The overall goals in this G-project are:
- To improve expression strenght of recombinant proteins since this is the major obstacle for economic production.
- To optimize conditions for cell growth in order to keep processes of production reproducible, stable and efficient.
- To engineer cell lines in order to secrete proteins with mammalian-type glycosyl residues.
- To improve biosafety and expression efficiency in baculovirus-mediated expression in insect cells.
- To construct artificial chromosomes in a variety of cell lines from different mammalian organisms.
Three particular cell types are being dealt with: CHO and BHK-21 of hamster origin and cell lines from insects.
A meeting of personnel from all 32 participating laboratories in this G-Project was held in Barcelona in October, 1995. Progress was assessed, milestones checked and, based on difficulties, appropriate modifications in the programs of the 7 groups were discussed.
The current progress report summarizes the main achievements and obstacles recognized in the various groups.
By request of the participants, the EC extended the project which was initially set up for 24 months to 30 months (cost neutral).
PROJECT: CHO-cells for high levels of foreign gene product.
Using the second intron of the CAD gene as a target for homologous recombination, no true recombinants were found, leaving two possibilities: a) the CAD locus is a cold-spot for recombination, or b) CHO cells are refractory to homologous recombination. To bypass possibility a) and directly test possibility b), targeted integration within Hmg1, a housekeeping, ubiquitous and strongly expressed gene will be undertaken. As positive control, the Hmg1 gene was targeted at high efficiency in mouse ES cells.
During the past 12 months, we cloned the Hmg1 gene from the same CHO cell line (CHO-DUKX) that is being used as the recipient from targeted recombination, to insure against a possible sequence drift between different CHO sub-lines.
The Hmg1 gene from a CHO cDNA bank was cloned. Several vectors for targeted recombination have been constructed so far.
In order to delay apoptosis in CHO cells, antiapoptotic genes from hamster cells were isolated. Different probes encoding mRNA of known apoptosis preventing mRNAs were isolated. Using RT-PCR, some probably new genes were cloned. Some of the full length clones were transferred into CHO cells.
Cell clones that stably express the FcRIIIB receptor in high copy numbers were adapted to grow under serum-free conditions and used for mass cell production. The receptor was cleaved from the cells by phospolipase C treatment as well as purified by affinity chromatography.
The glycosylation profiles of immunoglobulins and immunoglobulin receptors are analysed using recombinant proteins expressed in CHO cells that were cultured under various defined culture conditions. The glycosylation profile of proteins is markedly influenced by the cell line in use for production. Analysis of the diverse structures on glycoproteins requires high resolution chromatographic techniques and sensitive detection methods. The glycosylation profile of the humanized monoclonal antibody D1.3 (anti-lyzozyme mAb) expressed in CHO cells was analysed. In fact, three different chains were characterized described as G2-, G1 and G0-biantennaries. In contrast to glycosylation in human cells, no bisecting structures and no sialylation were found.
A new culture system was developed where the cells are grown up in fermenters under well defined serum-free culture conditions. In a second step, these cells are transferred to a newly developed cell culture chamber where they can be kept under extremely high cell densities. Prototypes of this newly developed system have been constructed and used for test runs. CHO cells transferred with the GPI-anchored FcRIII have been produced in mass cultures using the Super Spinner system.
PROJECT: Controlling the glycosylation of recombinant proteins.
The CHO cell line lacks a functional copy of the alpha2,6-sialyltransferase (alpha2,6-ST) gene. CHO-320 line (making IFN-gamma) and CHO-K1 line were both successfully transfected with the alpha2,6-ST gene, using a novel expression vector. The activity of the transfected alpha2,6-ST enzyme has been confirmed using enzyme assays and analysis of the oligosaccharides on recombinant IFN using mass spectrometry and fluorescent -HPLC. In the best clone the transfected alpha2,6-ST enzyme restored CHO cell sialylation to a human-type distribution of approximately 50% alpha2,3 & 50% alpha2,6 linked sialic acids. The overall level of sialylation can be improved by 56% compared to wild-type cells. Transfection of the recombinant enzyme had no adverse effects on the cell growth, metabolism, productivity or on other stages in the glycosylation pathway in CHO cells grown in batch culture. Enzyme activity was stable throughout batch culture of the cell line (175 hours).
PROJECT: Strategies for regulated production of pharmaceutical proteins.
A second transcriptional control system in which doxycycline, a tetracycline derivate functions as coactivator of the transactivator rtTA was developed. With this development, there are now two regulatory systems available which function in a mirror image-like fashion and which are controlled by tetracycline or derivates. In one system tetracyclines removal in the other tetracycline addition initiates expression of the gene of interest.
In a pilot experiment a mammalian cell line using site specific recombination was constructed. In this experiment, chromosomal loci with intrinsic high expression characteristics were identified by infection with a recombinant retrovirus containing double FRT sites and subsequent screening for overproduction of an encoded reporter gene. In a second step, these sites were targeted with an expression cassette that recombined with the aid of FLP recombinase from Saccharomyces cerevisiae yielding cells that had the second construct at the exact position of the first. By using retroviral constructs with double and single FRT sites, respectively, stable cell clones were created that no longer can be excised with FLP.
IgG-cytokine fusion proteins were expressed in BHK and CHO cell lines. Due to problems with stability of expression (transcription based) it was not possible to isolate highly overexpressing cell clones. Stabilization could be achieved by using tricistronic expression vectors (2 antibody genes and a selectable marker). Best cell clones were cultivated in scaleable devices and methods under different serum-free protein conditions. Examination of glycoprotein quality shows site specific protein degradation and terminal glykosylation variations in dependence of cultivation conditions.
PROJECT: Artificial chromosomes in mammalian cells.
Development of a linear vector for mammalian cells requires the definition of the elements essential for episomal maintenance of a linear molecule in mammalian cells. These are thought to include telomeres, a centromere and replication origins. The work concentrated both on the characterization of these elements and on the assembly of these elements into linear constructs. Approaches were made for the construction of linear vectors. These included the insertion of several viral origins of replication or mapped mammalian origins of replication into linear vectors carrying human telomeric sequences, the modification of existing YAC-vectors containing human DNA of a minimum size of 100 kb and the modification of naturally occurring human minichromosomes. Capped mammalian telomeres can be used to seed new telomeres in mammalian cells and the frequency of the generation of new telomeres is host dependent. The YAC integrated into host chromosomal DNA in each transformant. Retrofitted YACs do not integrate at random genomic locations. In 8 out of 10 transformants the YAC had integrated into telomeric regions.
To introduce a reporter gene into a human YAC, homologous recombination in yeast was used. Reporter genes were introduced into preexisting human minichromosomes derived from chromosomes and then modified chromosomes transferred to various cell types. They replicated as autonomous units, however, so far they are too large to be used as eucaryotic vectors.
PROJECT: Baculovirus vectors for foreign gene expression.
Baculovirus deletion mutants (single, double and triple gene deletions) have been further modified to incorporate reporter genes to enable detailed assessments of recombinant protein production.
Two genes encoding putative inhibitors of apoptosis can be removed from the Baculovirus genome without affecting replication in a number of cell lines; a third gene (p35) is absolutely required for replication in most insect cell lines.
Virus replication in different insect tissues can be monitored using an antibody to a virus-specific chitinase.
Yeast-based vector systems can be used to manipulate the baculovirus genome and provide a more rapid way of deleting virus genes than is currently possible with plaque purification techniques.
Baculovirus inhibitors of apoptosis can be overexpressed in the very late phase of virus infection to produce a moderate improvement in cell viability; it is unlikely that this will translate into increased foreign protein synthesis.
PROJECT: Construction of mammalian cells with new gycosylation properties.
The glycosylation characteristics of secretory recombinant human glycoproteins is governed by the host cell used for production. Previous work has shown that the decoration of outer chains of glycans can be reproducibly manipulated by applying controlled cell culture conditions. Using the beta-trace protein as a sectory target glycoprotein for the action of the Golgi-resident recombinant human alpha2-6 STase it was shown that the activity of this enzyme remained unchanged, irrespective of cell culture conditions.
The co-expression of human alpha2-6 STase and 'beta-trace' protein demonstrates for the first time that the in vivo substrate specificity for biantennary lactosamine-type oligosaccharides of the recombinant human glycosyltransferase is identical to that of its postulated in vitro activity. The branch-specificity of the recombinant enzyme expressed from BHK-21 cells allows the production of recombinant glycoproteins with oligosaccharide structures bearing terminal NeuAc which are identical with those found on natural human glycoproteins.
PROJECT: Translational modification of gene expression to promote secretion of intracellular proteins.
The conditions required for fractionationof the protein synthetic apparatus of CHO cells into free (FP), cytoskeletal-bound (CBP) and membrane-bound polysomes (MBP) have been determined. Polysome profiles indicate that all three polysome populations are highly active in protein synthesis. The levels of enrichment of 12 specific mRNAs in FP, CBP and MBP has been assessed. Glyceraldehyde-3-phosphate, enolase, beta-actin, vimentin and prothymosin mRNAs are enriched in CBP, glutathione peroxidase mRNA in FP and glucose transporter 1 mRNA in MBP.
Sets of dimeric gene constructs have been made, one based on the b-globin coding sequence. Three of these (globin coding region with its own 3'UTR, with no 3'UTR and with the c-myc 3'UTR) have been transfected into CHO cells and in situ hybridisation and fractionation studies of stable transfectants indicate that, as in fibroblasts, the c-myc 3'UTR can target a reporter mRNA sequence to the perinuclear cytoplasm and the cytoskeleton.
Funding SchemeCSC - Cost-sharing contracts
20521 Turku / Abo
59655 Villeneuve D'ascq
LE7 9SU Leicester
EH4 2XU Edinburgh
SP4 0JG Salisbury
OX1 3SR Oxford
EH25 9PP Roslin
WC1E 6BT London
6709 PD Wageningen