GB2237288A - Amplification of a heterologous gene in recombinant eukaryotic host cells - Google Patents

Abstract

A process, for increasing the copy number of a heterologous gene in a glutamine auxatrophic eukaryotic host cell line, comprises: transforming the host cell line with either (i) a vector containing a glutamine synthesase (GS) gene and the heterologous gene or (ii) a vector containing a GS gene and a separate vector containing the heteralogous gene; selecting glutamine-prototrophic transformants in a medium which does not contain glutamine but contains one or more metabolites required for growth in glutamine-free medium; culturing the transformed host cell line in a glutamine-free medium in which the level of the one or more metabolites is depleted. The copy number of the amplified heterologous gene is maintained by culturing the transformed host cell in the glutamine-free medium, defined above.

Description

AMPLIFICATION IN RECOMBINANT DNA HOST CELLS The present invention relates to a method of increasing the copy number of a heterologous gene inserted into a host cell line.

This process is commonly known as amplification. The present invention also relates to a method of maintaining the increased gene copy number during culture of the transformed cell line.

The ability of a cloned heterologous gene to function when introduced into host cell line cultures has proved to be invaluable in studies of gene expression. It has also provided a means of obtaining large quantities of the protein encoded by the heterologous gene which may otherwise be scarce or which may be a completely novel product of gene manipulation. It is advantageous to obtain such proteins from eukaryotic cells since such proteins are generally correctly folded, appropriately modified and completely functional, often in marked contrast to those proteins as expressed in bacterial cell lines.

Where large amounts of product are required, it is necessary to identify cell clones in which the heterologous gene is retained during cell proliferation. Such stable gene maintenance can be achieved either by use of a viral replicon or as a consequence of integration of the gene into the DNA of the host cell line.

Where the gene has been integrated into the host cell line's DNA, the copy number of the gene, and concomitantly the amount of product which could be expressed, can be increased by selecting for cell lines in which the gene has been amplified after integration into the host cell line's DNA.

A known method for carrying out such a selection procedure is to transform a host cell line with a vector comprising a gene which encodes an enzyme which is inhibited by a known drug. The vector may also comprise the heterologous gene. Alternatively the host cell line may be co-transformed with a second vector which comprises the heterologous gene.

The transformed or co-transformed host cells are then cultured in increasing concentrations of the known drug, thereby selecting drug-resistant cells. It has been found that one common mechanism leading to the appearance of mutant cells which can survive in the increased concentrations of the otherwise toxic drug is the over-production of the enzyme which is inhibited by the drug. This most commonly results from increased levels of its particular mRNA, which in turn is frequently caused by amplification of gene copies.

It has also been found that, where drug resistance is caused by an increase in copy number of the gene coding the inhibitable enzyme, there is a concomitant increase in the copy number of the heterologous gene in the host cell's DNA. There is thus an increased level of production of the desired protein.

The most commonly used system for such co-amplification uses the enzyme dihydrofolate reductase (DHFR). This enzyme can be inhibited by the drug methotrexate (MTX). To achieve co-amplification, it is usual to employ a host cell which lacks an active gene which encodes DHFR. Such a host cell is either transformed with a vector which comprises a DHFR gene and the heterologous gene or co-transformed with a vector comprising a DHFR gene and a vector comprising the heterologous gene. The transformed or co-transformed host cells are cultured in media containing increasing levels of MTX, and those cell lines which survive are selected.

Alternatively host cells which contain an active gene which encodes DHFR may be used if a mutant DHFR gene is used for transformation or co-transformation. For example, a gene coding for a mutant DHFR which binds MTX less strongly than native DHFR may be used, and thus permits selection of mutant DHFR transformed host cells.

Another system for producing co-amplification is set forth in WO 87/04462. The system described in WO 87/04462 relies on the use of a gene encoding glutamine synthetase (GS). This enzyme, which is essential to the viability of mammalian cell lines if glutamine is not provided in the culture medium, can be inhibited by methionine sulphoximine (Msx). A discussion of the function of the GS gene and its inhibitor is found in WO 87/04462, together with a description of the use of a cloned GS gene for amplification. In general, the amplification procedure involves culturing a transformed cell line in increasing concentrations of Msx.

Although the known amplification systems are to a large extent successful, they have disadvantages. The main disadvantage is that the agent used to select for amplification is toxic (both MTX and Msx are toxic). Thus, in order to obtain amplification, it is necessary to employ high concentrations of toxic agents, with the concomitant health hazard. It is desirable to be able to avoid such a health hazard.

Once amplification has been achieved using the toxic reagent, it is generally the practice to culture the amplified cells in a medium free of the toxic agent. However, this leads to a second disadvantage. In the absence of the toxic agent, there is no selection pressure for amplified number of copies of the enzyme gene. There is thus a tendency for the excess copies of the enzyme gene to be eliminated from the host cell line's DNA. It is often the case that, at the same time, the co-amplified heterologous gene is also eliminated, thus reversing the effects of amplification. The only way to avoid this is to carry out culture in a medium containing the toxic agent, but this again brings with it the health hazard referred to above. It is thus desirable to be able to maintain the level of amplification without the use of toxic agents.

According to a first aspect of the present invention, there is provided a method of increasing the copy number of a heterologous gene in a glutamine auxotrophic eukaryotic host cell line which comprises: transforming the host cell line with either a vector containing a GS gene and the heterologous gene or a vector containing a GS gene and separate vector containing the heterologous gene; selecting glutamine-prototrophic transformants in a medium which does not contain glutamine but contains one or more metabolites required for growth in glutamine-free medium; and culturing the transformed host cell line in glutamine-free medium in which the level of the said one or more metabolites is depleted.

It has been found that by selecting GS transfectants on a medium depleted in the said one or more metabolites, it is possible to achieve amplification of both the GS gene and heterologous vector sequences. It is believed that in the medium used, those cells which have only a low copy number of vector GS gene cannot produce enough glutamine to remain viable, and therefore die off. In transformed cells where the GS gene is amplified, there will be enough copies of the GS gene to ensure that sufficient glutamine is produced to avoid the requirement for the one or more metabolites, thereby enabling the cells to survive. It has also been found that heterologous vector sequences are amplified along with the GS gene.

Preferably, the one or more metabolites which is depleted to cause amplification is asparagine. Especially also the medium used lacks glutamate. It has been found surprisingly that a cell line transformed to glutamine independence using a GS gene can survive in a medium lacking glutamate as long as it contains asparagine.

The host cell line may contain an endogeneous GS gene. Such cell lines include the chinese hamster ovary (CHO) cell line and baby hamster kidney (BHK) cell line. Preferably, however, the cell line is auxotrophic for glutamine because the endogeneous GS activity is too low to permit survival in a glutamine-free medium. Such cell lines in particular include myeloma cell lines such as NSO or P3-X63-Ag8.653 or a GS-deficient variant of a cell line such as CHO. Where the cell line is dependent on exogenous glutamine for survival, it will be necessary to transform the cell line to glutamine independence prior to amplification.This can be achieved by firstly growing the transformed cell line in a medium containing glutamine and then continuing the growth of the cell line in a medium in which the glutamine is progressively depleted or in a medium lacking in glutamine but containing asparagine. Once the cell line has been grown to glutamine independence, amplification can be carried out by progressive depletion of asparagine or in a medium lacking asparagine.

The fact that it has been found surprisingly that the heterologous gene can be amplified by asparagine limitation using a host cell line transformed with a GS gene has led to the second aspect of the present invention.

According to this aspect of the present invention there is provided a method for maintaining the copy number of an amplified heterologous gene in a transformed glutamine independent eukaryotic host cell line wherein amplification was achieved by use of a co-transformed GS gene, which method comprises culturing the transformed host cell line in a glutamine-free medium in which the level of one or more metabolites required for growth in glutamine-free medium is depleted.

Preferably the one or more metabolites is asparagine.

The vectors and host cells which can be used in this aspect of the present invention are the same as those mentioned above, or any variants thereof as will be apparent to those skilled in the art.

The heterologous gene may be amplified by the method described in published International Patent application WO 87/04462 using increasing concentrations of a toxic GS inhibitor such as Msx.

However, preferably, the heterologous gene is amplified by the method of the first aspect of the invention.

The advantage of the present invention in either of its aspects is that the amplification and/or maintenance can be carried out without the use of toxic agents. This is a particularly advantageous for culturing the cell line to produce the desired product of the heterologous gene since the selection pressure will be maintained. It will therefore be possible to carry out the production of the desired product in a non-toxic medium without the risk that the heterologous gene (and the GS gene) will be lost during growth of the transformed cell lines.

The present invention is now described, by way of example only, with reference to the accompanying diagram which shows a photograph of a Southern blot giving results of GS copy number measurements.

Example The derivation of Cell Line B4.24 and C2.27 is described in EPA 8809129.3. Briefly each is an independent transfected clone of the NSO myeloma cell line into which has been inserted the plasmid pSV2GScLc. This plasmid contains a glutamine synthetase coding (GS) sequence under the control of the SV40 early promoter and splicing and polyadenylation signals also from SV40, together with a coding sequence for the chimaeric light chain of the B72.3 antibody under the control of the human cytomegalovirus (hCER) major-immediate early gene promoter-enhancer. The construction of p.SV2GScLc is also described in EPA 8809129.3 B4.24 and C2.27 were maintained in G-DMEM medium described in EPA 8809129.3.The cell lines were then subjected to selection for variants able to grow in amino-acid depleted medium (AAD Medium) consisting of Dulbecco's Modified Eagle Medium (DMEM) and 10% dialysed foetal calf serum (FCS), and penicillin and streptomycin. Thus this medium lacks non-essential amino acids and nucleosides present in G-DMEM.

Cell lines B4.24 and C2.27 were plated in AAD Medium in 24-well plates at a cell density of approximately 2-5X105 cells/well.

Both cell lines showed substantial cell death in this medium after about 5 days.

As described in EPA 8809129.3 it has been demonstrated that DMEM + 10% dialysed FCS + 500pM asparagine is sufficient to support growth of GS-transfected NSO cell lines. Thus the fact that AAD Medium is unable to support growth of these cell lines indicates the essential role of asparagine for cell growth under the conditions used.

The culture trays were incubated for 2-3 weeks in AAD medium and after this time, small asparagine-independent variant colonies were visible. The colonies obtained in this way from B4.24 were pooled and expanded in culture as were those from C2-27. Total genomic DNA was prepared from the parent B4.24 and C2.27 cell lines and from the asparagine-independent pools. The number of copies of the GS-genes in these cell lines could then be estimated by Southern blot analysis using a 32p-labelled GS-DNA fragment as a probe. DNA samples were digested with BglI and BglII restriction enzymes and a Southern blot of these samples was probed with the 5' Pst-l GS-DNA fragment of pGSC45 (Hayward, B.E., Hussain, A., Wilson, R.H., Lyons, A., Woodcock, V., McIntosh, B., and Harris, T.J.R., [1986] Nucleic Acids Research 14, 999-1008).

The result is shown in Figure 1. The probe cross-hybridised with the endogeneous mouse GS-gene in NSO cells and the presumed diploid complement of GS genes is seen as a 2.8kb fragment and an approximately 6kb fragment in each DNA sample. These bands serve as an internal control for loading of the same amount of DNA in each track on the gel. In the tracks corresponding to B4.24 and C2.27 DNA and their asparagine-independent variant derivatives, an additional 1.2kb BglI-BglII fragment is also detected which is of the size predicted for vector DNA.

Track 1 contains lO > g DNA from non-transfected NSO cells, showing just the 2.8kb and 6kb endogeneous GS-gene fragments.

Track 2 contains lOFg DNA from SV2GSNSO. SV2GSNSO is a pool of NSO transfectant cell lines containing the plasmid pSV2 BamGS (see EPA 8809129.3). Track 3 contains lO > g DNA from SV2GSNSO cells selected for resistance to lOOpM MSX. This demonstrates ampification of the vector DNA relative to the endogeneous genes: the copy-number of the DNA is estimated to have increased about 5-fold compared with the original SV2GSNSO transfectants (EPA 8809129.3). Track 4 contains lORg C2.27 DNA and track 5 contains lOWg DNA from MSX-resistant C2.27 variants. Track 6 contains lOFg DNA from the C2.27 asparagine-independent variant pool.The vector GS coy number in this pool has clearly increased approximately 2-4 fold, similar to the increase seen after selection with MSX in track 5, thus demonstrating vector amplification by removal of asparagine.

The degree of amplification is clearly the average for the pool and it is likely that particular clones within the pool will show a higher degree of vector amplification. The degree of amplification of vector copy number in C2-27 is similar whether MSX-selection or asparagine depletion is used (compare tracks 5 and 6 in Figure 1).

B4.24 asparagine-depleted variants were also analysed by Southern blot analysis but no increase in vector copy number was detected in this case. This is similar to the situation observed using MSX-selection: some transfectants amplify more readily than others. Hence it is necessary to screen a number of independent transfectants for those which amplify readily. Such screening can readily be carried out by Southern blot analysis of asparagine-depleted pooled cell lines or for instance by measuring the level of expression from a reporter gene carried on the same plasmid.

Claims (10)

1. A method, for increasing the copy number of a heterologous gene in a glutamine auxotrophic eukaryotic host cell line, which comprises: transforming the host cell line with either a vector containing a GS gene and the heterologous gene or a vector containing a GS gene and separate vector containing the heterologous gene; selecting glutamine-prototrophic transformants in a medium which does not contain glutamine but contains one or more metabolites required for growth in glutamine-free medium; and culturing the transformed host cell line in glutamine-free medium in which the level of the one or more metabolites is depleted.

2. The method of claim 1, wherein the one or more metabolites which is depleted to cause amplification is asparagine.

3. The method of claim 2, wherein the medium used lacks glutamate.

4. The method of any one of claims 1 to 3, wherein the host cell line contains an active endogenous GS gene.

5. The method of any one of claims 1 to 3, wherein the cell line is auxotrophic for glutamine because the activity of the endogenous GS gene is too low to permit survival in a glutamine-free medium.

6. The method of claim 5, wherein the cell line is a myeloma cell line such as NSO or P3-X63-Ag8.653.

7. The method of claim 5 or claim 6, wherein the cell line is transformed to glutamine independence prior to amplification.

8. The method of claim 7, wherein transformation to glutamine independence is achieved by firstly growing the transformed cell line in a medium containing glutamine and then continuing the growth of the cell line in a medium in which the glutamine is progressively depleted or in a medium lacking in glutamine but containing asparagine.

9. A method for maintaining the copy number of an amplified heterologous gene in a transformed glutamine independent eukaryotic host cell line wherein amplification was achieved by use of a co-transformed GS gene, which method comprises culturing the transformed host cell line in a glutamine-free medium in which the level of one or more metabolites required for growth in glutamine-free medium is depleted.

10. The method of claim 9, wherein the one or more metabolites is asparagine.