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

[0032]In addition to the functional membrane-bound folate receptor introduced into the cell line via an expression vector, the eukaryotic cell according to the present invention can comprise at least one endogenous functional unidirectional functional folate transport system, in particular one or more endogenous functional membrane-bound folate receptor(s). It is an advantage of the present invention that the method of selection as described herein below can be utilized even in the presence of such endogenous unidirectional functional folate transport system, i.e. where such endogenous system is retained. Accordingly, a further preferred embodiment relates to the eukaryotic cell of the present invention relates, comprising at least one endogenous unidirectional functional folate transport system, wherein such endogenous unidirectional functional folate transport system preferably comprises at least one endogenous functional membrane-bound folate receptor. In a preferred embodiment thereof, the endogenous functional membrane-bound folate receptor is selected from the group consisting of the folate receptor alpha (FRα) and the folate receptor beta (FRβ).

[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 for example due to genetic manipulation, i.e. cells are now unable to synthesize sufficient amounts of folates necessary for their cellular viability. For example, the capacity of such fungi or plant cells to endogenously biosynthesize folates, e.g. via an appropriate metabolic pathway, will be inactivated, e.g. by gene disruption or gene silencing of appropriate target genes, or inhibition of key enzymes, etc.

[0036]In a preferred embodiment thereof the eukaryotic cell is a mammalian cell. Preferably, such mammalian cell is selected from the group consisting of a rodent cell, a human cell and a monkey cell. Particularly preferred is a rodent cell, which preferably is selected from the group consisting of a CHO cell, a BHK cell, a NS0 cell, a mouse 3T3 fibroblast cell, and a SP2 / 0 cell. A most particularly preferred rodent cell is a CHO cell. Also preferred is a human cell, which, preferably, is selected from the group consisting of a HEK293 cell, a MCF-7 cell, a PerC6 cell, and a HeLa cell. Further preferred is monkey cell, which, preferably, is selected from the group consisting of a COS-1, a COS-7 cell and a Vero cell.

[0037]In another embodiment the present invention relates to a process for production of a eukaryotic cell according to the present invention, said process comprising providing an eukaryotic cell for which cellular viability is dependent upon folate uptake, and introducing a first polynucleotide located on an expression vector and encoding the functional membrane-bound folate receptor and a second polynucleotide located on an expression vector and encoding the product of interest, wherein the first polynucleotide and the second polynucleotide are located on the same expression vector or on separate an expression vectors. In a preferred embodiment, the first polynucleotide and the second polynucleotide are located on the same expression vector which, in a most preferred embodiment, is an expression vector according to the present invention, i.e. as disclosed herein.

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) that lack the DHFR gene (i.e. by targeted genomic deletion) can be used as recipients for the transfection of the DHFR gene in a medium that is free of nucleotides. After transfection, the cells can be subjected to a gradual increase in the concentrations of the antifolate MTX, a most potent DHFR inhibitor (Kd=1 pM), thereby forcing the cells to produce increased levels of DHFR. Upon multiple rounds of selection, the selectable marker DHFR frequently undergoes significant gene amplification. Furthermore, a mutant mouse DHFR with a major resistance to MTX has also been extensively used as a dominant selectable marker that markedly enhances the acquisition of high level MTX-resistance in transfectant cells. A major disadvantage of the DHFR / MTX selection system is that this technique utilizes a mutagenic cytotoxic agent, MTX, that can readily alter the genotype of the recipient cells. Additionally, specific safety measures may have to be taken to protect the persons handling such agents. This frequently results in MTX-resistant cell populations in which no expression of the target gene of interest is present due to loss of function mutations in the reduced folate carrier (RFC) and / or loss of RFC gene expression, both of which abolish MTX uptake. Another disadvantage is that the mutagenic drug MTX may readily contaminate the secreted overexpressed target product (e.g. a polypeptide like an antibody) contained in the growth medium thereby requiring labor intensive, time-consuming and expensive chromatographic methods necessary to rid off this mutagenic compound, MTX.

However, RFC displays a very poor affinity for the oxidized folate, folic acid.

There are several disadvantages for the RFC selection system: a) One must use RFC-null recipient cells in which the endogenous RFC locus has been knocked out or inactivated by targeted knockout or loss of function mutations. b) RFC has an extremely poor transport affinity for folic acid and thus this oxidized folate cannot be used for selection. c) As opposed to the current folate-receptor based system that is a unidirectional folate uptake system and which will be explained in detail below, RFC is a bi-directional folate transporter that exhibits equally potent import and export of folates.

This implies that under conditions of folate deprivation, RFC overexpression may be detrimental to the recipient cells that further export folate via the overexpressed RFC.