and scFv. In some embodiments, each Fc fusion is a fusion of Fc and cytokines.

[0039] In some embodiments, each Fc fusion is a direct fusion of Fc and VH. In some embodiments, each Fc fusion is a direct fusion of Fc and scFv. In some embodiments, each Fc fusion is a direct fusion of Fc and cytokines.

[0040] In some embodiments, each Fc fusion is a fusion of Fc and VH, wherein the fusion includes a linker between Fc and VH. In some embodiments, each Fc fusion is a fusion of Fc and scFv, wherein the fusion includes a linker between Fc and scFv. In some embodiments, each Fc fusion is a fusion of Fc and a cytokine, wherein the fusion includes a linker between Fc and the cytokine.

[0041] In the presence of two Fabs, although the Fabs are identical, there may be one or two heavy chain fusions, which may independently have scFv, VH, or cytokines fused at the N-terminus, C-terminus, between the CH1 and CH2 domains, or any combination thereof. In the presence of more than one heavy chain fusion, they may be identical or different.

[0042] In some embodiments, the multispecific antibody is a bispecific antibody. On one hand, the bispecific antibody has 1) a heavy chain; and 2) a heavy chain fusion or Fc fusion having scFv, VH, or cytokines, wherein the scFv, VH, or cytokines are fused to the N-terminus or C-terminus of the heavy chain portion of the heavy chain fusion or the Fc portion of the Fc fusion, or between CH1 and CH2 of the heavy chain portion of the heavy chain fusion.

[0043] In some embodiments, a multispecific antibody has only one Fab.

[0044] In some embodiments, the multispecific antibody has two identical Fabs.

[0045] In some embodiments, the multispecific antibody is a trispecific antibody. On one hand, the trispecific antibody has 1) a first heavy chain fusion body having a VH or cytokine fused to the N-terminus or C-terminus of the heavy chain portion of the first heavy chain fusion body; and 2) a second heavy chain fusion body having an scFv fused to the N-terminus of the heavy chain portion of the second heavy chain fusion body.

[0046] In some embodiments, the trispecific antibody has 1) a first heavy chain fusion body having a VH fused to the N-terminus or C-terminus of the heavy chain portion of the first heavy chain fusion body; and 2) a second heavy chain fusion body having an scFv fused to the N-terminus of the heavy chain portion of the second heavy chain fusion body. In some embodiments, the trispecific antibody has 1) a first heavy chain fusion body having a VH fused to the N-terminus of the heavy chain portion of the first heavy chain fusion body; and 2) a second heavy chain fusion body having an scFv fused to the N-terminus of the heavy chain portion of the second heavy chain fusion body. In some embodiments, the trispecific antibody has 1) a first heavy chain fusion body having a VH fused to the C-terminus of the heavy chain portion of the first heavy chain fusion body; and 2) a second heavy chain fusion body having an scFv fused to the N-terminus of the heavy chain portion of the second heavy chain fusion body.

[0047] In some embodiments, the trispecific antibody comprises 1) a first heavy chain fusion body having a cytokine fused to the N-terminus or C-terminus of the heavy chain portion of the first heavy chain fusion body; and 2) a second heavy chain fusion body having an scFv fused to the N-terminus of the heavy chain portion of the second heavy chain fusion body. In some embodiments, the trispecific antibody comprises 1) a first heavy chain fusion body having a cytokine fused to the N-terminus of the heavy chain portion of the first heavy chain fusion body; and 2) a second heavy chain fusion body having an scFv fused to the N-terminus of the heavy chain portion of the second heavy chain fusion body. In some embodiments, the trispecific antibody comprises 1) a first heavy chain fusion body having a cytokine fused to the C-terminus of the heavy chain portion of the first heavy chain fusion body; and 2) a second heavy chain fusion body having an scFv fused to the N-terminus of the heavy chain portion of the second heavy chain fusion body.

[0048] In some embodiments, each of the expression vectors contains an optional marker that may be the same or different. The optional marker may be selected from the group consisting of glutamine synthase and dihydrofolate reductase. The promoter operatively linked to the nucleotide sequence encoding the optional marker may be selected from the group consisting of mPGK, SRα, and SV40 promoters.

[0049] In some embodiments, each expression vector contains the same selectable biomarker. In some embodiments, each expression vector contains a glutamine synthase selectable biomarker. In some embodiments, each expression vector contains a dihydrofolate reductase selectable biomarker.

[0050] In some embodiments, each expression vector contains a different selectable biomarker. In some embodiments, one expression vector contains a glutamine synthase selectable biomarker, and another expression vector contains a dihydrofolate reductase selectable biomarker.

[0051] In some embodiments, the host cell is a mammalian host cell. The mammalian host cell may be a Chinese hamster ovary (CHO) cell. For example, the CHO cell may be a dihydrofolate reductase-deficient (dhfr-) or glutamine synthetase knockout (GSKO) CHO cell. In some embodiments, the CHO cell is a dihydrofolate reductase-deficient (dhfr-) CHO cell. In some embodiments, the CHO cell is a glutamine synthetase knockout (GSKO) CHO cell.

[0052] In some embodiments, the method further includes recovering antibodies from the culture. In some aspects, 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.

[0053] 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

[0054] Figure 1A-1B The diagrams show (A) schematic representations of various antibody configurations (where the Fab regions are identical or only one Fab region exists (left) and where the Fab regions are different (right)) and (B) schematic representations of vector configurations used to express a single light chain on one vector (top) and to express the same light chain on two different vectors (bottom). Figure 1A In the diagram at the top left, the recombinant protein contains two distinct heavy chains with identical Fabs targeting the same antigen, and the Fd and light chains (LCs) on either side are perfect homologous pairs. Figure 1A In the diagram at the top right, the recombinant protein contains two distinct heavy chains with different Fabs targeting two different antigens. In this diagram, the light chains in each Fab are identical, but the Fds are different, which means that at least one Fd:LC pair is non-homologous (i.e., not a perfect pair).

[0055] Figure 2A-2B A schematic diagram of (A) C1mAb and (B) carrier configuration used in Example 1 are shown.

[0056] Figures 3A-3BPool titers (in g / L) and cell productivity (qp) (in pg / cell / day) for 1xLC and 2xLC vector combinations of C1mAb-A are shown (A). Titers and qp are normalized relative to 1xLC 0 μM MSX conditions. Titers 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 the following vector configurations: one light chain (1x LC) or two light chains (2x LC). The MSX concentration used is indicated.

[0057] Figures 4A-4B Normalized effective titers (in g / L) and nrCE-SDS peak % for 1x LC and 2x LC vector combinations of C1mAb-A are shown (A) and (B). Effective titers were normalized relative to 1x LC 0 μM MSX conditions. Normalized effective titers were calculated on day 10 of fed-batch production. Data are expressed as the average of two independent transfections. Error bars represent the standard deviation (SD) of three technical replicates of fed-batch production. Data are stained according to the following vector configurations: one light chain (1x LC) or two light chains (2x LC). The MSX concentration used is indicated.

[0058] Figure 5 A schematic diagram of the carrier configuration used in Example 2 is shown.

[0059] Figures 6A-6B Normalized yields (in g / L) and nrCE-SDS peak percentages (B) of AmAb-A for combinations of 1x LC1, 1x LC2, and 2x LC carriers are shown. Yields were normalized relative to 1x LC1 MSX conditions. Normalized yields were calculated on day 10 of fed-batch production.

[0060] Figure 7 A schematic diagram of the carrier configuration used in Example 3 is shown.

[0061] Figures 8A-8B Normalized yields (in g / L) and nrCE-SDS main peak % for the 1x LC and 2x LC vector combinations of the trispecific antibody are shown (A) (B). Yields were normalized relative to 1x LC1 MSX conditions. Normalized yields were calculated on day 10 of fed-batch production.

[0062] Figure 9 A representative scheme of C1mAb asymmetric fusion is shown. Detailed Implementation

[0063] This disclosure is partly based on the finding that nucleotide sequences encoding a common or identical light chain on two expression vectors (i.e., two copies of the light chain coding sequence) and nucleotide sequences encoding two different heavy chains, heavy chain fusions, or Fc-fusions on separate vectors can be used to drive the expression of different antibody chains, thereby increasing the titer of multispecific antibodies having only one Fab or two identical Fab regions. Compared to existing methods for generating multispecific antibodies with different Fabs (i.e., different heavy chain variable regions), this disclosure provides a method for generating multispecific antibodies with only one Fab or identical Fabs. For example, a multispecific antibody may have only one light chain and an Fc fusion (and therefore only one Fab), or identical heavy chains (and thus two identical Fabs) but at least one heavy chain is modified to be a fusion with, for example, scFv, cytokines, VH, etc. Compared to expression vectors with only one copy of the light chain (where the other expression vector does not have a nucleotide sequence encoding the light chain), higher titers can be achieved by using bicistronic (not an optional marker) vectors to generate three chains of multispecific antibodies for expression in host cells.

[0064] 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 chains. Typically, for triple-chain molecules, a common light chain is used with two distinct heavy chains. The light chain is expressed along with one heavy chain on a bicistronic expression vector. The other heavy chain is expressed on a monocistronic vector. The inventors have unexpectedly discovered that by including a copy of the common or identical light chain on each expression vector, the titer of the resulting antibody structure can increase with improvements in product quality (e.g., reductions in aggregation, splicing, or the presence of undesirable isoforms).

[0065] By employing the methods, expression vectors, and host cells described herein, the yield of recombinant proteins (e.g., multispecific antibodies, such as triple-stranded molecules) can be increased while maintaining or improving product quality (e.g., as assessed by aggregation, splicing, or the presence of isotypes). Furthermore, such methods, expression vectors, and host cells can enable the production of biopharmaceuticals in a cheaper and more consistent manner. These innovations have particular utility in the commercial production of antibody forms with three distinct chains.

[0066] The methods described herein employ 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 two expression vectors (each containing nucleotide sequences encoding two antibody chains (having a common light chain)) 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).

[0067] definition

[0068] 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.

[0069] 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.

[0070] 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.

[0071] This disclosure provides tools for expressing a “target protein” (such as, for example, a multispecific antibody). A “target protein” includes recombinant proteins and engineered proteins (e.g., proteins that do not exist in nature and have been designed and / or produced by humans). The target protein may be, but does not have to be, a protein known or suspected of having therapeutic relevance.

[0072] As used herein, “antibody chain” or “chain” refers to an antibody light chain, antibody heavy chain, antibody heavy chain fusion protein, scFv-Fc fusion, VH fusion, cytokine fusion, etc. The terms “antibody heavy chain” and “antibody light chain” have their standard meaning in the art and include, for example, the various antibody heavy and light chains described elsewhere herein (e.g., heavy and light chains of IgG1, IgG2, IgG3, and IgG4 mAbs). The terms “antibody heavy chain” and “antibody light chain” include standard full-length antibody heavy and light chains. The terms “antibody heavy chain fusion” 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” can be an antibody heavy chain covalently linked to a cytokine. “Fusion” or “fused” means that the link can be direct or via a peptide linker (e.g., a glycine-serine linker). In an antibody heavy chain fusion protein, the antibody heavy chain can be linked to one or more additional proteins at the N-terminus or C-terminus (or both) of the heavy chain. Antibody heavy chains can also be linked to additional protein sequences, such as scFv, at internal amino acid residues or between Fab and Fc. As used herein, "antibody fusion protein" refers to an antibody as provided herein, covalently linked to one or more additional proteins or peptides (e.g., via the heavy or light chain of the antibody). Therefore, an antibody fusion protein contains at least one antibody heavy chain fusion protein or antibody light chain fusion protein as one of the peptides of the antibody fusion protein. Most commonly, an antibody fusion protein is a molecule 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.

[0073] For the purposes of the expression vector systems, host cells, and methods described herein, the triple-stranded molecule comprises 1) a common or identical light chain (i.e., a single light chain expressed on two bicistronic vectors) and a common heavy chain (where one or both of these heavy chains are part of a heavy chain fusion protein), and 2) a light chain, a heavy chain, and an Fc fusion that binds only to one arm of the antibody (i.e., a heavy chain lacking the Fd region but fused to the protein). scFv, cytokines, or VH regions may be added to either end of the heavy chain or the Fc (i.e., to the free end of the variable region or the free end of the constant region in the case of the heavy chain), or to either end of the Fc. Figure 1A The left side provides example antibody molecules that can be produced by the method of the present invention. Figure 1A The right side depicts different common LC methods for the variable region of the heavy chain.

[0074] 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 acid, as well as naturally occurring amino acid polymers. These terms may also cover amino acid polymers that have been modified, for example, by adding carbohydrate residues to form glycoproteins or by phosphorylation. Peptides and proteins may be produced by naturally occurring and non-recombinant cells, or by genetically engineered or recombinant cells. Peptides and proteins may comprise molecules having the amino acid sequence of a natural protein, or molecules having one or more amino acids with a natural sequence that have been omitted, added, and / or substituted.

[0075] 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.

[0076] 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.

[0077] 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.

[0078] 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.

[0079] 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).

[0080] 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.

[0081] 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.

[0082] "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.

[0083] "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.

[0084] “Growth” cell culture medium refers to a cell culture medium typically used during the exponential growth phase (“growth phase”) and sufficiently complete to support cell culture during this phase. Growth cell culture media may also contain selectants that confer resistance or viability to host cell lines using selectable markers. Such selectants include, but are not limited to, genimycin (G418), blast fungicide, neomycin, hygromycin B, puromycin, bleomycin, methionine sulfoxide, methotrexate, glutamine-free cell culture media, glycine-deficient cell culture media, hypoxanthine and thymidine, or thymidine alone.

[0085] "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.

[0086] “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.

[0087] 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.

[0088] 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.

[0089] 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.

[0090] "Serium-free" refers to cell media that do not contain animal serum, such as fetal bovine serum. Various tissue media (including defined media) are commercially available; for example, any one or a combination of the following cell media can be used: RPMI-1640, RPMI-1641, Dürbeco Modified Eagle Medium (DMEM), Eagle Minimum Essential Medium, F-12K, Ham F12, Iskov Modified Dürbeco Medium, McCoy 5A, Leibovitz L-15, and serum-free media such as EX-CELL. TM Examples of such media include the 300 series (JRH Biosciences, Lenexa, Kansas) and MCDB 302 (Sigma Aldrich Corp., St. Louis, MO). Serum-free forms of these media are also available. Depending on the needs of the cells being cultured and / or the desired cell culture parameters, cell culture media can be supplemented with additional or increased concentrations of components such as amino acids, salts, sugars, vitamins, hormones, growth factors, buffers, antibiotics, lipids, and trace elements. Custom-made cell culture media are also available.

[0091] "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.

[0092] 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.

[0093] 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”.

[0094] Expression vector configuration

[0095] Typical antibodies are Y-type 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 with three chains require alternative forms and are typically expressed by two different vectors. See, for example, Spiess et al. 2015, Mol. Immunol. [Molecular Immunology] 67:95-106; Brinkmann et al., 2017, MAbs [Monoclonal Antibodies] 9:192-212; and Ma et al., 2021, Frontiers in Immunology [Frontiers in Immunology] 12:626616.

[0096] Expression vectors typically include one or more promoters that are recognized by the host organism and operatively linked to a nucleotide sequence encoding a target protein. A promoter is a non-transcribed sequence (typically within approximately 100 to 1000 bp) located upstream (i.e., 5') of the start codon of a structural gene and controls the transcription of the structural gene.

[0097] Expression systems for triple-chain antibodies typically comprise two expression vectors: 1) a first vector having a first promoter driving the expression of a first nucleotide sequence encoding the common antibody light chain and a second promoter driving the expression of a second nucleotide sequence encoding the first antibody heavy chain (and a promoter driving the expression of a coding sequence encoding a selectable biomarker); and 2) a second vector having a third promoter driving the expression of the second antibody heavy chain (and a promoter driving the expression of a coding sequence encoding a selectable biomarker). This vector configuration is as follows: Figure 1B It is depicted as a 1x LC vector configuration. In some embodiments of this disclosure, the term "expression system" may be replaced with "expression vector system".

[0098] This disclosure provides an expression system for a three-chain antibody, comprising two expression vectors, wherein 1) a first vector has a first promoter driving the expression of a first nucleotide sequence encoding a common or identical antibody light chain and a second promoter driving the expression of a heavy chain, heavy chain fusion, or Fc fusion (and a third promoter driving the expression of a coding sequence encoding a selectable biomarker), and 2) a second vector has a first promoter driving the expression of a first nucleotide sequence encoding a common or identical antibody light chain and a second promoter driving the expression of a heavy chain, heavy chain fusion, or Fc fusion (and a third promoter driving the expression of a coding sequence encoding a selectable biomarker). This vector configuration is as follows: Figure 1B The diagram depicts an exemplary 2xLC vector configuration. Such a triple-chain antibody / molecule may include 1) a common or identical light chain (i.e., a single light chain expressed on two bicistronic vectors) and a common heavy chain, wherein one or both of these heavy chains are heavy chain fusion proteins, and 2) a light chain, a heavy chain, and a heavy chain lacking an Fd region that binds only to one arm of the antibody. scFv, cytokines, or VH / VHH regions may be added to either end of the heavy chain, i.e., to the free end of the variable region or the free end of the constant region.

[0099] The exemplary 2x LC vector configuration described above can be modified by different promoter selections. For example, a single promoter can be used for both the common light chain and the two heavy chains, i.e., the first and second promoters are the same on each expression vector. In some embodiments, the first promoter is the same on each vector, and the second promoter is the same on each vector, but the first and second promoters are different.

[0100] 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. An example of such a sequence is the adenovirus triplet leader sequence (ADL) (see Gingeras et al., 1982, J. Biol. Chem. 257:13475-91; GenBank accession number J01917).

[0101] In some embodiments, the first promoter on the first expression vector is a GAPDH promoter. In some embodiments, the second promoter on the first expression vector is a GAPDH promoter. In some embodiments, the first promoter on the first expression vector is a GAPDH promoter, and the second promoter on the first expression vector is a GAPDH promoter.

[0102] In some embodiments, the first promoter on the second expression vector is a GAPDH promoter. In some embodiments, the second promoter on the second expression vector is a GAPDH promoter. In some embodiments, the first promoter on the second expression vector is a GAPDH promoter, and the second promoter on the second expression vector is a GAPDH promoter.

[0103] In some embodiments, the first promoter on the first expression vector is a GAPDH promoter, the second promoter on the first expression vector is a GAPDH promoter, the first promoter on the second expression vector is a GAPDH promoter, and the second promoter on the second expression vector is a GAPDH promoter.

[0104] In some embodiments, the first and / or second promoter of each expression vector 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, and this combination can function as a better promoter than a single GAPDH promoter. In some embodiments, the CMV promoter is at the 5' of the GAPDH. Overall, CMV / GAPDH is the promoter.

[0105] In some embodiments, the CMV / GAPDH promoter comprises 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical polynucleotide sequences to the polynucleotide sequence of SEQ ID NO: 1. In some embodiments, the CMV / GAPDH promoter comprises the polynucleotide sequence of SEQ ID NO: 1.

[0106] In some embodiments, the first promoter on the first expression vector is a CMV / GAPDH promoter. In some embodiments, the second promoter on the first expression vector is a CMV / GAPDH promoter. In some embodiments, the first promoter on the first expression vector is a CMV / GAPDH promoter, and the second promoter on the first expression vector is a CMV / GAPDH promoter.

[0107] In some embodiments, the first promoter on the second expression vector is a CMV / GAPDH promoter. In some embodiments, the second promoter on the second expression vector is a CMV / GAPDH promoter. In some embodiments, the first promoter on the second expression vector is a CMV / GAPDH promoter, and the second promoter on the second expression vector is a CMV / GAPDH promoter.

[0108] In some embodiments, the first promoter on the first expression vector is a CMV / GAPDH promoter, the second promoter on the first expression vector is a CMV / GAPDH promoter, the first promoter on the second expression vector is a CMV / GAPDH promoter, and the second promoter on the second expression vector is a CMV / GAPDH promoter.

[0109] In some embodiments, the first promoter on the second expression vector is a CMV promoter. In some embodiments, the second promoter on the second expression vector is a CMV promoter. In some embodiments, the first promoter on the second expression vector is a CMV promoter, and the second promoter on the second expression vector is a CMV promoter.

[0110] In some embodiments, the first promoter on the first expression vector is a CMV promoter, the second promoter on the first expression vector is a CMV promoter, the first promoter on the second expression vector is a CMV promoter, and the second promoter on the second expression vector is a CMV promoter.

[0111] In the embodiments described herein, the heavy chains (HC1 and HC2) are different, but 1) have the same heavy chain Fd region, or 2) one heavy chain lacks an Fd region (i.e., an Fc). Therefore, in case 1), the different heavy chains have the same Fab targeting the same antigen when paired with a common light chain. Although the heavy chains have the same heavy chain variable region, one or both heavy chains fuse to an additional amino acid sequence. Such an additional amino acid sequence can be a heavy chain variable region, scFv, or a cytokine. In case 2), one heavy chain does not form a Fab because one of the chains lacks an Fd region. In both cases, only one Fab type exists in the molecule.

[0112] In the above expression vector, the polyA signal sequence can be after each gene (i.e., the coding sequences for the first and second antibody chains 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, second, and third polyA signal sequences on each expression vector 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.

[0113] In some embodiments, the first polyA signal sequence, the second polyA signal sequence, and the third polyA signal sequence on each expression vector are bovine growth hormone (BGH) polyA signal sequences.

[0114] In some embodiments, the first polyA signal sequence, the second polyA signal sequence, and the third polyA signal sequence on each expression vector are all thymidine kinase polyA (TKpA) signal sequences.

[0115] In some embodiments, the first polyA signal sequence, the second polyA signal sequence, and the third polyA signal sequence on each expression vector are all rabbit β-globin polyA signal sequences.

[0116] In some embodiments, the first polyA signal sequence, the second polyA signal sequence, and the third polyA signal sequence on each expression vector are all early polyA signal sequences of simian virus 40 (SV40).

[0117] In some embodiments, the BGH polyA signal sequence comprises 90%, 95%, 96%, 97%, 98%, 99%, or 100% the same polynucleotide sequence as SEQ ID NO: 5. In some embodiments, the BGH polyA signal sequence comprises the polynucleotide sequence of SEQ ID NO: 5.

[0118] In some embodiments, the rabbit β-globin polyA signal sequence comprises 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical polynucleotide sequences to the polynucleotide sequence of SEQ ID NO: 6. In some embodiments, the rabbit β-globin polyA signal sequence comprises the polynucleotide sequence of SEQ ID NO: 6.

[0119] In some embodiments, the early polyA signal sequence of SV40 comprises 90%, 95%, 96%, 97%, 98%, 99%, or 100% the same polynucleotide sequence as the polynucleotide sequence of SEQ ID NO: 7. In some embodiments, the early polyA signal sequence of SV40 comprises the polynucleotide sequence of SEQ ID NO: 7.

[0120] In some embodiments, the thymidine kinase polyA (TKpA) signal sequence comprises 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical polynucleotide sequence to the polynucleotide sequence of SEQ ID NO: 8. In some embodiments, the thymidine kinase polyA (TKpA) signal sequence comprises the polynucleotide sequence of SEQ ID NO: 8.

[0121] In one embodiment, the antibody produced by the method described herein is in the form of C1mAb (Fab-heterologous Fc-[scFv)). Asymmetric fusion, or {scFv-Fab [Fab]-heterologous Fc-[protein]). For example, in a triple-stranded form, one expression cassette contains coding sequences for one heavy chain and one light chain, and another expression cassette contains coding sequences for a heavy chain-scFv fusion and a common light chain. The first antibody chain in each expression cassette can be either 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 either a heavy chain or a heavy chain-scFv fusion.

[0122] A representative scheme for C1mAb asymmetric fusion is shown in Figure 9 middle.

[0123] The expression vectors provided in this article offer optimal expression of three-chain antibodies, likely due to the optimal chain ratio of the expressed peptide. The chain ratio can be measured using techniques well-known in the art.

[0124] It has been found that for multispecific antibodies with the same heavy chain variable region (e.g., triple-chain antibodies), expressing the common or the same light chain on two expression vectors unexpectedly leads to higher antibody yields.

[0125] Selectable markers

[0126] 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.

[0127] 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.

[0128] Other alternative gene amplification can be used to amplify the gene to be expressed. Amplification is the process of tandem replication of the gene required to produce proteins necessary for growth or cell survival 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] 2023 120:1159-1166), and promoterless thymidine kinase genes.

[0129] 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).

[0130] 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., 2012, Biotechnol Bioeng. 109(4):1007-1015). 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., 2020, Engineering in Life Sciences 20(3-4):112-125). Chain / vector expression can be affected by increasing strictness during pool recovery / selection through the addition of MSX.

[0131] 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.

[0132] In some embodiments, the optional biomarker in the expression vector (e.g., a first expression vector and / or a second expression vector) is glutamine synthase. Glutamine synthase (GS) catalyzes the biosynthesis of glutamine through the condensation of ammonia with glutamate.

[0133] 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.

[0134] As described above, this disclosure provides an expression system comprising: 1) a first expression vector comprising a polynucleotide sequence comprising, in a 5' to 3' order, a) a first promoter operably linked to a nucleotide sequence encoding a light chain, followed by a polyadenylation (polyA) signal sequence; b) a second promoter operably linked to a nucleotide sequence encoding a heavy chain, a heavy chain fusion, or an Fc fusion, followed by a second polyA signal sequence; and c) a third promoter operably linked to a nucleotide sequence encoding a selectable marker, followed by a third polyA signal sequence; and 2) a second expression vector comprising a polynucleotide sequence comprising, in a 5' to 3' order, a) a first promoter operably linked to a nucleotide sequence encoding an identical copy of a light chain, followed by a polyA signal sequence; b) a second promoter operably linked to a nucleotide sequence encoding a heavy chain, a heavy chain fusion, or an Fc fusion, followed by a second polyA signal sequence; and c) A third promoter, operatively linked to a nucleotide sequence encoding an selectable marker, followed by a third polyA signal sequence, wherein when expressed in a host cell, the expression system produces a multispecific antibody having only one Fab or having two identical Fabs.

[0135] In some embodiments, the selectable biomarker for the first expression vector is glutamine synthase. In some embodiments, the selectable biomarker for the second expression vector is glutamine synthase. In some embodiments, the selectable biomarker for both the first and second expression vectors is glutamine synthase.

[0136] In some embodiments, the third promoter on the first expression vector is an SRα promoter. In some embodiments, the third promoter on the second expression vector is an SRα promoter. In some embodiments, the third promoter on the first expression vector is an SRα promoter, and the third promoter on the second expression vector is an SRα promoter.

[0137] In some embodiments, the third promoter on the first expression vector is the mPGK promoter. In some embodiments, the third promoter on the second expression vector is the mPGK promoter. In some embodiments, the third promoter on the first expression vector is the mPGK promoter, and the third promoter on the second expression vector is the mPGK promoter.

[0138] In some embodiments, the third promoter on the first expression vector is the SV40 promoter. In some embodiments, the third promoter on the second expression vector is the SV40 promoter. In some embodiments, the third promoter on the first expression vector is the SV40 promoter, and the third promoter on the second expression vector is the SV40 promoter.

[0139] In some embodiments, the third promoter on the first expression vector is the SRα promoter, and the third promoter on the second expression vector is the mPGK promoter.

[0140] In some embodiments, the third promoter on the first expression vector is an SRα promoter, and the selectable biomarker of the first expression vector is glutamine synthase. In some embodiments, the third promoter on the second expression vector is an SRα promoter, and the selectable biomarker of the second expression vector is glutamine synthase. In some embodiments, the third promoter on both the first and second expression vectors is an SRα promoter, the selectable biomarker of the first expression vector is glutamine synthase, and the selectable biomarker of the second expression vector is glutamine synthase.

[0141] In some embodiments, the SRα promoter comprises 90%, 95%, 96%, 97%, 98%, 99%, or 100% the same polynucleotide sequence as SEQ ID NO: 2. In some embodiments, the SRα promoter comprises the polynucleotide sequence of SEQ ID NO: 2.

[0142] In some embodiments, the mPGK promoter comprises 90%, 95%, 96%, 97%, 98%, 99%, or 100% the same polynucleotide sequence as SEQ ID NO: 3. In some embodiments, the mPGK promoter comprises the polynucleotide sequence of SEQ ID NO: 3.

[0143] In some embodiments, the SV40 promoter comprises 90%, 95%, 96%, 97%, 98%, 99%, or 100% the same polynucleotide sequence as SEQ ID NO: 4. In some embodiments, the SV40 promoter comprises the polynucleotide sequence of SEQ ID NO: 4.

[0144] Additional expression vector components

[0145] The expression vectors described herein 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 the promoter / enhancer fragments, antibody chains, optional markers, and other sequences (e.g., polyadenylated sequences) specifically described above): one or more enhancer sequences, origin of replication, transcription and translation control sequences, transcription termination sequences, complete intron sequences containing donor and acceptor splicing sites, various pre-sequences / pro-sequences that improve glycosylation or yield, natural or heterologous signal sequences (lead sequences or signal peptides) for polypeptide secretion, ribosome binding sites, polyadenylated sequences, internal ribosome entry sites (IRES) sequences, expression enhancement sequence elements (EASE), triplet leader sequences (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.

[0146] 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.

[0147] 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.

[0148] 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.

[0149] 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 additional 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., globulins, elastases, albumins, alpha-fetoproteins, 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.

[0150] 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.

[0151] 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. Non-limiting examples of signal peptides that are functional in mammalian host cells include: the interleukin-7 signal sequence described in U.S. Patent No. 4,965,195; the interleukin-2 receptor signal sequence described in Cosman et al., 1984, Nature [Nature] 312:768; the interleukin-4 receptor signal peptide described in European Patent No. 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.

[0152] Additional control sequences shown to improve the expression of heterologous genes from mammalian expression vectors include elements such as expression-enhancing sequence elements (EASE) derived from CHO cells (Morris et al., in Animal Cell Technology, pp. 529-534 (1997); 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).

[0153] 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.

[0154] Table 1 provides non-limiting examples of synthetic nucleotide (DNA) sequences that can be used in certain expression vector components of the expression vectors disclosed herein.

[0155] Table 1. Non-limiting examples of expression vector component sequences

[0156]

[0157] Target protein

[0158] This disclosure provides expression systems comprising: 1) a first expression vector comprising a polynucleotide sequence comprising, in a 5' to 3' order, a) a first promoter operably linked to a nucleotide sequence encoding a light chain, followed by a polyadenylation (polyA) signal sequence; b) a second promoter operably linked to a nucleotide sequence encoding a heavy chain, a heavy chain fusion, or an Fc fusion, followed by a second polyA signal sequence; and c) a third promoter operably linked to a nucleotide sequence encoding a selectable marker, followed by a third polyA signal sequence; and 2) a second expression vector comprising a polynucleotide sequence comprising, in a 5' to 3' order, a) a first promoter operably linked to a nucleotide sequence encoding an identical copy of a light chain, followed by a polyA signal sequence; b) a second promoter operably linked to a nucleotide sequence encoding a heavy chain, a heavy chain fusion, or an Fc fusion, followed by a second polyA signal sequence; and c) A third promoter, operatively linked to a nucleotide sequence encoding a selectable marker, is followed by a third polyA signal sequence. When these expression systems are expressed in host cells (e.g., CHO cells), they produce a specific target protein, i.e., a multispecific antibody with only one Fab or two identical Fabs.

[0159] The target peptides and proteins produced using the expression system described above may have scientific or commercial significance, including protein-based therapeutics. Target proteins may include, in particular, secreted proteins, non-secreted proteins, intracellular proteins, or membrane-bound proteins. Target peptides and proteins may 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 may 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).

[0160] 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) Molecules. Non-limiting examples of antigen-binding proteins include human antibodies, humanized antibodies, chimeric antibodies, recombinant antibodies, single-chain antibodies; biantibodies; triantibodies; tetraantibodies; Fab fragments; F(ab')2 fragments; IgD antibodies; IgE antibodies; IgM antibodies; IgG1 antibodies; IgG2 antibodies; IgG3 antibodies; or IgG4 antibodies and fragments thereof. Also included are bispecific T-cell binders (BiTE). ® These include bispecific T-cell binders with extended durations (e.g., extended half-life), such as HLE BiTE molecules and heterologous Ig BITE molecules. Some of these antigen-binding protein forms can be generated using the expression vector system disclosed herein.

[0161] 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.

[0162] Antigen-binding proteins can have structures such as those of naturally occurring immunoglobulins. An immunoglobulin is a tetrameric molecule. In naturally occurring immunoglobulins, each tetramer consists of two pairs of identical polypeptide chains, each pair having a "light chain" (approximately 25 kDa) and a "heavy chain" (approximately 50-70 kDa). The amino-terminal portion of each chain includes a variable region of approximately 100 to 110 or more amino acids, which is primarily responsible for antigen recognition. The carboxyl-terminal portion of each chain defines a constant region primarily responsible for effector function. Human light chains are classified as κ light chains and λ light chains. Heavy chains are classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively.

[0163] 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).

[0164] 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 -10When M occurs, the antibody binds to the antigen with "extremely high affinity".

[0165] Unless otherwise stated, the term "antibody" includes any isotype or subclass of glycosylated and non-glycosylated immunoglobulin, or its antigen-binding region that competes with intact antibodies for specific binding. Additionally, unless otherwise stated, the term "antibody" refers to an intact immunoglobulin that competes with intact antibodies for specific binding. Unless otherwise stated, antibodies include human, humanized, chimeric, multispecific, monoclonal, polyclonal, heterologous IgG, bispecific antibodies, and oligomers. Antibodies include IgG1, IgG2, IgG3, or IgG4.

[0166] Antigen-binding fragments include Fab, Fab', F(ab')2, Fv, biantibody, Fd, dAb, macrobody, single-chain antibody molecule, 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.

[0167] 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 distinct binding sites. A standard nomenclature for multispecific antibody forms is VERITAS. See Biswas et al., 2023, mAbs [monoclonal antibodies] 15:1-9.

[0168] Fab fragments are those with V L V H C L and C H 1. A monovalent segment of a structural domain; F(ab')2 segment is a divalent segment having two Fab segments connected by a disulfide bridge in the hinge region; Fd segment has V H and C H 1. Structural domain; the Fv fragment has a V-shaped arm for the antibody. L and V H Structural domain; and the dAb fragment has V H Structural domain, V L structural domain, or V H or V LAntigen-binding fragments of the domain (US Patent Nos. 6,846,634, 6,696,245, US Patent Application Publication Nos. 2005 / 0202512, 2004 / 0202995, 2004 / 0038291, 2004 / 0009507, 2003 / 0039958, Ward et al., 1989, Nature 341:544-546).

[0169] Single-chain antibodies (scFv) are antibodies in which V L and V H Regions are linked via linkers (e.g., synthetic sequences of amino acid residues) to form continuous protein chains, where the linkers are long enough to allow the protein chains to fold back and form monovalent antigen-binding sites (see, for example, Bird et al., 1988, Science 242:423-26 and Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-83, U.S. Patents 7,741,465 and 6,319,494, and Eshhar et al., 1997, Cancer Immunol Immunotherapy 45:131-136). scFv retains the ability of the parent antibody to specifically interact with the target antigen.

[0170] 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.

[0171] 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.

[0172] 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.

[0173] 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.

[0174] In some embodiments, the target protein may include proteins that specifically bind to: one or more CD proteins, HER receptor family proteins, cell adhesion molecules, growth factors, nerve growth factor, fibroblast growth factor, transforming growth factor (TGF), insulin-like growth factor, bone-inducing factor, insulin and insulin-related proteins, coagulation and coagulation-related proteins, colony-stimulating factor (CSF), other blood and serum proteins, blood group antigens; receptors, receptor-related proteins, growth hormone, growth hormone receptor, T cell receptors; neurotrophic factors, neurotrophic proteins, relaxin, interferon, interleukin, viral antigens, lipoproteins, integrins, rheumatoid factor, immunotoxins, surface membrane proteins, transport proteins, homing receptors, addressins, regulatory proteins, and immunoadhesins.

[0175] 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.

[0176] 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 three domains are held together by hydrophobic interactions. “Fc” is a heavy chain segment of the Fc region.

[0177] Generation of mammalian host cells expressing the target protein

[0178] 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).

[0179] 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).

[0180] 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.

[0181] 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).

[0182] Vectors can be any molecule or entity (e.g., nucleic acids, plasmids, bacteriophages, transposons, granules, chromosomes, viruses, viral capsids, virions, naked DNA, complex DNA, etc.) suitable for transferring and / or transporting proteins encoding information to host cells and / or specific locations and / or compartments within host cells. 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 vector systems that can be used to express multispecific molecules, such as triple-stranded molecules, like molecules having the same heavy chain variable region. The expression vector systems of this disclosure can be introduced into host cells to allow replication of the vector and thereby amplify copies of the polynucleotides contained therein. As described 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.

[0183] 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).

[0184] 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."

[0185] 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.

[0186] 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.

[0187] cell lines

[0188] 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 vector system 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 serratus 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.

[0189] 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.

[0190] 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).

[0191] Cell culture process

[0192] 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), hollow fiber bioreactors (HFBs, which in some cases can be used for perfusion processes), and various other cell culture methods (see, for example, Tao et al., 2003, Biotechnol. Bioeng. [Biotechnology and Bioengineering] 82:751-65; Kuystermans and Al-Rubeai, (2011) “Bioreactor Systems for Producing Antibody from Mammalian Cells”). Antibody Expression and Production [ Antibody expression and productionIn Cell Engineering, 7:25-52, Al-Rubeai (ed.) Springer; Catapano et al., (2009) "Bioreactor Design and Scale-Up" Cell and Tissue Reaction Engineering: Principles and Practice [ Cellular and Tissue Reaction Engineering: Principles With practice [In Eibl et al. (edited) Springer-Verlag, which is incorporated herein by reference in its entirety].

[0193] 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.

[0194] 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.

[0195] 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.

[0196] 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).

[0197] 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.

[0198] 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.

[0199] "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.

[0200] 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.

[0201] 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.

[0202] 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%.

[0203] 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.

[0204] 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.

[0205] 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.

[0206] Cell cultures can be supplemented with concentrated feed media containing components (such as nutrients and amino acids) consumed during the cell culture production process.

[0207] 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.

[0208] 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.

[0209] 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.

[0210] 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.

[0211] 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.

[0212] 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.

[0213] 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.

[0214] 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.

[0215] 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.

[0216] 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.

[0217] Additional non-limiting exemplary embodiments

[0218] The non-limiting example embodiments disclosed herein also include:

[0219] E1. A method for preparing a multispecific antibody having only one Fab or having two identical Fabs, wherein the method comprises the following steps:

[0220] a) Introducing two different expression vectors into host cells, wherein the first expression vector encodes 1) the light chain and 2) the heavy chain, a heavy chain fusion, or an Fc-fusion; and the second expression vector encodes 1) the same light chain and 2) the heavy chain, a heavy chain fusion, or an Fc-fusion; and

[0221] b) Induce expression of the expression vector to obtain the multispecific antibody;

[0222] The multispecific antibody described therein has only one Fab or has two identical Fabs.

[0223] E2. The method as described in E1, wherein one or both of the heavy chain or Fc region are independently fused to scFv, VH, or cytokines.

[0224] E3. The method described in E1 or E2, wherein the multispecific antibody is a bispecific antibody.

[0225] E4. The method described in E1 or E2, wherein the multispecific antibody is a trispecific antibody.

[0226] E5. The method as described in E3, wherein the bispecific antibody has scFv, VH, or a cytokine, which is fused directly or via one or more adapter sequences to the N-terminus, C-terminus, or between CH1 and CH2 of a heavy chain.

[0227] E6. The method of any one of E1 to E5, wherein each of the expression vectors contains optional markers that may be the same or different.

[0228] E7. The method as described in E6, wherein the selectable marker is selected from the group consisting of glutamine synthase and dihydrofolate reductase.

[0229] E8. The method as described in E6 or E7, wherein the promoter operatively linked to the nucleotide sequence encoding the selectable marker is selected from the group consisting of mPGK, SRα, and SV40.

[0230] E9. The method of any one of E1 to E8, wherein the host cell is a mammalian host cell.

[0231] E10. As described in E9, wherein the mammalian host cell is a Chinese hamster ovary (CHO) cell.

[0232] E11. As described in E10, wherein the CHO cells are dhfr- or GSKO.

[0233] E12. The method of any one of E1 to E11, further comprising recovering the antibody from the culture.

[0234] E13. The method as described in E12, wherein the recovered antibody is purified and formulated into a pharmaceutically acceptable formulation.

[0235] Further non-limiting exemplary embodiments / features disclosed herein include:

[0236] F1. An expression system comprising:

[0237] 1) A first expression vector comprising a polynucleotide sequence containing the following elements in a 5' to 3' sequence:

[0238] a) A first promoter, which is operatively linked to a nucleotide sequence encoding a light chain, followed by a polyadenylation (poly-A) signal sequence;

[0239] b) A second promoter operatively linked to a nucleotide sequence encoding a heavy chain, a heavy chain fusion, or an Fc fusion, followed by a second polyA signal sequence; and

[0240] c) A third promoter operatively linked to a nucleotide sequence encoding a selectable marker, followed by a third poly-A signal sequence; and

[0241] 2) A second expression vector containing a multinucleotide sequence comprising the following elements in a 5' to 3' sequence:

[0242] a) A first promoter, which is operatively linked to the same copy of a nucleotide sequence encoding a light chain, followed by a polyA signal sequence;

[0243] b) A second promoter operatively linked to a nucleotide sequence encoding a heavy chain, a heavy chain fusion, or an Fc fusion, followed by a second polyA signal sequence; and

[0244] c) A third promoter, operatively linked to a nucleotide sequence encoding a selectable marker, followed by a third poly-A signal sequence.

[0245] When expressed in host cells, this expression system produces multispecific antibodies with only one Fab or with two identical Fabs.

[0246] F2. The expression system as described in F1, wherein each heavy chain fusion or Fc fusion is independently a fusion of a heavy chain or Fc with VH, scFv, or a cytokine, wherein the VH, scFv, or cytokine is fused to the N-terminus or C-terminus of the heavy chain portion of the heavy chain fusion or the Fc portion of the Fc fusion, or between CH1 and CH2 of the heavy chain portion of the heavy chain fusion.

[0247] F3. An expression system as described in F1 or F2, wherein the first expression vector encodes a heavy chain and the second expression vector encodes a heavy chain fusion compound having a VH, scFv, or cytokine fused to the C-terminus of the heavy chain portion of the heavy chain fusion compound.

[0248] F4. An expression system as described in F1 or F2, wherein the first expression vector encodes a heavy chain and the second expression vector encodes an Fc fusion having an N-terminus scFv fused to the Fc portion of the Fc fusion.

[0249] F5. An expression system as described in F1 or F2, wherein the first expression vector encodes a heavy chain fusion compound having a VH or cytokine fused to the C-terminus of the heavy chain portion of the heavy chain fusion compound, and the second expression vector encodes a heavy chain fusion compound having an scFv fused to the N-terminus of the heavy chain portion of the heavy chain fusion compound.

[0250] F6. An expression system comprising:

[0251] 1) A first expression vector comprising a polynucleotide sequence containing the following elements in a 5' to 3' sequence:

[0252] a) A first promoter, which is operatively linked to a nucleotide sequence encoding a light chain, followed by a polyadenylation (poly-A) signal sequence;

[0253] b) A second promoter operatively linked to a nucleotide sequence encoding the heavy chain, followed by a second poly-A signal sequence; and

[0254] c) A third promoter operatively linked to a nucleotide sequence encoding a selectable marker, followed by a third poly-A signal sequence; and

[0255] 2) A second expression vector containing a multinucleotide sequence comprising the following elements in a 5' to 3' sequence:

[0256] a) A first promoter, which is operatively linked to the same copy of a nucleotide sequence encoding a light chain, followed by a polyA signal sequence;

[0257] b) A second promoter operatively linked to a nucleotide sequence encoding the heavy chain fusion compound, followed by a second poly-A signal sequence; and

[0258] c) A third promoter, operatively linked to a nucleotide sequence encoding a selectable marker, followed by a third poly-A signal sequence.

[0259] When expressed in host cells, this expression system produces multispecific antibodies with two identical Fabs.

[0260] F7. An expression system as described in F6, wherein the heavy chain fusion is a fusion of the heavy chain with VH, scFv, or a cytokine.

[0261] F8. An expression system as described in F6 or F7, wherein the heavy chain fusion is a fusion of the heavy chain and VH.

[0262] F9. An expression system as described in F6 or F7, wherein the heavy chain fusion is a fusion of the heavy chain and scFv.

[0263] F10. An expression system as described in F6 or F7, wherein the heavy chain fusion is a fusion of a heavy chain and a cytokine.

[0264] F11. An expression system as described in F6 or F7, wherein the heavy chain fusion is a fusion of a heavy chain with VH, scFv, or a cytokine, the VH, scFv, or cytokine being fused to the N-terminus or C-terminus of the heavy chain portion of the heavy chain fusion, or between CH1 and CH2 of the heavy chain portion of the heavy chain fusion.

[0265] F12. An expression system as described in F6, F7, F8, or F11, wherein the heavy chain fusion is a fusion of a heavy chain and a VH, the VH being fused to the N-terminus of the heavy chain portion of the heavy chain fusion.

[0266] F13. An expression system as described in F6, F7, F8, or F11, wherein the heavy chain fusion is a fusion of a heavy chain and a VH, the VH being fused to the C-terminus of the heavy chain portion of the heavy chain fusion.

[0267] F14. An expression system as described in F6, F7, F8, or F11, wherein the heavy chain fusion is a fusion of a heavy chain and a VH, the VH being fused between CH1 and CH2 of the heavy chain portion of the heavy chain fusion.

[0268] F15. An expression system as described in F6, F7, F9, or F11, wherein the heavy chain fusion is a fusion of a heavy chain and an scFv, the scFv being fused to the N-terminus of the heavy chain portion of the heavy chain fusion.

[0269] F16. An expression system as described in F6, F7, F9, or F11, wherein the heavy chain fusion is a fusion of a heavy chain and an scFv, the scFv being fused to the C-terminus of the heavy chain portion of the heavy chain fusion.

[0270] F17. An expression system as described in F6, F7, F9, or F11, wherein the heavy chain fusion is a fusion of a heavy chain and an scFv, the scFv being fused between CH1 and CH2 of the heavy chain portion of the heavy chain fusion.

[0271] F18. An expression system as described in F6, F7, F10, or F11, wherein the heavy chain fusion is a fusion of a heavy chain and a cytokine, the cytokine being fused to the N-terminus of the heavy chain portion of the heavy chain fusion.

[0272] F19. An expression system as described in F6, F7, F10, or F11, wherein the heavy chain fusion is a fusion of a heavy chain and a cytokine, the cytokine being fused to the C-terminus of the heavy chain portion of the heavy chain fusion.

[0273] F20. An expression system as described in F6, F7, F10, or F11, wherein the heavy chain fusion is a fusion of a heavy chain and a cytokine, the cytokine being fused between CH1 and CH2 of the heavy chain portion of the heavy chain fusion.

[0274] F21. An expression system comprising:

[0275] 1) A first expression vector comprising a polynucleotide sequence containing the following elements in a 5' to 3' sequence:

[0276] a) A first promoter, which is operatively linked to a nucleotide sequence encoding a light chain, followed by a polyadenylation (poly-A) signal sequence;

[0277] b) A second promoter operatively linked to a nucleotide sequence encoding the heavy chain, followed by a second poly-A signal sequence; and

[0278] c) A third promoter operatively linked to a nucleotide sequence encoding a selectable marker, followed by a third poly-A signal sequence; and

[0279] 2) A second expression vector containing a multinucleotide sequence comprising the following elements in a 5' to 3' sequence:

[0280] a) A first promoter, which is operatively linked to the same copy of a nucleotide sequence encoding a light chain, followed by a polyA signal sequence;

[0281] b) A second promoter operatively linked to a nucleotide sequence encoding the Fc fusion compound, followed by a second poly-A signal sequence; and

[0282] c) A third promoter, operatively linked to a nucleotide sequence encoding a selectable marker, followed by a third poly-A signal sequence.

[0283] When expressed in host cells, this expression system produces multispecific antibodies with only one Fab.

[0284] F22. An expression system as described in F21, wherein the Fc fusion is a fusion of Fc with VH, scFv, or a cytokine.

[0285] F23. An expression system as described in F21 or F22, wherein the Fc fusion is a fusion of Fc and VH.

[0286] F24. An expression system as described in F21 or F22, wherein the Fc fusion is a fusion of Fc and scFv.

[0287] F25. An expression system as described in F21 or F22, wherein the Fc fusion is a fusion of Fc and a cytokine.

[0288] F26. The expression system as described in F21 or F22, wherein the Fc fusion is a fusion of Fc with VH, scFv, or a cytokine, the VH, scFv, or cytokine being fused to the N-terminus or C-terminus of the Fc portion of the Fc fusion.

[0289] F27. An expression system as described in F21, F22, F23, or F26, wherein the Fc fusion is a fusion of Fc and VH, the VH being fused to the N-terminus of the Fc portion of the Fc fusion.

[0290] F28. An expression system as described in F21, F22, F23, or F26, wherein the Fc fusion is a fusion of Fc and VH, the VH being fused to the C-terminus of the Fc portion of the Fc fusion.

[0291] F29. An expression system as described in F21, F22, F24, or F26, wherein the Fc fusion is a fusion of Fc and scFv, the scFv being fused to the N-terminus of the Fc portion of the Fc fusion.

[0292] F30. An expression system as described in F21, F22, F24, or F26, wherein the Fc fusion is a fusion of Fc and scFv, the scFv being fused to the C-terminus of the Fc portion of the Fc fusion.

[0293] F31. An expression system as described in F21, F22, F25, or F26, wherein the Fc fusion is a fusion of Fc with a cytokine, the cytokine being fused to the N-terminus of the Fc portion of the Fc fusion.

[0294] F32. An expression system as described in F21, F22, F25, or F26, wherein the Fc fusion is a fusion of Fc with a cytokine, the cytokine being fused to the C-terminus of the Fc portion of the Fc fusion.

[0295] F33. An expression system comprising:

[0296] 1) A first expression vector comprising a polynucleotide sequence containing the following elements in a 5' to 3' sequence:

[0297] a) A first promoter, which is operatively linked to a nucleotide sequence encoding a light chain, followed by a polyadenylation (poly-A) signal sequence;

[0298] b) A second promoter operatively linked to a nucleotide sequence encoding the heavy chain fusion compound, followed by a second poly-A signal sequence; and

[0299] c) A third promoter operatively linked to a nucleotide sequence encoding a selectable marker, followed by a third poly-A signal sequence; and

[0300] 2) A second expression vector containing a multinucleotide sequence comprising the following elements in a 5' to 3' sequence:

[0301] a) A first promoter, which is operatively linked to the same copy of a nucleotide sequence encoding a light chain, followed by a polyA signal sequence;

[0302] b) A second promoter operatively linked to a nucleotide sequence encoding the heavy chain fusion compound, followed by a second poly-A signal sequence; and

[0303] c) A third promoter, operatively linked to a nucleotide sequence encoding a selectable marker, followed by a third poly-A signal sequence.

[0304] When expressed in host cells, this expression system produces multispecific antibodies with two identical Fabs.

[0305] F34. An expression system as described in F33, wherein each heavy chain fusion is independently a fusion of the heavy chain with VH, scFv, or a cytokine.

[0306] F35. An expression system as described in F33 or F34, wherein each heavy chain fusion is independently a fusion of a heavy chain with VH, scFv, or a cytokine, the VH, scFv, or cytokine being fused to the N-terminus or C-terminus of the heavy chain portion of the heavy chain fusion, or between CH1 and CH2 of the heavy chain portion of the heavy chain fusion.

[0307] F36. An expression system as described in F33, F34, or F35, wherein the first expression vector encodes a heavy chain fusion compound having a VH, scFv, or cytokine fused to the C-terminus of the heavy chain portion of the heavy chain fusion compound, and the second expression vector encodes a heavy chain fusion compound having a VH, scFv, or cytokine fused to the N-terminus of the heavy chain portion of the heavy chain fusion compound.

[0308] F37. The expression system as described in any one of F33-F36, wherein the first expression vector encodes a heavy chain fusion compound having a VH or cytokine fused to the C-terminus of the heavy chain portion of the heavy chain fusion compound, and the second expression vector encodes a heavy chain fusion compound having an scFv fused to the N-terminus of the heavy chain portion of the heavy chain fusion compound.

[0309] F38. The expression system as described in any one of F33-F37, wherein the first expression vector encodes a heavy chain fusion having a VH fused at the C-terminus of the heavy chain portion of the heavy chain fusion, and the second expression vector encodes a heavy chain fusion having an scFv fused at the N-terminus of the heavy chain portion of the heavy chain fusion.

[0310] F39. The expression system as described in any one of F33-F37, wherein the first expression vector encodes a heavy chain fusion compound having a cytokine fused to the C-terminus of the heavy chain portion of the heavy chain fusion compound, and the second expression vector encodes a heavy chain fusion compound having an scFv fused to the N-terminus of the heavy chain portion of the heavy chain fusion compound.

[0311] F40. The expression system as described in any one of F1-F39, wherein the first expression vector and the second expression vector are both mammalian expression vectors.

[0312] F41. The expression system as described in any one of F1-F40, wherein the selectable marker on the first expression vector and the selectable marker on the second expression vector are independently glutamine synthase or dihydrofolate reductase.

[0313] F42. The expression system as described in any one of F1-F41, wherein the selectable marker on the first expression vector and the selectable marker on the second expression vector are both glutamine synthases.

[0314] F43. The expression system as described in any one of F1-F41, wherein the selectable marker on the first expression vector and the selectable marker on the second expression vector are both dihydrofolate reductase.

[0315] F44. The expression system as described in any one of F1-F43, 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 first polyA signal sequence, the second polyA signal sequence, and the third polyA signal sequence on the second expression vector 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.

[0316] F45. The expression system as described in any one of F1-F44, wherein the first polyA signal sequence, the second polyA signal sequence and the third polyA signal sequence on the first expression vector are identical.

[0317] F46. The expression system as described in any one of F1-F44, wherein the first polyA signal sequence, the second polyA signal sequence, and the third polyA signal sequence on the first expression vector are different.

[0318] F47. The expression system as described in any one of F1-F46, wherein the first polyA signal sequence, the second polyA signal sequence and the third polyA signal sequence on the first expression vector are identical.

[0319] F48. The expression system as described in any one of F1-F46, wherein the first polyA signal sequence, the second polyA signal sequence, and the third polyA signal sequence on the first expression vector are different.

[0320] F49. The expression system as described in any one of F1-F43, F45, or F47, 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 each of the first polyA signal sequence, the second polyA signal sequence, and the third polyA signal sequence on the second expression vector is identical.

[0321] F50. An expression system as described in any one of F1-F43, F45, F47, or F49, wherein the first polyA signal sequence, the second polyA signal sequence, and the third polyA signal sequence on the first expression vector, as well as the first polyA signal sequence, the second polyA signal sequence, and the third polyA signal sequence on the second expression vector, are all early polyA signal sequences of simian virus 40 (SV40).

[0322] F51. An expression system as described in any one of F1-F43, F45, F47, or F49, wherein

[0323] Each of the first polyA signal sequence, the second polyA signal sequence, and the third polyA signal sequence on the first expression vector, 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 identical; and

[0324] The selectable markers on both the first and second expression vectors are glutamine synthases.

[0325] F52. An expression system as described in any one of F1-F43, F45, F47, or F49-F51, wherein

[0326] The first, second, and third polyA signal sequences on the first expression vector, as well as the first, second, and third polyA signal sequences on the second expression vector, are all early polyA signal sequences of simian virus 40 (SV40); and

[0327] The selectable markers on both the first and second expression vectors are glutamine synthases.

[0328] F53. The expression system as described in any one of F1-F52, wherein the third promoter on the first expression vector and the third promoter on the second expression vector are independently selected from the group consisting of mPGK, SRα and SV40 promoters.

[0329] F54. The expression system as described in any one of F1-F53, wherein the third promoter on the first expression vector is the same as the third promoter on the second expression vector.

[0330] F55. The expression system as described in any one of F1-F54, wherein the third promoter on the first expression vector and the third promoter on the second expression vector are both mPGK promoters.

[0331] F56. The expression system as described in any one of F1-F54, wherein the third promoter on the first expression vector and the third promoter on the second expression vector are both SRα promoters.

[0332] F57. The expression system as described in any one of F1-F54, wherein the third promoter on the first expression vector and the third promoter on the second expression vector are both SV40 promoters.

[0333] F58. The expression system as described in any one of F1-F53, wherein the third promoter on the first expression vector is different from the third promoter on the second expression vector.

[0334] F59. The expression system as described in any one of F1-F40, wherein

[0335] Each of the first, second, and third polyA signal sequences on the first expression vector and the first, second, and third polyA signal sequences on the second expression vector 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;

[0336] The selectable biomarkers on the first expression vector and the second expression vector are independently glutamine synthase or dihydrofolate reductase; and

[0337] The third promoter on the first expression vector and the third promoter on the second expression vector are independently selected from the group consisting of mPGK, SRα and SV40 promoters.

[0338] F60. An expression system as described in any one of F1-F40 or F59, wherein

[0339] The first polyA signal sequence, the second polyA signal sequence, and the third polyA signal sequence on the first expression vector and the first polyA signal sequence, the second polyA signal sequence, and the third polyA signal sequence on the second expression vector are all identical, wherein the polyA signal sequence is 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;

[0340] The selectable biomarkers on both the first and second expression vectors are glutamine synthases; and

[0341] The third promoter on the first expression vector is the same as the third promoter on the second expression vector, wherein the third promoter is selected from the group consisting of mPGK, SRα and SV40 promoters.

[0342] F61. An expression system as described in any one of F1-F40, F59, or F60, wherein

[0343] Each of the first polyA signal sequence, the second polyA signal sequence, and the third polyA signal sequence on the first expression vector, 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).

[0344] The selectable biomarkers on both the first and second expression vectors are glutamine synthases; and

[0345] The third promoter on both the first expression vector and the second expression vector is an SRα promoter.

[0346] F62. The expression system as described in any one of F1-F61, wherein at least one of the first promoter on the first expression vector, the first promoter on the second expression vector, the second promoter on the first expression vector, and the second promoter on the second expression vector is a GAPDH promoter.

[0347] F63. The expression system as described in any one of F1-F62, wherein each of the first promoter on the first expression vector, the first promoter on the second expression vector, the second promoter on the first expression vector, and the second promoter on the second expression vector is a GAPDH promoter.

[0348] F64. The expression system as described in any one of F1-F63, wherein at least one of the first promoter on the first expression vector, the first promoter on the second expression vector, the second promoter on the first expression vector, and the second promoter on the second expression vector is a CMV / GAPDH promoter.

[0349] F65. The expression system as described in any one of F1-F64, wherein each of the first promoter on the first expression vector, the first promoter on the second expression vector, the second promoter on the first expression vector, and the second promoter on the second expression vector is a CMV / GAPDH promoter.

[0350] F66. The expression system as described in any one of F1-F40, wherein

[0351] Each of the first promoter on the first expression vector, the first promoter on the second expression vector, the second promoter on the first expression vector, and the second promoter on the second expression vector is a GAPDH promoter;

[0352] The first polyA signal sequence, the second polyA signal sequence, and the third polyA signal sequence on the first expression vector and the first polyA signal sequence, the second polyA signal sequence, and the third polyA signal sequence on the second expression vector are all identical, wherein the polyA signal sequence is 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;

[0353] The selectable biomarkers on both the first and second expression vectors are glutamine synthases; and

[0354] The third promoter on the first expression vector is the same as the third promoter on the second expression vector, wherein the third promoter is selected from the group consisting of mPGK, SRα and SV40 promoters.

[0355] F67. The expression system as described in any one of F1-F40, wherein

[0356] Each of the first promoter on the first expression vector, the first promoter on the second expression vector, the second promoter on the first expression vector, and the second promoter on the second expression vector is a GAPDH promoter;

[0357] Each of the first polyA signal sequence, the second polyA signal sequence, and the third polyA signal sequence on the first expression vector, 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).

[0358] The selectable biomarkers on both the first and second expression vectors are glutamine synthases; and

[0359] The third promoter on both the first expression vector and the second expression vector is an SRα promoter.

[0360] F68. The expression system as described in any one of F1-F40, wherein

[0361] Each of the first promoter on the first expression vector, the first promoter on the second expression vector, the second promoter on the first expression vector, and the second promoter on the second expression vector is a CMV / GAPDH promoter;

[0362] The first polyA signal sequence, the second polyA signal sequence, and the third polyA signal sequence on the first expression vector and the first polyA signal sequence, the second polyA signal sequence, and the third polyA signal sequence on the second expression vector are all identical, wherein the polyA signal sequence is 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;

[0363] The selectable biomarkers on both the first and second expression vectors are glutamine synthases; and

[0364] The third promoter on the first expression vector is the same as the third promoter on the second expression vector, wherein the third promoter is selected from the group consisting of mPGK, SRα and SV40 promoters.

[0365] F69. The expression system as described in any one of F1-F40, wherein

[0366] Each of the first promoter on the first expression vector, the first promoter on the second expression vector, the second promoter on the first expression vector, and the second promoter on the second expression vector is a CMV / GAPDH promoter;

[0367] Each of the first polyA signal sequence, the second polyA signal sequence, and the third polyA signal sequence on the first expression vector, 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).

[0368] The selectable biomarkers on both the first and second expression vectors are glutamine synthases; and

[0369] The third promoter on both the first expression vector and the second expression vector is an SRα promoter.

[0370] F70. A mammalian host cell comprising an expression system as described in any one of F1-F69.

[0371] F71. The mammalian host cell as described in F70, which is the Chinese hamster ovary (CHO) cell.

[0372] F72. The mammalian host cell as described in F71, wherein the CHO cell is a dihydrofolate reductase-deficient (DHFR-) CHO cell or a glutamine synthase knockout (GSKO) CHO cell.

[0373] F73. The mammalian host cell as described in F71 or F72, wherein the CHO cell is a dihydrofolate reductase-deficient (DHFR-) CHO cell.

[0374] F74. A mammalian host cell as described in F71 or F72, wherein the CHO cell is a glutamine synthase knockout (GSKO) CHO cell.

[0375] F75. A method for generating a multispecific antibody having only one Fab or having two identical Fabs, wherein the method comprises:

[0376] a) Introducing the expression system as described in any one of F1-F69 into mammalian host cells; and

[0377] b) Culture the mammalian host cells to produce multispecific antibodies.

[0378] F76. The method as described in F75, further comprising isolating the multispecific antibody.

[0379] F77. A method for generating a multispecific antibody having only one Fab or having two identical Fabs, wherein the method comprises the following steps:

[0380] a) Culture mammalian host cells, such as any one of F70-F74, to produce the multispecific antibody; and

[0381] b) Isolate the multispecific antibody.

[0382] F78. The method described in F76 or F77, further comprising purifying the multispecific antibody and formulating it into a pharmaceutically acceptable formulation.

[0383] F79. A method for preparing a multispecific antibody having only one Fab or having two identical Fabs, wherein the method comprises:

[0384] a) Introducing two different expression vectors into host cells, wherein the first expression vector encodes 1) the light chain and 2) the heavy chain, a heavy chain fusion, or an Fc fusion; and the second expression vector encodes 1) the same light chain and 2) the heavy chain, a heavy chain fusion, or an Fc fusion; and

[0385] b) The host cells are cultured in a mammalian cell culture to express the expression vector, thereby producing the multispecific antibody, wherein the multispecific antibody has only one Fab or has two identical Fabs.

[0386] F80. The method as described in F79, wherein each of the heavy chain fusion complex or Fc fusion complex is a heavy chain or Fc independently fused to scFv, VH, or a cytokine.

[0387] F81. The method described in F79 or F80, wherein the multispecific antibody is a bispecific antibody.

[0388] F82. The method as described in F81, wherein the bispecific antibody has 1) a heavy chain; and 2) a heavy chain fusion or Fc fusion having scFv, VH, or a cytokine, the scFv, VH, or cytokine being fused to the N-terminus or C-terminus of the heavy chain portion of the heavy chain fusion or the Fc portion of the Fc fusion, or between CH1 and CH2 of the heavy chain portion of the heavy chain fusion.

[0389] F83. The method described in F79 or F80, wherein the multispecific antibody is a trispecific antibody.

[0390] F84. The method as described in F83, wherein the trispecific antibody has 1) a first heavy chain fusion body having a VH or cytokine fused to the N-terminus or C-terminus of the heavy chain portion of the first heavy chain fusion body; and 2) a second heavy chain fusion body having an scFv fused to the N-terminus of the heavy chain portion of the second heavy chain fusion body.

[0391] F85. The method of any one of F79 to F84, wherein each of the expression vectors contains optional markers that may be the same or different.

[0392] F86. The method as described in F85, wherein each selectable marker is selected from the group consisting of glutamine synthase and dihydrofolate reductase.

[0393] F87. The method as described in F85 or F86, wherein each of the expression vectors further comprises a promoter operatively linked to a nucleotide sequence encoding the selectable marker, wherein each promoter is independently selected from the group consisting of mPGK, SRα, and SV40 promoters.

[0394] F88. The method of any one of F79-F87, wherein the host cell is a mammalian host cell.

[0395] F89. The method described in F88, wherein the mammalian host cell is a Chinese hamster ovary (CHO) cell.

[0396] F90. The method as described in F89, wherein the CHO cell is a dihydrofolate reductase-deficient (dhfr-) CHO cell or a glutamine synthase knockout (GSKO) CHO cell.

[0397] F91. The method as described in any one of F79-F90, further comprising recovering the multispecific antibody from the culture.

[0398] F92. The method as described in F91, wherein the recovered antibody is purified and formulated into a pharmaceutically acceptable preparation. Example

[0399] Example 1. Carrier engineering strategies for improving C1mAb productivity

[0400] Overview

[0401] 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).

[0402] This example describes how a 2xLC strategy using a triple-chain C1mAb antibody was employed to further optimize the vector system by modulating the expression levels of HC1, HC2-scFv, and co-LC, as well as the GS gene, in combination with different MSX concentrations, to improve productivity in CHO cells. The vector engineering strategies described herein can be extended to improve and optimize the productivity of other recombinant proteins typically expressed in stable pools.

[0403] Materials and Methods

[0404] Plasmid generation. The coding sequences for LC, HC1, and HC2-ScFv were inserted into the pPBGS plasmid backbone using Golden Gate cloning to generate polycistronic vectors. In short, the CMV / GAPDH promoter / enhancer fragments were used to control LC and HC with the SV40-polyA fragment, subsequently driving the SRα promoter (in sequence) for mGS-polyA expression. The vector configuration is shown in [Figure / Formula would be inserted here]. Figure 2B In the middle section, all fragments were unidirectionally assembled using a combination of protruding end sequences to facilitate Golden Gate cloning. MSX was used at 0 and 12.5 µM.

[0405] 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 C1mAb. 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.

[0406] 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.

[0407] Selection and recovery. Seventy-two hours after transfection, cells were centrifuged and transferred to selection medium containing 0 and 25 µM MSX, 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.

[0408] 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).

[0409] result

[0410] Figures 3A-3B The results show higher titers (A) and cell unit productivity (B) achieved using the 2xLC strategy at different MSX levels.

[0411] in addition, Figures 4A-4B The data shows higher effective titers (A) and higher nrCE-SDS-main peak (B) for the carrier configuration during feed-and-batch production. The colors correspond to MSX concentration levels.

[0412] Example 2. Carrier engineering strategies for improving AmAb bispecificity molecular productivity

[0413] Overview

[0414] 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).

[0415] This example describes further vector optimization to improve the production of AmAb 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 MSX. The vector engineering strategies described herein can be extended to improve and optimize the production of other recombinant proteins typically expressed in stable pools.

[0416] Materials and Methods

[0417] AmAb plasmid generation. The coding sequences for LC, HC1, and scFv-Fc were inserted into the pPBGS plasmid backbone using Golden Gate cloning to generate a polycistronic vector. In short, the CMV / GAPDH promoter / enhancer fragments were used to control HC, scFv-Fc, and LC with the SV40-polyA fragment, subsequently driving the SRα promoter (in sequence) for mGS-polyA expression. The vector configuration is shown in [Figure / Formula would be inserted here]. Figure 5 In the middle section, all fragments were unidirectionally assembled using a combination of protruding end sequences to facilitate Golden Gate cloning. MSX was used at 25 µM.

[0418] 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 AmAb. In proprietary medium, at 130 rpm, 36°C, and 5% CO2, in shake flasks, at 0.3–0.4 × 10⁻⁶ every 3–4 days. 6 Host cells were passaged at a seeding density of 1 × 10⁶ cells / mL. Twenty-four hours before transfection, host cells were seeded at a density of 1 × 10⁶ cells / mL. 6 10 cells / mL were seeded into the host cells to ensure that the cells were in the exponential growth phase at the time of transfection.

[0419] Stable pools expressing AmAb were generated using Lipofectamine LTX (Gibco, Billings, Montana) and Opti-MEM I serum-depleted medium (Gibco, Billings, Montana) according to the manufacturer's protocol. Single transfections were performed for each vector conformation. Briefly, 2 µg of each plasmid was added in combination with 2 µg of proprietary piggybac transposase to a concentration of 4 × 10⁻⁶. 6Transfected cells were collected in 4 mL of growth medium in 6-well plates at 225 rpm, 36°C and 5% CO2.

[0420] Selection and recovery. Seventy-two hours after transfection, cells were centrifuged and transferred to selection medium containing 25 µM MSX and free of glutamine. 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 0.3-0.4 × 10⁻⁶ cells / mL. 6 Cells / mL.

[0421] 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.

[0422] result

[0423] Figures 6A-6B The results show that the 2xLC configuration yields a higher normalized yield (A) and nrMCE MP% (B) compared to the 1xLC configuration.

[0424] Example 3. Carrier engineering strategies for improving the productivity of trispecific molecules

[0425] Overview

[0426] 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).

[0427] This example describes how further vector optimization was performed by using promoters of varying strengths to modulate the expression levels of the HC, LC, and GS genes in combination with different MSX concentrations to improve the production of trispecific 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.

[0428] Materials and Methods

[0429] Three-specific plasmid generation. The coding sequences of LC and HC were inserted into the pPBGS plasmid backbone using Golden Gate cloning to generate a polycistronic vector. In short, the CMV / GAPDH promoter / enhancer fragment was used to control LC and HC with the SV40-polyA fragment, subsequently driving the SRα promoter of mGS-polyA expression (in sequence). The vector configuration is shown in... Figure 7 In the middle section, all fragments were unidirectionally assembled using a combination of protruding end sequences to facilitate Golden Gate cloning. MSX was used at 25 µM.

[0430] 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 trispecific antibodies. In proprietary basal medium, at 130 rpm, 36°C, and 5% CO2, in shake flasks, at 0.3–0.4 × 10⁻⁶ every 3–4 days. 6 Host cells were passaged at a seeding density of 1 × 10⁶ cells / mL. Twenty-four hours before transfection, host cells were seeded at a density of 1 × 10⁶ cells / mL. 6 10 cells / mL were seeded into the host cells to ensure that the cells were in the exponential growth phase at the time of transfection.

[0431] Stable pools expressing trispecific antibodies were generated using Lipofectamine LTX (Gibco, Billings, Montana) and Opti-MEM I serum-depleted medium (Gibco, Billings, Montana) according to the manufacturer's protocol. Single transfection was performed for each vector conformation. Briefly, 2 µg of each 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 4 mL of growth medium in 6-well plates at 225 rpm, 36°C and 5% CO2.

[0432] Selection and recovery. Seventy-two hours after transfection, cells were centrifuged and transferred to selection medium containing 25 µM MSX and free of glutamine. Cells were then rotated every 3–4 days at approximately 1–2 × 10⁶ cells / day. 6Pass the cells at a seeding density of 100 cells / mL until viability exceeds 90%, at which point the seeding density is reduced to 0.3-0.4 × 10⁻⁶ cells / mL. 6 Cells / mL.

[0433] 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.

[0434] result

[0435] Figures 8A-8B The results show that the 2xLC configuration has a higher normalized yield (A) and nrMCE MP% (B) compared to the 1xLC configuration.

Claims

1. An expression system comprising: 1) A first expression vector comprising a polynucleotide sequence containing the following elements in a 5' to 3' sequence: a) A first promoter, which is operatively linked to a nucleotide sequence encoding a light chain, followed by a polyadenylation (poly-A) signal sequence; b) A second promoter operatively linked to a nucleotide sequence encoding a heavy chain, a heavy chain fusion, or an Fc fusion, followed by a second poly-A signal sequence; and c) A third promoter operatively linked to a nucleotide sequence encoding a selectable marker, followed by a third poly-A signal sequence; and 2) A second expression vector containing a polynucleotide sequence comprising the following elements in a 5' to 3' sequence: a) A first promoter, which is operatively linked to the same copy of a nucleotide sequence encoding a light chain, followed by a polyA signal sequence; b) A second promoter operatively linked to a nucleotide sequence encoding a heavy chain, a heavy chain fusion, or an Fc fusion, followed by a second poly-A signal sequence; and c) A third promoter, operatively linked to a nucleotide sequence encoding a selectable marker, followed by a third poly-A signal sequence. When expressed in host cells, this expression system produces multispecific antibodies with only one Fab or with two identical Fabs.

2. The expression system of claim 1, wherein each heavy chain fusion or Fc fusion is independently a fusion of a heavy chain or Fc with VH, scFv, or a cytokine, wherein the VH, scFv, or cytokine is fused to the N-terminus or C-terminus of the heavy chain portion of the heavy chain fusion or the Fc portion of the Fc fusion, or between CH1 and CH2 of the heavy chain portion of the heavy chain fusion.

3. The expression system of claim 1 or claim 2, wherein the first expression vector encodes a heavy chain and the second expression vector encodes a heavy chain fusion compound having a VH, scFv, or cytokine fused to the C-terminus of the heavy chain portion of the heavy chain fusion compound.

4. The expression system of claim 1 or claim 2, wherein the first expression vector encodes a heavy chain and the second expression vector encodes an Fc fusion having an N-terminus scFv fused to the Fc portion of the Fc fusion.

5. The expression system of claim 1 or claim 2, wherein the first expression vector encodes a heavy chain fusion compound having a VH or cytokine fused to the C-terminus of the heavy chain portion of the heavy chain fusion compound, and the second expression vector encodes a heavy chain fusion compound having an scFv fused to the N-terminus of the heavy chain portion of the heavy chain fusion compound.

6. The expression system according to any one of claims 1-5, wherein the first expression vector and the second expression vector are both mammalian expression vectors.

7. The expression system according to any one of claims 1-6, wherein the selectable marker on the first expression vector and the selectable marker on the second expression vector are independently glutamine synthase or dihydrofolate reductase.

8. The expression system according to any one of claims 1-7, wherein the selectable marker on the first expression vector and the selectable marker on the second expression vector are both glutamine synthases.

9. The expression system according to any one of claims 1-8, 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 first polyA signal sequence, the second polyA signal sequence, and the third polyA signal sequence on the second expression vector 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.

10. The expression system according to any one of claims 1-9, wherein the first polyA signal sequence, the second polyA signal sequence, and the third polyA signal sequence on the first expression vector and the first polyA signal sequence, the second polyA signal sequence, and the third polyA signal sequence on the second expression vector are identical.

11. The expression system according to any one of claims 1-10, 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 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).

12. The expression system according to any one of claims 1-11, wherein Each of the first, second, and third polyA signal sequences on the first expression vector, 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); and The selectable markers on both the first and second expression vectors are glutamine synthases.

13. The expression system of any one of claims 1-12, wherein the third promoter on the first expression vector and the third promoter on the second expression vector are independently selected from the group consisting of mPGK, SRα and SV40 promoters.

14. The expression system according to any one of claims 1-13, wherein the third promoter on the first expression vector is the same as the third promoter on the second expression vector.

15. The expression system according to any one of claims 1-14, wherein the third promoter on the first expression vector and the third promoter on the second expression vector are both SRα promoters.

16. The expression system of any one of claims 1-15, wherein each of the first promoter on the first expression vector, the first promoter on the second expression vector, the second promoter on the first expression vector, and the second promoter on the second expression vector is a GAPDH promoter.

17. The expression system of any one of claims 1-16, wherein each of the first promoter on the first expression vector, the first promoter on the second expression vector, the second promoter on the first expression vector, and the second promoter on the second expression vector is a CMV / GAPDH promoter.

18. A mammalian host cell comprising the expression system as described in any one of claims 1-17.

19. The mammalian host cell as described in claim 18, wherein it is a Chinese hamster ovary (CHO) cell.

20. The mammalian host cell of claim 18 or claim 19, wherein the CHO cell is a glutamine synthase knockout (GSKO) CHO cell.

21. A method for generating a multispecific antibody having only one Fab or having two identical Fabs, wherein the method comprises: a) Introducing the expression system as described in any one of claims 1-17 into a mammalian host cell; as well as b) Culture the mammalian host cells to produce the multispecific antibody.

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