Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Method of regulating cell metabolism or physiology
Reexamination Certificate
1999-02-03
2002-06-04
LeGuyader, John L. (Department: 1635)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Method of regulating cell metabolism or physiology
C435S320100, C435S325000, C536S023100
Reexamination Certificate
active
06399377
ABSTRACT:
This application is a 371 of PCT/US95/15098, filed Nov. 27, 1995.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention is generally in the field of mammalian gene expression systems used for the cell culture production of proteins.
In particular, the present invention concerns new gene expression systems for increasing the efficiency and the amount of protein production in eukaryotic cell lines and, in particular, the well-known chinese hamster ovary (CHO) cell culture systems which utilize the dihydrofolate reductase (DHFR)—methotrexate (MTX) gene expression amplification system for the production of a wide variety of medically and veterinarily important proteins on a commercial scale. These new expression systems which employ new gene-expression enhancing vectors containing an anti-sense DHFR gene sequence, permit higher levels of protein production in eukaryotic cells at significantly lower levels of MTX, i.e. these eukaryotic cells are rendered more MTX-sensitive.
2. Description of the Background Art
DHFR/TX Mediated Gene Amplification
Many medically and veterinarily important proteins are produced using the CHO cell culture system in which gene expression is amplified by the DHFR/MTX system (Kaufman, R. J., Methods in Enzymol. 185, 537-566 (1990)). This system was developed in the early 1980's (see Axel, U.S. Pat. No. 4,399,216; Ringold et al., J. Mol. Appl. Genet. 1:165-175 (1981); Kaufman et al., J. Mol. Biol. 15:601-621 (1982)). Examples of proteins produced by this system are interleukins, interferons, receptors, human factor IX, human factor VIII, bovine luteinizing hormone, and others.
In the known CHO DHFR/MTX protein production systems, the CHO cell lines are usually DHFR
−
and as such, they can only grow in the essential absence of methotrexate. These CHO cell lines are then transformed or co-transformed with a plasmid/vector carrying a gene sequence encoding the protein of choice to be expressed together with the DHFR gene carried either on the same vector or on a different one. When expressed, the enzyme DHFR serves as a selectable marker for these transformed cells. The vector is usually of the type which can undergo stable recombination and hence subsequent stable incorporation into the genome of CHO DHFR
−
cells thereby rendering such cells DHFR
+
in a stable and constitutive manner.
The positively transformed DHFR
+
cells are selected by growing the cells in a standard culture medium, in which DHFR
−
cells cannot grow, and subjecting the cell cultures to a number of culture cycles or passages. Such a medium usually contains sufficient methotrexate to kill DHFR cells but which is generally not lethal to the DHFR
+
cells. This gives rise to stably transformed DHFR
+
cells, i.e., in which the entire transforming vector or co-transforming vectors or the essential portions thereof such as the DHFR
+
sequence and sequences adjacent thereto which include the gene encoding the protein of choice are stably integrated into the host chromosome. These stably transformed DHFR
+
cells are further cultured and cloned, i.e., individual colonies of cells are taken and cultured separately to provide a number of cloned, transformed DHFR
+
cell lines. These DHFR
+
cell lines are then examined for their ability to express the desired protein and those cell lines showing good expression (i.e., expressing the protein of choice in its expected form either as an intact protein or as an intact fusion product, depending on how the gene encoding the protein of choice was originally constructed on the transforming vector) are selected.
Axel et al., U.S. Pat. No. 4,399,216, discloses a system for co-transforming eukaryotic cells with a foreign DNA encoding a desired proteinaceous material and with an unlinked DNA encoding a selectable phenotype such as DHFR conferring methotrexate resistance. Alternatively, Axel et al. discloses amplifying a gene encoding a desired protein linked to the DNA encoding a selectable phenotype by challenging with successively higher amounts of the selecting agent.
The addition of MTX during cell culture causes gene amplification of gene sequences at and around the DHFR sequence, such as large stretches of flanking DNA that include the gene sequence encoding the desired protein (when unlinked DNAs are cotransfected into a cell, they tend to form a cointegrate that link the DNAs prior to integration into the host genome by non-homologous recombination). This results in an increased number of copies of these sequences and consequently, also results in elevated levels of both DHFR and the desired protein. The degree of amplification is regulated by the MTX inhibitory effect of DHFR.
However, the above CHO DHFR/MTX system has a number of drawbacks, the major one being that the necessary constitutive expression of the DHFR gene during cell culture results in increased levels of DHFR which act to inhibit the effect of MTX. Thus, as the cell culture progresses through successive stages of amplifying stable transfectants from the previous stage, more and more MTX is required for gene amplification until a limit is reached whereby the elevated MTX levels become toxic to the cells; in other words, the upper concentration limit of MTX to which the cells are still MTX-resistant is reached. Accordingly, the current CHO protein production systems have an upper limit as to the amount of desired protein that can be produced. The level of constitutive heterologous (desired) protein expression is relatively limited; for example, only production levels of as high as 10-30 mg/l of culture can be obtained after MTX treatment. Consequently, in order to further increase the amounts of protein produced in these systems, either additional cultures are required or larger cultures need to be grown, which adds considerably to the production costs. As mentioned above, current CHO protein production systems are employed for the production of medically and veterinarily important proteins on a commercial scale. There has therefore been a long-felt need to improve these systems to increase the amount of desired protein produced, on the one hand, and on the other hand, to reduce the costs for producing this increased amount of protein.
Anti-Sense DNA
Anti-sense RNA is transcribed from an upstream promoter of a coding sequence oriented in the anti-sense direction, i.e., opposite the normal or sense direction of the DNA and its transcribed sense RNA. The expression of anti-sense RNA complementary to the sense RNA is a powerful way of regulating the biological function of RNA molecules. Through the formation of a stable duplex between the sense RNA and anti-sense RNA, the normal or sense RNA transcript can be rendered inactive and untranslatable.
In prokaryotes, anti-sense RNA is believed to control plasmid COLE1 replication (Tomizawa et al., Proc. Nat'l. Acad. Sci. USA 78:1421, 1981; Lacatena et al., Nature 294:623,1981) and regulation of outer membrane protein production (Mizuno et al., Proc. Nat'l Acad. Sci. USA 81:1966,1984) as well as many others. Izant et al., Cell 36:1007 (1984), showed that anti-sense RNA also inhibits gene expression in eukaryotes. They constructed a plasmid with a promoter directing the transcription of an anti-sense RNA complementary to the normal thymidine kinase (tk) transcript which substantially reduced expression of the normal thymidine kinase gene.
Besides the thymidine kinase gene and the outer membrane protein genes OmpF and OmpC, anti-sense DNA sequences have been used to express anti-sense RNA complementary to normal or sense RNA transcripts of numerous genes. As an example, Kaufman et al., U.S. Pat. No. 4,912,040, discloses a system for expressing an anti-sense GRP78 DNA sequence capable of hybridizing to part or all of the endogenous GRP78 (similar to immunoglobulin heavy chain binding protein)—encoding mRNA transcript and thereby preventing its translation into GRP78 protein.
Anti-sense DNA to DHFR or DHFR-TS complex has also been reported. Wang et
Cooper Iver P.
LeGuyader John L.
Schmidt M
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