WO2007081336A1 - Mammalian vectors for high-level expression of recombinant proteins - Google Patents

Abstract

Expression vectors that provide high-level expression of recombinant proteins, such as therapeutic proteins, are described. The invention also relates to expression vectors having a coding sequence, such as a recombinant DNA molecule encoding a therapeutic agent, and to mammalian cells transformed with the expression vector. Further provided are host cells transfected with expression vectors for expressing recombinant proteins.

Description

MAMMALIAN VECTORS FOR HIGH-LEVEL EXPRESSION OF RECOMBINANT PROTEINS

FIELD OF THE INVENTION

The present invention relates to expression vectors useful in the high-level expression of recombinant proteins. The invention also relates to expression vectors for transfection of a host cell and to host cells for expressing recombinant proteins. The invention further relates to host cells transfected with the expression vectors.

BACKGROUND OF THE INVENTION

The development of expression systems for the production of recombinant proteins is important for providing a source of a given protein for research and/or therapeutic use. Expression systems have been developed for both prokaryotic cells, such as E. coll, and for eukaryotic cells, such as yeast (i.e., Saccharomyces, Pichia and Kluyveromyces spp) and mammalian cells. Expression in mammalian cells is often preferred for manufacturing of therapeutic proteins as post-translational modifications in such expression systems are more likely to resemble those occurring on endogenous proteins in a mammal, than the type of post-translational modifications that occur in a microbial expression system.

Expression vectors are available for expression in mammalian hosts that contain various combinations of cis- and in some cases trans-regulatory elements to achieve high levels of recombinant protein in a minimal time frame. However, despite the availability of these expression vectors, the level of expression of a recombinant protein achieved in mammalian systems is often lower than that obtained with a microbial expression system. Additionally, because only a small percentage of transfected mammalian cells express high levels of the protein of interest, it can often take a considerably longer time to isolate and develop useful high-producing stable mammalian cell lines than it takes for microbial systems. In the expression of recombinant proteins, it is important to select regulatory

DNA sequences that include promoters and additional regulatory elements that are compatible with a host cell's transcriptional machinery. For this reason, regulatory DNA endogenous to the host cell of choice is generally preferred. Alternatively, considerable success has been achieved using regulatory DNA derived from viral genomic sequences in view of the broad host range of viruses in general and the demonstrated activity of viral regulatory DNA in different cell types.

SUMMARY OF THE INVENTION

In one aspect of the invention, an expression vector comprising, in the following operative order, a first regulatory element, a promoter sequence, a leader sequence, a multiple cloning site, a polyadenylation signal and a second regulatory element, wherein the first and second regulatory elements comprise sequences from a mammalian elongation factor- 1 is provided.

In another aspect of the invention, the promoter sequence comprises a CMV promoter and the leader sequence comprises a tripartite leader sequence. Also, an expression vector of the invention may further comprise a splicing donor site and a splicing acceptor site inserted between the tripartite leader sequence and the multiple cloning site wherein the multiple cloning site comprises a Pmel, Notl and a BamHl restriction site.

In yet another aspect, an expression vector of the invention further comprises an S V40 promoter, a neomycin resistance gene, an S V40 early polyadenylation signal, a pUC origin site, and an ampicillin resistance gene inserted between the first and second regulatory element. In certain embodiments, an expression vector of the invention comprises a mammalian elongation factor- 1 sequence that is chosen from among a human, a Chinese hamster, a cat or a chimpanzee elongation factor- 1 sequence. In certain embodiments, the mammalian elongation factor- 1 sequence comprises at least a portion of a Chinese hamster elongation factor- 1. In another aspect, an expression vector of the invention comprises, in the following operative order, SEQ ID NO:1 and SEQ ID NO:3. The expression vector may further comprise SEQ ID NO.2 inserted between SEQ ID NO:1 and SEQ ID NO:3. Additionally, the expression vector may further comprise SEQ ID NO:4 inserted adjacent SEQ ID NO:3. The present invention further provides a recombinant host cell that is transfected with the any of the expression vectors disclosed herein. Host cells of the invention preferably comprise mammalian cells, for example, mammalian cells chosen from among COS cells, BHK cells, CHO cells, HeLa cells, NS-I cells, or CHO cells, COS cells, or 293 cells. In yet another aspect of the invention, a composition comprising recombinant host cells, as described herein, further comprise a coding sequence that encodes a ^■ recombinant protein. Preferably the coding sequence encodes a therapeutic agent chosen from among any one of a member of the FGF family, a member of the IGF family, a member of the EGF family, a member of the NGF family, a member of the PDGF family, a member of the TNF family, a member of the VEGF family, a member of the TGF family, a member of the interleukin family (IL), epigen, amphiregulin, oncostatin M, betacellulin, epiregulin, a member of the trefoil factor family, or leukemia inhibitory factor (LIF). These compositions may optionally contain a coding sequence that comprises a therapeutic agent and a fusion molecule. Such fusion molecules include, but are not limited to molecules such as a polymer, a polypeptide, a succinyl group, fetuin A, fetuin B₅ albumin, a leucine zipper domain, a tetranectin trimerization domain, a mannose binding protein, a macrophage scavenger protein, an Fc region, or an- active fragment of any of these.

DESCRIPTION OF THE FIGURES

FIG. 1 is a general schematic of an expression vector of the instant invention. FIG. 2A represents a SEQ ID NO. Table. Column 1 shows an internal designation ID number; column 2 shows the nucleotide sequence identification number for the nucleic acid sequence (Nl); and column 3 shows the identification for the source clone or sequence. FIG. 2B provides SEQ ID NO:1. FIG. 2C provides SEQ ID NO:2. FIG. 2D provides SEQ ID NO:3. FIG. 2E provides SEQ ID NO:4.

DETAILED DESCRIPTION OF THE INVENTION Thus, a need exists in the art to identify promoter/enhancer regulatory DNA sequences that function in homologous and heterologous cell types to increase recombinant protein expression and provide a high yield of a desired recombinant protein product. Of particular importance is the need to identify such promoter/enhancer regulatory DNA that can be utilized most efficiently in mammalian cells in order to increase production of recombinant proteins in vitro that are glycosylated in a manner akin to glycosylation patterns that result from in vivo protein expression. Proteins expressed in this manner and administered therapeutically or prophylactically are typically less likely to be antigenic and more likely to be physiologically active. Regulatory DNA sequences of this type are also amenable to being inserted into host cells in order to increase expression of genes endogenous to the host cells or genes previously introduced into the genome of the host cell by techniques well known and routinely practiced in the art. The present invention may be more clearly understood in light of the following definitions. Generally, the terms used herein have their ordinary meaning and the meanings given them specifically below.

The term "expression vector" is understood to describe a vector that comprises various regulatory elements, described in detail below, that are important for the expression of recombinant, heterologous proteins in cells. The expression vector can include signals appropriate for maintenance in prokaryotic or eukaryotic cells, and/or the expression vector can be integrated into a chromosome. The expression vector can be part of a plasmid, virus, or nucleic acid fragment, of viral or non- viral origin. Typically, the expression vector includes an "expression cassette," which comprises a nucleic acid to be transcribed operably linked to a promoter. The term expression vector also encompasses naked DNA operably linked to a promoter.

By "nucleotide sequence" of a nucleic acid molecule or polynucleotide is intended, for a DNA molecule or polynucleotide, a sequence of deoxyribonucleotides, and for an RNA molecule or polynucleotide, the corresponding sequence of ribonucleotides (A, G, C and U), where each thymidine deoxyribonucleotide (T) in the specified deoxyribonucleotide sequence is replaced by the ribonucleotide uridine (U). A "nucleic acid" molecule can include both double- and single-stranded sequences and refers to, but is not limited to, cDNA from viral, prokaryotic or eukaryotic mRNA, genomic DNA sequences from viral (e.g., DNA viruses and retroviruses) or prokaryotic DNA, and especially synthetic DNA sequences. The term also captures sequences that include any of the known base analogs of DNA and RNA.

By "isolated" nucleic acid molecule(s) is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment. For example, recombinant DNA molecules contained in a vector are considered isolated for the purposes of the present invention. Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the DNA molecules of the present invention. Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically.

A "modulator" of the polypeptides or polynucleotides or an "agent" herein is an agonist or antagonist that interferes with the binding or activity of such polypeptides or polynucleotides. Such modulators or agents include, for example, polypeptide variants, whether agonist or antagonist; antibodies, whether agonist or antagonist; soluble receptors, usually antagonists; small molecule drugs, whether agonist or antagonist; RNAi, usually an antagonist; antisense molecules, usually an antagonist; and ribozymes, usually an antagonist. In some embodiments, an agent is a subject polypeptide, where the subject polypeptide itself is administered to an individual. In some embodiments, an agent is an antibody specific for a subject "target" polypeptide. In some embodiments, an agent is a chemical compound such as a small molecule that may be useful as an orally available drug. Such modulation includes the recruitment of other molecules that directly effect the modulation. For example, an antibody that modulates the activity of a subject polypeptide that is a receptor on a cell surface may bind to the receptor and fix complement, activating the complement cascade and resulting in lysis of the cell. An agent which modulates a biological activity of a subject polypeptide or polynucleotide increases or decreases the activity or binding at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 50%, at least about 80%, or at least about 2-fold, at least about 5-fold, or at least about 10-fold or more when compared to a suitable control.

"Modulating a level of active subject polypeptide" includes increasing or decreasing activity of a subject polypeptide, increasing or decreasing a level of active polypeptide protein, and increasing or decreasing a level of mRNA encoding active subject polypeptide.

"Operably linked" refers to an arrangement of elements wherein the components so described are configured so as to perform their desired function. Thus, a given promoter operably linked to a coding sequence is capable of effecting the expression of the coding sequence when the proper transcription factors, etc., are present. The promoter need not be contiguous with the coding sequence, so long as it functions to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between the promoter sequence and the coding sequence, as can translated introns, and the promoter sequence can still be considered "operably linked" to the coding sequence.

A "regulatory and/or control element" refers to a polynucleotide sequence that aids in the expression of a coding sequence to which it is linked. The term includes promoters, transcription termination sequences, upstream regulatory domains, polyadenylation signals, and when appropriate, leader sequences and enhancers, which collectively provide for the transcription and translation of a coding sequence in a host cell.

A "promoter" as used herein is a DNA regulatory region capable of binding RNA polymerase in a mammalian cell and initiating transcription of a downstream (3' direction) coding sequence operably linked thereto. For purposes of the present invention, a promoter sequence includes the minimum number of bases or elements necessary to initiate transcription of a gene of interest at levels detectable above background. Within the promoter sequence is a transcription initiation site, as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. Eucaryotic promoters will often, but not always, contain "TATA" boxes and "CAT" boxes. Promoters further include those that are naturally contiguous to a nucleic acid molecule and those that are not naturally contiguous to a nucleic acid molecule. Additionally, a promoter includes inducible promoters, conditionally active promoters, such as a cre-lox promoter, constitutive promoters and a tissue specific promoters.

A "leader sequence" is a sequence at the 5' end of an mRNA that is not translated into protein. It is the length of untranslated mRNA from the 5' end to the initiation codon AUG." By "selectable marker" is meant a gene which confers a phenotype on a cell expressing the marker, such that the cell can be identified under appropriate conditions. Generally, a selectable marker allows selection of transformed cells based on their ability to thrive in the presence or absence of a chemical or other agent that inhibits an essential cell function. Suitable markers, therefore, include genes coding for proteins which confer drug resistance or sensitivity thereto, impart color to, or change the antigenic characteristics of those cells transfected with a molecule encoding the selectable marker, when the cells are grown in an appropriate selective medium. For example, selectable markers include: cytotoxic markers and drug resistance markers, whereby cells are selected by their ability to grow on media containing one or more of the cytotoxins or drugs; auxotrophic markers by which cells are selected by their ability to grow on defined media with or without particular nutrients or supplements, such as thymidine and hypoxanthine; metabolic markers by which cells are selected for, e.g., their ability to grow on defined media containing the appropriate sugar as the sole carbon source, or markers which confer the ability of cells to form colored colonies on chromogenic substrates or cause cells to fluoresce.

"Transformation," as used herein, refers to the insertion of an exogenous polynucleotide into a host cell, irrespective of the method used for insertion: for example, transformation by direct uptake, transfection, infection, and the like. For particular methods of transfection, see further below. The exogenous polynucleotide may be maintained as a nonintegrated vector, for example, an episome, or alternatively, may be integrated into the host genome.

The terms "polypeptide" and "protein" refer to a polymer of amino acid residues and are not limited to a minimum length of the product. Thus, peptides, oligopeptides, dimers, multimers, and the like, are included within the definition. Both full-length proteins and fragments thereof are encompassed by the definition. The terms also include post-expression modifications of a polypeptide, for example, glycosylation, acetylation, phosphorylation and the like. Furthermore, for purposes of the present invention, a "polypeptide" includes modifications, such as deletions, additions and substitutions (generally conservative in nature), to the native sequence, as long as the protein maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification. A "gene," for the purposes of the present disclosure, includes a DNA region encoding a gene product, as well as all DNA regions which regulate the production of the gene product, whether or not such regulatory sequences are adjacent to coding and/or transcribed sequences. Accordingly, a gene includes, but is not necessarily limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites and locus control regions.

"Gene expression" refers to the conversion of the information, contained in a gene, into a gene product. A gene product can be the direct transcriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA or any other type of RNA) or a protein produced by translation of an mRNA. Gene products also include RNAs which are modified, by processes such as capping, polyadenylation, methylation, and editing, and proteins modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristilation, and glycosylation.

A "coding sequence" or a sequence which "encodes" a selected polypeptide, is a nucleic acid molecule which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A coding sequence can include, but is not limited to, cDNA from viral, procaryotic or eucaryotic mRNA, genomic DNA sequences from viral (e.g. DNA viruses and retroviruses) or procaryotic DNA, and especially synthetic DNA sequences. A transcription termination sequence may be located 3' to the coding sequence.

By "isolated" is meant, when referring to a polynucleotide or polypeptide of the invention, that the indicated molecule is substantially separated, e.g., from the whole organism in which the molecule is found or from the cell culture in which the antibody is produced, or is present in the substantial absence of other biological macromolecules of the same type.

By "fragment" is intended a polypeptide consisting of only a part of the intact full-length polypeptide sequence and structure. The fragment can include a C- terminal deletion an N-terminal deletion, and/or an internal deletion of the native polypeptide. A fragment of a protein will generally include at least about 5-10 contiguous amino acid residues of the full-length molecule, preferably at least about 15-25 contiguous amino acid residues of the full-length molecule, and most preferably at least about 20-50 or more contiguous amino acid residues of the full-length molecule, or any integer between 5 amino acids and the full-length sequence.

"Transformation" or "transfection" describes a process of genetic modification by which heterologous (i.e., foreign or exogenous) DNA enters and renders a recipient cell capable of expressing the heterologous DNA. Transformation may occur according to various methods well known in the art, for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method is selected based on the type of host cell being transformed and may include, but is not limited to, viral infection, electroporation, heat shock, lipofection, and particle bombardment. The terms "transformed cells" or "transfected cells" include stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, as well as transiently transformed or transfected cells which express the inserted DNA or RNA for limited periods of time. All of such transformed or transfected cells are referred to as "transgenic."

A "fusion molecule" is a sequence of amino acids corresponding to any therapeutic agent or fragment thereof, or a polynucleotide encoding such a sequence, and a fusion partner. A fusion molecule can be a product resulting from splicing strands of recombinant DNA and expressing the hybrid gene. A fusion molecule can be made by genetic engineering. It can be made by removing the stop codon from the DNA sequence of the first protein, then appending the DNA sequence of the second protein in frame. That DNA sequence will then be expressed by a cell as a single protein. Typically this is accomplished by cloning a cDNA into an expression vector in frame with an existing gene. A therapeutic protein fusion molecule may be a fusion protein, comprising a fusion partner comprising amino acids that represent all of or fragments of more than one gene. A therapeutic agent fusion molecule may also comprise a fusion partner which is not a polypeptide. A "fusion partner" is any component of a fusion molecule in addition to a therapeutic agent or fragment thereof. A fusion partner may comprise a polypeptide or a non-polypeptide moiety for example, polyethylene glycol.

The "fragment crystallizable" (Fc) fragment is the portion of an antibody molecule that interacts with effector molecules and cells. It comprises the carboxy- terminal portions of the immunoglobulin heavy chains. The functional differences between heavy-chain isotypes lie mainly in the Fc fragment.

A "hinge domain" is a short amino acid sequence in an antibody heavy chain which lies between the Fab and Fc domains and permits one to bend in relation to the other, in some instances, upon antigen binding. It is typically proline-rich. "Recombinant," as used herein to describe a nucleic acid molecule, means a polynucleotide of genomic, cDNA, viral, semisynthetic, and/or synthetic origin, which, by virtue of its origin or manipulation, is not associated with all or a portion of the polynucleotide with which it is associated in nature. The term "recombinant" as used with respect to a protein or polypeptide, means a polypeptide produced by expression of a recombinant polynucleotide. The term "recombinant" as used with respect to a host cell means a host cell into which a recombinant polynucleotide has been introduced.

A "growth factor" is an extracellular hormone or polypeptide signaling molecule that stimulates a cell to grow or proliferate. Many types of growth factors exist, including protein hormones and steroid hormones. Growth factors of the invention include variants and muteins. Examples of growth factors include fibroblast growth factors (FGF), epidermal growth factors (EGF), and platelet-derived growth factors (PDGF). These include, but are not limited to, FGF-2, FGF-4, FGF-9, IGF, IGF-I, PDGF, PDGF-BB, amphiregulin, epiregulin, Epigen, EGF, HB-EGF, and betacellulin.

A "member of the EGF family" is a growth factor that has a conserved domain known as the EGF motif, typically characterized by six conserved cysteine residues.

A "member of the FGF family" is a growth factor that interacts with heparin sulfate glycosaminoglycans and the extracellular domains of FGF cell surface receptors (FGFRs) to trigger receptor activation and biological responses, for example, as described in Olsen et al., J. Biol. Chem. (2003) 278(36):34,226-34,236 and Ornitz et al., Genome Biol. 2001 2:3005.1-3005.12.

A "member of the PDGF family" is a growth factor that binds to a PDGF receptor. Examples of members of the PDGF family include PDGF-BB and PDGF- DD, which are composed of two polypeptide B chains and D chains, respectively.

Expression Control Sequences

In the present invention, suitable expression control sequences may be derived from a variety of mammalian sources. Selection of appropriate regulatory elements, described herein, is dependent on the host cell selected, and may be readily accomplished by one of ordinary skill in the art. Some examples of regulatory elements include: a transcriptional promoter and enhancer or RNA polymerase binding sequence, splice signals, polyadenylation signals, including a translation initiation signal. Additionally, depending on the host cell chosen and the expression vector employed, other genetic elements, such as an origin of replication, additional DNA restriction sites, enhancers, sequences conferring inducibility of transcription, and selectable markers, may be incorporated into the expression vector. Strong promoters (or enhancer/promoters) are preferably selected. A strong promoter is one that directs the transcription of a gene whose product is abundant in the cell. The relative strength and specificities of a promoter/enhancer may be compared in comparative transient transfection assays. In one embodiment, a promoter may be selected that has little cell-type or species preference and which can therefore be strong when transfected into a variety of cell types. In preferred embodiments of the invention, an elongation factor- 1 regulator element is employed with a cytomegalovirus promoter.

Regulatory Elements

As used herein, regulatory elements and/or regulatory sequences are nucleotide sequences that enhance or otherwise modulate transcription and/or translation or that stabilize transcription and/or translation products. Thus, for example, promoters operably linked to a coding sequence of an expression construct enhance transcription of that coding sequence and polyadenylation sequences operably linked to a coding sequence modulate polyadenylation of the gene transcript. Exemplary regulatory elements can include, without limitation, promoters, enhancers, introns, termination sequences, polyadenylation sequences, stabilization sequences and the like. Some suitable regulatory sequences useful in the present invention will include, but are not limited to constitutive promoters, tissue-specific promoters, development-specific promoters, inducible promoters and viral promoters. Specific regulatory elements of the instant invention include, for example, mammalian regulatory elements comprising elongation factor-1 ("EF-I"). Sources of EF-I include, for example, human (Swissprot P68104), Chimpanzee (Swissprot Q5R.1X2), cat (Swissprot Q66RN5), and Chinese hamster (Swissprot P62629).

In certain embodiments, the nucleic acid encoding a gene product is operably linked and under transcriptional control of a promoter. A "promoter" refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene. The phrase "under transcriptional control" means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene.

The term promoter will be used here to refer to a group of transcriptional control modules that are clustered around the initiation site for RNA polymerase II. Much of the thinking about how promoters are organized derives from analyses of several viral promoters, including those for the HSV thymidine kinase (tk) and SV40 early transcription units. These studies, augmented by more recent work, have shown that promoters are typically composed of discrete functional modules, each consisting of approximately 7-20 bp of DNA, and containing one or more recognition sites for transcriptional activator or repressor proteins.

At least one module in each promoter functions to position the start site for RNA synthesis. The best known example of this is the TATA box, but in some promoters lacking a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation. Additional promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the tk promoter, for example, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either co-operatively or independently to activate transcription.

In certain embodiments of the invention, the human cytomegalovirus (CMV) promoter, the SV40 early promoter, the Rous sarcoma virus (RSV) long terminal repeat, a Chinese hamster, chimpanzee, cat or human elongation factor- 1 promoter, rat insulin promoter or glyceraldehyde-3-phosphate dehydrogenase promoter can be used to obtain high-level expression of the coding sequence of interest. The use of other viral or mammalian cellular or bacterial phage promoters which are well known in the art to achieve expression of a coding sequence of interest is contemplated as well, provided that the levels of expression are sufficient for a given purpose.

By employing a promoter with well-known properties, the level and pattern of expression of the protein of interest following transfection or transformation can be optimized. By way of illustration, a ubiquitous, strong (i.e., high activity) promoter may be employed to provide abundant gene expression in a group of host cells, or a tissue-specific promoter may be employed to target gene expression to one or more specific cell types. Further, selection of a promoter that is regulated in response to specific physiologic signals can permit inducible expression of the gene product.

Enhancers are genetic elements that increase transcription from a promoter located at a distant position on the same molecule of DNA. Enhancers are organized much like promoters. That is, they are composed of many individual elements, each of which binds to one or more transcriptional proteins.

The basic distinction between enhancers and promoters is operational. An enhancer region as a whole is typically able to stimulate transcription at a distance; this need not be true of a promoter region or its component elements. On the other hand, a promoter typically has one or more elements that direct initiation of RNA synthesis at a particular site and in a particular orientation, whereas enhancers generally lack these specificities. Promoters and enhancers are often overlapping and contiguous, often seeming to have a very similar modular organization.

Other promoter/enhancer combinations (see, e.g., the Eukaryotic Promoter Data Base EPDB) could also be used to drive expression of the gene. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided, either as part of the delivery complex or as an additional genetic expression construct. In one aspect, tissue-specific promoters, e.g., cardiac-specific and/or fibroblast-specific promoters, are of particular interest. By way of illustration, cardiac-specific promoters include the myosin light chain-2 promoter (Franz et al., 1994; Kelly et al., 1995), the alpha actin promoter (Moss et al., 1996), the troponin 1 promoter (Bhavsar et al, 1996); the Na⁺/Ca²⁺ exchanger promoter (Barnes et al., 1997), the dystrophin promoter (Kimura et al., 1997), the creatine kinase promoter (Ritchie, M. E., 1996), the alpha7 integrin promoter (Ziober & Kramer, 1996), the brain natriuretic peptide promoter (LaPointe et al, 1996) and the alpha B-crystallin/small heat shock protein promoter (Gopal-Srivastava, R., 1995), alpha myosin heavy chain promoter (Yamauchi-Takihara et al., 1989) and the ANF promoter (LaPointe et al., 1988).

Where a cDNA insert is employed, one will typically desire to include a polyadenylation signal to effect proper polyadenylation of the gene transcript. The nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and any such sequence may be employed such as human growth hormone and SV40 polyadenylation signals. Also contemplated as an element of the expression cassette is a terminator. These elements can serve to enhance message levels and to minimize read through from the cassette into other sequences.

Selectable Markers In certain embodiments of the invention, in which cells contain nucleic acid constructs of the present invention, a cell may be identified in vitro or in vivo by including a marker in the expression construct. Such markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression construct. Usually the inclusion of a drug selection marker aids in cloning and in the selection of transformants, for example, genes that confer resistance to ampicillin, neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol are useful selectable markers. Alternatively, enzymes such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may be employed. Immunologic markers also can be employed. The selectable marker employed is not believed to be important, so long as it is capable of being expressed simultaneously with the nucleic acid encoding a gene product. Further examples of selectable markers are well known to one of skill in the art. Expression vectors that retain the utility of a selectable, amplifϊable marker such as ampicilin resistant gene, while increasing the proportion of mRNAs encoding a desired recombinant protein, are described herein. Because many transcripts encode only the gene of interest and not the selectable marker, the inventive vectors produce less selectable marker protein, and only those transfectants that integrate into more transcriptionally active sites survive the selection process. Accordingly, use of the inventive expression vectors facilitates isolation of transfected pools and clones that express high levels of recombinant protein using lower levels of a selection agent than is possible in the absence of the internal polyadenylation signal.

Polyadenylation Signals

In expression of recombinant proteins, one will typically include a polyadenylation signal to effect proper polyadenylation of the transcript. The nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and any of a number of such sequences may be employed. Exemplary embodiments include the S V40 polyadenylation signal, the bovine growth hormone polyadenylation signal and others which are convenient and/or known to function well in various target cells. Also contemplated as an element of the expression cassette is a transcriptional termination site. These elements can serve to enhance message levels and/or to minimize read through from the cassette into other sequences.

Many polyadenylation signals are known in the art, and will also be useful in the instant invention. Examples include those shown in Table 1 below.

TABLE 1 Polyadenylation Signals

Additional polyadenylation sites can be identified or constructed using methods that are known in the art.

Vectors

The term "vector" is used to refer to carrier molecules with which a nucleic acid sequence can be associated for introduction into a cell. The nucleic acid sequence can be "exogenous," (e.g., foreign to the cell into which it is introduced) or "endogenous" (e.g., the same as a sequence in the cell into which it is introduced. Exemplary vectors include plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs), lipid-based vectors (e.g., liposomes) and other macromolecular complexes capable of mediating delivery of a polynucleotide to a host cell. One of skill in the art would be well equipped to construct a vector through standard techniques, for example standard recombinant techniques such as described in Sambrook et al., 1989 and Ausubel et al., 1994, both incorporated herein by reference.

A large number of viral and non- viral vectors (including lipid-based and other synthetic delivery systems known in the art) can likewise be employed to deliver polynucleotides of the present invention. Such vectors may be modified, as known to those of skill in the art, to confer or enhance cell specificity. By way of illustration, the surface of viral vectors may be modified such that they preferentially or exclusively bind to and/or infect a particular target cell population.

Among vectors useful in the present invention are the herein described vectors employing a pTT vector backbone, see, for example, Durocher et al., Nucl. Acid Res., 30(2) (2002). The pTT2p vector includes, inter alia, murine polyoma signals to make an episomal pTT2-gateway vector.

Briefly, the pTT vector backbone may be prepared by obtaining pIRESpuro/EGFP (pEGFP) and pSEAP basic vector(s), for example from Clontech (Palo Alto, CA), and ρcDNA3.1, pCDNA3.1/Myc-(His)₆ (His₆ tag disclosed as SEQ ID NO: 5) and pCEP4 vectors can be obtained from, for example, Invitrogen.

SuperGlo GFP variant (sgGFP) can be obtained from Q-Biogene (Carlsbad, CA). Preparing a pCEP5 vector can be accomplished by removing the CMV promoter and polyadenylation signal of pCEP4 by sequential digestion and self-ligation using Sail and Xbal enzymes resulting in plasmid pCEP4Δ. A GbIII fragment from pAdCMV5 (Massie et al., (1998)), encoding the CMV5-poly(A) expression cassette ligated in i?g/II-linearized pCEP4Δ, resulting in pCEP5 vector. The pTT vector can be prepared by deleting the hygromycin (Bsml and Sail excision followed by fill-in and ligation) and EBNAl (Clal and Nsϊl excision followed by fill-in and ligation) expression cassettes. The CoIEI origin {Fspl-Saϊl fragment, including the 3' end of β-lactamase ORF) can be replaced with a Fspl-Sall fragment from pcDNA3.1 containing the pMBI oring (and the same 3' end of β-lactamase ORF). A Myc-(His)₆ C-terminal fusion tag can be added to SEAP (Hindlll-Hpal fragment from pSEAP-basic) following in-frame ligation in pcDNA3.1/Myc-His digested with Hindlϊl and EcoRV. Plasmids can subsequently be amplified in Escherichia coli (DH5α) grown in LB medium and purified using MAXI prep columns (Qiagen, Mississauga, Ontario, Canada). To quantify, plasmids can be subsequently diluted in 50 mM Tris-HCl pH 7.4 and absorbencies can be measured at 260nm and 280nm. Preferably, plasmid preparations with A₂₆o/A₂₈o ratios between about 1.75 and about 2.00 are used. As described herein, an expression vector of the invention includes a vector containing a nucleic acid sequence coding for at least part of a gene product capable of being transcribed. In some cases, the transcription product(s) are then translated into a protein, polypeptide, or peptide. In other cases, these sequences are not translated, for example, in the production of antisense molecules or ribozymes. Expression vectors can contain a variety of "regulatory elements and/or control sequences," which refer to nucleic acid sequences that regulate the transcription and possibly translation of an operably linked coding sequence in a particular host organism. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well for example as described herein.

Recombinant expression vectors may include a coding sequence encoding a protein of interest, e.g., a therapeutic agent, (or fragment thereof), ribozymes, ribosomal mRNAs, antisense RNAs and the like. Preferably, the coding sequence encodes a therapeutic protein or peptide. The coding sequence may be synthetic, a cDNA-derived nucleic acid fragment or a nucleic acid fragment isolated by polymerase chain reaction (PCR).

Expression vectors may also comprise non-transcribed elements such as a suitable promoter and/or enhancer linked to the gene to be expressed, other 5' or 3' flanking non-transcribed sequences, 5' or 3' non-translated sequences such as ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, and transcriptional termination sequences. An origin of replication that confers the ability to replicate in a host, and a selectable gene to facilitate recognition of transfectants, may also be incorporated.

DNA regions are operably linked when they are functionally related to each other. For example, DNA for a signal peptide (secretory leader) is operably linked to DNA for a polypeptide if it is expressed as a precursor which participates in the secretion of the polypeptide; thus, in the case of DNA encoding secretory leaders, operably linked means contiguous and in reading frame. A promoter is operably linked to a coding sequence if it controls the transcription of the sequence; and a rϊbosome binding site is operably linked to a coding sequence if it is positioned so as to permit translation.

The transcriptional and translational control sequences in expression vectors to be used in transfecting cells may be provided by viral sources. For example, commonly used promoters and enhancers are derived from Polyoma, Adenovirus 2, Simian Virus 40 (SV40), and human cytomegalovirus. Viral genomic promoters, control and/or signal sequences may be utilized to drive expression, provided such control sequences are compatible with the host cell chosen. Examples of such vectors can be constructed as disclosed by Okayama and Berg (MoI. Cell. Biol. 3:280, 1983). Non-viral cellular promoters can also be used (i.e., the beta-globin and the EF-lα promoters), depending on the cell type in which the recombinant protein is to be expressed.

DNA sequences derived from the SV40 viral genome, for example, SV40 origin, early and late promoter, enhancer, splice, and polyadenylation sites may be used to provide the other genetic elements required for expression of a heterologous DNA sequence. The early and late promoters are particularly useful because both are obtained easily from the virus as a fragment which also contains the S V40 viral origin of replication (Fiers et al, Nature 273:113, 1978). Smaller or larger SV40 fragments may also be used, provided the approximately 250 bp sequence extending from the Hind III site toward the BgII site located in the viral origin of replication is included.

An additional technique that can be used in conjunction with the expression vectors described herein is described by Lucas et al. (Nucleic Acids Res. 24:1774; 1996). In an effort to increase production of a desired protein, Lucas et al. utilized mRNA splice donor and acceptor sites to develop stable clones that produced both a selectable marker and recombinant proteins. According to these investigators, the vectors they prepared resulted in the transcription of a high proportion of mRNA encoding the desired protein, and a fixed, relatively low level of the selection marker that allowed selection of stable transfectants.

Host Cells

As used herein, the terms "cell," "cell line," and "cell culture," may be used interchangeably. These terms also include their progeny, which is any and all subsequent generations. It is understood that all progeny may not be identical due to deliberate or inadvertent mutations. La the context of expressing a heterologous nucleic acid sequence, "host cell" refers to a prokaryotic or eukaryotic cell, and it includes any transformable organisms that is capable of replicating a vector and/or expressing a heterologous gene encoded by a vector. A host cell can, and has been, used as a recipient for vectors. A host cell may be "transfected" or "transformed," which refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A transformed cell includes the primary subject cell and its progeny.

Some expression vectors of the present invention may employ control sequences that allow it to be replicated and/or expressed in both prokaryotic and eukaryotic cells. One of skill in the art would further understand conditions under which to incubate such host cells to maintain them and to permit replication of a vector. Also understood and known are techniques and conditions that would allow large-scale production of vectors, as well as production of the nucleic acids encoded by vectors and their cognate polypeptides, proteins, or peptides.

Transfected host cells are cells which have been transfected (sometimes referred to as transformed) with heterologous DNA. Many techniques for transfecting cells are known; in one approach, cells are transfected with expression vectors constructed using recombinant DNA techniques and which contain sequences encoding recombinant proteins. Expressed proteins will preferably be secreted into the culture supernatant, but may be associated with the cell membrane, depending on the particular polypeptide that is expressed. Mammalian host cells are preferred for the instant invention. Various mammalian cell culture systems can be employed to express recombinant protein. Examples of suitable mammalian host cell lines include, but are not limited to, the COS-7 lines of monkey kidney cells, described by Gluzman (Cell 23:175, 1981), CV-1/EBNA (ATCC CRL 10478), L cells, C127, 3T3, Chinese hamster ovary (CHO), HeLa and BHK cell lines.

A commonly used cell line is DHFR- CHO cells which are auxotrophic for glycine, thymidine and hypoxanthine, and can be transformed to the DHFR+ phenotype using DHFR cDNA as an amplifϊable dominant marker. One such DHFR- CHO cell line, DXBl 1, was described by Urlaub and Chasin (Proc. Natl. Acad. Sci. USA 77:4216, 1980). Another example of a DBFR- CHO cell line is DG44 (see, for example, Kaufman, R. J., Meth. Enzymology 185:537 (1988)). Other cell lines developed for specific selection or amplification schemes will also be useful with the invention. Numerous other eukaryotic cells will also be useful in the present invention, including cells from other vertebrates, and insect cells. Those of skill in the art will be able to select appropriate vectors, regulatory elements, transfection and culture schemes according to the needs of their preferred culture system.

Expression Systems

Mammalian cells suitable for carrying out the present invention include, but are not limited to, COS cells, BHK cells, CHO cells, HeLa cells, and NS-I cells. As noted above, suitable expression vectors for directing expression in mammalian cells generally include a promoter, as well as other transcriptional and translational regulatory and/or control sequences. Representative methods include calcium phosphate mediated gene transfer, electroporation, retroviral, and protoplast fusion- mediated transfection (see, for example, Sambrook et al.).

Numerous expression systems exist that comprise at least a part or all of the compositions described herein. Prokaryote- and/or eukaryote-based systems can be employed for use with the present invention to produce nucleic acid sequences, or their cognate polypeptides, proteins and peptides. Many such systems are commercially and widely available.

The insect cell/baculovirus system can produce a high level of protein expression of a heterologous nucleic acid segment, such as described in U.S. Pat. Nos. 5,871,986, 4,879,236, both herein incorporated by reference, and which can be bought, for example, under the name MAXBAC® 2.0 from INVITROGEN® and BACP ACK™ baculovirus expression system from CLONTECH®.

Other examples of expression systems include STRAT AGENE®'s COMPLETE CONTROL™ Inducible Mammalian Expression System, which involves a synthetic ecdysone-inducible receptor, or its pET Expression System, an E. coli expression system. Another example of an inducible expression system is available from INVITROGEN®, which carries the T-REX™ (tetracycline-regulated expression) System, an inducible mammalian expression system that uses the full- length CMV promoter. INVITROGEN® also provides a yeast expression system called the Pichia methanolica Expression System, which is designed for high-level production of recombinant proteins in the methylotrophic yeast Pichia methanolica. One of skill in the art would know how to express a vector, such as an expression construct, to produce a nucleic acid sequence or its cognate polypeptide, protein, or peptide.

Both the early and late polyadenylation signals of S V40 can be employed in the instant invention. These sequences are encoded within the 237-base pair fragment between the BamnHI site at nucleotide 2533 and the BcII site at nucleotide 2770 of the SV40 genome (Carswell and Alwine, MoI. Cell. Biol. 9:4248; 1989). Carswell and Alwine concluded that, of the two SV40 polyadenylation signals, the late signal was more efficient, most likely because it comprises both downstream and upstream sequence elements that facilitate efficient cleavage and polyadenylation.

Preparation of Transfected Mammalian Cells

Several transfection protocols are known in the art, and are reviewed in Kaufman, R. J. The transfection protocol chosen will depend on the host cell type and the nature of the protein of interest, and can be chosen based upon routine experimentation. The basic requirements of any such protocol are first to introduce DNA encoding the protein of interest into a suitable host cell, and then to identify and isolate host cells which have incorporated the heterologous DNA in a stable, expressible manner.

One commonly used method of introducing heterologous DNA is calcium phosphate precipitation, for example, as described by Wigler et al. (Proc. Natl. Acad. Sci. USA 77:3567, 1980). DNA introduced into a host cell by this method frequently undergoes rearrangement, making this procedure useful for cotransfection of independent genes.

Polyethylene-induced fusion of bacterial protoplasts with mammalian cells (Schaffher et al., Proc. Natl. Acad. Sci. USA 77:2163, 1980) is another useful method of introducing heterologous DNA. Protoplast fusion protocols frequently yield multiple copies of the plasmid DNA integrated into the mammalian host cell genome. This technique requires the selection and amplification marker to be on the same plasmid as the gene of interest. Electroporation can also be used to introduce DNA directly into the cytoplasm of a host cell, as described by Potter et al. (Proc. Natl. Acad. Sci. USA 81:7161, 1988) or Sbigekawa and Dower (BioTechniques 6:742, 1988). Unlike protoplast fusion, electroporation does not require the selection marker and the gene of interest to be on the same plasmid. More recently, several reagents useful for introducing heterologous DNA into a mammalian cell have been described. These include Lipofectin.RTM. Reagent and Lipofectamine.TM. Reagent (Gibco BRL, Gaithersburg, Md.). Both of these reagents are commercially available reagents used to form lipid-nucleic acid complexes (or liposomes) which, when applied to cultured cells, facilitate uptake of the nucleic acid into the cells.

Transfection of cells with heterologous DNA and selection for cells that have taken up the heterologous DNA and express the selectable marker results in a pool of transfected cells. Individual cells in these pools will vary in the amount of DNA incorporated and in the chromosomal location of the transfected DNA. After repeated passage, pools frequently lose the ability to express the heterologous protein. To generate stable cell lines, individual cells can be isolated from the pools and cultured (a process referred to as cloning), a laborious time consuming process. However, in some instances, the pools them selves may be stable (i.e., production of the heterologous recombinant protein remains stable). The ability to select and culture such stable pools of cells would be desirable as it would allow rapid production of relatively large amounts of recombinant protein from mammalian cells.

A method of amplifying the gene of interest is also desirable for expression of the recombinant protein, and typically involves the use of a selection marker. Resistance to cytotoxic drugs is the characteristic most frequently used as a selection marker, and can be the result of either a dominant trait (i.e., can be used independent of host cell type) or a recessive trait (i.e., useful in particular host cell types that are deficient in whatever activity is being selected for). Several amplifiable markers are suitable for use in the inventive expression vectors (for example, as described in Maniatis, Molecular Biology: A Laboratory Manual, Cold Spring Harbor Laboratory, NY, 1989; pgs 16.9-16.14).

Useful selectable markers for gene amplification in drug-resistant mammalian cells are shown in Table 1 of Kaufman, R. J., supra, and include DHFR-MTX resistance, P-glycoprotein and multiple drug resistance (MDR)-various lipophilic cytoxic agents (i.e., adriamycin, colchicine, vincristine), and adenosine deaminase (ADA)-XyI-A or adenosine and 2'-deoxycoformycin. Specific examples of genes that encode selectable markers are those that encode antimetabolite resistance such as the DHFR protein, which confers resistance to methotrexate (Wigler et al., 1980, Proc. Natl. Acad. Sci. USA 77:3567; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); the GPT protein, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072), the neomycin resistance marker, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin et al., 1981, J. MoL Biol. 150:1); the Hygro protein, which confers resistance to hygromycin (Santerre et al., 1984, Gene 30:147); and the Zeocin™ resistance marker (available commercially from Invitrogen). In addition, the herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes can be employed in tk-, hgprt- and aprt-cells, respectively.

Other dominant selectable markers include microbially derived antibiotic resistance genes, for example neomycin, kanamycin or hygromycin resistance. However, these selection markers have not been shown to be amplifiable (Kaufman, R. J., supra,). Several suitable selection systems exist for mammalian hosts (Maniatis supra, pgs 16.9-16.15). Co-transfection protocols employing two dominant selectable markers have also been described (Okayama and Berg, MoI Cell Biol 5:1136, 1985). A particularly useful selection and amplification scheme utilizes DHFR-MTX resistance. MTX is an inhibitor of DHFR that has been shown to cause amplification of endogenous DHFR genes (Alt F. W., et al., J Biol Chem 253:1357, 1978) and transfected DHFR sequences (Wigler M., et al., Proc. Natl. Acad. Sci. USA 77:3567, 1980). Cells are transfected with DNA comprising the gene of interest and DNA encoding DHFR in a dicistronic expression unit (Kaufman et al., 1991 supra and Kaufman R. J., et al., EMBO J 6:187, 1987). Transfected cells are grown in media containing successively greater amounts of MTX, resulting in greater expression of the DHFR gene, as well as the gene of interest.

Useful regulatory elements, described previously, can also be included in the plasmids or expression vectors used to transfect mammalian cells. The transfection protocol chosen, and the elements selected for use therein, will depend on the type of host cell used. Those of skill in the art are aware of numerous different protocols and host cells, and can select an appropriate system for expression of a desired protein, based on the requirements of their selected cell culture system(s).

Uses of the Invention The inventive expression vectors will find use for the expression of a wide variety of recombinant therapeutic polypeptides. In addition to those described above, additional examples of such polypeptides include cytokines and growth factors, such as Interleukins 1 through 18, the interferons, RANTES, lymphotoxin-.beta., Fas ligand, flt-3 ligand, ligand for receptor activator of NF-kappa B (RANKL), TNF- related apoptosis-inducing ligand (TRAIL), CD40 ligand, Ox40 ligand, 4-1BB ligand (and other members of the TNF family), thymic stroma-derived lymphopoietin, granulocyte colony stimulating factor, granulocyte-macrophage colony stimulating factor, mast cell growth factor, stem cell growth factor, epidermal growth factor, growth hormone, tumor necrosis factor, leukemia inhibitory factor, oncostatin-M, and hematopoietic factors such as erythropoietin and thrombopoietin.

Also included are neurotrophic factors such as brain-derived neurotrophic factor, ciliary neurotrophic factor, glial cell-line derived neurotrophic factor and various ligands for cell surface molecules Elk and Hek (such as the ligands for eph- related kinases, or LERKS). Descriptions of proteins that can be expressed according to the inventive methods may be found in, for example, Human Cytokines: Handbook for Basic and Clinical Research, Vol. II (Aggarwal and Gutterman, eds. Blackwell Sciences, Cambridge Mass., 1998); Growth Factors: A Practical Approach (McKay and Leigh, eds., Oxford University Press Inc., New York, 1993) and The Cytokine Handbook (A W Thompson, ed.; Academic Press, San Diego Calif.; 1991).

Receptors for any of the aforementioned proteins may also be expressed using the inventive vectors and methods, including both forms of tumor necrosis factor receptor (referred to as p55 and p75), Interleukin-1 receptors (type 1 and 2), Interleukin-4 receptor, Interleukin-15 receptor, Interleukin-17 receptor, Interleukin-18 receptor, granulocyte-macrophage colony stimulating factor receptor, granulocyte colony stimulating factor receptor, receptors for oncostatin-M and leukemia inhibitory factor, receptor activator of NF-kappa B (RANK), receptors for TRAIL, and receptors that comprise death domains, such as Fas or Apoptosis-inducing Receptor (AIR).

Other proteins that can be expressed using the inventive vectors and methods include cluster of differentiation antigens (referred to as CD proteins), for example, those disclosed in Leukocyte Typing VI (Proceedings of the VIth International Workshop and Conference; Kishimoto, Kikutani et al., eds.; Kobe, Japan, 1996), or CD molecules disclosed in subsequent workshops. Examples of such molecules include CD27, CD30, CD39, CD40; and ligands thereto (CD27 ligand, CD30 ligand and CD40 ligand). Several of these are members of the TNF receptor family, which also includes 41BB and OX40; the ligands are often members of the TNF family (as are 4-1BB ligand and OX40 ligand); accordingly, members of the TNF and TNFR families can also be expressed using the present invention. Proteins that are enzymatically active can also be expressed according to the instant invention. Examples include metalloproteinase-disintegrin family members, various kinases (including streptokinase and tissue plasminogen activator as well as Death Associated Kinase Containing Ankyrin Repeats, and IKR 1 and 2), TNF-alpha Converting Enzyme, and numerous other enzymes. Ligands for enzymatically active proteins can also be expressed by applying the instant invention.

The inventive expression vectors are also useful for expression of other types of recombinant proteins, including immunoglobulin molecules or portions thereof, and chimeric antibodies (i.e., an antibody having a human constant region couples to a murine antigen binding region) or fragments thereof. Numerous techniques are known by which DNA encoding immunoglobulin molecules can be manipulated to yield DNAs capable of encoding recombinant proteins such as single chain antibodies, antibodies with enhanced affinity, or other antibody-based polypeptides (see, for example, Larrick et al., Biotechnology 7:934-938, 1989; Reichmann et al., Nature 332:323-327, 1988; Roberts et al., Nature 328:731-734, 1987; Verhoeyen et al., Science 239:1534-1536, 1988; Chaudhary et al., Nature 339:394-397, 1989).

Various fusion proteins can also be expressed using the inventive methods and vectors. Examples of such fusion proteins include proteins expressed as fusion with a portion of an immunoglobulin molecule, proteins expressed as fusion proteins with a zipper moiety, and novel polyfunctional proteins such as a fusion proteins of a cytokine and a growth factor (i.e., GM-CSF and IL-3, MGF and IL-3). WO 93/08207 and WO 96/40918 describe the preparation of various soluble oligomeric forms of a molecule referred to as CD40L, including an immunoglobulin fusion protein and a zipper fusion protein, respectively; the techniques discussed therein are readily applicable to other proteins. The invention provides that the compositions of the invention may comprise a therapeutic agent which is a fusion molecule. In an embodiment, the fusion molecule comprises a polypeptide having growth factor activity and a fusion partner. In an embodiment, the fusion partner confers a half-life to the growth factor that is longer in the subject than the half-life of the growth factor in the subject in the absence of the fusion partner. For example, the half-life of the long-acting therapeutic agent is at least one-half hour, one hour, two hours, three hours, four hours, or five hours, or more, longer in the subject than the half-life of the therapeutic agent of the first component in the absence of the fusion partner. Fusion partners suitable for use in the invention may comprise a polymer, a polypeptide, a succinyl group, fetuin A, fetuin B, albumin, a leucine zipper domain, a tetranectin trimerization domain, a mannose binding protein, a macrophage scavenger protein, an Fc region, or an active fragment of any of these. In an embodiment, the polymer is a polyethylene glycol moiety. In an embodiment, the polymer is a polypeptide. In an embodiment, the polymer is a polypeptide and the polyethylene glycol moiety is attached to the polypeptide through an amino group of an amino acid of the polypeptide. Polyethylene glycol moieties of the invention may be branched or linear chain polymers.

In one embodiment, the fusion molecule comprises at least a portion of an Fc region. In an embodiment, the fusion molecule comprises albumin, which comprises an albumin molecule, one or more fragments of albumin, a peptide that binds albumin, an albumin molecule that conjugates with a lipid, or an albumin molecule that binds to another molecule. In an embodiment, the fusion molecule comprises an oligomerization domain, for example, an oligomerization domain that comprises a coiled-coil domain (for example, a tetranectin coiled-coil domain, a coiled-coil domain found in a cartilage oligomeric matrix protein, an angiopoietin coiled-coil domain, or a leucine zipper domain), a collagen domain, a collagen-like domain, or a dimeric immunoglobulin domain. Suitable collagen or collagen-like domains comprise, for example, a collagen or collagen-like domain found in collagen, mannose-binding lectin, lung surfactant protein A, lung surfactant protein D, adiponectin, ficolin, conglutinin, macrophage scavenger receptor, or emilin. In an embodiment, the dimeric immunoglobulin domain comprises an antibody CH3 domain.

As additional examples, DNAs based on one or more expressed sequence tag (EST) from a library of ESTs can be prepared, inserted into the inventive vector and expressed to obtain recombinant polypeptide. Moreover, DNAs isolated by use of ESTs (i.e., by PCR or the application of other cloning techniques) can also be expressed by applying the instant invention. Information on the aforementioned polypeptides, as well as many others, can be obtained from a variety of public sources, including electronic databases such as GenBank. A particularly useful site is the website of the National Center for Biotechnology Information/National Library of Medicine/National Institutes of Health. Those of ordinary skill in the art are able to obtain information needed to express a desired polypeptide and apply the techniques described herein by routine experimentation.

The relevant disclosures of all references cited herein are specifically incorporated by reference. The following examples are intended to illustrate particular embodiments, and not limit the scope, of the invention. Those of ordinary skill in the art will readily recognize that additional embodiments are encompassed by the invention.

It will be clear that the invention may be practiced otherwise than as particularly described in the foregoing description and examples. Numerous modifications and variations of the present invention are possible in light of the above teachings and, therefore, are within the scope of the appended claims. AU publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety.

It must be noted that, as used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the content clearly dictates otherwise.

Claims

Claims

1. An expression vector comprising, in the following operative order, a first regulatory element, a promoter sequence, a leader sequence, a multiple cloning site, a polyadenylation signal and a second regulatory element, wherein the first and second regulatory element comprise sequences from a mammalian elongation factor- 1.

2. The expression vector of claim 1 , wherein the promoter sequence comprises a CMV promoter.

3. The expression vector of claim 2, wherein the leader sequence comprises a tripartite leader sequence.

4. The expression vector of claim 3, further comprising a splicing donor site and a splicing acceptor site inserted between the tripartite leader sequence and the multiple cloning site.

5. The expression vector of 4, wherein the multiple cloning site comprises a Pmel, Notl and aBamHl restriction site.

6. The expression vector of claim 5, further comprising an S V40 promoter, a neomycin resistance gene, an SV40 early polyadenylation signal, a pUC origin site, and an ampicillin resistance gene inserted between the first and second regulatory element.

7. The expression vector of claim 1 , wherein the mammalian elongation factor- 1 sequence is chosen from among a human, a Chinese hamster, a cat or a chimpanzee elongation factor- 1 sequence.

8. The expression vector of claim 1, wherein the mammalian elongation factor- 1 sequence comprises a Chinese hamster elongation factor- 1.

9. An expression vector comprising, in the following operative order, a first regulatory element, a promoter sequence, a leader sequence, a multiple cloning site, a polyadenylation signal, a second regulatory element an S V40 promoter, a neomycin resistance gene, an S V40 early polyadenylation signal, a pUC origin site, and an ampicillin resistance gene.

10. The expression vector of claim 9, wherein the promoter sequence comprises a CMV promoter.

11. The expression vector of claim 9, wherein the leader sequence comprises a tripartite leader sequence.

12. The expression vector of claim 11 , further comprising a splicing donor site and a splicing acceptor site inserted between the tripartite leader sequence and the multiple cloning site.

13. The expression vector of 9, wherein the multiple cloning site comprises a Pmel, Notl and a BamHl restriction site.

14. The expression vector of claim 9, wherein the first and second regulatory element comprise a sequence from a mammalian elongation factor- 1.

15. The expression vector of claim 14, wherein the mammalian elongation factor- 1 sequence is chosen from among a human, a Chinese hamster, a cat or a chimpanzee elongation factor- 1 sequence.

16. The expression vector of claim 14, wherein the mammalian elongation factor- 1 sequence comprises a Chinese hamster elongation factor- 1.

17. An expression vector comprising, in the following operative order, SEQ ID NO:l and SEQ IDNO:3.

18. The expression vector of claim 17, further comprising SEQ ID NO:2 inserted between SEQ ID NO:1 and SEQ ID NO:3.

19. The expression vector of claim 18, further comprising SEQ ID NO:4 inserted adjacent SEQ ID NO:3.

20. An expression vector comprising, in the following operative order, SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4.

21. A recombinant host cell transfected with the any of the expression vectors of claims 1-20.

22. The recombinant host cells of claim 21, wherein the cell is a mammalian cell.

23. The mammalian cells of claim 22, wherein the mammalian cells are chosen from among COS cells, BHK cells, CHO cells, HeLa cells, NS-I cells, or CHO cells, COS cells, or 293 cells.

24. The recombinant host cells of claim 22, wherein the cells are CHO cells.

25. A composition comprising the recombinant host cells of claim 21 , wherein the expression vectors further comprise a coding sequence that encodes a recombinant protein.

26. The composition of claim 25, wherein the recombinant host cells comprise mammalian cells.

27. The composition of claim 26, wherein the mammalian cells are chosen from among COS cells, BHK cells, CHO cells, HeLa cells, NS-I cells, CHO cells, COS cells, or 293 cells.

28. The composition of claim 26, where in the mammalian cells are CHO cells.

29. The composition of claim 25, wherein the coding sequence encodes a therapeutic agent.

30. The composition of 29, wherein the therapeutic agent is chosen from among any one of a member of the FGF family, a member of the IGF family, a member of the EGF family, a member of the NGF family, a member of the PDGF family, a member of the TNF family, a member of the VEGF family, a member of the TGF family, a member of the interleukin family (IL-I -IL 18), epigen, amphiregulin, oncostatin M, betacellulin, epiregulin, a member of the trefoil factor family, or leukemia inhibitory factor (LIF).

31. The composition of 30, wherein the therapeutic agent further comprises a fusion molecule.

32. The composition of 31, wherein the fusion molecule comprises a polymer, a polypeptide, a succinyl group, fetuin A, fetuin B, albumin, a leucine zipper domain, a tetranectin trimerization domain, a mannose binding protein, a macrophage scavenger protein an Fc region, or an active fragment of any of these.