Method for enhancing the transfection rate of a mammalian expression vector in CHO cells

The mammalian protein expression system that Medicilon provides is based on a serum-free cultivated system to provide the gene expression optimization, transient expression, stable expression and antibody production and other eukaryotic protein services.

Researchers of Medicilon established a well-developed mammalian protein expression system and purification services platform, which offers the expression and purification services of a variety of recombinant protein , antibody or fragment of antibody. Email:marketing@medicilon.com.cn web:www.medicilon.com

The present application is a continuation of U.S. application Ser. No. 11/962,261 (pending), which was filed on Dec. 21, 2007 (published as US 2008-0138902 A1 on Rine 12, 2008), which is a divisional of U.S. application Ser. No. 10/521,768, filed Jan. 19, 2005 (abandoned), which is a 371 U.S. national phase of International Application No. PCT/EP2003/007946, filed Jul. 21, 2003, which designated the U.S. and claims benefit of GB 0216648.6, filed Jul. 19, 2002, the entire contents of each of which are incorporated herein by reference.

The present invention relates to a method for expressing a recombinant product gene in a CHO cell line as well as to recombinant CHO host cells and to novel expression vector constructs.

The Chinese Hamster ovary cell (CHO) mammalian expression system is widely used in production of recombinant protein. Apart from lymphoid cell lines such as hybridorna cell lines, it is one of the few cell types allowing for simple and efficient high-density to suspension batch culture of animal cell. Furthermore, they allow for very high product yields and are comparatively robust to metabolic stresses whereas lymphoid cells are more difficult to culture at an industrial scale. Given considerable cost of production, it is of utmost importance to maximize the yield of recombinant protein per bioreactor run. Choice of culture medium composition and bioreactor design and operation are parameters that impact yield and may be quite complex to optimize. More predictably, increases in the strength or transcriptional activity of the promoter controlling expression of product protein enhance yield. Incremental increases at the single cell level will translate into considerable improvements of product yield in high-density batch or fed-batch culture showing stationary phase gene expression at cell densities in the range of 106 to 107 cells/ml.

U.S. Pat. No. 5,866,359 describes a method of enhancing expression from an already strong hCMV promoter in CHO and NSE cells by co-expressiong adenoviral E1A protein from a weak promoter. E1A is a multifunctional transcription factor which may act on cell cycle regulation and has both independent transcriptional activating and repressing functional domains. The finetunizig of E1A expression to appropriate low level expression is crucial for success of the co-expression approach in order to achieve the ideal balance in between gene transactivation whilst avoiding any negative impact on cell cycle progression. As a disadvantage, apart from careful choice of the promoter driving E1A expression, this system blocks part of the protein synthesis capacity of the cell with E1A expression rather than expressing the recombinant protein of interest.

WO 95/17516 describes use of the murine immunoglobulin gamma 2A locus for targeting an expression vector construct to a highly active gene locus in lymphoid cells of the B-cell lineage, e.g. widely used NSO myeloma cells. NSO cells essentially are a tumor cell line of murine plasma or B-cells. Only in B-cells, the chromatin harboring the immunoglobulin loci is in its fully active, open state, allowing for high transcriptional activity of native immunoglobulin promoters or recombinant expression constructs integrated into those gene loci.

As a disadvantage, due to the principle of homologous recombination, the targetting sequence will target efficiently in murine cell lines only matching the sequence of the gamma 2 A targetting sequence harboring a recombinatorial hot spot; for high level expression, the gamma 2A locus region must be a transcriptionally active genomic region, limiting its effectiveness for homologous recombination to B-cell types.

It is an object of the present invention to devise another expression system for CHO protein expression in biotechnology which allows for enhanced expression from a standard promoter. According to the present invention, this aim is surprisingly achieved by equipping a gene expression vector for CHO cells with a gene targetting sequence having been originally devised for homologous recombination in murine B-cells.

Possible embodiments of the invention are shown in the figures. What is shown is:

According to the present invention, a DNA sequence for expression of a recombinant gene in a mammalian cell comprises a recombinant product gene and a promoter for expressing the recombinant product gene, preferably a CMV promoter, and further comprises a murine immunoglobulin gamma 2A locus DNA sequence or fragments or sequence variants thereof capable of enhancing expression from the promoter. According to the present invention, such a DNA sequence is useful expression vector construct for expression of recombinant product gene in CHO cells.

According to the present invention, the method of expressing a recombinant protein comprises the steps of

a. culturing a CHO cell transfected with an expression vector comprising a promoter active in CHO cells driving expression of a recombinant product protein and further comprising the murine IgG 2 A gene locus DNA or a DNA sequence variant or DNA fragment thereof which is enhancing activity of said promoter, and b. harvesting the product protein A recombinant product gene according, to the present invention is the product protein that is sought to be expressed and harvested in high amount. It may be any protein of interest, e.g. therapeutic proteins such as interleukins or enzymes or subunits of multimeric proteins such as antibodies or fragments thereof. The recombinant product gene may include a signal sequence coding sequence portion allowing secretion of the once expressed polypeptide from the host producer cell. In a further preferred embodiment of the present invention, the product protein is a secreted protein. More preferably, the first or product protein is an antibody or engineered antibody or a fragment thereof, most preferably it is an Immunoglobulin G (IgG) antibody.

The DNA sequence of the murine immunoglobulin gamma 2A gene locus (IgG 2A) has originally been devised in WO 95/17516 for use as a genomic targetting sequence for generating stably recombinant lymphoid B-cell lines that show high expression of the recombinant gene product. B lymphocytes or plasma cells normally express extremely high levels of immunoglobulin RNA from the Ig heavy chain locus, probably due to cell-type specific enhancer/transcription factor activity and open chromatin structure. The preferred murine immunoglobuline gamma 2A gene sequence of the present invention is the same as the targetting sequence used in WO 95/17516. It is a 5.1 kb BamHI genomic fragment which includes all of the coding region of murine Ig gamma 2A except the most 5′ part of the CH1 exon (Yamawaki-Kataoka, Y. et al., Proc. Natl. Acad. Sci. U.S.A. (1982) 79: 2623-2627; Hall, B. et al., Molecular Immunology (1989) 26:819-826; Yamawaki-Kataoka, Y. et al., Nucleic Acid Research (1981) 9: 1365-1381). According to the present invention, promotion of site-directed, homologous recombination is not the relevant property of the immunoglobulin gamma 2A gene sequence (IgG 2A).

Accordingly, any sequence variant of said IgG 2A gene sequence or sequence fragment or variant sequence fragment that is functional in or capable of enhancing recombinant product gene expression from the promoter, preferably from a hCMV promoter as set forth below, both under condition of transient or stable expression in CHO cells is also encompassed by the present invention.

Such ‘functional’ variants encompass e.g. base insertions, deletions or point mutations and be generated by methods well-known in the art, e.g. by primer-directed PCR, ‘error-prone’ PCR, ‘gene-shuffling’ termed PCR-reassembly of overlapping DNA fragments or by in-vivo random mutagenesis of bacterial clones followed by library transfection and functional selection in CHO cells. For instance, random mutagenesis can be achieved by alkylating chemicals or UV-irradiations as described in Miller, J., Experiments in Molecular Genetics, Cold Spring Harbor Laboratory 1972). Optionally, a natural mutator-strain of a host bacterium may be used.

Preferably, such variant sequence or sequence fragment is at least 65%, more preferably 75%, most preferably 90% homologous in DNA sequence to the corresponding part of the natural murine immunoglobulin gamma 2A gene locus. For instance, it is possible to insert a Sal I restriction site at the naturally occurring Stu I site present 39 by upstream of membrane exon 2 (M2) to provide a unique site for linearization within the murine immunoglobulin gamma 2A sequence; such sequence variant was originally devised for site-specific recombination targetting, but can as well be employed in the context of the present invention.