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  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/67—General methods for enhancing the expression
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
  • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts

Definitions

• the present invention generally relates to methods for increasing the secretory capacities of cells, and more particularly to the use of genetically altered eukaryotic cells having an increased expression of ribosome receptors so as to exhibit increased secretory (or intracellular protein transport) capacity.
• TPA tissue plasminogen activator
• EPO erythropoeitin
• U.S. Patent 5,759,810 inventors Honjo et al., issued June 2, 1998, discloses a method for secreting a human growth hormone in an E. coli host cell by expressing a genetically engineered E. coli host cell to produce enhanced amounts of glutathione reductase with the target recombinant protein.
• US. Patent 5,679,543, issued October 21, 1997, inventor Lawlis describes fusion DNA sequences which when expressed in a filamentous fungus, are said to result in increased levels of secretion of the desired polypeptide, such as human tissue plasminogen activator, human growth hormone, human interferon, etc.
• mammalian cells such as CHO (Chinese Hamster Ovary)
• CHO Choinese Hamster Ovary
• mammalian cell systems are more expensive due to the limited number of cells that can be grown per liter of medium, and the expensive nature of the components of the growth medium itself.
• a process for increasing the intracellular transport or secretion of a desired protein in a eukaryotic host cell comprises expressing a gene encoding the desired protein in a eukaryotic host cell that has been genetically altered.
• the genetic alteration is effective to express an increased amount of at least a portion of a ribosome receptor so as to increase either the intracellular transport (if the desired protein is a membrane-bound receptor or enzyme) or the secretion of the desired protein with respect to wild-type eukaryotic host cells.
• inventive cells having the genetically engineered proliferation of ribosome receptors or fragments thereof are for the production of proteins, such as proteins useful for therapeutic purposes. Yields of the desired proteins ⁇ usually, but not always recombinant proteins — can be increased through this technology.
• the invention provides a process to increase the secretory capacities of cells. In this way cells can be created with an increased ability to transport or secrete a recombinant protein of interest, or, cells already expressing a desired protein can be transformed by practice of the invention to reach previously unattainable levels of transport and secretion.
• Figure 1 A is the primary structure (amino acid sequence) of the canine ribosome receptor
• Figure IB is a diagrammatic representation of constructs used in the studies outlined below;
• Figure 1C illustrates the alignment of amino acid sequences deduced from cDNA clones encoding ribosome receptor homologues from murine and human sources (dashes indicate identities, letters in mouse and human indicate variance from the canine, dots indicate putative deletions);
• Figure 2 is an electron micrograph of wild-type yeast, where proniinently shown are three major organelles, the nucleus (N), mitochondria (M) and the vacuole (V);
• Figure 3 is an electron micrograph of yeast showing rough membranes that are elaborated in response to the expression of the full-length ribosome receptor;
• Figure 4 is an electron micrograph of yeast expressing a construct
• Figure 5 is an electron micrograph of yeast expressing a construct that contains only the ribosome binding domain and the membrane anchor ( ⁇ CT in Fig. IB) that resulted in the most dramatic a proliferation of rough membranes;
• Figure 6 is a Northern blot showing that stimulation of membrane proliferation induces the expression of genes encoding proteins specific to organelles along the secretory pathway, where the gene that has been transfected into each strain is indicated at the top of the figure, and each row represents the expression levels of a gene that encodes a protein known to participate in different organelle-specific aspects of the secretory process;
• Figure 7 is an indirect immunofluorescence of control yeast (top row) as well as cells expressing the ⁇ CT construct (bottom row), and shown is staining with an anti-ribosome receptor antibody (left panels), an antibody against the resident ER protein Sec ⁇ lp (middle panels), and an antibody against Gdalp, a resident protein of the Golgi complex (right panels);
• Figure 8 is a growth assay demonstrating that cells expressing high levels of a gene encoding bovine pancreatic trypsin inhibitor (BPTI) are unable to grow unless transfected with genes encoding ⁇ CT or full-length ribosome receptor;
• BPTI bovine pancreatic trypsin inhibitor
• Figure 9 is an assay whereby levels of BPTI are measured in the culture medium of control yeast cells or of cells expressing ⁇ CT;
• Figure 10 is an electron micrograph of control monkey COS-7 cells (Panel A, lower cell, and Panel C) and COS-7 cells transiently transfected with a plasmid encoding the full-length ribosome receptor (Panel A, upper cell, and Panel
• Figure 11 shows immunofluorescent detection of the ribosome receptor in Chinese hamster ovary (CHO) cells that have been stably transfected with cDNA encoding the ribosome receptor;
• Figure 12 shows immunofluorescent detection of the ribosome receptor in Chinese hamster ovary (CHO) cells that have been stably transfected with a cDNA construct that enables inducible expression of the ribosome receptor;
• Figure 13 illustrates results of an assay for secreted alkaline phosphatase where cells described in Fig. 12 were transiently transfected with a plasmid encoding a secreted form of the enzyme alkaline phosphatase.
• one aspect of this invention is a process for increasing the intracellular transport or secretion of a desired protein in a eukaryotic host cell.
• intracellular transport it is meant that the desired protein is conveyed by cellular processes toward the cell membrane.
• membrane- bound proteins that could be desirably increased in transport by practicing this invention are ones which carry out covalent modifications on proteins of commercial importance.
• Many processing steps are being carried out enzymatically by these proteins in vitro.
• Some illustrative, important enzymes whose production would be increased through a stimulated proliferation of the secretory pathway would include ones involved in glycosylation (core and terminal), tyrosine sulfation, proteolytic cleavage, or the addition of lipid anchors.
• the desired protein is a secreted protein
• practice of the inventive process increases secretion of that desired protein through the cell membrane.
• suitable candidates are recombinant proteins such as growth factors, clotting factors, lymphokines and other cytokines, hormones, protease inhibitors, and other serum proteins.
• the subject invention can be carried out in substantially any eukaryotic host cell, whether derived from fungi, plant, protozoan, invertebrate, or vertebrate cells.
• Expression of genes encoding desired proteins in genetically altered eukaryotic host cells can be accomplished by genetically engineered constructions and methods now well known to practitioners in this field.
• Yeasts have been employed in large scale fermentations for centuries, can often be grown to higher densities than bacteria, and are readily adaptable to continuous fermentation processing.
• Suitable cultured cell systems for production of recombinant proteins in yeast systems are well known, as described, for example, by U.S. Patent 5,618,676.
• vertebrate cells particularly mammalian cells
• insect cells derived from Drosophila or moths are preferred and where viral vectors (baculovirus) can yield high levels of expression.
• viral vectors baculovirus
• Patent 5,770,192 issued June 23, 1998, inventors Cayley et al., describe recombinant baculoviruses which express a foreign protein that is secreted from insect cells infected with the recombinant baculovirus.
• the most widely used mammalian cell line for protein production continues to be CHO (Chinese hamster ovary), although some companies rely on baby hamster kidney (BHK) lines for the production of certain proteins.
• BHK baby hamster kidney
• hybridomas derived from fusions of plasma cell lines such as SP2/0 with antibody producing spleen cells
• hybridomas have been developed where hybridomas have been fused to human B cells as part of the "humanization” process of recombinant antibodies.
• SV-40 SV-40 promoters are common in systems using gene "amplification" technology. In this case constructs usually contain the dihydrofolate reductase (DHFR) gene, which allows selection for amplification using methotrexate.
• DHFR dihydrofolate reductase
• genetically altered cells with increased secretory capacities can be obtained by practice of the present invention.
• Such genetically altered eukaryotic host cells will be altered, or engineered, to express an increased amount of a ribosome receptor or a fragment thereof with respect to wild-type eukaryotic host cells.
• the totality of the secretory apparatus, including all of the processing enzymes can be upregulated.
• the most striking feature of the amino acid sequence is a 10-amino acid consensus motif that is repeated 54 times in tandem without interruption in the amino-terminal positively charged region. Wanker et al., J. CellBiol, 130, pp. 29-39 (1995), incorporated herein by reference.
• RR amino acid sequence
• MA membrane-anchoring region
• RBD ribosome binding domain
• CT carboxyl-terminal region
• Shown as ⁇ CT is a truncation of the carboxyl-terminal domain
• ⁇ NT is a construct harboring a deletion of the RBD. Further details, such as a restriction map, are described in Wanker et al. supra.
• Fig. 1 A The primary structure of the ribosome receptor shown in Fig. 1 A is deduced from the canine cDNA sequence. This protein appears very highly conserved in other species where sequence data is available. There is an ever- increasing number of snippets of cDNA, known as expressed sequence tags (EST), appearing in the databases. The most commonly posted mammalian sequences are from mouse and human sources. When translated into amino acids, a number of them align quite nicely with the sequence of the canine ribosome receptor. Due to its repetitive nature, it is difficult to align short ESTs within the N-terminal repeat- containing domain. However, as can be seen in Fig.
• EST expressed sequence tags
• the secretory capacity of eukaryotic cells can be increased through a stimulated membrane biogenetic event.
• a ribosome receptor, or portions of the ribosome receptor is expressed in a stable fashion in the desired cell type. These cells take on the attributes of highly differentiated secretory tissues such as pancreas, liver or plasma cells. These engineered cells then become the host for the high level production of correctly processed secretory or membrane proteins of commercial importance. Operationally speaking, host cells may be transfected with a gene encoding the protein of interest whose expression is optimized to the limit of processing and secretory capacity of the host cell.
• eukaryotic cells from yeast to human programmed with foreign DNA to produce a given protein for commercial use may be manipulated, by way of the invention's process, to achieve an increase in their ability to transport, process and potentially secrete such proteins.
• these cells may be transfected with a gene, or parts thereof, encoding a ribosome receptor.
• the cells will, as noted above, take on the attributes of secretory tissues and thereby result in increased transport, processing and secretion when compared to the unmanipulated original cell. Expression levels of the ribosome receptor constructs can be adjusted to be optimal for the transport, processing and secretion of the protein of interest.
• recombinant proteins whose expression is based on the transcription of cloned cDNA driven by a suitable promoter. Based on current technology, such constructs are most often, but not always, integrated into the genome of the host cell for more stable and reproducible expression levels.
• the cDNAs encoding the recombinant proteins are often derived from sources other than the host cell's complement of transcripts and would be defined as “heterologous” (e.g., a human cDNA expressed in CHO cells). Through the use of human host cells, homologous expression of human proteins could also be achieved.
• the transport, processing and secretion of artificial proteins as exemplified by "humanized” monoclonal antibodies, can also be improved over existing technology through the use of the invention.
• the expression of an increased amount of the desired protein may be inducible or non-inducible. Constitutive expression would typically be preferred in the routine commercial situation. However, as there must be an optimization between the synthesis of a recombinant protein and its ability to be transported, processed and secreted, placing either the recombinant protein, or the secretory apparatus under regulated control may help to facilitate such optimization.
• the desired protein is obtained in increased amounts with respect to wild-type eukaryotic host cell, then it preferably will be purified either from the growth medium of genetically engineered host cells or by extraction and purification from the host cells directly.
• known technologies for isolation from growth media can be utilized.
• intracellular proteins, or membrane bound enzymes many biochemical purifications have been worked out.
• the recombinant DNA molecule introduced into the genetic material of the eukaryotic host cell can be incorporated into the genome of the eukaryotic host cell or not.
• the ability to create cell lines that achieve stable expression of proteins over long periods of time, a necessary prerequisite for FDA approval, can only be achieved by integration into the host cell's chromosomes.
• Episomal (plasmid-based) transfection can lead to higher levels of expression, but using present technologies it is inherently unstable. Advances may come about in the future whereby episomal expression can be stabilized. In such cases, the increased levels of production of recombinant proteins would benefit from a host cell's having increased secretory capacity.
• promoters can also be used in preparing constructs useful in practicing the invention.
• ADH is another gene whose promoter could enable a high level of gene expression and represents a suitable equivalent. It may also be possible to induce equivalent, or even higher, levels of membrane proliferation by manipulating the constructs to contain only the ribosome binding (repeat-containing) domain and a membrane anchor. Alternatively, constructs with fewer or more repeats may enhance membrane production, and thus secretory activity.
• ribosome receptor cDNAs as well as the N-terminal half of the receptor (amino acids 1-826) were cloned into a pcDNA 3.1/Zeo vector (Invitrogen, San Diego, CA), where constitutive gene expression is controlled by the CMV promoter.
• Transient transfections of mammalian cells were carried out through electroporation, prior to growth on DMEM (+10% FBS) in 10 cm dishes.
• CHO cells were transfected by electroporation with Seal linearized expression constructs to enable integration into the host cell genome.
• Cell lines stably expressing proteins cloned into the pcDNA 3.1 vector were selected through the inclusion of the antibiotic zeocin (250 ⁇ g/ml) in the growth medium.
• Other animal cells successfully responding to transfection by increasing their endoplasmic reticulums (ER) complement include HeLa (human cervical carcinoma), COS-7
• Inducible expression constructs A ⁇ CT cDNA fragment was ligated into the pIND expression vector (Invitrogen) by standard techniques. In the resultant constructs, transcription was directed from the ecdysone/glucocorticoid response elements and the minimal heat shock promoter.
• Secreted protein construct Expression vectors encoding Secretory
• Alkaline Phosphatase (pSEAP2-Control) were obtained from Clonetech (Palo Alto, CA). These constructs were introduced into the appropriate cell type using lipofection techniques (Fugene ⁇ , Boehringer, Mannheim; Lipofectamine Plus, Life
• CHO/Kl cells were grown in Ham's/F12 medium with 10% FBS.
• CHO-Ecr ecdysone receptor-expressing cells were grown in the same way as CHO/Kl but zeocin was added to a concentration of 250 ⁇ g/ml. 48 hours prior to transfection, cells were plated at 25% confluency, at 16 hours prior to transfection fresh medium was added, and in the case of CHO-Ecr, zeocin was omitted.
• Clone selection was carried out using high dilution and seeding in 96-well plates or by cylinder cloning discrete foci on 150 mm plates.
• Fig. 4 where the ribosome binding domain has been deleted from the receptor that is being expressed, large quantities of membranes are still produced, as in Fig. 3, however there are no densely-stained particles (ribosomes) associated with the membranes.
• Fig. 5 where yeast have been transfected with a construct encoding little more than the repeat domain and the membrane anchor, the greatest proliferation of rough membranes has occurred, leaving little if any unoccupied space in the cytoplasm.
• Figs. 3 and 5 represent rough ER. Further evidence supporting the notion that these membranes represent bona fide rough ER is shown in Fig. 6.
• the expression of constructs that induce rough membranes induces the co-expression of genes encoding rough ER proteins.
• the expression of genes encoding marker proteins further along the secretory pathway occurs in the case of membrane induction by the ⁇ CT construct.
• the uniqueness of the ability of ⁇ CT to selectively induce these genes is demonstrated by the relatively low ability of other constructs — some of which induce smooth membrane proliferation in yeast (e.g., ⁇ NT) ⁇ to enable similar changes.
• Fig. 7 documents the fact that the proteins whose genes are expressed at higher levels in induced cells (Fig. 6) are also synthesized and incorporated into the correct organelle is demonstrated in Fig. 7.
• Indirect immunofluorescence using the appropriate antibodies show an upregulation in the appearance of ER, complete with one of its resident proteins (Sec ⁇ lp), as well as an increase in the number of Golgi-like structures in the transfected cells containing the resident guanosine diphosphatase (Gdal) protein.
• the data presented in Figs. 6 and 7 indicate that genes of the secretory pathway are expressed and incorporated correctly into organelles during membrane proliferation induced by the expression of (part of) the ribosome receptor.
• Fig. 8 is a growth assay demonstrating that cells expressing high levels of a gene encoding the secretory protein bovine pancreatic trypsin inhibitor (BPTI) are unable to grow unless transfected with genes encoding ⁇ CT or full- length ribosome receptor.
• BPTI is expressed in these cells in a regulated manner; the use of a GAL promoter enables expression when cells are grown in galactose- containing medium, but not on glucose.
• Fig. 9 is an assay whereby levels of BPTI are measured in the culture medium of control yeast cells or of cells expressing ⁇ CT. BPTI was expressed under GAL control as described in the legend to Fig. 9. Quantification of BPTI was carried out as described in the text below.
• BPTI block was relieved by its secretion from the cells that had proliferated the secretory pathway is demonstrated in Fig. 9.
• a sensitive assay for BPTI's ability to inhibit the proteolytic cleavage of a substrate by trypsin indicated that a 400% increase in BPTI secretion had taken place in ⁇ CT-containing cells, compared to vector-only controls.
• This value may represent a minimum value in the cell's ability to upregulate its secretory capacity as neither the levels of BPTI, nor of ⁇ CT have been optimized as yet. That which was observed in yeast can also occur in mammalian cells.
• CHO Chinese hamster ovary cells
• constructs encoding ⁇ CT as the inducer of membrane proliferation.
• a vector was used that places ⁇ CT under control of the constitutive human cytomegalovirus (CMV) promoter, whereas the second construct placed ⁇ CT behind an inducible hybrid promoter that is stimulated through including the insect hormone ecdysone or ecdysone-analogs in the growth medium.
• CMV human cytomegalovirus
• FIG. 11 shows immunofluorescent detection of the ribosome receptor in Chinese hamster ovary (CHO) cells that have been stably transfected with cDNA encoding the ribosome receptor. Note extensive membrane proliferation in transfected cells. Left panel: Cells transfected with cDNA encoding the ribosome receptor. Right panel: Cells transfected with vector alone.
• Fig. 12 shows immunofluorescent detection of the ribosome receptor in Chinese hamster ovary (CHO) cells that have been stably transfected with a cDNA construct that enables inducible expression of the ribosome receptor. Left panel: Cells that have not been induced to express the ribosome receptor. Right panel: Cells grown for 24 hours in the presence of inducer. Fig.
• FIG. 13 illustrates results of an assay for secreted alkaline phosphatase where cells described in Fig. 12 were transiently transfected with a plasmid encoding a secreted form of the enzyme alkaline phosphatase. Quantities of the enzyme appearing the culture medium at 24 hours after induction were measured colorimetrically for control cells and for ones whose expression of the ribosome receptor had been induced.
• Figs. 11 and 12 document the proliferation of the endoplasmic reticulum in cells that express ⁇ CT.
• a similar picture was obtained in the case of cells stably transfected with ⁇ CT under control of the inducible promoter. In this instance, striking upregulation of rough ER was observed after 24 hours of growth in the presence of the inducer when compared to uninduced control cells (Fig. 12).

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• 1998
  • 1998-07-02 JP JP50742799A patent/JP2002507894A/ja not_active Withdrawn
  • 1998-07-02 AU AU82873/98A patent/AU744484B2/en not_active Ceased
  • 1998-07-02 EP EP98933141A patent/EP0991774A4/en not_active Withdrawn
  • 1998-07-02 CA CA002295310A patent/CA2295310A1/en not_active Abandoned
  • 1998-07-02 WO PCT/US1998/013870 patent/WO1999001565A1/en not_active Application Discontinuation

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