Expression vector element combination, novel method for producing production cells and use thereof in the recombinant production of polypeptides

Abstract

This article reports that for transient transfection, the use of the human elongation factor 1α promoter (containing intron A) provides enhanced productivity (in LC-HC-SM tissues), the use of the bovine growth hormone polyA signal sequence provides enhanced productivity compared to the use of the SV40 polyA signal sequence, in vectors containing the hCMV promoter, adding HGT to the bGH PolyA signal sequence results in increased productivity, and the vector tissue LC(3`-5′)-HC-SM produces improved expression. For stable libraries, it is reported that libraries generated with vectors containing the hEF1α promoter show enhanced productivity in batch analysis, clones generated with vectors containing the hEF1α promoter show a reduced number of low-producing clones, and clones generated with vectors containing the hEF1α promoter show higher IgG expression stability. For monoclonal antibodies, it is reported that vector tissues (LC-HC-SM) with the selection marker placed downstream have a positive effect on the productivity of monoclonal antibodies, and clones generated with vectors containing the bGH polyA signal sequence and hGT have higher productivity.

Description

[0001] The present application is a divisional application of Chinese patent application No. 201280062793.9 filed by the applicant on December 19, 2012, entitled “Combination of expression vector elements, novel method for producing production cells and use thereof in recombinant production of polypeptides”. Technical Field

[0002] Reported herein are novel combinations of expression vector elements such as promoters, polyA signal sequences and transcription terminators, expression vector organizations, combinations thereof, and novel methods for generating producer cell lines, such as novel transfection or selection methods, and the use of these expression vectors and producer cell lines for the recombinant production of polypeptides of interest. Background Art

[0003] The transcription level of a gene can have a strong influence on its expression level and thus determines the productivity of the cell. It is mainly influenced by three vector elements: the promoter, the polyA signal sequence and (if present) the transcription terminator.

[0004] The nucleic acid encoding the antibody heavy chain is generally included in a leader sequence (signal sequence) (about 57 bp / 19 aa) that is removed when the protein matures, a variable region VH (about 350 bp / 115 aa) and a constant region CH (about 990 bp / 330 aa). The nucleic acid encoding the antibody light chain is generally composed of a leader sequence (about 66 bp / 22 aa) that is removed when the protein matures, a variable region VK or VL (about 350 bp / 115 aa) and a constant region CK or CL (about 321 bp / 107 aa).

[0005] Recombinant production of antibodies in eukaryotic cells involves the creation of expression systems (see McCafferty, J. et al. (eds.), Antibody Engineering, A Practical Approach., IRL Press (1997)). To develop an antibody expression system, an expression cassette is generated comprising a light chain encoding nucleic acid flanked by a promoter and polyadenylation (polyA) region. Similarly, a heavy chain expression cassette is generated comprising a heavy chain encoding nucleic acid flanked by a promoter and polyA region. The heavy chain expression cassette can be combined into the light chain expression cassette in a single vector comprising both heavy and light chain expression cassettes, or can be integrated into two separate vectors.

[0006] US 7,053,202 reports immunoglobulin DNA cassette molecules, monomeric (monobody) constructs, methods for producing and using the same. In US 5,168,062, transfer vectors and microorganisms containing human cytomegalovirus immediate early promoter regulatory DNA sequences were reported. US 5,225,348 reports a DNA fragment containing the promoter region of human polypeptide chain elongation factor-1α, its base sequence, and an expression plasmid containing a DNA fragment that is highly suitable for a wide range of host cells with high expression capacity. In US 5,266,491, an expression plasmid containing an SV40 origin of replication and a DNA fragment having the promoter region of human polypeptide chain elongation factor-1α gene was reported. In US 5,122,458, the expression of recombinant DNA compounds and polypeptides such as tPA was reported. In US 7,422,874, an expression vector for animal cells was reported.

[0007] Sanna Pietro, P. reported the expression of antibody Fab fragments and whole immunoglobulins in mammalian cells (Meth. Mol. Biol. 178 (2002) 389-395). Higuchi, K. et al. (J. Immunol. Meth. 202 (1997) 193-204) reported a cell display library for gene cloning of the variable region of human antibodies against hepatitis B surface antigen. Kim, D. reported an improved mammalian expression system by manipulating the transcription termination region (Biotechnol. Progress 19 (2003) 1620-1622). Costa, RA et al. (Eur. J. Pharmaceut. Biopharmaceut. 74 (2010) 127-138) reported guidance for cell engineering for monoclonal antibody production. Kim, DW et al. reported the use of the human elongation factor 1α promoter as a universal and efficient expression system (Gene 91 (1990) 217-223). Buchman, AR et al. (Mol. Cell. Biol. 8 (1988) 4395-4405) reported a comparison of intron-dependent and intron-independent gene expression. Wang, F. et al. reported antibody expression in mammalian cells (in Therapeutic monoclonal antibodies – From bench to clinic, Wiley (2009) pp. 557-572). Li et al. (J. Immunol. Meth. 318 (2007) 113-124) reported a comparative study of different vector designs for mammalian expression of recombinant IgG antibodies. Ho, SCL et al. reported an IRES-mediated tricistronic vector for enhancing the production of a high monoclonal antibody-expressing CHO cell line (J. Biotechnol. 157 (2011) 130-139). Hotta, A. et al. (J. Biosci. Bioeng. 98 (2004) 298-303) reported the production of anti-CD2 chimeric antibodies by recombinant animal cells. Lee, JC. et al. reported efficient protein expression mediated by the glycosome entry site within enterovirus 71 (Biotechnol. Bioeng. 90 (2005) 656-662). In WO 2008 / 142124, Avian Recombinant protein production in cells. SUMMARY OF THE INVENTION

[0008] It has been found that the performance of an expression vector depends primarily on its intended use, with different optimal vectors being used for transient transfection, stable libraries, and monoclonal selection.

[0009] To highlight the main findings: For transient transfection, bidirectional expression of antibody light and heavy chains and the use of the full-length hCMV promoter including intron A is advantageous. However, for stable transfection, it has been shown that the arrangement of 1) antibody light chain, 2) antibody heavy chain and 3) selection marker in a row is advantageous.

[0010] While the hEF1α promoter clearly outperformed the hCMV promoter in stable pools, a strikingly opposite effect was observed at the monoclonal level. Here, the highest productivity was achieved using the human cytomegalovirus immediate early promoter / enhancer (hCMV) clone.

[0011] In addition, hCMV promoter performance can be further improved by combining it with the bGH polyA signal and the terminator sequence of the human gastrin gene (hGT), which increases both productivity and expression stability.

[0012] It has been found that the use of expression vectors comprising an antibody heavy chain expression cassette and an antibody light chain expression cassette, each comprising a promoter, a structural gene and a polyA signal sequence and, optionally, a terminator sequence, results in a higher number of antibody-producing / secreting cell clones following transfection if i) the promoter is the human cytomegalovirus promoter (hCMV), the polyA signal sequence is the bovine growth hormone polyA signal sequence (bGH polyA), and the terminator sequence is the human gastrin gene transcription terminator sequence (hGT), or 2) the promoter is the human elongation factor 1 alpha promoter (hEF1α), the polyA signal sequence is the bovine growth hormone polyA signal sequence (bGH polyA), and the terminator sequence is absent.

[0013] By using the above-described expression vectors, higher numbers of antibody-producing / secreting cells can be obtained after transfection, thereby reducing the effort required to identify high-producing cells suitable for large-scale recombinant antibody production.

[0014] Thus, one aspect as reported herein is a method for selecting a recombinant mammalian cell comprising the following steps:

[0015] a) Transfecting mammalian cells with an expression vector comprising

[0016] a first expression cassette comprising, in 5' to 3' direction, an hCMV promoter, a nucleic acid encoding an antibody light chain, a bGH polyA signal sequence and an hGT terminator sequence,

[0017] - a second expression cassette comprising, in 5' to 3' direction, an hCMV promoter, a nucleic acid encoding an antibody heavy chain, a bGH polyA signal sequence and an hGT terminator sequence, and

[0018] Thus, a large number of recombinant mammalian cells are obtained.

[0019] b) selecting a (single) recombinant mammalian cell from the plurality of recombinant mammalian cells.

[0020] In one embodiment, the nucleic acid encoding the antibody light chain and / or the nucleic acid encoding the antibody heavy chain comprises at least one intron.

[0021] In one embodiment, the nucleic acid encoding the antibody light chain and / or the nucleic acid encoding the antibody heavy chain is cDNA.

[0022] In one embodiment, the first expression cassette and the second expression cassette are arranged unidirectionally for selection of stably transfected cells.

[0023] In one embodiment, the first expression cassette and the second expression cassette are arranged bidirectionally for selection of transiently transfected cells.

[0024] In one embodiment, the expression plasmid further comprises a selection marker. In one embodiment, the expression cassette and the selection marker are arranged in a unidirectional manner. In one embodiment, the expression cassette is arranged in the order LC-HC-SM.

[0025] In one embodiment, the mammalian cell is selected from CHO cells, HEK cells, BHK cells, NS0 cells and SP2 / 0 cells. In one embodiment, the mammalian cell is a CHO cell for selecting stably transfected cells. In one embodiment, the mammalian cell is a HEK cell for selecting transiently transfected cells.

[0026] One aspect as reported herein is a method for producing an antibody comprising the following steps:

[0027] a) Cultivating mammalian cells comprising

[0028] a first expression cassette comprising, in 5' to 3' direction, an hCMV promoter, a nucleic acid encoding an antibody light chain, a bGH polyA signal sequence and an hGT terminator sequence,

[0029] - a second expression cassette comprising, in 5' to 3' direction, an hCMV promoter, a nucleic acid encoding an antibody heavy chain, a bGH polyA signal sequence and an hGT terminator sequence, and

[0030] b) recovering the antibody from the cells or culture medium.

[0031] In one embodiment, the nucleic acid encoding the antibody light chain and / or the nucleic acid encoding the antibody heavy chain comprises at least one intron.

[0032] In one embodiment, the nucleic acid encoding the antibody light chain and / or the nucleic acid encoding the antibody heavy chain is cDNA.

[0033] In one embodiment, the first expression cassette and the second expression cassette are arranged unidirectionally for stable production of the antibody.

[0034] In one embodiment, the first expression cassette and the second expression cassette are arranged bidirectionally for transient production of antibodies.

[0035] In one embodiment, the expression plasmid further comprises a selection marker. In one embodiment, the expression cassette and the selection marker are arranged in a unidirectional manner. In one embodiment, the expression cassette is arranged in the order LC-HC-SM.

[0036] In one embodiment, the mammalian cell is selected from CHO cells, HEK cells, BHK cells, NS0 cells and SP2 / 0 cells. In one embodiment, the mammalian cell is a CHO cell for stably producing an antibody. In one embodiment, the mammalian cell is a HEK cell for transiently producing an antibody.

[0037] One aspect as reported herein is an expression vector comprising:

[0038] a first expression cassette comprising, in 5' to 3' direction, an hCMV promoter, a nucleic acid encoding an antibody light chain, a bGH polyA signal sequence and an hGT terminator sequence,

[0039] - a second expression cassette comprising, in 5' to 3' direction, the hCMV promoter, the nucleic acid encoding the antibody heavy chain, the bGH polyA signal sequence and the hGT terminator sequence.

[0040] In one embodiment, the nucleic acid encoding the antibody light chain and / or the nucleic acid encoding the antibody heavy chain comprises at least one intron.

[0041] In one embodiment, the nucleic acid encoding the antibody light chain and / or the nucleic acid encoding the antibody heavy chain is cDNA.

[0042] In one embodiment, the first expression cassette and the second expression cassette are arranged unidirectionally for selection of stably transfected cells.

[0043] In one embodiment, the first expression cassette and the second expression cassette are arranged bidirectionally for selection of transiently transfected cells.

[0044] In one embodiment, the expression plasmid further comprises a selection marker. In one embodiment, the expression cassette and the selection marker are arranged in a unidirectional manner. In one embodiment, the expression cassette is arranged in the order LC-HC-SM.

[0045] It has been found that for the generation of stable recombinant antibody expressing / secreting cell lines, the presence of the hGT terminator sequence reduces the achievable expression yields when the human elongation factor 1 alpha promoter (hEF1α) is used in combination with the bGH polyA signal sequence.

[0046] One aspect as reported herein is a method for selecting a recombinant mammalian cell comprising the following steps:

[0047] a) Transfecting mammalian cells with an expression vector comprising

[0048] a first expression cassette comprising, in 5' to 3' direction, the hEF1α promoter, the nucleic acid encoding the antibody light chain and the bGH polyA signal sequence,

[0049] - a second expression cassette comprising, in 5' to 3' direction, the hEF1α promoter, the nucleic acid encoding the antibody heavy chain and the bGH polyA signal sequence, and

[0050] Thus, a large number of recombinant mammalian cells are obtained.

[0051] b) selecting a (single) recombinant mammalian cell from the plurality of recombinant mammalian cells.

[0052] In one embodiment, the nucleic acid encoding the antibody light chain and / or the nucleic acid encoding the antibody heavy chain comprises at least one intron.

[0053] In one embodiment, the nucleic acid encoding the antibody light chain and / or the nucleic acid encoding the antibody heavy chain is cDNA.

[0054] In one embodiment, the first expression cassette and the second expression cassette are arranged unidirectionally for selection of stably transfected cells.

[0055] In one embodiment, the first expression cassette and the second expression cassette are arranged bidirectionally for selection of transiently transfected cells.

[0056] In one embodiment, the expression plasmid further comprises a selection marker. In one embodiment, the expression cassette and the selection marker are arranged in a unidirectional manner. In one embodiment, the expression cassette is arranged in the order LC-HC-SM.

[0057] In one embodiment, the human elongation factor 1 alpha promoter comprises intron A.

[0058] In one embodiment, the expression vector does not contain any transcription terminator sequence.In one embodiment, the terminator sequence is an hGT sequence.

[0059] In one embodiment, the mammalian cell is selected from CHO cells, HEK cells, BHK cells, NS0 cells and SP2 / 0 cells. In one embodiment, the mammalian cell is a CHO cell for selecting stably transfected cells. In one embodiment, the mammalian cell is a HEK cell for selecting transiently transfected cells.

[0060] One aspect as reported herein is a method for producing an antibody comprising the following steps:

[0061] a) Cultivating mammalian cells comprising

[0062] a first expression cassette comprising, in 5' to 3' direction, the hEF1α promoter, the nucleic acid encoding the antibody light chain and the bGH polyA signal sequence,

[0063] - a second expression cassette comprising, in 5' to 3' direction, the hEF1α promoter, the nucleic acid encoding the antibody heavy chain and the bGH polyA signal sequence, and

[0064] b) recovering the antibody from the cells or culture medium.

[0065] In one embodiment, the nucleic acid encoding the antibody light chain and / or the nucleic acid encoding the antibody heavy chain comprises at least one intron.

[0066] In one embodiment, the nucleic acid encoding the antibody light chain and / or the nucleic acid encoding the antibody heavy chain is cDNA.

[0067] In one embodiment, the first expression cassette and the second expression cassette are arranged unidirectionally for selection of stably transfected cells.

[0068] In one embodiment, the first expression cassette and the second expression cassette are arranged bidirectionally for selection of transiently transfected cells.

[0069] In one embodiment, the expression plasmid further comprises a selection marker. In one embodiment, the expression cassette and the selection marker are arranged in a unidirectional manner. In one embodiment, the expression cassette is arranged in the order LC-HC-SM.

[0070] In one embodiment, the human elongation factor 1 alpha promoter comprises intron A.

[0071] In one embodiment, the expression vector does not contain any transcription terminator sequence.In one embodiment, the terminator sequence is an hGT sequence.

[0072] In one embodiment, the mammalian cell is selected from CHO cells, HEK cells, BHK cells, NS0 cells and SP2 / 0 cells. In one embodiment, the mammalian cell is a CHO cell for stably producing an antibody. In one embodiment, the mammalian cell is a HEK cell for transiently producing an antibody.

[0073] One aspect as reported herein is an expression vector comprising:

[0074] - a first expression cassette comprising, in 5' to 3' direction, the hEF1α promoter, the nucleic acid encoding the antibody light chain and the bGH polyA signal sequence, and

[0075] - a second expression cassette comprising, in 5' to 3' direction, the hEF1α promoter, the nucleic acid encoding the antibody heavy chain and the bGH polyA signal sequence.

[0076] In one embodiment, the nucleic acid encoding the antibody light chain and / or the nucleic acid encoding the antibody heavy chain comprises at least one intron.

[0077] In one embodiment, the nucleic acid encoding the antibody light chain and / or the nucleic acid encoding the antibody heavy chain is cDNA.

[0078] In one embodiment, the first expression cassette and the second expression cassette are arranged unidirectionally for selection of stably transfected cells.

[0079] In one embodiment, the first expression cassette and the second expression cassette are arranged bidirectionally for selection of transiently transfected cells.

[0080] In one embodiment, the expression plasmid further comprises a selection marker. In one embodiment, the expression cassette and the selection marker are arranged in a unidirectional manner. In one embodiment, the expression cassette is arranged in the order LC-HC-SM.

[0081] In one embodiment, the human elongation factor 1 alpha promoter comprises intron A.

[0082] In one embodiment, the expression vector does not contain any transcription terminator sequence.In one embodiment, the terminator sequence is an hGT sequence.

[0083] It has been found that for the stable recombinant production of antibodies, the use of an expression vector comprising:

[0084] a first expression cassette comprising, in 5' to 3' direction, a first promoter, a nucleic acid encoding an antibody light chain, a first polyA signal sequence and optionally a first transcription terminator sequence,

[0085] a second expression cassette comprising, in 5' to 3' direction, a second promoter, the nucleic acid encoding the antibody heavy chain, a second polyA signal sequence and optionally a second transcription terminator sequence, and

[0086] a third expression cassette comprising, in 5' to 3' direction, a third promoter, a nucleic acid conferring resistance to a selection agent, a third polyA signal sequence and optionally a third transcription terminator sequence,

[0087] The three expression cassettes are thus organized unidirectionally and in the order first expression cassette - second expression cassette - third expression cassette.

[0088] In contrast to the above, it has been found that for the transient recombinant production of antibodies, the use of an expression vector comprising:

[0089] a first expression cassette comprising, in 5' to 3' direction, a first promoter, a nucleic acid encoding an antibody light chain, a first polyA signal sequence and optionally a first transcription terminator sequence,

[0090] a second expression cassette comprising, in 5' to 3' direction, a second promoter, the nucleic acid encoding the antibody heavy chain, a second polyA signal sequence and optionally a second transcription terminator sequence, and

[0091] a third expression cassette comprising, in 5' to 3' direction, a third promoter, a nucleic acid conferring resistance to a selection agent, a third polyA signal sequence and optionally a third transcription terminator sequence,

[0092] The expression cassettes are thereby organized bidirectionally, whereby the first expression cassette and the second expression cassette are arranged in opposite directions.

[0093] The term "in opposite directions" means that one expression cassette is transcribed in a 5'->3' direction and one expression cassette is transcribed in a 3'->5' direction.

[0094] Thus, one aspect as reported herein is the use of an expression vector for the stable recombinant production of an antibody in mammalian cells comprising:

[0095] a first expression cassette comprising, in 5' to 3' direction, a first promoter, a nucleic acid encoding an antibody light chain, a first polyA signal sequence and optionally a first transcription terminator sequence,

[0096] a second expression cassette comprising, in 5' to 3' direction, a second promoter, the nucleic acid encoding the antibody heavy chain, a second polyA signal sequence and optionally a second transcription terminator sequence, and

[0097] a third expression cassette comprising, in 5' to 3' direction, a third promoter, a nucleic acid conferring resistance to a selection agent, a third polyA signal sequence and optionally a third transcription terminator sequence,

[0098] The three expression cassettes are thus organized unidirectionally and in the order first expression cassette - second expression cassette - third expression cassette.

[0099] In one embodiment, the nucleic acid encoding the antibody light chain and / or the nucleic acid encoding the antibody heavy chain comprises at least one intron.

[0100] In one embodiment, the nucleic acid encoding the antibody light chain and / or the nucleic acid encoding the antibody heavy chain is cDNA.

[0101] In one embodiment, the first and second promoters are hCMV promoters, the first and second polyA signal sequences are bGH polyA signal sequences, and the transcription termination sequence is present and is an hGT terminator sequence.

[0102] In one embodiment, the first and second promoters are hEF1α promoters, the first and second polyA signal sequences are bGH polyA signal sequences, and the expression cassette does not contain a transcription terminator sequence.

[0103] In one embodiment, the mammalian cell is selected from the group consisting of a CHO cell, a HEK cell, a BHK cell, a NSO cell, and a SP2 / 0 cell. In one embodiment, the mammalian cell is a CHO cell.

[0104] One aspect as reported herein is an expression vector comprising:

[0105] a first expression cassette comprising, in 5' to 3' direction, a first promoter, a nucleic acid encoding an antibody light chain, a first polyA signal sequence and optionally a first transcription terminator sequence,

[0106] a second expression cassette comprising, in 5' to 3' direction, a second promoter, the nucleic acid encoding the antibody heavy chain, a second polyA signal sequence and optionally a second transcription terminator sequence, and

[0107] a third expression cassette comprising, in 5' to 3' direction, a third promoter, a nucleic acid conferring resistance to a selection agent, a third polyA signal sequence and optionally a third transcription terminator sequence,

[0108] The three expression cassettes are thus organized unidirectionally and in the order first expression cassette - second expression cassette - third expression cassette.

[0109] In one embodiment, the nucleic acid encoding the antibody light chain and / or the nucleic acid encoding the antibody heavy chain comprises at least one intron.

[0110] In one embodiment, the nucleic acid encoding the antibody light chain and / or the nucleic acid encoding the antibody heavy chain is cDNA.

[0111] In one embodiment, the first and second promoters are hCMV promoters, the first and second polyA signal sequences are bGH polyA signal sequences, and the transcription termination sequence is present and is an hGT terminator sequence.

[0112] In one embodiment, the first and second promoters are hEF1α promoters, the first and second polyA signal sequences are bGH polyA signal sequences, and the expression cassette does not contain a transcription terminator sequence.

[0113] One aspect as reported herein is the use of an expression vector for the transient recombinant production of an antibody in mammalian cells comprising:

[0114] a first expression cassette comprising, in 5' to 3' direction, a first promoter, a nucleic acid encoding an antibody light chain, a first polyA signal sequence and optionally a first transcription terminator sequence,

[0115] a second expression cassette comprising, in 5' to 3' direction, a second promoter, the nucleic acid encoding the antibody heavy chain, a second polyA signal sequence and optionally a second transcription terminator sequence, and

[0116] a third expression cassette comprising, in 5' to 3' direction, a third promoter, a nucleic acid conferring resistance to a selection agent, a third polyA signal sequence and optionally a third transcription terminator sequence,

[0117] The expression cassettes are thereby organized bidirectionally, whereby the first expression cassette and the second expression cassette are arranged in opposite directions.

[0118] In one embodiment, the nucleic acid encoding the antibody light chain and / or the nucleic acid encoding the antibody heavy chain comprises at least one intron.

[0119] In one embodiment, the nucleic acid encoding the antibody light chain and / or the nucleic acid encoding the antibody heavy chain is cDNA.

[0120] In one embodiment, the first and second promoters are hCMV promoters, the first and second polyA signal sequences are bGH polyA signal sequences, and the transcription termination sequence is present and is an hGT terminator sequence.

[0121] In one embodiment, the first and second promoters are hEF1α promoters, the first and second polyA signal sequences are bGH polyA signal sequences, and the expression cassette does not contain a transcription terminator sequence.

[0122] In one embodiment, the mammalian cell is selected from the group consisting of CHO cells, HEK cells, BHK cells, NS0 cells and SP2 / 0 cells. In one embodiment, the mammalian cell is a HEK cell.

[0123] One aspect as reported herein is an expression vector comprising:

[0124] a first expression cassette comprising, in 5' to 3' direction, a first promoter, a nucleic acid encoding an antibody light chain, a first polyA signal sequence and optionally a first transcription terminator sequence,

[0125] a second expression cassette comprising, in 5' to 3' direction, a second promoter, the nucleic acid encoding the antibody heavy chain, a second polyA signal sequence and optionally a second transcription terminator sequence, and

[0126] a third expression cassette comprising, in 5' to 3' direction, a third promoter, a nucleic acid conferring resistance to a selection agent, a third polyA signal sequence and optionally a third transcription terminator sequence,

[0127] The expression cassettes are thereby organized bidirectionally, whereby the first expression cassette and the second expression cassette are arranged in opposite directions.

[0128] In one embodiment, the nucleic acid encoding the antibody light chain and / or the nucleic acid encoding the antibody heavy chain comprises at least one intron.

[0129] In one embodiment, the nucleic acid encoding the antibody light chain and / or the nucleic acid encoding the antibody heavy chain is cDNA.

[0130] In one embodiment, the first and second promoters are hCMV promoters, the first and second polyA signal sequences are bGH polyA signal sequences, and the transcription termination sequence is present and is an hGT terminator sequence.

[0131] In one embodiment, the first and second promoters are hEF1α promoters, the first and second polyA signal sequences are bGH polyA signal sequences, and the expression cassette does not contain a transcription terminator sequence.

[0132] Furthermore, it has been found that, among other things, expression vectors comprising the hCMV promoter containing intron A and the human EF1α promoter, rather than the short human CMV promoter without intron A, enhance transient and reservoir gene expression.

[0133] One aspect as reported herein is an expression plasmid comprising:

[0134] a first expression cassette comprising in 5' to 3' direction a first promoter, a nucleic acid encoding an antibody light chain and a first polyA signal sequence,

[0135] a second expression cassette comprising in 5' to 3' direction a second promoter, the nucleic acid encoding the antibody heavy chain and a second polyA signal sequence,

[0136] One or both of the expression cassettes further comprises a human gastrin terminator sequence after the polyA signal sequence.

[0137] In one embodiment, the first and second polyA signal sequences are independently selected from the group consisting of an SV40 polyA signal sequence and a bovine growth hormone polyA signal sequence.

[0138] In one embodiment, the first and second promoters are independently selected from the group consisting of human CMV promoter, SV40 promoter and human elongation factor 1α promoter.

[0139] In one embodiment, the nucleic acid encoding the antibody light chain and / or the nucleic acid encoding the antibody heavy chain comprises at least one intron.

[0140] In one embodiment, the nucleic acid encoding the antibody light chain and / or the nucleic acid encoding the antibody heavy chain is cDNA.

[0141] In one embodiment, the first expression cassette and the second expression cassette are arranged unidirectionally.

[0142] In one embodiment, the expression plasmid further comprises a selection marker.In one embodiment, the expression cassette and the selection marker are arranged in two directions.

[0143] One aspect as reported herein is the use of an expression plasmid as reported herein for transient or stable expression of an antibody.

[0144] One aspect as reported herein is a eukaryotic cell comprising an expression plasmid as reported herein.

[0145] One aspect as reported herein is a method for producing an antibody comprising the steps of:

[0146] - culturing a eukaryotic cell comprising the expression plasmid as reported herein or a cell as reported herein,

[0147] - Recovering the antibody from the eukaryotic cells or culture medium.

[0148] In one embodiment, the eukaryotic cell is a mammalian cell. In one embodiment, the mammalian cell is selected from the group consisting of a CHO cell, a HEK cell, a BHK cell, a NSO cell, and a SP2 / 0 cell.

[0149] One aspect as reported herein is an expression plasmid comprising:

[0150] a first expression cassette comprising in 5' to 3' direction a first promoter, a nucleic acid encoding an antibody light chain and a first polyA signal sequence,

[0151] a second expression cassette comprising in 5' to 3' direction a second promoter, the nucleic acid encoding the antibody heavy chain and a second polyA signal sequence,

[0152] The first and / or second promoter is human elongation factor 1α promoter.

[0153] In one embodiment, one or both of the expression cassettes does not comprise the human gastrin terminator sequence after the polyA signal sequence.

[0154] In one embodiment, one or both of the expression cassettes does not contain the human gastrin terminator sequence.

[0155] In one embodiment, the human elongation factor 1 alpha promoter comprises intron A.

[0156] In one embodiment, the first and second polyA signal sequences are independently selected from the group consisting of an SV40 polyA signal sequence and a bovine growth hormone polyA signal sequence.

[0157] In one embodiment, the first and second promoters are independently selected from the group consisting of human CMV promoter, SV40 promoter and human elongation factor 1α promoter.

[0158] In one embodiment, the nucleic acid encoding the antibody light chain and / or the nucleic acid encoding the antibody heavy chain comprises at least one intron.

[0159] In one embodiment, the nucleic acid encoding the antibody light chain and / or the nucleic acid encoding the antibody heavy chain is cDNA.

[0160] In one embodiment, the first expression cassette and the second expression cassette are arranged unidirectionally.

[0161] In one embodiment, the expression plasmid further comprises a selectable marker.

[0162] In one embodiment, the expression cassette and the selectable marker are arranged bidirectionally.

[0163] One aspect as reported herein is the use of an expression plasmid as reported herein for transient or stable expression of an antibody.

[0164] One aspect as reported herein is a eukaryotic cell comprising an expression plasmid as reported herein.

[0165] One aspect as reported herein is a method for producing an antibody comprising the steps of:

[0166] - culturing a eukaryotic cell comprising the expression plasmid as reported herein or a cell as reported herein,

[0167] - Recovering the antibody from the eukaryotic cells or culture medium.

[0168] In one embodiment, the eukaryotic cell is a mammalian cell. In one embodiment, the mammalian cell is selected from the group consisting of a CHO cell, a HEK cell, a BHK cell, a NSO cell, and a SP2 / 0 cell.

[0169] One aspect as reported herein is an expression plasmid comprising in 5' to 3' direction a promoter sequence, a nucleic acid encoding an antibody heavy chain or an antibody light chain, an IRES element, a nucleic acid sequence encoding a selectable marker and a polyA signal sequence, whereby the IRES element is an EMCV-IRES element.

[0170] In one embodiment, the nucleic acid encodes an antibody heavy chain.

[0171] In one embodiment, the selectable marker is a fusion protein of the formula ACS, whereby A is a detectable polypeptide, C is a proteolytic signal sequence, and S is a selectable marker.

[0172] In one embodiment, the proteolytic signal sequence is the PEST sequence of ornithine decarboxylase.

[0173] In one embodiment, the detectable polypeptide is green fluorescent protein.

[0174] In one embodiment, the selectable marker is neomycin.

[0175] One aspect reported herein is a nucleic acid encoding a polypeptide comprising, in N-terminal to C-terminal direction, a green fluorescent protein, a PEST sequence for ornithine decarboxylase, and neomycin.

[0176] One aspect as reported herein is the use of a nucleic acid encoding a polypeptide comprising in N-terminal to C-terminal direction a green fluorescent protein, a PEST sequence for ornithine decarboxylase and neomycin for selecting antibody secreting cells.

[0177] One aspect as reported herein is the use of an expression cassette comprising in 5' to 3' direction a promoter sequence, a nucleic acid encoding an antibody heavy chain or an antibody light chain, an IRES element, a nucleic acid sequence encoding a selectable marker and a polyA signal sequence for selecting antibody-producing cells, whereby the IRES element is an EMCV-IRES element.

[0178] In one embodiment, the nucleic acid encodes an antibody heavy chain.

[0179] In one embodiment, the selectable marker is a fusion protein of the formula ACS, whereby A is a detectable polypeptide, C is a proteolytic signal sequence, and S is a selectable marker.

[0180] In one embodiment, the proteolytic signal sequence is the PEST sequence of ornithine decarboxylase.

[0181] In one embodiment, the detectable polypeptide is green fluorescent protein.

[0182] In one embodiment, the selectable marker is neomycin.

[0183] One aspect as reported herein is a method for selecting eukaryotic cells expressing an antibody comprising the following steps:

[0184] - culturing eukaryotic cells comprising i) the expression plasmid as reported in this aspect and ii) a nucleic acid encoding the respective additional antibody chain not encoded by the expression plasmid as reported in this aspect,

[0185] - Selecting cells expressing the detectable polypeptide.

[0186] One aspect as reported herein is an expression plasmid comprising in 5' to 3' direction a promoter sequence, a nucleic acid encoding an antibody light chain, an IRES element, a nucleic acid encoding an antibody heavy chain and a polyA signal sequence, whereby the IRES element is an EV71-IRES element.

[0187] In one embodiment, the promoter sequence is selected from the group consisting of a human CMV promoter sequence with or without intron A, an SV40 promoter sequence, and a human elongation factor 1α promoter sequence with or without intron A.

[0188] In one embodiment, the polyA signal sequence is selected from the group consisting of a bovine growth hormone polyA signal sequence and an SV40 polyA signal sequence.

[0189] In one embodiment, the plasmid comprises the human gastrin terminator sequence 3' to the polyA signal sequence.

[0190] One aspect as reported herein is the use of an expression plasmid comprising in 5' to 3' direction a promoter sequence, a nucleic acid encoding an antibody light chain, an IRES element, a nucleic acid sequence encoding an antibody heavy chain and a polyA signal sequence for expressing an antibody, whereby the IRES element is an EV71-IRES element.

[0191] In one embodiment, the promoter sequence is selected from the group consisting of a human CMV promoter sequence with or without intron A, an SV40 promoter sequence, and a human elongation factor 1α promoter sequence with or without intron A.

[0192] In one embodiment, the polyA signal sequence is selected from the group consisting of a bovine growth hormone polyA signal sequence and an SV40 polyA signal sequence.

[0193] In one embodiment, the plasmid comprises the human gastrin terminator sequence 3' to the polyA signal sequence.

[0194] One aspect as reported herein is a method for producing an antibody comprising the following steps:

[0195] - culturing eukaryotic cells comprising the expression plasmid as reported herein,

[0196] - Recovering the antibody from the cell or culture medium, thereby producing the antibody.

[0197] In one embodiment, the hCMV promoter has the sequence of SEQ ID NO: 01. This is the hCMV promoter without intron A and without 5'UTR.

[0198] In one embodiment, the hCMV promoter has the sequence of SEQ ID NO: 02. This is the hCMV promoter without intron A and containing a 5'UTR.

[0199] In one embodiment, the hCMV promoter has the sequence of SEQ ID NO: 03. This is the full-length hCMV promoter including intron A.

[0200] In one embodiment, the human elongation factor 1α promoter has the sequence of SEQ ID NO: 04. This is the hEF1α promoter without intron A.

[0201] In one embodiment, the human elongation factor 1α promoter has the sequence of SEQ ID NO: 05. This is the hEF1α promoter containing intron A.

[0202] In one embodiment, the human elongation factor 1α promoter has the sequence of SEQ ID NO: 06. This is a short hEF1α promoter containing intron A and a 5'UTR.

[0203] In one embodiment, the rat CMV promoter has the sequence of SEQ ID NO:07.

[0204] In one embodiment, the SV40 polyA signal sequence has the sequence of SEQ ID NO:08.

[0205] In one embodiment, the bovine growth hormone polyA signal sequence has the sequence of SEQ ID NO:09.

[0206] In one embodiment, the human gastrin terminator has the sequence of SEQ ID NO:10.

[0207] In one embodiment, the SV40 promoter has the sequence of SEQ ID NO:11.

[0208] In one embodiment, the PEST sequence of ornithine decarboxylase is encoded by the sequence of SEQ ID NO:12.

[0209] In one embodiment, the GFP sequence is encoded by the sequence of SEQ ID NO:13.

[0210] In one embodiment, the neomycin selectable marker has the sequence of SEQ ID NO:14.

[0211] In one embodiment, the GFP-PEST-NEO fusion polypeptide is encoded by the sequence of SEQ ID NO:15.

[0212] In one embodiment, the EMCV-IRES has the sequence of SEQ ID NO:16.

[0213] In one embodiment, the EV71-IRES has the sequence of SEQ ID NO:17.

[0214] In one embodiment of all aspects as reported herein the antibody is a bispecific antibody.

[0215] In one embodiment, the bispecific antibody has a first binding specificity or binding site that specifically binds to a first antigen or a first epitope on an antigen, and the bispecific antibody has a second binding specificity or binding site that specifically binds to a second antigen or a second epitope on an antigen.

[0216] In one embodiment, the expression vector comprises:

[0217] a first expression cassette comprising, in 5' to 3' direction, a promoter, a nucleic acid encoding a first antibody light chain, a polyA signal sequence and an optional terminator sequence,

[0218] a second expression cassette comprising, in 5' to 3' direction, a promoter, a nucleic acid encoding a second antibody light chain, a polyA signal sequence and optionally a terminator sequence,

[0219] a third expression cassette comprising, in 5' to 3' direction, a promoter, the nucleic acid encoding the heavy chain of the first antibody, a polyA signal sequence and optionally a terminator sequence,

[0220] a fourth expression cassette comprising in 5' to 3' direction a promoter, the nucleic acid encoding the second antibody heavy chain, a polyA signal sequence and optionally a terminator sequence,

[0221] or

[0222] a first expression cassette comprising, in 5' to 3' direction, a promoter, a nucleic acid encoding an antibody light chain, a polyA signal sequence and an optional terminator sequence,

[0223] - a second expression cassette comprising, in 5' to 3' direction, a promoter, the nucleic acid encoding the first antibody heavy chain, a polyA signal sequence and optionally a terminator sequence, and

[0224] a third expression cassette comprising, in 5' to 3' direction, a promoter, the nucleic acid encoding the second antibody heavy chain, a polyA signal sequence and optionally a terminator sequence,

[0225] This antibody light chain is thus the common light chain of two antibody heavy chains.

[0226] In one embodiment of all aspects as reported herein the expression vector comprises:

[0227] -Antibody light chain expression cassette,

[0228] -First antibody heavy chain expression cassette,

[0229] - a second antibody heavy chain expression cassette, and

[0230] - a selectable marker expression cassette,

[0231] wherein at least one of the antibody heavy chain expression cassette, the antibody light chain expression cassette and the selectable marker expression cassette are arranged in a unidirectional manner, and

[0232] The unidirectional expression cassette is arranged in the 5' to 3' order of the antibody heavy chain expression cassette, the antibody light chain expression cassette and the selection marker expression cassette, or the unidirectional expression cassette is arranged in the 5' to 3' order of the antibody light chain expression cassette, the antibody heavy chain expression cassette and the selection marker expression cassette.

[0233] In one embodiment of all aspects as reported herein the expression vector comprises:

[0234] -First antibody light chain expression cassette,

[0235] - Second antibody light chain expression cassette,

[0236] -First antibody heavy chain expression cassette,

[0237] - a second antibody heavy chain expression cassette, and

[0238] - a selectable marker expression cassette,

[0239] wherein one of the antibody heavy chain expression cassettes, one of the antibody light chain expression cassettes and the selectable marker expression cassette are arranged in a unidirectional manner, and

[0240] The unidirectional expression cassette is arranged in the 5' to 3' order of the antibody heavy chain expression cassette, the antibody light chain expression cassette and the selection marker expression cassette, or the unidirectional expression cassette is arranged in the 5' to 3' order of the antibody light chain expression cassette, the antibody heavy chain expression cassette and the selection marker expression cassette.

[0241] In one embodiment, one of the antibody heavy chain expression cassettes encodes an antibody heavy chain comprising a hole mutation.

[0242] In one embodiment, one of the antibody heavy chain expression cassettes encodes an antibody heavy chain comprising a knob mutation.

[0243] In one embodiment, one of the antibody light chain expression cassettes encodes an antibody light chain comprising an antibody light chain variable domain and an antibody heavy chain CH1 domain as a constant domain, and / or one of the antibody light chain expression cassettes encodes an antibody light chain comprising an antibody light chain variable domain and an antibody light chain CL domain as a constant domain.

[0244] In one embodiment, one of the antibody heavy chain expression cassettes encodes an antibody heavy chain comprising an antibody light chain constant domain (CL) as the first constant domain, and / or one of the antibody heavy chain expression cassettes encodes an antibody heavy chain comprising an antibody heavy chain CH1 domain as the first constant domain. Detailed Description of the Invention

[0245] I. General

[0246] As known to those skilled in the art, the use of recombinant DNA technology enables the production of many derivatives of nucleic acids and / or polypeptides. Such derivatives can be modified in a single or several positions, for example, by substitution, change, exchange, deletion or insertion. The modification or derivatization can be carried out, for example, by means of site-directed mutagenesis. Those skilled in the art can easily carry out such modifications (see, for example, Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, USA (1999)). The use of recombinant technology enables those skilled in the art to transform a variety of host cells with one or more heterologous nucleic acids. Although the transcription and translation (i.e., expression) machinery of different cells uses the same elements, among other things, cells belonging to different species can have different so-called codon selections. Thus the same polypeptide (with respect to amino acid sequence) can be encoded by one or more different nucleic acids. In addition, due to the degeneracy of the genetic code, different nucleic acids can encode the same polypeptide.

[0247] The use of recombinant DNA technology enables the production of many derivatives of nucleic acids and / or polypeptides. Such derivatives can be modified in a single or several positions, for example, by substitution, alteration, exchange, deletion or insertion. The modification or derivatization can be performed, for example, by means of site-directed mutagenesis. Those skilled in the art can easily carry out such modifications (see, for example, Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, USA (1999); Hames, BD and Higgins, SJ, Nucleic acid hybridization—a practical approach, IRL Press, Oxford, England (1985)).

[0248] The use of recombinant technology enables the transformation of a variety of host cells with one or more heterologous nucleic acids. Although the transcription and translation (i.e., expression) machinery of different cells uses the same components, cells belonging to different species may, among other things, have different so-called codon usages. Thus, the same polypeptide (in terms of amino acid sequence) can be encoded by one or more different nucleic acids. In addition, due to the degeneracy of the genetic code, different nucleic acids can encode the same polypeptide.

[0249] definition

[0250] An "affinity matured" antibody is one with one or more alterations in one or more hypervariable regions (HVRs) which result in an improvement in the affinity of the antibody for antigen, compared to a parent antibody which does not possess such alteration(s).

[0251] The term "antibody" herein is used in the broadest sense and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0252] "Antibody fragments" refer to molecules other than intact antibodies that comprise a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0253] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0254] The "class" of an antibody refers to the type of constant domain or region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, several of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0255] The term "expression" as used herein refers to the transcription and / or translation process that occurs in a cell. The level of transcription of a target nucleic acid sequence in a cell can be determined based on the amount of the corresponding mRNA present in the cell. For example, mRNA transcribed from the target sequence can be quantified by RT-PCR or by Northern hybridization (see Sambrook et al., 1999, supra). The polypeptide encoded by the target nucleic acid can be quantified by a variety of methods, such as by ELISA, by measuring the biological activity of the polypeptide, or by utilizing an assay independent of such activity, such as Western blotting or radioimmunoassay using immunoglobulins that recognize and bind to the polypeptide (see Sambrook et al., 1999, supra).

[0256] An "expression cassette" refers to a construct comprising regulatory elements, such as a promoter and a polyadenylation site, necessary for expression of at least the contained nucleic acid in a cell.

[0257] An "expression vector" is a nucleic acid that provides all the elements necessary to express one or more structural genes contained therein in a host cell. Typically, an expression plasmid comprises: a prokaryotic plasmid propagation unit, such as for Escherichia coli (E. coli), comprising an origin of replication and a selectable marker; a eukaryotic selectable marker; and one or more expression cassettes for expressing one or more structural genes of interest, each comprising a promoter, a structural gene, and a polyadenylation signal (polyA signal sequence). Gene expression is typically placed under the control of a promoter, and such a structural gene is referred to as being "operably linked" to the promoter. Similarly, a regulatory element is operably linked to a core promoter if it modulates the activity of the core promoter.

[0258] The term "Fc region" herein is used to define the C-terminal region of an immunoglobulin heavy chain, which comprises at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) in the Fc region may be present or absent. Unless otherwise indicated herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system described in Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242, also referred to as the EU index.

[0259] "Fc region" is a well-known term, defined according to the papain cleavage of the antibody heavy chain. In one embodiment, the complex reported herein can include a human Fc region or an Fc region derived from a human source as an antibody heavy chain hinge region polypeptide. In another embodiment, the Fc region is the Fc region of a human antibody of the IgG4 subclass, or the Fc region of a human antibody of the IgG1, IgG2 or IgG3 subclass, which is modified in such a way that no Fc gamma receptor (e.g., Fc gamma RIIIa) is bound and / or no C1q is detected. In one embodiment, the Fc region is a human Fc region, and especially a mutant Fc region from a human IgG4 subclass or from a human IgG1 subclass. In one embodiment, the Fc region is from a human IgG1 subclass with mutations L234A and L235A. IgG4 shows reduced Fc gamma receptor (Fc gamma RIIIa) binding, but antibodies of other IgG subclasses show strong binding. However, Pro238, Asp265, Asp270, Asn297 (loss of Fc carbohydrate), Pro329, Leu234, Leu235, Gly236, Gly237, Ile253, Ser254, Lys288, Thr307, Gln311, Asn434 or / and His435 are residues that, when altered, also provide reduced Fcγ receptor binding (Shields, RL et al., J. Biol. Chem. 276 (2001) 6591-6604; Lund, J. et al., FASEB J. 9 (1995) 115-119; Morgan, A. et al., Immunology 86 (1995) 319-324; EP 0 307 434). In one embodiment, the antibody to be expressed in one aspect as reported herein is for Fcγ receptor binding of the IgG4 subclass or the IgG1 or IgG2 subclass, has a mutation in L234, L235 and / or D265, and / or comprises the PVA236 mutation. In one embodiment, the mutation is S228P, L234A, L235A, L235E and / or PVA236 (PVA236 represents the substitution of the amino acid sequence ELLG (given in the single-letter amino acid code) from amino acid positions 233 to 236 of IgG1 or EFLG of IgG4 with PVA). In one embodiment, the mutation is S228P of IgG4 and L234A and L235A of IgG1. The Fc region of the antibody is directly involved in ADCC (antibody-dependent cellular cytotoxicity) and CDC (complement-dependent cytotoxicity). Complexes that do not bind to Fcγ receptors and / or complement factor C1q do not elicit antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC). The knob modification represents the mutation T366W (Kabat numbering) in the antibody CH3 domain.Hole modifications refer to the mutations T366S, L368A and Y407V in the CH3 domain of the antibody. In addition to the knob and hole modifications, the mutation S354C may be present in one CH3 domain and the mutation Y349C may be present in the other CH3 domain.

[0260] "Framework region" or "FR" refers to the variable domain residues outside the hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences generally appear in the following order in VH (or VL): FR1-H1 (L1)-FR2-H2 (L2)-FR3-H3 (L3)-FR4.

[0261] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody that has a structure substantially similar to a native antibody structure or has heavy chains that comprise an Fc region as defined herein.

[0262] "Gene" refers to a nucleic acid that is a segment on a chromosome or plasmid that can affect the expression of a peptide, polypeptide or protein. In addition to the coding region (i.e., structural gene), a gene contains other functional elements such as a signal sequence, one or more promoters, introns and / or terminators.

[0263] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably to refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the originally transformed cell and progeny derived therefrom, without regard to the number of passages. The nucleic acid content of the progeny may not be identical to that of the parent cell, but may contain mutations. Mutant progeny that have the same function and biological activity as screened or selected for in the originally transformed cell are included herein.

[0264] A "human antibody" is an antibody having an amino acid sequence corresponding to an antibody produced by a human or human cell or derived from a non-human source utilizing human antibody libraries or other human antibody encoding sequences. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues.

[0265] "Humanized" antibodies refer to chimeric antibodies comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one and typically two variable domains, wherein all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has been humanized.

[0266] The term "hypervariable region" or "HVR" as used herein refers to each of the regions of an antibody variable domain that are highly variable in sequence and / or form structurally defined loops ("hypervariable loops"). Typically, a natural four-chain antibody comprises six HVRs; three in VH (H1, H2, H3) and three in VL (L1, L2, L3). HVRs generally comprise amino acid residues from hypervariable loops and / or from "complementarity determining regions" (CDRs), which have the highest sequence variability and / or are involved in antigen recognition. Exemplary hypervariable loops are present at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia, C. and Lesk, AM, J. Mol. Biol. 196 (1987) 901-917). Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) are present at amino acid residues 24-34 of L1, 50-56 of L2, 89-97 of L3, 31-35B of H1, 50-65 of H2, and 95-102 of H3 (Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH publication 91-3242). Except for CDR1 in VH, CDRs generally comprise amino acid residues that form hypervariable loops. CDRs also comprise "specificity determining residues" or "SDRs," which are residues that contact antigen. SDRs are contained in regions of CDRs called shortened CDRs or α-CDRs. Exemplary α-CDRs (α-CDR-L1, α-CDR-L2, α-CDR-L3, α-CDR-H1, α-CDR-H2, and α-CDR-H3) occur at amino acid residues 31-34 of L1, 50-55 of L2, 89-96 of L3, 31-35B of H1, 50-58 of H2, and 95-102 of H3 (Almagro, JC and Fransson, J., Front. Biosci. 13 (2008) 1619-1633). Unless otherwise indicated, HVR residues and other residues (e.g., FR residues) in the variable domain are numbered herein according to Kabat et al., supra.

[0267] "Internal ribosome entry site" or "IRES" describes a sequence that functionally promotes translation initiation 5' of a gene independent of the IRES and allows translation of two cistrons (open reading frames) from a single transcript in animal cells. An IRES provides an independent ribosome entry site for translation of the open reading frame immediately downstream thereof (downstream is used interchangeably with 3' herein). Unlike bacterial mRNAs, which can be polycistronic, i.e., encoding several different polypeptides that are translated sequentially from the mRNA, most mRNAs of animal cells are monocistronic, encoding the synthesis of only one protein. For polycistronic transcripts in eukaryotic cells, translation will initiate from the translation initiation site closest to the 5' site, terminate at the first stop codon, and the transcript will be released from the ribosome, resulting in translation of only the first encoded polypeptide in the mRNA. In eukaryotic cells, polycistronic transcripts with an IRES operably linked to a second or subsequent open reading frame in a transcript allow the downstream open reading frames to be translated sequentially to produce two or more polypeptides encoded by the same transcript. The use of IRES elements in vector construction has been described previously, see, for example, Pelletier, J. et al., Nature 334 (1988) 320-325; Jang, SK et al., J. Virol. 63 (1989) 1651-1660; Davies, MV et al., J. Virol. 66 (1992) 1924-1932; Adam, MA et al., J. Virol. 65 (1991) 4985-4990; Morgan, RA et al., Nucl. Acids Res. 20 (1992) 1293-1299; Sugimoto, Y et al., Biotechnology 12 (1994) 694-698; Ramesh, N. et al., Nucl. Acids Res. 24 (1996) 2697-2700; and Mosser, DD et al., BioTechniques 22(1997)150-152).

[0268] The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous antibody population, i.e., except for example comprising naturally occurring mutations or possible variant antibodies (such variants are generally present in smaller amounts) that occur during the production of monoclonal antibody preparations, the single antibody comprising the population is identical and / or binds to the same epitope. Unlike polyclonal antibody preparations that generally comprise different antibodies against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is against a single determinant on the antigen. Therefore, the modifier "monoclonal" refers to the feature that an antibody is obtained from a substantially homogeneous antibody population, and is not interpreted as requiring antibody production by any specific method. For example, the monoclonal antibody to be used in accordance with the present invention can be prepared by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of human immunoglobulin loci, and this paper describes such methods and other exemplary methods for preparing monoclonal antibodies.

[0269] "Natural antibodies" refer to naturally occurring immunoglobulin molecules with different structures. For example, natural IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons consisting of two identical light chains and two identical heavy chains that form disulfide bonds. From N-terminus to C-terminus, each heavy chain has a variable region (VH), also referred to as a variable heavy chain domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from N-terminus to C-terminus, each light chain has a variable region (VL), also referred to as a variable light chain domain or a light chain variable domain, followed by a constant light chain (CL) domain. Based on the amino acid sequence of its constant domain, the light chain of an antibody can be assigned to one of two types, referred to as κ and λ.

[0270] As used herein, "nucleic acid" refers to a polymer molecule composed of the mononucleotides (also referred to as bases) a, c, g, and t (or u in RNA), such as DNA, RNA, or modifications thereof. This polynucleotide molecule can be a naturally occurring polynucleotide molecule or a synthetic polynucleotide molecule or a combination of one or more naturally occurring polynucleotide molecules and one or more synthetic polynucleotide molecules. This definition also encompasses naturally occurring polynucleotide molecules in which one or more nucleotides have been altered (e.g., by mutagenesis), deleted, or added. A nucleic acid can be isolated or integrated into another nucleic acid (e.g., an expression cassette), a plasmid, or a chromosome of a host cell. A nucleic acid is also characterized by its nucleic acid sequence consisting of mononucleotides.

[0271] The processes and methods for converting, for example, an amino acid sequence of a polypeptide into a corresponding nucleic acid sequence encoding the amino acid sequence are well known to those skilled in the art. Thus, a nucleic acid is characterized as a nucleic acid sequence consisting of mononucleotides and is also characterized as the amino acid sequence of the polypeptide encoded thereby.

[0272] As used herein, "nucleic acid" refers to a naturally occurring or partially or completely non-naturally occurring nucleic acid encoding a polypeptide that can be recombinantly produced. Nucleic acids can be constructed from isolated or chemically synthesized DNA fragments. Nucleic acids can be integrated into another nucleic acid, such as an expression plasmid or the genome / chromosome of a eukaryotic host cell. Plasmids include shuttle plasmids and expression plasmids. Typically, the plasmid will also contain a prokaryotic propagation unit containing a replication origin (e.g., ColE1 replication origin) and a selectable marker (e.g., ampicillin or tetracycline resistance gene) for replication and selection, respectively, of a plasmid in a prokaryotic organism.

[0273] "Operably linked" refers to the juxtaposition of two or more components, wherein the components are in a relationship that allows them to function in their intended manner. For example, if a promoter and / or enhancer acts in cis to control or regulate the transcription of the sequence to which it is linked, it is operably linked to a coding sequence. Typically, but not necessarily, "operably linked" DNA sequences are adjacent and, when necessary to connect two protein coding regions (such as a secretory leader and a polypeptide), are adjacent and in (readable) frame. However, although an operably linked promoter is typically located upstream of a coding sequence, it is necessarily adjacent to the coding sequence. An enhancer need not be adjacent. If an enhancer increases the transcription of a coding sequence, then the enhancer is operably linked to the coding sequence. An operably linked enhancer can be located upstream of the coding sequence, within the coding sequence, or downstream of the coding sequence, and at a considerable distance from the promoter. If a polyadenylation site is located at the downstream end of the coding sequence, such that transcription passes through the coding sequence and enters the polyadenylation sequence, then it is operably linked to the coding sequence. If the translation stop codon is located at the downstream end (3' end) of the coding sequence, such that translation proceeds to the stop codon through the coding sequence and terminates there, it is effectively linked to the exon nucleic acid sequence. Linking is achieved by recombinant methods known in the art, for example, using PCR methods and / or by linking at convenient restriction sites. If there is no convenient restriction site, synthetic oligonucleotide adapters or joints are used as per conventional practice.

[0274] A "polycistronic transcription unit" is a transcription unit in which more than one structural gene is under the control of the same promoter.

[0275] As used herein, the term "polyadenylation signal" refers to a nucleic acid sequence used to induce cleavage and polyadenylation of the primary transcript of a specific nucleic acid sequence segment. The 3' untranslated region comprising the polyadenylation signal can be selected from the 3' untranslated region containing the polyadenylation signal derived from SV40, bovine growth hormone (bGH) gene, immunoglobulin gene, and thymidine kinase gene (tk, e.g., herpes simplex virus thymidine kinase polyadenylation signal).

[0276] "Promoter" refers to a polynucleotide sequence that controls the transcription of a gene / structural gene or nucleic acid sequence that is operatively connected to it. The promoter comprises signals for RNA polymerase binding and transcription initiation. The promoter used will be functional in the cell type of the host cell in which the selected sequence is to be expressed. A large number of promoters, including constitutive, inducible and repressible promoters from a variety of different sources, are well known in the art (and identified in databases such as GenBank) and can be obtained as cloned polynucleotides or within cloned polynucleotides (e.g., from depositories such as ATCC and other commercial or personal sources).

[0277] A "promoter" comprises a nucleotide sequence that directs transcription of a structural gene. Typically, a promoter is located in the 5' non-coding or untranslated region of a gene, near the transcription start site of the structural gene. Sequence elements within a promoter that play a role in initiating transcription are often characterized as a consensus nucleotide sequence. These promoter elements include RNA polymerase binding sites, TATA sequences, CAAT sequences, differentiation-specific elements (DSEs; McGehee, R. E. et al., Mol. Endocrinol. 7 (1993) 551), cyclic AMP response elements (CREs), serum response elements (SREs; Treisman, R., Seminars in Cancer Biol. 1 (1990) 47), glucocorticoid response elements (GREs), and others such as CRE / ATF (O'Reilly, M. A. et al., J. Biol. Chem. 267 (1992) 1993-8), AP2 (Ye, J. et al., J. Biol. Chem. 269 (1994) 257-28), SP1, cAMP response element binding protein (CREB; Loeken, M. R., Gene Expr. 3 (1993) 253), and octanucleotide factors (see generally Watson et al., (eds.), Molecular Biology of the Gene, 4th ed. Benjamin / Cummings Publishing Company, Inc. (1987)) and Lemaigre, FP and Rousseau, GG, Biochem. J. 303 (1994) 1-14). If the promoter is an inducible promoter, the transcription rate increases in response to the inducer. In contrast, if the promoter is a constitutive promoter, the transcription rate is not regulated by the inducer. Repressible promoters are also known. For example, the c-fos promoter is specifically activated when growth hormone binds to its receptor on the cell surface. Tetracycline (tet)-regulated expression can be achieved by an artificial hybrid promoter consisting of, for example, the CMV promoter followed by two Tet operator sites. The Tet repressor binds to the two Tet operator sites and blocks transcription. When the inducer tetracycline is added, the Tet repressor is released from the Tet operator sites and transcription continues (Gossen, M. and Bujard, H., PNAS 89 (1992) 5547-5551). For other inducible promoters including metallothionein and heat shock promoters, see, eg, Sambrook et al. (supra) and Gossen et al., Curr. Opin. Biotech. 5 (1994) 516-520.Among the eukaryotic promoters that have been identified as strong promoters for high-level expression are the SV40 early promoter, the adenovirus major late promoter, the mouse metallothionein-I promoter, the Rous sarcoma virus long terminal repeat, the Chinese hamster elongation factor 1 alpha (CHEF-1, see e.g., U.S. Pat. No. 5,888,809), human EF-1 alpha, ubiquitin, and the human cytomegalovirus immediate early promoter (CMV IE).

[0278] A "promoter" can be constitutive or inducible. Enhancers (i.e., cis-acting DNA elements that act on a promoter to increase transcription) may necessarily function in conjunction with a promoter to increase the expression level achieved with the promoter alone and may be included as transcriptional regulatory elements. Typically, a polynucleotide segment containing a promoter will also include an enhancer sequence (e.g., CMV or SV40).

[0279] The term "stably transformed", "stably transfected" or "stable" as used herein refers to the heritable and stable integration of exogenous nucleic acid into the host cell genome / chromosome. Stably transfected cells are obtained after a cell selection process under selective growth conditions (i.e., in the presence of one or more selection markers).

[0280] "Structural gene" refers to the region of a gene that does not contain a signal sequence, ie, the coding region.

[0281] The term "transcription terminator" refers to a DNA sequence of 50-750 base pairs in length that signals RNA polymerase to terminate mRNA synthesis. Especially when using strong promoters, it is advisable to use a very effective (strong) terminator at the 3' end of the expression cassette to prevent RNA polymerase from reading through. Ineffective transcription terminators can lead to the formation of operon-like mRNAs, which can be the cause of undesirable (e.g., plasmid-encoded) gene expression.

[0282] In the scope of the present invention, it is possible to obtain transfected cells with any of the transfection methods known in the art. For example, nucleic acid can be introduced into cells by means of electroporation or microinjection. Alternatively, a lipid transfection reagent such as FuGENE 6 (Roche Diagnostics GmbH, Germany), X-tremeGENE (Roche Diagnostics GmbH, Germany) and LipofectAmine (Invitrogen Corp., the U.S.) can be used. Also alternatively, nucleic acid can be introduced into cells (Singer, O., Proc. Natl. Acad. Sci. USA 101 (2004) 5313-5314) by a suitable viral vector system based on retrovirus, slow virus, adenovirus or adeno-associated virus.

[0283] As used herein, the term "transient transfection" refers to a method in which a nucleic acid introduced into a cell is not integrated into the genome or chromosomal DNA of the cell. Instead, it is maintained in the cell as an extrachromosomal element (e.g., as an episome). The transcription process of the episomal nucleic acid is not affected, and, for example, the protein encoded by the episomal nucleic acid is produced. Transient transfection produces "transiently transfected" cells.

[0284] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs) (see, e.g., Kindt, TJ et al., Kuby Immunology, 6th ed., WH Freeman and Co., NY (2007), p. 91). A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, antibodies that bind to a specific antigen can be isolated by screening libraries of complementary VL or VH domains, respectively, using VH or VL domains from antibodies that bind to the antigen (see, e.g., Portolano, S. et al., J. Immunol. 150 (1993) 880-887; Clackson, T. et al., Nature 352 (1991) 624-628).

[0285] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are autonomously replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0286] Antibody

[0287] The methods and compositions provided herein are used to produce recombinant monoclonal antibodies.Antibodies can have a variety of structures, such as, but not limited to, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, monovalent antibodies, multivalent antibodies (e.g., bivalent antibodies).

[0288] In certain embodiments, the antibody is an antibody fragment. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv, and scFv fragments, i.e., other fragments described below. For a review of certain antibody fragments, see Hudson, PJ et al., Nat. Med. 9 (2003) 129-134. For a review of scFv fragments, see, for example, Plueckthun, A., In: The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore (eds.), Springer-Verlag, New York (1994), pp. 269-315; see also WO 1993 / 16185, US 5,571,894, and US 5,587,458. For a discussion of Fab and F(ab')2 fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see U.S. Pat. No. 5,869,046.

[0289] Diabodies are antibody fragments with two antigen-binding sites that can be bivalent or bispecific (see, e.g., EP 0 404 097; WO 1993 / 01161; Hudson, PJ et al., Nat. Med. 9 (2003) 129-134; and Holliger, P. et al., Proc. Natl. Acad. Sci. USA 90 (1993) 6444-6448). Triabodies and tetrabodies are also described in Hudson, PJ et al., Nat. Med. 9 (2003) 129-134.

[0290] Single-domain antibodies are antibody fragments that contain all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody.In certain embodiments, the single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., US 6,248,516 Bl).

[0291] Antibody fragments can be prepared by a variety of techniques including, but not limited to, proteolytic digestion of intact antibodies as described herein, and production by recombinant host cells (eg, E. coli or phage).

[0292] In certain embodiments, the antibody is a chimeric antibody. Certain chimeric antibodies are described in, for example, US 4,816,567; and Morrison, SL et al., Proc. Natl. Acad. Sci. USA 81 (1984) 6851-6855. In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate (such as a monkey)) and a human constant region. In another example, a chimeric antibody is a "class-switched" antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0293] In certain embodiments, chimeric antibodies are humanized antibodies. Generally, humanized non-human antibodies reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. Generally, humanized antibodies include one or more variable domains, wherein HVR, such as CDR (or part thereof) are derived from non-human antibodies, and FR (or part thereof) are derived from human antibody sequences. Humanized antibodies will also optionally include at least some human constant regions. In some embodiments, some FR residues in humanized antibodies are replaced with corresponding residues from non-human antibodies (such as antibodies from which HVR residues are derived), such as to restore or improve antibody specificity or affinity.

[0294] Humanized antibodies and methods for making them are reviewed, for example, in Almagro, JC and Fransson, J., Front. Biosci. 13 (2008) 1619-1633, and further described, for example, in Riechmann, I. et al., Nature 332 (1988) 323-329; Queen, C. et al., Proc. Natl. Acad. Sci. USA 86 (1989) 10029-10033; US 5,821,337, US 7,527,791, US 6,982,321 and US 7,087,409; Kashmiri, SV et al., Methods 36 (2005) 25-34 (describing SDR(a-CDR) grafting); Padlan, EA, Mol. Immunol. 28 (1991) 489-498 (describing “resurfacing”); Dall'Acqua, WF et al., Methods 36 (2005) 43-60 (describing “FR shuffling”); Osbourn, J. et al., Methods 36 (2005) 61-68; and Klimka, A. et al., Br. J. Cancer 83 (2000) 252-260 (describing the “guided selection” approach to FR shuffling).

[0295] The human framework regions that can be used for humanization include, but are not limited to, framework regions selected by the "optimal" method (see, e.g., Sims, MJ et al., J. Immunol. 151 (1993) 2296-2308); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter, P. et al., Proc. Natl. Acad. Sci. USA 89 (1992) 4285-4289; and Presta, LG et al., J. Immunol. 151 (1993) 2623-2632); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro, JC and Fransson, J., Front. Biosci. 13 (2008) 1619-1633); and framework regions derived from screening FR libraries (see, e.g., Baca, M. et al., J. Biol. Chem. 272 ​​(1997) 10678-10684 and Rosok, MJ et al., J. Biol. Chem. 271 (19969 22611-22618).

[0296] In certain embodiments, the antibody is a human antibody. Human antibodies can be produced using a variety of techniques known in the art. Human antibodies are generally described in van Dijk, MA and van de Winkel, JG, Curr. Opin. Pharmacol. 5 (2001) 368-374 and Lonberg, N., Curr. Opin. Immunol. 20 (2008) 450-459.

[0297] Human antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified to produce complete human antibodies or complete antibodies with human variable regions in response to antigenic attack. Such animals typically contain all or part of a human immunoglobulin locus that replaces the endogenous immunoglobulin locus, or it is present extrachromosomally or randomly integrated into the chromosomes of the animal. In such transgenic mice, the endogenous immunoglobulin locus is typically inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, N., Nat. Biotech. 23 (2005) 1117-1125, and also see, for example, the description of the XENOMOUSE TM US 6,075,181 and US 6,150,584 of the technology; description US 5,770,429 of the technology; describes KM US 7,041,870; Description of the technology Human variable regions from intact antibodies produced by such animals can be further modified, for example, by combining with different human constant regions.

[0298] Human antibodies can also be prepared by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for producing human monoclonal antibodies have been described (see, for example, Kozbor, D., J. Immunol. 133 (1984) 3001-3005; Brodeur, BR et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York (1987), pp. 51-63; and Boerner, P. et al., J. Immunol. 147 (1991) 86-95). Human antibodies produced by human B cell hybridoma technology are also described in Li, J. et al., Proc. Natl. Acad. Sci. USA 103 (2006) 3557-3562. Other methods include those described in, for example, US 7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, J., Xiandai Mianyixue 26 (2006) 265-268 (describing human-human hybridomas). Human hybridoma technology (triad technology) is also described in Vollmers, HP and Brandlein, S., Histology and Histopathology 20 (2005) 927-937 and Vollmers, HP and Brandlein, S., Methods and Findings in Experimental and Clinical Pharmacology 27 (2005) 185-191.

[0299] Human antibodies can also be produced by separating the Fv clone variable domain sequences selected from the phage display libraries derived from humans. Such variable domain sequences can then be combined with the human constant region of desire. The technology for selecting human antibodies from antibody libraries is described below.

[0300] Combinatorial libraries can be screened for antibodies with one or more desired activities to isolate antibodies. For example, various methods are known in the art for generating phage libraries and screening such libraries for antibodies with desired binding characteristics. Such methods are reviewed in, for example, Hoogenboom, HR et al., Methods in Molecular Biology 178 (2001) 1-37, and further described in, for example, McCafferty, J. et al., Nature 348 (1990) 552-554; Clackson, T. et al., Nature 352 (1991) 624-628; Marks, JD et al., J. Mol. Biol. 222 (1992) 581-597; Marks, JD and Bradbury, A., Methods in Molecular Biology 248 (2003) 161-175; Sidhu, SS et al., J. Mol. Biol. 338 (2004) 299-310; Lee, CV et al., J. Mol. Biol. 340 (2004) 1073-1093; Fellouse, FA, Proc. Natl. Acad. Sci. USA 101 (2004) 12467-12472; and Lee, CV et al., J. Immunol. Methods 284 (2004) 119-132.

[0301] In some phage display methods, libraries of VH and VL genes are cloned separately by polymerase chain reaction (PCR) and randomly combined in a phage library, which is then screened for phage binding to antigen as described in Winter, G. et al., Ann. Rev. Immunol. 12 (1994) 433-455. Phage typically display antibody fragments as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immune sources provide high-affinity antibodies against immunogens without the need to construct hybridomas. Alternatively, a library used for the first experiment (e.g., from humans) can be cloned as described in Griffiths, AD et al., EMBO J. 12 (1993) 725-734 to provide a single source of antibodies against a wide range of non-self and self antigens without any immunization. Finally, the library for the first experiment can also be prepared by cloning unrearranged V gene segments from stem cells and encoding highly variable CDR3 regions with PCR primers containing random sequences and achieving in vitro rearrangement, as described in Hoogenboom, HR and Winter, G., J. Mol. Biol. 227 (1992) 381-388. Patents describing human antibody phage libraries include, for example, US 5,750,373, US 2005 / 0079574, US 2005 / 0119455, US 2005 / 0266000, US 2007 / 0117126, US 2007 / 0160598, US 2007 / 0237764, US 2007 / 0292936 and US 2009 / 0002360.

[0302] Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments herein.

[0303] In certain embodiments, the antibody is a multispecific antibody, such as a bispecific antibody. A multispecific antibody is a monoclonal antibody that has binding specificities for at least two different sites. In certain embodiments, one of the binding specificities is for a first antigen and the other is for a different second antigen. In certain embodiments, a bispecific antibody can bind to two different epitopes of the same antigen. Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing the antigen. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0304] Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs of different specificities (see Milstein, C. and Cuello, AC, Nature 305 (1983) 537-540; WO 93 / 08829; and Traunecker, A. et al., EMBO J. 10 (1991) 3655-3659) and "knob-in-hole" engineering (see, e.g., U.S. Pat. No. 5,731,168). Multispecific antibodies can also be prepared by engineering electrostatic guidance for preparing antibody Fc-heterodimer molecules (WO 2009 / 089004); cross-linking two or more antibodies or fragments (see, for example, US 4,676,980 and Brennan, M. et al., Science 229 (1985) 81-83); using leucine zippers to produce bispecific antibodies (see, for example, Kostelny, SA et al., J. Immunol. 148 (1992) 1547-1553); using "diabody" technology for preparing bispecific antibody fragments (see, for example, Holliger, P. et al., Proc. Natl. Acad. Sci. USA 90 (1993) 6444-6448); using single-chain Fv (scFv) dimers (see, e.g., Gruber, M. et al., J. Immunol. 152 (1994) 5368-5374); preparing trispecific antibodies as described, e.g., in Tutt, A. et al., J. Immunol. 147 (1991) 60-69.

[0305] Also included herein are engineered antibodies having three or more functional antigen-binding sites, including "octopus antibodies" (see, e.g., US 2006 / 0025576).

[0306] The antibody may be a "dual-acting Fab" or "DAF" that contains an antigen-binding site that binds to a first antigen as well as a second, different antigen (see, eg, US 2008 / 0069820).

[0307] The antibody or fragment may also be a multispecific antibody as described in WO 2009 / 080251, WO 2009 / 080252, WO 2009 / 080253, WO 2009 / 080254, WO 2010 / 112193, WO 2010 / 115589, WO 2010 / 136172, WO 2010 / 145792 or WO 2010 / 145793.

[0308] method

[0309] In certain embodiments, the methods provided herein are used to alter (ie, increase or decrease) the extent of glycosylation of an antibody.

[0310] When the antibody comprises an Fc region, the carbohydrate attached to the Fc region can be changed. Natural antibodies produced by mammalian cells typically contain branched biantennary oligosaccharides, which are generally attached to Asn297 of the CH2 domain of the Fc region via an N-link (see, for example, Wright, A. and Morrison, SL, TIBTECH 15 (1997) 26-32). Oligosaccharides can include a variety of carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, as well as fucose attached to GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modification of the oligosaccharides in the antibodies of the present invention can be performed to produce antibody variants with certain improved properties.

[0311] In one embodiment, the provided methods result in the production of antibodies having carbohydrate structures that lack fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297 relative to the sum of all sugar structures (e.g., complex, hybrid, and high mannose structures) attached to Asn 297 as measured by MALDI-TOF mass spectrometry as described in, for example, WO 2008 / 077546. Asn297 refers to the asparagine residue located at approximately position 297 in the Fc region (Kabat EU numbering of Fc region residues); however, due to small sequence variations in antibodies, Asn297 can also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylation variants may have improved ADCC function (see, eg, US 2003 / 0157108; US 2004 / 0093621). Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include: US 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US 2003 / 0115614; US 2002 / 0164328; US 2004 / 0093621; US ​​2004 / 0132140; US 2004 / 0110704; US 2004 / 0110282; US 2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; WO2005 / 053742; WO2002 / 031140; Okazaki, A. et al., J. Mol. Biol. 336 (2004) 1239-1249; Yamane-Ohnuki, N. et al., Biotech. Bioeng. 87 (2004) 614-622.Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells deficient in protein fucosylation (Ripka, J. et al., Arch. Biochem. Biophys. 249 (1986) 533-545; US 2003 / 0157108; and WO 2004 / 056312, particularly in Example 11) and knockout cell lines, such as CHO cells in which the α-1,6-fucosyltransferase gene FUT8 is knocked out (see, e.g., Yamane-Ohnuki, N. et al., Biotech. Bioeng. 87 (2004) 614-622; Kanda, Y. et al., Biotechnol. Bioeng. 94 (2006) 680-688; and WO 2003 / 085107).

[0312] In certain embodiments, provided method can be used to produce antibodies with bisected oligosaccharides, for example, biantennary oligosaccharides wherein attached to the antibody Fc region are bisected by GlcNAc. Such antibody variants can have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described in, for example, WO 2003 / 011878, US 6,602,684 and US 2005 / 0123546. Antibody variants having at least one galactose residue in the oligosaccharides attached to the Fc region can also be produced. Such antibody variants can have improved CDC function. Such variants are described in, for example, WO 1997 / 30087, WO 1998 / 58964 and WO 1999 / 22764.

[0313] Antibodies can be produced using, for example, the recombinant methods and compositions described in US 4,816,567. The nucleic acid can encode an amino acid sequence comprising the VL of the antibody and / or an amino acid sequence comprising the VH of the antibody (e.g., a light chain and / or heavy chain of the antibody). In another embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In another embodiment, a host cell comprising such nucleic acids is provided. In one such embodiment, the host cell comprises (or has been transformed with) the following: (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody; or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell or a lymphoid cell (e.g., Y0, NS0, Sp2 / 0). In one embodiment, a method of producing an antibody is provided, wherein the method comprises culturing a host cell containing a nucleic acid encoding the antibody as provided above under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0314] In order to recombinantly produce an antibody, the nucleic acid encoding the antibody is separated and inserted into one or more vectors for further cloning and / or expression in a host cell. This nucleic acid can be easily separated and sequenced using conventional methods (e.g., by using oligonucleotide probes that can specifically bind the heavy chain and light chain of the encoding antibody).

[0315] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, especially when glycosylation and Fc effector functions are not required. Expression of antibody fragments and polypeptides in bacteria is described, for example, in US 5,648,237, US 5,789,199, and US 5,840,523; see also Charlton, KA, In: Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2003), pp. 245-254, which describes expression of antibody fragments in E. coli. After expression, the antibody can be isolated from the bacterial paste in the soluble fraction and can be further purified.

[0316] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains whose glycosylation pathways have been "humanized" to produce antibodies with partially or fully human glycosylation patterns (see Gerngross, TU, Nat. Biotech. 22 (2004) 1409-1414; and Li, H. et al., Nat. Biotech. 24 (2006) 210-215).

[0317] Suitable host cells for expressing glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Many baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfecting Spodoptera frugiperda cells.

[0318] Plant cell cultures can also be used as hosts (see, for example, US 5,959,177, US 6,040,498, US 6,420,548, US 7,125,978, and US 6,417,429 (describing PLANTIBODIES for producing antibodies in transgenic plants)). TM technology)).

[0319] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to grow in suspension can be used. Other examples of useful mammalian host cell lines are monkey kidney CV1 cell line (COS-7) transformed by SV40; human embryonic kidney cell line (described in, for example, Graham, FL et al., 293 or 293 cells in J. Gen Virol. 36 (1977) 59-74); baby hamster kidney cells (BHK); mouse supporting cells (described in, for example, Mather, JP, TM4 cells in Biol. Reprod. 23 (1980) 243-252); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat liver cells (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor (MMT 060562); described in, for example, Mather, JP et al., Annals of TRI cells in NY Acad. Sci. 383 (1982) 44-68; MRC 5 cells; and FS4 cells. Other useful mammalian cell lines include Chinese hamster ovary (CHO) cells, including DHFR negative (DHFR - ) CHO cells (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA 77 (1980) 4216-4220); and myeloma cell lines, such as Y0, NS0, and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki, P. and Wu, AM, Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268.

[0320] II. Specific Aspects of the Invention

[0321] It has been found that, depending on the vector organization, the performance of expression vectors (1) varies according to the vector design and (2) differs between transient and stable transfections of the same vector.

[0322] It has been found that the optimal vector organization for transient and stable transfection can be significantly different. Without being bound by this theory, several factors may contribute to these differences: 1) the phenomenon of transcriptional interference between integrated vector copies in the host genome, which depends on and is specific to each vector design and does not exist in transient systems; 2) the influence of the selection method and selection stringency, which depends on each vector organization and plays a more important role in stable systems than in transient systems; and 3) the optimal LC to HC polypeptide ratio, which is significantly different for transient and stable expression of monoclonal antibodies and is lower in transient than in stable transfections.

[0323] It was found that for transient transfection, bidirectional expression of LC and HC achieved the highest product titers in all tissues tested with the vectors. Without being bound by theory, it is hypothesized that this vector design meets two criteria for optimal IgG expression: A) high expression levels of both LC and HC polypeptides and B) an optimal "transient" LC:HC polypeptide ratio, and C) bidirectional expression of LC and HC also minimizes transcriptional interference mechanisms, such as read-through by RNA polymerase.

[0324] However, it was found that bidirectional expression of LC and HC was inferior to the bidirectional organization LC-HC-SM for stable transfection. Without being bound by theory: 1) the convergent organization of the expression cassettes for LC, HC, and SM may reduce transcriptional interference between integrated vector copies; 2) placement of the LC upstream of the HC clearly facilitates an optimal (better) LC:HC polypeptide ratio for stable transfection; and 3) downstream placement of the selectable marker significantly increases the stringency of selection. Furthermore, the percentage of IgG-producing cells and the productivity of the cell line were found to be increased. For vectors containing bidirectional selectable markers upstream of the antibody expression cassette, although the stringency of selection was significantly increased, increasing the concentration of the selective pressure did not improve the productivity of stable pools or clones (data not shown).

[0325] It was found that simultaneous exchange of the hCMV promoter and SV40 poly signal with the hEF1α promoter and bGH polyA signal significantly increased transient product titers when the antibody expression cassette was organized unidirectionally, but decreased product titers when the LC and HC expression cassettes were placed bidirectionally.

[0326] The hEF1α promoter has been found to produce a large number of well-producing clones and very few non- or low-producing clones. However, the best single clone from the hEF1α promoter exhibited lower product titers in fed-batch assays than clones from the hCMV promoter. However, the total number of top-ranked clones from the hCMV promoter is relatively low, and their identification typically requires a high screening effort.

[0327] The use of hGT has been found to significantly improve productivity in both transient and stable transfections for vectors containing the hCMV promoter when combined with either the SV40 or bGH polyA signal. However, for vectors containing the hEf1α promoter, its effect on product titer was negligible when combined with the bGH polyA signal. Thus, the effect of hGT on vector performance has been found to be dependent on the promoter used.

[0328] The selection of appropriate clones for the final evaluation in the large-scale fermentation for full control is usually based on batch or fed-batch analysis in shake flasks. It has been found that there are differences between the performance and sorting of some expression vectors or elements in batch and fed-batch analysis. In batch analysis rather than in fed-batch analysis, for the vector containing the hCMV promoter, replacing SV40 polyA with bGH polyA signal has improved productivity. In batch analysis rather than in fed-batch analysis, hGT has no significant effect on the titer of the cloned product. The placement of the selective marker or the difference between the different vectors of the promoter (hEf1α or hCMV promoter) are moderately significant in batch analysis, but strongly manifest in fed-batch analysis. Expression level and specific production rate are higher in fed-batch mode than in batch mode.

[0329] Good correlation of performance in fed-batch analysis and 2 L fermentation was found for most clones, not only at the level of absolute product titer, but also in terms of ranking between different vectors and clones.

[0330] It has been found that placing the selectable marker downstream slightly reduces productivity loss compared to placing the selectable marker bidirectionally upstream of the antibody expression cassette. Without being bound by theory, this may be due to increased selection stringency, resulting in higher mRNA levels or improved LC:HC mRNA or polypeptide ratios. Both factors may lead to a higher tolerance to productivity variations.

[0331] Compared to the SV40 polyA signal, the bGH polyA signal significantly reduced the stability of antibody expression in clones. However, inserting hGT downstream of the bGH polyA signal significantly improved expression stability. The positive effect of hGT on stability was most pronounced in the absence of selective pressure.

[0332] Stability analysis of stable pools revealed that cells rapidly lost productivity when produced with hCMV, but not when produced with the hEf1α promoter. Surprisingly, for vectors containing the hEf1α promoter, hGT reduced the productivity of clones but slightly increased their stability.

[0333] Only a small number of clones significantly produce antibodies after the selection process. However, the modification of the vector organization and / or the modification of the element significantly improve the ratio of the clone that produces IgG to the clone that does not produce IgG. Different vector organizations and therefore the different expression levels of the selective markers that determine the selection stringency also significantly affect the percentage of cells that produce IgG. Statistical simulations based on the data of the screening process have shown that some expression vectors also have the potential to significantly reduce the workload in the screening process. This fact has a major impact on the cost of biopharmaceutical companies.

[0334] Improvement of the transcription process

[0335] Transcription initiation of promoter

[0336] Promoters determine the level of gene transcription and therefore have a strong influence on the productivity and stability of cell lines:

[0337] - Promoter-mediated transcription initiation determines the amount of mRNA that can be translated into recombinant protein;

[0338] - despite stable integration, the long-term productivity of clones can decrease rapidly due to inherited gene silencing (e.g., by promoter methylation);

[0339] - Promoter activity is cell type dependent.

[0340] Promoters that have been shown to be strong promoters in a variety of cell lines are described. It is known that introns can contain enhancer-like elements. Typically, the first intron (intron A) contains the majority of regulatory elements. This intron A is the first intron to occur within the full-length native promoter sequence / tissue.

[0341] PolyA signal-mediated mRNA polyadenylation

[0342] Polyadenylation of mRNA has multiple functions. It strongly affects nuclear export of mRNA, translation initiation, and mRNA stability. Therefore, the efficiency of the polyadenylation process has a strong impact on gene expression levels and therefore productivity.

[0343] Several publications have shown that polyadenylation signals can have a strong effect on protein expression. It has been reported that a polyA signal derived from bovine growth hormone (bGH polyA signal) can result in enhanced protein expression when replacing the SV40 polyA signal.

[0344] It has been discovered that the bovine growth hormone polyA signal may have improved properties in the recombinant production of antibody chains.

[0345] Transcription termination supported by transcription terminators

[0346] Elements contained in an expression vector can strongly interfere with each other (transcriptional interference). This is due to competition during transcription. For example, due to space limitations and reduced availability of resources for the transcription machinery, suboptimal promoters are brought into proximity with transcription factors and / or RNA polymerase. Interference can also occur between different, closely adjacent expression cassettes. For example, readthrough of RNA polymerase from a first expression unit through a second expression unit can occur due to inefficient transcription termination.

[0347] It is reported that ineffective transcription termination can lead to transcription interference, which can lead to ineffective expression of genes. The use of the human gastrin gene transcription terminator (hGT) can improve transcription termination, thereby facilitating the enhanced expression of recombinant proteins. This effect depends on the promoter combined with the hGT. Other effective transcription terminators are known.

[0348] It has been found that by inserting the sequence of the human gastrin transcription terminator after the early SV40 polyA signal of the antibody expression cassette, it is possible to enhance transcription termination and prevent transcriptional interference between, for example, the expression cassettes of the antibody light chain and the antibody heavy chain.

[0349] result

[0350] Reported herein are expression vectors for enhancing the expression of one or more encoding nucleic acids, eg, structural genes encoding antibody chains.

[0351] It has been discovered that when the genetic elements required for transcription are correctly selected and arranged, the productivity of recombinant cells expressing the structural gene is improved.

[0352] -polyA signal sequence and transcription terminator sequence

[0353] In vector p5068, the expression cassette is terminated by the SV40 polyA signal.

[0354] In vector px6001, the expression cassette also contains the human gastrin transcription terminator (hGT) placed downstream of the SV40 PolyA signal.

[0355] In vector px6007, the expression cassette is terminated by the bGH polyA signal and the hGT transcriptional terminator.

[0356] In vector px6008, the expression cassette is terminated by the bGH polyA signal without an additional transcription terminator.

[0357] These vectors were transiently transfected into CHO-K1 cells. Six days after transfection, the cell culture supernatants were collected and the amount of antibodies produced in the culture supernatants was measured by ELISA.

[0358] Compared to the control vector p5068, the replacement of the SV40 polyA signal with the bGH polyA signal and the addition / insertion of the human gastrin terminator (hGT) after the SV40 polyA signal resulted in an approximately 45% to 30% increase in transient expression productivity. The combination of the bGH polyA signal and the human gastrin terminator (hGT) resulted in the greatest increase in titer (+58%) compared to the control vector p5068.

[0359] carrier Amount of antibody in supernatant [μg / ml] carrier element p5068 4.6 SV40 polyA only px6001 6.1 SV40 polyA and hGT terminator px6007 7.4 bGH polyAl+hGT terminator px6008 6.8 bGH polyA only

[0360] Thus, it has been found that polyadenylation of the bGH polyA signal and improved transcription termination by addition of the human gastrin transcription terminator enhances antibody secretion in a transient expression system.

[0361] Vectors p5068, px6001, px6007, and px6008 were used to generate stable antibody-expressing cell lines.

[0362]

[0363] The average productivity of the top 15 monoclonal clones generated with vector p5068 in batch antibody production was 624 μg / ml. Compared to vector p5068, clones generated with vector px6001 (containing the human gastrin terminator (hGT)) or vector px6007 (containing a combination of the bGH polyA signal and hGT) had 23% and 40% higher productivity (770 μg / ml for vector px6001 and 872 μg / ml for vector px6007), respectively (see Figure 1 This increase in productivity was also reflected at the level of the top clones (+23% for vector px6001 (939 μg / ml) and +31% for vector px6007 (1001 μg / ml); values ​​were calculated for the three best clones obtained with each vector).

[0364] The clones generated with the vector px6008 had an average productivity of 576 μg / ml for the 15 best clones.

[0365] The average productivity of the three best clones of vector px6008 was 760 μg / ml.

[0366] -Promoter sequence

[0367] In vector px6051, the expression cassette contains the full-length human CMV promoter including intron A and the SV40 polyA signal.

[0368] In vector px6052, the expression cassette contains the human Ef1α promoter and the SV40 polyA signal.

[0369] In vector px6062, the expression cassette contains the full-length human CMV promoter including intron A, the bGH polyA signal and the human gastrin terminator.

[0370] In the vector px6063, the expression cassette contains the human Ef1α promoter, the bGH polyA signal and the human gastrin terminator.

[0371] carrier promoter polyA signal Transcription terminator px6051 Full-length hCMV containing intron A SV40 polyA signal No transcription terminator px6052 Ef1α promoter containing intron A SV40 polyA signal No transcription terminator px6062 Full-length hCMV containing intron A bGH polyA signal Human gastrin transcription terminator (hGT) px6063 Ef1α promoter containing intron A bGH polyA signal Human gastrin transcription terminator (hGT)

[0372] The best 18 stable clones obtained for each vector were identified and tested for productivity in batch analysis.

[0373] The combined exchange of the bGH polyA signal and the hGT polyA signal for the SV40 polyA signal enhanced the average productivity by approximately 19% in the case of the hEF1α promoter (505 μg / ml for vector px6063 vs. 426 μg / ml for vector 6052) and by approximately 71% in the case of the full-length hCMV promoter (333 μg / ml for vector px6062 vs. 195 μg / ml for vector px6051).

[0374] An increase in productivity was also observed at the level of the top clones (+36% for vector px6063 (693 μg / ml) vs. vector px6052 (511 μg / ml) and +102% for vector px6062 (529 μg / ml) vs. vector px6051 (262 μg / ml); calculated for the three best clones for each vector) (see Figure 2 ).

[0375] Thus, it has been found that the combination of the hEF1α promoter with the bGH polyA signal and hGT enhances productivity in stable transfections using a bidirectional vector organization (see px6052) with a selectable marker expression cassette in one transcriptional orientation and light chain expression cassettes upstream of the heavy chain expression cassette, each in the other transcriptional orientation.

[0376] Furthermore, it has been found that the combination of the bGH polyA signal and hGT enhances productivity in stable transfections using either a hCMV promoter with a unidirectional vector organization (5'->3' orientation of the LC-HC-SM expression cassette) in which the light chain expression cassette is upstream of the heavy chain expression cassette, which in turn is positioned upstream of the selectable marker expression cassette (see px9005), or a bidirectional vector organization (see px6007) in which the selectable marker expression cassette is in one transcriptional orientation and the light chain expression cassettes upstream of the heavy chain expression cassette are each in the other transcriptional orientation.

[0377] The combination of bGH polyA signal and hGT was also used in transient transfection methods. Vectors px6062 and px6063 (containing the full-length hCMV promoter or hEF1α promoter with a combination of bGH polyA signal and hGT) were directly compared with vectors px6051 and px6052 (containing the full-length hCMV promoter or hEF1α promoter with an SV40 polyA signal).

[0378] Unlike stable transfection, the combination of the bGH polyA signal and the human gastrin terminator (hGT) did not increase the productivity of transient transfection (2.38 μg / ml for vector px6052 vs. 2.32 μg / ml for vector px6063) in the case of the full-length human CMV promoter containing intron A and the human EF1α promoter containing intron A. In the case of the full-length human CMV promoter containing intron A, the combination of the bGH polyA signal and the human gastrin terminator (hGT) resulted in a productivity of 2.74 μg / ml for vector px6062A and 3.64 μg / ml for vector px6051.

[0379] In cells obtained by transient transfection, it has been found that the combination of a short hCMV promoter with the bovine growth hormone polyA signal sequence and the human gastrin terminator results in improved antibody expression yields.

[0380] In cell lines obtained by transfection and selection of stable cell clones, it was found that the combination of the bovine growth hormone polyA signal sequence and the human gastrin terminator resulted in improved antibody expression yields independently of the promoter used.

[0381] In vector p5068, expression of genes encoding both the light chain (LC) and heavy chain (HC) of the antibody is driven by the short human CMV promoter (hCMV). This promoter is active in a wide range of cell types and is commonly used in mammalian expression plasmids. Since promoter activity is strongly dependent on cell type and the hCMV promoter is known to be sensitive to gene silencing, promoter methylation may be stronger and / or there may be promoters that are more resistant in CHO-K1 cells.

[0382] Expression vectors containing different promoters were constructed: i) a short hCMV promoter without intron A; ii) a full-length hCMV promoter containing intron A; iii) a full-length rat CMV promoter containing intron A; and iv) a hEF1α promoter containing intron A.

[0383] Vectors containing the full-length hCMV promoter (px6051), the hEF1α promoter (px6052), and the rat CMV promoter (px6053) were constructed and used for transient and stable transfections.

[0384]

[0385]

[0386] Expression vectors p5068, px6051, px6052, and px6053 were transiently transfected into CHO-K1 cells by nucleofection. Four days after transfection, cell culture supernatants were collected and productivity was determined by ELISA.

[0387] Compared to the expression vector p5068, the vectors containing the hEF1α promoter containing intron A (px6052) or the full-length hCMV promoter containing intron A (px6051) had productivity increased by 53% and 134%, respectively (3.64 μg / ml for vector px6051, 2.38 μg / ml for vector px6052, and 1.56 μg / ml for vector p5068). However, the vector p6053 containing the rat CMV promoter showed a decrease in productivity by approximately 50% (see Figure 3 ).

[0388] Stable transfections were also performed with vectors containing the full-length human CMV promoter (vectors px6051 and px6062) or the hEF1α promoter containing intron A (vectors px6052 and px6063).

[0389] Expression vectors p5068, px6051, px6052, and px6053 were transfected into CHO-K1 cells by nucleofection and stable pools were selected. The productivity of the pools was analyzed in batch assays.

[0390] Batch analysis of stable pools showed that the antibody titer from cells transfected with a vector containing the hEF1α promoter containing intron A (vector px6052) was more than 4-fold higher than that from cells transfected with a vector containing a short hCMV promoter (vector p5068) or a vector containing the full-length hCMV promoter containing intron A (vector px6051) (78 μg / ml for vector px6052, 14 μg / ml for vector p5068, 17 μg / ml for vector px6051, and 8.46 μg / ml for vector px6053) ( Figure 4 ).

[0391] Stable clones were generated using expression vectors p5068, px6051 and px6052. In addition, vectors px6062 and px6063 (both containing a combination of the bGH polyA signal and the human gastrin terminator in place of the SV40 polyA signal) were used.

[0392]

[0393] Batch analysis of the best 54 clones obtained for each vector showed an average productivity of 316 μg / ml from cells transfected with vector px6052 and 341 μg / ml for vector p5068. Clones generated with vectors px6051 and px6062 containing the full-length hCMV promoter had an average productivity of 141 μg / ml for vector px6051 and 220 μg / ml for vector px6062.

[0394] The average productivity of the 54 clones tested, transfected with the vector px6063 (containing the hEF1α promoter combined with the bGH polyA signal and hGT), in batch analysis was 367 μg / ml. The best clones with the highest titers were derived from the vector px6063 (748 and 731 μg / ml, respectively).

[0395] It was found that clones generated using vectors containing the human EF1α promoter (px6052 and px6063) showed a reduced number of low-producing clones.

[0396] The clones were tested for stability of antibody production. Twelve clones obtained with vectors p5068 and px6052 were cultured for three generations in the presence and absence of hygromycin B.

[0397] In the absence of selection pressure (hygromycin B) for three generations, a 35% reduction in the average productivity of the 12 clones tested was observed for the short hCMV promoter lacking intron A (p5068; 264 μg / ml vs. 410 μg / ml). In contrast, only an 18% reduction in average productivity was observed for the hEF1α promoter containing intron A (px6052; 256 μg / ml vs. 312 μg / ml).

[0398] In the presence of selection pressure, clones obtained with vector p5068 showed an average productivity reduction of 27% (298 μg / ml vs. 410 μg / ml), and the titers from clones obtained with vector px6052 containing the hEF1α promoter containing intron A were reduced by 15% (266 μg / ml vs. 312 μg / ml).

[0399] The number of stable clones transfected with the vector p5068 containing a short hCMV promoter lacking intron A or with the vector px6052 containing the hEF1α promoter containing intron A after three generations with or without selection pressure was determined. To determine whether a clone was "stable" after three generations, the IgG titer from passage 0 was set to 100% and a threshold of 80% was defined. Clones that showed a relative IgG titer of 80% of the IgG titer at passage 0 (passage 0) after three generations with or without selection pressure were defined as unstable.

[0400] After three passages, the relative antibody titers of all clones transfected with p5068 decreased to less than 80% compared to the antibody titers at passage 0, with or without selection pressure. Stable cell clones are clonal cell populations that produce antibody titers of 80% or more compared to the antibody titers at passage 0, both in the presence and absence of selection pressure.

[0401] After three passages, relative titers of clones transfected with px6052 fell below 80% in 8 of 12 cases compared to IgG titers at passage 0, with or without selection pressure. However, 4 clones were defined as "stable." Unlike clones generated with the p5068 vector, 8 of the 12 clones generated with the px6052 vector were "stable" in the presence of selection pressure. In contrast, only one of the clones derived from the p5068 vector was stable in the presence of selection pressure.

[0402] Vectors px6014, px6014A, and px6014B contain the hEF1α promoter in front of the antibody light chain and a short hCMV promoter in front of the heavy chain. These vectors differ in the 5′UTR of the light chain (px6014: 5′UTR of the hEF1α promoter containing a PmeI restriction site; px6014A: 5′UTR of the hEF1α promoter without a PmeI restriction site; px6014B: 5′UTR of the hEF1α promoter plus the 5′UTR of the vector p5068). The vectors were transiently transfected into CHO-K1 cells using nucleofection.

[0403] Compared to vector px6014, vectors px6014A and px6014B showed 20% and 40% enhancement in productivity, respectively (2.01 μg / ml for vector px6014B, 1.71 μg / ml for vector px6014A, 1.41 μg / ml for vector px6014).

[0404] It was found that vectors comprising the human EF1α promoter or the full-length human CMV promoter (both containing intron A) showed improved productivity compared to vector p5068 in transient transfections.

[0405] It was found that stable pools generated with a vector comprising the human EF1α promoter showed increased productivity in batch analysis compared to vector p5068.

[0406] It has been found that cell clones obtained by stable transfection with a vector comprising the human EF1α promoter containing intron A show a reduced number of low-producing clones.

[0407] It was found that cell clones obtained by stable transfection with a vector comprising the full-length human CMV promoter including intron A showed a strongly reduced productivity both at the level of the mean and the top ranked clones.

[0408] Thus, it has been found that using a bidirectional vector organization with a selectable marker expression cassette in one transcriptional direction and a light chain expression cassette upstream of the heavy chain expression cassette each in the other transcriptional direction (see px6052), the combination of the bGH polyA signal and hGT compared to the SV40 polyA enhances productivity in stable transfections independently of the promoter used.

[0409] Furthermore, it has been found that the combination of the bGH polyA signal and hGT enhances productivity in stable transfections using the hCMV promoter in a unidirectional vector organization (5'->3' orientation of the LC-HC-SM expression cassette) in which the light chain expression cassette is upstream of the heavy chain expression cassette, which in turn is positioned upstream of the selectable marker expression cassette.

[0410] Improvements in selection methods

[0411] Cell line development is currently very time-consuming and costly due to the high number of non- and low-producing clones and the low stability of gene expression. Among thousands of clones, you usually only find a few stable "high producers".

[0412] Without being bound by theory, a possible reason for the low selectivity and low stringency of the selection strategy could be the separate expression of the antibody and the selection marker in the vector system used, which does not exert selection pressure for antibody expression (see e.g. vector p5069).

[0413] It has been found that this problem can be overcome by using IRES elements.

[0414] IRES elements are DNA elements that function as internal ribosome entry sites (at the mRNA level), thus allowing the expression of two genes from one mRNA.

[0415] IRES-linked expression of the selection marker and antibody chain exerts selection pressure on total antibody expression (ie, one mRNA encodes both the selection marker and the antibody chain), thereby ensuring the selectivity of the selection. The stability of gene expression can be improved.

[0416] The use of an IRES element with weak IRES activity allows for weak expression of a selection marker. This weak expression of a selection marker can increase the stringency of selection.

[0417] By co-expressing the selectable marker and the antibody chain via IRES linkage, the selection method can be improved by:

[0418] - Increase the number of producing cells,

[0419] - Enhance the stability of gene expression, and

[0420] - Enhance the rigor of selection methods.

[0421] For IRES-linked co-expression of antibodies and selectable markers, the IRES element used must meet two requirements:

[0422] - the IRES element may have no or only minimal impact on mRNA stability / antibody expression, and

[0423] The -IRES element must show weak IRES activity so that the selection marker is only slightly expressed.

[0424] The light and heavy chain encoding nucleic acids were linked by IRES elements: EV71-IRES, ELF4G-IRES, and EMCV-IRES (vectors px6015A, px6015B, and px6015C). The expression cassette contained, in a 5' to 3' orientation, a human CMV promoter, light chain encoding nucleic acid, IRES, heavy chain encoding nucleic acid, and a polyA site.

[0425] The reference vector p5068, which does not contain an IRES element, and the vectors px6015A, px6015B, and px6015C were transiently transfected into CHO-K1 cells, and the productivity was determined by ELISA.

[0426] Vectors px6015B and px6015C (containing ELF4G and EMCV-IRES, respectively) showed IgG expression of 0.1 μg / ml to 0.15 μg / ml. Vector p5068 showed IgG expression of 2 μg / ml. Vector px6015A containing EV71-IRES showed a productivity of 1.7 μg / ml.

[0427] Alternatively, the selectable marker neomycin can be directly linked to the heavy chain of the antibody via an IRES element. The vector comprises, in 5' to 3' direction, the elements human CMV promoter, light chain encoding nucleic acid, polyA site, human CMV promoter, heavy chain encoding nucleic acid, IRES element, neomycin selectable marker nucleic acid, and polyA site.

[0428] Vectors px6010A, px6010B and px6010C were transiently transfected into CHO-K1 cells as described and productivity was determined by ELISA.

[0429] The EMCV-IRES-containing vector px6010C showed a productivity of 13 μg / ml. The p5069 vector showed a productivity of 14 μg / ml. The vectors containing EV71-IRES (vector px6010A) or ELF4G-IRES (vector px6010B) showed productivity of 4.3 μg / ml and 2.4 μg / ml, respectively.

[0430] Thus, it has been found that the EMCV-IRES element meets the necessary requirements for IRES-linked expression of a selectable marker:

[0431] - weak IRES activity (resulting in only slight expression of the selection marker),

[0432] - The IRES element has at most a minimal influence on antibody expression (expression of the respective IRES-linked selection marker).

[0433] Thus, the selectable marker neomycin (mediating resistance to G418) can be linked to the heavy chain encoding nucleic acid via the EMCV-IRES.

[0434] Vectors p5069 and px6010C were tested for productivity and stability in stable transfections, both at the pool level and at the single clone level.

[0435] The vector was transfected into CHO-K1 cells by nucleofection. A stable pool was selected and its productivity was analyzed in a batch assay.

[0436] The pool generated with vector px6010C showed a productivity of 14 μg / ml in batch analysis. The pool generated with vector p5069 showed a productivity of 5.4 μg / ml in batch analysis ( Figure 5 ).

[0437] It was found that libraries generated with vector px6010C consist of more producer cells (or more good producer cells) and / or have an enhanced stability of IgG expression (compared to vector p5069).

[0438] It has been found that IRES-linked expression of the selection marker neomycin in the vector px6010C also leads to enhanced library stability. Stable libraries were cultured for 30 generations in the presence and absence of selective pressure. The productivity of the library was determined in batch analysis at the beginning (= generation 0) and end (= generation 30) of the stability test.

[0439] Compared to libraries produced with vector p5069, libraries produced with vector px6010C showed enhanced IgG expression stability in batch analysis. After 30 generations, the productivity of libraries obtained with vector p5069 was strongly reduced in batch analysis (>80%; values ​​below the detection limit) in the presence and absence of selective pressure. In the absence of selective pressure, the productivity of libraries produced with vector px6010C also decreased by approximately 70%. However, in the presence of selective pressure, the productivity of libraries produced with vector px6010C decreased by only 10% ( Figure 6 ).

[0440] Vector p5069 and vector px6010C were transfected into CHO-K1 cells by nucleofection, and stable clones were generated as described above. Clones were screened, and the productivity of the top 15 clones from each transfection was analyzed in batch analysis. Two independent transfections were performed for each vector.

[0441] The clones generated with the vector px6010C showed an average productivity of 348 μg / ml. The clones generated with the vector p5069 showed an average productivity of 239 μg / ml. The increase in average productivity of the clones generated with the vector px6010C was not due to better ranked clones, but rather to a significant reduction in the number of low-producing clones. Unlike the vector p5069, no clones generated with the vector px6010C showed a productivity below 200 μg / ml in batch analysis ( Figure 7 ).

[0442] By cultivating 45 generations in the presence and absence of selective pressure, the stability of antibody expression was tested for 17 clones (p5069) and 14 clones (px6010C), respectively. At the beginning of the stability test (generation 0) and at generation 45, the productivity of the clones was measured in batch analysis. The IgG titer at generation 45 was compared with the productivity of the clones at the beginning of the stability test.

[0443] The average productivity of 17 clones generated using vector p5069 decreased after 45 generations (approximately 43% loss of productivity) in both the presence and absence of selective pressure. In the absence of selective pressure, the average productivity of 14 clones generated using vector px6010C decreased by approximately 45%. However, in the presence of selective pressure, the productivity of these clones decreased by 4%.

[0444] In the presence of selection pressure, only 6 of the 17 clones tested produced by the vector p5069 showed a productivity still above 80% of the productivity of generation 0. In contrast, 10 of the 14 clones tested produced with the vector px6010C showed a productivity above 80% of the productivity of generation 0.

[0445] In the absence of selective pressure, there was no significant difference in stability between clones generated using vector p5069 and vector px6010C, respectively.

[0446] Improvements in selection and screening methods

[0447] Cell line development is currently very time-consuming and costly due to intensive screening efforts where among thousands of clones you typically only find a few stable high producers.

[0448] Currently, several screening strategies exist for identifying high-producing clones:

[0449] - ELISA-based screening strategies for direct detection of produced proteins (time and cost consuming);

[0450] - Fluorescence-based screening strategies for direct detection of produced proteins (time and cost consuming);

[0451] - FACS-based sorting of cells co-expressing a quantitative screening marker (surface protein or fluorescent marker, such as GFP) for indirect detection / quantification of produced proteins.

[0452] In most cases, the expression of the fluorescent marker is not linked to the expression of the antibody. This limits the dependence of fluorescence intensity and productivity, resulting in poor correlation between fluorescence intensity and productivity.

[0453] GFP protein (as an example of a fluorescent marker) is stable and tends to accumulate. There is (in most cases) no good correlation between the expression level (fluorescence intensity) of fluorescent markers in cells and their productivity.

[0454] It has been discovered that by linking antibody expression and fluorescent marker expression, a combinatorial selection and screening strategy to identify cell clones has enhanced stability and productivity while being simple, thus allowing for rapid and easy identification of high producers.

[0455] IRES-linked expression of fusion proteins has been found to function as both a selectable marker and a quantitative screening marker.

[0456] A fusion protein of green fluorescent protein (GFP) and the selectable marker neomycin directly linked to the antibody heavy chain via an IRES element was constructed. The green fluorescent protein and the selectable marker neomycin in this fusion protein are separated by a PEST sequence (a sequence corresponding to codons 423-449 of the mouse ornithine decarboxylase gene (mODC)). This PEST sequence acts as a strong proteolytic signal sequence at the protein level, thereby significantly reducing the half-life of the protein.

[0457] The GFP-PEST-Neo fusion protein serves not only as a selection marker but also as a quantitative screening marker.

[0458] The use of PEST sequences provides improved clonal selection due to the PEST-mediated reduction in the half-life of the fusion protein (increased selection stringency).

[0459] The use of PEST sequences provides a good correlation between the GFP fluorescence intensity of the fusion protein and the antibody expression level due to IRES-linked co-expression of the antibody and the selectable marker.

[0460] The use of PEST sequences reduces the accumulation of fusion proteins due to the reduced half-life of the protein.

[0461] GFP expression levels of stable single clones were analyzed by FACS and correlated with productivity in batch analysis. Populations with different GFP expression levels from the stable pool were sorted by FACS and the productivity of the sorted populations was analyzed.

[0462] IRES elements that meet the requirements of this method:

[0463] - should provide weak IRES activity so that the fusion protein is only slightly expressed;

[0464] - Should not affect antibody expression levels.

[0465] IRES activity or strength was determined by expressing light and heavy chains linked to the EV71-IRES, ELF4G-IRES, and EMCV-IRES elements (see vectors px6015A, px6015B, and px6015C). The reference vector p5068 (without an IRES element) and vectors px6015A, px6015B, and px6015C were transiently transfected into CHO-K1 cells, and productivity was measured by ELISA.

[0466] Vectors px6015B and px6015C (containing ELF4G and EMCV-IRES, respectively) showed IgG expression of 0.1 to 0.15 μg / ml. Vector p5068 showed IgG expression of 2 μg / ml. Vector px6015A containing EV71-IRES showed a productivity of 1.7 μg / ml, indicating that the EV71-IRES element has strong IRES activity in CHO-K1 cells.

[0467] The GFP-PEST-Neo fusion protein was directly linked to the heavy chain encoding nucleic acid of the antibody via an IRES element (see Figure 8 ).

[0468] Constructs in which an IRES element linked the selection marker to the antibody heavy chain contained in vectors px6011A, px6011B and px6011C were transiently transfected into CHO-K1 cells as described, and productivity was determined by ELISA.

[0469] The EMCV-IRES-containing vector px6011C showed 8.7 μg / ml IgG productivity, while the p5059 vector showed 10.8 μg / ml IgG productivity. The EV71-IRES-containing vector px6011A showed 3.9 μg / ml IgG productivity. The ELF4G-IRES-containing vector px6011B showed no relevant productivity.

[0470] Vector px6011C, vector p5069, and vector px6010C containing an IRES-linked GFP-PEST-Neo fusion protein were tested in batch analysis of stable pools and stable clones.

[0471] Vectors p5069, px6010C, and px6011C were transfected into CHO-K1 cells by nucleofection. Stable pools were selected as described and analyzed for productivity in batch assays.

[0472] The stable pool generated with the vector px6010C containing an IRES-linked selection marker showed an IgG productivity of 9.5 μg / ml. The stable pool generated with the vector p5069 showed an IgG productivity of 5.4 μg / ml. The pool generated with the vector px6011C containing an IRES-linked fusion protein showed a productivity of 36.3 μg / ml ( Figure 9 ).

[0473] These data indicate that the pools produced with vectors px6010C and px6011C consisted of significantly more or even better producing cells than the pools produced with vector p5069. The enhanced IgG expression stability in / with the px6010C and px6011C pools may also contribute to the increased productivity.

[0474] To compare the productivity of the expression vector p5068 with that of the vectors px6010C and px6011C in stable clones, the vectors were transfected into CHO-K1 cells by nucleofection. Stable clones were selected and clones were screened as described. The productivity of the top 15 clones for each vector was analyzed in batch analysis.

[0475] For vector p5068, 95 of a total of 3072 wells in a 384-well plate showed IgG production above 2 μg / ml (background). IRES-linked expression of the selectable marker (px6010C) doubled the number of producing cells / well to 195, and in the case of an IRES-linked fusion protein (px6011C), the number of producing cells / well was further increased to over 280.

[0476] Furthermore, not only the number of producing cells, but also the average productivity of IRES-containing clones generated by vectors px6010C and px6011C was higher (for the best 100 clones for each vector, 3.4 μg / ml for vector p5069, 4.2 μg / ml for vector px6010C, and 8.1 μg / ml for vector px6011C; for the best 250 clones for each vector, 2.2 mg / ml for vector p5069, 3.0 μg / ml for vector px6010C, and 5.1 μg / ml for vector px6011C).

[0477] The clones produced with the IRES vectors px6010C and px6011C and p5059 showed an average productivity of 212 μg / ml for the vector px6011C, 178 μg / ml for the vector px6010C and 118 μg / ml for the vector p5069 in a 24-well screening (measured in 24-well cultures with undetermined cell counts 4 days after cell splitting). A reduced number of non-producing or low-producing clones and an increased number of good producing clones were observed for the vectors px6010C and px6011C ( Figure 10 ).

[0478] Batch analysis of single clones showed that the average productivity of the 15 best clones generated with the px6010C vector was 348 μg / ml, while that with the p5069 vector was 239 μg / ml. The average productivity of the 15 best clones generated with the px6010C vector was 404 μg / ml. The clone with the highest overall titer was derived from a transfectant with the px6011C vector. No clone generated with either the px6010C or px6011C vector exhibited a productivity below 200 μg / ml in batch analysis.

[0479] To test the antibody expression stability of the clones, 14 to 19 clones obtained with each vector were cultured for 45 generations in the presence and absence of selection pressure (G418). The productivity of the clones was measured in batch analysis at the beginning of the stability test (generation 0) and at generation 45. The IgG titer at generation 45 was compared with the productivity of the clone at generation 0 at the beginning of the stability test (value set to 100%).

[0480] After 45 generations, the average productivity of 17 clones generated using vector p5069 decreased in both the presence and absence of selective pressure (about 43% loss of productivity). In the absence of selective pressure, the average productivity of 14 to 19 clones generated using vectors px6010C and px6011C, respectively, also decreased (about 45-35% loss of productivity), but in the presence of selection marker G418, the average productivity of these clones decreased by only 0-4% after 45 generations.

[0481] In the presence of the selection pressure G418, 6 of the 17 clones tested produced by the vector p5069 showed a productivity greater than 80% of the productivity of generation 0. In contrast, 10 of the 14 clones tested produced with the vector px6010C and 17 of the 19 clones tested produced with the vector px6011C showed a productivity greater than 80% of the productivity of generation 0.

[0482] In the absence of selection pressure, clones generated with vectors p5069, px6010C and px6011C showed comparable behavior.

[0483] It has been found that vectors comprising an IRES, and hence clones obtained therefrom, have enhanced stability in the presence of a selectable marker.

[0484] However, in the absence of a selectable marker, there was no difference in stability between clones obtained with vectors containing or without an IRES.

[0485] The GFP-PEST-Neo fusion protein was used as a quantitative selection marker. The GFP fluorescence level of clones generated with the vector px6011C was predictive of their productivity.

[0486] The GFP expression level / fluorescence intensity of individual clones was determined and the results were correlated with the productivity of the clones in batch analysis. Populations with different GFP expression levels / fluorescence intensities were sorted from the stable pool by FACS (1,000 cells per vector) and the productivity of these different populations was analyzed in batch analysis. Three different populations were sorted:

[0487] Group 1: No GFP expression, geometric mean (GM) of FL1-H (=GFP) 0-4

[0488] Population 2: low GFP expression level, GM 4-5.5

[0489] Group 3: high GFP expression, GM 5.5-7

[0490] Batch and FACS analysis showed that there was a good correlation between the GFP fluorescence of clones / pools and their productivity at both the single clone and pool levels. Clones or pools with high GFP fluorescence generally showed higher productivity than cells showing low GFP fluorescence. In general, the productivity of clones / pools increased with increasing fluorescence intensity ( Figure 11 and 12 ).

[0491] High-producing clones can be directly identified by FACS-based sorting of single clones with high GFP fluorescence from stable pools. Single clones showing no or high GFP expression are sorted by FACS. Clones are expanded into shaken 6-well plates and the productivity of the clones is determined in batch analysis.

[0492] It has been found that EMCV-IRES linked expression of a selection marker or GFP-PEST-Neo fusion protein significantly enhances the selectivity of selection (producing more producing clones) and it also enhances the stringency of selection (higher average productivity of producing clones). Thus, the screening effort is significantly reduced.

[0493] The stronger effect of the IRES-linked fusion protein (px6011C) compared to the IRES-linked selection marker (px6010C) may be due to the PEST sequence in the fusion protein, which mediates a reduced half-life of the fusion protein and / or a lower affinity of the fusion protein for the selection agent - both factors that significantly increase the stringency of selection.

[0494] Vector elements combined with carrier tissue

[0495] The following vectors were tested in the CHO-K1 host cell line in transient transfections, in stable pools and some at the monoclonal level.

[0496] carrier organize promoter polyA signal Transcription terminator px9001 SM(3′-5`)-LC-HC hCMV SV40 polyA does not exist px9002 LC-HC-SM hCMV SV40 polyA does not exist px9003 LC-HC-SM hEF1α SV40 polyA does not exist px9004 LC-HC-SM hCMV bGH polyA does not exist px9005 LC-HC-SM hCMV bGH polyA hGT px9006 LC-HC-SM hEF1α bGH polyA does not exist px9007 LC-HC-SM hEF1α bGH polyA hGT px9010 LC(3′-5′)-HC-SM hEF1α bGH polyA does not exist px9011 LC(3′-5′)-HC-SM hCMV SV40 polyA hGT

[0497] The performance of different vectors in transient transfection was tested after nucleofection into CHO-K1 cells.

[0498] Compared to using the hCMV promoter, vectors containing the human elongation factor 1α promoter (hEF1α) (based on the vector organization LC-HC-SM) had approximately +34% (px9003 vs. px9002; SV40 polyA signal sequence) and +30% (px9006 vs. px9004; bGH polyA signal sequence) increased productivity, respectively, depending on the polyA signal sequence used.

[0499] Addition of the human gastrin terminator (hGT) to the bGH polyA signal sequence had a positive effect on the productivity of the hCMV-containing promoter (px9005 vs. px9004; +13%).

[0500] Expression vectors based on bidirectional expression of the antibody's light and heavy chains showed improved performance. Depending on the promoter used (hEF1α or hCMV) and the polyA signal sequence used (SV40 or bGH polyA signal sequence), product titers were increased by approximately 2.7 to 3.4 times compared to the control vector px9001.

[0501] It was found that the use of the human elongation factor 1α promoter and the bGH polyA signal sequence had a positive effect on productivity in the carrier tissue LC-HC-SM (+50%; compare px9006 and px9002), but not in the carrier tissue LC(3'-5')-HC-SM (-28%; compare px9010 and px9011).

[0502] To compare the productivity of expression vector px9002 and vector px9003-9007, the vectors were transfected into CHO-K1 cells by nucleofection and stable pools were selected. The productivity of the pools was analyzed in batch analysis (see Figure 10 ).

[0503] Batch analysis of stable pools showed that antibody titers from pools transfected with vectors containing the human elongation factor 1α promoter (px9003, px9006, px9007) were approximately 7-8 times higher than antibody titers from cells transfected with a reference vector containing the short hCMV promoter (vector px9002) (97.5 μg / ml, 112.5 μg / ml and 95.6 μg / ml for vectors px9003, px9006 and px9007 compared to 14.0 μg / ml for vector px9002).

[0504] Vectors px9001, px9002, and px9004 to px9007 were transfected into CHO-K1 cells by nucleofection, and the best single clones were identified by classical screening. The productivity of the best 36 clones for each vector was analyzed in a batch assay, and the best 15 clones from the batch assay were tested in a fed-batch assay.

[0505] The average productivity of the top 36 individual clones generated with vector px9001 in batch analysis was 356 μg / ml. Compared to the control vector px9001, clones generated with vector px9002 or with vectors px9004 and 9005 (which also contained the bGH polyA signal (alone or in combination with HGT) in place of the SV40 polyA signal) showed productivity increases of 37% (px9002), 61% (px9004), and 53% (px9005), respectively. Clones generated with vectors px9006 and px9007 showed productivity increases of approximately 19% and 7%, respectively.

[0506] In fed-batch experiments, the best 15 clones obtained with each vector in batch analysis were tested in fed-batch analysis over 14 days.

[0507] The average productivity of the top 15 individual clones (generated with vector px9001) in a fed-batch assay was 1345 μg / ml. Compared to the control vector px9001, clones generated with vector px9002 or with vectors px9004 and px9005 (which also contained the bGH PolyA signal sequence (alone or in combination with hGT) in place of the SV40 PolyA signal sequence) showed increases in productivity of 80% (px9002), 58% (px9004), and 92% (px9005), respectively.

[0508] In addition to the average productivity increase (see above), the performance of the top clones was also strongly improved. In terms of productivity of the top 5 clones, vectors px9002, px9004 and px9005 showed an increase of about 64% (px9002), 50% (px9004) and 88% (px9005) compared to the control vector px9001.

[0509] Below, the percentage of producing or non-producing cells for each of the different vectors is directly compared. 14.2% of clones generated with the vector px9001 produced antibody, while in the remaining resistant clones, antibody expression was silent or the clones had other defects.

[0510] Vector organization with vector px9002 nearly doubled the percentage of producing cells (to 26.0%). Vectors that also contained the bGH PolyA signal sequence in place of the SV40 polyA signal sequence, either alone (vector px9004) or in combination with hGT (vector px9005), showed an approximately three-fold increase in the percentage of producing cells (39% and 43%, respectively).

[0511] Replacing the hCMV promoter with the human elongation factor 1 alpha promoter increased the number of producing cells up to 5-fold (more than 70% of the clones obtained after the selection procedure actually produced antibody).

[0512] The 15 best clones (based on fed-batch results) obtained by transfection with vectors px9001-9007 were cultured for 15 generations (= approximately 60 generations) in the presence and absence of hygromycin B. The product titers of the clones after 15 generations in batch analysis were compared with the product titers of the clones in batch analysis at the beginning of the stability test.

[0513] In the presence of selection pressure, after 15 generations, the variation in product titer among the 15 clones for each vector ranged from -14.7% for vector px9007 to +0.2% for vector px9002.

[0514] In the absence of selective pressure, the reduction in product titer ranged from 25.5% for vector px9004 to 5.9% for vector px9005.

[0515] The number of clones meeting the defined stability criteria (e.g., product titer >80% in batch analysis compared to the starting point (GO) value in the presence and absence of the selection marker) varied from 4 to 10. Also in the presence and absence of selection pressure / selection marker, vectors px9005, px9007, and px9002 produced the highest number of stable clones (px9005: 10; px9007: 7; px9002: 6).

[0516] Thus, it has been found that, in particular in the absence of selection pressure, organization of the vector px9005 shows a positive effect on stability and increases the number of stable clones.

[0517] It has been found that the combination of bGH polyA and hGT significantly increases the productivity of stable clones compared to SV40 polyA without the transcription terminator (hGT), independently of the promoter used.

[0518] transient transfection

[0519] - Use of the human elongation factor 1α promoter (containing intron A) provides enhanced productivity (in LC-HC-SM tissue)

[0520] - Use of the bovine growth hormone polyA signal sequence provides enhanced productivity compared to use of the SV40 polyA signal sequence

[0521] - HGT addition to the bGH PolyA signal sequence results in increased productivity in vectors containing the hCMV promoter

[0522] - Vector organization LC(3`-5′)-HC-SM leads to improved expression

[0523] Stable library

[0524] - Libraries generated with vectors containing the hEF1α promoter showed enhanced productivity in batch analysis

[0525] - Clones generated using vectors containing the hEF1α promoter showed a reduced number of low-producing clones

[0526] - Clones generated using vectors containing the hEF1α promoter showed greater IgG expression stability

[0527] Monoclonal

[0528] - Vector organization with downstream selection marker (LC-HC-SM) has a positive effect on the productivity of stable monoclonal clones

[0529] - Clones generated using vectors containing the bGH polyA signal sequence and hGT have higher productivity and stability Summary

[0530] Several different transcription-related genetic elements and combinations thereof have been compared with reference genetic element combinations. Based on comparative transient experiments, the following results (see table below) were obtained for a bidirectional vector organization in which the selectable marker expression cassette was in the opposite direction to the expression cassettes of the light and heavy chains (the light chain expression cassette was upstream of the heavy chain expression cassette).

[0531]

[0532] Different promoters were combined with the bGH polyA signal and the hGT transcription terminator (see table below).

[0533]

[0534] Several transcription-related genetic elements and their combinations were compared to a reference vector (px9001, vector organization SM (3'-5' orientation)-LC-HC (5'-3' orientation). Based on comparative experiments, the following results were obtained for unidirectional vector organization, in which the expression cassettes for the light and heavy chains (light chain expression cassette upstream of the heavy chain expression cassette) and the selectable marker were in the same orientation, compared to the reference vector (px9001, bidirectional vector organization SM (3'-5')-LC-HC (5'-3')) (see the table below).

[0535]

[0536] Promoter used

[0537] carrier promoter polyA signal Transcription terminator px5068 Short hCMV without intron A SV40 polyA signal No transcription terminator px6001 Short hCMV without intron A SV40 polyA signal hGT transcription terminator px6008 Short hCMV without intron A bGH polyA signal No transcription terminator px6007 Short hCMV without intron A bGH polyA signal hGT transcription terminator px6051 Full-length hCMV containing intron A SV40 polyA signal No transcription terminator px6052 hEF1α promoter containing intron A SV40 polyA signal No transcription terminator px6053 Rat CMV promoter containing intron A rat CMV SV40 polyA signal No transcription terminator px6062 Full-length hCMV containing intron A bGH polyA signal hGT transcription terminator px6063 hEF1α promoter containing intron A bGH polyA signal hGT transcription terminator

[0538] It has been found that increased expression (productivity) can be achieved with the vector elements / combinations of elements as reported herein:

[0539]

[0540] Human CMV promoter:

[0541] Xu et al., J. Control. Release, 81 (2002) 155-163.

[0542] Xia et al., Prot. Expr. Purif. 45 (2006) 115-124.

[0543] Rat CMV promoter:

[0544] Xia et al., Prot. Expr. Purif. 45 (2006) 115-124.

[0545] Human EF1α promoter:

[0546] Teschendorf et al., Anticancer Res. 22 (2002) 3325-3330.

[0547] Li et al., J. Immunol. Methods 318 (2007) 113-124.

[0548] MPSV promoter:

[0549] Xia et al., Prot. Expr. Purif. 45 (2006) 115-124.

[0550] Artelt et al., Gene 68 (1988) 213-219.

[0551] Stocking et al., Proc. Natl. Acad. Sci. USA 82 (1985) 5746-5750.

[0552] Lin et al., Gene 147 (1994) 287-292.

[0553] MPSV-CMV hybrid promoter:

[0554] Liu et al., Anal. Biochem. 246 (1996) 150-152.

[0555] By expressing the selectable marker using an IRES-linked expression cassette, highly selective and stringent selection methods can be provided:

[0556] -Selective pressure on antibody expression leads to high selectivity

[0557] - Linked expression of antibody and selection marker leads to high selectivity

[0558] - It has been found that the use of IRES elements with weak activity leads to high stringency, i.e. high antibody production and low selection marker production

[0559] - Expression of antibodies and selectable markers linked via IRES elements

[0560] - Identification of weakly active IRES elements (EMCV / Gtx) that do alter IgG expression to a greater or lesser degree

[0561] -Using fusion proteins as selection and screening markers

[0562] - Bifunctional GFP-neomycin fusion protein

[0563] -The PEST sequence of ornithine decarboxylase is a strong proteolytic signal sequence that confers a reduced half-life on the protein

[0564] -IRES-linked expression of fusion proteins results in high selectivity

[0565] - Short half-life of the proteolytic signal sequence leads to high stringency

[0566] - Due to the weak expression and short half-life of fusion proteins, strong expression is required

[0567] - Rapid identification of high producers by FACS (sorting of high GFP expressing clones provides selection of high producers)

[0568] The selectable marker is linked to the antibody heavy chain via an IRES element

[0569]

[0570]

[0571] GFP-Neo fusion protein is linked to the antibody heavy chain via different IRES elements

[0572]

[0573] Gtx-IRES:

[0574] Komuro et al., EMBO J. 12 (1993) 1387-1401.

[0575] EMCV-IRES:

[0576] Mountford et al., Proc. Natl. Acad. Sci. USA 91 (1991) 4303-4307.

[0577] EV71-IRES:

[0578] Lee et al., Biotechnol. Bioeng. 90 (2005) 656-662.

[0579] ELF4G-IRES:

[0580] Wong et al., Gene Ther. 9 (2002) 337-344.

[0581] Gtx(synthetic)-IRES:

[0582] Chappell et al., Proc. Natl. Acad. Sci. USA 97 (2000) 1536-1541.

[0583] It has been found that increased expression (productivity) can be achieved by linking a light chain expression cassette to a heavy chain expression cassette via the EV71-IRES element:

[0584]

[0585] One aspect as reported herein is an optimized human elongation factor 1 alpha promoter comprising an optimized 5'UTR having the sequence of SEQ ID NO: 06.

[0586] The following examples, figures and sequences are provided to aid the understanding of the present invention, the true scope of which is indicated in the appended claims. It should be understood that modifications can be made in the procedures set forth without departing from the spirit of the invention.

[0587] sequence

[0588] SEQ ID NO: 01 Short CMV promoter without intron A

[0589] SEQ ID NO: 02 Short human CMV promoter containing 5'UTR without intron A

[0590] SEQ ID NO: 03 Full-length human CMV promoter containing intron A

[0591] SEQ ID NO: 04 Full-length human EF1α promoter without intron A

[0592] SEQ ID NO: 05 Full-length human EF1α promoter containing intron A

[0593] SEQ ID NO: 06 Short human EF1α promoter containing 5'UTR and intron A

[0594] SEQ ID NO: 07 Full-length rat CMV promoter containing intron A

[0595] SEQ ID NO: 08SV40 polyA signal sequence

[0596] SEQ ID NO: 09bGH polyA signal sequence

[0597] SEQ ID NO: 10 hGT terminator sequence

[0598] SEQ ID NO:11SV40 promoter

[0599] SEQ ID NO: 12 PEST sequence of ornithine decarboxylase

[0600] SEQ ID NO: 13 Nucleic acid sequence encoding GFP

[0601] SEQ ID NO: 14 Neomycin selection marker

[0602] SEQ ID NO: 15 GFP-PEST-NEO fusion protein encoding nucleic acid

[0603] SEQ ID NO:16EMCV-IRES

[0604] SEQ ID NO:17EV71-IRES Sequence Listing <110> F. Hoffmann-La Roche AG <120> Combination of expression vector elements, novel method for producing production cells and their use in recombinant production of polypeptides <130> 30789 WO <150> EP11195361 <151> 2011-12-22 <160> 17 <170> PatentIn Version 3.5 <210> 1 <211> 608 <212> DNA <213> Human cytomegalovirus <400> 1 gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata 60 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 120 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 180 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac 240 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg 300 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg 360 tattagtcat cgctattagc atggtgatgc ggttttggca gtacatcaat gggcgtggat 420 agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt 480 tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc 540 aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctccg tttagtgaac 600 gtcagatc 608 <210> 2 <211> 696 <212> DNA <213> Human cytomegalovirus <400> 2 gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata 60 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 120 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 180 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac 240 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg 300 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg 360 tattagtcat cgctattagc atggtgatgc ggttttggca gtacatcaat gggcgtggat 420 agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt 480 tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc 540 aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctccg tttagtgaac 600 gtcagatcta gctctgggag aggagcccag cactagaagt cggcggtgtt tccattcggt 660 gatcagcact gaacacagag gaagcttgcc gccacc 696 <210> 3 <211> 2125 <212> DNA <213> Human cytomegalovirus <400> 3 ctgcagtgaa taataaaatg tgtgtttgtc cgaaatacgc gttttgagat ttctgtcgcc 60 gactaaattc atgtcgcgcg atagtggtgt ttatcgccga tagagatggc gatattggaa 120 aaatcgatat ttgaaaatat ggcatattga aaatgtcgcc gatgtgagtt tctgtgtaac 180 tgatatcgcc atttttccaa aagtgatttt tgggcatacg cgatatctgg cgatagcgct 240 tatatcgttt acgggggatg gcgatagacg actttggtga cttgggcgat tctgtgtgtc 300 gcaaatatcg cagtttcgat ataggtgaca gacgatatga ggctatatcg ccgatagagg 360 cgacatcaag ctggcacatg gccaatgcat atcgatctat acattgaatc aatattggcc 420 attagccata ttattcattg gttatatagc ataaatcaat attggctatt ggccattgca 480 tacgttgtat ccatatcata atatgtacat ttatattggc tcatgtccaa cattaccgcc 540 atgttgacat tgattattga ctagttatta atagtaatca attacggggt cattagttca 600 tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc 660 gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat 720 agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt 780 acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc 840 cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta 900 cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg 960 atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt 1020 gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac 1080 gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctc gtttagtgaa 1140 ccgtcagatc gcctggagac gccatccacg ctgttttgac ctccatagaa gacaccggga 1200 ccgatccagc ctccgcggcc gggaacggtg cattggaacg cggattcccc gtgccaagag 1260 tgacgtaagt accgcctata gagtctatag gcccaccccc ttggcttctt atgcatgcta 1320 tactgttttt ggcttggggt ctatacaccc ccgcttcctc atgttatagg tgatggtata 1380 gcttagccta taggtgtggg ttattgacca ttattgacca ctcccctatt ggtgacgata 1440 ctttccatta ctaatccata acatggctct ttgccacaac tctctttatt ggctatatgc 1500 caatacactg tccttcagag actgacacgg actctgtatt tttacaggat ggggtctcat 1560 ttattattta caaattcaca tatacaacac caccgtcccc agtgcccgca gtttttatta 1620 aacataacgt gggatctcca cgcgaatctc gggtacgtgt tccggacatg ggctcttctc 1680 cggtagcggc ggagcttcta catccgagcc ctgctcccat gcctccagcg actcatggtc 1740 gctcggcagc tccttgctcc taacagtgga ggccagactt aggcacagca cgatgcccac 1800 caccaccagt gtgccgcaca aggccgtggc ggtagggtat gtgtctgaaa atgagctcgg 1860 ggagcgggct tgcaccgctg acgcatttgg aagacttaag gcagcggcag aagaagatgc 1920 aggcagctga gttgttgtgt tctgataaga gtcagaggta actcccgttg cggtgctgtt 1980 aacggtggag ggcagtgtag tctgagcagt actcgttgct gccgcgcgcg ccaccagaca 2040 taatagctga cagactaaca gactgttcct ttccatgggt cttttctgca gtcaccgtcc 2100 ttgacacggt ttaaacgccg ccacc 2125 <210> 4 <211> 575 <212> DNA <213> Human (Homo sapiens) <400> 4 cccgggctgg gctgagaccc gcagaggaag acgctctagg gatttgtccc ggactagcga 60 gatggcaagg ctgaggacgg gaggctgatt gagaggcgaa ggtacaccct aatctcaata 120 caacctttgg agctaagcca gcaatggtag agggaagatt ctgcacgtcc cttccaggcg 180 gcctccccgt caccaccccc cccaacccgc cccgaccgga gctgagagta attcatacaa 240 aaggactcgc ccctgccttg gggaatccca gggaccgtcg ttaaactccc actaacgtag 300 aacccagaga tcgctgcgtt cccgccccct cacccgcccg ctctcgtcat cactgaggtg 360 gagaagagca tgcgtgaggc tccggtgccc gtcagtgggc agagcgcaca tcgcccacag 420 tccccgagaa gttgggggga ggggtcggca attgaaccgg tgcctagaga aggtggcgcg 480 gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag ggtgggggag 540 aaccgtatat aagtgcagta gtcgccgtga acgtt 575 <210> 5 <211> 1571 <212> DNA <213> Human <400> 5 cccgggctgg gctgagaccc gcagaggaag acgctctagg gatttgtccc ggactagcga 60 gatggcaagg ctgaggacgg gaggctgatt gagaggcgaa ggtacaccct aatctcaata 120 caacctttgg agctaagcca gcaatggtag agggaagatt ctgcacgtcc cttccaggcg 180 gcctccccgt caccaccccc cccaacccgc cccgaccgga gctgagagta attcatacaa 240 aaggactcgc ccctgccttg gggaatccca gggaccgtcg ttaaactccc actaacgtag 300 aacccagaga tcgctgcgtt cccgccccct cacccgcccg ctctcgtcat cactgaggtg 360 gagaagagca tgcgtgaggc tccggtgccc gtcagtgggc agagcgcaca tcgcccacag 420 tccccgagaa gttgggggga ggggtcggca attgaaccgg tgcctagaga aggtggcgcg 480 gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag ggtgggggag 540 aaccgtatat aagtgcagta gtcgccgtga acgttctttt tcgcaacggg tttgccgcca 600 gaacacaggt aagtgccgtg tgtggttccc gcgggcctgg cctctttacg ggttatggcc 660 cttgcgtgcc ttgaattact tccacgcccc tggctgcagt acgtgattct tgatcccgag 720 cttcgggttg gaagtgggtg ggagagttcg aggccttgcg cttaaggagc cccttcgcct 780 cgtgcttgag ttgaggcctg gcctgggcgc tggggccgcc gcgtgcgaat ctggtggcac 840 cttcgcgcct gtctcgctgc tttcgataag tctctagcca tttaaaattt ttgatgacct 900 gctgcgacgc tttttttctg gcaagatagt cttgtaaatg cgggccaaga tctgcacact 960 ggtatttcgg tttttggggc cgcgggcggc gacggggccc gtgcgtccca gcgcacatgt 1020 tcggcgaggc ggggcctgcg agcgcggcca ccgagaatcg gacgggggta gtctcaagct 1080 ggccggcctg ctctggtgcc tggcctcgcg ccgccgtgta tcgccccgcc ctgggcggca 1140 aggctggccc ggtcggcacc agttgcgtga gcggaaagat ggccgcttcc cggccctgct 1200 gcagggagct caaaatggag gacgcggcgc tcgggagagc gggcgggtga gtcacccaca 1260 caaaggaaaa gggcctttcc gtcctcagcc gtcgcttcat gtgactccac ggagtaccgg 1320 gcgccgtcca ggcacctcga ttagttctcg atcttttgga gtacgtcgtc tttaggttgg 1380 ggggaggggt tttatgcgat ggagtttccc cacactgagt gggtggagac tgaagttagg 1440 ccagcttggc acttgatgta attctccttg gaatttgccc tttttgagtt tggatcttgg 1500 ttcattctca agcctcagac agtggttcaa agtttttttc ttccatttca ggtggtttaa 1560 acgccgccac c 1571 <210> 6 <211> 1653 <212> DNA <213> Artificial sequence <220> <223> Human elongation factor-1α promoter containing intron A and optimal 5'UTR <400> 6 cccgggctgg gctgagaccc gcagaggaag acgctctagg gatttgtccc ggactagcga 60 gatggcaagg ctgaggacgg gaggctgatt gagaggcgaa ggtacaccct aatctcaata 120 caacctttgg agctaagcca gcaatggtag agggaagatt ctgcacgtcc cttccaggcg 180 gcctccccgt caccaccccc cccaacccgc cccgaccgga gctgagagta attcatacaa 240 aaggactcgc ccctgccttg gggaatccca gggaccgtcg ttaaactccc actaacgtag 300 aacccagaga tcgctgcgtt cccgccccct cacccgcccg ctctcgtcat cactgaggtg 360 gagaagagca tgcgtgaggc tccggtgccc gtcagtgggc agagcgcaca tcgcccacag 420 tccccgagaa gttgggggga ggggtcggca attgaaccgg tgcctagaga aggtggcgcg 480 )]]gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag ggtgggggag 540 aaccgtatat aagtgcagta gtcgccgtga acgttctttt tcgcaacggg tttgccgcca 600 gaacacaggt aagtgccgtg tgtggttccc gcgggcctgg cctctttacg ggttatggcc 660 cttgcgtgcc ttgaattact tccacgcccc tggctgcagt acgtgattct tgatcccgag 720 cttcgggttg gaagtgggtg ggagagttcg aggccttgcg cttaaggagc cccttcgcct 780 cgtgcttgag ttgaggcctg gcctgggcgc tggggccgcc gcgtgcgaat ctggtggcac 840 cttcgcgcct gtctcgctgc tttcgataag tctctagcca tttaaaattt ttgatgacct 900 gctgcgacgc tttttttctg gcaagatagt cttgtaaatg cgggccaaga tctgcacact 960 ggtatttcgg tttttggggc cgcgggcggc gacggggccc gtgcgtccca gcgcacatgt 1020 tcggcgaggc ggggcctgcg agcgcggcca ccgagaatcg gacgggggta gtctcaagct 1080 ggccggcctg ctctggtgcc tggcctcgcg ccgccgtgta tcgccccgcc ctgggcggca 1140 aggctggccc ggtcggcacc agttgcgtga gcggaaagat ggccgcttcc cggccctgct 1200 gcagggagct caaaatggag gacgcggcgc tcgggagagc gggcgggtga gtcacccaca 1260 caaaggaaaa gggcctttcc gtcctcagcc gtcgcttcat gtgactccac ggagtaccgg 1320 gcgccgtcca ggcacctcga ttagttctcg atcttttgga gtacgtcgtc tttaggttgg 1380 ggggaggggt tttatgcgat ggagtttccc cacactgagt gggtggagac tgaagttagg 1440 ccagcttggc acttgatgta attctccttg gaatttgccc tttttgagtt tggatcttgg 1500 ttcattctca agcctcagac agtggttcaa agtttttttc ttccatttca ggtgtcgtga 1560 ggaattagct ctgggagagg agcccagcac tagaagtcgg cggtgtttcc attcggtgat 1620 cagcactgaa cacagaggaa gcttgccgcc acc 1653 <210> 7 <211> 2473 <212> DNA <213> Rattus norvegicus <400> 7 gatattttta tggaaatttt aaaaaattct ggtaagctat ttaaaaaaat gaactttatt 60 atgaaactat tgcccttttc tctaaaaaac aacacaattt cacggaatat cctatgatta 120 attatgacct tttagccagt tcccatatta agaatgagtt atagatgact ctctttaaaa 180 aattattcga tttaaaccat ctgttttaaa gcacagcatt tgtgaataat gtgaagaact 240 tagaagtata atctactcca aggtctgatg tattttcaa ggccacgtta aagtgtatgc 300 ttgtaacaga gtgcttacat tcaagccaaa tgttaatata acaatcctga attcgtacat 360 aatgtgaata agacactcaa ctctatttaa atccagatct aaatagttac tttatctaa 420 atgtcaccat ctgtttctac ttagaataat aaacttctta aaggtcacgt atcgggctga 480 ttataaatca ttataattat aacaaaacag atgatttgtt taaaggtcac atcccgttcc 540 gtggtctttt tagtcgaaat aactattaat cttcatattag tttctgagaa agtttaaata 600 tcacgatttc caccataac agtcattatg agtcagtggg agtcatactg aatcagggta 660 ttttaactgg aaattttttg aaaaacatga gtttttctta aggtcaacat ctggtcttat 720 aaacagaact gagatttatg gccggtaatt accactggac gatttcccgg gaaatcgcta 780 tgggaacggc ccgttttgca acttctttga ccaaaatata tcgagttaag caacttttaa 840 ggccaagtca ctatgactat gccaaataaa gcaactatta aggtcatttc actatggaaa 900 cacccaattc agcaacattg taagccaaat ctccatagaa acctcataag tcagccaaaa 960 gtcaacgacc taccatctgt ttctgcttat ttctctaatt ttaattgcag actttgtcat 1020 tttatgttcc tcttattctg agaatacgtg acgcccgctc gttaaggaca ccgaaactgc 1080 ataagagtca cgttgactca gatgacctcg acatctggtc tggttttct gccaattttt 1140 cgtctaaact gtggaaaatc cccacagatg acctacaaaa ctccgatttc tattggacga 1200 tgaccgtcag acgtaggtat aaatctccta acgccgttcg ggcagtcaca gtcttcggat 1260 cggacgccgt ggaacgcagt tctcagcgaa gaaggacacc gcccgactcc agaagacacc 1320 gctgcccgaa gaagagaaga cttcatcggt aagagaccca gcttctcctc cccggagctt 1380 cggccacgcc gctccacacc cgggaaccga ggcttcggag cccgataccc ggacagaagc 1440 ttctccccgg ccgctccaca tcagggagcc ttgaccggcg agcctgctat ccgggtagag 1500 actgtcctgc ggccgcttca gcagctccac gatcgacgac tgtgaccgtt gagcccgccg 1560 tttaggcaga ggctccgctt caactaccct accgacacat tcgcggttct tcctccagaa 1620 catcttaccc tctactcggc cactctacaa ggaccggtaa gcaattttta tatactagac 1680 ttaaatgttt ctatgatcat tatgtggtga tggttctgtg tatgaagaga gctaggtgga 1740 ggctatcttt cgcttcggtg atggaacact actcttacaa tggcggctct aatgacggtt 1800 ttctcaacat cggtggcggc tctaattacg gttctctcaa catcggtggt ggtcttcgca 1860 tgcgagctct agatttttt tatctgtaaa ataagattga agatggttga ctgtgtatca 1920 attctttttc ataggcatca gatcttgtca accgttatta atctttagga tcagatgaac 1980 ttgcgagctc gatatctaga atagaatccc cgtgactgct aagatcatct ccgttcatac 2040 accagatgtt acaggccacg gctaccatta tgaatccaaa catgaacaga attgccagaa 2100 tggtgctcaa tggttgtatc catctcgctg gtctattttc tctcaccgac gagaccccaa 2160 catcgagagt tccgtttatt tcatgagtcg accttttagt tcgtgattta ttttctgtgt 2220 taagaaaatc agtgagatca attattgtca gtctatacga ttacaataat gtctgaatta 2280 tcgacgtgca taagatcgtc tcacccggcg cagattccaa cagatctttg tcgccatgcc 2340 ttccgttaga aaggtagtat agtaatatga taccagcaat gcacagaatc gaacatttga 2400 taacaatttt gttgatgtcg tatatctgtt aaaaattaat aaatatatta cagtcagttt 2460 aaacgccgcc acc 2473 <210> 8 <211> 129 <212> DNA <213> Simian virus 40 <400> 8 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca 60 aataaagcat ttttttcacc attctagttg tggtttgtcc aaactcatca atgtatctta 120 tcatgtctg 129 <210> 9 <211> 225 <212> DNA <213> Cattle (Bos taurus)[[ID=--]] <400> 9 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc 60 tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc 120 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt 180<-- gggaagacaa tagcaggcat gctggggatg cggtgggctc tatgg 225 <210> 10 <211> 73 <212> DNA <213> Human <400> 10 Please note that there seems to be a formatting issue with the tag in the original text which might be a typo. I've left it as is in the translation. Also, the tag <-- seems incorrect in the original, but again, I've translated it as it is.caggataata tatggtaggg ttcatagcca gagtaacctt tttttttaat ttttatttta 60 ttttattttt gag 73 <210> 11 <211> 288 <212> DNA <213> Simian virus 40 <400> 11 agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca 60 tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa 120 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag 180 aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag 240 gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcg 288 <210> 12 <211> 81 <212> DNA <213> Human <400> 12 catggcttcc cgccggaggt ggaggagcag gatgatggca cgctgcccat gtcttgtgcc 60 caggagagcg ggatggaccg t 81 <210> 13 <211> 798 <212> DNA <213> Artificial Sequence <220> <223> Green Fluorescent Protein Encoding Nucleic Acid <400> 13 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtcc 720 ggactcagat ctcgagctca agcttcgaat tctgcagtcg acggtaccgc gggcccggga 780 tccaccggat ctagatga 798 <210> 14 <211> 795 <212> DNA <213> Artificial sequence <220> <223> Neomycin selection marker <400> 14 atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc 60 ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca 120 gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg 180 caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg 240 ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag 300 gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg 360 cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc 420 atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa 480<00016六百二十二>gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac 540 ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat 600 ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat 600 ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac 660 ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac 660 atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc 720 atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc 720 ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt 780 ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt 780 gacgagttct tctga 795 gacgagttct tctga 795 <210> 15<210> 15 <211> 1677<211> 1677 <212> DNA<212> DNA <213> Artificial sequence<213> Artificial sequence <220><220> <223> Nucleic acid encoding GFP-PEST-NEO fusion polypeptide<223> Nucleic acid encoding GFP-PEST-NEO fusion polypeptide <400> 15<400> 15 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtcc 720 ggactcagat ctcgagctca agcttcgaat tctgcagtcg acggtaccgc gggcccggga 780 tccaccggat ctagacatgg cttcccgccg gaggtggagg agcaggatga tggcacgctg 840 cccatgtctt gtgcccagga gagcgggatg gaccgtagtt taaacattga acaagatgga 900 ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa 960 cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt 1020 ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg 1080 ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa 1140 gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac 1200 cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt 1260 gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact 1320 cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg 1380 ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg 1440 acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc 1500 atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt 1560 gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc 1620 gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctga 1677 <210> 16 <211> 583 <212> DNA <213> Encephalomyocarditis virus <400> 16 ggcgcgcccc cctctccctc cccccccct aacgttactg gccgaagccg cttggaataa 60 ggccggtgtg cgtttgtcta tatgtgattt tccaccatat tgccgtcttt tggcaatgtg 120 agggcccgga aacctggccc tgtcttcttg acgagcattc ctaggggtct ttcccctctc 180 gccaaaggaa tgcaaggtct gttgaatgtc gtgaaggaag cagttcctct ggaagcttct 240 tgaagacaaa caacgtctgt agcgaccctt tgcaggcagc ggaacccccc acctggcgac 300 aggtgcctct gcggccaaaa gccacgtgta taagatacac ctgcaaagc ggcacaaccc 360 cagtgccacg ttgtgagttg gatagttgtg gaaagagtca aatggctctc ctcaagcgta 420 ttcaacaagg ggctgaagga tgcccagaag gtaccccatt gtatgggatc tgatctgggg 480 cctcggtgca catgctttac atgtgtttag tcgaggttaa aaaaacgtct aggccccccg 540 aaccacgggg acgtggtttt cctttgaaaa acacgatgga tcc 583 <210> 17 <211> 655 <212> DNA <213> Pages 71 <400> 17 ggcgcgcccc cgaagtaact tagaagctgt aaatcaacga tcaatagcag gtgtggcaca 60 ccagtcatac cttgatcaag cacttctgtt tccccggact gagtatcaat aggctgctcg 120 cgcggctgaa ggagaaaacg ttcgttaccc gaccaactac ttcgagaagc ttagtaccac 180 catgaacgag gcagggtgtt tcgctcagca caaccccagt gtagatcagg ctgatgagtc 240 actgcaaccc ccatgggcga ccatggcagt ggctgcgttg gcggcctgcc catggagaaa 300 tccatgggac gctctaattc tgacatggtg tgaagagcct attgagctag ctggtagtcc 360 tccggcccct gaatgcggct aatcctaact gcggagcaca tgctcacaaa ccagtgggtg 420 gtgtgtcgta acgggcaact ctgcagcgga accgactact ttgggtgtcc gtgtttcctt 480 ttatcctat attggctgct tatggtgaca atcaaaaagt tgttaccata tagctattgg 540 attggccatc cggtgtgcaa cagggcaatt gtttacctat ttattggttt tgtaccatta 600 tcactgaagt ctgtgatcac tctcaaattc attttgaccc tcaacacaat caaac 655 BRIEF DESCRIPTION OF THE DRAWINGS

[0605] Figure 1 Productivity of stable clones generated with vectors p5068, px6001, px6008 and px6007. Shown is the average productivity of the best 15 clones obtained with each vector in batch analysis from a total of three independent transfections.

[0606] Figure 2Productivity of stable clones generated with vectors px6051, px6062, px6052 and px6063. Shown is the average productivity of the best 18 clones for each vector from a total of three independent transfections in batch analysis.

[0607] Figure 3 Productivity of vectors p5068, px6051, px6052 and px6053 in transient transfection using the 96-well shuttle system from Amaxa. Shown is the average productivity of eight independent transfections of each vector measured by ELISA on day 4 after transfection.

[0608] Figure 4 Productivity of different stable pools generated with vectors p5068, px6051, px6052 and px6053 in batch analysis. Shown is the average productivity of three pools for each vector at day 7.

[0609] Figure 5 Productivity of stable pools generated with vectors p5069 and px6010C. Shown are the average productivity of two (px5069) or three (px6010C) different pools for each vector at day 7 of the batch analysis.

[0610] Figure 6 Stability of gene expression in stable pools generated with vectors p5069 and px6010C. Shown are the average productivity of two different pools for each vector at generation 0 (set to 100%, black bars) and generation 30 with selection pressure (G418) (white bars) and without selection pressure (patterned bars) on day 7 of batch analysis.

[0611] Figure 7 Productivity of the best 15 clones generated by vectors p5069 and px6010C. Average productivity of the best 15 clones for each vector in batch analysis totaling two independent transfections.

[0612] Figure 8 Schematic diagram of the vector design for the px6011C vector, which mediates IRES-mediated expression of a GFP-PEST-NEO fusion protein. The GFP-PEST-Neo fusion protein is linked to the antibody heavy chain via the EMCV-IRES. The coding sequences for the antibody heavy chain and the fusion protein are transcribed into a single mRNA from a short human CMV promoter. Translation of this mRNA produces the antibody heavy chain and the GFP-PEST-NEO fusion protein.

[0613] Figure 9Productivity of different stable pools generated by vectors p5069, px6011C and px6010C in batch analysis. Shown is the average productivity of two different pools for each vector at day 7.

[0614] Figure 10 Productivity of the top 15 clones generated by the vectors p5069, px6010C (a vector expressing the selectable marker neomycin via an EMCV-IRES element linked to the heavy chain of an antibody), and px6011C (a vector expressing a fusion protein of GFP-PEST-neomycin via an EMCV-IRES element linked to the heavy chain of an antibody). (A) Distribution of productivity of the top 15 clones for each vector in a batch analysis from two independent transfections. (B) Average productivity of the top 15 clones for each vector in a batch analysis from two independent transfections.

[0615] Figure 11 Shown are the dependence of GPF expression levels / fluorescence intensity and productivity in batch analysis for 11 clones generated using the vector px6011C. Clones were randomly selected in a 24-well screening format, expanded, and analyzed in batch analysis. The geometric mean (GM) of GFP fluorescence intensity and the percentage of GFP-positive cells for each clone were determined by FACS. (A) Dependence of GFP fluorescence intensity and productivity in batch analysis for 11 individual clones. (B) Dependence of the percentage of GFP-positive cells for each of the 11 individual clones and productivity in batch analysis.

[0616] Figure 12 Productivity of stable pools with different GFP fluorescence intensities in batch analysis. Cells with different GFP expression levels / fluorescence intensities (low (1), medium (2), and high (3)) were sorted by FACS. Pools were expanded and their productivity was determined on day 7 of the batch analysis.

[0617] Figure 13 .Plasmid map of px6007.

[0618] Figure 14 .Plasmid map of px6053.

[0619] Figure 15 .Plasmid map of px6062. Example

[0620] Expression vectors p5068 and p5069

[0621] Expression plasmids p5068 and p5069 contain the expression cassette for expressing the anti-P-selectin antibody reported in WO 2005 / 100402 (genomic organization expression cassette retaining the exon-intron organization).

[0622] The anti-P-selectin HuMab light chain and heavy chain encoding genes were separately loaded into mammalian cell expression vectors.

[0623] The gene segments encoding the anti-P-selectin HuMab light chain variable region (VL) and the human kappa light chain constant region (CL) are thereby connected, as are the gene segments encoding the anti-P-selectin HuMab heavy chain variable region (VH) and the human gamma 1 heavy chain constant region or the human gamma 4 heavy chain constant region (CH1-hinge-CH2-CH3).

[0624] General information regarding the nucleotide sequences of human light and heavy chains from which codon usage can be inferred is provided in Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication No 91-3242.

[0625] The transcription unit of the anti-P-selectin HuMab kappa light chain consists of the following elements:

[0626] - the immediate early enhancer and promoter from human cytomegalovirus (hCMV),

[0627] -Synthetic 5'-UT including Kozak sequence,

[0628] - a murine immunoglobulin heavy chain signal sequence including a signal sequence intron,

[0629] - cloned anti-P-selectin HuMab variable light chain cDNA with a unique BsmI restriction site placed at the 5' end, a splice donor site and a unique NotI restriction site placed at the 3' end,

[0630] - the genomic human kappa gene constant region, including intron 2 mouse Ig-kappa enhancer (Picard, D. and Schaffner, W. Nature 307 (1984) 80-82), and

[0631] -Human immunoglobulin kappa polyadenylation ("polyA") signal sequence

[0632] The transcription unit of the anti-P-selectin HuMabγl heavy chain consists of the following elements:

[0633] - the immediate early enhancer and promoter from human cytomegalovirus (hCMV),

[0634] -Synthetic 5'-UT including Kozak sequence,

[0635] - a modified murine immunoglobulin heavy chain signal sequence including a signal sequence intron,

[0636] - cloned anti-P-selectin HuMab variable heavy chain cDNA with a unique BsmI restriction site placed at the 5' end, a splice donor site and a unique NotI restriction site placed at the 3' end,

[0637] - the genomic human γl heavy chain gene constant region, including the mouse Igμ enhancer (Neuberger, MS, EMBO J. 2 (1983) 1373-1378), and

[0638] - Human gamma 1 immunoglobulin polyadenylation ("polyA") signal sequence.

[0639] In addition to the anti-P-selectin HuMab kappa light chain or gamma 1 heavy chain expression cassette, these plasmids also contain:

[0640] - Hygromycin resistance gene,

[0641] -oriP, the origin of replication of Epstein-Barr virus (EBV),

[0642] - an origin of replication from the vector pUC18, which allows this plasmid to replicate in E. coli, and

[0643] -β-lactamase gene that confers ampicillin resistance in Escherichia coli.

[0644] Recombinant DNA technology

[0645] Cloning was performed using standard cloning techniques as described in Sambrook et al., 1999 (supra). All molecular biology reagents were commercially available (unless otherwise stated) and used according to the manufacturer's instructions.

[0646] Nucleic acid synthesis

[0647] The DNA of the different genetic elements was synthesized by Geneart AG, Regensburg.

[0648] Nucleic acid sequence determination

[0649] DNA sequences were determined by double-strand sequencing performed at SequiServe (SequiServe GmbH, Germany).

[0650] DNA and protein sequence analysis and sequence data management

[0651] Sequence creation, mapping, analysis, annotation, and interpretation were performed using Vector NTI Advance suite version 9.0.

[0652] Cell culture technology

[0653] CHO-K1 cells were cultured in 1x HT supplement (Invitrogen Corp., CD-CHO medium (Invitrogen Corp., Catalog number 10743-011).

[0654] For selection of stably transfected CHO-K1 pools / cell lines, 400 to 800 μg / ml G418 or 200 to 400 μg / ml hygromycin (Roche Diagnostics GmbH, Roche Applied Sciences, Germany, cat. no. 843555) were added.

[0655] All cell lines were maintained at 37°C in a humidified incubator with 5% CO2 and constant agitation at 120 to 140 rpm. Cells were split into fresh medium every 3 to 4 days. Culture density and viability were determined using a Casey TT or Cedex Hires cell counter (Rocheinnovates AG, Bielefeld). Cells were transfected using the Amaxa nucleofection technique (Lonza GmbH, Germany).

[0656] In addition, standard cell culture techniques are applied as described in, for example, Bonifacino, JS et al., (eds.), Current Protocols in Cell Biology, John Wiley and Sons, Inc. (2000).

[0657] Cell counting and cell viability determination

[0658] a) Cellular System with Electric Field (CASY)

[0659] The TT Technology Cell Counter (Roche Innovatis AG, Bielefeld) uses electrical current to count cells. Pulse area analysis is used to obtain information from the signal generated when cells pass through a measuring aperture in a low-voltage electric field. The structural integrity of the cell membrane is a measure of cell viability. Therefore, viability determination does not require dyes such as trypan blue.

[0660] b) Automated trypan blue exclusion (Cedex)

[0661] The Cedex HiRes system (Roche Innovatis AG, Bielefeld) was used to determine cell viability during library selection and to perform automated cell counting.

[0662] Trypan blue is a dye that cannot enter cells through intact cell membranes. Only cells with damaged cell membranes are stained and marked as dead. The staining process, cell count, and graphical analysis of the results are automatically performed by the Cedex system using digital image recognition. Other measured parameters are cell size, morphology, and aggregation rate. Using a multisampler, up to 20 samples can be measured continuously.

[0663] Plasmid preparation and quality control for accurate comparison of plasmids used in transfection

[0664] Several factors, such as the amount and quality of DNA, strongly influence transfection efficiency and thus productivity. To ensure equivalent starting conditions for each vector, the DNA amount and quality of all vectors were centrally tested before transfection.

[0665] -Simultaneous preparation of expression vectors

[0666] All vectors were prepared simultaneously by the High Speed ​​Maxi Plasmid Isolation Kit (Qiagen GMBH, Hilden) according to the manufacturer's instructions.

[0667] - Phenol / chloroform purification and ethanol precipitation

[0668] All vectors were purified simultaneously by phenol / chloroform purification. 500 μg of each linearized plasmid DNA was mixed with 200 μl of a Tris-buffered solution of 50% (v / v) phenol, 48% (v / v) chloroform, and 2% (v / v) isoamyl alcohol and centrifuged at 13,000 rpm for 1 minute. The upper aqueous phase was then transferred to a new tube and mixed with 200 μl of 96% (v / v) chloroform and 4% (v / v) isoamyl alcohol and centrifuged at 13,000 rpm for 1 minute. The upper phase was again transferred to a new tube and mixed with 1 / 10 (total volume) of 3M sodium acetate (pH 5.2) and 2.5 times (total volume) of 100% ethanol. After mixing and incubating the reaction at room temperature for 5 minutes, the mixture was centrifuged at 13,000 rpm for 5 minutes to precipitate the DNA. The supernatant was discarded, and the pellet was washed with 900 μl of 70% (v / v) ethanol and incubated at room temperature for 5 minutes. After a final centrifugation step at maximum speed for 5 minutes, the supernatant was discarded and the pellet was dried and resuspended in sterile water.

[0669] -DNA testing

[0670] The DNA amount of each vector was determined using a BioPhotometer (Eppendorf; Hamburg). DNA measurements were always performed in triplicate using a 1:20 dilution in Tris pH 8.0.

[0671] -Agarose gel

[0672] The DNA quality of each plasmid was checked on a 0.8% agarose gel. DNA degradation, vector conformation, and DNA concentration were determined. Transient and stable transfections were performed with vectors that showed comparable quantity and quality (no DNA degradation on the gel, similar supercoiled (ccc) forms, and similar DNA amounts).

[0673] transient transfection

[0674] All vectors were transfected into CHO-K1 cells using the Amaxa 96-well shuttle system (Lonza GmbH, Germany) according to the manufacturer's instructions. Each vector was transfected eight times. The amount of DNA in the transfected vector was normalized to an equimolar amount per copy number using 1 μg of a reference expression plasmid (p5068 or p5069). To determine productivity, cell-free cell culture supernatants were analyzed for IgG titers on days 4 to 7 after transfection by a one-step universal ELISA (Dianova).

[0675] Amaxa 96-well shuttle system:

[0676] CHO-K1 cells cultured in cell culture flasks were pelleted by centrifugation at 850 rpm for 5 minutes and resuspended in culture medium. Circular plasmids were plated in 96-well nucleofection plates at an equimolar concentration of 1 μg of the reference expression vector p5068 or p5069. 4 × 10 5 Cells were plated at a concentration of 10 μg / mL. Transfection was performed using Amaxa program DN-137. After transfection, cells were incubated for 10 minutes and then transferred to a 96-well flat-bottom plate containing 200 μl of culture medium. The cells were then allowed to incubate statically. IgG levels were measured using a one-step universal ELISA four to six days after transfection.

[0677] Stable transfection and generation of recombinant CHO cell lines

[0678] Stable transfection was performed using the nucleofection technique (Amaxa Biosystems, Lonza Cologne AG) according to the manufacturer's instructions. Prior to transfection, the transfection plasmids were linearized using the restriction enzyme SgrA I. Each plasmid was transfected twice or three times. 5 x 10 6 Cells and 1.2 pmol of linearized plasmid were added (Nucleofector Kit T, Amaxa program A33).

[0679] For transfection, resuspend the cells in Nucleofector solution T and aliquot into 2 ml tubes. After adding the plasmid, transfection is performed by applying a pulse. The cells are then transferred to a T25 tissue culture flask containing 4 ml of pre-warmed fresh medium and 4 ml of conditioned medium. 24 hours after transfection, selective pressure is applied by adding 250 μg / ml hygromycin B.

[0680] Generation of stable libraries

[0681] Vectors were transfected into CHO-K1 cells using Amaxa nucleofection technology, and stable pools were selected using either hygromycin B or G418. Each transfection was performed in triplicate. To generate stable pools, all plasmids were uniformly linearized by restriction digestion with SgrA I. Stable transfections were performed using Amaxa's Nucleofector Kit T, with each plasmid transfected in triplicate.

[0682] Stable pools were prepared as follows: 5 x 10 6 Cells and 1.2 pmol of linearized plasmid were added. Resuspend the cells in Solution T and aliquot into 2 ml tubes. After adding the plasmid, transfection was performed by pulse application (Amaxa program A33). The transfected cell pool was cultured statically in a T25 tissue culture flask containing 4 ml of prewarmed fresh medium and 4 ml of conditioned medium.

[0683] Twenty-four hours after transfection, selection pressure was applied: cells were centrifuged at 800 rpm for 5 minutes and resuspended in 3 ml of culture medium containing 300 μg / ml hygromycin B. Three days after transfection, cells were transferred to flat-bottomed 6-well plates. Cells were then cultured for two weeks, until cell viability reached a minimum and then returned to above 99%. Cell number and viability were frequently determined using the Cedex HiRes system (Innovatis, Bielefeld). During the culture period, cell debris was removed by centrifugation, and cells were always resuspended in 3 ml of fresh culture medium.

[0684] Generation of stable clones using the Caliper robotic system

[0685] The vectors were transfected into CHO-K1 cells as described above. 48 hours after transfection, selection pressure (hygromycin B or G418) was applied and cells were seeded at a concentration of 350 to 700 cells per well in 384-well flat-bottom plates using an automated high-throughput cloning and isolation system (Sciclone ALH 3000 workstation, Caliper LifeSciences GmbH, Mainz).

[0686] After 10 to 14 days, 384-well plates were screened for IgG levels using an ELISA-based ultra-high throughput screen (ELSIA uHTS). The best production clones were selected from the preliminary screening and transferred to flat-bottom 96-well plates. After 3 to 6 days, cells were screened for IgG levels in a second round. The best production clones were selected again and manually transferred to flat-bottom 24-well plates. After another ELISA-based screening step, the best clones were selected and transferred to flat-bottom 6-well plates. IgG levels in the 6-well plates were measured by ProtA to identify the final best clones for batch culture in shaking 6-well plates.

[0687] Batch analysis of libraries / single clones

[0688] To detect differences in productivity and stability, the cell number of clones / pools was counted using a Casey cell counter and the number of cells was calculated as 3 x 10 5 Cells were plated uniformly in a 3.0 ml volume at a concentration of 10 cells / ml into flat-bottom 6-well plates. All batch cultures were grown for 12 days, and cell culture supernatants were screened for human IgG levels on day 4, 7, 9, 11, or 12.

[0689] IgG quantification

[0690] IgG titers were determined in transient experiments and in screening formats (384-well to 24-well) using a one-step universal ELISA. Productivity of stable pools and stable monoclonals in batch experiments was determined by Protein A HPLC.

[0691] One-step universal ELISA

[0692] Human IgG levels in cell culture supernatants were determined using a one-step universal ELISA (Dianova). A standard curve was prepared using serial dilutions of anti-P-selectin antibody (F. Hoffmann-La Roche AG, Basle, Switzerland) ranging from 0.3125 to 20 ng / ml using dilution buffer (PBS + 5% (w / v) RPLA1). To a streptavidin-coated 96-well MTP plate (StreptaWell, Roche Diagnostics GmbH) was added 95 μl of an antibody cocktail containing 0.5 μg / ml biotinylated F(ab')2 anti-human Fc antibody (Jackson Laboratories) and 0.1 μg / ml peroxidase-conjugated F(ab')2 anti-human Fcγ antibody (Jackson Laboratories; Suffolk). 5 μl of a 1:20,000 dilution of cell culture supernatant was added to the plate and incubated for 1 hour. The antibody-coated plates were washed three times with 200 μl of wash buffer (PBS + 0.05% (v / v) Tween 20). 100 μl of ABTS (Roche Diagnostics GmbH, Mannheim, Germany) was added to the plates and the absorbance was measured at 405 nm with a reference wavelength of 492 nm.

[0693] ProtA measurements

[0694] IgG titers in batch analysis were determined by Protein A using HPLC-based chromatography in combination with a one-step universal ELISA.

[0695] FACS

[0696] Fluorescence activated cell sorting was used to determine the transfection efficiency (based on cells expressing GFP) or GFP expression level of stably or transiently transfected cells. Typically, 5 x 10 cells of each clone or pool were measured using a FACSCalibur flow cytometer (BD Biosciences, San Diego, CA). 6 Forward and side scatter data were used to determine cell size, viability, and cell morphology.

[0697] Some embodiments of the present invention:

[0698] 1. A method for selecting recombinant transiently transfected mammalian cells, comprising the following steps:

[0699] a) transfecting mammalian cells with an expression vector comprising:

[0700] a first expression cassette comprising, in 5' to 3' direction, an hCMV promoter, a nucleic acid encoding an antibody light chain, a bGH polyA signal sequence, and an hGT terminator sequence;

[0701] a second expression cassette comprising, in 5' to 3' direction, an hCMV promoter, nucleic acid encoding an antibody heavy chain, a bGH polyA signal sequence, and an hGT terminator sequence; and

[0702] Thus, the first expression cassette and the second expression cassette are arranged bidirectionally,

[0703] Thus, a large number of recombinant mammalian cells are obtained;

[0704] b) selecting a (single) transiently transfected recombinant mammalian cell from said plurality of recombinant mammalian cells.

[0705] 2. The method according to embodiment 1, characterized in that the nucleic acid encoding the antibody light chain and / or the nucleic acid encoding the antibody heavy chain comprises at least one intron.

[0706] 3. The method according to any one of embodiments 1 to 2, characterized in that the nucleic acid encoding the antibody light chain and / or the nucleic acid encoding the antibody heavy chain is cDNA.

[0707] 4. The method according to any one of embodiments 1 to 3, characterized in that the expression plasmid further comprises a selection marker.

[0708] 5. The method according to any one of embodiments 1 to 4, characterized in that the expression cassettes are arranged in the order LC-HC-SM.

[0709] 6. The method according to any one of embodiments 1 to 5, characterized in that the mammalian cells are selected from CHO cells, HEK cells, BHK cells, NSO cells and SP2 / 0 cells.

[0710] 7. The method according to any one of embodiments 1 to 6, characterized in that the mammalian cells are HEK cells for selecting transiently transfected cells.

[0711] 8. The method according to any one of embodiments 1 to 7, characterized in that the expression vector comprises:

[0712] a first expression cassette comprising, in 5' to 3' direction, an hCMV promoter, a nucleic acid encoding a first antibody light chain, a bGH polyA signal sequence, and an hGT terminator sequence;

[0713] a second expression cassette comprising, in 5' to 3' direction, an hCMV promoter, a nucleic acid encoding a second antibody light chain, a bGH polyA signal sequence, and an hGT terminator sequence;

[0714] a third expression cassette comprising, in 5' to 3' direction, an hCMV promoter, the nucleic acid encoding the heavy chain of the first antibody, a bGH polyA signal sequence, and an hGT terminator sequence;

[0715] a fourth expression cassette comprising, in 5' to 3' direction, an hCMV promoter, the nucleic acid encoding the second antibody heavy chain, the bGH polyA signal sequence, and the hGT terminator sequence;

[0716] or

[0717] a first expression cassette comprising, in 5' to 3' direction, an hCMV promoter, a nucleic acid encoding an antibody light chain, a bGH polyA signal sequence, and an hGT terminator sequence;

[0718] - a second expression cassette comprising, in 5' to 3' direction, an hCMV promoter, the nucleic acid encoding the heavy chain of the first antibody, a bGH polyA signal sequence, and an hGT terminator sequence; and

[0719] a third expression cassette comprising, in 5' to 3' direction, an hCMV promoter, the nucleic acid encoding the second antibody heavy chain, the bGH polyA signal sequence, and the hGT terminator sequence;

[0720] The antibody light chain is thus the common light chain of two antibody heavy chains.

[0721] 9. The method according to any one of embodiments 1 to 8, characterized in that the expression vector encodes a bispecific antibody.

[0722] 10. The method of embodiment 8 or 9, characterized in that the bispecific antibody has a first binding specificity or binding site that specifically binds to a first antigen or a first epitope on an antigen, and the bispecific antibody has a second binding specificity or binding site that specifically binds to a second antigen or a second epitope on an antigen.

[0723] 11. The method according to any one of embodiments 8 to 10, characterized in that the expression vector comprises:

[0724] -Antibody light chain expression cassette;

[0725] -First antibody heavy chain expression cassette;

[0726] - a second antibody heavy chain expression cassette; and

[0727] - Selectable marker expression cassette.

[0728] 12. The method according to any one of embodiments 8 to 11, characterized in that the expression vector comprises:

[0729] -First antibody light chain expression cassette;

[0730] - Second antibody light chain expression cassette;

[0731] -First antibody heavy chain expression cassette;

[0732] - a second antibody heavy chain expression cassette; and

[0733] - Selectable marker expression cassette.

[0734] 13. The method according to any one of embodiments 8 to 12, characterized in that one of the antibody heavy chain expression cassettes encodes an antibody heavy chain comprising a hole mutation.

[0735] 14. The method according to any one of embodiments 8 to 13, characterized in that one of the antibody heavy chain expression cassettes encodes an antibody heavy chain comprising a knob mutation.

[0736] 15. The method according to any one of embodiments 8 to 14, characterized in that one of the antibody light chain expression cassettes encodes an antibody light chain variant comprising an antibody light chain variable domain and an antibody heavy chain CH1 domain as a constant domain, and / or one of the antibody light chain expression cassettes encodes an antibody light chain comprising an antibody light chain variable domain and an antibody light chain CL domain as a constant domain.

[0737] 16. The method according to any one of embodiments 8 to 15, characterized in that one of the antibody heavy chain expression cassettes encodes an antibody heavy chain variant comprising an antibody light chain constant domain (CL) as the first constant domain, and / or one of the antibody heavy chain expression cassettes encodes an antibody heavy chain comprising an antibody heavy chain CH1 domain as the first constant domain.

[0738] 17. A method for producing an antibody, comprising the steps of:

[0739] a) culturing mammalian cells comprising:

[0740] a first expression cassette comprising, in 5' to 3' direction, an hCMV promoter, a nucleic acid encoding an antibody light chain, a bGH polyA signal sequence, and an hGT terminator sequence;

[0741] - a second expression cassette comprising, in 5' to 3' direction, an hCMV promoter, nucleic acid encoding an antibody heavy chain, a bGH polyA signal sequence, and an hGT terminator sequence; and

[0742] b) recovering the antibody from the cells or culture medium,

[0743] The first expression cassette and the second expression cassette are arranged bidirectionally for transient production of antibodies.

[0744] 18. The method according to embodiment 17, characterized in that the nucleic acid encoding the antibody light chain and / or the nucleic acid encoding the antibody heavy chain comprises at least one intron.

[0745] 19. The method according to embodiment 17 or 18, characterized in that the nucleic acid encoding the antibody light chain and / or the nucleic acid encoding the antibody heavy chain is cDNA.

[0746] 20. The method according to any one of embodiments 17 to 19, characterized in that the expression plasmid further comprises a selection marker.

[0747] 21. The method according to any one of embodiments 17 to 20, characterized in that the expression cassettes are arranged in the order LC-HC-SM.

[0748] 22. The method according to any one of embodiments 17 to 21, characterized in that the mammalian cells are selected from the group consisting of CHO cells, HEK cells, BHK cells, NSO cells and SP2 / 0 cells.

[0749] 23. The method according to any one of embodiments 17 to 22, characterized in that the mammalian cells are HEK cells for transient production of antibodies.

[0750] 24. An expression vector comprising:

[0751] a first expression cassette comprising, in 5' to 3' direction, an hCMV promoter, a nucleic acid encoding an antibody light chain, a bGH polyA signal sequence, and an hGT terminator sequence;

[0752] a second expression cassette comprising, in 5' to 3' direction, a hCMV promoter, the nucleic acid encoding the antibody heavy chain, the bGH polyA signal sequence and the hGT terminator sequence,

[0753] The first expression cassette and the second expression cassette are arranged bidirectionally for selecting transiently transfected cells.

[0754] 25. The expression vector of embodiment 24, characterized in that the nucleic acid encoding the antibody light chain and / or the nucleic acid encoding the antibody heavy chain comprises at least one intron.

[0755] 26. The expression vector of embodiment 24 or 25, characterized in that the nucleic acid encoding the antibody light chain and / or the nucleic acid encoding the antibody heavy chain is cDNA.

[0756] 27. The expression vector according to any one of embodiments 24 to 26, characterized in that the expression plasmid further comprises a selection marker.

[0757] 28. The expression vector according to any one of embodiments 24 to 27, characterized in that the expression cassettes are arranged in the order LC-HC-SM.

[0758] 29. The expression vector according to any one of embodiments 24 to 28, characterized in that the expression vector comprises:

[0759] a first expression cassette comprising, in 5' to 3' direction, an hCMV promoter, a nucleic acid encoding a first antibody light chain, a bGH polyA signal sequence, and an hGT terminator sequence;

[0760] a second expression cassette comprising, in 5' to 3' direction, an hCMV promoter, a nucleic acid encoding a second antibody light chain, a bGH polyA signal sequence, and an hGT terminator sequence;

[0761] a third expression cassette comprising, in 5' to 3' direction, an hCMV promoter, the nucleic acid encoding the heavy chain of the first antibody, a bGH polyA signal sequence, and an hGT terminator sequence;

[0762] a fourth expression cassette comprising, in 5' to 3' direction, an hCMV promoter, the nucleic acid encoding the second antibody heavy chain, the bGH polyA signal sequence, and the hGT terminator sequence;

[0763] or

[0764] a first expression cassette comprising, in 5' to 3' direction, an hCMV promoter, a nucleic acid encoding an antibody light chain, a bGH polyA signal sequence, and an hGT terminator sequence;

[0765] - a second expression cassette comprising, in 5' to 3' direction, an hCMV promoter, the nucleic acid encoding the heavy chain of the first antibody, a bGH polyA signal sequence, and an hGT terminator sequence; and

[0766] a third expression cassette comprising, in 5' to 3' direction, an hCMV promoter, the nucleic acid encoding the second antibody heavy chain, the bGH polyA signal sequence, and the hGT terminator sequence;

[0767] The antibody light chain is thus the common light chain of two antibody heavy chains.

[0768] 30. The expression vector according to any one of embodiments 24 to 29, characterized in that the expression vector encodes a bispecific antibody.

[0769] 31. The expression vector of any one of embodiments 24 to 30, characterized in that the bispecific antibody has a first binding specificity or binding site that specifically binds to a first antigen or a first epitope on an antigen, and the bispecific antibody has a second binding specificity or binding site that specifically binds to a second antigen or a second epitope on an antigen.

[0770] 32. The expression vector according to any one of embodiments 24 to 31, characterized in that the expression vector comprises:

[0771] -Antibody light chain expression cassette;

[0772] -First antibody heavy chain expression cassette;

[0773] - a second antibody heavy chain expression cassette; and

[0774] - Selectable marker expression cassette.

[0775] 33. The expression vector according to any one of embodiments 24 to 32, characterized in that the expression vector comprises:

[0776] -First antibody light chain expression cassette;

[0777] - Second antibody light chain expression cassette;

[0778] -First antibody heavy chain expression cassette;

[0779] - a second antibody heavy chain expression cassette; and

[0780] - Selectable marker expression cassette.

[0781] 34. The expression vector according to any one of embodiments 24 to 33, characterized in that one of the antibody heavy chain expression cassettes encodes an antibody heavy chain comprising a hole mutation.

[0782] 35. The expression vector according to any one of embodiments 24 to 34, characterized in that one of the antibody heavy chain expression cassettes encodes an antibody heavy chain comprising a knob mutation.

[0783] 36. The expression vector of any one of embodiments 24 to 35, characterized in that one of the antibody light chain expression cassettes encodes an antibody light chain variant comprising an antibody light chain variable domain and an antibody heavy chain CH1 domain as a constant domain, and / or one of the antibody light chain expression cassettes encodes an antibody light chain comprising an antibody light chain variable domain and an antibody light chain CL domain as a constant domain.

[0784] 37. The expression vector of any one of embodiments 24 to 36, characterized in that one of the antibody heavy chain expression cassettes encodes an antibody heavy chain variant comprising an antibody light chain constant domain (CL) as the first constant domain, and / or one of the antibody heavy chain expression cassettes encodes an antibody heavy chain comprising an antibody heavy chain CH1 domain as the first constant domain.

[0785] 38. A method for selecting recombinant transiently transfected mammalian cells comprising the steps of:

[0786] a) transfecting mammalian cells with an expression vector comprising:

[0787] - a first expression cassette comprising, in 5' to 3' direction, the hEF1α promoter, the nucleic acid encoding the antibody light chain, and the bGH polyA signal sequence;

[0788] - a second expression cassette comprising, in 5' to 3' direction, the hEF1α promoter, the nucleic acid encoding the antibody heavy chain, and the bGH polyA signal sequence; and

[0789] The first expression cassette and the second expression cassette are arranged bidirectionally for selecting transiently transfected cells,

[0790] Thus, a large number of recombinant mammalian cells are obtained;

[0791] b) selecting a (single) transiently transfected recombinant mammalian cell from said plurality of recombinant mammalian cells.

[0792] 39. The method according to embodiment 38, characterized in that the nucleic acid encoding the antibody light chain and / or the nucleic acid encoding the antibody heavy chain comprises at least one intron.

[0793] 40. The method according to embodiment 38 or 39, characterized in that the nucleic acid encoding the antibody light chain and / or the nucleic acid encoding the antibody heavy chain is cDNA.

[0794] 41. The method according to any one of embodiments 38 to 40, characterized in that the expression plasmid further comprises a selection marker.

[0795] 42. The method according to any one of embodiments 38 to 41, characterized in that the expression cassettes are arranged in the order LC-HC-SM.

[0796] 43. The method according to any one of embodiments 38 to 42, characterized in that the human elongation factor 1 alpha promoter contains intron A.

[0797] 44. The method according to any one of embodiments 38 to 43, characterized in that the expression vector does not contain any transcription terminator sequence.

[0798] 45. The method according to embodiment 44, characterized in that the terminator sequence is an hGT sequence.

[0799] 46. ​​The method according to any one of embodiments 38 to 45, characterized in that the mammalian cells are selected from the group consisting of CHO cells, HEK cells, BHK cells, NSO cells and SP2 / 0 cells.

[0800] 47. The method according to embodiment 46, characterized in that the mammalian cells are HEK cells for selecting transiently transfected cells.

[0801] 48. A method for producing an antibody comprising the steps of:

[0802] a) Cultivating transiently transfected mammalian cells comprising:

[0803] - a first expression cassette comprising, in 5' to 3' direction, the hEF1α promoter, the nucleic acid encoding the antibody light chain, and the bGH polyA signal sequence;

[0804] - a second expression cassette comprising, in 5' to 3' direction, the hEF1α promoter, the nucleic acid encoding the antibody heavy chain, and the bGH polyA signal sequence; and

[0805] The first expression cassette and the second expression cassette are arranged bidirectionally for selecting transiently transfected cells,

[0806] b) recovering the antibody from the transiently transfected cells or culture medium.

[0807] 49. The method according to embodiment 48, characterized in that the nucleic acid encoding the antibody light chain and / or the nucleic acid encoding the antibody heavy chain comprises at least one intron.

[0808] 50. The method according to embodiment 48 or 49, characterized in that the nucleic acid encoding the antibody light chain and / or the nucleic acid encoding the antibody heavy chain is cDNA.

[0809] 51. The method according to any one of embodiments 48 to 50, characterized in that the expression plasmid further comprises a selection marker.

[0810] 52. The method according to any one of embodiments 48 to 51, characterized in that the expression cassettes are arranged in the order LC-HC-SM.

[0811] 53. The method according to any one of embodiments 48 to 52, characterized in that the human elongation factor 1 alpha promoter contains intron A.

[0812] 54. The method according to any one of embodiments 48 to 53, characterized in that the expression vector does not contain any transcription terminator sequence.

[0813] 55. The method according to embodiment 54, characterized in that the terminator sequence is an hGT sequence.

[0814] 56. The method according to any one of embodiments 48 to 55, characterized in that the mammalian cells are selected from the group consisting of CHO cells, HEK cells, BHK cells, NSO cells and SP2 / 0 cells.

[0815] 57. The method according to embodiment 56, characterized in that the mammalian cells are HEK cells for transient production of antibodies.

[0816] 58. An expression vector comprising:

[0817] - a first expression cassette comprising, in 5' to 3' direction, the hEF1α promoter, the nucleic acid encoding the antibody light chain, and the bGH polyA signal sequence;

[0818] - a second expression cassette comprising, in 5' to 3' direction, the hEF1α promoter, the nucleic acid encoding the antibody heavy chain and the bGH polyA signal sequence,

[0819] The first expression cassette and the second expression cassette are arranged bidirectionally for selecting transiently transfected cells.

[0820] 59. The expression vector of embodiment 58, characterized in that the nucleic acid encoding the antibody light chain and / or the nucleic acid encoding the antibody heavy chain comprises at least one intron.

[0821] 60. The expression vector of embodiment 58 or 59, characterized in that the nucleic acid encoding the antibody light chain and / or the nucleic acid encoding the antibody heavy chain is cDNA.

[0822] 61. The expression vector according to any one of embodiments 58 to 60, characterized in that the expression plasmid further comprises a selection marker.

[0823] 62. The expression vector according to any one of embodiments 58 to 61, characterized in that the expression cassettes are arranged in the order LC-HC-SM.

[0824] 63. The expression vector according to any one of embodiments 58 to 62, characterized in that the human elongation factor 1α promoter contains intron A.

[0825] 64. The expression vector according to any one of embodiments 58 to 63, characterized in that the expression vector does not contain any transcription terminator sequence.

[0826] 65. The expression vector of embodiment 64, characterized in that the terminator sequence is an hGT sequence.

[0827] 66. Use of an expression vector comprising:

[0828] a first expression cassette comprising, in 5' to 3' direction, a first promoter, a nucleic acid encoding an antibody light chain, a first polyA signal sequence, and optionally a first transcription terminator sequence;

[0829] a second expression cassette comprising, in 5' to 3' direction, a second promoter, the nucleic acid encoding the antibody heavy chain, a second polyA signal sequence, and optionally a second transcription terminator sequence; and

[0830] a third expression cassette comprising, in 5' to 3' direction, a third promoter, a nucleic acid conferring resistance to a selection agent, a third polyA signal sequence, and optionally a third transcription terminator sequence;

[0831] Thereby the expression cassettes are bidirectionally organized, whereby the first expression cassette and the second expression cassette are arranged in opposite directions.

[0832] 67. The use according to embodiment 66, characterized in that the nucleic acid encoding the antibody light chain and / or the nucleic acid encoding the antibody heavy chain comprises at least one intron.

[0833] 68. The use according to any one of embodiments 66 to 67, characterized in that the nucleic acid encoding the antibody light chain and / or the nucleic acid encoding the antibody heavy chain is cDNA.

[0834] 69. Use according to any one of embodiments 66 to 68, characterized in that the first and second promoters are hCMV promoters, the first and second polyA signal sequences are bGH polyA signal sequences, and the transcription terminator sequence is present and is an hGT terminator sequence.

[0835] 70. Use according to any one of embodiments 66 to 69, characterized in that the first and second promoters are hEF1α promoters, the first and second polyA signal sequences are bGH polyA signal sequences, and the expression cassette does not contain a transcription terminator sequence.

[0836] 71. The use according to any one of embodiments 66 to 69, characterized in that the mammalian cells are selected from CHO cells, HEK cells, BHK cells, NSO cells and SP2 / 0 cells.

[0837] 72. The use according to any one of embodiments 66 to 71, characterized in that the mammalian cells are HEK cells.

[0838] 73. The use according to any one of embodiments 66 to 72, characterized in that the expression vector comprises:

[0839] a first expression cassette comprising, in 5' to 3' direction, an hCMV promoter, a nucleic acid encoding a first antibody light chain, a bGH polyA signal sequence, and an hGT terminator sequence;

[0840] a second expression cassette comprising, in 5' to 3' direction, an hCMV promoter, a nucleic acid encoding a second antibody light chain, a bGH polyA signal sequence, and an hGT terminator sequence;

[0841] a third expression cassette comprising, in 5' to 3' direction, an hCMV promoter, the nucleic acid encoding the heavy chain of the first antibody, a bGH polyA signal sequence, and an hGT terminator sequence;

[0842] a fourth expression cassette comprising, in 5' to 3' direction, an hCMV promoter, the nucleic acid encoding the second antibody heavy chain, the bGH polyA signal sequence, and the hGT terminator sequence;

[0843] or

[0844] a first expression cassette comprising, in 5' to 3' direction, an hCMV promoter, a nucleic acid encoding an antibody light chain, a bGH polyA signal sequence, and an hGT terminator sequence;

[0845] - a second expression cassette comprising, in 5' to 3' direction, an hCMV promoter, the nucleic acid encoding the heavy chain of the first antibody, a bGH polyA signal sequence, and an hGT terminator sequence; and

[0846] a third expression cassette comprising, in 5' to 3' direction, an hCMV promoter, the nucleic acid encoding the second antibody heavy chain, the bGH polyA signal sequence, and the hGT terminator sequence;

[0847] The antibody light chain is thus the common light chain of two antibody heavy chains.

[0848] 74. The use according to any one of embodiments 66 to 73, characterized in that the expression vector encodes a bispecific antibody.

[0849] 75. The use of any one of embodiments 66 to 74, characterized in that the bispecific antibody has a first binding specificity or binding site that specifically binds to a first antigen or a first epitope on an antigen, and the bispecific antibody has a second binding specificity or binding site that specifically binds to a second antigen or a second epitope on an antigen.

[0850] 76. The use according to any one of embodiments 66 to 75, characterized in that the expression vector comprises:

[0851] -Antibody light chain expression cassette;

[0852] -First antibody heavy chain expression cassette;

[0853] - a second antibody heavy chain expression cassette; and

[0854] - Selectable marker expression cassette.

[0855] 77. The use according to any one of embodiments 66 to 76, characterized in that the expression vector comprises:

[0856] -First antibody light chain expression cassette;

[0857] - Second antibody light chain expression cassette;

[0858] -First antibody heavy chain expression cassette;

[0859] - a second antibody heavy chain expression cassette; and

[0860] - Selectable marker expression cassette.

[0861] 78. The use according to any one of embodiments 66 to 77, characterized in that one of the antibody heavy chain expression cassettes encodes an antibody heavy chain comprising a hole mutation.

[0862] 79. The use according to any one of embodiments 66 to 78, characterized in that one of the antibody heavy chain expression cassettes encodes an antibody heavy chain comprising a knob mutation.

[0863] 80. The use of any one of embodiments 66 to 79, characterized in that one of the antibody light chain expression cassettes encodes an antibody light chain variant comprising an antibody light chain variable domain and an antibody heavy chain CH1 domain as a constant domain, and / or one of the antibody light chain expression cassettes encodes an antibody light chain comprising an antibody light chain variable domain and an antibody light chain CL domain as a constant domain.

[0864] 81. The use of any one of embodiments 66 to 80, characterized in that one of the antibody heavy chain expression cassettes encodes an antibody heavy chain variant comprising an antibody light chain constant domain (CL) as the first constant domain, and / or one of the antibody heavy chain expression cassettes encodes an antibody heavy chain comprising an antibody heavy chain CH1 domain as the first constant domain.

[0865] 82. An expression vector comprising:

[0866] a first expression cassette comprising, in 5' to 3' direction, a first promoter, a nucleic acid encoding an antibody light chain, a first polyA signal sequence, and optionally a first transcription terminator sequence;

[0867] a second expression cassette comprising, in 5' to 3' direction, a second promoter, the nucleic acid encoding the antibody heavy chain, a second polyA signal sequence, and optionally a second transcription terminator sequence; and

[0868] a third expression cassette comprising, in 5' to 3' direction, a third promoter, a nucleic acid conferring resistance to a selection agent, a third polyA signal sequence, and optionally a third transcription terminator sequence;

[0869] Thereby the expression cassettes are bidirectionally organized, whereby the first expression cassette and the second expression cassette are arranged in opposite directions.

[0870] 83. The expression vector of embodiment 82, characterized in that the nucleic acid encoding the antibody light chain and / or the nucleic acid encoding the antibody heavy chain comprises at least one intron.

[0871] 84. The expression vector of embodiment 82 or 83, characterized in that the nucleic acid encoding the antibody light chain and / or the nucleic acid encoding the antibody heavy chain is cDNA.

[0872] 85. The expression vector according to any one of embodiments 82 to 84, characterized in that the first and second promoters are hCMV promoters, the first and second polyA signal sequences are bGH polyA signal sequences, and the transcription terminator sequence is present and is an hGT terminator sequence.

[0873] 86. The expression vector according to any one of embodiments 82 to 84, characterized in that the first and second promoters are hEF1α promoters, the first and second polyA signal sequences are bGH polyA signal sequences, and the expression cassette does not contain a transcription terminator sequence.

[0874] 87. An expression plasmid comprising, in 5' to 3' direction, a promoter sequence, a nucleic acid encoding an antibody heavy chain or an antibody light chain, an IRES element, a nucleic acid sequence encoding a selectable marker, and a polyA signal sequence, whereby the IRES element is an EMCV-IRES element.

[0875] 88. Use of an expression cassette comprising, in 5' to 3' direction, a promoter sequence, a nucleic acid encoding an antibody heavy chain or an antibody light chain, an IRES element, a nucleic acid sequence encoding a selectable marker and a polyA signal sequence for selecting antibody-producing cells, wherein the IRES element is an EMCV-IRES element.

[0876] 89. A method for selecting eukaryotic cells expressing an antibody, comprising the steps of:

[0877] - culturing a eukaryotic cell comprising i) an expression plasmid of embodiment 87 and ii) a nucleic acid encoding another antibody chain not encoded by the expression plasmid of embodiment 87;

[0878] - selecting cells expressing said detectable polypeptide.

[0879] 90. An expression plasmid comprising, in 5' to 3' direction, a promoter sequence, a nucleic acid encoding an antibody light chain, an IRES element, a nucleic acid sequence encoding an antibody heavy chain, and a polyA signal sequence, wherein the IRES element is an EV71-IRES element.

[0880] 91. Use of an expression plasmid comprising, in 5' to 3' direction, a promoter sequence, a nucleic acid encoding an antibody light chain, an IRES element, a nucleic acid sequence encoding an antibody heavy chain and a polyA signal sequence, for expressing an antibody, wherein the IRES element is an EV71-IRES element.

[0881] 92. A method for selecting eukaryotic cells expressing an antibody, comprising the steps of:

[0882] - culturing a eukaryotic cell comprising i) an expression plasmid of embodiment 90 and ii) a nucleic acid encoding another antibody chain not encoded by the expression plasmid of embodiment 90;

[0883] - selecting cells expressing said detectable polypeptide.

Claims

1. Use of an expression vector comprising the following for transient recombinant production of an antibody in mammalian cells: - a first expression cassette comprising, in the 5' to 3' direction, a first promoter, a nucleic acid encoding an antibody light chain, a first polyA signal sequence, and optionally a first transcription terminator sequence; - a second expression cassette comprising, in the 5' to 3' direction, a second promoter, a nucleic acid encoding an antibody heavy chain, a second polyA signal sequence, and optionally a second transcription terminator sequence; and - a third expression cassette comprising, in the 5' to 3' direction, a third promoter, a nucleic acid conferring resistance to a selection agent, a third polyA signal sequence, and optionally a third transcription terminator sequence; wherein the expression cassettes are organized bidirectionally, and wherein the first expression cassette and the second expression cassette are arranged in opposite directions, wherein the first and second promoters are hCMV promoters, the first and second polyA signal sequences are bGH polyA signal sequences, and the transcription terminator sequence is present and is an hGT terminator sequence; or wherein the first and second promoters are hEF1α promoters, the first and second polyA signal sequences are bGH polyA signal sequences, and the expression cassette does not contain a transcription terminator sequence.

2. The use according to claim 1, characterized in that the nucleic acid encoding the antibody light chain and / or the nucleic acid encoding the antibody heavy chain comprises at least one intron.

3. The use according to claim 1, characterized in that the nucleic acid encoding the antibody light chain and / or the nucleic acid encoding the antibody heavy chain is cDNA.

4. The use according to claim 1, characterized in that the first and second promoters are hCMV promoters, the first and second polyA signal sequences are bGH polyA signal sequences, and the transcription terminator sequence is present and is an hGT terminator sequence.

5. The use according to claim 1, characterized in that the first and second promoters are hEF1α promoters, the first and second polyA signal sequences are bGH polyA signal sequences, and the expression cassette does not contain a transcription terminator sequence.

6. The use according to claim 1, characterized in that the mammalian cells are selected from CHO cells, HEK cells, BHK cells, NS0 cells, and SP2 / 0 cells.

7. The use according to claim 1, characterized in that the mammalian cells are HEK cells.

8. The use according to claim 4, characterized in that the expression vector comprises: - a first expression cassette comprising, in the 5' to 3' direction, an hCMV promoter, a nucleic acid encoding a first antibody light chain, a bGH polyA signal sequence, and an hGT terminator sequence; - a second expression cassette comprising, in the 5' to 3' direction, an hCMV promoter, a nucleic acid encoding a second antibody light chain, a bGH polyA signal sequence, and an hGT terminator sequence; - a third expression cassette comprising, in the 5' to 3' direction, an hCMV promoter, a nucleic acid encoding a first antibody heavy chain, a bGH polyA signal sequence, and an hGT terminator sequence; - A fourth expression cassette containing an hCMV promoter, a nucleic acid encoding the heavy chain of a second antibody, a bGH polyA signal sequence, and an hGT terminator sequence in the 5' to 3' direction; or - A first expression cassette containing an hCMV promoter, a nucleic acid encoding the light chain of an antibody, a bGH polyA signal sequence, and an hGT terminator sequence in the 5' to 3' direction; - A second expression cassette containing an hCMV promoter, a nucleic acid encoding the heavy chain of a first antibody, a bGH polyA signal sequence, and an hGT terminator sequence in the 5' to 3' direction; and - A third expression cassette containing an hCMV promoter, a nucleic acid encoding the heavy chain of a second antibody, a bGH polyA signal sequence, and an hGT terminator sequence in the 5' to 3' direction; wherein the antibody light chain is the common light chain of the two antibody heavy chains.

9. Use according to claim 1, characterized in that the expression vector encodes a bispecific antibody.

10. Use according to claim 9, characterized in that the bispecific antibody has a first binding specificity or binding site that specifically binds to a first antigen or a first epitope on an antigen, and the bispecific antibody has a second binding specificity or binding site that specifically binds to a second antigen or a second epitope on an antigen.

11. Use according to any one of claims 1 to 10, characterized in that the expression vector comprises: - An antibody light chain expression cassette; - A first antibody heavy chain expression cassette; - A second antibody heavy chain expression cassette; and - A selectable marker expression cassette.

12. Use according to any one of claims 1 to 10, characterized in that the expression vector comprises: - A first antibody light chain expression cassette; - A second antibody light chain expression cassette; - A first antibody heavy chain expression cassette; - A second antibody heavy chain expression cassette; and - A selectable marker expression cassette.

13. Use according to claim 11, characterized in that one of the antibody heavy chain expression cassettes encodes an antibody heavy chain containing a epsilon mutation.

14. Use according to claim 12, characterized in that one of the antibody heavy chain expression cassettes encodes an antibody heavy chain containing a epsilon mutation.

15. Use according to claim 11, characterized in that one of the antibody heavy chain expression cassettes encodes an antibody heavy chain containing a kappa mutation.

16. Use according to claim 12, characterized in that one of the antibody heavy chain expression cassettes encodes an antibody heavy chain containing a kappa mutation.

17. Use according to claim 12, characterized in that one of the antibody light chain expression cassettes encodes an antibody light chain variant containing an antibody light chain variable domain and an antibody heavy chain CH1 domain as a constant domain, and / or one of the antibody light chain expression cassettes encodes an antibody light chain containing an antibody light chain variable domain and an antibody light chain CL domain as a constant domain.

18. Use according to claim 12, characterized in that one of the antibody heavy chain expression cassettes encodes an antibody heavy chain variant containing an antibody light chain constant domain (CL) as a first constant domain, and / or one of the antibody heavy chain expression cassettes encodes an antibody heavy chain containing an antibody heavy chain CH1 domain as a first constant domain.

19. An expression vector, which comprises: - A first expression cassette comprising, in the 5' to 3' direction, a first promoter, a nucleic acid encoding an antibody light chain, a first polyA signal sequence, and optionally a first transcription terminator sequence; - A second expression cassette comprising, in the 5' to 3' direction, a second promoter, a nucleic acid encoding an antibody heavy chain, a second polyA signal sequence, and optionally a second transcription terminator sequence; and - A third expression cassette comprising, in the 5' to 3' direction, a third promoter, a nucleic acid conferring resistance to a selection agent, a third polyA signal sequence, and optionally a third transcription terminator sequence; wherein the expression cassettes are organized bidirectionally, and wherein the first expression cassette and the second expression cassette are arranged in opposite directions, wherein the first and second promoters are hCMV promoters, the first and second polyA signal sequences are bGH polyA signal sequences, and the transcription terminator sequence is present and is the hGT terminator sequence; or wherein the first and second promoters are hEF1α promoters, the first and second polyA signal sequences are bGH polyA signal sequences, and the expression cassette does not contain a transcription terminator sequence.

20. The expression vector of claim 19, wherein the nucleic acid encoding the antibody light chain and / or the nucleic acid encoding the antibody heavy chain comprises at least one intron.

21. The expression vector of claim 19, wherein the nucleic acid encoding the antibody light chain and / or the nucleic acid encoding the antibody heavy chain is cDNA.

22. The expression vector of claim 19, wherein the first and second promoters are hCMV promoters, the first and second polyA signal sequences are bGH polyA signal sequences, and the transcription terminator sequence is present and is the hGT terminator sequence.

23. The expression vector of claim 19, wherein the first and second promoters are hEF1α promoters, the first and second polyA signal sequences are bGH polyA signal sequences, and the expression cassette does not contain a transcription terminator sequence.

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