# Patent

## Title
Mammalian expression vector pUHAB

## Abstract

The present invention relates to the construction and utilization of a new mammalian expression vector that contains a unique multiple cloning site (MCS), designated pUHAB. The pUHAB vector comprises a high copy replication origin (ColE1), a drug resistance gene (TK-Hygromycin), and a human cytomegalovirus promoter operably associated with a unique intron (hCMV/intron). Further, pUHAB comprises a selectable marker conferring resistance to kanamycin in bacterial cells, and a phage f1(+) region. pUHAB can be used to transiently or stably express cloned genes when transfected into mammalian cells. The invention also encompasses kits and host cells and cell lines comprising pUHAB, and methods of producing a recombinant protein using pUHAB.

## Background
This application is a divisional of U.S. patent application Ser. No. 12/617,497; filed Nov. 12, 2009; which claims the benefit of U.S. provisional patent application No. 61/113,824; filed Nov. 12, 2008; each of which is herein incorporated by reference in its entirety.


FIELD OF THE INVENTION
The present invention relates to the construction and utilization of a new mammalian expression vector that contains a unique multiple cloning site (MCS), designated pUHAB. The pUHAB vector comprises a high copy replication origin (ColE1), a drug resistance gene (TK-Hygromycin), and a human cytomegalovirus promoter operably associated with a unique intron (hCMV/intron). Further, pUHAB comprises a selectable marker conferring resistance to kanamycin in bacterial cells, and a phage f1(+) region. pUHAB can be used to transiently or stably express cloned genes when transfected into mammalian cells. The invention also encompasses kits and host cells and cell lines comprising pUHAB, and methods of producing a recombinant protein using pUHAB.
BACKGROUND OF THE INVENTION
Culturing cells for the commercial production of therapeutic proteins is a costly process. The equipment required is expensive and research and development and production costs are high. Development of cell culture processes which maximize the quantity of therapeutic protein produced per liter of cell culture will minimize the resources necessary to produce a given quantity of the protein. It is, thus, desirable to use commercially viable reagents which produce large quantities of proteins.
Many naturally occurring cells do not produce large quantities of desired proteins, under standard culture conditions. Rather, extensive research and development of cell culture processes, which coax cells in culture to generate large quantities of therapeutic protein, must be performed. Typically, identifying plasmid vectors useful for expressing a protein at a high level requires a significant amount of inventive input.
SUMMARY OF THE INVENTION
The present invention provides, in part, a new expression vector. In one embodiment, the invention provides a vector characterized by a long and unique multiple cloning site. The multiple cloning site may comprise 10, 11, 12, 13, 14 or 15 restriction sites selected from the group consisting of: AflII, HpaI, AvrII, EcoRV, Acc651, KpnI, PacI, NotI, BstZ17I, SrfI, ApaI, NheI, BglII, SphI and BamHI. In one embodiment, the multiple cloning site comprises all of said restriction sites, e.g., in the order: 5′-AflII-HpaI-AvrII-EcoRV-Acc651-KpnI-PacI-NotI-BstZ17I-SrfI-ApaI-NheI-BglII-SphI-BamHI-3′. In an exemplary embodiment, the multiple cloning site comprises the nucleotide sequence set forth in SEQ ID NO: 2.
The vector may further comprise a promoter located upstream of or within the multiple cloning site. In one embodiment, the promoter is the human cytomegalovirus (hCMV) promoter. The promoter may be operably associated with an intron that enhances expression from the promoter. In one embodiment, the nucleotide sequence of the intron is comprised by the nucleotide sequence of SEQ ID NO: 1.
The vector may further comprise at least 1, 2, 3 or 4 elements selected from the group consisting of: a selectable marker for eukaryotic cells (e.g., a TK-Hygromycin gene); a prokaryotic origin of replication (e.g., a ColE1 origin of replication); a bacterial drug resistance marker (e.g., a kanamycin resistance gene); and a phage f1 region (e.g., a phage f1(+) region). In one embodiment, the vector comprises all of these elements. The vector may further comprise a terminator/polyA addition site.
The invention also provides a vector comprising an hCMV promoter operably associated with an intron that enhances expression from said promoter, wherein the nucleotide sequence of the intron is comprised by the nucleotide sequence of SEQ ID NO: 1. The vector may further comprise at least 1, 2, 3, 4 or 5 elements selected from the group consisting of: a multiple cloning site (e.g., the multiple cloning site of SEQ ID NO: 2); a selectable marker for eukaryotic cells (e.g., a TK-Hygromycin gene); a prokaryotic origin of replication (e.g., a ColE1 origin of replication); a bacterial drug resistance marker (e.g., a kanamycin resistance gene); and a phage f1 region (e.g., a phage f1(+) region). In one embodiment, the vector comprises all of these elements. The vector may further comprise a terminator/polyA addition site.
In an exemplary embodiment, the vector comprises the nucleotide sequence set forth in SEQ ID NO: 1.
The vector of the invention may be an expression vector for use in expression of a recombinant polypeptide in a mammalian host cell or organism. In this embodiment, the vector comprises a heterologous DNA sequence encoding the recombinant polypeptide, wherein the heterologous DNA sequence is operably linked to the promoter sequence.
The invention also provides a host cell comprising the vector of the invention. Also encompassed is a method for producing a recombinant polypeptide in a mammalian host cell, comprising introducing the vector of the invention into the host cell under conditions which allow for expression of the polypeptide. The recombinant polypeptide may then be purified.
The invention also provides a kit comprising the vector of the invention.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1. Nucleotide sequence of pUHAB (SEQ ID NO: 1).
FIG. 2. Plasmid map of pUHAB.










Feature Map











Element
Start
End














Beta Globin polyA signal
5
243



TK-Hygromycin
289
2298



f1(+) origin
2467
2923



KN(R) (Tn903 type I)
3523
4339



ColE1 origin
4884
5824



hCMV/intron
6084
6925



MCS
6926
5









FIG. 3. Nucleotide sequence of Vector A (SEQ ID NO: 3).
FIG. 4. Plasmid map of Vector A.













Feature Map











Element
Start
End














hCMV/intron
5
846



(complementary)





ColE1 origin
1106
2046



(complementary)





KN(R) (Tn903 type I)
2591
3407



(complementary)





f1(+) origin
4007
4463



(complementary)





TK-Hygromycin
4632
6641



Beta Globin polyA signal
6687
6925



(complementary)





IgG1 constant
6950
7930



(complementary)





VDJ (variable region)
7942
8391



(complementary)









DETAILED DESCRIPTION OF THE INVENTION
The present invention provides an expression vector useful for recombinant protein expression in any cell, for example in a mammalian cell, a bacterial cell, a yeast cell or an insect cell. The vector may be used to transiently or stably express a broad range of recombinant proteins. The multiple cloning site of the vector offers many common and rare restriction sites to accommodate a variety of expression cassettes.
The present invention includes a vector comprising or consisting of the nucleotide sequence of SEQ ID NO: 1.
Molecular Biology
In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein “Sambrook, et al., 1989”); DNA Cloning: A Practical Approach, Volumes I and II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. (1985)); Transcription And Translation (B. D. Hames & S. J. Higgins, eds. (1984)); Animal Cell Culture (R. I. Freshney, ed. (1986)); Immobilized Cells And Enzymes (IRL Press, (1986)); B. Perbal, A Practical Guide To Molecular Cloning (1984); F. M. Ausubel, et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994).
A “polynucleotide,” “nucleic acid” or “nucleic acid molecule” includes the polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; “RNA molecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; “DNA molecules”). Polynucleotides of the invention can be in any form, including circular, linear, double-stranded or single-stranded.
A “polynucleotide sequence,” “nucleic acid sequence” or “nucleotide sequence” is a series of nucleotides in a nucleic acid, such as DNA or RNA, and means any chain of two or more nucleotides.
A “coding sequence” or a sequence “encoding” an expression product, such as a RNA or peptide, is a nucleotide sequence that, when expressed, results in production of the product.
The term “gene” means a DNA sequence that codes for or corresponds to a particular sequence of ribonucleotides or amino acids which comprise all or part of one or more RNA molecules or proteins, and may or may not be operably linked to regulatory DNA sequences, such as promoter sequences, which determine, for example, the conditions under which the gene is expressed. Genes may be transcribed from DNA to RNA which may or may not be translated into an amino acid sequence.
As used herein, the term “oligonucleotide” refers to a nucleic acid, generally of no more than about 300 nucleotides (e.g., 30, 40, 50, 60, 70, 80, 90, 150, 175, 200, 250 or 300), that may be hybridizable to a genomic DNA molecule, a cDNA molecule, or an mRNA molecule encoding a gene, mRNA, cDNA, or other nucleic acid of interest. Oligonucleotides are usually single-stranded, but may be double-stranded. Oligonucleotides can be labeled, e.g., by incorporation of 32P-nucleotides, 3H-nucleotides, 14C-nucleotides, 35S-nucleotides or nucleotides to which a label, such as biotin, has been covalently conjugated. In one embodiment, a labeled oligonucleotide can be used as a probe to detect the presence of a nucleic acid. In another embodiment, oligonucleotides (one or both of which may be labeled) can be used as PCR primers, either for cloning full length or a fragment of the gene, or to detect the presence of nucleic acids. Generally, oligonucleotides are prepared synthetically, e.g., on a nucleic acid synthesizer.
A “protein sequence,” “peptide sequence” or “polypeptide sequence,” or “amino acid sequence” refers to a series of two or more amino acids in a protein, peptide or polypeptide.
“Protein,” “peptide” or “polypeptide” includes a contiguous string of two or more amino acids.
The term “isolated polynucleotide” or “isolated polypeptide” includes a polynucleotide (e.g., RNA or DNA molecule, or a mixed polymer) or a polypeptide, respectively, which is partially or fully separated from other components that are normally found in cells or in recombinant DNA expression systems or any other contaminant. These components include, but are not limited to, cell membranes, cell walls, ribosomes, polymerases, serum components and extraneous genomic sequences.
An isolated polynucleotide or polypeptide will, preferably, be an essentially homogeneous composition of molecules but may contain some heterogeneity.
As used herein, the term “functional variant” refers to a variant nucleotide or polypeptide that produces substantially the same biological effect as the original nucleotide or polypeptide.
Variants included in the invention may contain individual substitutions, deletions or additions to the original nucleic acid or polypeptide sequences. Such changes will alter, add or delete a single amino acid or a small percentage of amino acids (e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%) in the encoded sequence. Variants are referred to as “conservatively modified variants” where the alteration results in the substitution of an amino acid with a chemically similar amino acid.
“PCR amplification” of DNA as used herein includes the use of polymerase chain reaction (PCR) to increase the concentration of a particular DNA sequence within a mixture of DNA sequences. For a description of PCR see Saiki et al., Science (1988) 239:487.
The term “host cell” includes any cell of any organism that is selected, modified, transfected, transformed, grown, or used or manipulated in any way, for the production of a substance by the cell, for example the expression or replication, by the cell, of a gene, a DNA or RNA or a protein. For example, a host cell may be a mammalian cell, a bacterial cell, a yeast cell or an insect cell.
A further aspect of the present invention relates to a host cell or host cell line comprising the vector of the invention. In one embodiment, the host cell is a mammalian cell. Examples of mammalian host cells include, by way of nonlimiting example, Chinese hamster ovary (CHO) cells, CHO-K1 cells, CHO-DXB-11 cells, CHO-DG44 cells, bovine mammary epithelial cells, mouse Sertoli cells, canine kidney cells, buffalo rat liver cells, human lung cells, mouse mammary tumor cells, rat fibroblasts, bovine kidney (MDBK) cells, NSO cells, SP2 cells, TRI cells, MRC 5 cells, FS4 cells, HEK-293T cells, NIH-3T3 cells, HeLa cells, baby hamster kidney (BHK) cells, African green monkey kidney (COS) cells, human hepatocellular carcinoma (e.g., Hep G2) cells, A549 cells, etc. In one embodiment, the mammalian host cell is a human host cell. Mammalian host cells can be cultured according to methods known in the art (see, e.g., J. Immunol. Methods 56:221 (1983), Animal Cell Culture: A Practical Approach 2nd Ed., Rickwood, D. and Hames, B. D., eds. Oxford University Press, New York (1992)).
Vectors of the invention can also be introduced into a bacterial cell. In one embodiment, competent E.coli are transformed. Examples of suitable E. coli include DH1, DH5α, XL1-Blue, SURE, SCS110, OneShot Top 10, and HB101.
Vectors of the invention may be introduced into host cells according to any of the many techniques known in the art, e.g., dextran-mediated transfection, polybrene-mediated transfection, protoplast fusion, electoporation, calcium phosphate co-precipitation, lipofection, direct microinjection of the vector into nuclei, or any other means appropriate for a given host cell type.
A “cassette” or an “expression cassette” refers to a DNA coding sequence or segment of DNA that codes for an expression product (e.g., peptide or RNA) that can be inserted into a vector, e.g., at defined restriction sites. The expression cassette may comprise a promoter and/or a terminator and/or polyA signal operably linked to the DNA coding sequence.
The sequence of a nucleic acid may be determined by any method known in the art (e.g., chemical sequencing or enzymatic sequencing). “Chemical sequencing” of DNA includes methods such as that of Maxam and Gilbert (Proc. Natl. Acad. Sci. USA (1977) 74:560), in which DNA is randomly cleaved using individual base-specific reactions. “Enzymatic sequencing” of DNA includes methods such as that of Sanger (Sanger et al., Proc. Natl. Acad. Sci. USA (1977) 74:5463).
In general, a “promoter” or “promoter sequence” is a DNA regulatory region capable of binding an RNA polymerase in a cell (e.g., directly or through other promoter-bound proteins or substances) and initiating transcription of a coding sequence. A promoter sequence is, in general, bounded at its 3′ terminus by the transcription initiation site and extends upstream (5′ direction) to include the minimum number of bases or elements necessary to initiate transcription at any level. Within the promoter sequence may be found a transcription initiation site (conveniently defined, for example, by mapping with nuclease S1), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. The promoter may be operably associated with or operably linked to other expression control sequences, including enhancer and repressor sequences or with a nucleic acid to be expressed. An expression control sequence is operably associated with or operably linked to a promoter if it regulates expression from said promoter.
Promoters which may be used to control gene expression include, but are not limited to, SRα promoter (Takebe et al., Molec. and Cell. Bio. 8:466-472 (1988)), the human CMV immediate early promoter (Boshart et al., Cell 41:521-530 (1985); Foecking et al., Gene 45:101-105 (1986)), the mouse CMV immediate early promoter, the SV40 early promoter region (Benoist et al., Nature 290:304-310 (1981)), the Orgyia pseudotsugata immediate early promoter, the herpes thymidine kinase promoter (Wagner et al., Proc. Natl. Acad. Sci. USA 78:1441-1445 (1981)), the regulatory sequences of the metallothionein gene (Brinster et al., Nature 296:39-42 (1982)); prokaryotic expression vectors such as the β-lactamase promoter (Villa-Komaroff et al., Proc. Natl. Acad. Sci. USA 75:3727-3731 (1978)), or the tac promoter (DeBoer et al., Proc. Natl. Acad. Sci. USA 80:21-25 (1983)); and promoter elements from yeast or other fungi such as the GAL1, GAL4 or GAL10 promoter, the ADH (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter or the alkaline phosphatase promoter.
Viral long terminal repeat promoters such as the mouse mammary tumor virus long terminal repeat (MMTV-LTR) (Fasel et al., EMBO J. 1(1):3-7 (1982)), the moloney murine sarcoma virus long terminal repeat (Reddy et al., Proc. Natl. Acad. Sci. USA 77(9): 5234-5238 (1980)), the moloney murine leukemia virus long terminal repeat (Van Beveren et al., Proc. Natl. Acad. Sci. USA 77(6): 3307-3311 (1980)), the HIV LTR (Genbank Accession No. AB100245), the bovine foamy virus LTR (Genbank Accession No. NC—001831), RSV 5′-LTR (Genbank Accession No. K00087), the HIV-2 LTR (Genbank Accession No. NC—001722), an avian retroviral LTR (Ju et al., Cell 22: 379-386 (1980)) and the human herpes virus LTR (Genbank Accession No. NC—001806) may be included in the vectors of the present invention.
Other acceptable promoters include the human CMV promoter, the human CMV5 promoter, the murine CMV promoter, the EF1α promoter, the SV40 promoter, a hybrid CMV promoter for liver specific expression (e.g., made by conjugating CMV immediate early promoter with the transcriptional promoter elements of either human α1-antitrypsin (HAT) or albumin (HAL) promoter), or promoters for hepatoma specific expression (e.g., wherein the transcriptional promoter elements of either human albumin (HAL; about 1000 bp) or human α1-antitrypsin (HAT, about 2000 bp) are combined with a 145 bp long enhancer element of human α1-microglobulin and bikunin precursor gene (AMBP); HAL-AMBP and HAT-AMBP).
In addition, bacterial promoters, such as the T7 RNA Polymerase promoter or the tac promoter, may be used to control expression.
In one embodiment, the promoter is the human CMV (hCMV) promoter. The hCMV promoter provides a high level of expression in a variety of mammalian cell types.
A coding sequence is “under the control of”, “functionally associated with”, “operably linked to” or “operably associated with” transcriptional and translational control sequences in a cell when the sequences direct or regulate expression of the sequence. For example, a promoter operably linked to a gene will direct RNA polymerase mediated transcription of the coding sequence into RNA, preferably mRNA, which may then be spliced (if it contains introns) and, optionally, translated into a protein encoded by the coding sequence. A terminator/polyA signal operably linked to a gene terminates transcription of the gene into RNA and directs addition of a polyA signal onto the RNA.
The terms “express” and “expression” mean allowing or causing the information in a gene, RNA or DNA sequence to become manifest; for example, producing a protein by activating the cellular functions involved in transcription and translation of a corresponding gene. “Express” and “expression” include transcription of DNA to RNA and of RNA to protein. A DNA sequence is expressed in or by a cell to form an “expression product” such as an RNA (e.g., mRNA) or a protein. The expression product itself may also be said to be “expressed” by the cell.
The term “transformation” means the introduction of a nucleic acid into a cell. The introduced gene or sequence may be called a “clone”. A host cell that receives the introduced DNA or RNA has been “transformed” and is a “transformant” or a “clone.” The DNA or RNA introduced to a host cell can come from any source, including cells of the same genus or species as the host cell, or from cells of a different genus or species. Examples of transformation methods which are very well known in the art include liposome delivery, electroporation, CaPO4 transformation, DEAE-Dextran transformation, microinjection and viral infection.
The present invention includes vectors which comprise polynucleotides of the invention. The term “vector” may refer to a vehicle (e.g., a plasmid) by which a DNA or RNA sequence can be introduced into a host cell, so as to transform the host and, optionally, promote expression and/or replication of the introduced sequence.
The polynucleotides of the invention may be expressed in an expression system. The term “expression system” means a host cell and compatible vector which, under suitable conditions, can express a protein or nucleic acid which is carried by the vector and introduced to the host cell. Common expression systems include E. coli host cells and plasmid vectors, insect host cells and baculovirus vectors, and mammalian host cells and vectors such as plasmids, cosmids, BACs, YACs and viruses such as adenovirus and adenovirus associated virus (AAV).
Vectors
The invention provides a new expression vector. In one embodiment, the vector comprises a unique and long multiple cloning site that provides the vector with the flexibility to incorporate a variety of recombinant genes in complex arrangements for expression in a mammalian cell or organism. In one embodiment, the vector comprises a multiple cloning site comprising restriction sites selected from the group consisting of: AflII, HpaI, AvrII, EcoRV, Acc651, KpnI, PacI, NotI, BstZ17I, SrfI, ApaI, NheI, BglII, SphI, and BamHI. The multiple cloning site may comprise 10, 11, 12, 13, 14 or 15 of said restriction sites. The multiple cloning site may comprise, for example, the nucleotide sequence of SEQ ID NO: 2 as shown below:








(SEQ ID NO: 2)



                                         Kpnl



AflllI    Hpal       Avrll     EcoRV     Acc651   Pacl


-------  ------     ------    -------   -------   ----


CTTAAGAGTG TTAACGCGAC CTAGGTAAGA TATCCTTGGT ACCGTGTTAA


 


Pacl      NotI      BstZ171     Srfl       Apal     Nhel


----   ---------   --------   ---------   -------   -----


TTAACTGGCG GCCGCTGTGT ATACGTGGCC CGGGCTGGGG GCCCATAGCT


 


 


Nhel   Bglll     Sphl     BamHI


----  -------   -------   ------


AGCGTTAGATC TCTGGCATG CGCTGGATCC






The present invention contemplates vectors comprising the above-indicated multiple cloning site in the orientation shown or in the opposite orientation.
In one embodiment of the invention, the vector comprises a promoter which is essentially any RNA polymerase-dependent promoter, e.g., an RNA polymerase II-dependent promoter.
In certain embodiments, the vector may comprise a combination of a promoter and an intron that increases gene expression from said promoter. The intron may be synthetic or naturally-derived. In one embodiment, the intron increases gene expression from the hCMV promoter. In an exemplary embodiment, the intron is a unique intron including the nucleotide sequence:









(SEQ ID NO: 4)



CA GGTAAGTTTA AAGCTCAGGT CGAGACCGGG CCTTTGTCCG



 



GCGCTCCCTT GGAGCCTACC TAGACTCAGC CGGCTCTCCA



 



CGCTTTGCCT GACCCTGCTT GCTCAACTCT ACGTCTTTGT



 



TTCGTTTTCT GTTCCTTTCT CTCCACAGGC.






The combination of the human CMV promoter and the intron (hCMV/intron) provides higher expression levels than those achieved by using hCMV alone.
The vector may also include one or more further regulatory elements in addition to the promoter and the intron, such as enhancer elements, splicing signals, polyadenylation signals, termination signals, RNA export elements, secretion signals, internal ribosome entry sites, and the like.
In one embodiment, the vector of the invention comprises a selective marker which allows for selection of eukaryotic (e.g., mammalian) host cells into which the vector has been introduced. The selective marker may, for example, confer resistance to drugs such as G418, hygromycin or methotrexate. In one embodiment of the invention, the selective marker is a gene providing positive selection for hygromycin resistance in both prokaryotes and eukaryotes, fused to the thymidine kinase promoter (TK-Hygromycin).
In one embodiment, the vector of the invention comprises a prokaryotic antibiotic resistance marker such as the ampicillin resistance gene or the kanamycin resistance gene.
In one embodiment, the vector of the invention comprises a phage f1 region comprising the origin of replication from the f1 filamentous phage, allowing rescue of single-stranded DNA upon co-infection with helper phage. This single-stranded DNA may be used for, e.g., dideoxynucleotide sequencing or site-directed mutagenesis. In one embodiment, the phage f1 region is a phage f1(+) region.
In certain embodiments of the invention, the vector is a plasmid. The plasmid vector may comprise a prokaryotic origin of replication to allow autonomous replication within a prokaryotic host cell. In one embodiment, the prokaryotic origin of replication is the high copy ColE1 origin of replication, which allows the vector to produce the high levels of plasmid DNA required for large scale transient transfections.
In an embodiment of the invention, the vector comprises a multiple cloning site of SEQ ID NO: 2, a ColE1 high copy origin of replication, a TK-Hygromycin drug resistance gene, a human CMV promoter operably associated with an intron, a kanamycin resistance marker, and a phage f1(+) region. The invention also encompasses vectors wherein any or all of these elements are replaced by functional variants of said elements. In one embodiment, the vector is described by the plasmid map of FIG. 2.
In one embodiment of the invention, the vector is an expression vector capable of expressing a recombinant polypeptide in a host cell or organism. In an exemplary embodiment, the host cell or organism is a mammalian host cell or organism.
The expression vector comprises, in an embodiment of the invention, a terminator sequence to terminate transcription. Further, the expression vector may comprise a polyadenylation (polyA) signal for stabilization and processing of the 3′ end of an mRNA transcribed from the promoter. PolyA signals include, for example, the rabbit beta globin polyA signal or the bovine growth hormone polyA signal, as well as polyA signals of viral origin, such as the SV40 late polyA region. In one embodiment of the invention, the vector comprises a chicken beta globin terminator/polyA signal. The multiple cloning site of the vector may be located between the promoter and the polyA signal. In some embodiments, restriction sites may also be included downstream of the polyA signal.
The vector may contain more than one expression cassette to allow for expression of multiple recombinant polypeptides from a single vector. In certain embodiments, the vector comprises 1, 2, 3, 4 or 5 expression cassettes.
In an exemplary embodiment, the vector is pUHAB. The expression “pUHAB” refers to a vector comprising of the nucleotide sequence of SEQ ID NO: 1 (as shown in FIG. 1). The multiple cloning site of the pUHAB vector is flexible enough to incorporate a variety of recombinant genes in complex, multicistronic arrangements for expression in mammalian cells. Further, two expression cassettes containing the gene of interest and the TK-Hygromycin drug resistance gene can be transferred together from the vector of the invention to a different vector using either Eco571 or BssHII restriction enzymes to generate a new vector containing more than two mammalian expression cassettes.
Transfection may result in transiently transfected cell lines, in which the vector is maintained episomally and has not integrated into the genome. Transfection may also result in stably transfected cell lines, in which parts of the vector are stably integrated into the genome of the host cell, e.g., by random, non-homologous recombination events. A stable transfection may result in loss of parts of the vector sequence that are not directly related to expression of the recombinant gene product, e.g., bacterial copy number control regions. Accordingly, a stably transfected host cell is defined as a host cell that has integrated at least part or different parts of the expression vector into its genome.
Genes
Any of several genes may be inserted into the plasmids of the present invention, for example, immunoglobulins, e.g., which bind specifically to IGF1R. Plasmids of the present invention encoding any of the following target immunoglobulin amino acid sequences form part of the present invention.












19D12/15H12 Light Chain (SEQ ID NO: 5)


 








 


19D12/15H12 Heavy Chain (SEQ ID NO: 6)


 








 


19D12/15H12 Light Chain-C (LCC) (SEQ ID NO: 7)


 








 


19D12/15H12 Light Chain-D (LCD) (SEQ ID NO: 8)


 








 


19D12/15H12 Light Chain-E (LCE) (SEQ ID NO: 9)


 








 


19D12/15H12 Light Chain-F (LCF) (SEQ ID NO: 10)


 








 


19D12/15H12 heavy chain-A (HCA) (SEQ ID NO: 11)


 








 


19D12/15H12 heavy chain-B (HCB) (SEQ ID NO: 12)


 















See international application publication no. WO2003/100008 which is incorporated herein by reference in its entirety.








2C6 heavy chain



(SEQ ID NO: 13)



MELGLSWIFLLAILKGVQC



EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSKGYVDSVKGRFTIS


RDNAKNSLYLQMNSLRAEDTALYYCAKDIRIGVAASYYFGMDVWGHGTTVTVSS


 










2C6 CDR-H1:
GFTFDDYAMH
(SEQ ID NO: 14)



2C6 CDR-H2:
GISWNSGSKGYVDSVKG
(SEQ ID NO: 15)


2C6 CDR-H3:
DIRIGVAASYYFGMDV
(SEQ ID NO: 16)


 








2C6 Light chain



(SEQ ID NO: 17)



MDMRVPAQLLGLLLLWLPGARC



AIQLTQSPSSLSASVGDRVTITCRASQGISSVLAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDF


TLTISSLQPEDFATYYCQQFNSYPYTFGQGTKLEIK


 










2C6 CDR-L1:
RASQGISSVLA
(SEQ ID NO: 18)



2C6 CDR-L2:
DASSLES
(SEQ ID NO: 19)


2C6 CDR-L3:
QQFNSYPYT
(SEQ ID NO: 20)


 








9H2 Heavy chain



(SEQ ID NO: 21)



MDWTWRILFLVAAATGAHS



QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYVMHWVRQAPGQRLEWMGWINAGNGNTKYSQKFQGRVTIT


RDTSASTVYMELSSLRSEDTAVYYCARGGMPVAGPGYFYYYGMDVWGQGTTVTVSS


 










9H2 CDR-H1:
GYTFTSYVMH
(SEQ ID NO: 22)



9H2 CDR-H2:
WINAGNGNTKYSQKFQG
(SEQ ID NO: 23)


9H2 CDR-H3:
GGMPVAGPGYFYYYGMDV
(SEQ ID NO: 24)


 








9H2 Light chain



(SEQ ID NO: 25)



METPAQLLFLLLLWLPDTTG



EIVLTQSPGTLSLSPGERATLSCRASQSVSRSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTD


FTLTISRLEPEDFAVYCCQQYGSSPWTFGQGTKVEIKRT


 










PH2 CDR-L1:
RASQSVSRSYLA
(SEQ ID NO: 26)



9H2 CDR-L2:
GASSRAT
(SEQ ID NO: 27)


9H2 CDR-L3:
QQYGSSPWT
(SEQ ID NO: 28)


 








Heavy chain immunoglobulin variable region # 1.0 sequence



(SEQ ID NO: 29)



E VQLLESGGGL VQPGGSLRLS CTASGFTFSS YAMNWVRQAP GKGLEWVSAI



SGSGGTTFYA DSVKGRFTIS RDNSRTTLYL QMNSLRAEDT AVYYCAKDLG


WSDSYYYYYG MDVWGQGTTV TVSS;


 


Light chain immunoglobulin variable region # 1.0 sequence


(SEQ ID NO: 30)



DIQMTQFP SSLSASVGDR VTITCRASQG IRNDLGWYQQ KPGKAPKRLI



YAASRLHRGV PSRFSGSGSG TEFTLTISSL PQEDFATYYC LQHNSYPCSF


GQGTKLEIKR;






Embodiments of the invention include those wherein the plasmid includes more than one immunoglobulin, for example, a combination of any of those set forth herein (e.g., heavy chain Ig. #1.0 and light chain Ig. #1.0; or LCC and HCA; or LCF and HCA; or LCC and HCB).
Protein Expression and Purification
A further aspect of the present invention relates to a method for the production of a recombinant protein (e.g., anti-TGFβ, anti-IGF1R, anti-IL-23, anti-EGFR, anti-IL-17, anti-PD1, anti-HGF), comprising the steps of: a) transfecting a host cell or host cell line (e.g., a mammalian host cell or host cell line) with an expression vector according to the invention; b) culturing the cell under appropriate conditions to enable growth and/or propagation of the cell and expression/production of the recombinant protein; and, optionally c) harvesting the recombinant protein produced. Methods for harvesting (isolating and/or purifying) a given protein from, e.g., a cell, a cell culture or the medium in which cells have been cultured are well known in the art. By way of nonlimiting example, proteins can be isolated and/or purified from biological material by salt or alcohol precipitation (e.g., ammonium sulfate precipitation or ethanol precipitation), affinity chromatography (e.g., used in conjunction with a purification tag); fractionation on immunoaffinity or ion-exchange columns; high pressure liquid chromatography (HPLC); reversed-phase HPLC; chromatography on silica or on a cation-exchange resin such as DEAE; chromatofocusing;); isoelectric focusing; countercurrent distribution; SDS-PAGE; gel filtration (using, e.g., Sephadex G-75); and protein A Sepharose columns to remove contaminants such as IgG. Such purification methods are well known in the art and are disclosed, e.g., in “Guide to Protein Purification”, Methods in Enzymology, Vol. 182, M. Deutscher, Ed., 1990, Academic Press, New York, N.Y.
Growth of mammalian cells in liquid aqueous culture is well known in the art. Examples of mammalian cell culture growth media which are known in the art include EX-CELL ACF, CHO medium (Sigma-Aldrich (St. Louis, Mo.); discussed further below), DMEM, DMEM/F-12, F-10 Nutrient Mixture, RPMI Medium 1640, F-12 Nutrient Mixture, Medium 199, Eagle's MEM, RPMI, 293 media, and Iscove's Media.
Cell growth can be performed in any of several systems. For example, cell growth can be done in a simple flask, e.g., a glass shake flask. Other systems include tank bioreactors, bag bioreactors and disposable bioreactors. A tank bioreactor includes, typically, a metal vessel (e.g., a stainless steel jacketed vessel) in which cells are growth in a liquid medium. Tank bioreactors can be used for a wide range of culture volumes (e.g., 100 l, 150 l, 10000 l, 15000 l). Tank bioreactors often have additional features for controlling cell growth conditions, including means for temperature control, medium agitation, controlling sparge gas concentrations, controlling pH, controlling O2 concentration, removing samples from the medium, reactor weight indication and control, cleaning hardware, sterilizing the hardware, piping or tubing to deliver all services, adding media, control pH, control solutions, and control gases, pumping sterile fluids into the growth vessel and, supervisory control and a data acquisition. Classifications of tank bioreactor include stirred tank reactors wherein mechanical stirrers (e.g., impellers) are used to mix the reactor to distribute heat and materials (such as oxygen and substrates). Bubble column reactors are tall reactors which use air alone to mix the contents. Air lift reactors are similar to bubble column reactors, but differ by the fact that they contain a draft tube. The draft tube is typically an inner tube which improves circulation and oxygen transfer and equalizes shear forces in the reactor. In fluidized bed reactors, cells are “immobilized” on small particles which move with the fluid. The small particles create a large surface area for cells to stick to and enable a high rate of transfer of oxygen and nutrients to the cells. In packed bed reactors cells are immobilized on large particles. These particles do not move with the liquid. Packed bed reactors are simple to construct and operate but can suffer from blockages and from poor oxygen transfer. A disposable bioreactor is a disposable, one-time use bioreactor. Often, disposable bioreactors possess features similar to non-disposable bioreactors (e.g., agitation system, sparge, probes, ports, etc.).
Particularly where a polypeptide is isolated from a cellular or tissue source, it is preferable to include one or more inhibitors of proteolytic enzymes in the assay system, such as phenylmethanesulfonyl fluoride (PMSF), Pefabloc SC, pepstatin, leupeptin, chymostatin and EDTA.
In some embodiments, the protein of interest is with a second polypeptide or polynucleotide moiety, which may be referred to as a “tag” or “marker”. A tag may be used, for example, to facilitate purification or detection of the polypeptide after expression. A fused polypeptide may be constructed, for example, by in-frame insertion of a polynucleotide encoding the tag on the 5′ or 3′ end of the polynucleotide encoding the polypeptide to be expressed. The fused polynucleotide may then be expressed in the expression system of the invention. Such tags include glutathione-S-transferase (GST), hexahistidine (His6) tags, maltose binding protein (MBP) tags, haemagglutinin (HA) tags, cellulose binding protein (CBP) tags and myc tags. Detectable tags such as 32P, 35S, 3H, 99mTc, 123I, 111In, 68Ga, 18F, 125I, 131I, 113mIn, 76Br, 67Ga, 99mTc, 123I, 111In and 68Ga may also be used to label the polypeptides and polynucleotides of the invention. Methods for constructing and using such fusions are very conventional and well known in the art.
One skilled in the art appreciates that purification methods suitable for the polypeptide of interest may require modification to account for changes in the character of the polypeptide upon expression in recombinant cell culture.
Kits
The vectors of the invention may be provided in a kit. The kits of the invention may include, in addition to one or more vectors, any reagent which may be employed in the use of the vector. In one embodiment, the kit includes reagents necessary for transformation of the vectors into mammalian cells. For example, the kit may include reagents for a calcium phosphate transformation procedure: calcium chloride, buffer (e.g., 2× HEPES buffered saline), and sterile, distilled water. In another embodiment, the kit includes reagents for a DEAE-Dextran transformation: Chloroquine in PBS, DEAE-dextran in PBS and Phosphate buffered saline. In yet another embodiment, reagents for a liposome transformation are included in the kit: Liposomes extruded from DOTAP/cholesterol extruded liposomes. For example, the kit may include the cationic lipid-based transfection reagent LipofectamineTM (Invitrogen Life Technologies; Carlsbad, Calif.).
The kit may include reagents required for bacterial transformation of the vectors of the invention. For example, the kit may include transformation competent bacteria (e.g., DH1, DH5, DH5α, XL1-Blue, SURE, SCS110, OneShot Top 10, or HB101).
The kit may include growth media or reagents required for making growth media. For example, in one embodiment, the kit can include fetal calf serum or DMEM (Dulbecco/Vogt modified Eagle's (Harry Eagle) minimal essential medium) for growth of mammalian cells. In another embodiment, the kit can contain powdered Luria broth media or Luria broth plates containing an appropriate antibiotic (e.g., ampicillin or kanamycin) for growing bacteria.
Components supplied in the kit may be provided in appropriate vials or containers (e.g., plastic or glass vials). The kit can include appropriate label directions for storage, and appropriate instructions for usage.
EXAMPLE
The following example is provided to further describe the present invention and should not be construed as a limitation thereof. The scope of the present invention includes any and all of the methods which are set forth below in the following example.
Example 1
Construction of pUHAB
The multiple cloning site of pUHAB, as shown in SEQ ID NO: 2, was synthesized using PCR. The PCR product and the vector of SEQ ID NO: 3 (Vector A; see FIG. 3) were digested by the restriction enzymes AflII and BamHI. The digested PCR product was then ligated to the digested Vector A to form the pUHAB vector (SEQ ID NO: 1). pUHAB was transformed into cells, which were positively selected for presence of the vector. Finally, the integrity of the pUHAB multiple cloning site was confirmed by sequencing.
The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.
Patents, patent applications, publications, product descriptions, and protocols are cited throughout this application, the disclosures of which are incorporated herein by reference in their entireties for all purposes.

## Claims

1. An isolated vector comprising a multiple cloning site comprising the nucleotide sequence set forth in SEQ ID NO: 2.

2. The vector of claim 1, comprising a promoter located upstream of or within the multiple cloning site.

3. The vector of claim 2, wherein the promoter is human cytomegalovirus (hCMV) promoter.

4. The vector of claim 2, wherein said promoter is operably associated with an intron that enhances expression from said promoter.

5. The vector of claim 4, wherein the nucleotide sequence of said intron comprises the nucleotide sequence of SEQ ID NO: 4.

6. The vector of claim 1, further comprising at least three elements selected from the group consisting of:
(a) a selectable marker for eukaryotic cells;
(b) a prokaryotic origin of replication;
(c) a bacterial drug resistance marker; and
(d) a phage f1(+) region.

7. The vector of claim 6, wherein said vector comprises all of said elements.

8. The vector of claim 6, wherein said selectable marker for eukaryotic cells is a hygromycin (TK-Hygromycin) drug resistance gene operably linked to a thymidine kinase promoter.

9. The vector of claim 6, wherein said prokaryotic origin of replication is the ColE1 origin of replication.

10. The vector of claim 6, wherein said bacterial drug resistance marker is a kanamycin resistance gene.

11. The vector of claim 1, comprising a terminator/polyA addition site.

12. The vector of claim 1, comprising the nucleotide sequence set forth in SEQ ID NO: 1.

13. The vector of claim 1, comprising a heterologous DNA sequence encoding a polypeptide, which DNA sequence is operably linked to a promoter and an intron.

14. The vector of claim 13 wherein the polypeptide is an antibody immunoglobulin chain.

15. The vector of claim 14 wherein the antibody immunoglobulin chain is from an antibody that binds specifically to IGFIR.

16. The vector of claim 15 wherein the immunoglobulin chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5-30.

17. An isolated plasmid vector by having the plasmid map of FIG. 2.

18. An isolated host cell comprising the vector of claim 1.

19. A host cell line comprising the host cell of claim 18.

20. A kit comprising the vector of claim 1 and instructions for use of the vector.