EA043386B1 - DIRECT SELECTION OF CELLS EXPRESSING HIGH LEVELS OF HETEROMERIC PROTEINS USING GLUTAMINE SYNTHETASE VECTORS FOR INTRAGENIC COMPLEMENTATION - Google Patents

Description

Cross references to related applications

This application claims priority to US Provisional Application No. 62/334,966, filed May 11

2016, which is incorporated herein by reference in its entirety.

Description of the text file submitted electronically

This application contains a Sequence Listing that was filed electronically in ASCII format and is incorporated herein by reference in its entirety. A copy of the Sequence List in machine-readable form that was created on May 8, 2017 is named A-2011-WO-PCT_SEQ_ST25.txt and is 79.1 kilobytes in size.

Field of technology

This invention relates to the general field of recombinant expression of polypeptides in mammalian cell culture. More specifically, the invention relates to improved selection in cells of recombinantly designed vectors for the expression of heteromeric polypeptides.

State of the art

Expression of heteromeric recombinant proteins usually requires similar expression of the component chains to avoid unnecessary synthesis of chains that cannot be incorporated into mature proteins. In addition, most methods for producing heteromeric recombinant proteins require screening many clones to identify rare high-expressing clones that express high levels of each chain of the mature heteromeric protein. A selectable separate marker system (in which a selectable marker is divided into two parts and each fragment is associated with the expression of one heteromeric protein chain) can be used to identify those cells that express both selectable marker fragments, and therefore both heteromeric protein chains.

Glutamine synthetase (GS) catalyzes glutamine biosynthesis by condensing ammonia with glutamate. The mammalian GS enzyme is a decamer consisting of two connected pentameric rings, with ten active sites located at the junctions of the subunits. Each active site is formed by residues from the N-terminal domain (β-grasp domain, consisting of residues 25-112) of one subunit and residues from the C-terminal domain (catalytic domain, consisting of residues 113-373) of the adjacent subunit. Genetic studies in S. cerevisiae and E. coli have shown that some GS mutations exhibit intragenic complementarity, in which some mutations that are located at the 5' end of the GS gene can suppress those that are at the 3' end (Mitchell, A. P. Genetics 111, 243-258 (1985); MacNeil, T., et al. Journal of Bacteriology 150, 1302-1313 (1982)). Accordingly, the identification of a metabolic enzyme-based selection system, such as that involving intragenic complementation at glutamine synthetase, would be useful for the expression of molecules with more than two unique polypeptide chains (eg, monoclonal antibodies and other recombinant proteins).

The essence of the invention

In one embodiment of the invention, a vector is provided containing:

a) a first nucleic acid encoding a first polypeptide, and

b) a second nucleic acid encoding a second polypeptide, wherein the second polypeptide is the original mutant subunit of a selectable marker, wherein the transcription of the first nucleic acid is operably linked to the transcription of the second nucleic acid, further comprising: d

c) a third nucleic acid encoding a third polypeptide, wherein the third polypeptide is capable of binding to the first polypeptide to form a heteromeric complex; and

d) a fourth nucleic acid that encodes a fourth polypeptide, wherein the fourth polypeptide is a complementary mutant subunit of a selectable marker, wherein the transcription of the third nucleic acid is operably linked to the transcription of the fourth nucleic acid, wherein the parent mutant subunit and the complementary mutant subunit of the selectable marker interact to provide selectable activity and, in addition, the vector is capable of being transfected into mammalian cells and enhancing the selection of transfected cells.

In another embodiment of the invention, a vector is provided containing:

e) a first nucleic acid encoding a first polypeptide, and

f) a second nucleic acid encoding a second polypeptide, wherein the second polypeptide is the original fragment of a marker suitable for selection, wherein the transcription of the first nucleic acid is operably linked to the transcription of the second nucleic acid, further comprising:

g) a third nucleic acid encoding a third polypeptide, wherein the third polypeptide is capable of binding to the first polypeptide to form a heteromeric complex; and

h) a fourth nucleic acid that encodes a fourth polypeptide, the fourth

- 1 043386 the polypeptide is a complementary fragment of a selectable marker, wherein the transcription of a third nucleic acid is operably linked to the transcription of a fourth nucleic acid, wherein the original mutant subunit and the complementary mutant subunit of the selectable marker interact to provide selectable activity, and, in addition Moreover, the vector is able to be transfected into mammalian cells and improve the selection of transfected cells.

In a further embodiment of the invention, the heteromeric complex is an immunoglobulin. In one embodiment, the first nucleic acid encodes an immunoglobulin heavy chain and the third nucleic acid encodes an immunoglobulin light chain; in an alternative embodiment, the first nucleic acid encodes an immunoglobulin light chain and the third nucleic acid encodes an immunoglobulin heavy chain. In the above embodiments, the selectable marker is a metabolic enzyme selected from the group consisting of glutamine synthetase, threonine dehydratase, adenylosuccinate synthetase and glutamate dehydrogenase.

The invention may include an internal ribosome binding site (IRES); in one embodiment of the invention, the IRES is located at a region selected from the group consisting of: a region between the first nucleic acid and the second nucleic acid; a region between the third nucleic acid and a fourth nucleic acid and regions between both the first and second and between the third and fourth nucleic acids. In a further embodiment, the internal ribosome binding site comprises SEQ ID NO: 23.

For any of the embodiments described herein, where the parent mutant selectable marker subunit is an N-terminal mutant of glutamine synthetase (mutGS-NT), the complementary mutant selectable marker subunit is a C-terminal mutant of glutamine synthetase (mutGS-CT ). In one such aspect of the invention, mutGS-NT contains two or more mutations selected from the group consisting of W60A N61A D63A D63R S66A and D76A, and mutGS-CT contains one or more mutations selected from the group consisting of E134A E136A E196A E203A N248A H253A N255A R319A and R324A. In one embodiment, mutGS-NT is selected from the group consisting of W60A N61A D63A (mutGS-NT 1, SEQ ID NO: 6), W60A N61A D63A S66A (mutGS-NT2; SEQ ID NO: 10) W60A N61A D63A S66A D76A (mutGSNT3, SEQ ID NO: 25) and W60A N61A D63R S66A (mutGS-NT4; SEQ ID NO: 19); and mutGS-CT is selected from the group consisting of E134A E136A E196A E203A (mutGS-CT 1, SEQ ID NO: 21) and N248A H253A N255A (mutGS-CT2; SEQ ID NO: 22).

In other embodiments, N-terminal and C-terminal glutamine synthetase fragments are created by protein cleavage at one or more amino acid positions of the glutamine synthetase protein. In one such aspect of the invention, the N-terminal fragment and/or C-terminal fragments of glutamine synthetase are cleaved at an amino acid position of glutamine synthetase selected from the group consisting of: E110, Y104, S125, N126, E264, T111, N105, N126, Q127 and N265 .

The invention also relates to an isolated host cell that has been transfected, transformed or transduced with a vector as described above. The host cell may be selected from the group consisting of CHO, VERO, BHK, HeLa, Cos, MDCK, 293, 3T3, myeloma cell line and WI38 cells. In one aspect of the invention, there is provided a method for producing a heteromeric complex, comprising the step of culturing such a host cell under conditions where the heteromeric complex is expressed by the host cell. In one embodiment of the invention, the heteromeric complex is an antibody. The claimed methods according to the invention may also include isolation of the heteromeric complex.

In one embodiment, the invention provides an expression system comprising:

a) a first vector encoding a bicistronic transcript containing a first nucleic acid encoding a first polypeptide that is operably linked to a second nucleic acid encoding a second polypeptide, wherein the second polypeptide is the original mutant subunit of the selectable marker, and

b) a second vector encoding a bicistronic transcript containing a third nucleic acid encoding a third polypeptide that is operably linked to a fourth nucleic acid encoding a fourth polypeptide, wherein the fourth polypeptide is a complementary mutant subunit of a selectable marker that is capable of binding to the original mutant subunit of the marker suitable for selection to provide selectable activity, in addition, the third polypeptide is capable of binding to the first polypeptide to form a heteromeric complex;

In addition, the expression system can be transfected into mammalian cells and improve the selection of these cells.

In another embodiment, the invention provides an expression system comprising:

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c) a first vector encoding a bicistronic transcript containing a first nucleic acid encoding a first polypeptide that is operably linked to a second nucleic acid encoding a second polypeptide, wherein the second polypeptide is the original fragment of a selectable marker, and

d) a second vector encoding a bicistronic transcript containing a third nucleic acid encoding a third polypeptide that is operably linked to a fourth nucleic acid encoding a fourth polypeptide, wherein the fourth polypeptide is a complementary fragment of a selectable marker that is capable of binding to the original mutant fragment of the selectable marker for selection to provide selectable activity, wherein the third polypeptide is capable of binding to the first polypeptide to form a heteromeric complex;

In addition, the expression system can be transfected into mammalian cells and improve the selection of these cells.

In a further embodiment of the invention, the heteromeric complex is an immunoglobulin. In one embodiment, the first nucleic acid encodes an immunoglobulin heavy chain and the third nucleic acid encodes an immunoglobulin light chain; in an alternative embodiment, the first nucleic acid encodes an immunoglobulin light chain and the third nucleic acid encodes an immunoglobulin heavy chain. In the above embodiments, the selectable marker is a metabolic enzyme selected from the group consisting of glutamine synthetase, threonine dehydratase, adenylosuccinate synthetase and glutamate dehydrogenase.

The expression system described above according to the present invention may include an internal ribosome binding site (IRES); in one embodiment of the invention, the IRES is located at a region selected from the group consisting of: a region between the first nucleic acid and the second nucleic acid; a region between the third nucleic acid and a fourth nucleic acid and regions between both the first and second and between the third and fourth nucleic acids. In a further embodiment, the internal ribosome binding site comprises SEQ ID NO: 23.

In the expression system described herein, when the original mutant selectable marker subunit is an N-terminal mutant of glutamine synthetase (mutGS-NT), the complementary mutant selectable marker subunit is a C-terminal mutant of glutamine synthetase (mutGS-CT). In one such aspect of the invention, mutGS-NT contains two or more mutations selected from the group consisting of W60A N61A D63A D63R S66A and D76A, and mutGS-CT contains one or more mutations selected from the group consisting of E134A E136A E196A E203A N248A H253A N255A R319A and R324A. In one embodiment, mutGS-NT is selected from the group consisting of W60A N61A D63A (mutGS-NT 1, SEQ ID NO: 6), W60A N61A D63A S66A (mutGS-NT2; SEQ ID NO: 10) W60A N61A D63A S66A D76A (mutGS-NT3, SEQ ID NO: 25) and W60A N61A D63R S66A (mutGS-NT4; SEQ ID NO: 19); and mutGS-CT is selected from the group consisting of E134A E136A E196A E203A (mutGS-CTl, SEQ ID NO: 21) and N248A H253A N255A (mutGS-CT2; SEQ ID NO: 22).

In other embodiments, N-terminal and C-terminal glutamine synthetase fragments are created by protein cleavage at one or more amino acid positions of the glutamine synthetase protein. In one such aspect of the invention, the N-terminal fragment and/or C-terminal fragments of glutamine synthetase are cleaved at an amino acid position of glutamine synthetase selected from the group consisting of: E110, Y104, S125, N126, E264, T111, N105, N126, Q127 and N265 .

The invention also relates to an isolated host cell that has been transfected, transformed or transduced with a vector as described above. The host cell may be selected from the group consisting of CHO, VERO, BHK, HeLa, Cos, MDCK, 293, 3T3, myeloma cell line and WI38 cells. In one aspect of the invention, a method for producing a heteromeric complex is provided, comprising the step of culturing such a host cell under conditions where the heteromeric complex is expressed by the host cell. In one embodiment of the invention, the heteromeric complex is an antibody. The claimed methods according to the invention may also include isolation of the heteromeric complex.

In one embodiment, the invention provides an expression system comprising a first vector comprising a first nucleic acid encoding an antibody heavy chain operably linked to a second nucleic acid encoding N-terminal mutated glutamine synthetase (mutGS-NT) and a second vector comprising a third nucleic acid encoding an antibody light chain operably linked to a fourth nucleic acid encoding a C-terminal mutated glutamine synthetase (mutGS-CT), wherein each mutant glutamine synthetase subunit has no selectable activity when expressed alone, but when co-expressed mutGS-NT with mutGS-CT provides glutamine synthetase activity, and the expression system is capable of transfection into mammalian cells and improves the selection of transfected cells.

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In one embodiment, the invention provides an expression system comprising a first vector containing a first nucleic acid encoding an antibody heavy chain operably linked to a second nucleic acid encoding an N-terminal fragment of glutamine synthetase, and a second vector containing a third nucleic acid encoding a light chain. an antibody chain operatively linked to a fourth nucleic acid that encodes the C-terminal fragment of glutamine synthetase, wherein each mutant subunit of glutamine synthetase does not have selectable activity if expressed individually, and the co-expression of the N-terminal and C-terminal fragments of glutamine synthetase provides glutamine synthetase activity, with This expression system can be transfected into mammalian cells and improve the selection of transfected cells.

In another embodiment, the invention provides an expression system comprising a first vector comprising a first nucleic acid encoding an antibody light chain operably linked to a second nucleic acid encoding N-terminal mutated glutamine synthetase (mutGS-NT) and a second vector comprising a third nucleic acid encoding an antibody heavy chain operably linked to a fourth nucleic acid encoding a C-terminal mutated glutamine synthetase (mutGS-CT), wherein each mutant glutamine synthetase subunit has no selectable activity when expressed alone, but when co-expressed mutGS-NT with mutGS-CT provides glutamine synthetase activity, and the expression system is capable of transfection into mammalian cells and improves the selection of transfected cells.

In another embodiment, the invention provides an expression system comprising a first vector containing a first nucleic acid encoding an antibody light chain operably linked to a second nucleic acid encoding an N-terminal fragment of glutamine synthetase, and a second vector containing a third nucleic acid encoding a heavy chain. an antibody chain operatively linked to a fourth nucleic acid that encodes the C-terminal fragment of glutamine synthetase, wherein each glutamine synthetase fragment has no activity suitable for selection if expressed separately, and the co-expression of the N-terminal and C-terminal fragments of glutamine synthetase provides glutamine synthetase activity, while the expression system is able to be transfected into mammalian cells and improve the selection of transfected cells.

For any of the above embodiments, in one aspect of the invention, mutGS-NT contains two or more mutations selected from the group consisting of W60A N61A D63A D63R S66A and D76A, and mutGS-CT contains one or more mutations selected from the group consisting of E134A E136A E196A E203A N248A H253A N255A R319A and R324A. In one embodiment, mutGS-NT is selected from the group consisting of W60A N61A D63A (mutGS-NT1, SEQ ID NO: 6), W60A N61A D63A S66A (mutGS-NT2; SEQ ID NO: 10) W60A N61A D63A S66A D76A ( mutGS-NT3, SEQ ID NO: 25) and W60A N61A D63R S66A (mutGS-NT4; SEQ ID NO: 19); and mutGS-CT is selected from the group consisting of E134A E136A E196A E203A (mutGS-CT1, SEQ ID NO: 21) and N248A H253A N255A (mutGS-CT2; SEQ ID NO: 22).

In other embodiments, N-terminal and C-terminal glutamine synthetase fragments are created by protein cleavage at one or more amino acid positions of the glutamine synthetase protein. In one such aspect of the invention, the N-terminal fragment and/or C-terminal fragments of glutamine synthetase are cleaved at an amino acid position of glutamine synthetase selected from the group consisting of: E110, Y104, S125, N126, E264, T111, N105, N126, Q127 and N265 .

Likewise, the invention also relates to an isolated host cell that has been transfected, transformed or transduced with a vector as described above. The host cell may be selected from the group consisting of CHO, VERO, BHK, HeLa, Cos, MDCK, 293, 3T3, myeloma cell line and WI38 cells. In one aspect of the invention, there is provided a method for producing a heteromeric complex, comprising the step of culturing such a host cell under conditions where the heteromeric complex is expressed by the host cell. In one embodiment of the invention, the heteromeric complex is an antibody. The claimed methods according to the invention may also include isolation of the heteromeric complex.

Additional aspects of the invention include an expression system comprising a first vector encoding a bicistronic transcript containing a first nucleic acid encoding a desired polypeptide operably linked to a second nucleic acid encoding the original mutant subunit of a selectable marker, and a second vector encoding a bicistronic transcript containing a third a nucleic acid operably linked to a fourth nucleic acid encoding a complementary mutant subunit of a selectable marker, wherein the first and complementary mutant subunits of the selectable marker are associated with obtaining selectable activity, and wherein the expression system is capable of being transfected into mammalian cells and improving the selection of transfected cells, and in addition the first nucleic acid encodes the heavy chain of the antibody, and the third nucleic acid encodes

- 4 043386 antibody light chain. In one embodiment of the invention, the marker suitable for selection is glutamine synthetase.

Other aspects of the invention include an expression system comprising a first vector encoding a bicistronic transcript containing a first nucleic acid encoding a desired polypeptide operably linked to a second nucleic acid encoding the original fragment of a selectable marker, and a second vector encoding a bicistronic transcript containing a third nucleic acid an acid operably linked to a fourth nucleic acid encoding a complementary fragment of a selectable marker, wherein the first and complementary fragments of the selectable marker are associated with producing selectable activity and wherein the expression system is capable of being transfected into mammalian cells and enhancing selection of transfected cells, and in addition, the first nucleic acid encodes the heavy chain of the antibody, and the third nucleic acid encodes the light chain of the antibody. In one embodiment of the invention, the selectable marker is glutamine synthetase.

In one such aspect of the invention, glutamine synthetase contains two complementary mutations; thus, mutGS-NT contains two or more mutations selected from the group consisting of W60A N61A D63A D63R S66A and D76A, and mutGS-CT contains one or more mutations selected from the group consisting of E134A E136A E196A E203A N248A H253A N255A R319A and R324A. In one embodiment, mutGS-NT is selected from the group consisting of W60A N61A D63A (mutGS-NT1, SEQ ID NO: 6), W60A N61A D63A S66A (mutGS-NT2, SEQ ID NO: 10), W60A N61A D63A S66A D76A (mutGS-NT3; SEQ ID NO: 25) and W60A N61A D63R S66A (mutGS-NT4; SEQ ID NO: 19); and mutGS-CT is selected from the group consisting of E134A E136A E196A E203A (mutGS-CT1; SEQ ID NO: 21) and N248A H253A N255A (mutGS-CT2; SEQ ID NO: 22).

In other such embodiments, N-terminal and C-terminal glutamine synthetase fragments are created by protein cleavage at one or more amino acid positions of the glutamine synthetase protein. In one such aspect of the invention, the N-terminal fragment and/or C-terminal fragments of glutamine synthetase are cleaved at an amino acid position of glutamine synthetase selected from the group consisting of: E110, Y104, S125, N126, E264, T111, N105, N126, Q127 and N265.

The invention also relates to an isolated host cell that has been transfected, transformed or transduced with a vector as described above. The host cell may be selected from the group consisting of CHO, VERO, BHK, HeLa, Cos, MDCK, 293, 3T3, myeloma cell line and WI38 cells. In one aspect of the invention, there is provided a method for producing a heteromeric complex, comprising the step of culturing such a host cell under conditions where the heteromeric complex is expressed by the host cell. In one embodiment of the invention, the heteromeric complex is an antibody. The claimed methods according to the invention may also include isolation of the heteromeric complex.

Brief description of graphic materials

In fig. 1 is a schematic representation of the nucleic acid constructs used in Examples 1-5, each of which contains a selectable marker subunit and expresses different polypeptides that can be associated to form a heteromeric complex in a cell. Abbreviations represent the following: muGS, mutant glutamine synthetase; Ampr, ampicillin resistance. In fig. 1 (A) shows vector A (LC:mutGS-CT), and FIG. 1 (B) shows vector B (HC:mutGS-NT).

In fig. 2 is a graph showing the results of Survival Experiment 1. Constructs A and B for each version (1-5) were transfected into CHO-K1 GS knockout cells and tested for their ability to support cell viability in media lacking glutamine. A plasmid expressing the full-length GS enzyme was used as a control.

In fig. 3 is a graph showing that neither fragment A or B, taken alone, is able to ensure viability of the KO cell line, confirming that both fragments are required to restore GS enzyme activity.

In fig. 4 is a schematic representation of the expression vectors used in Example 6, each containing an antibody light chain or heavy chain, an IRES, and a glutamine synthetase fragment.

In fig. 5 is a graph showing the use of GS fragment vectors downstream of the LC and HC genes. The combination of 2A-LC and 2B-HC vectors were able to provide viability to GS knockout cells.

In fig. 6 is a graph showing the cell productivity of selected pools of Design 2 in a performance assay in fed-batch culture for 10 days and compared to a control pool that was selected using a vector expressing the full-length glutamine synthetase enzyme.

Detailed description of the invention

The efficient production of recombinant heteromeric complexes in cells is improved,

- 5 043386 if each component of the complex is expressed in proportional and large quantities. This invention improves the selection of transfected cells by providing methods and compositions for selecting recombinantly modified cells that express more than one heterologous polypeptide in proportional amounts such that the polypeptides can efficiently bind to form a heteromeric complex with higher expression levels than in the case of heteromeric complexes obtained by traditional methods. This invention also provides the advantage that it reduces the time required to select cells expressing high levels of the desired recombinant heteromeric polypeptide complex, another aspect of improving the selection of transfected cells is proposed in this invention.

Although the terminology used in this invention is standard in the art, definitions of certain terms are provided herein to provide clarity and certainty within the meaning of the claims. Units, prefixes and symbols may be designated in the accepted SI (International System of Units) form. The numeric ranges given herein include numbers that define the range and include and validate every integer within the stated range. The methods and techniques described herein are generally performed in accordance with conventional methods well known in the art and described in various general and more specific references cited and discussed herein unless otherwise indicated. See, for example, Sambrook, et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001) and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992), and Harlow and Lane Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990).

The disclosed methods are applicable to adherent or suspension cultures grown in stirred tank reactors (including traditional semi-continuous and fed-batch cell cultures that may, but not necessarily, contain a centrifugal filter), perfusion systems (including cultures grown using variable tangential flow (ATF), acoustic perfusion systems, depth filter perfusion systems and other systems), hollow fiber bioreactors (HFB, which in some cases can be used in perfusion processes), as well as various other cell culture methods (see ., for example, Tao et al., (2003) Biotechnol. Bioeng. 82:751-65; Kuystermans & Al-Rubeai, (2011) Bioreactor Systems for Producing Antibody from Mammalian Cells, in Antibody Expression and Production. Cell Engineering 7: 25-52, Al-Rubeai (ed) Springer; Catapano et al., (2009) Bioreactor Design and Scale-Up, in Cell and Tissue Reaction Engineering: Principles and Practice. Eibl et al. (eds) Springer-Verlag, incorporated herein by reference in its entirety).

As used herein, singular noun terms mean one or more unless specifically stated otherwise. In addition, unless the context otherwise indicates, terms in the singular include the plural and terms in the plural include the singular. In general, the nomenclatures used in connection with the technologies of cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are well known and widely used in the art.

This disclosure provides methods for modulating the properties of cell cultures expressing a protein of interest, the protein of interest includes naturally occurring proteins, recombinant proteins, and engineered proteins (eg, proteins that do not occur in nature and that have been designed and/or created by humans). The protein of interest may, but need not, be a protein that is known or suspected to be therapeutically significant. Specific examples of a protein of interest include antigen-binding proteins (as described and defined herein), peptide-associated antibodies (i.e., a molecule containing peptide(s) fused either directly or indirectly to other molecules, such as an Fc domain antibodies, wherein the peptide fragment specifically binds to the desired target, the peptide(s) may be fused to either an Fc region, inserted into an Fc loop, or into a modified Fc molecule, for example, as described in US Patent Application Publication No. US 2006 /0140934, incorporated herein by reference in its entirety), fusion proteins (e.g., Fc fusion proteins, wherein an Fc fragment is fused to a protein or peptide, including a peptide-associated antibody), cytokines, growth factors, hormones, and others found in naturally secreted proteins, as well as mutant forms of naturally occurring proteins.

The term heteromeric complex means a molecular complex formed by the association of at least two different molecules. The association may be a non-covalent interaction or a covalent connection, such as disulfide bonds. The two different molecules are typically two different polypeptides, but the invention contemplates heteromeric complexes between polypeptides and nucleic acids and between different nucleic acids. In one embodiment of the invention, the heteromeric complex provides functional activity, such as the ability to bind to a substrate (eg, an immunoglobulin capable of binding to a corresponding antigen), enzymatic activity, or the like. In one embodiment of the invention, the heteromeric complex according to the invention is secreted into the culture medium of the host cell in which it is produced.

As used herein, the terms association or interaction are intended to describe a relationship between at least two molecules, with one molecule binding to and/or influencing the activity of the others. The interaction may involve direct or indirect binding of two polypeptides (or a polypeptide and a nucleic acid), or the functional activation or inhibition of the activity of a molecule by another molecule.

In a specific embodiment of the invention, the heteromeric complex is an immunoglobulin molecule. An immunoglobulin in vertebrate systems is an antibody consisting of two identical light chains and two identical heavy chains. The four chains are linked together by disulfide bonds such that each light chain is connected to a heavy chain and the heavy chains are linked together at their tails, overall forming a Y-shaped heteromeric complex. Numerous methods are known by which DNA encoding immunoglobulin molecules can be manipulated to produce DNA capable of encoding recombinant proteins such as enhanced affinity antibodies or other antibody-based polypeptides (see, for example, Larrick et al. (1989), Biotechnology 7:934-938; Reichmann et al. (1988), Nature 332:323-327; Roberts et al. (1987), Nature 328:731-734; Verhoeyen et al. (1988), Science 239:1534-1536 ;Chaudhary et al. (1989), Nature 339:394-397).

Recombinant cells producing human antibodies (eg, produced using antibody libraries and/or transgenic animals and, optionally, further modified in vitro), as well as humanized antibodies can also be used in the invention. See, for example, Cabilly et al., US Patent No. 4,816,567; Cabilly et al., European Patent No. 0,125,023 Bl; Boss et al., US Patent No. 4816397; Boss et al., European Patent No. 0,120,694 B1; Neuberger et al., WO 86/01533; Neuberger et al., European Patent No. 0,194,276 B1; Winter, US Patent No. 5,225,539; Winter, European Patent No. 0,239,400 B1; Queen et al., European Patent No. 0,451,216 B1; and Padlan et al., European Patent No. 0,519,596 A1. For example, the invention can be used to induce the expression of human and/or humanized antibodies that immunospecifically recognize specific cellular targets, for example human EGF receptor, her-2/neu antigen, CEA antigen, prostate-specific membrane antigen (PSMA), Cd5, CD11a, CD18 , NGF, CD20, CD45, Epcam, other cancer cell surface molecules, TNF-alpha, TGF-b 1, VEGF, other cytokines, alpha 4 beta 7 integrin, IgE, viral proteins (eg, cytomegalovirus), etc. , thus listing only a few.

Examples of heteromeric complexes other than immunoglobulins include, but are not limited to, any heterodimeric or hetero-oligomeric protein, e.g. BMP2/BMP7, osteogenic protein, interleukin-1 converting enzyme (ICE), various interleukin receptors (e.g. IL-18 receptor , IL-13 receptor, IL-4 receptor and IL-7 receptor), nuclear receptors such as retinoid receptors, T cell receptors, integrins such as cell adhesion molecules, integrins, tumor necrosis factor receptor and soluble and membrane bound forms of major histocompatibility complex class I and class II (MHC) proteins. For heteromeric complexes that are receptors, the invention covers both soluble and membrane-bound forms of the polypeptides. Descriptions of additional heteromeric proteins that can be prepared in accordance with the invention can be found, for example, in Human Cytokines: Handbook for Basic and Clinical Research, Vol. II (Aggarwal and Gutterman, eds. Blackwell Sciences, Cambridge Mass., 1998); Growth Factors: A Practical Approach (McKay and Leigh, Eds. Oxford University Press Inc., New York, 1993) and The Cytokine Handbook (A W Thompson, ed.; Academic Press, San Diego Calif.; 1991).

As used herein, the term fusion protein refers to a protein or protein domain (eg, a soluble extracellular domain) fused to a heterologous protein or peptide. Examples of such fusion proteins include proteins expressed fused to a portion of an immunoglobulin molecule, proteins expressed fused to a portion of a zipper, and novel multifunctional proteins such as cytokine and growth factor fusion proteins (ie, GM-CSF and IL- 3, MGF and IL-3). WO 93/08207 and WO 96/40918 describe the preparation of various soluble oligomeric forms of the molecule designated CD40L, including an immunoglobulin fusion protein and a zipper fusion protein, respectively; the methods discussed therein are applicable to other proteins. Any of the molecules described herein can be expressed as a fusion protein, including, but not limited to, the extracellular domain of a cell receptor molecule, an enzyme, a hormone, a cytokine, a moiety of an immunoglobulin molecule, a zipper domain, and an epitope.

The term antigen binding protein is used in its broadest sense and means a protein containing a region that binds an antigen or target and, optionally, a scaffold or backbone that allows the antigen binding portion to adopt a conformation that facilitates the binding of the antigen binding protein to the antigen. Examples of antigen binding proteins include human antibody, humanized antibody; chimeric antibody; recombinant antibody; single-chain 7 043386 antibody; diabody; triatelo; tetrabody; Fab fragment; fragment F(ab')2; IgD antibody; IgE antibody; IgM antibody; IgG1 antibody; IgG2 antibody; IgG3 antibody; or IgG4 antibody, and fragments thereof. The antigen binding protein may comprise, for example, an alternative protein scaffold or an artificial scaffold grafted with CDRs or CDR derivatives. Such scaffolds include, but are not limited to, antibody-derived scaffolds containing mutations introduced, for example, to stabilize the three-dimensional structure of an antigen-binding protein, as well as fully synthetic scaffolds containing, for example, a biocompatible polymer. See, for example, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 53(1): 121–129 (2003); Roque et al., Biotechnol. Prog. 20:639-654 (2004). Additionally, peptide antibody mimetics (PAMs) can be used, as well as antibody mimetics-based scaffolds using fibronectin components as the scaffold.

The antigen binding protein may have, for example, the structure of a naturally occurring immunoglobulin. Immunoglobulin is a tetrameric molecule. In naturally occurring immunoglobulin, each tetramer consists of two identical pairs of polypeptide chains, with each pair having one light chain (about 25 kDa) and one heavy chain (about 50-70 kDa). The amino terminal portion of each chain contains a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain represents a constant region primarily responsible for effector function. Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon and define the antibody isotype as IgM, IgD, IgG, IgA, and IgE, respectively.

Naturally occurring immunoglobulin chains have the same general structure of relatively conserved framework regions (FRs) connected by three hypervariable regions, also called complementarity determining regions or CDRs. From the N-terminus to the C-terminus, both the light and heavy chains contain the FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 domains. The amino acid designation for each domain can be determined according to the definitions of Kabat et al. in Sequences of Proteins of Immunological Interest, ^5th Ed., US Dept., of Health and Human Services, PHS, NIH, NIH Publication no. 91-3242, (1991). If desired, the CDR can also be redefined according to an alternative nomenclature scheme such as Chothia (see Chothia & Eesk, (1987) J. Mol. Biol. 196:901-917; Chothia et al., (1989) Nature 342: 878-883 or Honegger & Pluckthun, (2001) J Mol Biol 309:657-670).

In the context of this disclosure, an antigen binding protein is said to specifically bind or selectively bind to its target antigen when the dissociation constant (KD) is <10'8 M. The antibody specifically binds to the antigen with high affinity when the KD is <5x10' 9 M and very high affinity in the case where KD is <5x10' ¹⁰ M.

The term antibody includes reference to glycosylated and non-glycosylated immunoglobulins of any isotype or subclass or to the antigen-binding region thereof that competes with the intact antibody for specific binding, unless otherwise noted. Additionally, the term antibody refers to an intact immunoglobulin or an antigen-binding portion thereof that competes with the intact antibody for specific binding unless otherwise noted. Antigen binding sites can be obtained by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies and can form a protein element of interest. Antigen binding moieties include, but are not limited to, Fab, Fab', F(ab') ₂ , Fv, domain antibodies (dAbs), fragments including complementarity determining regions (CDRs), single chain antibodies (scFv), chimeric antibodies, diabodies, tribodies , tetrabodies and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to specifically bind an antigen to the polypeptide.

The term antibody refers to a molecule in which the structure and/or function is based on the structure and/or function of an antibody, for example, a full-length or whole immunoglobulin molecule and/or derived from variable heavy chain (VH) and/or variable light chain (VL) domains. antibody or fragment thereof. Thus, the antibody construct is capable of binding to its specific target or antigen. In addition, the binding domain of an antibody construct in accordance with the invention contains the minimum structural requirements for the antibody that ensure target binding. This minimum requirement may, for example, be determined by the presence of at least three light chain CDRs (i.e., VL region CDR1, CDR2, and CDR3) and/or three heavy chain CDRs (i.e., VH region CDR1, CDR2, and CDR3) , preferably all six CDRs. An alternative approach to determining the minimum requirements for an antibody structure is to define the antibody epitope in the structure of a particular target, respectively, the protein domain of the target protein constituting the epitope region (epitope cluster), or by reference to a specific antibody that competes with the epitope of a particular antibody. Antibodies on which the constructs of the invention are based include, for example, monoclonal, recombinant, chimeric, deimmunized, humanized and human antibodies.

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The Fab fragment is a monovalent fragment having V _L , VH, C _L and _CH 1 domains; the F(ab') ₂ fragment is a bivalent fragment having two Fab fragments connected by a disulfide bridge at the hinge region; The Fd fragment has VH and CH1 domains; The Fv fragment has the V _L and VH domains of one antibody arm; the dAb fragment has a _VH domain, a _VL domain, or an antigen binding fragment of a VH or _VL domain; isolated complementarity determining region (CDR); and single chain Fv (scFv) (US Patent Nos. 6,846,634, 6,696,245, US Patent Applications No. 05/0202512, 04/0202995, 04/0038291, 04/0009507, 03/0039958, Ward et al., (1989) Nature 34 1: 544-546).

A single chain antibody (scFv) is an antibody in which the V _L and V H regions are joined by a linker (e.g., a synthetic sequence of amino acid residues) to form a continuous protein chain in which the linker is long enough to allow the protein chain to fold so as to form monovalent antigen binding site (see, for example, Bird et al., Science 242:423-26 (1988) and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-83). Diabodies are divalent antibodies containing two polypeptide chains, where each polypeptide chain contains VH and VL domains connected by a linker that is too short to allow pairing between two domains on the same chain, allowing each domain to pair with a complementary one. domain on another polypeptide chain (see, for example, Holliger et al., (1993) Proc. Natl. Acad. Sci. USA 90:6444-48; and Poljak et al., (1994) Structure 2:1121-23). If the two polypeptide chains of a diabody are identical, then the diabody resulting from their pairing will have two identical antigen-binding sites. Polypeptide chains having different sequences can be used to form a diabody with two different antigen binding sites. Similarly, tribodies and tetrabodies are antibodies containing three and four polypeptide chains, respectively, and form three and four antigen binding sites, respectively, which may be the same or different.

In addition, the term antibody includes monovalent, divalent and polyvalent/multivalent constructs and, thus, bispecific constructs that specifically bind to only two antigenic structures, as well as polyspecific/multispecific constructs that specifically bind to more than two antigenic structures, for example, three , four or more, using separate binding domains. In addition, the term antibody includes molecules consisting of only one polypeptide chain, as well as molecules consisting of more than one polypeptide chain, the chains being either identical (homodimers, homotrimers, or homooligomers) or different (heterodimers, heterotrimers, or heterooligomers). ). Examples of the above antibodies and variants or derivatives thereof are described in particular in Harlow and Lane, Antibodies a laboratory manual, CSHL Press (1988) and Using Antibodies: a laboratory manual, CSHL Press (1999), Kontermann and Dribel, Antibody Engineering, Springer, 2nd ed. 2010 and Little, Recombinant Antibodies for Immunotherapy, Cambridge University Press 2009.

As used herein, the term bispecific refers to an antibody that is at least bispecific, that is, it contains at least a first binding domain and a second binding domain, wherein the first binding domain binds to a single antigen or target (in this case: surface antigen of the target cell), and the second binding domain binds to another antigen or target. Accordingly, bispecific antibody constructs have specificity for at least two different antigens or targets. Bispecific antibodies may also include multispecific antibody constructs, such as trispecific antibody constructs, the latter of which have three binding domains, or constructs having specificity for more than three targets (eg, four, five...).

One or more CDRs may be included in a molecule either covalently or non-covalently to make the molecule an antigen-binding protein. An antigen binding protein may contain a CDR as part of a longer polypeptide chain, may covalently link a CDR to another polypeptide chain, or may contain a CDR non-covalently. CDRs allow an antigen binding protein to specifically bind to a particular antigen of interest.

An antigen binding protein may have one or more binding sites. If there is more than one binding site, the binding sites may be identical to each other or may be different. For example, a naturally occurring human immunoglobulin typically has two identical binding sites, whereas a bispecific or bifunctional antibody has two different binding sites.

For clarity, and as described herein, it should be noted that the antigen binding protein may, but need not, be of human origin (eg, a human antibody) and in some cases will contain a protein of non-human origin, such as a rat or mouse protein, and in In other cases, the antigen binding protein may comprise a hybrid of human and non-human proteins (eg, a humanized antibody).

The protein of interest may include/imply a human antibody. The term human antibody includes all antibodies that have one or more variable and constant regions derived from human immunoglobulin sequences. In one embodiment

- 9 043386 tions of the invention, all variable and constant domains are derived from human immunoglobulin sequences (fully human antibody). Such antibodies can be produced in a variety of ways, including by immunizing a genetically modified mouse with an antigen of interest to express antibodies derived from human heavy and/or light chain genes, such as a mouse derived from the Xenomouse®, UltiMab™ or Velocimmune® systems. Phage-based approaches can also be used.

Alternatively, the protein of interest may comprise a humanized antibody. A humanized antibody has a sequence that differs from an antibody sequence derived from a non-human species by one or more amino acid substitutions, deletions and/or insertions such that the humanized antibody is less likely to induce an immune response and/or induces a less pronounced immune response when administered to a human, compared to an antibody obtained from a non-human species. In one embodiment of the invention, certain amino acids in the backbone and constant domains of the heavy and/or light chains of antibodies from non-human species are mutated to form a humanized antibody. In another embodiment, the constant domain(s) from a human antibody are fused to the variable domain(s) of a non-human species. Examples of how to make humanized antibodies can be found in US Patent Nos. 6,054,297, 5,886,152 and 5,877,293.

The Fc region, as used herein, contains two heavy chain fragments containing the antibody domains CH2 and CH3. The two heavy chain fragments are held together by two or more disulfide bonds and hydrophobic interactions of C _H 3 domains. Proteins of interest containing the Fc region, including antigen binding proteins and Fc fusion proteins, represent another aspect of this disclosure.

A hemibody is an immunologically functional immunoglobulin construct comprising a complete heavy chain, a complete light chain, and a second heavy chain Fc region linked to a complete heavy chain Fc region. A linker may, but is not required to, be used to attach a heavy chain Fc region to a second heavy chain Fc region. In specific embodiments, the hemibody is a monovalent form of the antigen binding protein disclosed herein. In other embodiments, pairs of charged residues may be used to link one Fc region to a second Fc region. The hemibody may be a protein of interest in the context of this disclosure.

The term host cell means a cell that has been genetically modified to express a polypeptide of commercial or scientific interest. Genetic modification of a cell line involves transfection, transformation, or transduction of cells with a nucleic acid encoding a recombinant polynucleotide molecule (gene of interest) and/or other modification (for example, by homologous recombination and gene activation or fusion of a recombinant cell with a non-recombinant cell) to provide expression host cell of the desired recombinant polypeptide. Methods and vectors for genetically modifying cells and/or cell lines to express a polypeptide of interest are well known to those skilled in the art; for example, various methods are illustrated in Current Protocols in Molecular Biology. Ausubel et al., eds. (Wiley & Sons, New York, 1988, and quarterly updates); Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Laboratory Press, 1989); Kaufman, R.J., Large Scale Mammalian Cell Culture, 1990, pp. 15-69. The term includes the progeny of a parent cell, regardless of whether the progeny is morphologically or genetically identical to the original parent cell, as long as the gene of interest is present. The cell culture may contain one or more host cells.

The term hybridoma means a cell or progeny of a cell resulting from the fusion of an immortalized cell and an antibody-producing cell. The resulting hybridoma is an immortalized cell that produces antibodies. The individual cells used to create a hybridoma can be obtained from any mammalian source, including, but not limited to, hamster, rat, pig, rabbit, sheep, goat and human. The term also covers trioma cell lines that are produced when the progeny of heterohybrid myeloma fusions, which are the product of a fusion between human cells and a mouse myeloma cell line, are subsequently fused with a plasma cell. The term refers to any immortalized hybrid cell line that produces antibodies, such as, for example, Quadromas (see, for example, Milstein et al., (1983) Nature, 537:3053).

The terms culture and cell culture are used interchangeably and refer to a population of cells that is maintained in a medium under conditions suitable for the survival and/or growth of the cell population. As will be clear to those skilled in the art, these terms also refer to the combination comprising a population of cells and a medium in which the culture is maintained.

The terms polypeptide and protein (eg, as used in the context of a protein of interest or a polypeptide of interest) are used interchangeably herein to refer to a polymer of amino acid residues. The terms also apply to amino acids

- 10 043386 polymers in which one or more amino acid residues are an analog or mimetic of the corresponding naturally occurring amino acid, as well as naturally occurring amino acid polymers. The terms may also cover amino acid polymers that have been modified, for example, by the addition of carbohydrate residues to form glycoproteins or phosphorylated. The polypeptides and proteins can be produced using a naturally occurring and non-recombinant cell, or the polypeptides and proteins can be produced using a genetically modified or recombinant cell. Polypeptides and proteins may include molecules having the amino acid sequence of a native protein, or molecules having deletions, insertions and/or substitutions of one or more amino acids of the native sequence.

The terms polypeptide and protein cover molecules containing only naturally occurring amino acids, as well as molecules that contain non-naturally occurring amino acids. Examples of non-naturally occurring amino acids (which may be replaced by any naturally occurring amino acid found in any sequence disclosed herein, as appropriate) include (but are not limited to): 4-hydroxyproline, γ-carboxyglutamate, ε-N, N, N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3methylhistidine, 5-hydroxylysine, σ-N-methylarginine and other similar amino acids and imino acids (for example, 4-hydroxyproline). In the polypeptide nomenclature used herein, the left direction is towards the amino terminus and the right direction is towards the carboxy terminus according to standard usage and convention.

Cell culture or culture refers to the growth and propagation 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, D. Rickwood, ed., Oxford University Press, New York (1992). Mammalian cells can be cultured in suspension or on the surface of a solid substrate. Fluidized bed bioreactors, hollow fiber bioreactors, spin flasks, shake flasks, or stirrer bioreactors can be used, with or without microcarriers. In one embodiment of the invention, bioreactors with a volume of 500-2000 liters are used. In one embodiment of the invention, bioreactors with a volume of 1000-2000 liters are used.

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

The nutrient solution may optionally be supplemented with additional optional components to optimize cell growth, such as hormones and other growth factors, for example, insulin, transferrin, epidermal growth factor, serum and the like; salts, eg calcium, magnesium and phosphate, and buffers, eg HEPES; nucleosides and bases, for example adenosine, thymidine, hypoxanthine; and protein and tissue hydrolysates, for example, hydrolyzed animal or vegetable protein (peptone or peptone mixtures, which can be obtained from animal by-products, purified gelatin or plant material); antibiotics, such as gentamicin; cell protectants or surfactants such as Pluronic®F68 (also called Lutrol® F68 and Kolliphor® P188, nonionic triblock copolymers consisting of a central hydrophobic polyoxypropylene (poly(propylene oxide)) chain flanked by two hydrophilic polyoxyethylene (polyethylene oxide) chains )); polyamines, for example putrescine, spermidine and spermine (see, for example, WIPO publication No. WO 2008/154014) and pyruvate (see, for example, US patent No. 8053238) depending on the needs of the cultured cells and/or the parameters of the desired cell culture.

Cell culture media include those commonly used and/or known for use with any cell culture process, such as, but not limited to, semi-continuous, semi-continuous extended, fed-batch and/or flow or continuous cell culture.

Basic (or semi-continuous) 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.

Growth culture medium refers to a cell culture medium that is typically used in cell cultures during the exponential growth period, growth phase, and is complete enough to

- 11 043386 cell culture support during this phase. The cell growth culture medium may also contain selective agents that confer resistance or survival to selectable markers included in the host cell line.

Such selective agents include, but are not limited to, geneticin (G4118), neomycin, hygromycin B, puromycin, zeocin, methionine sulfoximine, methotrexate, cell culture medium free of glutamine, cell culture medium free of glycine, hypoxanthine and thymidine, or only Thymidine

Cell culture production medium refers to the cell culture medium that is typically used in cell cultures during the transition when exponential growth ends and protein production enters the transition and/or product phases and is sufficiently complete to maintain the required cell density, viability and /or product titer during this phase.

Flow-through cell culture medium refers to a cell culture medium that is commonly used in cell cultures that are maintained by flow-through or continuous culture methods and is sufficiently complete to support the cell culture during this process. Flow cell culture medium preparations may be more enriched or more concentrated than base cell culture medium preparations to tailor the method used to remove the spent medium. Flow cell culture medium can be used at both the growth and production stages.

The concentrated cell culture medium may contain some or all of the nutrients necessary to maintain the cell culture; in particular, the concentrated medium may contain nutrients that are determined or known to be consumed during the production phase of the cell culture. The concentrated medium can be based on any cell culture medium preparation. Such concentrated culture medium may contain some or all of the cell culture medium components in amounts of, for example, 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 of their usual amount.

The components used to prepare the cell culture medium can be completely ground to form a powdered medium; partially ground with liquid additives added to the cell culture medium if necessary; or added in completely liquid form to cell culture.

Cell cultures can also be supplemented with independent concentrated nutrient mixtures of certain nutrients that are difficult to incorporate into the preparation or are quickly depleted in cell cultures. Such nutrients may be amino acids such as tyrosine, cysteine and/or cystine (see, for example, WIPO Publication No. 2012/145682). In one embodiment of the invention, a concentrated solution of tyrosine is separately supplied to a cell culture grown in a cell culture medium containing tyrosine such that the concentration of tyrosine in the cell culture does not exceed 8 mM. In another embodiment of the invention, a concentrated solution of tyrosine and cystine is separately supplied to a cell culture grown in a cell culture medium without tyrosine, cystine or cysteine. Separate nutrient mixtures can be added before or at the beginning of the production phase. Separate nutrient mixtures may be added in fed-batch culture to the cell culture medium on the same or different days as the concentrated nutrient medium. Individual nutrients may also be added through the flow method on the same or different days as the flow through medium.

Serum-free refers to a cell culture medium that does not contain animal serum, such as fetal bovine serum. Various tissue culture media, including the following culture media, are commercially available, for example, any combination of the following cell culture media may be used: RPMI-1640 medium, RPMI-1641 medium, Dulbecco's modified Eagle's medium (DMEM), Eagle's minimal essential medium , F-12K medium, Ham's F12 medium, Iscove's modified Dulbecco's medium, McCoy's 5A medium, Leibovitz's L-15 medium, and serum-free medium such as EX-CELL™ 300 series (JRH Biosciences, Lenexa, KS) and others . Serum-free versions of such culture media are also available. Cell culture media may be supplemented with additional or increased concentrations of components such as amino acids, salts, sugars, vitamins, hormones, growth factors, buffers, antibiotics, lipids, trace elements and the like, depending on the needs of the cells to be cultured and/or or desired cell culture parameters.

The term bioreactor means any container suitable for the growth of a cell culture. Cell cultures according to this disclosure can be grown in a bioreactor, which can be selected based on the use of a protein of interest that is produced by cells growing in the bioreactor. The bioreactor can be of any size as long as it is suitable for cell culture; Typically, the size of the bioreactor corresponds to the volume of the cell culture grown inside it. Typically, the bioreactor will have a volume of at least 1 liter and the volume can be 2, 5, 10, 50, 100,

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200, 250, 500, 1000, 1500, 2000, 2500, 5000, 8000, 10000, 12000 liters or more, or any intermediate volume between these values. The internal conditions of the bioreactor, including, but not limited to, pH and temperature, can be controlled during the culture period. Those skilled in the art will know and be able to select suitable bioreactors for use in the practice of this invention based on appropriate considerations.

Cell density refers to the number of cells in a given volume of culture medium. Viable cell density refers to the number of living cells in a given volume of culture medium, as determined by standard viability analytical methods (such as the trypan blue exclusion method).

The term cell viability refers to the ability of cells in culture to survive a given set of culture conditions or experimental changes. The term also refers to the proportion of cells that are alive at a particular time relative to the total number of cells, living and dead, in the culture at that time.

Cell volume (PCV), also called percentage cell volume (%PCV), is the ratio of the volume occupied by cells to the total volume of a cell culture, expressed as a percentage (see Stettler, et al., (2006) Biotechnol Bioeng. Dec 20:95(6): 1228-33). The volume of cell mass depends on cell density and cell diameter; the increase in cell mass volume may be due to an increase in cell density or cell diameter, or both. Cell mass volume is a measure of the solid content of a cell culture. Solids are removed during collection and further purification. More solids means more effort to separate the solid material from the desired product during collection and subsequent purification steps. Additionally, the desired product may be trapped in solids and lost during the harvesting process, resulting in reduced product yield. Because host cells vary in size and cell cultures also contain dead and dying cells and other cellular debris, cell mass volume is a more accurate way of describing the solid content of a cell culture than cell density or viable cell density. For example, a 2000 L culture having a cell density of 50 x 106 cells/ml would have significantly different volumes of cell mass depending on cell size. In addition, some cells in a growth arrest state will increase in size, so the volume of cell mass before and after growth arrest is likely to differ due to the increase in biomass resulting from increased cell size.

Growth arrest, which may also be referred to as cell growth arrest, is the point at which cells stop increasing in number or when progression through the cell cycle stops. Growth arrest can be monitored by determining the density of viable cells in the cell culture. Some cells in a state of growth arrest may increase in size, but not in number, so the volume of cell mass of a culture in a state of growth arrest may increase. The growth arrest can be partially reversed if the cells are not in a state of deterioration, reversing the conditions that lead to the growth arrest.

The term titer means the total amount of polypeptide or protein of interest (which may be a naturally occurring or recombinant protein of interest) produced by a cell culture in a given volume of medium. The titer may be expressed in units of milligrams or micrograms of polypeptide or protein per milliliter (or other measure of volume) of medium. The cumulative titer is the titer produced by cells when grown in culture and can be determined, for example, by measuring daily titer values and using these values to calculate the cumulative titer.

The term fed-batch culture refers to a form of suspension culture and refers to a method of culturing cells in which additional components are added to the culture after the culture process has begun one or more times. The added components typically contain nutritional supplements for cells that have been depleted during the culture process. Additionally or alternatively, additional components may include additional components (eg, compounds that inhibit cell cycle progression). The fed-batch culture is typically stopped at some point, and cells and/or components in the medium are collected and optionally purified.

The terms integrated viable cell density or IVCD are used interchangeably and mean the average viable cell density during culture multiplied by the amount of time the culture took place.

Cumulative viable cell density (CVCD) is calculated by multiplying the average viable cell density (VCD) between two time points by the amount of elapsed time between the two time points. CVCD is the area under the curve obtained by plotting VCD versus time.

The invention uses selectable markers that can be subjected to intragenic complementation to directly select expression constructs that encode heteromeric proteins, such as antibodies, using a two-vector system. Intragenic complementation refers to complementation between pieces of genetic material (ie, nucleic acids), each of which has a different defect within the same locus. A single protein encoded by genetic material is defective and nonfunctional, but two separate defective proteins can be associated to form an active protein. If individual defective proteins are able to associate and exhibit activity, the individual defective proteins are considered complementary. For selectable marker proteins that are composed of multiple identical subunits (such as GS), intragenic complementation can occur when the mutant subunits are complementary.

Intragenic complementation can also occur between individual protein fragments encoded by genetic material that are associated with the formation of an active protein. As described herein, the mature GS enzyme in higher eukaryotes exists as a decameric complex consisting of two pentameric rings. This invention demonstrates that intragenic GS complementation can be successfully used as part of a selection system to generate cell lines expressing recombinant proteins. The system is useful for expressing recombinant antibody at high levels in CHO cells that are genetically modified to be glutamine synthetase deficient.

A particular protein encoded by genetic material becomes defective and nonfunctional as a result of the introduction of one or more mutations in the encoding nucleic acid, the mutation(s) altering the structure of the protein sufficiently to render it nonfunctional. The mutation or mutations can be introduced by any method known in the field of molecular biology. Alternatively, a protein fragment that is damaged and nonfunctional may be used. In this case, two protein fragments can associate to form an active protein by intragenic complementation.

Nucleic acid molecules have been created that encode a polypeptide and a mutant subunit of a marker suitable for selection, arranged in such a way that the expression of the mutant subunit correlates with the expression of the polypeptide. Thus, when nucleic acid molecules encoding complementary mutant subunits are introduced into cells and selective conditions are applied, approximately equal and high levels of expression of each of the complementary mutant subunits provide maximum selectable activity. In addition, functionally related polypeptides will be expressed in nearly equal and high amounts, thereby optimizing the selection of cells expressing equal and high levels of the desired polypeptides.

In one embodiment, the selectable marker is a glutamine synthetase (GS), the active site of which is located at the junction of two GS subunits such that coexpression of complementary, functionally disrupted mutant subunits should result in a number of intact functionally active sites. The individual subunits of the mutants do not have significant selectable activity individually, but provide selectable activity when coexpressed with their complementary mutant subunit. The optimal activity of the subunits may depend on their interaction and thus may be mediated by the interaction of the domains. Such interacting domains may be endogenous to the subunit or may be heterologous to the subunit. Additional selectable markers that are useful in this invention include arginosuccinate lyase, adenylosuccinate synthetase and glutamate dehydrogenase, for which complementary mutants can be identified. For example, intragenic complementation in argininosuccinate lyase mutants can be confirmed by growth in the absence of arginine, whereas intragenic complementation in adenylosuccinate synthetase mutants can be confirmed by growth in the absence of adenosine.

In one non-limiting embodiment, the invention entails the use of two selectable marker fragments, each of which can be expressed as a fusion protein in the interaction domain. When expressed, the interaction domain promotes association or dimerization of two moieties, thereby allowing the subunits to function and provide selectable activity (e.g., but not limited to, as described in Pelletier et al. (1998), Proc. Natl. Acad. Sci., 95:12141-12146). Suitable selectable markers include GS and other selectable markers described above, as well as selectable markers such as those that confer resistance to specific drugs that are typically toxic, and metabolic enzymes that promote cell survival or induce cell death under certain conditions, as disclosed herein.

An interaction domain is a domain, including but not limited to polypeptides, capable of facilitating the interaction or association of two or more homologous or heterologous polypeptides. In one embodiment of the invention, the interaction domain is a dimerization domain. The dimerization domain may be a polypeptide capable of inducing interaction or association of two polypeptides. There are two types of dimers, those

- 14 043386 which are capable of forming homodimers (having the same sequence) or heterodimers (having a different sequence).

In one illustrative, but non-limiting, embodiment of the invention, the interaction domain is a coiled-coil polypeptide with a leucine zipper. The leucine zipper typically contains about 35 amino acids, having a characteristic repeat of seven amino acids with hydrophobic residues in the first and fourth residues of the repeat (Harbury et al. (1993), Science 262:1401). Thus, the leucine zipper is applicable to fusion with a polypeptide to oligomerize the polypeptide because it is a small molecule and is less likely to disrupt the normal function of polypeptides than a larger interaction domain. Examples of leucine zippers include, but are not limited to, leucine zipper domains from polypeptides such as GCN4, C/EBP, c-Fos, c-Jun, c-Myc, and c-Max.

Additional examples of dimerization domains include helix-loop-helix domains (Murre et al. (1989), Cell 58:537-544). Retinoic acid receptor, thyroid hormone receptor, other nuclear hormone receptors (Kurokawa et al. (1993), Genes Dev. 7:1423-1435) and yeast transcription factors GAL4 and HAP1 (Marmonstein et al. (1992), Nature 356:408 -414; Zhang et al. (1993), Proc. Natl. Acad. Sci. USA 90: 2851-2855; US Patent No. 5,624,818), all have regions of dimerization with this motif.

In yet another embodiment, the interaction domain is a tetramerization domain, which is a polypeptide capable of binding to three other tetramerization domains to form a tetramer complex. Examples of proteins containing tetramerization domains include, but are not limited to, the E. coli lactose repressor (amino acid 46360, Chakerian et al. (1991), J. Biol. Chem. 266:1371; Alberti et al. (1993), EMBO J. 12:3227; and Lewis et al. (1996), Nature 271:1247) and the p53 tetramerization domain formed by residues 322-355 (Clore et al. (1994), Science 265:386; Harbury et al. (1993 ), Science 262:1401; US Patent No. 5,573,925).

In an alternative embodiment, the invention entails the use of three selectable marker subunits, each of which is expressed as a fusion protein in an interaction domain, thereby enhancing the association to provide selectable activity. In this embodiment of the invention there are three components of the heteromeric complex. In the expressed vector(s), the coding sequences are each operably linked to the coding sequences of each of the corresponding subunits of a selectable marker, for example, a bispecific antibody expressed as one heavy chain and two different light chains, both light chains being capable of binding to the heavy chain. chain. The invention also covers the use of selectable markers, known or still to be studied, which have four or even more subunits.

The present invention will also be useful for the preparation of heteromultimeric (or heterooligomeric) proteins that contain three or more subunits, such as those described above. This includes bispecific antibodies and other heteromultimeric proteins that are known in the art. For example, a bispecific antibody may be expressed using a first selectable marker that can be subject to intragenic complementation as described herein (e.g., GS) to express the heavy chain and light chain of the first antibody and another selectable marker such as a separated metabolic enzyme or resistance marker, or a second selectable marker that is different from the first selectable marker and can undergo intragenic complementation, expressing the heavy chain and light chain of a second antibody that is capable of binding to the first antibody to form a bispecific antibody.

Alternatively, a first selectable marker that is capable of intragenic complementation as described herein (eg, GS) can be used to express two different heavy chains of a bispecific antibody, where the heavy chains are capable of binding to each other. The light chains can then be expressed using another marker suitable for selection. In some cases, the light chain may be the same for both arms of a bispecific antibody, indicating that a single selectable marker (metabolic enzyme or resistance marker or other form of selectable marker) can be used to express the light chains. Alternatively, a different light chain may be associated with each different heavy chain of the bispecific antibody, in which case another selectable marker, such as a separated metabolic enzyme or resistance marker, or a second selectable marker that is different from the first selectable marker and may undergo intragenic complementation, used to express the first and second light chains.

As will be shown in the Examples below, it has been found that the methods and compositions of the invention reduce the time required to select desired cells expressing high levels of heteromultimeric protein. Thus, in yet another embodiment, the invention encompasses the selection of cells expressing high levels of recombinant polypeptide, in particular heteromultimeric polypeptides. In addition, the invention has particular utility in improving the production of heteromeric complexes through cell culture processes.

In some embodiments, nucleic acids encoding mutant marker subunits are fused in frame to a nucleic acid encoding a linker, which is further fused in frame to a nucleic acid encoding an interaction domain. Linkers can include any relatively short flexible sequence that allows the interacting domain to interact and the subunits to function to provide selectable activity. Examples of linkers are widely known in the art and may include GGPGG, GPGGG, where in single letter amino acid codes G represents glycine and P represents proline. In one embodiment, the linker is a series of glycine and serine residues, such as those described by Curtis et al. (1991; Proc Natl Acad Sci 88(13):5809–5813).

In one embodiment of the invention, two complementary mutant subunits are expressed from two vectors, where the first vector contains a first nucleic acid encoding a first polypeptide, and where the first nucleic acid is operably linked to a second nucleic acid encoding a mutant subunit of a selectable marker. The second vector contains a third nucleic acid encoding a polypeptide that is capable of binding to a first polypeptide encoded by the first nucleic acid, the third nucleic acid being operably linked to a fourth nucleic acid encoding a complementary mutant subunit of a selectable marker. Thus, both vectors are simultaneously introduced into a population of cells, and selection for intragenic complementation is carried out.

In another embodiment of the invention, two complementary fragments are expressed from two vectors, where the first vector contains a first nucleic acid encoding a first polypeptide, and where the first nucleic acid is operably linked to a second nucleic acid encoding a selectable marker fragment. The second vector contains a third nucleic acid encoding a polypeptide that is capable of binding to a first polypeptide encoded by the first nucleic acid, the third nucleic acid being operably linked to a fourth nucleic acid encoding a complementary fragment of a selectable marker. Thus, both vectors are simultaneously introduced into a population of cells, and selection for intragenic complementation is carried out.

For example, a two-vector system for antibody expression is constructed by constructing a first vector containing a first nucleic acid encoding one antibody chain, the first nucleic acid operably linked to a second nucleic acid encoding mutGS-NT. The second vector contains a third nucleic acid encoding another antibody chain, the third nucleic acid being operably linked to a fourth nucleic acid encoding mutGS-CT. Thus, in one embodiment of the invention, the first nucleic acid encodes an immunoglobulin heavy chain and the third nucleic acid encodes an immunoglobulin light chain. Alternatively, the first nucleic acid encodes an immunoglobulin light chain and the third nucleic acid encodes an immunoglobulin heavy chain.

In another embodiment of the invention, two complementary fragments are expressed from two vectors, where the first vector contains a first nucleic acid encoding a first polypeptide, and where the first nucleic acid is operably linked to a second nucleic acid encoding a selectable marker fragment. The second vector contains a third nucleic acid encoding a polypeptide that is capable of binding to a first polypeptide encoded by the first nucleic acid, the third nucleic acid being operably linked to a fourth nucleic acid encoding a complementary fragment of a selectable marker. Thus, both vectors are simultaneously introduced into a population of cells, and selection for intragenic complementation is carried out.

In such a case, a two-vector system for antibody expression is constructed by constructing a first vector containing a first nucleic acid encoding one chain of the antibody, the first nucleic acid operably linked to a second nucleic acid encoding an N-terminal fragment of glutamine synthetase. The second vector contains a third nucleic acid encoding another antibody chain, the third nucleic acid being operably linked to a fourth nucleic acid encoding a C-terminal fragment of glutamine synthetase. Thus, in one embodiment of the invention, the first nucleic acid encodes an immunoglobulin heavy chain and the third nucleic acid encodes an immunoglobulin light chain. Alternatively, the first nucleic acid encodes an immunoglobulin light chain and the third nucleic acid encodes an immunoglobulin heavy chain.

An additional embodiment of the invention includes the use of one or more IRES, allowing the expression of one or both mutGS subunits. IRES in the context of this invention means an internal ribosome binding site that facilitates the initiation of translation of mRNA from an internal site (ie, a site different from the 5' end of the mRNA).

One example of a suitable IRES is the encephalomyocarditis virus (ECMV) IRES, as

- 16 043386 described in Jang and Wimmer Genes & Development 4 1560 (1990) and Jang, Davies, Kaufman and Wimmer J.

Vir. 63 1651 (1989). Residues 335–848 of EMCV form a suitable IRES; other options or parts

ECMV IRES are known and will be suitable for use in this invention. A suitable portion or variant of an IRES is one that will facilitate sufficient translation of the second open reading frame (OPC).

In addition, the 3' end of the IRES can be altered (or mutated) to reduce translation efficiency, thereby providing a means to improve selection and/or amplification methods. For example, the efficiency of an IRES can be reduced by using a sequence that has previously been known to provide efficient selection and amplification (Aldrich et al., Biotechnol Prog 19, 1433; 2003). Alternative sequences are known or can be determined by one skilled in the art.

In another embodiment, the invention further includes a nucleic acid encoding another functional selectable marker in addition to the selectable marker subunit and a heteromeric complex polypeptide. As used herein, the other functional selectable marker is not a mutant subunit of a selectable marker that is capable of intragenic complementation, but is a protein with other selectable activity. Well-known markers such as zeomycin, neomycin, puromycin, blasticidin S or GPT, which confers resistance to mycophenolic acid, etc., can be used as other functional markers suitable for selection. Additionally, complementary mutants of another selectable marker that is capable of intragenic complementation can also be used.

In this embodiment, the invention includes two vectors, wherein each of the vectors contains a first nucleic acid encoding a polypeptide that can form a heteromeric complex, operably linked to a second nucleic acid encoding at least one mutant subunit of a selectable marker, and a nucleic acid , encoding another functional marker suitable for selection. In addition, the corresponding polypeptides encoded by the first nucleic acid of each vector may be associated with the formation of a complex, and the subunit or subunits encoded by the second nucleic acids of each vector may be associated with providing selection activity, and the polypeptides encoded by third nucleic acids provide selection activity. selection activity different from the selectable activity of the subunits encoded by the second nucleic acids.

For example, the first vector may encode neomycin resistance and the second vector may encode zoomycin resistance, or only one vector may contain an additional other functional selectable marker. Thus, one vector is transfected into the cell line and selection is performed (ie, the G418 drug is added to neomycin-resistant cells). Once selected, conventional methods can be used to determine the presence of the vector and the level of expression of the polypeptides encoded by the nucleic acids of the vector, for example, by PCR, Southern blotting, ELISA, Western blotting and the like. Once a high level of expression is obtained, the second vector is transfected into the cell line. As selection continues with respect to the first vector, selection is carried out with respect to the second marker suitable for selection (i.e., resistance to zeomycin) and the presence of the second vector and the expression of the corresponding proteins encoded by the vector are assessed. In this embodiment, once it has been determined that both vectors are present, selection is made regarding the expression of subunits that associate in the cell to provide selectable activity as described herein.

In an alternative embodiment, both nucleic acids of the invention encoding independent selectable activities are transfected simultaneously and selection is carried out simultaneously. Once it has been determined that both vectors are present, selection is made regarding the expression of subunits that associate in the cell to provide selectable activity as described above.

In yet another embodiment, the vectors of the invention encoding independent selectable activities are each transfected into separate cell lines. After selecting and identifying clones that express high levels of the proteins encoded by each desired vector, the cells are fused as described in Hori et al. (US Patent No. 5,916,771). After completion of the fusion, selection is carried out regarding the selectable activity provided by the subunits.

In yet another embodiment, the nucleic acids of the invention, optionally not containing a separate selectable activity, are transfected simultaneously with the third vector. The third vector encodes a distinct selectable activity, such as, for example, neomycin or beta-galactosidase resistance, which may allow preselection of cells that have been successfully transfected. Once this preliminary selection has been completed, selection can be made for the selectable activity of the mutant subunits. In this embodiment, equal amounts of two expression vectors are used for transfection, while the amount of the third transfection vector is one third of the concentration of the first two vectors (for example, a ratio of 3:3:1 or 6:6:1 or the like) . One skilled in the art will appreciate that changes in ratios are within the scope of the invention.

Nucleic acids encoding a component of the desired heteromeric complex can be obtained as cDNA or as genomic DNA by methods known in the art. For example, messenger RNA encoding the desired component can be isolated from a suitable source using standard RNA isolation techniques and using oligo-dTcellulose chromatography to separate poly-A mRNA. In the case where the heteromeric complex to be expressed is an antibody, suitable sources of the desired nucleic acids can be isolated from mature B cells or hybridoma culture. In addition, nucleic acids for use in the invention can be obtained by chemical synthesis.

In addition to the nucleic acid encoding the desired component of the heteromeric complex, the vector constructs may contain additional components to facilitate replication in prokaryotic and/or eukaryotic cells, integration of the construct into the eukaryotic chromosome, and markers for selection and/or screening of cells containing the construct. The vectors of the invention are recombinant DNA vectors, including, but not limited to, plasmids, phages, phagemids, cosmids, viruses, retroviruses and the like, which introduce the desired nucleic acid into a cell.

A nucleic acid is operably linked when it is operably linked to another nucleic acid. More specifically, functional linkage means that two different nucleic acids encoding different polypeptides are transcribed when induced simultaneously. Functional coupling must also mean that the associated nucleic acids can coexist in a single transcription unit, while translation begins at one or more ribosome binding sites (eg, an internal ribosome binding site). Functionally linked transcription units that encode two different polypeptides are called bicistronic transcripts.

The methods of the invention can also be used in combination with known or as yet undiscovered methods for inducing the production of recombinant proteins. By inducing conditions is meant a method of increasing the relative production of a desired recombinant protein in a cell. Such methods, to name just a few examples, include cold temperature shift and addition of chemicals, and combinations of any known or as yet undiscovered methods, as well as any as yet undescribed and/or undiscovered induction methods. Typically, a high-density batch or flow culture of cells is induced to produce recombinant protein. Often other cellular processes (such as growth and division) are inhibited in order to direct most of the cells' energy to the production of the recombinant protein.

The methods and compositions of the invention can use any selectable marker having subunits. As used herein, the term subunit in the context of a selectable marker refers to a portion of a selectable marker. In addition, a first mutant subunit (referred to herein as the original mutant subunit) of a selectable marker may be expressed with a second complementary mutant subunit (referred to herein as a complementary mutant subunit) of the same selectable marker to provide a level of selectable marker selection of activity that is not characteristic of any of the mutant subunits individually. A subunit may also refer to a polypeptide having mutations that are complemented by another mutated polypeptide, which is also another subunit of the selectable marker.

Selectable markers that confer resistance to specific drugs that are generally toxic to an animal cell can be used in the methods and compositions of the invention. For example, the following are non-limiting examples of resistance markers suitable for selection: zeomycin (zeo); puromycin (PAC); blasticidin S (BlaS), a GPT that confers resistance to mycophenolic acid (Mulligan & Berg (1981), Proc. Natl. Acad. Sci. USA, 78:2072); the neomycin resistance gene, which confers resistance to aminoglycoside G-418 (Colberre-Garapin et al. (1981), J. Mol. Biol. 150:1); and a hygromycin resistance gene, which confers resistance to hygromycin (Santerre et al. (1984), Gene 30:147).

Metabolic enzymes that promote cell survival or induce cell death under specified conditions may also be used in the methods and compositions of the invention. Examples include, but are not limited to: dihydrofolate reductase (DHFR); herpes simplex virus thymidine kinase (TK) (Wigler et al. (1977), Cell 11:223), hypoxanthine guanine phosphoribosyltransferase (HGPRT) (Szybalska & Szybalski (1962), Proc. Natl. Acad. Sci. USA

- 18 043386

48:2026) and adenine phosphoribosyltransferase (APRT) (Lowy et al. (1980), Cell 22:817), which are genes that can be used in cells lacking TK, HGPRT or APRT, respectively.

In one embodiment, glutamine synthetase (GS) is a suitable selection marker for use in the methods and compositions of the invention. GS contains two connected pentameric rings of identical subunits with ten active sites formed by the N-terminal residues of one subunit and the C-terminal residues of the adjacent subunit. N-terminal (mutGS-NT) and C-terminal (mutGS-CT) mutant constructs were generated and assessed for complementation. Neither mutGS-NT nor mutGS-CT allows cell growth to occur in glutamine-free media, however, when GS mutant cells are transfected with both constructs, those cells that are transfected with both constructs at similar levels survive in glutamine-free media. When two such mutants are co-transfected, the mutants are called complementary. Alternatively, cells expressing endogenous GS can be used and transfectants can be selected to confer increased resistance to toxic levels of methionine sulfoximine (MSX).

Selectable markers based on color selection may also be used in the methods and compositions of the invention. In a specific example, betagalactosidase (Blau et al., WO 98/44350) can be used. Fluorescent markers can also be used in the methods of this invention, for example, GFP has been used for clonal selection of cells to measure protein interactions in protein fragment complementation studies (Remy and Michnick, 1999), Proc. Natl. Acad. Sci., 96:5394-5399). Likewise, fluorescein-conjugated methotrexate can be used to detect cells expressing complementary DHFR fragments (Remy and Michnick, 2001), Proc. Natl. Acad. Sci., 98:7678-83). The advantage of fluorescent markers is that this selection can be performed on any type of animal cell and is not limited to those that have a defect in the metabolic pathway or do not require drug sensitivity, such as neomycin.

As used herein, the term polypeptide includes naturally occurring or recombinantly expressed proteins, including pre- and post-translational processing, or fragments thereof, which typically retain secondary structure. Proteins are large molecules with high molecular weight (from about 10,000 for small (50 to 100 amino acids) to more than 1,000,000 for some forms); they are made up of varying amounts of the same 20 amino acids, which in the intact protein are held together by covalent chemical bonds called peptide bonds. Amino acids linked together form linear, unbranched polymer structures called polypeptide chains; such chains can contain hundreds of amino acid residues; they are arranged in a specific order for given types of protein. The term peptide includes short fragments of polypeptides or proteins, usually less than 20 amino acids in length.

The term cell culture refers to the growth and propagation of cells outside a multicellular organism or tissue. Typically, cell culture is grown under sterile, temperature-controlled and atmospheric conditions in tissue culture vessels (e.g., 10 cm dishes, 96-well plates, etc.) or in other adherent culture (e.g., microcarrier beads) or suspension culture, for example in bottles with stirring. Cultures can be grown in shake tanks, small bioreactors, and/or large-scale bioreactors. A bioreactor is a device used for culturing cells in which environmental conditions such as temperature, atmosphere, agitation and/or pH can be monitored and adjusted. A number of companies (e.g., ABS Inc., Wilmington, Del., Cell Trends, Inc., Middletown, Md.), as well as university and/or government-funded organizations (e.g., The Cell Culture Center, Minneapolis, Minnesota) offer cell cultivation on a contract basis.

Optimal periods for which cultures are in contact with agents that select for selectable activity are longer than the typical period of one normal growth cycle (for example, for Chinese hamster ovary cells (CHO cells), for which a single cycle duration has been reported growth approximately 20-22 hours (Rasmussen et al. (1998), Cytotechnology, 28:31-42)). Thus, in one embodiment of the invention, the crops include suitable conditions for selection, for example, drugs, metabolites or color substrates, preferably for at least about one day, more preferably for at least about 3 days, and even more preferably for for at least about 7 days.

A wide variety of animal cell lines suitable for growth in culture are available, for example, from the American Type Culture Collection (ATCC, Manassas, VA) and NRRL (Peoria, IL). Some of the more well-known cell lines commonly used in an industrial or scientific laboratory include CHO, VERO, BHK, HeLa, Cos, CV1, MDCK, 293, 3T3, PC12, myeloma (eg NSO) and WI38 cell lines, to name a few examples. In other embodiments, cell lines other than animal cell lines may be

- 19 043386 used in the methods according to the invention, for example, plant cell lines, insect cell lines (eg sf9), yeast cells or bacterial cells such as E. coli.

One embodiment of the invention uses GS-deficient CHO cells, such as CHOZN® GS -/- ZFN-modified CHO cells (Sigma Aldrich Fine Chemicals, St. Louis, MO). GS-deficient CHO cells (or other mammalian cells) can also be obtained using methods known in the art. In addition, CHO cells are easy to manipulate as adherent or suspension cultures and exhibit relatively good genetic stability. In addition, new animal cell lines can be generated using methods well known to those skilled in the art (eg, transformation, viral infection and/or selection).

As noted above, a variety of host-expressed vector systems can be used to express the heteromeric complexes of the invention. In the case where the heteromeric complex is soluble, the peptide or polypeptide can be isolated from the culture, that is, from the host cell in cases where the heteromeric complexes are not secreted, and from the culture medium in cases where the heteromeric complexes are secreted by the cells. However, expression systems also include modified host cells that express heteromeric complexes anchored in the cell membrane.

Purification or enrichment of heteromeric complexes from such expression systems can be accomplished using suitable detergents and lipid micelles and methods well known to those skilled in the art. However, such modified host cells can be used in situations where it is important not only to preserve the structural and functional parameters of heteromeric complexes, but also to evaluate biological activity, for example, in drug screening assays.

The protein expressed according to the methods of the invention can be selected. In addition, the protein may be purified or partially purified from such a culture or component (eg, from culture medium or cell extracts or body fluid) using known methods. The phrase partially purified means that some fractionation procedure or procedure has been performed, but more polypeptide species (at least 10%) are present than the desired protein. By purified it is meant that the protein is essentially homogeneous, i.e. Less than 1% contaminating proteins are present. Fractionation procedures may include, but are not limited to, one or more steps of filtration, centrifugation, sedimentation, phase separation, affinity purification, gel filtration, ion exchange chromatography, size exclusion chromatography (SEC), hydrophobic interaction chromatography (HIC; using such resins as phenyl ether, butyl ether or propyl ether), HPLC or some combination of the above.

The invention also does not necessarily include further preparation of proteins. By formulation it is meant that the proteins may be buffer exchanged, sterilized, packaged in multi-dose form, and/or packaged for the end user. For purposes of the invention, the term sterile multi-dose form means that the preparation is free or substantially free of microbial contamination (to the extent acceptable for food and/or medicinal purposes) and has a defined composition and concentration.

The term sterile unit dosage form means a form suitable for administration or consumption by a client and/or patient. Such compositions may contain an effective amount of protein in combination with other components, such as a physiologically acceptable diluent, carrier or excipient. The term physiologically acceptable means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient(s).

In accordance with the present invention, sets of inactivating mutations have been developed that encode changes in the N-terminus (mutGS-NT) of a mammalian GS protein, as well as sets of inactivating mutations that encode changes in the C-terminus (mutGS-CT) of the protein. The GS catalytic site was subjected to an alanine scan in which mutations were made individually and in combination to all evolutionarily conserved alanine residues that are involved in glutamate, ATP and/or ammonia binding. Most residues were selected based on published X-ray diffraction and biochemical studies of GS enzymes, but all residues within 4.5 A of substrates/ligands/metals in the active site were assessed as potential candidates. Combinations of N-terminal and C-terminal catalytically disrupted but structurally intact GS mutants were identified and assessed for the ability to complement each other to restore some GS activity when coexpressed.

A two-vector system was created by combining the expression of one antibody chain with mutGSNT or an N-terminal GS fragment and the other with mutGS-CT or a C-terminal GS fragment. A single expression vector was constructed with a promoter/enhancer leading to expression of the antibody heavy chain (HC) on a single mRNA with an IRES allowing expression of mutGS-NT or the N-terminal fragment of GS. Separately, antibody light chain (LC) expression was coupled to mutGS-CT or C-terminal GS fragment by constructing a vector with promoter/enhancer-controlled LC expression and an IRES that allows expression of mutGS-CT or C-terminal GS fragment . Neither mutGS-NT, nor mutGS-CT, nor the N-terminal nor C-terminal fragments of GS, by themselves allow cells to grow in glutamine-free media. However, when GS mutant cells were transfected with both constructs, those cells that were transfected with both constructs at similar levels were able to survive in glutamine-free media. These cells also produced similar amounts of LC and HC antibody of interest. The strength of selection can be further increased by reducing the efficiency of the IRES, resulting in reduced translation of the selectable marker mutGS, or potentially by adding a GS inhibitor, Lmethionine sulfoximine (MSX).

The present invention is not to be limited in scope to the specific embodiments described herein, which are intended merely to illustrate certain aspects of the invention, and functionally equivalent methods and components are within the scope of the invention. Indeed, various modifications of the invention, in addition to those shown and described herein, will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are to be considered within the scope of the appended claims.

Examples

Example 1.

This example describes the selection and preparation of glutamine synthetase (GS) mutants that are likely to be useful in intragenic complementation. Four N-terminal residues of mouse GS (W60, N61, D63 and S66) and 11 C-terminal residues (E134, E136, E196, E203, N248, H253, N255, R319, R324, E338 and R340) were selected for evaluation in during alanine mutation scanning. Individual and combined mutant constructs were generated in pcDNA3.3 (neomycin resistance, Invitrogen Grand Island, NY) vectors, resulting in 9 N-terminal mutant constructs and 16 C-terminal mutant constructs.

Mutant constructs were assessed for expression of intact GS protein in CHO-K1 cell lines. Cells were cultured in non-selective chemically defined growth media containing glutamine in vented shake flasks at 36°C, 5% CO ₂ , 70% relative humidity and shaking speed of 150-160 rpm in humidified chambers. Viable cell density (VCD) and viability were measured using a ViCell automated cell counter (Beckman-Coulter, Inc., Brea, CA). Cultures were passaged by dilution every 3-4 days.

One million cells for each condition were transfected with 10 micrograms of linearized DNA by electroporation (biological triplicates). DNA was linearized using the restriction enzyme PvuI-HF (New England Biolabs, Ipswich, MA) overnight at 37°C according to the manufacturer's recommendations. Agarose gel electrophoresis was used to confirm complete linearization of each plasmid. After restriction digestion, DNA was precipitated overnight at −70°C by adding 3 M sodium acetate at 10% of the reaction volume and 100% ethanol at 200% of the reaction volume to each digestion product. After ethanol precipitation, the DNA was pelleted, the pellet was washed once with 70% ethanol, air dried, and resuspended in HEPES-buffered saline. DNA concentrations were measured using a NanoDrop 2000 (Thermo Scientific, Wilmington, DE) and adjusted to 1 microgram/microliter using HEPES-buffered saline.

To test for intragenic complementation, 5 micrograms of each of the two plasmids were used, as well as 10 micrograms of hydrodynamically fragmented salmon milt DNA (Invitrogen). Transfections were performed by pelleting 1 million cells for each condition from a culture grown for 3 days, resuspending the cells in 150 μl of HEPES-buffered saline, adding a total of 10 μg of plasmid DNA (5 μg of each plasmid and 10 micrograms of transferring the mixture into the wells of a 96-well electroporation plate (Bio-Rad Laboratories, Inc., Hercules, CA).

Electroporation was performed using Gene Pulser MXcell™ 96-well plates (BioRad Laboratories, Inc., Hercules, CA) with the following settings: exponential wave, voltage = 290 V, capacitance = 950 μF, resistance = 950 ohms. After electroporation, cells were transferred to prewarmed nonselective growth medium in ventilated 24-well plates (24DWP) with a volume of 2 ml/well and shaking at 220 rpm in Kuhner humidified chambers (Kuhner AG, Basel, Switzerland).

Three days after transfection, cells were counted and transferred to selective media (chemically defined growth media without glutamine) at a density of 1x106 viable cells/ml. Stable pools were obtained by sequential passage of cells in selective medium every 3-4 days until culture viability was restored above 90%. Cell numbers were counted on a Guava EasyCyte™ HT flow cytometer (EMD Millipore, Billerica, MA) using the ViaCount assay module after 15 minutes of cell incubation in the reagent (1:10 culture dilution in ViaCount FLEX reagent (EMD Millipore , Billerica, MA) at working concentration (1:50 dilution in PBS)).

All pools recovered viability to at least 90% prior to testing in the functionality studies. Following pool formation, Western blot analysis of the triplicate pools was performed to determine the level of GS protein overexpression for each construct and to determine whether any mutation affected the production of intact GS protein.

For Western blot analysis, 3x10 ⁶ cells were lysed in 100 μl of RIPA buffer (Thermo Scientific Pierce, Rockford, IL) supplemented with EDTA-free Protease and Phosphatase Inhibitor Mixture (Thermo Scientific, Rockford, IL) for 30 min on ice. Lysates were cleared by centrifugation at 13,000 rpm, 4× loading buffer (Invitrogen, Grand Island, NY) was added, and samples were boiled at 95°C for 5 min. 15 μl of each sample was run on E-Page 48 gels (Invitrogen, Grand Island, NY) and transferred to PVDF membranes using iBlot (Invitrogen, Grand Island, NY). Each membrane was probed at 1:500 with anti-glutamine synthetase antibody (Cat #610518, BD biosciences, San Jose, CA) overnight at 4°C, followed by incubation at 1:2000 with Alexa Fluor 680 anti-mouse secondary antibody ( Life Technologies, Grand Island, New York). Blots were scanned using an Odyssey infrared imaging system (LI-COR, Lincoln, NY) and quantified using ImageJ software.

Table 1

Mouse glutamine synthetase mutants studied

Selected GS mutants (alanine scan) Class Mutant Intact GS protein ID sequences W60A N-terminus Yes 2 N61A N-terminus Yes 3 D63A N-terminus Yes 4 S66A N-terminus Yes 5 W60A N61A D63 A (NT 1) N-terminus Yes 6 W60A N61AS66A N-terminus Yes 7 W60A D63A S66A N-terminus Yes 8 N61AD63AS66A N-terminus Yes 9 W60A N61A D63A S66A (NT2) N-terminus Yes 10 E134A C-end Yes AND E136A C-end Yes 12 E196A C-end Yes 13 E203A C-end Yes 14 N248A C-end Yes 15 H253A C-end Yes 16 N255A C-end Yes 17 R319A C-end Yes 18 R324A C-end Yes 20 E134A E136A E196A E203A C-end Yes 21 N248A H253A N255A C-end Yes 22

Although the initial analysis identified residues E388 and R340 as candidate residues for mutation, overexpression of the GS mutants E388A and R340A (as single mutations or in combination with other mutations) did not result in intact GS protein. Therefore, constructs containing these mutations were not considered further.

Because stable pools of GS mutants were generated from the parental wild-type CHO-K1 cell line, which has a functional GS enzyme, assessment of mutant activity was performed using a growth/viability screen in selective media lacking glutamine and supplemented with 25 μM of the GS inhibitor, L-methionine sulfoximine (MSX). ). Wild-type CHO-ΚΙ cells died after 4 passages of soot in a selective medium. Certain N-terminal and C-terminal mutations prevented culture growth in selective media, but even pools with reduced growth retained relatively high viability.

Cells transfected with individual N-terminal GS mutants grew similarly to the wild-type construct, so these constructs were not tested further. However, two N-terminal constructs with multiple mutations W60A N61A D63A (mutGS-NT1; SEQ ID NO: 6) and W60A N61A D63A S66A (mutGS-NT2; SEQ ID NO: 10) provided high levels of overexpressed GS protein and behaved most similarly with the empty negative control vector in the functional assay and were selected for further analysis. In addition, two other N-terminal mutant constructs, W60A N61A D63A S66A D76A (mutGS-NT3; SEQ ID NO: 25) and W60A N61A D63R S66A (mutGSNT4; SEQ ID NO: 19) were generated to increase the stringency of the complementation effect, and appreciated. In the latter construct, aspartate-63 was mutated from alanine to agrginine, since a similar mutant in Anabena azollae completely eliminated biosynthetic activity and was expressed similarly to the wild type, whereas the alanine mutation resulted in retention of 6.5% biosynthetic activity ( Eur J Biochem 266, 1202 (1999) Crespo et al.

Although several constructs with single C-terminal mutations underwent functional evaluation, two constructs that contained multiple mutations, E134A E136A E196A E203A (mutGS-CT1; SEQ ID NO: 21) and N248A H253A N255A (mutGS-CT2; SEQ ID NO: 22) were selected for further analysis.

Example 2.

This example describes the effect of mutations in GS on the expression of a recombinant heterodimeric protein, that is, a monoclonal antibody (mAb). Selected N-terminal and C-terminal mutants described in Example 1 were cloned into mAb expression vectors to construct multiple combinations of two-vector systems, where light chain (LC) expression is linked by an IRES to mutGS-CT and heavy chain (HC) is linked with mutGS-NT. The GS knockout cell line CHO-K1 was generated using CompoZr® Knockout ZFN Kit-CHO GS according to the manufacturer's instructions (SigmaAldrich St. Louis, MO). Glutamine synthetase knockout CHO-K1 cell lines (CHO-K1 GS-KO) were cultured as described above.

Triplicate transfections of CHO-K1 GS knockouts were performed using vectors containing LC or HC mAbs coupled to either wild-type GS (positive control) or selected N- and C-terminal mutants and cultured in glutamine-free media. The original pcDNA3.3 vector containing wild-type GS was used as an additional positive control. Both pcDNA3.3 wild-type GS pools and mAb-bound wild-type GS pools provided greater than 90% recovery of viability at day 20 of passage in glutamine-free media. In contrast, pools transfected as a negative control without DNA and all mAb-bound GS mutant pools died after 17 days in glutamine-free medium, confirming that the selected mutants produce a functional GS protein product.

Finally, selected N-terminal and C-terminal mutants were evaluated for intragenic complementation of GS in a two-vector expression system to generate mAb-producing pools in the CHO-K1 GS knockout cell line. These cells were transfected with intragenic complementation vectors A (LC:mutGS-CT) and B (HC:mutGS-NT) with the indicated mutations in the GS sequence (see Table 2 below). All mutGS-CT/mutGS-NT combinations evaluated were able to achieve recovery to approximately 90% viability within 25 days of passaging in glutamine-free media. Dual-vector LC:mutGS-CT/HC:mutGS-NT pools had a greater reduction in viability and took longer to recover in selective media than single-vector positive controls containing wild-type GS.

The resulting triplicate stable pools were further assessed for mAb expression using a 10-day fed-batch production assay. Production studies were performed using cultures grown for 4 days at a 1:5 split by dilution in chemically defined production medium in 24DWP (-0.5x106 viable cells/ml). Culture growth and viability were monitored on days 3, 6, 8, and 10 using the Guava ViaCount assay (Millipore, Billerica, MA). Bolus administration of nutrients was carried out on days 3, 6 and 8 with a chemically determined nutrient medium. Glucose concentrations in the media were measured on days of nutrient addition using PolyChem colorimetric reagent (Polymedco, Cortlandt Manor, NY) and adjusted to 12 g/L using a 50% glucose stock solution. The final product titer was determined using day 10 culture supernatant by high performance liquid chromatography (HPLC) using a POROS A/20 Protein A column. All pools generated by the GS two-vector intragenic complementation system containing single mAb chains linked via IRES with individual GS mutants produced detectable levels of antibodies; values are shown in table. 2 (example 3) below.

- 23 043386

Example 3.

This example describes a method for increasing antibody titers in transfected pools by increasing the stringency of selection by reducing the efficiency of GS and IRES binding. The sequence in the original constructs is the wild-type EMCV sequence up to ATG-12, which was fused to the GS start codon (Davies and Kaufman, J Virol 66, 1924; 1992). The efficiency of the IRES was reduced by using a previously shown sequence to ensure efficient selection and amplification (Aldrich et al., Biotechnol Prog 19, 1433; 2003). Below, the DNA sequence of the compounds in the more effective (ED3) compound is compared with the DNA sequence of the less effective compound (317). The numbers below indicate the ED3 sequences of the ATGs found in EMCV WT. The last ATG defines the GS start codon.

317 GTZGTZTAZTCCCTCGAGATCCGTGCCATCATG (SEQ ID NO:23)

ED3 GTTGATAATATGGCCACAACCATG (SEQ ID NO:24)

11 12

The expression vectors were identical to those shown in FIG. 1, except for the IRES and GS junction sequence. Transfection of cells with both plasmids with the less efficient IRES-GS compound resulted in lower post-transfection survival and longer recovery times. Only two of three CT1-NT4 transfections recovered, and none of the other mutIRES combinations recovered. However, for those mutIRES combinations that did recover, 10 days of fed-batch culture resulted in 0.55 g/L for one pool and 0.17 g/L for the other. The specific productivity of these pools was 16 p/c/d and 6.9 p/c/d, respectively. The 0.55 g/L titer is superior to those previously reported and does not require FACS sorting to achieve their level (Ye, J. et al. Biotechnol Prog 26, 1431; 2010).

table 2

Expression of intragenic GS transfectants with ____________different IRES-GS____________ compounds

Design Average titer (n=3) g/l Average qP (η = 3) p/c/d CT1-NT1 0.08 1.17 CT1-NT2 0.05 0.76 CT1-NT3 0.05 0.82 CT1-NT4 0.07 0.82 CT2-NT1 0.04 0.59 CT2-NT2 0.05 0.72 CT2-NT3 0.06 0.87 CT2-NT4 0.04 0.56 CT2-NT4 MSX 1 0.29 9.18 mut IRES CT1-NT4 1 0.55 15.69 mut IRES CT1-NT4 3 0.17 6.86

The samples designated CT1-NT1-4 and CT2-NT1-4 used compound pED3 (SEQ ID NO: 24). Samples annotated as mutIRES have a less potent IRES present in SEQ ID NO: 23.

Example 4.

This example describes another strategy for increasing the selectivity of these crops. Transfections were carried out as described in Example 2, but the GS inhibitor, MSX, was added to the cultures during selection to select for relatively higher levels of GS expression. One of the three CT2-NT4 cultures with the more efficient (i.e., non-mutant) IRES was recovered, while none of the CT1-NT4 or any of the transfectants with the mutant IRES were recovered. For the only crop that recovered, the 10-day fed-batch crop, a yield of 0.29 grams/L was obtained with a specific productivity of 9.18 p/c/d (see Table 2 above).

Example 5.

This example describes the results of a large number of transfections of the CT1-NT4 IRES. These designs have the less effective IRES specified in SEQ ID NO: 23. In total, there were

- 24 043386 88 transfections of CHO-K1, knockout relative to GS, were carried out using vectors containing the mut IRES CT1-NT4. A total of 10 of 88 transfections survived selection and resulted in stable growing transfection pools after approximately 25 days in culture and behaved similarly to the smaller number of transfections in Example 2. GS pools transfected as a negative control without DNA died after 17 days in glutamine-free environment.

Duplicate stable pools were further assessed for mAb expression using a 10-day fed-batch production assay. Productivity studies were conducted using a 4-day culture grown in a chemically defined production medium in 24DWPs at 0.6x106 viable cells/ml. Culture growth and viability were monitored on days 3, 6, 8, and 10 using the Guava ViaCount assay (Millipore, Billerica, MA). Bolus administration of nutrients was carried out on days 3, 6 and 8 with a chemically determined nutrient medium. Glucose concentrations in the media were measured on days of nutrient addition using PolyChem colorimetric reagent (Polymedco, Cortlandt Manor, NY) and adjusted to 12 g/L using a 50% glucose stock solution. The final product titer was determined using the day 10 culture supernatant by high performance liquid chromatography (HPLC) using a POROS A/20 Protein A column. All pools generated by the GS two-vector intragenic complementation system containing single mAb chains linked by IRES with individual GS mutants produced relatively high levels of antibodies, with two pools having an average value above 1 g/L; values are given in table. 3 below.

Table 3

Expression of CT1-NT4 GS intragenic transfectants with mut IRES-GS junctions

Design Average titer (n=2) g/l Average qP (n=2) p/c/d mut IRES CT1-NT4 4 0.425 14.10 mut IRES CT1-NT4 5 0.76 13.02 mut IRES CT1-NT4 6 0.57 11.05 mut IRES CT1-NT4 7 1.025 11.64 mut IRES CT1-NT4 8 0.44 6.73 mut IRES CT1-NT4 9 1.055 11.74 mut IRES CT1-NT4 10 0.6 12.16 mut IRES CT1-NT4 eleven 0.375 7.61 mut IRES CT1-NT4 12 0.56 7.21 mut IRES CT1-NT4 13 0.81 14.06

Example 6.

This example describes a molecular complementation approach for the enzyme glutamine synthetase. The glutamine synthetase enzyme was expressed as two separate fragments, and the enzyme activity was completely restored by genetic complementation of the two fragments. A series of constructs were created in which the glutamine synthetase enzyme was separated at different residue positions. The selection of these points was based on information about the molecular structure of the enzyme. These constructs were tested in a cell-based assay for their ability to provide survival to a cell line that is defective in glutamine synthetase activity. This testing identified several fragments that were capable of fully promoting the survival of glutamine synthetase-deficient cells and therefore demonstrated that these fragments could bind to create a fully functional glutamine synthetase enzyme.

Much of the previous work on protein fragment complementation has used proteins that exist as functional monomers and their assembly requires only the presence of designed fragments. This work demonstrated the ability to use molecular complementation to recapitulate the activity of both the GS monomer and the subsequent formation of a decamer

- 25 043386 functional enzyme complex. Complementation of protein fragments was identified using a molecular modeling approach to search for regions in GS monomers that would allow protein partitioning. A GCN4 leucine zipper (LZ) and a flexible linker were also added to individual fragments to enable assembly. The tested fragments are shown in table. 4, and the sequences of the mouse GS protein used in these studies are given in table. 5. GS fragments were cloned into a mammalian expression vector and transfected into CHO cells that lacked GS activity. These cells can usually only survive in cell cultures supplemented with glutamine. The ability of these constructs to promote survival of these cells was tested by removing glutamine from the medium.

Table 4

Construction of GS molecular complementation fragments

A-chain 1 B-circuit 2 1. N-end - E110 - LZ LZ - Till - C-end 2. N-end-Y104-LZ LZ - N105 - C-end 3. N-end - S125 - LZ LZ - N126 - C-end 4. N-terminus - N126 - LZ Q127 - C-end - LZ 5. N-end - E264 - LZ LZ - N265 - C-end

N end LZ = MSDRMKQLEDKVEELLSKVYHLENEVARLKKLVGERGGGGSGGGGGS C end LZ = GGGGSGGGGGSSDRMKQLEDKVEELLSKVYHLENEVARLKKLVGER STOP

Table 5 Mouse GS enzyme sequence used in these studies and highlighted breakpoints

MATS AS SHLNKGIKQMYMSLPQGEK VQ AMYIW VDGTGEGLRCKTRTLDCE

PKCVEELPEWNFDGSSTFQSEGSNSDMYLHPVAMFRDPFRKDPNKLVLCE

101 VFKYNRKPAETNLRHICKRIMDMVSNQHPWFGMEQEYTLMGTDGHPFGWP

151 SNGFPGPQGPYYCGVGADKAYGRDIVEAHYRACLYAGVKITGTNAEVMPA

201 QWEFQIGPCEGIRMGDHLWIARFILHRVCEDFGVIATFDPKPIPGNWNGA

251 GCHTNFSTKAMREENGLKCIEEAIDKLSKRHQYHIRAYDPKGGLDNARRL

301 TGFHETSNINDFSAGVANRGASIRIPRTVGQEKKGYFEDRRPSANCDPYA

351 VTEAIVRTCLLNETGDEPFQYKN*

Y 104 E 110 S 125 N 126 E 264

As shown in FIG. 2, constructs 1, 2, 3 and 5 were able to successfully ensure the survival of cells deficient in glutamine synthetase. This demonstrates that these fragments are capable of forming a fully functional glutamine synthetase enzyme in vivo. A summary of the survival results is shown in FIG. 6. As a control, it was assessed whether either fragment A or B succeeded in ensuring the survival of a cell line deficient in glutamine synthetase. As shown in FIG. 4, neither fragment A or B was able to provide survival to a glutamine synthetase-deficient cell line, confirming that both fragments are required to restore glutamine synthetase enzyme activity.

Table 6

Dynamics of survival by GS fragments described in Table. 4

W.T. 4/4 14 1. 2/4 ~30 2. 4/4 18 3. 4/4 ~24 4. 0/4 No recovery 5. 4/4 ~24

-

Claims (23)

These molecular complementation constructs were then tested in the context of expression vectors containing antibody light chain and heavy chain genes to assess their utility in a cell lineage developmental selection system. Two expression vectors were adapted to contain complementary glutamine synthetase fragments, as shown in Table. 4, one of which contains the antibody light chain genes, and the second contains the antibody heavy chain genes (Fig. 4). The IRES sequences have also been replaced by a version that is attenuated by an in-frame ATG mutation (BioTechniques 20:102-110 January 1996). This led to a further reduction in the expression levels of glutamine synthetase fragments, compared to antibody genes, and an increase in the stringency of selection of the system to achieve a level of selection pressure that would be prone to integration into the genome at sites with high transcriptional activity. The results of this study are depicted in Fig. 6. Constructs 1, 2, 3, and 5 were tested in this study and also linked the heavy chain and light chain genes in both orientations to each of the glutamine synthetase fragments A and B. Vectors containing the glutamine synthetase fragments from construct 2 were able to provide survival under selection in a glutamine-free environment. The cell productivity of the two selected construct 2 pools was assessed in a 10-day fed-batch productivity assay and compared with a control pool that was selected using a vector expressing the full-length GS enzyme. As shown in FIG. 6, cells of design 2 demonstrated high productivity. CLAIM 1. A vector capable of being transfected into mammalian cells and improving the selection of transfected cells, containing: a) a first nucleic acid encoding a first polypeptide, and b) a second nucleic acid encoding a second polypeptide, wherein the second polypeptide is a mammalian glutamine synthetase N-terminal mutant (mutGS-NT), wherein mutGS-NT contains two or more mutations selected from the group consisting of W60A, N61A, D63A, D63R , S66A and D76A, wherein the transcription of the first nucleic acid is operably linked to the transcription of the second nucleic acid, further comprising: c) a third nucleic acid encoding a third polypeptide, wherein the third polypeptide is capable of binding to the first polypeptide to form a heteromeric complex, and d) a fourth nucleic acid that encodes a fourth polypeptide, wherein the fourth polypeptide is a mammalian glutamine synthetase C-terminal mutant (mutGS-CT), where mutGS-CT contains one or more mutations selected from the group consisting of E134A, E136A, E196A, E203A, N248A, H253A, N255A, R319A and R324A, wherein the transcription of the third nucleic acid is operably linked to the transcription of the fourth nucleic acid, wherein the N-terminal glutamine synthetase mutant and the C-terminal glutamine synthetase mutant cooperate to provide selection-appropriate activity. 2. Vector according to claim 1, characterized in that the heteromeric complex is an immunoglobulin. 3. The vector according to claim 2, characterized in that the first nucleic acid encodes an immunoglobulin heavy chain, and the third nucleic acid encodes an immunoglobulin light chain. 4. A vector according to any one of claims 1 to 3, characterized in that the internal ribosome binding site (IRES) is located at a site selected from the group consisting of: a) a region between the first nucleic acid and the second nucleic acid; b) the region between the third nucleic acid and the fourth nucleic acid, c) the regions between the first and second and third and fourth nucleic acids. 5. The vector according to claim 1, characterized in that mutGS-NT is selected from the group consisting of SEQ ID NO: 6; SEQ ID NO: 10, SEQ ID NO: 25; SEQ ID NO: 19; and mutGS-CT is selected from the group consisting of SEQ ID NO: 21 and SEQ ID NO: 22. 6. An isolated host cell that has been transfected, transformed or transduced with a vector according to any one of claims 1 to 5. 7. The host cell of claim 6, which is selected from the group consisting of CHO, VERO, BHK, HeLa, Cos, MDCK, 293, 3T3, myeloma cell line and WI38 cells. 8. A method for producing a heteromeric complex, comprising the step of culturing a host cell according to claim 7 under conditions under which the heteromeric complex is expressed by the host cell. 9. The method of claim 8, wherein the heteromeric complex is an antibody. 10. The method according to claim 9, further comprising isolating the heteromeric complex. 11. Expression system containing: - 27 043386 a) a first vector encoding a bicistronic transcript containing a first nucleic acid encoding a first polypeptide that is operably linked to a second nucleic acid encoding a second polypeptide, wherein the second polypeptide is a mammalian glutamine synthetase N-terminal mutant (mutGS-NT), where mutGS- NT contains two or more mutations selected from the group consisting of W60A, N61A, D63A, D63R, S66A and D76A, and b) a second vector encoding a bicistronic transcript containing a third nucleic acid encoding a third polypeptide that is operably linked to a fourth nucleic acid encoding a fourth polypeptide, wherein the fourth polypeptide is a C-terminal mutant of mammalian glutamine synthetase (mutGS-CT), where mutGS-CT contains one or more mutations selected from the group consisting of E134A, E136A, E196A, E203A, N248A, H253A, N255A, R319A and R324A, which is capable of binding to an N-terminal mutant of glutamine synthetase to provide selectable activity, wherein the third polypeptide is capable of bind to the first polypeptide to form a heteromeric complex; In addition, the expression system can be transfected into mammalian cells and improve the selection of these cells. 12. The expression system according to claim 11, characterized in that the heteromeric complex is an immunoglobulin. 13. The expression system according to claim 12, characterized in that the first nucleic acid encodes an immunoglobulin heavy chain, and the third nucleic acid encodes an immunoglobulin light chain. 14. An expression system according to any one of claims 11 to 13, characterized in that the internal ribosome binding site (IRES) is located at a site selected from the group consisting of: a) a region between the first nucleic acid and the second nucleic acid; b) the region between the third nucleic acid and the fourth nucleic acid, c) regions between the first and second and third and fourth nucleic acids. 15. The expression system according to claim 12, characterized in that mutGS-NT is selected from the group consisting of SEQ ID NO: 6; SEQ ID NO: 10, SEQ ID NO: 25; SEQ ID NO: 19; and mutGS-CT is selected from the group consisting of SEQ ID NO: 21 and SEQ ID NO: 22. 16. An isolated host cell transfected, transformed or transduced with the expression system according to any one of claims 11-15. 17. The host cell of claim 16, which is selected from the group consisting of CHO, VERO, BHK, HeLa, Cos, MDCK, 293, 3T3, myeloma cell line and WI38 cells. 18. A method for producing a heteromeric complex, comprising the step of culturing a host cell according to claim 17 under conditions under which the heteromeric complex is expressed by the host cell. 19. The method according to claim 18, further comprising isolating the heteromeric complex. 20. An expression system comprising a first vector containing a first nucleic acid encoding an antibody heavy chain operably linked to a second nucleic acid encoding mammalian glutamine synthetase with a mutation at the N-terminus (mutGS-NT), and a second vector containing a third nucleic acid , encoding an antibody light chain operably linked to a fourth nucleic acid that encodes a mammalian glutamine synthetase with a mutation at the C-terminus (mutGS-CT), wherein mutGS-NT contains two or more mutations selected from the group consisting of W60A, N61A, D63A , D63R, S66A, and D76A, and mutGS-CT contains one or more mutations selected from the group consisting of E134A, E136A, E196A, E203A, N248A, H253A, N255A, R319A, and R324A, each mutant glutamine synthetase subunit lacking selection activity if expressed separately, and co-expression of mutGS-NT with mutGS-CT provides glutamine synthetase activity, while the expression system is able to be transfected into mammalian cells and improve the selection of transfected cells. 21. The expression system as specified in claim 20, characterized in that mutGS-NT is selected from the group consisting of SEQ ID NO: 6; SEQ ID NO: 10, SEQ ID NO: 25 and SEQ ID NO: 19; and mutGS-CT is selected from the group consisting of SEQ ID NO: 21 and SEQ ID NO: 22. 22. An isolated host cell transfected, transformed or transduced with the expression system of any one of claims 20 or 21. 23. An expression system comprising a first vector encoding a bicistronic transcript containing a first nucleic acid encoding a desired polypeptide operably linked to a second nucleic acid encoding a mammalian glutamine synthetase N-terminal mutant (mutGS-NT) having mutations selected from the group consisting of W60A, N61A, D63A, S66A and combinations thereof, and a second vector encoding a bicistronic transcript containing a third nucleic acid operably linked to a fourth nucleic acid encoding a C-terminal mutant of mammalian glutamine synthetase (mutGS-CT) having mutations selected from group consisting of E134A, E136A, E196A, E203A, N248A, H253A, N255A, R319A, R324A, wherein the N-terminal mutant of glutamine synthetase and the C-terminal mutant of glutamine synthetase are associated with obtaining activity suitable for selection, and the expression system is capable of transfection into mammalian cells and improve the selection of transfected cells, and, in addition, the first nucleic acid -