Expression vector element combinations, novel production cell production methods and their use in the recombinant production of polypeptides
阅读说明：本技术 表达载体元件组合、新的生产用细胞产生方法及其在重组产生多肽中的用途 (Expression vector element combinations, novel production cell production methods and their use in the recombinant production of polypeptides ) 是由 P·M·许尔斯曼 H·克内根 于 2012-12-19 设计创作，主要内容包括：本文报道,对于瞬时转染,人延伸因子1α启动子(含有内含子A)的使用提供增强的生产力(在LC-HC-SM组织中),与使用SV40polyA信号序列相比,牛生长激素polyA信号序列的使用提供增强的生产力,在包含hCMV启动子的载体中,向bGH PolyA信号序列加入HGT产生提高的生产力,载体组织LC(3`-5′)-HC-SM产生改善的表达。对于稳定库,据报道,用包含hEF1α启动子的载体产生的库在分批分析中显示增强的生产力,用包含hEF1α启动子的载体产生的克隆显示减少的低产克隆数,且用包含hEF1α启动子的载体产生的克隆显示更高的IgG表达稳定性。对于单克隆,据报道,选择标记放置在下游的载体组织(LC-HC-SM)对单克隆的生产力具有积极作用,且用包含bGH polyA信号序列和hGT的载体产生的克隆具有更高的生产力。(Herein is reported that for transient transfection, the use of the human elongation factor 1 alpha promoter (containing intron a) provides enhanced productivity (in LC-HC-SM organization), the use of the bovine growth hormone polyA signal sequence provides enhanced productivity compared to the use of the SV40polyA signal sequence, the addition of HGT to the bGH polyA signal sequence in a vector comprising the hCMV promoter results in enhanced productivity, and the vector organization LC (3 '-5') -HC-SM results in improved expression. For stable pools, it was reported that pools produced with vectors containing the hEF1 α promoter showed enhanced productivity in batch analysis, clones produced with vectors containing the hEF1 α promoter showed reduced numbers of low-producing clones, and clones produced with vectors containing the hEF1 α promoter showed higher stability of IgG expression. For the monoclonal, it was reported that the vector organization (LC-HC-SM) with the selectable marker placed downstream had a positive effect on the productivity of the monoclonal, and that clones generated with the vector containing the bGH polyA signal sequence and hGT had higher productivity.)
1. A method for selecting a recombinant transiently transfected mammalian cell, comprising the steps of:
a) transfecting a mammalian cell with an expression vector comprising:
-a first expression cassette comprising in 5 'to 3' direction the hEF 1a promoter, a nucleic acid encoding an antibody light chain and a bGH polyA signal sequence;
-a second expression cassette comprising in 5 'to 3' direction the hEF1 α promoter, a nucleic acid encoding the heavy chain of an antibody and a bGH polyA signal sequence; and
the first expression cassette and the second expression cassette are arranged bi-directionally for selection of transiently transfected cells,
thereby obtaining a plurality of recombinant mammalian cells;
b) (iii) selecting (single) transiently transfected recombinant mammalian cells from the plurality of recombinant mammalian cells.
2. The method of claim 1, characterized in that said nucleic acid encoding the light chain of the antibody and/or said nucleic acid encoding the heavy chain of the antibody comprises at least one intron.
3. Method according to claim 1 or 2, characterized in that said nucleic acid encoding the light chain of an antibody and/or said nucleic acid encoding the heavy chain of an antibody is a cDNA.
4. Method according to any one of claims 1 to 3, characterized in that the expression plasmid further comprises a selection marker.
5. Method according to any one of claims 1 to 4, characterized in that the expression cassettes are arranged in LC-HC-SM order.
6. Method according to any one of claims 1 to 5, characterized in that the human elongation factor 1 alpha promoter contains intron A.
7. Method according to any one of claims 1 to 6, characterized in that the expression vector does not contain any transcription terminator sequence.
8. The method of claim 7, wherein said terminator sequence is the hGT sequence.
9. Method according to any one of claims 1 to 8, characterized in that the mammalian cells are selected from the group consisting of CHO cells, HEK cells, BHK cells, NS0 cells and SP2/0 cells.
10. Method according to claim 9, characterized in that the mammalian cells are HEK cells for selection of transiently transfected cells.
11. A method for producing an antibody comprising the steps of:
a) culturing a mammalian cell comprising the following transient transfections:
-a first expression cassette comprising in 5 'to 3' direction the hEF 1a promoter, a nucleic acid encoding an antibody light chain and a bGH polyA signal sequence;
-a second expression cassette comprising in 5 'to 3' direction the hEF1 α promoter, a nucleic acid encoding the heavy chain of an antibody and a bGH polyA signal sequence; and
the first expression cassette and the second expression cassette are arranged bi-directionally for selection of transiently transfected cells,
b) recovering the antibody from the transiently transfected cells or culture medium.
12. Method according to claim 11, characterized in that said nucleic acid encoding the light chain of an antibody and/or said nucleic acid encoding the heavy chain of an antibody comprise at least one intron.
13. Method according to claim 11 or 12, characterized in that said nucleic acid encoding the light chain of an antibody and/or said nucleic acid encoding the heavy chain of an antibody is a cDNA.
14. Method according to any one of claims 11 to 13, characterized in that the expression plasmid further comprises a selection marker.
15. Method according to any one of claims 11 to 14, characterized in that the expression cassettes are arranged in LC-HC-SM order.
16. Method according to any one of claims 11 to 15, characterized in that the human elongation factor 1 α promoter contains intron a.
17. Method according to any one of claims 11 to 16, characterized in that the expression vector does not contain any transcription terminator sequence.
18. The method of claim 17, wherein said terminator sequence is the hGT sequence.
19. Method according to any one of claims 11 to 18, characterized in that the mammalian cells are selected from the group consisting of CHO cells, HEK cells, BHK cells, NS0 cells and SP2/0 cells.
20. The method of claim 19, characterized in that the mammalian cells are HEK cells for transient antibody production.
21. An expression vector comprising:
-a first expression cassette comprising in 5 'to 3' direction the hEF 1a promoter, a nucleic acid encoding an antibody light chain and a bGH polyA signal sequence;
-a second expression cassette comprising in 5 'to 3' direction the hEF1 alpha promoter, a nucleic acid encoding the heavy chain of an antibody and a bGH polyA signal sequence,
the first expression cassette and the second expression cassette are arranged bi-directionally for selection of transiently transfected cells.
22. The expression vector of claim 21, characterized in that said nucleic acid encoding an antibody light chain and/or said nucleic acid encoding an antibody heavy chain comprises at least one intron.
23. The expression vector according to claim 21 or 22, characterized in that said nucleic acid encoding the antibody light chain and/or said nucleic acid encoding the antibody heavy chain is a cDNA.
24. The expression vector according to any one of claims 21 to 23, characterized in that the expression plasmid further comprises a selectable marker.
25. The expression vector according to any one of claims 21 to 24, characterized in that the expression cassettes are arranged in LC-HC-SM order.
26. The expression vector according to any one of claims 21 to 25, characterized in that the human elongation factor 1 alpha promoter contains intron a.
27. The expression vector according to any one of claims 21 to 26, characterized in that the expression vector does not contain any transcription terminator sequence.
28. The expression vector of claim 27, characterized in that the terminator sequence is the hGT sequence.
Technical Field
Herein is reported a novel combination of expression vector elements such as promoters, polyA signal sequences and transcription terminators, expression vector tissues, combinations thereof, as well as novel methods for generating production cell lines, such as novel transfection or selection methods, and the use of these expression vectors and production cell lines for the recombinant production of polypeptides of interest.
Background
The level of transcription of a gene can have a strong influence on its expression level and thus determines the productivity of the cell. It is mainly affected by three vector elements: a promoter, a polyA signal sequence, and (if present) a transcription terminator.
The nucleic acid encoding the heavy chain of an antibody typically comprises a leader sequence (signal sequence) (about 57bp/19aa), a variable region VH (about 350bp/115aa) and a constant region CH (about 990bp/330aa) removed during protein maturation. The nucleic acid encoding the light chain of an antibody usually consists of a leader sequence (about 66bp/22aa), a variable region VK or VL (about 350bp/115aa) and a constant region CK or CL (about 321bp/107aa) which are removed during maturation of the protein.
Recombinant production of antibodies in eukaryotic cells involves the production 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 a polyadenylation (polyA) region. Likewise, a heavy chain expression cassette is produced comprising a heavy chain encoding nucleic acid flanked by a promoter and a polyA region. The heavy chain expression cassette may be combined into the light chain expression cassette in a single vector containing both heavy and light chain expression cassettes, or may be integrated into two separate vectors.
Immunoglobulin DNA cassette molecules, monomeric constructs, methods of production and methods of use thereof are reported in US 7,053,202. In US 5,168,062, transfer vectors and microorganisms containing human cytomegalovirus immediate early promoter regulatory DNA sequences are reported. In US 5,225,348, a DNA fragment containing a promoter region of human polypeptide chain elongation factor-1 α, its base sequence, and an expression plasmid containing the DNA fragment highly suitable for a wide range of host cells having high expression ability are reported. In US 5,266,491, an expression plasmid containing a SV40 origin of replication and a DNA fragment having a promoter region of a human polypeptide chain elongation factor-1 α gene is reported. Expression of recombinant DNA compounds and polypeptides such as tPA is reported in US 5,122,458. In US 7,422,874, expression vectors for animal cells are reported.
Sanna Pietro, p. reports the expression of antibody Fab fragments and whole immunoglobulins in mammalian cells (meth. mol. biol.178(2002) 389-. Higuchi, K. et al (J.Immunol. meth.202(1997)193-204) report cell display libraries for gene cloning of the variable regions of human antibodies against hepatitis B surface antigen. Kim, D. reports a mammalian expression system modified by manipulation of the transcription termination region (Biotechnol. progress 19(2003) 1620-. Costa, r.a. et al (eur.j. pharmaceut.biopharmaceut.74(2010)127-138) report guidance on cell engineering for monoclonal antibody production. Kim, D.W. et al reported the human elongation factor 1. alpha. promoter as a universal and efficient expression system (Gene 91(1990) 217-223). Buchman, A.R. 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 clinical, Wiley (2009) 557) -572). Li et al (j. immunological. meth.318(2007)113-124) reported comparative studies of different vector designs for mammalian expression of recombinant IgG antibodies. Ho, S.C.L. et al report on methods for enhancing the production of high monoclonal antibody expression CHO cell linesIRES-mediated tricaistronic vectors (J.Biotechnol.157(2011) 130-139). Hotta, A. et al (J.biosci.Bioeng.98(2004)298-303) report the production of anti-CD 2 chimeric antibodies by recombinant animal cells. Lee, J-C, et al report high-level protein expression mediated by sugar entry sites within enterovirus 71 (Biotechnol. Bioeng.90(2005) 656-. In WO 2008/142124, Avian is reportedRecombinant protein production in a cell.
Summary of The Invention
It has been found that the performance of an expression vector is largely dependent on its intended use, with the best vectors for transient transfection, stable pools and monoclonal selection differing.
To highlight the main findings: for transient transfection, bidirectional expression of antibody light and heavy chains and use of a full-length hCMV promoter including intron a are advantageous. However, for stable transfection, it has been shown that a row arrangement of 1) antibody light chain, 2) antibody heavy chain and 3) selectable marker is advantageous.
Although the hEF 1a promoter was clearly superior to the hCMV promoter in the stable pool, a clear opposite effect on the monoclonal level was found. Here, the highest productivity was obtained with the human cytomegalovirus immediate early promoter/enhancer (hCMV) clone.
Furthermore, 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.
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, leads to a higher number of antibody producing/secreting cell clones after 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 α promoter (hEF1 α), the polyA signal sequence is the bovine growth hormone polyA signal sequence (bGH polyA), and the terminator sequence is absent.
By using the above expression vectors, a higher number 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.
Thus, one aspect as reported herein is a method for selecting a recombinant mammalian cell comprising the steps of:
a) transfecting a mammalian cell with an expression vector comprising
A first expression cassette comprising in the 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,
-a second expression cassette comprising in the 5 'to 3' direction an hCMV promoter, a nucleic acid encoding the heavy chain of an antibody, a bGH polyA signal sequence and an hGT terminator sequence, and
thereby obtaining a plurality of recombinant mammalian cells,
b) (individual) recombinant mammalian cells are selected from the plurality of recombinant mammalian cells.
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.
In one embodiment, the nucleic acid encoding the antibody light chain and/or the nucleic acid encoding the antibody heavy chain is cDNA.
In one embodiment, the first expression cassette and the second expression cassette are arranged unidirectionally for selection of stably transfected cells.
In one embodiment, the first expression cassette and the second expression cassette are arranged bi-directionally for selection of transiently transfected cells.
In one embodiment, the expression plasmid further comprises a selectable marker. In one embodiment, the expression cassette and the selectable marker are arranged unidirectionally. In one embodiment, the expression cassettes are arranged in LC-HC-SM order.
In one embodiment, the mammalian cell is selected from the group consisting of a CHO cell, a HEK cell, a BHK cell, a NS0 cell, and a SP2/0 cell. In one embodiment, the mammalian cell is a CHO cell used to select for stably transfected cells. In one embodiment, the mammalian cell is a HEK cell used to select for transiently transfected cells.
One aspect as reported herein is a method for producing an antibody comprising the steps of:
a) culturing a mammalian cell comprising
A first expression cassette comprising in the 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,
-a second expression cassette comprising in the 5 'to 3' direction an hCMV promoter, a nucleic acid encoding the heavy chain of an antibody, a bGH polyA signal sequence and an hGT terminator sequence, and
b) recovering the antibody from the cell or the culture medium.
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.
In one embodiment, the nucleic acid encoding the antibody light chain and/or the nucleic acid encoding the antibody heavy chain is cDNA.
In one embodiment, the first expression cassette and the second expression cassette are arranged unidirectionally for stable production of the antibody.
In one embodiment, the first expression cassette and the second expression cassette are arranged bi-directionally for transient antibody production.
In one embodiment, the expression plasmid further comprises a selectable marker. In one embodiment, the expression cassette and the selectable marker are arranged unidirectionally. In one embodiment, the expression cassettes are arranged in LC-HC-SM order.
In one embodiment, the mammalian cell is selected from the group consisting of a CHO cell, a HEK cell, a BHK cell, a NS0 cell, and a SP2/0 cell. In one embodiment, the mammalian cell is a CHO cell for stable production of antibodies. In one embodiment, the mammalian cell is a HEK cell for transient antibody production.
One aspect as reported herein is an expression vector comprising:
a first expression cassette comprising in the 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,
-a second expression cassette comprising in the 5 'to 3' direction an hCMV promoter, a nucleic acid encoding the heavy chain of an antibody, a bGH polyA signal sequence and an hGT terminator sequence.
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.
In one embodiment, the nucleic acid encoding the antibody light chain and/or the nucleic acid encoding the antibody heavy chain is cDNA.
In one embodiment, the first expression cassette and the second expression cassette are arranged unidirectionally for selection of stably transfected cells.
In one embodiment, the first expression cassette and the second expression cassette are arranged bi-directionally for selection of transiently transfected cells.
In one embodiment, the expression plasmid further comprises a selectable marker. In one embodiment, the expression cassette and the selectable marker are arranged unidirectionally. In one embodiment, the expression cassettes are arranged in LC-HC-SM order.
It has been found that the presence of the hGT terminator sequence reduces the obtainable expression yield when the human elongation factor 1 alpha promoter (hEF1 alpha) is used in combination with the bGH polyA signal sequence for the production of stable recombinant antibody expressing/secreting cell lines.
One aspect as reported herein is a method for selecting a recombinant mammalian cell comprising the steps of:
a) transfecting a mammalian cell with an expression vector comprising
-a first expression cassette comprising in 5 'to 3' direction the hEF1 alpha promoter, a nucleic acid encoding the light chain of an antibody and a bGH polyA signal sequence,
-a second expression cassette comprising in 5 'to 3' direction the hEF1 alpha promoter, a nucleic acid encoding the heavy chain of an antibody and a bGH polyA signal sequence, and
thereby obtaining a plurality of recombinant mammalian cells,
b) (individual) recombinant mammalian cells are selected from the plurality of recombinant mammalian cells.
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.
In one embodiment, the nucleic acid encoding the antibody light chain and/or the nucleic acid encoding the antibody heavy chain is cDNA.
In one embodiment, the first expression cassette and the second expression cassette are arranged unidirectionally for selection of stably transfected cells.
In one embodiment, the first expression cassette and the second expression cassette are arranged bi-directionally for selection of transiently transfected cells.
In one embodiment, the expression plasmid further comprises a selectable marker. In one embodiment, the expression cassette and the selectable marker are arranged unidirectionally. In one embodiment, the expression cassettes are arranged in LC-HC-SM order.
In one embodiment, the human elongation factor 1 α promoter comprises intron a.
In one embodiment, the expression vector does not contain any transcription terminator sequences. In one embodiment, the terminator sequence is the hGT sequence.
In one embodiment, the mammalian cell is selected from the group consisting of a CHO cell, a HEK cell, a BHK cell, a NS0 cell, and a SP2/0 cell. In one embodiment, the mammalian cell is a CHO cell used to select for stably transfected cells. In one embodiment, the mammalian cell is a HEK cell used to select for transiently transfected cells.
One aspect as reported herein is a method for producing an antibody comprising the steps of:
a) culturing a mammalian cell comprising
-a first expression cassette comprising in 5 'to 3' direction the hEF1 alpha promoter, a nucleic acid encoding the light chain of an antibody and a bGH polyA signal sequence,
-a second expression cassette comprising in 5 'to 3' direction the hEF1 alpha promoter, a nucleic acid encoding the heavy chain of an antibody and a bGH polyA signal sequence, and
b) recovering the antibody from the cell or the culture medium.
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.
In one embodiment, the nucleic acid encoding the antibody light chain and/or the nucleic acid encoding the antibody heavy chain is cDNA.
In one embodiment, the first expression cassette and the second expression cassette are arranged unidirectionally for selection of stably transfected cells.
In one embodiment, the first expression cassette and the second expression cassette are arranged bi-directionally for selection of transiently transfected cells.
In one embodiment, the expression plasmid further comprises a selectable marker. In one embodiment, the expression cassette and the selectable marker are arranged unidirectionally. In one embodiment, the expression cassettes are arranged in LC-HC-SM order.
In one embodiment, the human elongation factor 1 α promoter comprises intron a.
In one embodiment, the expression vector does not contain any transcription terminator sequences. In one embodiment, the terminator sequence is the hGT sequence.
In one embodiment, the mammalian cell is selected from the group consisting of a CHO cell, a HEK cell, a BHK cell, a NS0 cell, and a SP2/0 cell. In one embodiment, the mammalian cell is a CHO cell for stable production of antibodies. In one embodiment, the mammalian cell is a HEK cell for transient antibody production.
One aspect as reported herein is an expression vector comprising:
-a first expression cassette comprising in 5 'to 3' direction the hEF1 alpha promoter, a nucleic acid encoding the light chain of an antibody and a bGH polyA signal sequence, and
-a second expression cassette comprising in 5 'to 3' direction the hEF1 α promoter, a nucleic acid encoding the heavy chain of an antibody and a bGH polyA signal sequence.
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.
In one embodiment, the nucleic acid encoding the antibody light chain and/or the nucleic acid encoding the antibody heavy chain is cDNA.
In one embodiment, the first expression cassette and the second expression cassette are arranged unidirectionally for selection of stably transfected cells.
In one embodiment, the first expression cassette and the second expression cassette are arranged bi-directionally for selection of transiently transfected cells.
In one embodiment, the expression plasmid further comprises a selectable marker. In one embodiment, the expression cassette and the selectable marker are arranged unidirectionally. In one embodiment, the expression cassettes are arranged in LC-HC-SM order.
In one embodiment, the human elongation factor 1 α promoter comprises intron a.
In one embodiment, the expression vector does not contain any transcription terminator sequences. In one embodiment, the terminator sequence is the hGT sequence.
It has been found that for stable recombinant production of antibodies, the use of an expression vector comprising:
a first expression cassette comprising in the 5 'to 3' direction a first promoter, a nucleic acid encoding a light chain of an antibody, a first polyA signal sequence and optionally a first transcription terminator sequence,
-a second expression cassette comprising in 5 'to 3' direction a second promoter, a nucleic acid encoding the heavy chain of the antibody, 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,
whereby the three expression cassettes are organized unidirectionally and in the order of first expression cassette-second expression cassette-third expression cassette.
In contrast to the above, it has been found that for transient recombinant production of antibodies, the use of an expression vector comprising:
a first expression cassette comprising in the 5 'to 3' direction a first promoter, a nucleic acid encoding a light chain of an antibody, a first polyA signal sequence and optionally a first transcription terminator sequence,
-a second expression cassette comprising in 5 'to 3' direction a second promoter, a nucleic acid encoding the heavy chain of the antibody, 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,
whereby the expression cassettes are organized bidirectionally, whereby the first expression cassette and the second expression cassette are arranged in opposite directions.
The term "in the opposite direction" means that one expression cassette is transcribed in the 5'- > 3' direction and one expression cassette is transcribed in the 3 '- > 5' direction.
Thus, one aspect as reported herein is the use of an expression vector comprising:
a first expression cassette comprising in the 5 'to 3' direction a first promoter, a nucleic acid encoding a light chain of an antibody, a first polyA signal sequence and optionally a first transcription terminator sequence,
-a second expression cassette comprising in 5 'to 3' direction a second promoter, a nucleic acid encoding the heavy chain of the antibody, 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,
whereby the three expression cassettes are organized unidirectionally and in the order of first expression cassette-second expression cassette-third expression cassette.
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.
In one embodiment, the nucleic acid encoding the antibody light chain and/or the nucleic acid encoding the antibody heavy chain is cDNA.
In one embodiment, the first and second promoters are hCMV promoters, the first and second polyA signal sequences are bGH polyA signal sequences, the transcription termination sequence is present and is an hGT terminator sequence.
In one embodiment, the first and second promoters are hEF 1a promoters, the first and second polyA signal sequences are bGH polyA signal sequences, and the expression cassette does not contain a transcription terminator sequence.
In one embodiment, the mammalian cell is selected from the group consisting of a CHO cell, a HEK cell, a BHK cell, a NS0 cell, and a SP2/0 cell. In one embodiment, the mammalian cell is a CHO cell.
One aspect as reported herein is an expression vector comprising:
a first expression cassette comprising in the 5 'to 3' direction a first promoter, a nucleic acid encoding a light chain of an antibody, a first polyA signal sequence and optionally a first transcription terminator sequence,
-a second expression cassette comprising in 5 'to 3' direction a second promoter, a nucleic acid encoding the heavy chain of the antibody, 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,
whereby the three expression cassettes are organized unidirectionally and in the order of first expression cassette-second expression cassette-third expression cassette.
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.
In one embodiment, the nucleic acid encoding the antibody light chain and/or the nucleic acid encoding the antibody heavy chain is cDNA.
In one embodiment, the first and second promoters are hCMV promoters, the first and second polyA signal sequences are bGH polyA signal sequences, the transcription termination sequence is present and is an hGT terminator sequence.
In one embodiment, the first and second promoters are hEF 1a promoters, the first and second polyA signal sequences are bGH polyA signal sequences, and the expression cassette does not contain a transcription terminator sequence.
One aspect as reported herein is the use of an expression vector comprising: :
a first expression cassette comprising in the 5 'to 3' direction a first promoter, a nucleic acid encoding a light chain of an antibody, a first polyA signal sequence and optionally a first transcription terminator sequence,
-a second expression cassette comprising in 5 'to 3' direction a second promoter, a nucleic acid encoding the heavy chain of the antibody, 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,
whereby the expression cassettes are organized bidirectionally, whereby the first expression cassette and the second expression cassette are arranged in opposite directions.
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.
In one embodiment, the nucleic acid encoding the antibody light chain and/or the nucleic acid encoding the antibody heavy chain is cDNA.
In one embodiment, the first and second promoters are hCMV promoters, the first and second polyA signal sequences are bGH polyA signal sequences, the transcription termination sequence is present and is an hGT terminator sequence.
In one embodiment, the first and second promoters are hEF 1a promoters, the first and second polyA signal sequences are bGH polyA signal sequences, and the expression cassette does not contain a transcription terminator sequence.
In one embodiment, the mammalian cell is selected from the group consisting of a CHO cell, a HEK cell, a BHK cell, a NS0 cell, and a SP2/0 cell. In one embodiment, the mammalian cell is a HEK cell.
One aspect as reported herein is an expression vector comprising:
a first expression cassette comprising in the 5 'to 3' direction a first promoter, a nucleic acid encoding a light chain of an antibody, a first polyA signal sequence and optionally a first transcription terminator sequence,
-a second expression cassette comprising in 5 'to 3' direction a second promoter, a nucleic acid encoding the heavy chain of the antibody, 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,
whereby the expression cassettes are organized bidirectionally, whereby the first expression cassette and the second expression cassette are arranged in opposite directions.
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.
In one embodiment, the nucleic acid encoding the antibody light chain and/or the nucleic acid encoding the antibody heavy chain is cDNA.
In one embodiment, the first and second promoters are hCMV promoters, the first and second polyA signal sequences are bGH polyA signal sequences, the transcription termination sequence is present and is an hGT terminator sequence.
In one embodiment, the first and second promoters are hEF 1a promoters, the first and second polyA signal sequences are bGH polyA signal sequences, and the expression cassette does not contain a transcription terminator sequence.
Furthermore, it has been found that expression vectors comprising, among other things, the hCMV promoter containing intron a and the human EF 1a promoter, rather than the short human CMV promoter without intron a, enhance transient gene expression and sink gene expression.
One aspect as reported herein is an expression plasmid comprising:
-a first expression cassette comprising in the 5 'to 3' direction a first promoter, a nucleic acid encoding a light chain of an antibody and a first polyA signal sequence,
-a second expression cassette comprising in 5 'to 3' direction a second promoter, a nucleic acid encoding a heavy chain of an antibody and a second polyA signal sequence,
wherein one or both of the expression cassettes further comprises a human gastrin terminator sequence following the polyA signal sequence.
In one embodiment, the first and second polyA signal sequences are independently selected from the group consisting of the SV40polyA signal sequence and the bovine growth hormone polyA signal sequence.
In one embodiment, the first and second promoters are independently selected from the group consisting of the human CMV promoter, the SV40 promoter and the human elongation factor 1 α promoter.
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.
In one embodiment, the nucleic acid encoding the antibody light chain and/or the nucleic acid encoding the antibody heavy chain is cDNA.
In one embodiment, the first expression cassette and the second expression cassette are arranged unidirectionally.
In one embodiment, the expression plasmid further comprises a selectable marker. In one embodiment, the expression cassette and the selectable marker are arranged bi-directionally.
One aspect as reported herein is the use of an expression plasmid as reported herein for the transient expression or for the stable expression of an antibody.
One aspect as reported herein is a eukaryotic cell comprising an expression plasmid as reported herein.
One aspect as reported herein is a method for producing an antibody comprising the steps of:
-culturing a eukaryotic cell comprising an expression plasmid as reported herein or a cell as reported herein,
-recovering the antibody from the eukaryotic cell or culture medium.
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 NS0 cell, and a SP2/0 cell.
One aspect as reported herein is an expression plasmid comprising:
-a first expression cassette comprising in the 5 'to 3' direction a first promoter, a nucleic acid encoding a light chain of an antibody and a first polyA signal sequence,
-a second expression cassette comprising in 5 'to 3' direction a second promoter, a nucleic acid encoding a heavy chain of an antibody and a second polyA signal sequence,
wherein the first and/or second promoter is a human elongation factor 1 alpha promoter.
In one embodiment, one or both of the expression cassettes does not comprise a human gastrin terminator sequence following the polyA signal sequence.
In one embodiment, one or both of the expression cassettes do not contain a human gastrin terminator sequence.
In one embodiment, the human elongation factor 1 α promoter comprises intron a.
In one embodiment, the first and second polyA signal sequences are independently selected from the group consisting of the SV40polyA signal sequence and the bovine growth hormone polyA signal sequence.
In one embodiment, the first and second promoters are independently selected from the group consisting of the human CMV promoter, the SV40 promoter and the human elongation factor 1 α promoter.
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.
In one embodiment, the nucleic acid encoding the antibody light chain and/or the nucleic acid encoding the antibody heavy chain is cDNA.
In one embodiment, the first expression cassette and the second expression cassette are arranged unidirectionally.
In one embodiment, the expression plasmid further comprises a selectable marker.
In one embodiment, the expression cassette and the selectable marker are arranged bi-directionally.
One aspect as reported herein is the use of an expression plasmid as reported herein for the transient expression or for the stable expression of an antibody.
One aspect as reported herein is a eukaryotic cell comprising an expression plasmid as reported herein.
One aspect as reported herein is a method for producing an antibody comprising the steps of:
-culturing a eukaryotic cell comprising an expression plasmid as reported herein or a cell as reported herein,
-recovering the antibody from the eukaryotic cell or culture medium.
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 NS0 cell, and a SP2/0 cell.
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 selection marker and a polyA signal sequence, whereby the IRES element is an EMCV-IRES element.
In one embodiment, the nucleic acid encodes an antibody heavy chain.
In one embodiment, the selectable marker is a fusion protein of the formula A-C-S, whereby A is a detectable polypeptide, C is a proteolytic signal sequence, and S is a selectable marker.
In one embodiment, the proteolytic signal sequence is the PEST sequence of ornithine decarboxylase.
In one embodiment, the detectable polypeptide is green fluorescent protein.
In one embodiment, the selectable marker is neomycin.
One aspect as reported herein is a nucleic acid encoding a polypeptide comprising in N-terminal to C-terminal direction green fluorescent protein, PEST sequence of ornithine decarboxylase and neomycin.
One aspect as reported herein is the use of a nucleic acid encoding a polypeptide comprising in N-terminal to C-terminal direction green fluorescent protein, PEST sequence of ornithine decarboxylase and neomycin for selecting antibody secreting cells.
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 selection marker and a polyA signal sequence for selecting an antibody producing cell, whereby the IRES element is an EMCV-IRES element.
In one embodiment, the nucleic acid encodes an antibody heavy chain.
In one embodiment, the selectable marker is a fusion protein of the formula A-C-S, whereby A is a detectable polypeptide, C is a proteolytic signal sequence, and S is a selectable marker.
In one embodiment, the proteolytic signal sequence is the PEST sequence of ornithine decarboxylase.
In one embodiment, the detectable polypeptide is green fluorescent protein.
In one embodiment, the selectable marker is neomycin.
One aspect as reported herein is a method for selecting eukaryotic cells expressing an antibody comprising the steps of:
-culturing a eukaryotic cell comprising i) the expression plasmid reported in this aspect and ii) a nucleic acid encoding each further antibody chain not encoded by the expression plasmid reported in this aspect,
-selecting a cell expressing the detectable polypeptide.
One aspect as reported herein is an expression plasmid comprising in 5 'to 3' direction a promoter sequence, a nucleic acid encoding the antibody light chain, an IRES element, a nucleic acid encoding the antibody heavy chain and a polyA signal sequence, whereby the IRES element is an EV71-IRES element.
In one embodiment, the promoter sequence is selected from the group consisting of a human CMV promoter sequence with or without intron a, a SV40 promoter sequence, and a human elongation factor 1 α promoter sequence with or without intron a.
In one embodiment, the polyA signal sequence is selected from the bovine growth hormone polyA signal sequence and the SV40polyA signal sequence.
In one embodiment, the plasmid comprises a human gastrin terminator sequence 3' to the polyA signal sequence.
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 a light chain of an antibody, an IRES element, a nucleic acid sequence encoding a heavy chain of an antibody and a polyA signal sequence for expressing an antibody, whereby the IRES element is an EV71-IRES element.
In one embodiment, the promoter sequence is selected from the group consisting of a human CMV promoter sequence with or without intron a, a SV40 promoter sequence, and a human elongation factor 1 α promoter sequence with or without intron a.
In one embodiment, the polyA signal sequence is selected from the bovine growth hormone polyA signal sequence and the SV40polyA signal sequence.
In one embodiment, the plasmid comprises a human gastrin terminator sequence 3' to the polyA signal sequence.
One aspect as reported herein is a method for producing an antibody comprising the steps of:
-culturing a eukaryotic cell comprising an expression plasmid as reported herein,
-recovering the antibody from the cell or culture medium, thereby producing the antibody.
In one embodiment, the hCMV promoter has the sequence of SEQ ID NO: 01. This is the hCMV promoter without intron a and without the 5' UTR.
In one embodiment, the hCMV promoter has the sequence of SEQ ID NO 02. This is the hCMV promoter without intron a and with a 5' UTR.
In one embodiment, the hCMV promoter has the sequence of SEQ ID NO 03. This is the full-length hCMV promoter containing intron a.
In one embodiment, the human elongation factor 1 α promoter has the sequence of SEQ ID NO 04. This is the hEF 1a promoter without intron a.
In one embodiment, the human elongation factor 1 α promoter has the sequence of SEQ ID NO. 05. This is the hEF1 α promoter containing intron a.
In one embodiment, the human elongation factor 1 α promoter has the sequence of SEQ ID NO 06. This is a short hEF 1a promoter containing intron a and containing the 5' UTR.
In one embodiment, the rat CMV promoter has the sequence of SEQ ID NO: 07.
In one embodiment, the SV40polyA signal sequence has the sequence of SEQ ID NO: 08.
In one embodiment, the bovine growth hormone polyA signal sequence has the sequence of SEQ ID NO 09.
In one embodiment, the human gastrin terminator has the sequence of SEQ ID NO 10.
In one embodiment, the SV40 promoter has the sequence of SEQ ID NO. 11.
In one embodiment, the PEST sequence of the ornithine decarboxylase is encoded by the sequence of SEQ ID NO 12.
In one embodiment, the GFP sequence is encoded by the sequence of SEQ ID NO 13.
In one embodiment, the neomycin selection marker has the sequence of SEQ ID NO 14.
In one embodiment, the GFP-PEST-NEO fusion polypeptide is encoded by the sequence of SEQ ID NO. 15.
In one embodiment, the EMCV-IRES has the sequence of SEQ ID NO 16.
In one embodiment, the EV71-IRES has the sequence of SEQ ID NO 17.
In one embodiment of all aspects reported herein, the antibody is a bispecific antibody.
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.
In one embodiment, the expression vector comprises:
a first expression cassette comprising in the 5 'to 3' direction a promoter, a nucleic acid encoding a light chain of a first antibody, a polyA signal sequence and optionally a terminator sequence,
a second expression cassette comprising in the 5 'to 3' direction a promoter, a nucleic acid encoding a light chain of a second antibody, a polyA signal sequence and optionally a terminator sequence,
a third expression cassette comprising in 5 'to 3' direction a promoter, a nucleic acid encoding the heavy chain of the first antibody, a polyA signal sequence and optionally a terminator sequence,
a fourth expression cassette comprising in 5 'to 3' direction a promoter, a nucleic acid encoding a heavy chain of a second antibody, a polyA signal sequence and optionally a terminator sequence,
or
A first expression cassette comprising in the 5 'to 3' direction a promoter, a nucleic acid encoding a light chain of an antibody, a polyA signal sequence and optionally a terminator sequence,
-a second expression cassette comprising in 5 'to 3' direction a promoter, a nucleic acid encoding a heavy chain of a first antibody, a polyA signal sequence and optionally a terminator sequence, and
a third expression cassette comprising in 5 'to 3' direction a promoter, a nucleic acid encoding a heavy chain of a second antibody, a polyA signal sequence and optionally a terminator sequence,
whereby the antibody light chain is the common light chain of the two antibody heavy chains.
In one embodiment of all aspects reported herein, 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 selection marker expression cassette,
wherein at least one of the antibody heavy chain expression cassette, the antibody light chain expression cassette and the selection marker expression cassette are arranged unidirectionally, and
wherein 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.
In one embodiment of all aspects reported herein, 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 selection marker expression cassette,
wherein one of the antibody heavy chain expression cassettes, one of the antibody light chain expression cassettes and the selection marker expression cassette are arranged unidirectionally, and
wherein 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.
In one embodiment, one of the antibody heavy chain expression cassettes encodes an antibody heavy chain comprising a hole (hole) mutation.
In one embodiment, one of the antibody heavy chain expression cassettes encodes an antibody heavy chain comprising a knob (knob) mutation.
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.
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.
Brief Description of Drawings
FIG. 1. Productivity of stable clones generated with vectors p5068, px6001, px6008 and px 6007. Shown is the average productivity in batch analysis of the best 15 clones obtained with each vector for a total of three independent transfections.
FIG. 2 Productivity of stable clones generated with vectors px6051, px6062, px6052 and px 6063. Shown is the average productivity of the best 18 clones of each vector in a batch analysis for a total of three independent transfections.
FIG. 3 Productivity of vectors p5068, px6051, px6052 and px6053 in transient transfections using a 96-well shuttle system from Amaxa. Shown is the average productivity of eight independent transfections of each vector as measured by ELISA at day 4 post-transfection.
FIG. 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 per vector at day 7.
FIG. 5 Productivity of the stable library generated with vectors p5069 and px 6010C. Shown are the average productivity of two (px5069) or three (px6010C) different pools per vector on day 7 of the batch analysis.
FIG. 6 stability of gene expression in the stable pools generated with vectors p5069 and px 6010C. Shown are the average productivity of two different pools of each vehicle on day 7 of the batch analysis at passage 0 (set to 100%, black bars) and passage 30 with selective pressure (G418) (white bars) and without selective pressure (patterned bars).
FIG. 7 Productivity of the best 15 clones produced by vectors p5069 and px 6010C. The best 15 clones of each vector for a total of two independent transfections averaged productivity in the batch analysis.
FIG. 8 schematic representation of the vector design of vector px6011C mediating IRES-mediated expression of GFP-PEST-NEO fusion protein. The GFP-PEST-Neo fusion protein is linked to the heavy chain of the antibody by EMCV-IRES. The coding sequence for the antibody heavy chain and the coding sequence for the fusion protein are transcribed in one mRNA by a short human CMV promoter. Translation of this mRNA produced the heavy chain of the antibody and the GFP-PEST-NEO fusion protein.
FIG. 9 Productivity of different stable pools generated by vectors p5069, px6011C and px6010C in batch analysis. Shown is the average productivity of two different pools per vector at day 7.
FIG. 10. Productivity of the best 15 clones generated by vectors p5069, px6010C (vector expressing selection marker neomycin by EMCV-IRES element linked to the heavy chain of antibody) and px6011C (vector expressing GFP-PEST-neomycin fusion protein by EMCV-IRES element linked to the heavy chain of antibody). (A) The productivity profiles of the best 15 clones of each vector in a batch analysis amounted to two independent transfections. (B) The best 15 clones of each vector for a total of two independent transfections averaged productivity in the batch analysis.
FIG. 11 shows the GPF expression level/fluorescence intensity and productivity dependence in batch analysis of 11 clones produced with vector px 6011C. Clones were randomly picked in a 24-well screening format, then expanded, and finally analyzed in a batch assay. Geometric Mean (GM) of GFP fluorescence intensity and percentage of GFP positive cells per clone were determined by FACS. (A) Dependence of GFP fluorescence intensity of 11 monoclonals and productivity in batch analysis. (B) Percentage of GFP positive cells per clone of 11 monoclonals and dependence of productivity in batch analysis.
FIG. 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. The pools were expanded and their productivity was determined on day 7 of the batch analysis.
FIG. 13 plasmid map of px 6007.
FIG. 14 plasmid map of px 6053.
FIG. 15 plasmid map of px 6062.
Examples
Expression vectors p5068 and p5069
The expression plasmids P5068 and P5069 contain expression cassettes for expression of anti-P-selectin antibodies reported in WO 2005/100402 (genomic tissue expression cassettes retaining exon-intron tissue).
anti-P-selectin HuMab light and heavy chain encoding genes were separately loaded into mammalian cell expression vectors.
Thus joining gene segments encoding the anti-P-selectin HuMab light chain variable region (VL) and the human kappa light chain constant region (CL) just as the anti-P-selectin HuMab heavy chain variable region (VH) and the human gamma l heavy chain constant region or the human gamma 4 heavy chain constant region (CH 1-hinge-CH 2-CH 3).
General information on the nucleotide Sequences of human light and heavy chains from which codon usage can be deduced is provided in Kabat, E.A. et al, Sequences of Proteins of Immunological Interest, 5 th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication No. 91-3242.
The transcription unit of the anti-P-selectin HuMab kappa light chain consists of the following elements:
immediate early enhancer and promoter from human cytomegalovirus (hCMV),
-synthetic 5' -UTs comprising a Kozak sequence,
a murine immunoglobulin heavy chain signal sequence comprising an intron of the signal sequence,
cloned anti-P-selectin HuMab variable light chain cDNA with a unique BsmI restriction site placed at the 5 'end and a splice donor site and a unique NotI restriction site placed at the 3' end,
genomic human kappa gene constant region, including intron 2 mouse Ig-kappa enhancer (Picard, D. and Schaffner, W.Nature307(1984)80-82), and
human immunoglobulin kappa polyadenylation ("polyA") signal sequence
The transcription unit of the heavy chain of the anti-P-selectin HuMab γ l consists of the following elements:
immediate early enhancer and promoter from human cytomegalovirus (hCMV),
-synthetic 5' -UTs comprising a Kozak sequence,
-a modified murine immunoglobulin heavy chain signal sequence comprising a signal sequence intron,
cloned anti-P-selectin HuMab variable heavy chain cDNA with a unique BsmI restriction site placed at the 5 'end and a splice donor site and a unique NotI restriction site placed at the 3' end,
genomic human γ l heavy chain gene constant region, including the mouse Ig μ enhancer (Neuberger, M.S., EMBO J.2(1983)1373-
-a human γ l immunoglobulin polyadenylation ("polyA") signal sequence.
In addition to the anti-P-selectin HuMab kappa light chain or gamma 1 heavy chain expression cassettes, these plasmids also contain:
-a gene for resistance to hygromycin,
the origin of replication oriP of the EB virus (EBV),
an origin of replication from the vector pUC18, which allows replication of this plasmid in E.coli, and
-a beta-lactamase gene conferring ampicillin resistance in E.coli.
Recombinant DNA technology
Cloning was performed using standard cloning techniques as described in Sambrook et al, 1999 (supra). All molecular biological reagents were commercially available (unless otherwise indicated) and used as per the manufacturer's instructions.
Nucleic acid synthesis
DNA of different genetic elements was synthesized by Geneart AG, Regensburg.
Nucleic acid sequence determination
The DNA sequence was determined by double-strand sequencing performed in SequiServe (SequiServe GmbH, Germany).
DNA and protein sequence analysis and sequence data management
Sequence creation, mapping, analysis, annotation, and description were performed using Vector NTI Advance suite version 9.0.
Cell culture technique
CHO-K1 cells were cultured in culture supplemented with 1x HT supplement (Invitrogen Corp.,catalog number 11067-030) CD-CHO medium (Invitrogen Corp.,directory number 10743-.
To select the stably transfected CHO-K1 pool/cell line, 400 to 800. mu.g/ml G418 or 200 to 400. mu.g/ml hygromycin (Roche Diagnostics GmbH, Roche Applied Sciences, Germany, Cat. No. 843555) was added.
All cell lines were maintained at 37 ℃ with constant stirring at 120 to 140 rpm with 5% CO2In a humidified incubator. Cells were divided into fresh medium every 3 to 4 days. The density and viability of the cultures were determined using a Casey TT or Cedex Hires cell counter (Roche innovaties AG, Bielefeld). Transfection of cells was performed by the Amaxa nuclear transfection technique (Lonza GmbH, germany).
In addition, standard Cell culture techniques are used as described, for example, in Bonifacino, j.s., et al, (ed.), Current Protocols in Cell Biology, John Wiley and Sons, inc. (2000).
Cell count and cell viability assay
a) Electric field cell technology system (CASY)
Technology TT type cell counter (Roche Innovatis AG, Bielefeld) performs cell counting with electric current. Pulse Area Analysis (Pulse Area Analysis) was used to obtain information from the signals generated when cells passed through measurement wells in low voltage electric fields. The structural integrity of the cell membrane is the degree of cell viability. Thus, a dye such as trypan blue is not required for the determination of the viability.
b) Automatic trypan blue exclusion (Cedex)
Cell viability was determined during bank selection using the Cedex HiRes system (Roche Innovatis AG, Bielefeld) and automated cell counting was performed.
Trypan blue is a dye that cannot enter cells through intact cell membranes. Only those cells with damaged cell membranes were stained and marked dead. The staining process, cell counting and graphical analysis of the results were performed automatically by the Cedex system by digital image recognition. Other measured parameters are cell size, morphology and aggregation rate. Up to 20 samples were measured in succession using a multiplex injector.
Plasmid preparation and quality testing for precise comparison of plasmids in transfection
Several factors such as the amount and quality of DNA strongly influence the transfection efficiency and thus the productivity. To ensure equal initial conditions for each vector, the DNA quantity and quality of all vectors were tested collectively before transfection.
Simultaneous preparation of expression vectors
All vectors were prepared simultaneously by High Speed Maxi plasmid isolation kit (Qiagen GMBH, High den) according to the manufacturer's instructions.
Phenol/chloroform purification and ethanol precipitation
All vectors were purified simultaneously by phenol/chloroform purification. Mu.g each of the linearized plasmid DNA was mixed with 200. mu.l of Tris-buffered 50% (v/v) phenol, 48% (v/v) chloroform, 2% (v/v) isoamyl alcohol solution and centrifuged at 13,000 rpm for 1 minute. The upper aqueous phase was then transferred to a new tube and mixed with 200. mu.l of 96% (v/v) chloroform, 4% (v/v) isoamyl alcohol, and centrifuged at 13,000 rpm for 1 minute. The upper phase was transferred again 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. mu.l 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, the pellet dried, and resuspended in sterile water.
DNA assay
The amount of DNA in each vector was determined using a BioPhotometer (Eppendorf; Hamburg). DNA measurements were always performed in triplicate by 1:20 dilution in Tris pH8.0.
-agarose gel
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 exhibiting comparable amounts and quality (no DNA degradation on gel, similar supercoiled (ccc) format, similar DNA amount).
Transient transfection
All vectors were transfected into CHO-K1 cells by the Amaxa 96-well shuttle system (Lonza GmbH, Germany) according to the manufacturer's instructions. Each vector was transfected 8 times in duplicate. The amount of DNA transfected into the vector was normalized to equimolar amounts per copy number according to 1. mu.g of the reference expression plasmid (p5068 or p 5069). To determine productivity, cell-free cell culture supernatants were analyzed for IgG titer by one-step universal elisa (dianova) on days 4 to 7 post-transfection.
Amaxa96 well shuttle system:
CHO-K1 cells cultured in cell culture flasks were pelleted by centrifugation at 850 rpm for 5 minutes and resuspended in culture medium. The circular plasmids were plated at an equimolar concentration of 1. mu.g of the reference expression vector p5068 or p5069 in 96-well nuclear transfection plates. Then 4x10 per well5Concentration of cells were added to the plate. Transfection was performed by Amaxa program DN-137. Cells were incubated for 10 minutes after transfection and then transferred to 96-well flat-bottom incubation plates containing 200. mu.l of medium. Then, the cells were cultured by standing. IgG levels were determined by one-step universal ELISA on days 4 to 6 post transfection.
Stable transfection and Generation of recombinant CHO cell lines
Stable transfection was performed by nuclear transfection technique (Amaxa Biosystems, Lonza colongene AG) according to the manufacturer's instructions. Before transfection, the transfection plasmid was linearized by the restriction enzyme SgrA I. Each plasmid was transfected twice or three times. 5X10 was used for each transfection6Cells and 1.2pmol linearized plasmid. (Nucleofector Kit T, Amaxa program A33).
For transfection, cells were resuspended in Nucleofector solution T and dispensed into 2ml tubes. After the addition of the plasmid, transfection was performed by applying a pulse. The cells were then transferred to a T25 tissue culture flask containing 4ml of fresh medium and 4ml of conditioned medium, which were pre-warmed. Selection pressure was applied 24 hours after transfection by adding 250. mu.g/ml hygromycin B.
Generation of stable pools
Vectors were transfected into CHO-K1 cells by Amaxa nuclear transfection technique and stable pools were selected using hygromycin B or G418 as selection agents. Each transfection was performed in triplicate. To generate a stable pool, all plasmids were linearized uniformly by restriction digestion with SgrA I. Stable transfections were performed with the Nucleofector Kit T from Amaxa, three transfections per plasmid.
The stable library was established as follows: 5X10 was used for each transfection6Cells and 1.2pmol linearized plasmid. Cells were resuspended in solution T and dispensed into 2ml tubes. After addition of the plasmid, transfection was performed by applying pulses (Amaxa program a 33). The transfected cell bank was cultured still in a T25 tissue culture flask containing 4ml of fresh medium and 4ml of conditioned medium, which were pre-warmed.
24 hours after transfection, selection pressure was applied: the cells were centrifuged at 800 rpm for 5 minutes and resuspended in 3ml of medium containing 300. mu.g/ml hygromycin B. Cells were transferred to flat bottom 6-well plates 3 days after transfection. The cells were then cultured for two weeks until the cell viability decreased to a minimum and increased again to above 99%. Cell number and viability are often determined using the Cedex HiRes system (Innovatis, Bielefeld). During the culture, cell debris was removed by centrifugation and the cells were always resuspended in 3ml of fresh medium.
Generation of stable clones with Caliper robot System
The vector was 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 on 384-well flat bottom plates using an automated high throughput clonal separation system (Sciclone ALH 3000 workstation, Caliper Life Sciences GmbH, Mainz).
After 10 to 14 days, 384-well plates were screened for IgG levels using ELISA-based ultra-high throughput screening (ELSIA uHTS). The best producing clones were selected from the primary screen and transferred to flat bottom 96-well plates. After 3 to 6 days, cells were screened for IgG levels in the second round. The best producing clones were selected again and transferred manually into flat bottom 24-well plates. After a further ELISA-based screening step, the best clones were selected and transferred into flat bottom 6-well plates. IgG levels in 6-well plates were determined by ProtA measurements to identify the final best clones for batch culture in shaken 6-well plates.
Batch analysis of pools/monoclonals
To examine the differences in productivity and stability, the number of cells of the clones/pools was counted by Casey cytometry and expressed at 3X105The concentration of cells/ml and the total volume of 3.0ml were uniformly seeded into flat bottom 6-well plates. All batch cultures were cultured for 12 days and cell culture supernatants were screened for human IgG levels on days 4, 7, 9, 11, or 12.
IgG quantitation
IgG titers were determined in transient experiments and in screening formats (384-24 wells) by using a one-step universal ELISA. The productivity of stable pools and stable monoclonals in batch experiments was determined by protein a HPLC.
One-step universal ELISA
Human IgG levels from cell culture supernatants were determined by one-step universal elisa (dianova). A standard curve was prepared using dilution buffer (PBS + 5% (w/v) RPLA1) with serial dilutions ranging from 0.3125 to 20ng/ml of anti-P-selectin antibody (F. Hoffmann-La Roche AG, Basle, Switzerland). To a streptavidin-coated 96-well MTP (StreptaWell, Roche Diagnostics GmbH) was added 95. mu.l of biotinylated F (ab') containing 0.5. mu.g/ml2Anti-human Fc antibodies (Jackson laboratories) and 0.1. mu.g/ml peroxidase-conjugated F (ab')2A mixture of antibodies to anti-human Fc gamma antibodies (Jackson laboratories; Suffolk). To the plate, 5. mu.l of 1:20.000 diluted cell culture supernatant was added and incubated for 1 hour. The antibody-coated plates were washed three times with 200. mu.l of wash buffer (PBS + 0.05% (v/v) Tween 20). To the plate was added 100. mu.l ABTS (Roche Diagnostics GmbH, Mannheim, Germany) and the absorbance was measured at 405nm with a reference wavelength of 492 nm.
ProtA measurement
In combination with the one-step universal ELISA, IgG titers in the batch analysis were determined by protein a using HPLC-based chromatography.
FACS
Fluorescence activated cell sorting was used to determine the transfection efficiency (based on GFP expressing cells) or GFP expression levels of stably or transiently transfected cells. Usually, using FACSCalibur flow cytometer (BD Biosciences, San Diego, Calif.) measures 5x10 of each clone or pool6And (4) cells. Forward and side scatter data were used to determine cell size, viability and cell morphology.
Some embodiments of the invention:
1. a method for selecting a recombinant transiently transfected mammalian cell, comprising the steps of:
a) transfecting a mammalian cell with an expression vector comprising:
-a first expression cassette comprising in the 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;
-a second expression cassette comprising in the 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
whereby the first expression cassette and the second expression cassette are arranged bi-directionally,
thereby obtaining a plurality of recombinant mammalian cells;
b) (iii) selecting (single) transiently transfected recombinant mammalian cells from the plurality of recombinant mammalian cells.
2. The method of embodiment 1, characterized in that said nucleic acid encoding the light chain of an antibody and/or said nucleic acid encoding the heavy chain of an antibody comprises at least one intron.
3. The method according to any one of embodiments 1 to 2, characterized in that said nucleic acid encoding the light chain of the antibody and/or said nucleic acid encoding the heavy chain of the antibody is a cDNA.
4. The method according to any one of embodiments 1 to 3, characterized in that the expression plasmid further comprises a selectable marker.
5. The method according to any of embodiments 1 to 4, characterized in that the expression cassettes are arranged in LC-HC-SM order.
6. The method according to any of embodiments 1 to 5, characterized in that the mammalian cells are selected from the group consisting of CHO cells, HEK cells, BHK cells, NS0 cells and SP2/0 cells.
7. The method according to any of embodiments 1 to 6, characterized in that the mammalian cells are HEK cells for selection of transiently transfected cells.
8. The method according to any one of embodiments 1 to 7, 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 light chain of a second antibody, 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 the heavy chain of the first antibody, a bGH polyA signal sequence, and an hGT terminator sequence;
-a fourth expression cassette comprising in the 5 'to 3' direction an hCMV promoter, a nucleic acid encoding a heavy chain of a second antibody, a bGH polyA signal sequence, and an hGT terminator sequence;
or
-a first expression cassette comprising in the 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;
-a second expression cassette comprising in the 5 'to 3' direction an hCMV promoter, a nucleic acid encoding a heavy chain of a first antibody, a bGH polyA signal sequence, and an hGT terminator sequence; and
-a third expression cassette comprising in the 5 'to 3' direction an hCMV promoter, a nucleic acid encoding a heavy chain of a second antibody, a bGH polyA signal sequence, and an hGT terminator sequence;
whereby the antibody light chain is a common light chain of two antibody heavy chains.
9. The method according to any one of embodiments 1 to 8, characterized in that the expression vector encodes a bispecific antibody.
10. The method of embodiment 8 or 9, characterized in that said 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 said 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. The method according to any one of embodiments 8 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 selection marker expression cassette.
12. The method according to any one of embodiments 8 to 11, 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 selection marker expression cassette.
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.
14. The method of 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.
15. The method of 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 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 constant domain.
16. The method according to any one of embodiments 8 to 15, characterized in that one of said antibody heavy chain expression cassettes encodes an antibody heavy chain variant comprising an antibody light chain constant domain (CL) as a first constant domain, and/or one of said antibody heavy chain expression cassettes encodes an antibody heavy chain comprising an antibody heavy chain CH1 domain as a first constant domain.
17. A method for producing an antibody comprising the steps of:
a) culturing a mammalian cell comprising:
-a first expression cassette comprising in the 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;
-a second expression cassette comprising in the 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
b) recovering the antibody from the cells or the culture medium,
the first expression cassette and the second expression cassette are arranged bi-directionally for transient production of antibodies.
18. The method of embodiment 17, characterized in that said nucleic acid encoding a light chain of an antibody and/or said nucleic acid encoding a heavy chain of an antibody comprises at least one intron.
19. The method of embodiment 17 or 18, characterized in that said nucleic acid encoding the light chain of an antibody and/or said nucleic acid encoding the heavy chain of an antibody is a cDNA.
20. The method according to any one of embodiments 17 to 19, characterized in that the expression plasmid further comprises a selectable marker.
21. The method according to any one of embodiments 17 to 20, characterized in that the expression cassettes are arranged in LC-HC-SM order.
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, NS0 cells and SP2/0 cells.
23. The method according to any of embodiments 17 to 22, characterized in that the mammalian cells are HEK cells for transient antibody production.
24. An expression vector comprising:
-a first expression cassette comprising in the 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;
a second expression cassette comprising in the 5 'to 3' direction an hCMV promoter, a nucleic acid encoding the heavy chain of an antibody, a bGH polyA signal sequence and an hGT terminator sequence,
the first expression cassette and the second expression cassette are arranged bi-directionally for selection of transiently transfected cells.
25. The expression vector of embodiment 24, characterized in that said nucleic acid encoding an antibody light chain and/or said nucleic acid encoding an antibody heavy chain comprises at least one intron.
26. The expression vector of embodiment 24 or 25, characterized in that said nucleic acid encoding the antibody light chain and/or said nucleic acid encoding the antibody heavy chain is a cDNA.
27. The expression vector according to any one of embodiments 24 to 26, characterized in that the expression plasmid further comprises a selectable marker.
28. The expression vector according to any one of embodiments 24 to 27, characterized in that the expression cassettes are arranged in LC-HC-SM order.
29. The expression vector according to any one of embodiments 24 to 28, characterized in that it 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 light chain of a second antibody, 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 the heavy chain of the first antibody, a bGH polyA signal sequence, and an hGT terminator sequence;
-a fourth expression cassette comprising in the 5 'to 3' direction an hCMV promoter, a nucleic acid encoding a heavy chain of a second antibody, a bGH polyA signal sequence, and an hGT terminator sequence;
or
-a first expression cassette comprising in the 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;
-a second expression cassette comprising in the 5 'to 3' direction an hCMV promoter, a nucleic acid encoding a heavy chain of a first antibody, a bGH polyA signal sequence, and an hGT terminator sequence; and
-a third expression cassette comprising in the 5 'to 3' direction an hCMV promoter, a nucleic acid encoding a heavy chain of a second antibody, a bGH polyA signal sequence, and an hGT terminator sequence;
whereby the antibody light chain is a common light chain of two antibody heavy chains.
30. The expression vector according to any one of embodiments 24 to 29, characterized in that the expression vector encodes a bispecific antibody.
31. The expression vector according to any one of embodiments 24 to 30, characterized in that the bispecific antibody has a first binding specificity or binding site specifically binding to a first antigen or a first epitope on an antigen and the bispecific antibody has a second binding specificity or binding site specifically binding to a second antigen or a second epitope on an antigen.
32. The expression vector according to any one of embodiments 24 to 31, characterized in that it comprises:
-an antibody light chain expression cassette;
-a first antibody heavy chain expression cassette;
-a second antibody heavy chain expression cassette; and
-a selection marker expression cassette.
33. The expression vector according to any one of embodiments 24 to 32, 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 selection marker expression cassette.
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.
35. The expression vector of 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.
36. The expression vector according to 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 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 constant domain.
37. The expression vector according to any one of embodiments 24 to 36, characterized in that one of said antibody heavy chain expression cassettes encodes an antibody heavy chain variant comprising an antibody light chain constant domain (CL) as a first constant domain and/or one of said antibody heavy chain expression cassettes encodes an antibody heavy chain comprising an antibody heavy chain CH1 domain as a first constant domain.
38. A method for selecting a recombinant transiently transfected mammalian cell, comprising the steps of:
a) transfecting a mammalian cell with an expression vector comprising:
-a first expression cassette comprising in 5 'to 3' direction the hEF 1a promoter, a nucleic acid encoding an antibody light chain and a bGH polyA signal sequence;
-a second expression cassette comprising in 5 'to 3' direction the hEF1 α promoter, a nucleic acid encoding the heavy chain of an antibody and a bGH polyA signal sequence; and
the first expression cassette and the second expression cassette are arranged bi-directionally for selection of transiently transfected cells,
thereby obtaining a plurality of recombinant mammalian cells;
b) (iii) selecting (single) transiently transfected recombinant mammalian cells from the plurality of recombinant mammalian cells.
39. The method of embodiment 38, characterized in that said nucleic acid encoding a light chain of an antibody and/or said nucleic acid encoding a heavy chain of an antibody comprises at least one intron.
40. The method of embodiment 38 or 39, characterized in that said nucleic acid encoding a light chain of an antibody and/or said nucleic acid encoding a heavy chain of an antibody is a cDNA.
41. The method of any one of embodiments 38 to 40, characterized in that the expression plasmid further comprises a selectable marker.
42. The method according to any one of embodiments 38 to 41, characterized in that the expression cassettes are arranged in LC-HC-SM order.
43. The method according to any one of embodiments 38 to 42, characterized in that the human elongation factor 1 α promoter contains intron a.
44. The method according to any of embodiments 38 to 43, characterized in that the expression vector does not contain any transcription terminator sequence.
45. The method of embodiment 44, characterized in that said terminator sequence is the hGT sequence.
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, NS0 cells and SP2/0 cells.
47. The method of embodiment 46, characterized in that said mammalian cells are HEK cells for selection of transiently transfected cells.
48. A method for producing an antibody comprising the steps of:
a) culturing a mammalian cell comprising the following transient transfections:
-a first expression cassette comprising in 5 'to 3' direction the hEF 1a promoter, a nucleic acid encoding an antibody light chain and a bGH polyA signal sequence;
-a second expression cassette comprising in 5 'to 3' direction the hEF1 α promoter, a nucleic acid encoding the heavy chain of an antibody and a bGH polyA signal sequence; and
the first expression cassette and the second expression cassette are arranged bi-directionally for selection of transiently transfected cells,
b) recovering the antibody from the transiently transfected cells or culture medium.
49. The method of embodiment 48, characterized in that said nucleic acid encoding a light chain of an antibody and/or said nucleic acid encoding a heavy chain of an antibody comprises at least one intron.
50. The method of embodiment 48 or 49, characterized in that said nucleic acid encoding a light chain of an antibody and/or said nucleic acid encoding a heavy chain of an antibody is a cDNA.
51. The method of any one of embodiments 48 to 50, characterized in that the expression plasmid further comprises a selectable marker.
52. The method according to any of embodiments 48 to 51, characterized in that the expression cassettes are arranged in LC-HC-SM order.
53. The method according to any one of embodiments 48 to 52, characterized in that the human elongation factor 1 α promoter contains intron a.
54. The method according to any of embodiments 48 to 53, characterized in that the expression vector does not contain any transcription terminator sequence.
55. The method of embodiment 54, characterized in that said terminator sequence is the hGT sequence.
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, NS0 cells and SP2/0 cells.
57. The method of embodiment 56, characterized in that said mammalian cells are HEK cells for transient production of antibodies.
58. An expression vector comprising:
-a first expression cassette comprising in 5 'to 3' direction the hEF 1a promoter, a nucleic acid encoding an antibody light chain and a bGH polyA signal sequence;
-a second expression cassette comprising in 5 'to 3' direction the hEF1 alpha promoter, a nucleic acid encoding the heavy chain of an antibody and a bGH polyA signal sequence,
the first expression cassette and the second expression cassette are arranged bi-directionally for selection of transiently transfected cells.
59. The expression vector of embodiment 58, characterized in that said nucleic acid encoding an antibody light chain and/or said nucleic acid encoding an antibody heavy chain comprises at least one intron.
60. The expression vector of embodiment 58 or 59, characterized in that said nucleic acid encoding an antibody light chain and/or said nucleic acid encoding an antibody heavy chain is a cDNA.
61. The expression vector according to any one of embodiments 58 to 60, characterized in that the expression plasmid further comprises a selectable marker.
62. The expression vector of any one of embodiments 58 to 61, characterized in that the expression cassettes are arranged in LC-HC-SM order.
63. The expression vector according to any one of embodiments 58 to 62, characterized in that the human elongation factor 1 α promoter contains intron a.
64. The expression vector according to any one of embodiments 58 to 63, characterized in that said expression vector does not contain any transcription terminator sequence.
65. The expression vector of embodiment 64, characterized in that said terminator sequence is the hGT sequence.
66. Use of an expression vector for transient recombinant production of an antibody in a mammalian cell comprising:
-a first expression cassette comprising in the 5 'to 3' direction a first promoter, a nucleic acid encoding a light chain of an antibody, a first polyA signal sequence and optionally a first transcription terminator sequence;
-a second expression cassette comprising in 5 'to 3' direction a second promoter, a nucleic acid encoding a heavy chain of an antibody, a second polyA signal sequence and optionally a second transcription terminator sequence; and
-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;
whereby said expression cassettes are organized bidirectionally, whereby said first expression cassette and said second expression cassette are arranged in opposite directions.
67. The use of embodiment 66, characterized in that said nucleic acid encoding a light chain of an antibody and/or said nucleic acid encoding a heavy chain of an antibody comprises at least one intron.
68. The use according to any one of embodiments 66 to 67, characterized in that said nucleic acid encoding the light chain of an antibody and/or said nucleic acid encoding the heavy chain of an antibody is a cDNA.
69. The use according to any one of embodiments 66 to 68, characterized in that said first and second promoters are hCMV promoters, said first and second polyA signal sequences are bGH polyA signal sequences, said transcription terminator sequence is present and is an hGT terminator sequence.
70. The use of any one of embodiments 66 to 69, characterized in that said first and second promoters are the hEF 1a promoter, said first and second polyA signal sequences are the bGH polyA signal sequence, and said expression cassette does not contain a transcription terminator sequence.
71. The use according to any one of embodiments 66 to 69, characterized in that the mammalian cell is selected from the group consisting of CHO cells, HEK cells, BHK cells, NS0 cells and SP2/0 cells.
72. The use according to any of embodiments 66 to 71, characterized in that said mammalian cells are HEK cells.
73. The use according to any one of embodiments 66 to 72, characterized in that said 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 light chain of a second antibody, 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 the heavy chain of the first antibody, a bGH polyA signal sequence, and an hGT terminator sequence;
-a fourth expression cassette comprising in the 5 'to 3' direction an hCMV promoter, a nucleic acid encoding a heavy chain of a second antibody, a bGH polyA signal sequence, and an hGT terminator sequence;
or
-a first expression cassette comprising in the 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;
-a second expression cassette comprising in the 5 'to 3' direction an hCMV promoter, a nucleic acid encoding a heavy chain of a first antibody, a bGH polyA signal sequence, and an hGT terminator sequence; and
-a third expression cassette comprising in the 5 'to 3' direction an hCMV promoter, a nucleic acid encoding a heavy chain of a second antibody, a bGH polyA signal sequence, and an hGT terminator sequence;
whereby the antibody light chain is a common light chain of two antibody heavy chains.
74. The use according to any of embodiments 66 to 73, characterized in that the expression vector encodes a bispecific antibody.
75. The use according to any one of embodiments 66 to 74, characterized in that said bispecific antibody has a first binding specificity or binding site specifically binding to a first antigen or a first epitope on an antigen and said bispecific antibody has a second binding specificity or binding site specifically binding to a second antigen or a second epitope on an antigen.
76. The use according to any one of embodiments 66 to 75, characterized in that said 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 selection marker expression cassette.
77. The use according to any one of embodiments 66 to 76, characterized in that said 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 selection marker expression cassette.
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.
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.
80. The use according to any one of embodiments 66 to 79, characterized in that one of said 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 constant domain, and/or one of said 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 constant domain.
81. The use according to any one of embodiments 66 to 80, characterized in that one of said antibody heavy chain expression cassettes encodes an antibody heavy chain variant comprising an antibody light chain constant domain (CL) as a first constant domain, and/or one of said antibody heavy chain expression cassettes encodes an antibody heavy chain comprising an antibody heavy chain CH1 domain as a first constant domain.
82. An expression vector comprising:
-a first expression cassette comprising in the 5 'to 3' direction a first promoter, a nucleic acid encoding a light chain of an antibody, a first polyA signal sequence and optionally a first transcription terminator sequence;
-a second expression cassette comprising in 5 'to 3' direction a second promoter, a nucleic acid encoding a heavy chain of an antibody, a second polyA signal sequence and optionally a second transcription terminator sequence; and
-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;
whereby said expression cassettes are organized bidirectionally, whereby said first expression cassette and said second expression cassette are arranged in opposite directions.
83. The expression vector of embodiment 82, characterized in that said nucleic acid encoding an antibody light chain and/or said nucleic acid encoding an antibody heavy chain comprises at least one intron.
84. The expression vector of embodiment 82 or 83, characterized in that said nucleic acid encoding the light chain of an antibody and/or said nucleic acid encoding the heavy chain of an antibody is a cDNA.
85. The expression vector of any one of embodiments 82 to 84, characterized in that said first and second promoters are hCMV promoters, said first and second polyA signal sequences are bGH polyA signal sequences, said transcription terminator sequence is present and is an hGT terminator sequence.
86. The expression vector of any one of embodiments 82 to 84, characterized in that said first and second promoters are the hEF 1a promoter, said first and second polyA signal sequences are the bGH polyA signal sequence, and said expression cassette does not contain a transcription terminator sequence.
87. An expression plasmid comprising in a 5 'to 3' orientation 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 said IRES element is an EMCV-IRES element.
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 cells producing an antibody, whereby said IRES element is an EMCV-IRES element.
89. A method for selecting eukaryotic cells expressing an antibody comprising the steps of:
-culturing a eukaryotic cell comprising i) the expression plasmid of embodiment 87 and ii) a nucleic acid encoding another antibody chain not encoded by the expression plasmid of embodiment 87;
-selecting a cell expressing said detectable polypeptide.
90. An expression plasmid comprising in a 5 'to 3' orientation a promoter sequence, a nucleic acid encoding a light chain of an antibody, an IRES element, a nucleic acid sequence encoding a heavy chain of an antibody and a polyA signal sequence, whereby said IRES element is an EV71-IRES element.
91. Use of an expression plasmid comprising in a 5 'to 3' orientation a promoter sequence, a nucleic acid encoding a light chain of an antibody, an IRES element, a nucleic acid sequence encoding a heavy chain of an antibody and a polyA signal sequence for expressing an antibody, whereby said IRES element is an EV71-IRES element.
92. A method for selecting eukaryotic cells expressing an antibody comprising the steps of:
-culturing a eukaryotic cell comprising i) the expression plasmid of embodiment 90 and ii) a nucleic acid encoding another antibody chain not encoded by the expression plasmid of embodiment 90;
-selecting a cell expressing said detectable polypeptide.
Sequence listing
<110> Fuffmann-Rarosch Co., Ltd (F. Hoffmann-La Roche AG)
<120> combination of expression vector elements, novel method for producing cells for production and use thereof for 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
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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
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<210> 3
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<213> human cytomegalovirus
<400> 3
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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-1 alpha 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> Brown rat (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 tatttttcaa ggccacgtta aagtgtatgc 300
ttgtaacaga gtgcttacat tcaagccaaa tgttaatata acaatcctga attcgtacat 360
aatgtgaata agacactcaa ctctatttaa atccagatct aaatagttac ttttatctaa 420
atgtcaccat ctgtttctac ttagaataat aaacttctta aaggtcacgt atcgggctga 480
ttataaatca ttataattat aacaaaacag atgatttgtt taaaggtcac atcccgttcc 540
gtggtctttt tagtcgaaat aactattaat cttcattatg tttctgagaa agtttaaata 600
tcacgatttc cacccataac 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 tggtttttct 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 agattttttt 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)
<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
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
gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac 540
ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat 600
ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac 660
atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc 720
ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt 780
gacgagttct tctga 795
<210> 15
<211> 1677
<212> DNA
<213> Artificial sequence
<220>
<223> GFP-PEST-NEO fusion polypeptide-encoding nucleic acids
<400> 15
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 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 ccccccccct 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 ctgcaaaggc 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> Enterovirus 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
ttattcctat attggctgct tatggtgaca atcaaaaagt tgttaccata tagctattgg 540
attggccatc cggtgtgcaa cagggcaatt gtttacctat ttattggttt tgtaccatta 600
tcactgaagt ctgtgatcac tctcaaattc attttgaccc tcaacacaat caaac 655
Detailed Description
I. General aspects
As known to those skilled in the art, the use of recombinant DNA techniques enables the production of many derivatives of nucleic acids and/or polypeptides. Such derivatives may be modified in single or several positions, for example by substitution, alteration, exchange, deletion or insertion. This modification or derivatization can be carried out, for example, by means of site-directed mutagenesis. Such modifications can be readily made by those skilled in the art (see, e.g., Sambrook, J. et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, USA (1999)). The use of recombinant techniques enables one of skill 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, cells belonging to different species may have different so-called codon usage, among others. Whereby the same polypeptide (in terms of amino acid sequence) may be encoded by one or more different nucleic acids. In addition, due to the degeneracy of the genetic code, different nucleic acids may encode the same polypeptide.
The use of recombinant DNA technology enables the production of many derivatives of nucleic acids and/or polypeptides. Such derivatives may be modified in single or several positions, for example by substitution, alteration, exchange, deletion or insertion. This modification or derivatization can be carried out, for example, by means of site-directed mutagenesis. Such modifications can be readily made by those skilled in the art (see, e.g., Sambrook, J. et al, Molecular Cloning: Alabortory Manual, Cold Spring Harbor Laboratory Press, New York, USA (1999); Hames, B.D. and Higgins, S.J., Nucleic acid hybridization-a practical proproach, IRL Press, Oxford, England (1985)).
The use of recombinant techniques 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 elements, cells belonging to different species may have different so-called codon usage, among others. Whereby the same polypeptide (in terms of amino acid sequence) may be encoded by one or more different nucleic acids. In addition, due to the degeneracy of the genetic code, different nucleic acids may encode the same polypeptide.
Definition of
An "affinity matured" antibody refers to an antibody having one or more alterations in one or more hypervariable regions (HVRs) which result in an improvement in the affinity of the antibody for an antigen compared to a parent antibody not having such alterations.
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 (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.
An "antibody fragment" refers to a molecule other than an intact antibody, which comprises a portion of an intact antibody that binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab '-SH, F (ab')2(ii) a A diabody; a linear antibody; single chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.
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.
The "class" of an antibody refers to the type of constant domain or constant region that its heavy chain has. There are five main classes of antibodies: IgA, IgD, IgE, IgG and IgM, several of which may be further divided into subclasses (isotypes), e.g. IgG1、IgG2、IgG3、IgG4、IgA1And IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.
The term "expression" as used herein refers to the process of transcription and/or translation that occurs within a cell. The level of transcription of a nucleic acid sequence of interest in a cell can be determined based on the amount of the corresponding mRNA present in the cell. For example, mRNA transcribed from a sequence of interest can be quantified by RT-PCR or by Northern hybridization (see Sambrook et al, 1999, supra). The polypeptide encoded by the nucleic acid of interest can be quantified by a variety of methods, e.g., by ELISA, by assaying the biological activity of the polypeptide, or by using assays independent of such activity, such as Western blots or radioimmunoassays using immunoglobulins that recognize and bind to the polypeptide (see Sambrook et al, 1999, supra).
An "expression cassette" refers to a construct comprising regulatory elements such as a promoter and polyadenylation site necessary for expression of at least the contained nucleic acid in a cell.
An "expression vector" is a nucleic acid that provides all of the elements necessary for expression of one or more structural genes contained in a host cell. Typically, the expression plasmid comprises: prokaryotic plasmid propagation units, for example for e.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 said to be "operably linked" to the promoter. Similarly, a regulatory element is operably linked to a core promoter if the regulatory element modulates the activity of the core promoter.
The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, which comprises at least part 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 carboxy-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. 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, E.A. et al, Sequences of Proteins of Immunological Interest, 5 th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242, also known as EU index.
The "Fc region" is a well-known term defined in terms of papain cleavage of antibody heavy chains. In one embodiment, the complex as reported herein may comprise 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 of a human antibody of the subclass IgG4, or of a human antibody of the subclass IgG1, IgG2 or IgG3, modified in such a way that no fcgamma receptor (e.g., fcyriiia) binding and/or no C1q binding can be detected. In one embodiment, the Fc region is a human Fc region, and in particular a mutant Fc region from the human IgG4 subclass or from the human IgG1 subclass. In one embodiment, the Fc region is from the human IgG1 subclass with mutations L234A and L235A. IgG4 showed reduced Fc γ receptor (Fc γ RIIIa) binding, but antibodies of the other IgG subclasses showed 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 which when altered also provide reduced Fc γ receptor binding (Shields, R.L. et al, J.biol.chem.276(2001)6591 and 6604; Lund, J.et al, FASEB J.9(1995)115 and 119; Morgan, A. et al, Immunology 86(1995)319 and 324; EP 0307434). In one embodiment, the antibody to be expressed in one aspect as reported herein is directed to Fc γ receptor binding of the subclass IgG4 or IgG1 or IgG2, has a mutation in L234, L235 and/or D265, and/or comprises a PVA236 mutation. In one embodiment, the mutation is S228P, L234A, L235A, L235E and/or PVA236(PVA236 denotes the replacement of the amino acid sequence ELLG (given in the single letter amino acid code) from amino acid positions 233 to 236 of IgG1 with PVA or the EFLG of IgG 4). In one embodiment, the mutations are S228P for IgG4 and L234A and L235A for IgG 1. The Fc region of an antibody is directly involved in ADCC (antibody-dependent cytotoxicity) and CDC (complement-dependent cytotoxicity). Complexes that do not bind Fc γ receptor 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 domain of antibody CH 3. Hole modifications represent mutations T366S, L368A and Y407V in the domain of antibody CH 3. 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.
"framework region" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FRs of a variable domain generally consist of four FR domains: FR1, FR2, FR3 and FR 4. Thus, HVR and FR sequences typically occur in the VH (or VL) in the following order: FR1-H1(L1) -FR2-H2(L2) -FR3-H3(L3) -FR 4.
The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a native antibody structure or having a heavy chain comprising an Fc region as defined herein.
"Gene" means a nucleic acid, which is a segment on, for example, a chromosome or on a plasmid that can affect the expression of a peptide, polypeptide, or protein. In addition to the coding region (i.e.the structural gene), the gene comprises further functional elements such as signal sequences, one or more promoters, introns and/or terminators.
The terms "host cell," "host cell line," and "host cell culture" are used interchangeably to refer to a cell into which an exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include the originally transformed cell and progeny derived therefrom, regardless of the number of passages. The nucleic acid content of the progeny may not be identical to the parent cell, but may comprise mutations. Included herein are mutant progeny that have the same function and biological activity as screened or selected in the originally transformed cell.
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 using a human antibody repertoire or other human antibody coding sequences. This definition of human antibodies specifically excludes humanized antibodies comprising non-human antigen binding residues.
A "humanized" antibody is a chimeric antibody 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, in which 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. The humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. "humanized forms" of antibodies (e.g., non-human antibodies) refer to antibodies that have been humanized.
The term "hypervariable region" or "HVR" as used herein refers to each of the regions of an antibody variable domain which are hypervariable in sequence and/or form structurally defined loops ("hypervariable loops"). Typically, a native four-chain antibody comprises six HVRs; three in VH (H1, H2, H3) and three in VL (L1, L2, L3). HVRs typically comprise amino acid residues from hypervariable loops and/or from "complementarity determining regions" (CDRs) that 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, A.M., 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, amino acid residues 50-56 of L2, amino acid residues 89-97 of L3, amino acid residues 50-65 of amino acid residues 31-35B, H2 of H1 and amino acid residues 95-102 of H3 (Kabat, E.A. et al, Sequences of Proteins of Immunological Interest, published Health Service 5, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242). In addition to CDR1 in VH, the CDRs generally comprise amino acid residues that form hypervariable loops. CDRs also contain "specificity determining residues" or "SDRs," which are residues that contact antigen. SDR is contained within a region of a CDR called the shortened CDR or a-CDR. Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2 and a-CDR-H3) are present at amino acid residues 31-34 of L1, amino acid residues 50-55 of L2, amino acid residues 89-96 of L3, amino acid residues 50-58 of amino acid residues 31-35B, H2 of H1 and amino acid residues 95-102 of H3 (Almagro, J.C. and Fransson, J., Front.biosci.13(2008) 1619-1633). Unless otherwise indicated, HVR residues and other residues (e.g., FR residues) in the variable domains are numbered herein above according to Kabat et al.
An "internal ribosome entry site" or "IRES" describes a sequence that functionally facilitates translation initiation 5' of a gene independent of the IRES and allows translation of two cistrons (open reading frames) from a single transcript within an animal cell. The IRES provides an independent ribosome entry site for translation of open reading frames just downstream (downstream is used interchangeably herein with 3'). Unlike bacterial mrnas (which may be polycistronic, i.e., encoding several different polypeptides that are sequentially translated from the mRNA), most mrnas of animal cells are monocistronic, encoding only the synthesis of one protein. For polycistronic transcripts in eukaryotic cells, translation will start from the translation start site closest to 5' and stop 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 having an IRES operably linked to a second or subsequent open reading frame in the transcript allow for the sequential translation of the downstream open reading frame to produce two or more polypeptides encoded by the same transcript. The use of IRES elements in vector construction has been described previously, see, e.g., Pelletier, J. et al, Nature 334(1988) 320-; jang, S.K., et al, J.Virol.63(1989) 1651-1660; davies, M.V. et al, J.Virol.66(1992) 1924-; adam, M.A. et al, J.Virol.65(1991) 4985-4990; morgan, R.A. 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-; and Mosser, D.D., et al, BioTechniques 22(1997)150- "152).
The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., a single antibody comprising the population is identical and/or binds the same epitope, except, for example, for antibodies comprising naturally occurring mutations or possible variants that occur during the production of monoclonal antibody preparations (such variants are typically present in smaller amounts). Unlike polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies 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 that utilize transgenic animals containing all or part of a human immunoglobulin locus, such methods and other exemplary methods for preparing monoclonal antibodies are described herein.
"native antibody" refers to a naturally occurring immunoglobulin molecule having a different structure. For example, a native IgG antibody is a heterotetrameric glycoprotein 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 known as a variable heavy chain domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH 3). Similarly, from N-terminus to C-terminus, each light chain has a variable region (VL), also known as a variable light chain domain or light chain variable domain, followed by a constant light Chain (CL) domain. The light chain of an antibody can be assigned to one of two types, called kappa and lambda, depending on the amino acid sequence of its constant domain.
As used herein, "nucleic acid" refers to a polymer molecule, such as DNA, RNA or modifications thereof, that is composed of mononucleotides (also referred to as bases) a, c, g, and t (or u in RNA). The polynucleotide molecule may 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 are altered (e.g., by mutagenesis), deleted, or added. The nucleic acid may be isolated, or integrated into another nucleic acid (e.g., an expression cassette), a plasmid, or the chromosome of the host cell. Nucleic acids are also characterized as nucleic acid sequences consisting of single nucleotides.
Procedures and methods for converting, for example, an amino acid sequence of a polypeptide into a corresponding nucleic acid sequence encoding such an amino acid sequence are well known to those skilled in the art. Thus, a nucleic acid is characterized by its nucleic acid sequence consisting of a single nucleotide and likewise by the amino acid sequence of the polypeptide encoded thereby.
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. The nucleic acid may be integrated into another nucleic acid, e.g., an expression plasmid or the genome/chromosome of a eukaryotic host cell. Plasmids include shuttle plasmids and expression plasmids. Typically, the plasmid will also comprise a prokaryotic reproductive unit containing the origin of replication (e.g., the ColE1 origin of replication) and a selectable marker (e.g., an ampicillin or tetracycline resistance gene) of the plasmid in prokaryotes for replication and selection, respectively.
"operably linked" refers to the juxtaposition of two or more components wherein the components are in a relationship permitting them to function in their intended manner. For example, a promoter and/or enhancer is operably linked to a coding sequence if it acts in cis to control or regulate the transcription of the linked sequence. Typically, but not necessarily, the "operably linked" DNA sequences are contiguous and, where necessary to join two protein coding regions (e.g., a secretory leader and a polypeptide), contiguous and in (open) frame. However, although an operably linked promoter is typically located upstream of a coding sequence, it must be located adjacent to the coding sequence. Enhancers need not be contiguous. An enhancer is operably linked to a coding sequence if it increases the transcription of the coding sequence. An operably linked enhancer may be located upstream of, within or downstream of a coding sequence and at a considerable distance from the promoter. A polyadenylation site is operably linked to a coding sequence if it is located downstream of the coding sequence such that transcription proceeds through the coding sequence into the polyadenylation sequence. A translation stop codon is operably linked to an exonic nucleic acid sequence if it is located at the downstream end (3' end) of the coding sequence such that translation proceeds through the coding sequence to the stop codon and terminates there. Ligation is achieved by recombinant methods known in the art, e.g., using PCR methods and/or by ligation at convenient restriction sites. If no convenient restriction sites exist, synthetic oligonucleotide adaptors or linkers are used as is conventional practice.
A "polycistronic transcription unit" is a transcription unit in which more than one structural gene is under the control of the same promoter.
The term "polyadenylation signal" as used within this application refers to a nucleic acid sequence used to induce cleavage and polyadenylation of a primary transcript of a particular nucleic acid sequence segment. The 3 'untranslated region comprising a polyadenylation signal may be selected from the 3' untranslated regions comprising polyadenylation signals derived from SV40, the bovine growth hormone (bGH) gene, the immunoglobulin gene, and the thymidine kinase gene (tk, e.g., herpes simplex virus thymidine kinase polyadenylation signal).
"promoter" refers to a polynucleotide sequence that controls the transcription of a gene/structural gene or nucleic acid sequence to which it is operably linked. The promoter contains 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 contemplated to be expressed. Numerous 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 are available as or within cloned polynucleotides (e.g., from depositories such as ATCC and other commercial or personal sources).
A "promoter" comprises a nucleotide sequence that directs the transcription of a structural gene. Typically, a promoter is located in the 5' non-coding or untranslated region of a gene, near the transcriptional start site of a structural gene. Sequence elements within a promoter that play a role in initiating transcription are often characterized as consensus nucleotide sequences. These promoter elements include RNA polymerase binding sites, TATA sequences, CAAT sequences, differentiation specific elements (DSE; McGehe, R.E. et al, mol.Endocrinol.7(1993)551), cyclic AMP response elements (CREs), serum response elements (SRE; Treisman, R., Seninars in Cancer biol.1(1990)47), Glucocorticoid Response Elements (GRE), and others such as CRE/ATF (O' Reilly, M.A. et al, J.biol.Chem.267(1992)19938), AP2(Ye, J.et al, J.biol.Chem.269(1994)25728), SP1, cAMP response element binding protein (CREB; Loeken, M.R., Gene Expr.3(1993)253), and octanucleotide factors (see generally, editor et al, (editions), Molecular of The transcription of The Gene promoter sequences (E.7, Inc. 7, Inc., and Company, Inc. 7, Inc. 7, Inc., rice, Inc. 7, Inc., and Leigh. If the promoter is an inducible promoter, the rate of transcription increases in response to an inducing agent. Conversely, if the promoter is a constitutive promoter, the rate of transcription is not regulated by an inducing agent. 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, a CMV promoter followed by two Tet operator sites. The Tet repressor binds to both Tet operator sites and blocks transcription. The Tet repressor is released from the Tet operator site upon addition of the inducer tetracycline, and transcription continues (Gossen, M. and Bujard, H., PNAS 89(1992) 5547-. For other inducible promoters including metallothionein and heat shock promoters, see, e.g., Sambrook et al (supra) and Gossen et al, curr. Opin. Biotech.5(1994) 516-. Among the eukaryotic promoters that have been identified as strong promoters for high levels of expression are the SV40 early promoter, the adenovirus major late promoter, the mouse metallothionein-I promoter, the Rous sarcoma virus long terminal repeat, Chinese hamster elongation factor 1 α (CHEF-1, see, e.g., US 5,888,809), human EF-1 α, ubiquitin, and the human cytomegalovirus immediate early promoter (CMV IE).
A "promoter" may be constitutive or inducible. Enhancers (i.e., cis-acting DNA elements that act on a promoter to increase transcription) may be necessary to act in conjunction with a promoter to increase the level of expression obtained 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 SV 40).
The terms "stably transformed", "stably transfected" or "stable" as used within this application refer to the genetic and stable integration of an exogenous nucleic acid into the host cell genome/chromosome. Stably transfected cells are obtained following a cell selection process under selective growth conditions (i.e., in the presence of one or more selectable markers).
"structural gene" refers to a region of a gene that does not contain a signal sequence, i.e., a coding region.
The term "transcription terminator" refers to a DNA sequence of 50 to 750 base pairs in length from which RNA polymerase sends a signal to terminate mRNA synthesis. Especially when using strong promoters, it is advisable to prevent read-through by the RNA polymerase with a very efficient (strong) terminator at the 3' end of the expression cassette. A defective transcription terminator may lead to the formation of operon-like mRNA, which may be responsible for undesirable (e.g., plasmid-encoded) gene expression.
Within the scope of the present invention, transfected cells may be obtained using essentially any of the transfection methods known in the art. For example, nucleic acids can be introduced into cells by electroporation or microinjection. Alternatively, lipofection reagents such as FuGENE 6(Roche Diagnostics GmbH, Germany), X-tremeGENE (Roche Diagnostics GmbH, Germany) and LipofectAmine (Invitrogen Corp., USA) can be used. Still alternatively, the nucleic acid may be introduced into the cells by a suitable viral vector system based on retroviruses, lentiviruses, adenoviruses or adeno-associated viruses (Singer, o., proc.natl.acad.sci.usa101(2004) 5313-.
The term "transient transfection" as used within this application refers to a method in which a nucleic acid introduced into a cell does not integrate into the genomic or chromosomal DNA of the cell. It is actually maintained in the cell as an extrachromosomal element (e.g., as an episome). The transcription process of the nucleic acid of the episome is not affected and, for example, a protein encoded by the nucleic acid of the episome is produced. Transient transfection results in "transiently transfected" cells.
The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding of 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 (FR) and three hypervariable regions (HVRs) (see, e.g., Kindt, t.j. et al, Kuby Immunology, 6 th edition, w.h.freeman and co., n.y. (2007), page 91). A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, libraries of complementary VL or VH domains can be screened from VH or VL domains, respectively, of antibodies that bind to the antigen to isolate antibodies that bind to a particular antigen (see, e.g., Portolano, S. et al, J.Immunol.150(1993) 880-.
The term "vector" as used herein refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes a vector which is an autonomously replicating nucleic acid construct, as well as a vector which is incorporated into the genome of a host cell into which it is 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".
Antibodies
The methods and compositions provided herein are useful for the production of 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).
In certain embodiments, the antibody is an antibody fragment. Antibody fragments include, but are not limited to, Fab '-SH, F (ab')2Fv and scFv fragments are the other fragments described below. For a review of certain antibody fragments see Hudson, P.J., et al, nat. Med.9(2003) 129-. For reviews 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), p.269-315; see also WO 1993/16185, US 5,571,894 and US 5,587,458. Fab and F (ab') comprising salvage receptor binding epitope residues and having increased half-life in vivo2See U.S. Pat. No. 5,869,046 for a discussion of fragments.
Diabodies are antibody fragments with two antigen-binding sites, which may be bivalent or bispecific (see, e.g., EP 0404097; WO 1993/01161; Hudson, P.J. et al, nat. Med.9(2003) 129-. Tri-and tetrabodies are also described in Hudson, P.J., et al, nat. Med.9(2003) 129-134.
A single domain antibody is an antibody fragment comprising all or part of the heavy chain variable domain or all or part of the light chain variable domain of the antibody. In certain embodiments, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, e.g., US 6,248,516B 1).
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 (e.g., E.coli or phage).
In certain embodiments, the antibody is a chimeric antibody. Certain chimeric antibodies are described, for example, in US 4,816,567; and Morrison, S.L., 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 (e.g., monkey)) and a human constant region. In another example, a chimeric antibody is a "class switch" antibody, wherein the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.
In certain embodiments, the chimeric antibody is a humanized antibody. Typically, non-human antibodies are humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. Typically, a humanized antibody comprises one or more variable domains in which HVRs, e.g., CDRs (or portions thereof), are derived from a non-human antibody and FRs (or portions thereof) are derived from a human antibody sequence. The humanized antibody will also optionally comprise at least partially human constant regions. In some embodiments, some FR residues in the humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., an antibody from which HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.
Humanized antibodies and methods for their preparation are reviewed, for example, in Almagro, J.C. 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, S.V. et al, Methods 36(2005)25-34 (describing SDR (a-CDR) grafting); padlan, e.a., mol.immunol.28(1991)489-498 (described "resurfacing"); dall' Acqua, w.f. 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 (the" guide selection "pathway describing FR shuffling).
Human framework regions that can be used for humanization include, but are not limited to: framework regions selected by the "best-fit" method (see, e.g., Sims, M.J., et al, J.Immunol.151(1993) 2296-; framework regions derived from consensus sequences of human antibodies for a particular subset of light or heavy chain variable regions (see, e.g., Carter, P. et al, Proc. Natl. Acad. Sci. USA 89 (1992)) 4285-; human mature (somatic mutation) or germline framework regions (see, e.g., Almagro, J.C. 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-.
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, m.a. and van de Winkel, j.g., curr. opin. pharmacol.5(2001) 368-.
Human antibodies can be made by administering an immunogen to a transgenic animal that has been modified to produce whole human antibodies or whole antibodies with human variable regions in response to an antigen challenge. Such animals typically contain all or part of a human immunoglobulin locus, which replaces an endogenous immunoglobulin locus, or which is present extrachromosomally or randomly integrated into the chromosome of the animal. In such transgenic mice, the endogenous immunoglobulin locus has typically been inactivated. For reviews of methods for obtaining human antibodies from transgenic animals, see Lonberg, N., Nat. Biotech.23(2005)1117-TMTechnical US 6,075,181 and US 6,150,584; description of the inventionUS 5,770,429 of the art; description of K-MUS 7,041,870 of the art; description of the inventionUS 2007/0061900 of the art. May be further modified, for example, by combination with different human constant regionsHuman variable regions that complement intact antibodies produced by such animals.
Human antibodies can also be made by hybridoma-based methods. Human myeloma and mouse-human mixed myeloma cell lines useful for the Production of human Monoclonal antibodies have been described (see, e.g., Kozbor, D., J.Immunol.133(1984) 3001-3005; Brodeur, B.R. et al, Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York (1987), pp. 51-63; and Borner, 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, U.S. Pat. No. 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 (triple hybridoma technology) is also described in Vollmers, H.P. and Brandlein, S., Histology and Histopathology 20(2005) 927-.
Human antibodies can also be produced by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences can then be combined with the desired human constant regions. Techniques for selecting human antibodies from antibody libraries are described below.
Combinatorial libraries can be screened against antibodies having one or more desired activities to isolate the 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, for example, in Hoogenboom, H.R. et al, Methods in Molecular Biology 178(2001)1-37, and are further described, for example, in McCafferty, J. et al, Nature 348(1990) 552-; clackson, T.et al, Nature 352(1991) 624-; marks, J.D., et al, J.mol.biol.222(1992) 581-597; marks, J.D. and Bradbury, A., Methods in Molecular Biology 248(2003) 161-175; sidhu, S.S. et al, J.mol.biol.338(2004) 299-310; lee, C.V., et al, J.mol.biol.340(2004) 1073-1093; fellouse, F.A., Proc.Natl.Acad.Sci.USA101(2004) 12467-12472; and Lee, C.V., et al, J.Immunol.methods 284(2004) 119-132.
In some phage display methods, pools of VH and VL genes are separately cloned by Polymerase Chain Reaction (PCR) and randomly combined in phage libraries, which are then screened against antigen-binding phage as described in Winter, G.et al, Ann. Rev. Immunol.12(1994) 433-. Phage typically display antibody fragments as single chain fv (scfv) fragments or as Fab fragments. Libraries from immunized sources provide high affinity antibodies against the immunogen without the need to construct hybridomas. Alternatively, the first experimental pool (e.g., from humans) can be cloned as described by Griffiths, A.D., et al, EMBO J.12(1993)725-734 to provide a single source of antibodies against a wide range of non-self as well as self antigens without any immunization. Finally, libraries first used in the experiment can also be prepared synthetically by cloning unrearranged V gene segments from stem cells and using PCR primers comprising random sequences to encode the highly variable CDR3 regions and to effect in vitro rearrangement, as described by Hoogenboom, H.R., and Winter, G., J.mol.biol.227(1992) 381-. Patent publications 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.
Antibodies or antibody fragments isolated from a human antibody library are considered herein to be human antibodies or human antibody fragments.
In certain embodiments, the antibody is a multispecific antibody, e.g., a bispecific antibody. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites. In certain embodiments, one of the binding specificities is directed to a first antigen and the other is directed to a second, different antigen. In certain embodiments, a bispecific antibody can bind to two different epitopes of the same antigen. Bispecific antibodies can also be used to target cytotoxic agents to cells expressing the antigen. Bispecific antibodies can be prepared as full length antibodies or antibody fragments.
Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein, C. and Cuello, A.C., Nature 305(1983) 537-3659; WO 93/08829; and Traunecker, A. et al, EMBO J.10(1991)3655-3659) and "pestle-in-hole" engineering (see, e.g., U.S. Pat. No. 5,731,168). Multispecific antibodies can also be prepared by: engineered electrostatic priming for the preparation of antibody Fc-heterodimer molecules (WO 2009/089004); crosslinking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980 and Brennan, M. et al, Science 229(1985) 81-83); production of bispecific antibodies using leucine zippers (see, e.g., Kostelny, S.A., et al, J.Immunol.148(1992) 1547-1553); the use of the "diabody" technique for the preparation of bispecific antibody fragments (see, e.g., Holliger, P. et al, Proc. Natl. Acad. Sci. USA 90(1993) 6444-6448); the use of single chain fv (scFv) dimers (see, e.g., Gruber, M. et al, J.Immunol.152(1994) 5368-5374); trispecific antibodies were prepared as described, for example, in Tutt, A. et al, J.Immunol.147(1991) 60-69.
Also included herein are engineered antibodies having three or more functional antigen binding sites, including "octopus antibodies" (see, e.g., US 2006/0025576).
The antibody may be a "dual acting Fab" or "DAF" containing an antigen binding site that binds to a first antigen as well as to a different antigen (see e.g. US 2008/0069820).
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.
Method
In certain embodiments, the methods provided herein are used to alter (i.e., increase or decrease) the degree of glycosylation of an antibody.
Where the antibody comprises an Fc region, the carbohydrate attached to the Fc region may be altered. Natural antibodies produced by mammalian cells typically comprise branched biantennary oligosaccharides, which are typically N-linked to Asn297 of the CH2 domain attached to the Fc region (see, e.g., Wright, a. and Morrison, s.l., TIBTECH 15(1997) 26-32). Oligosaccharides may include a variety of saccharides, 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, modifications of oligosaccharides in the antibodies of the invention can be performed to produce antibody variants with certain improved properties.
In one embodiment, the provided methods result in the production of antibodies having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. For example, the amount of fucose in such an antibody may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose was determined by calculating the average amount of fucose within the sugar chain at Asn297 with respect to the sum of all sugar structures attached to Asn297 (e.g., complex, hybrid and high mannose structures) measured by MALDI-TOF mass spectrometry as described in, for example, WO 2008/077546. Asn297 refers to the asparagine residue located at about position 297 in the Fc region (Kabat EU numbering of Fc region residues); however, due to small sequence variations in antibodies, Asn297 may also be located about ± 3 amino acids upstream or downstream of position 297, i.e. between positions 294 and 300. Such fucosylated variants may have improved ADCC function (see e.g., 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; WO 2005/053742; WO 2002/031140; 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 protein fucosylation deficient Lec13 CHO cells (Ripka, J. et al, Arch. biochem. Biophys.249(1986) 533-.
In certain embodiments, the provided methods can be used to produce antibodies having bisected oligosaccharides, e.g., where the biantennary oligosaccharides attached to the Fc region of the antibody are bisected by GlcNAc. Such antibody variants may 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 an oligosaccharide attached to an Fc region may also be produced. Such antibody variants may have improved CDC function. Such variants are described, for example, in WO 1997/30087, WO 1998/58964 and WO 1999/22764.
Antibodies can be produced using recombinant methods and compositions such as those described in US 4,816,567. The nucleic acid may encode an amino acid sequence comprising a VL of an antibody and/or an amino acid sequence comprising a VH of an antibody (e.g., a light chain and/or a heavy chain of an antibody). In another embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In another embodiment, host cells comprising such nucleic acids are provided. In one such embodiment, the host cell comprises (or has been transformed with): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising a VL of an antibody and an amino acid sequence comprising a VH of an antibody; or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising a VL of an antibody and a second vector comprising a nucleic acid encoding an amino acid sequence comprising a VH of an antibody. In one embodiment, the host cell is a eukaryotic cell, such as a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NS0, Sp 2/0). In one embodiment, a method of producing an antibody is provided, wherein the method comprises culturing a host cell provided above containing a nucleic acid encoding the antibody under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).
For recombinant production of the antibody, the nucleic acid encoding the antibody is isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such nucleic acids can be readily isolated and sequenced using conventional methods (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of an antibody).
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, particularly where glycosylation and Fc effector function 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, K.A., In: Methods In Molecular Biology, volume 248, Lo, B.K.C. (eds.), Humana Press, Totowa, NJ (2003), pp.245-. After expression, the antibody can be isolated from the bacterial paste in the soluble fraction and can be further purified.
In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for vectors encoding antibodies, including fungi and yeast strains in which the glycosylation pathway has been "humanized" resulting in the production of antibodies with partially or fully human glycosylation patterns (see Gerngross, T.U., nat. Biotech.22(2004) 1409-.
Host cells suitable for expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. A number of baculovirus strains have been identified which can be used in conjunction with insect cells, particularly for transfecting Spodoptera frugiperda (Spodoptera frugiperda) cells.
Plant cell cultures may also be used as hosts (see, e.g., US 5,959,177, US 6,040,498, US 6,420,548, US 7,125,978 and US 6,417,429 (describe PLANTIBODIES for antibody production in transgenic plants)TMTechnique)).
Vertebrate cells can also be used as hosts. For example, mammalian cell lines domesticated to grow in suspension may be used. Other examples of useful mammalian host cell lines are SV40 transformed monkey kidney CV1 cell line (COS-7); human embryonic kidney cell lines (293 or 293 cells as described, for example, in Graham, f.l. et al, j.gen virol.36(1977) 59-74); baby hamster kidney cells (BHK); mouse support cells (TM 4 cells as described, for example, in Mather, J.P., biol. reprod.23(1980)243- & 252); monkey kidney cells (CV 1); VERO cells (VERO-76); human cervical cancer cells (HELA);canine kidney cells (MDCK); buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor (MMT 060562); TRI cells as described, for example, in Mather, J.P., et al, Annals N.Y.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. USA77(1980) 4216-4220); and myeloma cell lines, such as Y0, NS0 and Sp 2/0. For a review of certain mammalian host cell lines suitable for antibody production see, e.g., Yazaki, p. and Wu, a.m., Methods in Molecular Biology, volume 248, Lo, b.k.c. (eds.), Humana Press, Totowa, NJ (2004), page 255-268.
Specific aspects of the invention
It has been found that depending on the vector organization, the performance of the expression vector varies (1) by vector design, and (2) between transient and stable transfection of the same vector.
It has been found that the optimal vector tissues for transient and stable transfection can be significantly different. Without being bound by theory, several points may contribute to these differences: 1) transcriptional interference phenomena between integrated vector copies in the host genome, which depend on and are specific to each vector design, and which do not exist in transient systems; 2) the influence of the selection method and the selection stringency, which depends on the respective vector organization, and plays an important role in a stable system rather than in a transient system; and 3) optimal LC to HC polypeptide ratio, which for monoclonal antibody transient expression and stable expression is significantly different, and in transient transfection is lower than in stable transfection.
It was found that for transient transfection, bidirectional expression of LC and HC reached the highest product titer in all vector tissues tested. Without being bound by theory, it is hypothesized that this vector design meets the two criteria of optimal IgG expression, a) high expression levels of LC and HC polypeptides and B) optimal "transient" LC: HC polypeptide ratios, and C) bi-directional expression of LC and HC may also minimize transcriptional interference mechanisms, such as read-through by RNA polymerase.
However, it has been found that for stable transfection, bidirectional expression of LC and HC is worse than for the aligned tissue LC-HC-SM. Without being limited to this theory: 1) the convergent organization of the expression cassettes for LC, HC and SM reduces the phenomenon of transcriptional interference between the integrated vector copies; 2) LC upstream of HC clearly facilitates the optimal (better) LC: HC polypeptide ratio for stable transfection; and 3) downstream placement of a selectable marker significantly increases the stringency of selection. In addition, the percentage of IgG-producing cells and productivity of the cell line was found to be increased. For vectors containing the selectable marker bi-directionally upstream of the antibody expression cassette, while the stringency of selection was significantly increased, the concentrations that increased the selection pressure did not increase the productivity of the stable pool or clones (data not shown).
It has been found that the simultaneous exchange of the hCMV promoter and SV40poly signal with the hEF1 α promoter and the bGH polyA signal significantly increases the transient product titer when the antibody expression cassette is unidirectionally organized, but decreases the product titer when the LC and HC expression cassettes are placed in both directions.
It has been found that the hEF1 α promoter produces a large number of good clones and a very small number of non-producing or low producing clones. However, the best monoclonal hEF1 α promoter gave lower product titers in the fed-batch assay than clones of the hCMV promoter. However, the total number of top-ranked clones of the hCMV promoter is relatively low and their identification usually requires high screening efforts.
It has been found that the use of hGT significantly improves productivity when combined with SV40 or bGH polyA signals for vectors containing the hCMV promoter, both in transient transfection as well as in stable transfection. However, for vectors containing the hEf1 α promoter, when combined with the bGH polyA signal, it had negligible effect on product titer. Thus, it has been found that the effect of hGT on vector performance is dependent on the promoter used.
The selection of appropriate clones for final evaluation in fully controlled large-scale fermentations is usually based on batch or fed-batch analysis in shake flasks. It has been found that the performance and sequencing of some expression vectors or elements differs between batch and fed-batch assays. For vectors containing the hCMV promoter, replacing SV40polyA with the bGH polyA signal increased productivity in batch assays rather than fed-batch assays. In the batch assay, but not in the fed-batch assay, hGT had no significant effect on the product titer of the clones. The placement of the selection marker or the differences between the different vectors of the promoters (hEf1 α or hCMV promoter) were moderately significant in the batch analysis, but strongly visualized in the fed-batch analysis. The expression levels and specific production rates were higher in fed-batch mode than in batch mode.
It was found that the fed-batch analysis of most clones and the performance of 2L fermentations correlated well, not only at the level of absolute product titers, but also in the ordering between different vectors and clones.
It has been found that downstream placement of the selection marker slightly reduces productivity loss compared to bi-directional placement of the selection marker upstream of the antibody expression cassette. Without being bound by theory, this may be due to increased selection stringency, thus higher mRNA levels or improved LC: HC mRNA or polypeptide ratios. Both factors can lead to higher tolerance to changes in productivity.
The bGH polyA signal significantly reduced the stability of antibody expression in clones compared to the SV40polyA signal. However, the insertion of hGT downstream of the bGH polyA signal significantly improved the stability of expression. hGT the positive effect on stability is most pronounced in the absence of selective pressure.
Stability analysis of the stable pool revealed that cells rapidly lost productivity when produced with hCMV, but not when produced with the hEf 1a promoter. Surprisingly, hGT reduced the productivity of clones for vectors containing the hEf1 α promoter, but slightly improved their stability.
Only a small fraction of the clones produced significant antibody after the selection process. However, modification of the vector tissue and/or modification of the elements significantly increases the ratio of IgG-producing clones to non-IgG-producing clones. Different vector tissues and thus different expression levels of selection markers that determine the stringency of selection also significantly affect the percentage of IgG-producing cells. Statistical simulations based on data from the screening process demonstrated that some expression vectors also have the potential to significantly reduce the workload during the screening process. This fact has a major impact on the cost of biopharmaceutical companies.
Improvement of transcription process
Transcription initiation of promoters
Promoters determine the transcription level of a gene and therefore have a strong influence on the productivity and stability of cell lines:
promoter-mediated transcription initiation determines the amount of mRNA that can be translated into a recombinant protein;
long-term productivity of clones can be rapidly reduced despite stable integration (due to genetic gene silencing (e.g. by promoter methylation));
promoter activity is cell type dependent.
Promoters are described that exhibit strong promoters in a variety of cell lines. It is known that introns may contain enhancer-like elements. Typically, the first intron (intron a) contains most of the regulatory elements. This intron a is the first intron to appear within the full-length native promoter sequence/tissue.
PolyA signal mediated polyadenylation of mRNA
Polyadenylation of mRNA serves multiple functions. It strongly affects nuclear export of mRNA, translation initiation and mRNA stability. Thus, the efficiency of the polyadenylation process has a strong influence on the expression level of the gene and thus on productivity.
Several publications have shown that polyadenylation signals can have a strong effect on protein expression. It was reported that the bovine growth hormone-derived polyA signal (bGH polyA signal) could produce enhanced protein expression when substituted for the SV40polyA signal.
It has been found that the bovine growth hormone polyA signal may have improved properties in the recombinant production of antibody chains.
Transcription termination supported by transcription terminator
The elements contained in the expression vector may interfere strongly with each other (transcription interference). This is due to competition in the transcription process. For example, due to space limitations and reduced availability of resources of the transcription machinery, suboptimal promoters are close to transcription factors and/or RNA polymerases. Interference can also occur between different, closely adjacent expression cassettes. For example, read-through of RNA polymerase from a first expression unit through a second expression unit by an inefficient (ineffecific) transcription termination may occur.
It has been reported that inefficient transcription termination can lead to transcriptional interference, which can lead to inefficient expression of the gene. The use of a human gastrin gene transcription terminator (hGT) may improve transcription termination, resulting in enhanced expression of recombinant proteins. This effect is dependent on the promoter in combination with hGT. Other efficient transcription terminators are known.
It has been found that enhanced transcription termination and prevention of transcriptional interference between expression cassettes such as antibody light and antibody heavy chains can be achieved by inserting the sequence of the human gastrin transcription terminator after the early SV40polyA signal of the antibody expression cassette.
Results
Herein is reported an expression vector for enhancing the expression of one or more coding nucleic acids, e.g. a structural gene encoding an antibody chain.
It has been found that the productivity of recombinant cells expressing the structural gene is improved when the genetic elements required for transcription are correctly selected and aligned.
-a polyA signal sequence and a transcription terminator sequence
In vector p5068, the expression cassette is terminated by the SV40polyA signal.
In vector px6001, the expression cassette also contained a human gastrin transcription terminator (hGT) placed downstream of the SV40PolyA signal.
In vector px6007, the expression cassette was terminated by the bGH polyA signal and the hGT transcription terminator.
In vector px6008, the expression cassette was terminated by the bGH polyA signal without an additional transcription terminator.
These vectors were transiently transfected into CHO-K1 cells, and 6 days after transfection, cell culture supernatants were collected and the amount of antibody produced in the culture supernatants was measured by ELISA.
The addition/insertion of the human gastrin terminator (hGT) instead of the SV40polyA signal and SV40polyA signal followed by the bGH polyA signal resulted in an increase in transiently expressed productivity of about 45% to 30% compared to the control vector p 5068. The combination of the bGH polyA signal and the human gastrin terminator (hGT) resulted in the greatest increase in potency (+ 58%) compared to the control vector p 5068.
Carrier
Amount of antibody in supernatant [ μ g/ml [ ]]
Carrier element
p5068
4.6
SV40polyA alone
px6001
6.1
SV40polyA and hGT terminator
px6007
7.4
bGH polyA + hGT terminator
px6008
6.8
bGH PolyA Only
Thus, it has been found that polyadenylation of the bGH polyA signal and improved transcriptional termination by addition of a human gastrin transcriptional terminator enhances antibody secretion in transient expression systems.
Vectors p5068, px6001, px6007 and px6008 were used to generate stable antibody expressing cell lines.
The best 15 monoclonal produced with vector p5068 had an average productivity of 624 μ g/ml in batch antibody production. Clones generated with vector px6001 (containing the human gastrin terminator (hGT)) or vector px6007 (containing the combination of bGH polyA signal and hGT) had improved productivity by 23% and 40%, respectively, compared to vector p5068 (see fig. 1) (770 μ g/ml for vector px6001 and 872 μ g/ml for vector px 6007). This increase in productivity was also reflected in the level of the top-ranked clones (vector px6001 at + 23% (939 μ g/ml), vector px6007 at + 31% (1001 μ g/ml)); calculate the value of the three best clones obtained with each vector).
The clones produced with vector px6008 had an average productivity of the 15 best clones of 576. mu.g/ml.
The average productivity of the three best clones of vector px6008 was 760 μ g/ml.
-promoter sequence
In vector px6051, the expression cassette comprises the full-length human CMV promoter containing intron a and the SV40polyA signal.
In vector px6052, the expression cassette contains the human Ef1 α promoter and the SV40polyA signal.
In vector px6062, the expression cassette comprises the full-length human CMV promoter containing intron a, the bGH polyA signal, and the human gastrin terminator.
In vector px6063, the expression cassette comprises the human Ef1 α promoter, the bGH polyA signal, and the human gastrin terminator.
Carrier
Promoters
polyA signal
Transcription terminator
px6051
Full-length hCMV containing Intron A
SV40polyA Signal
Does not contain a transcription terminator
px6052
Ef1 alpha promoter containing intron A
SV40polyA Signal
Does not contain a transcription terminator
px6062
Full-length hCMV containing Intron A
bGH polyA Signal
Human gastrin transcription terminator (hGT)
px6063
Ef1 alpha promoter containing intron A
bGH polyA Signal
Human gastrin transcription terminator (hGT)
The best 18 stable clones obtained for each vector were identified and the productivity of the clones tested in a batch analysis.
The combined exchange of the bGH polyA signal and hGT for the SV40polyA signal enhanced average productivity: enhancement was about 19% in the case of hEF1 alpha promoter (505. mu.g/ml for vector px6063 vs 426. mu.g/ml for vector 6052) and about 71% in the case of full-length hCMV promoter (333. mu.g/ml for vector px6062 vs 195. mu.g/ml for vector px 6051).
The increase in productivity was also observed at the level of the top-ranked clones (+ 36% of vector px6063 (693. mu.g/ml) to vector px6052 (511. mu.g/ml) and + 102% of vector px6062 (529. mu.g/ml) to vector px6051 (262. mu.g/ml); the three best clone calculations for each vector) (see FIG. 2).
Thus, it has been found that the combination of the hEF 1a promoter with the bGH polyA signal and hGT enhances productivity in stable transfection with bidirectional vector tissue (see px6052) with a selectable marker expression cassette in one direction of transcription and a light chain expression cassette upstream of the heavy chain expression cassette in each other direction of transcription.
Furthermore, it has been found that the combination of the bphgh a signal and hGT enhances productivity in stable transfection of the hCMV promoter using either unidirectional vector tissue (5 '- > 3' direction of the LC-HC-SM expression cassette) (see px9005) with the light chain expression cassette upstream of the heavy chain expression cassette, which in turn is positioned upstream of the selectable marker expression cassette, or bidirectional vector tissue (see px6007) with the light chain expression cassette in one direction of transcription and the light chain expression cassette upstream of the heavy chain expression cassette each in the other direction of transcription.
The combination of the bGH polyA signal and hGT was also used for transient transfection methods. Vectors px6062 and px6063 (containing the full-length hCMV promoter with the combination of bGH polyA signal and hGT or the hEF1 α promoter) were directly compared to vectors px6051 and px6052 (containing the full-length hCMV promoter with the SV40polyA signal or the hEF1 α promoter).
Unlike stable transfection, the combination of bGH polyA signal and human gastrin terminator (hGT) did not improve productivity of transient transfection in both the case of the full-length human CMV promoter containing intron a and the case of the human EF 1a promoter containing intron a (2.38 μ g/ml for vector px6052 versus 2.32 μ g/ml for vector px 6063). 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. mu.g/ml for vector px6062A and 3.64. mu.g/ml for vector px 6051.
In cells obtained by transient transfection, it has been found that the combination of the short hCMV promoter with the bovine growth hormone polyA signal sequence and the human gastrin terminator results in improved antibody expression yields.
In cell lines obtained by transfection and selection of stable cell clones, it has been found that the combination of the bovine growth hormone polyA signal sequence and the human gastrin terminator is independent of the promoter used, resulting in improved antibody expression yields.
In vector p5068, expression of genes encoding both the Light Chain (LC) and the Heavy Chain (HC) of the antibody is driven by a 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 cell type dependent, and hCMV promoter is known to be sensitive to gene silencing, promoter methylation may be stronger and/or more resistant promoters are present in CHO-K1 cells.
Expression vectors containing different promoters were constructed: i) comprises short hCMV without intron a; ii) a full length hCMV promoter containing intron a; iii) full-length rat CMV promoter containing intron A; and iv) the hEF1 alpha promoter containing intron A.
Vectors containing the full-length hCMV promoter (px6051), hEF1 α promoter (px6052), and rat CMV promoter (px6053) were constructed and used for transient and stable transfection.
Expression vectors p5068, px6051, px6052 and px6053 were transiently transfected into CHO-K1 cells by nuclear transfection, four days after transfection, cell culture supernatants were collected and productivity was determined by ELISA.
The vectors containing the hEF1 alpha promoter containing intron A (px6052) or the full-length hCMV promoter containing intron A (px6051) had 53% and 134% improved productivity (3.64. mu.g/ml for vector px6051, 2.38. mu.g/ml for vector px6052, 1.56. mu.g/ml for vector p5068), respectively, compared to the expression vector p 5068. However, the vector p6053 containing the rat CMV promoter showed a reduction in productivity of about 50% (see fig. 3).
Stable transfection was also performed with vectors containing either the full-length human CMV promoter (vectors px6051 and px6062) or the hEF1 α promoter containing intron a (vectors px6052 and px 6063).
Expression vectors p5068, px6051, px6052 and px6053 were transfected into CHO-K1 cells by nuclear transfection and stable pools were selected. The productivity of the library was analyzed in a batch analysis.
Batch analysis of the stable pool showed that antibody titers from cells transfected with the vector containing the hEF 1a promoter containing intron a (vector px6052) were more than 4-fold higher than those of cells transfected with the vector containing the short hCMV promoter (vector p5068) or the vector containing the full-length hCMV promoter containing intron a (vector px6051) (78 μ g/ml for vector px6052, 14 μ g/ml for vector p5068, 17 μ g/ml for vector px6051, 8.46 μ g/ml for vector px6053) (fig. 4).
Stable clones were generated with expression vectors p5068, px6051 and px 6052. In addition, vectors px6062 and px6063 (both comprising a combination of the bGH polyA signal and the human gastrin terminator instead of the SV40polyA signal) were used.
Batch analysis of the best 54 clones obtained for each vector showed an average productivity of 316. mu.g/ml from cells transfected with vector px6052 and 341. mu.g/ml for vector p 5068. Clones generated with vectors px6051 and px6062 comprising the full-length hCMV promoter had an average productivity of 141 μ g/ml for vector px6051 and 220 μ g/ml for vector px 6062.
The average productivity of the 54 clones tested transfected with vector px6063 (containing the hEF1 α promoter combined with the bGH polyA signal and hGT) in batch analysis had a productivity of 367 μ g/ml. The best clone with the highest titer was derived from the vector px6063 (748 and 731. mu.g/ml, respectively).
It was found that clones produced with vectors comprising the human EF 1a promoter (px6052 and px6063) showed a reduced number of low-producing clones.
Clones were tested for stability of antibody production. Three generations of 12 clones obtained with vectors p5068 and px6052 were cultured in the presence and absence of hygromycin B.
In the absence of the 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 (p 5068; 264. mu.g/ml vs. 410. mu.g/ml). In contrast, for the hEF 1. alpha. promoter containing intron A, only an 18% reduction in average productivity was observed (px 6052; 256. mu.g/ml vs 312. mu.g/ml).
In the presence of selection pressure, the clones obtained with vector p5068 showed a 27% reduction in average productivity (298. mu.g/ml versus 410. mu.g/ml) and a 15% reduction in titer from clones obtained with vector px6052 comprising the hEF 1. alpha. promoter containing intron A (266. mu.g/ml versus 312. mu.g/ml).
After three generations with or without selective pressure, the number of stable clones transfected with vector p5068 containing the short hCMV promoter lacking intron a or with vector px6052 containing the hEF 1a promoter containing intron a was determined. To determine whether a clone is "stable" after three generations, the IgG titer from passage 0 was set to 100% and a threshold of 80% was defined. Clones showing a relative IgG titer of 80% lower than that of passage 0 (passage 0) after three generations with or without selective pressure were defined as unstable.
Relative antibody titers from all clones transfected with p5068 dropped to below 80% after three generations with or without selective pressure compared to those of passage 0. A stable cell clone is a population of clonal cells that produces an antibody titer of 80% or more, as compared to the antibody titer of passage 0, in the presence of and in the absence of selection pressure.
Relative titers from clones transfected with px6052 dropped to less than 80% in 8 of 12 cases, compared to IgG titers at passage 0, after three generations with or without selective pressure, but 4 clones were defined as "stable". Unlike clones produced with vector p5068, 8 of the 12 clones produced with vector px6052 were "stable" in the presence of selection pressure. In contrast, only one of the clones derived from vector p5068 was stable in the presence of selection pressure.
Vectors px6014, px6014A and px6014B contained the hEF 1a promoter in front of the antibody light chain and the short hCMV promoter in front of the heavy chain. These vectors differ in the 5'UTR of the light chain (px 6014: 5' UTR of hEF 1a promoter with a PmeI restriction site; px 6014A: 5'UTR of hEF 1a promoter without a PmeI restriction site; px 6014B: 5' UTR of hEF 1a promoter in p 5068). This vector was transiently transfected into CHO-K1 cells using nuclear transfection.
Compared with vector px6014, vectors px6014A and px6014B showed an enhancement in productivity of 20% and 40%, respectively (2.01 μ g/ml for vector px6014B, 1.71 μ g/ml for vector px6014A, 1.41 μ g/ml for vector px 6014).
It was found that in transient transfection, vectors comprising either the human EF 1a promoter or the full-length human CMV promoter (both containing intron a) showed increased productivity compared to vector p 5068.
It was found that the stable library generated with the vector comprising the human EF 1a promoter showed increased productivity in batch analysis compared to vector p 5068.
It has been found that cell clones obtained by stable transfection with a vector comprising the human EF 1a promoter containing intron a show a reduced number of low-producing clones.
It was found that the cell clones obtained by stable transfection with a vector comprising the full-length human CMV promoter containing intron a showed strongly reduced productivity at the level of the mean and the top-ranked clones.
Thus, it has been found that the combination of the bGH polyA signal and hGT enhances productivity in stable transfection independently of the promoter used, compared to SV40polyA, using a bidirectional vector organization with the selectable marker expression cassette in one direction of transcription and the light chain expression cassette upstream of the heavy chain expression cassette in each other direction of transcription (see px 6052).
Furthermore, it has been found that the combination of the bGH polyA signal and hGT enhances productivity in stable transfection of the hCMV promoter using a light chain expression cassette upstream of the heavy chain expression cassette which in turn is located in a unidirectional vector tissue (5 '- > 3' direction of the LC-HC-SM expression cassette) upstream of the selectable marker expression cassette.
Improvements in selection methods
Cell line development is currently very time consuming and costly due to the high number of non-productive and low-productive clones and the low stability of gene expression. Of the thousands of clones, you usually find only a few stable "high producers".
Without being bound by theory, a possible reason for the low selectivity and low stringency of the selection strategy may be the separate expression of the antibody and the selection marker in the vector system used, which does not exert selective pressure on antibody expression (see e.g. vector p 5069).
It has been found that this problem can be overcome by using IRES elements.
IRES elements are DNA elements that act as internal ribosome entry sites (at the mRNA level), thus allowing the expression of two genes from one mRNA.
IRES-linked expression of the selection marker and antibody chain exerts selective pressure on total antibody expression (i.e., one mRNA encodes both the selection marker and the antibody chain), thereby ensuring selectivity of selection. The stability of gene expression can be improved.
The use of IRES elements with weak IRES activity allows for weak expression of selection markers. This weak expression of the selection marker may increase the stringency of the selection.
By IRES-linked co-expression of the selection marker and antibody chain, the selection process can be improved by:
-increasing the number of producing cells,
enhancing the stability of gene expression, and
-increasing the stringency of the selection method.
For IRES-linked co-expression of antibodies and selectable markers, the IRES elements used must meet two requirements:
IRES elements may have no or only minimal effect on mRNA stability/antibody expression, and
the IRES element must exhibit weak IRES activity so that the selection marker is only slightly expressed.
Light and heavy chain encoding nucleic acids were linked by several of EV71-IRES, ELF4G-IRES and EMCV-IRES elements (vectors px6015A, px6015B and px 6015C). The expression cassette comprises in the 5 'to 3' direction a human CMV promoter, a light chain encoding nucleic acid, an IRES, a heavy chain encoding nucleic acid, and a polyA site.
The vector p5068, which did not contain IRES elements, and the vectors px6015A, px6015B, and px6015C, which were used as references, were transiently transfected into CHO-K1 cells, and productivity was measured by ELISA.
The vectors, px6015B and px6015C (comprising ELF4G and EMCV-IRES, respectively) showed IgG expression from 0.1. mu.g/ml to 0.15. mu.g/ml. Vector p5068 shows IgG expression at 2. mu.g/ml. The vector px6015A, which contains EV71-IRES, showed a productivity of 1.7. mu.g/ml.
Alternatively, the selection marker neomycin may be linked directly to the heavy chain of the antibody by an IRES element. The vector comprises in the 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.
Vectors px6010A, px6010B and px6010C were transiently transfected into CHO-K1 cells as described and productivity was determined by ELISA.
The vector px6010C, which contains EMCV-IRES, showed a productivity of 13. mu.g/ml. Vector p5069 showed a productivity of 14. mu.g/ml. Vectors containing EV71-IRES (vector px6010A) or ELF4G-IRES (vector px6010B) showed productivities of 4.3. mu.g/ml and 2.4. mu.g/ml, respectively.
Thus, EMCV-IRES elements have been found to meet the requirements necessary for IRES-linked expression of selectable markers:
weak IRES activity (so that the selection marker is only slightly expressed),
the IRES element has at most a minimal effect on antibody expression (selection marker for expression linked to the respective IRES).
Thus, the selection marker neomycin (mediating resistance to G418) can be linked to the heavy chain encoding nucleic acid by EMCV-IRES.
Vectors p5069 and px6010C were tested for productivity and stability in stable transfections, both at the library level and at the monoclonal level.
The vector was transfected into CHO-K1 cells by nuclear transfection technique. Stable pools were selected and the productivity of the pools was analyzed in a batch analysis.
The library generated with vector px6010C showed a productivity of 14 μ g/ml in the batch analysis. The library generated with vector p5069 showed a productivity of 5.4. mu.g/ml in the batch analysis (FIG. 5).
It was found that the library generated with vector px6010C consisted of more producer cells (or more good producer cells) and/or that the stability of IgG expression was enhanced (compared to vector p 5069).
It has been found that IRES-linked expression of the selection marker neomycin in vector px6010C also leads to enhanced stability of the library. Stable pools were cultured for 30 generations in the presence and absence of selection pressure. The productivity of the pools was determined in batch analysis at the beginning (═ passage 0) and at the end (═ passage 30) of the stability tests.
The library generated with vector px6010C showed enhanced stability of IgG expression in batch analysis compared to the library generated with vector p 5069. The productivity of the library obtained with vector p5069 in batch analysis after 30 generations was strongly reduced (> 80%; values below the detection limit) in the presence and absence of selection pressure. In the absence of selective pressure, the productivity of the library generated with vector px6010C was also reduced by about 70%. However, in the presence of selective pressure, the productivity of the library generated with vector px6010C decreased only by 10% (fig. 6).
Vector p5069 and vector px6010C were transfected into CHO-K1 cells by nuclear transfection, and stable clones were generated as described above. Clones were screened and the productivity of the best 15 clones for each transfection was analyzed in a batch analysis. Two independent transfections were performed for each vector.
Clones generated with vector px6010C showed an average productivity of 348 μ g/ml. Clones generated with vector p5069 showed an average productivity of 239 μ g/ml. The increase in average productivity of clones produced with vector px6010C was not due to better top-ranked clones, but due to a significant reduction in the number of low-yielding clones. Unlike vector p5069, the clone produced with vector px6010C did not show productivity below 200 μ g/ml in batch analysis (fig. 7).
Stability of antibody expression was tested against 17 clones (p5069) and 14 clones (px6010C), respectively, by culturing for 45 generations in the presence and absence of selection pressure. At the beginning of the stability test (passage 0) and at passage 45, the productivity of the clones was measured in a batch analysis. IgG titers at passage 45 were compared to the productivity of clones at the beginning of the stability test.
The average productivity of 17 clones produced with vector p5069 decreased after 45 generations (approximately 43% loss in productivity) in the presence and absence of selection pressure. In the absence of selective pressure, the average productivity of 14 clones produced with vector px6010C decreased by about 45%. However, in the presence of selection pressure, the productivity of these clones decreased by 4%.
In the presence of selection pressure, only 6 of the 17 clones tested generated by vector p5069 showed productivity still higher than 80% of the productivity of passage 0. In contrast, 10 of the 14 clones tested produced with vector px6010C showed productivity that was 80% higher than that of passage 0.
In the absence of selective pressure, there was no significant difference in stability between clones produced with vector p5069 and vector px6010C, respectively.
Improvements in selection and screening methods
Cell line development is currently very time and cost consuming due to intensive screening efforts. Of the thousands of clones, you usually find only a few stable high producers.
Currently, there are several screening strategies for identifying highly productive clones:
ELISA-based screening strategies for direct detection of the produced proteins (time and cost consuming);
fluorescence-based screening strategies for direct detection of the produced proteins (time and cost consuming);
co-expression quantification of the proteins produced-FACS-based sorting of cells for the quantitative screening of markers (surface proteins or fluorescent markers, such as GFP) for indirect detection/quantification.
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 on productivity, so that there is often no good correlation between fluorescence intensity and productivity.
GFP proteins (as an example of a fluorescent label) are stable and tend to accumulate. There is no (in most cases) good correlation between the expression level (fluorescence intensity) of fluorescent markers in cells and their productivity.
It has been found that by linking antibody expression and fluorescent marker expression, a selection and screening strategy that identifies combinations of cell clones has enhanced stability and productivity while being simple, thus allowing rapid and easy identification of high producers.
It has been found that IRES-linked expression of fusion proteins functions as a selectable marker and a quantitative screening marker.
A fusion protein of Green Fluorescent Protein (GFP) and the selectable marker neomycin was constructed that was directly linked to the antibody heavy chain by an IRES element. The green fluorescent protein and the selection marker neomycin in this fusion protein are separated by the PEST sequence (corresponding to the sequence of codons 423-449 of the mouse ornithine decarboxylase gene (mODC)). This PEST sequence acts as a strong proteolytic signal sequence at the protein level, thus significantly reducing the half-life of the protein.
The GFP-PEST-Neo fusion protein not only acts as a selection marker but also as a quantitative screening marker.
The use of PEST sequences provides improved clonal selection due to the reduced half-life of PEST-mediated fusion proteins (increased selective stringency).
The use of PEST sequences provides a good correlation between GFP fluorescence intensity of the fusion protein and antibody expression levels due to the linked co-expression of the antibody and IRES of the selectable marker.
The use of PEST sequences reduces the accumulation of fusion proteins due to the reduced half-life of the protein.
The GFP expression levels of the stable monoclonals were analyzed by FACS and correlated with productivity in batch analysis. Populations with different GFP expression levels from stable pools were sorted by FACS and the productivity of the sorted populations was analyzed.
IRES elements that meet the requirements of this method:
weak IRES activity should be provided so that the fusion protein is only slightly expressed;
should not affect the antibody expression level.
IRES activity or strength was determined by expression of several of EV71-IRES, ELF4G-IRES and EMCV-IRES elements (see vectors px6015A, px6015B and px6015C) linked the light and heavy chains. Vector p5068 (without IRES elements) and vectors px6015A, px6015B and px6015C, which were used as references, were transiently transfected into CHO-K1 cells, and productivity was measured by ELISA.
The vectors px6015B and px6015C (comprising ELF4G and EMCV-IRES, respectively) showed IgG expression from 0.1 to 0.15 μ g/ml. Vector p5068 shows IgG expression at 2. mu.g/ml. Vector px6015A, containing EV71-IRES, showed productivity of 1.7 μ g/ml, indicating that EV71-IRES element has strong IRES activity in CHO-K1 cells.
The GFP-PEST-Neo fusion protein is directly linked to the heavy chain encoding nucleic acid of the antibody by an IRES element (see FIG. 8).
Constructs in which an IRES element linked a selectable 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.
The vector px6011C, which contained EMCV-IRES, showed IgG productivity of 8.7. mu.g/ml, and the vector p5059 showed IgG productivity of 10.8. mu.g/ml. The vector px6011A, which contains EV71-IRES, showed an IgG productivity of 3.9. mu.g/ml. The vector px6011B comprising ELF4G-IRES showed no associated productivity.
Vector px6011C, vector p5069, and vector px6010C containing IRES-linked GFP-PEST-Neo fusion proteins were tested in batch assays of stable pools and stable clones.
Vectors p5069, px6010C and px6011C were transfected into CHO-K1 cells by nuclear transfection. The stable pool was selected as described and the productivity of the stable pool was analyzed in a batch analysis.
The stable pool generated with vector px6010C comprising an IRES-linked selection marker showed IgG productivity of 9.5 μ g/ml. The stable pool generated with vector p5069 showed IgG productivity of 5.4. mu.g/ml. The library generated with vector px6011C comprising IRES-linked fusion proteins showed productivity of 36.3 μ g/ml (fig. 9).
These data indicate that the libraries generated with vectors px6010C and px6011C clearly consist of more or even better producing cells than the library generated with vector p 5069. Enhanced IgG expression stability in/of the px6010C and px6011C pools may also contribute to increased productivity.
To compare the productivity of the expression vector p5068 with the vectors px6010C and px6011C in stable clones, the vectors were transfected into CHO-K1 cells by nuclear transfection. Stable clones were selected as described and clones were screened. The productivity of the best 15 clones of each vector was analyzed in a batch analysis.
For vector p5068, 95 out of a total of 3072 wells in 384 well plates showed IgG production above 2 μ g/ml (background). IRES-linked expression of the selectable marker (px6010C) doubled the number of producing cells/well to 195, and in the case of IRES-linked fusion protein (px6011C) the number of producing cells/well was further increased to over 280.
Furthermore, not only the number of cells produced, but also the average productivity of the clones containing IRES produced by the vectors px6010C and px6011C was higher (3.4. mu.g/ml for the vector p5069, 4.2. mu.g/ml for the vector px6010C, 8.1. mu.g/ml for the vector px6011C, 8.1. mu.g/ml for the best 250 clones for each vector, 2.2mg/ml for the vector p5069, 3.0. mu.g/ml for the vector px6010C, 5.1. mu.g/ml for the vector px 6011C).
Clones generated with IRES vectors pX6010C and pX6011C and p5059 showed average productivities of 212. mu.g/ml for vector pX6011C, 178. mu.g/ml for vector pX6010C and 118. mu.g/ml for vector p5069 in a 24-well screen (determined in 24-well cultures with undetermined cell counts after 4 days of cell division). For vectors px6010C and px6011C, a reduced number of non-producing or low producing clones and an increased number of good producing clones were observed (fig. 10).
Batch analysis of the single clones showed that the average productivity of the 15 best clones produced with vector px6010C was 348. mu.g/ml and vector p5069 was 239. mu.g/ml. The average productivity of the 15 best clones produced with vector px6010C was 404 μ g/ml. The clone with the highest overall titer was derived from the transfectant of vector px 6011C. The clones produced with vector px6010C and vector px6011C did not show productivity below 200 μ g/ml in batch analysis.
To test the antibody expression stability of the clones, 14 to 19 clones obtained with each vector were cultured for 45 passages 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 (passage 0) and at generation 45. IgG titers at passage 45 were compared to the productivity of clones at passage 0 (value set to 100%) at the beginning of the stability test.
The average productivity of 17 clones generated with vector p5069 was reduced after 45 generations (about 43% loss in productivity) in the presence and absence of selection pressure. The average productivity of 14 to 19 clones produced with the vectors px6010C and px6011C, respectively, also decreased in the absence of selection pressure (lost about 45-35% of productivity), but in the presence of the selection marker G418, after the 45 generations, the decrease in average productivity of these clones was only 0-4%.
In the presence of selection pressure G418, 6 of the 17 tested clones produced by vector p5069 showed a productivity of 80% higher than that of passage 0. In contrast, 10 of the 14 tested clones produced with vector px6010C and 17 of the 19 tested clones produced with vector px6011C showed productivity that was 80% higher than that of passage 0.
In the absence of selection pressure, clones generated with vectors p5069, px6010C and px6011C showed comparable behavior.
It has been found that vectors comprising IRES and thus clones obtained therefrom have enhanced stability in the presence of a selectable marker.
However, in the absence of a selectable marker, there was no difference in stability between clones obtained with vectors containing IRES or vectors without IRES.
GFP-PEST-Neo fusion protein was used as a quantitative screening marker. The level of GFP fluorescence of clones produced with vector px6011C is predictive of their productivity.
The GFP expression level/fluorescence intensity of a single clone was determined and the results were correlated with the productivity of the clones in a batch analysis, populations (1,000 cells per vector) with different GFP expression levels/fluorescence intensities were sorted from the stable pool by FACS, and then the productivity of these different populations was analyzed in a batch analysis. Three different populations were sorted:
population 1: geometric Mean (GM) of FL1-H (═ GFP) 0-4 without GFP expression
Population 2: low GFP expression level, GM 4-5.5
Population 3: high GFP expression, GM 5.5-7
Batch and FACS analysis showed that there is a good correlation between GFP fluorescence of clones/pools and their productivity at both the monoclonal and pool levels. Clones or pools with high GFP fluorescence typically show higher productivity than cells showing low GFP fluorescence. In general, the productivity of clones/pools increased with fluorescence intensity (FIGS. 11 and 12).
High producing clones can be identified directly by sorting high GFP fluorescence monoclonals from stable pools based on FACS. Monoclonals showing no or high GFP expression were sorted by FACS. Clones were expanded to 6-well plates with shaking and the productivity of the clones was determined in a batch analysis.
It has been found that EMCV-IRES linked expression of a selectable marker or GFP-PEST-Neo fusion protein significantly enhances the selectivity of selection (resulting in more producing clones), and it also enhances the stringency of selection (resulting in higher average productivity of clones). Thus, screening efforts are significantly reduced.
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 significantly enhance the stringency of selection.
Carrier elements combined with carrier tissue
The following vectors were tested in the CHO-K1 host cell line in transient transfections, in stable pools and some at the monoclonal level.
Carrier
Tissue of
Promoters
polyA signal
Transcription terminator
px9001
SM(3′-5`)-LC-HC
hCMV
SV40 polyA
Is absent from
px9002
LC-HC-SM
hCMV
SV40 polyA
Is absent from
px9003
LC-HC-SM
hEF1α
SV40 polyA
Is absent from
px9004
LC-HC-SM
hCMV
bGH polyA
Is absent from
px9005
LC-HC-SM
hCMV
bGH polyA
hGT
px9006
LC-HC-SM
hEF1α
bGH polyA
Is absent from
px9007
LC-HC-SM
hEF1α
bGH polyA
hGT
px9010
LC(3′-5′)-HC-SM
hEF1α
bGH polyA
Is absent from
px9011
LC(3′-5′)-HC-SM
hCMV
SV40 polyA
hGT
The performance of the different vectors in transient transfection was tested after nuclear transfection into CHO-K1 cells.
Vectors comprising the human elongation factor 1 alpha promoter (hEF1 alpha) (based on the vector tissue LC-HC-SM) have an increased productivity of about + 34% (px9003 vs. px 9002; SV40polyA signal sequence) and + 30% (px9006 vs. px 9004; bGH polyA signal sequence), respectively, depending on the polyA signal sequence used, compared to the use of the hCMV promoter.
The 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 px 9004; + 13%).
Expression vectors based on bidirectional expression of the light and heavy chains of the antibody show improved performance. Depending on the promoter used (hEF1 α or hCMV) and the polyA signal sequence used (SV40 or bGH polyA signal sequence), the product titer increased by about 2.7 to 3.4 fold compared to the control vector px 9001.
It was found that the use of the human elongation factor 1 alpha promoter and the bGH polyA signal sequence had a positive effect on productivity in vector tissue LC-HC-SM (+ 50%; compare px9006 and px9002) but not in vector tissue LC (3 '-5') -HC-SM (-28%; compare px9010 and px 9011).
To compare the productivity of the expression vector px9002 and the vector px9003-9007, the vectors were transfected into CHO-K1 cells by nuclear transfection, and stable pools were selected. The productivity of the library was analyzed in a batch analysis (see FIG. 10).
Batch analysis of stable pools showed that antibody titers from pools transfected with vectors containing the human elongation factor 1 alpha promoter (px9003, px9006, px9007) were about 7-8 times higher than those of cells transfected with a reference vector containing the short hCMV promoter (vector px9002) (97.5. mu.g/ml, 112.5. mu.g/ml and 95.6. mu.g/ml for vectors px9003, px9006 and px9007 compared with 14.0. mu.g/ml for vector px 9002).
The vectors px9001, px9002 and px9004 to px9007 were transfected into CHO-K1 cells by nuclear transfection, and the best monoclonals were identified by classical selection methods. The productivity of the best 36 clones per vector was analyzed in batch analysis and the best 15 clones in batch analysis were tested in fed-batch analysis.
The best 36 single clones produced with vector px9001 had an average productivity of 356 μ g/ml in the batch analysis. Clones generated with vector px9002 or with vectors px9004 and 9005 (also comprising the bGH PolyA signal (alone or in combination with HGT) instead of the SV40PolyA signal) showed an increase in productivity of 37% (px9002), 61% (px9004) to 53% (px9005), respectively, compared to the control vector px 9001. Cloning of the vectors px9006 and px9007 showed an increase in productivity of about 19% and 7%, respectively.
In fed-batch experiments, the best 15 clones obtained with each vector in batch analysis were tested in fed-batch analysis over 14 days.
The best 15 single clones (generated with vector px 9001) had an average productivity of 1345 μ g/ml in the fed-batch analysis. Clones generated with vector px9002 or with vectors px9004 and px9005 (also comprising the bGH PolyA signal sequence (alone or in combination with hGT) instead of the SV40PolyA signal sequence) showed improved productivity by 80% (px9002), 58% (px9004) to 92% (px9005), respectively, compared to the control vector px 9001.
In addition to the increase in average productivity (see above), the performance of the top-ranked clones is also strongly improved. In terms of productivity of the first 5 clones, the vectors px9002, px9004 and px9005 showed about 64% (px9002), 50% (px9004) and 88% (px9005) improvements compared to the control vector px 9001.
In the following, the percentage of producer or non-producer cells for each different vector is directly compared. 14.2% of the clones produced with vector px9001 produced antibodies, and in the remaining resistant clones, antibody expression was silent or clones had other defects.
The vector organization of vector px9002 almost doubled the percentage of cells produced (to 26.0%). Vectors further comprising the bGH PolyA signal sequence in place of the SV40PolyA signal sequence alone (vector px9004) or in combination with hGT (vector px9005) showed an approximately 3-fold increase in the percentage of cells produced (39% and 43% respectively).
Replacement of 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 process did produce antibodies).
The 15 best clones obtained by transfection with vector px9001-9007 (based on fed-batch results) were cultured for 15 passages (═ about 60 passages) in the presence and absence of hygromycin B. The product titer of the clones after 15 passages in the batch analysis was compared with the product titer of the clones in the batch analysis at the beginning of the stability test.
The variation in product titer between 15 clones for each vector varied from-14.7% for vector px9007 to + 0.2% for vector px9002 after 15 generations in the presence of selection pressure.
In the absence of selection pressure, the reduction in product titer ranged from 25.5% for vector px9004 to 5.9% for vector px 9005.
The number of clones meeting defined stability criteria (e.g. product titer > 80% in batch analysis compared to the value of the starting point (G0) in the presence and absence of selection marker) varied from 4 to 10. The vectors px9005, px9007 and px9002 gave the highest number of stable clones (px 9005: 10; px 9007: 7; px 9002: 6) also in the presence and absence of selection pressure/selection marker.
Thus, it has been found that, especially in the absence of selection pressure, the tissue of vector px9005 shows a positive effect on stability and on increasing the number of stable clones.
It was found that the combination of bGH polyA and hGT significantly improved the productivity of stable clones independent of the promoter used compared to SV40polyA without transcription terminator (hGT).
Transient transfection
Use of the human elongation factor 1 alpha promoter (containing intron A) provides enhanced productivity (in LC-HC-SM tissue)
The use of the bovine growth hormone polyA signal sequence provides enhanced productivity compared to the use of the SV40polyA signal sequence
Addition of HGT to the bGH PolyA signal sequence leads to increased productivity in vectors comprising the hCMV promoter
Vector tissue LC (3 '-5') -HC-SM leads to improved expression
Stable storehouse
Libraries generated with vectors containing the hEF1 α promoter showed enhanced productivity in batch analysis
Clones produced with vectors containing the hEF1 alpha promoter show a reduced number of low-yielding clones
Clones produced with vectors containing the hEF1 alpha promoter show higher IgG expression stability
Monoclonal antibody
The vector organization with the selectable marker located downstream (LC-HC-SM) has a positive effect on the productivity of stable monoclonals
Higher productivity and stability of clones produced with vectors containing the bGH polyA signal sequence and hGT
Several different transcription-associated genetic elements and combinations thereof have been compared to reference genetic element combinations. According to comparative transient experiments, the following results were obtained for a bidirectional vector organization with the selectable marker expression cassette in the opposite orientation to the expression cassettes for the light and heavy chains (light chain expression cassette upstream of the heavy chain expression cassette) (see table below).
Different promoters were combined with the bGH polyA signal and hGT transcription terminator (see table below).
Several transcription-associated genetic elements and combinations thereof have been compared to a reference vector (px9001, vector tissue SM (3 '-5' orientation) -LC-HC (5 '-3' orientation)). According to comparative experiments, the following results were obtained for the unidirectional vector tissue with the light chain and the heavy chain (light chain expression cassette upstream of the heavy chain expression cassette) and the expression cassette of the selection marker in the same orientation compared to the reference vector (px9001, bidirectional vector tissue SM (3 '-5') -LC-HC (5 '-3'))) (see table below).
Promoters used
Carrier
Promoters
polyA signal
Transcription terminator
px5068
Short hCMV without Intron A
SV40polyA Signal
Does not contain a transcription terminator
px6001
Short hCMV without Intron A
SV40polyA Signal
hGT transcription terminator
px6008
Short hCMV without Intron A
bGH polyA Signal
Does not contain a transcription terminator
px6007
Short hCMV without Intron A
bGH polyA Signal
hGT transcription terminator
px6051
Full-length hCMV containing Intron A
SV40polyA Signal
Does not contain a transcription terminator
px6052
hEF1 alpha promoter containing intron A
SV40polyA Signal
Does not contain a transcription terminator
px6053
Rat CMV promoter rat Ratty CMV containing Intron A
SV40polyA Signal
Does not contain a transcription terminator
px6062
Full-length hCMV containing Intron A
bGH polyA Signal
hGT transcription terminator
px6063
hEF1 alpha promoter containing intron A
bGH polyA Signal
hGT transcription terminator
It has been found that increased expression (productivity) can be achieved with the vector elements/elements combinations reported herein:
human CMV promoter:
xu et al, J.control.Release,81(2002)155-163.
Xia et al, prot.Expr.Purif.45(2006)115-124.
Rat CMV promoter:
xia et al, prot.Expr.Purif.45(2006)115-124.
Human EF1 α promoter:
teschendorf et al, Anticancer Res.22(2002) 3325-.
Li et al, J.Immunol.methods 318(2007)113-124.
MPSV promoter:
xia et al, prot.Expr.Purif.45(2006)115-124.
Artemit et al, Gene 68(1988)213-219.
Stoking et al, Proc.Natl.Acad.Sci.USA 82(1985) 5746-.
Lin et al, Gene 147(1994)287-292.
MPSV-CMV hybrid promoter:
liu et al, anal. biochem.246(1996) 150-.
By expressing the selection marker with an IRES-linked expression cassette, a highly selective and high stringency selection method can be provided:
selection pressure on antibody expression leads to high selectivity
Linked expression of antibody and selection marker results in high selectivity
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
Linking the expression of the antibody and the selection marker by an IRES element
Identification of IRES elements with a weak Activity (EMCV/Gtx) that indeed alter IgG expression to a greater or lesser extent
Use of the fusion protein as a selection marker and selection marker
-bifunctional GFP-neomycin fusion proteins
The PEST sequence of the ornithine decarboxylase is a strong proteolytic signal sequence conferring a reduced half-life to the protein
IRES-linked expression of the fusion protein leads to high selectivity
Short half-life of the proteolytic signal sequence leading to high stringency
Strong expression is required due to weak expression and short half-life of the fusion protein
Rapid identification of high producers by FACS (sorting of high GFP expressing clones provides selection of high producers)
The selectable marker is linked to the antibody heavy chain by an IRES element
GFP-Neo fusion proteins are linked to the antibody heavy chain by different IRES elements
Gtx-IRES：
Komuro et al, EMBO J.12(1993)1387-1401.
EMCV-IRES：
Mountford et al, Proc. Natl. Acad. Sci. USA 91(1991) 4303-.
EV71-IRES：
Lee et al, Biotechnol. Bioeng.90(2005) 656-.
ELF4G-IRES：
Wong et al, Gene ther.9(2002)337-344.
Gtx (synthetic) -IRES:
chappell et al, Proc. Natl. Acad. Sci. USA97(2000) 1536-.
It has been found that by EV71-IRES element, increased expression (productivity) can be achieved with ligation of a light chain expression cassette to a heavy chain expression cassette:
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.
The following examples, figures and sequences are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications may be made in the illustrated methods without departing from the spirit of the invention.
Sequence of
01 short CMV promoter without Intron A
02 short human CMV promoter without Intron A containing 5' UTR
03 full-Length human CMV promoter containing intron A
04 full-Length human EF1 alpha promoter without Intron A
SEQ ID NO 05 full-Length human EF1 alpha promoter containing intron A
SEQ ID NO 06 short human EF1 alpha promoter containing intron A containing the 5' UTR
SEQ ID NO 07 full-Length rat CMV promoter containing intron A
08SV40 polyA Signal sequence of SEQ ID NO
09bGH polyA Signal sequence of SEQ ID NO
10hGT terminator sequence of SEQ ID NO
11SV40 promoter
PEST sequence of Ornithine decarboxylase of SEQ ID NO 12
Nucleic acid sequence of SEQ ID NO 13 encoding GFP
14 neomycin selection marker of SEQ ID NO
15GFP-PEST-NEO fusion protein coding nucleic acid of SEQ ID NO
SEQ ID NO:16EMCV-IRES
SEQ ID NO:17EV71-IRES