Organic compounds

Abstract

The present invention relates to a novel selection system for use in a eukaryotic cell culture process and for expression of a recombinant product of interest. The selection system is based on the introduction of an exogenous functional membrane-bound folate receptor gene together with the polynucleotide or gene encoding the product of interest into a eukaryotic cell and can be widely utilized with eukaryotic cells for which cellular viability is dependent upon folic acid uptake.

Description

FIELD OF THE INVENTION[0001]The present invention relates to a novel selection system for use in a eukaryotic cell culture process and for expression of a recombinant product of interest. The selection system is based on the introduction of an exogenous functional membrane-bound folate receptor gene together with the polynucleotide or gene encoding the product of interest into a eukaryotic cell and can be widely utilized with eukaryotic cells for which cellular viability is dependent upon folic acid uptake.BACKGROUND OF THE INVENTION[0002]Selection markers and selection systems are widely used in genetic engineering, recombinant DNA technology and production of recombinant products, for example antibodies, hormones and nucleic acids, in eukaryotic cell culture. The primary goal of such dominant selection markers and selection systems is to introduce a selectable gene which upon exposure to selective growth conditions provides cells capable of high-level production of the recombinant...

AI Technical Summary

Benefits of technology

[0033]Another preferred embodiment relates to a eukaryotic cell according to the present invention, wherein the endogenous unidirectional functional folate transport system, for example comprising at least e.g. one endogenous functional membrane-bound folate receptor, is lacking full activity, i.e. is attenuated. Such attenuation can be provided for example by any type of mutagenesis of the endogenous folate transport system in question, e.g. the endogenous functional membrane-bound folate receptor, for example by point mutation, gene disruption, and the like. The attenuation can be a partial or complete. In the latter case the eukaryotic cell according to the present invention does not comprise an endogenous functional unidirectional functional folate transport system, e.g. an endogenous functional membrane-bound folate receptor. Accordingly, a preferred embodiment the present invention relates to such a eukaryotic cell wherein an expression vector of the present invention has been stably introduced, and which cell is lacking full activity of at least one endogenous functional membrane-bound folate receptor.

[0034]With respect to the expression vector introduced into said host cell any expression vector of the present invention, including its preferred embodiments, as described herein, can be utilized. In a preferred embodiment of the eukaryotic cell of the present invention the first polynucleotide encoding a functional membrane-bound folate receptor and the second polynucleotide encoding the product of interest are located on the same expression vector. Preferably, such expression vector is and expression vector according to the present invention, i.e. as described herein.

[0035]The eukaryotic cell according to the present invention is, preferably, selected from the group consisting of a mammalian cell, an insect cell, a plant cell and a fungi cell. With respect to fungi cells and plant cells, which usually are prototrophic for folates (i.e. such cells can autonomously synthesize their own folates necessary for their cellular viability, i.e. cellular growth and proliferation). The present invention encompasses in particular such fungi and plant cells which may become auxotrophic for folates. This may be fo...

Problems solved by technology

(a) The glutamine synthetase system: The enzyme glutamine synthetase (GS) is responsible for the biosynthesis of glutamine from glutamate and ammonia. This biosynthetic reaction provides the sole pathway for glutamine formation in mammalian cells. Thus, in the absence of glutamine in the growth medium, the enzyme GS is essential for the survival of mammalian cells in culture. Importantly, certain mammalian cell lines including mouse myeloma cells lack the expression of sufficient GS and thus cannot survive without exogenously added glutamine. Hence, such a cell line is an suitable acceptor for a transfected GS gene that in this system can function as a selectable marker that allows for cell growth in a medium lacking glutamine. In contrast, cell lines such as the widely used Chinese hamster ovary (CHO) cells express sufficient GS to support growth in glutamine-free medium. Therefore, if these CHO cells are to be used as the recipient cells for the transfection of the GS gene, the specific and potent GS inhibitor methionine sulfoximine (MSX) can be applied in order to inhibit endogenous GS activity such that only transfectants expressing high levels of the transfected GS gene can survive in a glutamine-free medium. A major disadvantage of the GS system is the relatively long time (i.e. 2-6 months) of selective growth in order to establish cells stably overexpressing the target gene of interest. Another disadvantage is the frequent utilization of the cytotoxic agent MSX for the augmentation of the selective pressure. The presence of such a cytotoxic agent along with a recombinant product of interest (e.g. a polypeptide like an antibody) may require additional purification steps to rid of this cytotoxic agent.

(b) The dihydrofolate reductase/MTX selection system: Dihydrofolate reductase (DHFR) catalyzes the NADP-dependent reduction of dihydrofolic acid to tetrahydrofolic acid (THF). THF is then interconverted to 10-formyl-THF and 5,10-methylene-THF which are used in the de novo biosynthesis of purines and thymidylate, respectively. DHF is the byproduct of the catalytic activity of thymidylate synthase (TS) which catalyzes the conversion of dUMP to dTMP in a 5,10-methylene-THF-dependent reaction. Thus, DHFR is crucial for the recycling of THF cofactors that are essential for the biosynthesis of purine and pyrimidine nucleotides that are necessary for DNA replication. Hence, cells (e.g. CHO cells...