JP2012506694A - Expression vector and method thereof - Google Patents

Abstract

The present invention relates to vectors and compounds for the expression of recombinant soluble proteins. In particular, the invention relates to nucleic acid molecules, expression vectors, and host cells for the expression of recombinant soluble tumor necrosis factor alpha receptor (TNFR) -human IgG Fc fusion protein. The invention further relates to a method for preparing recombinant soluble tumor necrosis factor alpha receptor (TNFR) -human IgG Fc fusion protein using host cells transfected with this expression vector.
[Selection figure] None

Description

  The present invention relates to vectors and compounds for the expression of recombinant soluble proteins. In particular, the invention relates to nucleic acid molecules, expression vectors, and host cells for the expression of recombinant soluble tumor necrosis factor alpha receptor (TNFR) -human IgG Fc fusion protein. The invention further relates to a method for preparing recombinant soluble tumor necrosis factor alpha receptor (TNFR) -human IgG Fc fusion protein using host cells transfected with this expression vector.

  In 1986, the US FDA approved the use of human tissue plasminogen activator (tPA; Genentech, CA, USA) protein from mammalian cells for therapeutic purposes. Advances in cell culture and recombinant DNA technology have facilitated the expression of various similar proteins of therapeutic or other economic value using genetically modified cells. Currently, there are several monoclonal antibodies that have been approved for therapeutic use by regulatory authorities, and hundreds are in various development and approval stages. Similar to tPA, most of these proteins are expressed in immortalized Chinese hamster ovary (CHO) cells, such as mouse myeloma (NS0), baby hamster kidney (BHK), human fetal kidney (HEK-293), etc. Other cell lines have also been approved for recombinant protein production.

  The expression of many bioactive therapeutics obtained from higher eukaryotic sources often requires post-translational modifications that do not occur naturally in lower eukaryotic or prokaryotic cells. It is forced to use cells obtained from higher eukaryotic sources. Thus, mammalian expression systems are usually preferred for the production of most therapeutic proteins since the required post-translational modifications are equally successful in cell lines. Currently, various mammalian cell expression systems are available for protein expression. Expression vectors typically use strong viral or cellular promoter / enhancers to facilitate expression of the recombinant gene. However, the expression levels of recombinant proteins obtained from these expression vectors / expression systems in mammalian cells are not commercially feasible. There are two important issues that need to be addressed during the production of therapeutics: (a) the time it takes to provide this substance, and (b) especially in developing or developing countries. To reduce the price of this material so that it can be used by the masses. Therefore, the biotechnology industry continues to explore new technology and process development strategies that reduce schedules and costs.

  ENBREL (etanercept), also known as a TNFR: Fc fusion protein, is a recombinant fusion protein comprising the extracellular domain of the human tumor necrosis factor receptor superfamily, member 1B (p75) and the Fc domain of human IgG1. ENBREL is a significant scientific advancement for the treatment of rheumatoid arthritis, which in many patients is a sign of rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, and psoriasis It has been shown to reduce symptoms. Tumor necrosis factor (TNF) is a natural cytokine involved in normal inflammatory and immune responses. TNF plays an important role in the inflammatory process of rheumatoid arthritis (RA), polyarticular juvenile rheumatoid arthritis (JRA), and the resulting joint lesions. Elevated TNF levels are seen in RA patient synovial fluid.

  Production of recombinant proteins such as TNFR: Fc in mammalian cells can be difficult because of the low genetic stability of the recombinant gene and / or because the recombinant gene is silenced. Several molecular mechanisms that can cause gene silencing have been reported, including DNA methylation, negative position effects, integration-dependent repression, and so on, to name just one example.

  Thus, there is a need in the art to overcome the deficiencies of known production methods for such proteins by providing an expression system with greater genetic stability during large-scale production of recombinant fusion proteins. is there. In order to facilitate the production of large amounts of TNFR: Fc from cell culture, a novel expression vector was developed. The use of this expression vector has been shown to increase the expression of therapeutic proteins. The cloning, expression and purification of TNFR: Fc is described in this application.

  This disclosure includes one or more of the following features, which may include patentable subject matter, alone or in any combination, as set forth in the appended claims.

  The present invention provides a novel scaffold / matrix binding region (S / MAR) sequence that can be used to increase and stabilize the expression yield of recombinant proteins in mammalian cells and other eukaryotic cells. Based on discovery. S / MAR sequences increase the genetic stability of nearby transcription cassettes and inhibit gene silencing by interfering with mechanisms such as DNA methylation. Furthermore, the presence of the S / MAR sequence is thought to reduce variability between clones by reducing the position effect.

  Accordingly, in one embodiment, the present invention provides SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, variants and functional fragments thereof, and at least 70% thereof. It relates to an isolated nucleic acid having one or more nucleotide sequences selected from the group consisting of homologous sequences or sequences that hybridize under stringent conditions to the isolated nucleic acid.

  In another embodiment, the invention relates to an expression vector carrying a scaffold / matrix binding region (S / MAR) sequence and any combination thereof. S / MAR sequences are SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, their complements, variants, and functional fragments, and BLAST (basic local alignment search). Tools) may be selected from the group consisting of sequences that are at least 70% homologous to them as determined by pairwise DNA sequence alignment using matching methods such as algorithms.

  In another embodiment, the present invention provides a tumor necrosis factor alpha receptor (operatingly linked to a scaffold / matrix binding region (S / MAR) sequence or any combination thereof and one or more expression control elements ( (TNFR) -expression vectors carrying sequences encoding IgG Fc fusion proteins.

  The S / MAR sequence may be upstream or downstream of the transcription promoter in the expression vector. Furthermore, the S / MAR sequence may be 0-10 kb away from the sequence encoding the tumor necrosis factor alpha receptor (TNFR) -human IgG Fc fusion protein.

  In a further embodiment, the present invention relates to a method for constructing an expression vector carrying a scaffold / matrix binding region (S / MAR) sequence or any combination thereof.

  In another embodiment, the invention relates to a host cell comprising an expression vector carrying a scaffold / matrix binding region (S / MAR) sequence or any combination thereof.

  In yet another embodiment, the present invention provides tumor necrosis factor alpha receptor (TNFR) -IgG by recombinant expression using an expression vector carrying a scaffold / matrix binding region (S / MAR) sequence or any combination thereof. It relates to a method for producing an Fc fusion protein.

  In yet a further embodiment, the invention relates to a tumor necrosis factor alpha receptor (TNFR) -IgG Fc fusion protein expressed by an expression vector carrying a scaffold / matrix binding region (S / MAR) sequence or any combination thereof. .

  In yet another embodiment, the present invention transfects mammalian cells with an expression construct carrying a gene encoding a tumor necrosis factor alpha receptor (TNFR) -IgG Fc fusion protein to produce a scaffold / matrix binding region (S / MAR) relates to a method for producing a tumor necrosis factor alpha receptor (TNFR) -IgG Fc fusion protein by co-transfecting the same mammalian cells with a plasmid carrying the sequence or any combination thereof.

  In yet a further embodiment, the invention relates to epigenetic and genetic factors that affect the biological activity of the scaffold / matrix binding region (S / MAR) sequence.

The invention will be further described with reference to the following drawings.
The construction of the plasmid vector pCDNA3.1 / TNFR: Fc is shown. The construction of the plasmid vector pCDNA3.1 / MAR1 / TNFR: Fc is shown. The construction of the plasmid vector pCDNA3.1 / MAR2 / TNFR: Fc is shown. The construction of the plasmid vector pCDNA3.1 / MAR3 / TNFR: Fc is shown. The construction of the plasmid vector pCDNA3.1 / MAR4 / TNFR: Fc is shown. The construction of the plasmid vector pCDNA3.1 / MAR5 / TNFR: Fc is shown. The construction of the plasmid vector pCDNA3.1 / MAR6 / TNFR: Fc is shown.

  As used herein, the term “scaffold / matrix binding region (S / MAR)” refers to about 60, 60 of the eukaryotic genomic nuclear DNA by periodic binding to the protein scaffold or matrix of the cell nucleus. Refers to non-consensus-like AT-rich DNA elements of several hundred base pairs (bp) in length, organized into 000 chromatin domains. They are usually found in non-coding regions such as flanking regions, chromatin border regions, and introns.

  By “at least 70% homology”, a DNA whose nucleotide sequence is at least 70% homologous to a defined sequence measured by pair-wise DNA sequence alignment using a matching method such as the BLAST (Basic Local Alignment Search Tool) algorithm. means.

  As used herein, the term “functional fragment of SEQ ID NOs: 1, 2, 3, 4, 5, and 6” means a fragment of the sequence that is large enough to have the desired effect on expression yield.

  As used herein, the term “flanking” means that the sequence in question is directly connected to an expression vector, or such a linking sequence does not interfere with the desired effect of these sequences. As long as it is either connected by a ligated DNA sequence that can be up to 10 kb in length or longer. The expression vector includes at least the gene of interest and an expression control element operably linked thereto.

  Expression control elements include conventional regulatory elements such as transcription promoters, transcription enhancers, transcription repressors, RNA polymerase binding sites, polyadenylation sites, translation initiation signals, and translation termination signals, which are easily accomplished by those skilled in the art. Can be done.

The term “complement” refers to a nucleic acid sequence that has a complementary nucleotide sequence and reverse orientation when compared to a reference nucleotide sequence. For example, the sequence 5'ATGCACGGGG 3 'is complementary to 5'CCCGTGCAT 3'.

  As used herein, the term “variant” refers to a specifically identified sequence in which one or more nucleotides or amino acid residues are deleted, substituted, or added. Refers to a different polynucleotide or polypeptide sequence. The mutant may be a naturally occurring allelic mutant or a non-naturally occurring mutant. Variants may be from the same species or from other species and may include homologs, paralogs, and orthologs. In certain embodiments, the variant has a biological activity that is equivalent to or similar to the biological activity of the sequence in question.

  A variant polynucleotide sequence is preferably at least 50%, more preferably at least 51%, more preferably at least 52%, more preferably at least 53%, more preferably at least 54%, more preferably with a sequence of the invention. Is at least 55%, more preferably at least 56%, more preferably at least 57%, more preferably at least 58%, more preferably at least 59%, more preferably at least 60%, more preferably at least 61%, more preferably At least 62%, more preferably at least 63%, more preferably at least 64%, more preferably at least 65%, more preferably at least 66%, more preferably at least 67%, more preferably at least 68%, more preferably At least 69%, more preferably at least 70%, more preferably at least 71%, more preferably at least 72%, more preferably at least 73%, more preferably at least 74%, more preferably at least 75%, more preferably At least 76%, more preferably at least 77%, more preferably at least 78%, more preferably at least 79%, more preferably at least 80%, more preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferred Or at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably Exhibits at least 97%, more preferably at least 98%, and most preferably at least 99% identity.

  The term “sequence motif” as used herein refers to a specific nucleotide sequence of at least 2 nucleotides contained within a larger oligonucleotide sequence. A sequence motif can appear once in an oligonucleotide sequence or it can appear multiple times. For example, the oligonucleotide 5′-AUCAUCAUG-3 ′ has the sequence motif 5′-AU-3 ′ that appears three times and the sequence motifs 5′-UC-3 ′ and 5′-CA-3 ′ that appear twice. And a sequence motif 5′-UG-3 ′ that appears once.

  As used herein, the term “epigenetic factor” refers to any external process or factor that manipulates the expression of a gene or set of genes, such as proteins, nucleic acids, and the like. Contrast with “genetic factor” which refers to any internal process or intrinsic factor involving an intrinsic factor.

  As used herein, the term “TNFR: Fc receptor protein” or “TNFR: Fc” is a protein having an amino acid sequence similar to the extracellular domain of human TNFRII (p75) protein, Refers to a protein that is capable of binding to TNF-alpha, a key ligand, thereby inhibiting TNF-alpha from binding to cell membrane bound TNFRI or TNFRII. Of the two different forms of TNFR known to exist, the preferred TNFR of the present invention is TNFRII (p75). Soluble TNFR constructs lack a transmembrane region to facilitate extracellular secretion. The soluble portion of TNFRII, the extracellular domain, is fused in-frame to the Fc region of human IgG1. The fusion protein of the present invention is biologically active, i.e. it can bind to TNF in solution.

  As used herein, the term “biomolecule” refers to a substance, compound, or component associated with a biological environment, including sugars, amino acids, peptides, proteins, oligonucleotides, polynucleotides, polypeptides, organics. Examples include, but are not limited to, molecules, haptens, epitopes, living cells, portions of living cells, vitamins, hormones, and the like.

  The term “expression system” refers to any in vivo or in vitro biological system used to produce one or more proteins encoded by a polynucleotide.

  As used herein, the term “recombinant” means that the protein is derived from a recombinant expression system, which is herein an expression system based on mammalian cells. .

  As used herein, the term “isolated DNA sequence” refers to a DNA polymer in the form of an independent fragment or as part of a larger DNA construct. Such a sequence could be cloned into an expression vector and the sequence isolated in large quantities for identification, manipulation and recovery of DNA fragments. Such sequences are provided in an open reading frame without any interruption by untranslated DNA regions or by introns.

  As used herein, the term “nucleotide sequence” refers to a heteropolymer of deoxyribonucleotides. To provide a synthetic gene capable of being expressed in a recombinant transcription unit by combining a DNA sequence encoding a protein provided by the present invention from a cDNA fragment and a short oligonucleotide linker or from a series of oligonucleotides. Can do.

  A “chromosome” is an organized structure of DNA and protein found inside a cell.

  “Chromatin” is a complex of DNA and protein found in the nucleus of a eukaryotic cell, and thereby constitutes a chromosome.

  “DNA” or “deoxyribonucleic acid” contains genetic information. It is composed of various nucleotides A, G, T, or C.

  As used herein, a “gene” refers to a deoxyribonucleotide (DNA) sequence that encodes a given mature protein. It does not include untranslated flanking regions such as RNA transcription initiation signals, polyadenylation addition sites, promoters, or enhancers.

  As used herein, the term “transcription promoter” refers to a nucleic acid sequence that controls the expression of a coding sequence or functional RNA. A promoter may be derived from a native gene or may be composed of various elements derived from various promoters found in nature. A promoter may be any nucleic acid sequence that exhibits transcriptional activity in a host cell, and may be derived from a gene that encodes either a homologous or heterologous protein for the host cell. Examples of suitable promoters are the SV40 promoter, the MT-1 (metallothionein gene) promoter, the human cytomegalovirus immediate early promoter, and the like.

  A “transcription enhancer” refers to a sequence of a gene that serves to initiate transcription of a gene, regardless of the location or orientation of the gene.

  A “transcriptional repressor” refers to a sequence of a gene that acts to inhibit transcription of the gene, regardless of the location or orientation of the gene.

  The term “signal peptide” refers to an amino terminal polypeptide preceding the secreted mature protein. Since the signal peptide is cleaved, it is not present in the mature protein.

  As used herein, the term “protein” refers to the carboxyl carbon atom of a carboxylic acid group attached to the α-carbon of an amino acid (or amino acid residue) attached to the α-carbon of an adjacent amino acid. Any polymer of two or more individual amino acids linked via peptide bonds (whether naturally occurring or not), such as occurs when it becomes covalently bonded to the amino nitrogen atom of the amino group Not). These peptide bond linkages, as well as the atoms that make them (ie, α-carbon and carboxyl carbon atoms (and their substituted oxygen atoms) and amino nitrogen atoms (and their substituted hydrogen atoms)) are attached to the “polypeptide backbone of the protein. ”. Further, as used herein, the term “protein” is understood to include the terms “polypeptide” and “peptide”, which can sometimes be used interchangeably herein. The

  As used herein, the term “vector” refers to a nucleic acid molecule capable of carrying another nucleic acid linked thereto. Vectors are usually derived from plasmids, which function like “molecular carriers” that carry DNA fragments into host cells.

  The vector can be any vector that can be conveniently subjected to recombinant DNA procedures, and the choice of vector often depends on the host cell into which it is to be introduced. Thus, the vector may be an autonomously replicating vector, ie a vector that exists as an extrachromosomal entity, for example a plasmid, whose replication is independent of chromosomal replication. Alternatively, a vector may be one that, when introduced into a host cell, is integrated into the host cell genome and replicated along with the chromosome into which it has been integrated. The vector is preferably an expression vector in which the coding DNA sequence is operably linked to additional portions required for transcription of the DNA. In general, expression vectors are derived from plasmid or viral DNA, or may contain elements of both. The term “operably linked” means that these portions are for their intended purpose, eg, transcription is initiated in a promoter and proceeds in a DNA sequence encoding a polypeptide. , Indicate that they are arranged to work in concert.

  As used herein, the term “plasmid” refers to a small circular double stranded polynucleotide structure of DNA found in bacteria and some other organisms. The plasmid can replicate independently of the host cell chromosome.

  The term “replication” refers to the synthesis of DNA from its template DNA strand.

  The term “transcription” refers to the synthesis of RNA from a DNA template.

  The term “translation” refers to the synthesis of a polypeptide from messenger RNA.

  The term “cis” refers to the placement of two or more linked DNA elements on the same plasmid.

  The term “trans” refers to the arrangement of two or more elements on two or more different plasmids.

  The term “directional” refers to the order of nucleotides in a DNA sequence.

  As used herein, the term “isolated nucleic acid fragment” refers to a polymer of DNA or RNA that is single-stranded or double-stranded. An isolated nucleic acid fragment in the form of a polymer of DNA may be composed of one or more portions of cDNA, genomic DNA, or synthetic DNA.

  As used herein, the term “gene amplification” refers to selective and repeated replication of a particular gene or genes without proportionally increasing the copy number of other genes. Gene amplification is an important developmental and evolutionary process that has become widespread in many organisms. Gene amplification is naturally occurring, such as (i) developmentally regulated gene expression as seen in Xenopus oocytes and (ii) amplification of the lac region reported in Escherichia coli. It can be divided into two categories of gene expression that occur. The best known gene amplification in mammalian cells is dihydrofolate reductase (DHFR).

  The term “transformation” refers to the transfer of a nucleic acid fragment into the genome of a host organism, resulting in a genetically stable inheritance. A host organism that contains a transformed nucleic acid fragment is referred to as a “transformed” organism.

  The term “eukaryotic cell” refers to any cell derived from a eukaryote whose cells are organized into a complex structure by the inner membrane or cytoskeleton. Any eukaryotic cell that can be used for gene / protein manipulation and that can be maintained under cell culture conditions and later transfected is included in the present invention. Particularly preferred cell types include stem cells, embryonic stem cells, Chinese hamster ovary cells (CHO), COS, BHK21, NIH3T3, HeLa, C2C12, HEK, MDCK, cancer cells, and primary or primary undifferentiated cells. . Mammalian cells can include CHO cells, HeLa cells, baby hamster kidney (BHK) cells, COS cells, HEK293 cells, or other immortalized cell lines.

  As used herein, the term “transfection” refers to the introduction of foreign substances, such as DNA, into eukaryotic cells by any means of transfer. Various transfection methods include, but are not limited to, calcium phosphate transfection, electroporation, lipofectamine transfection, and DEAE-dextran transfection.

  The term “transfected cell” refers to a eukaryotic cell into which exogenous DNA has been introduced into the eukaryotic cell. This DNA can be part of the host chromosome or can replicate as an extrachromosomal element.

  The term “cotransfection” refers to a method of co-transfecting a eukaryotic cell with two or more exogenous genes that are heterologous to the cell.

  The term “transient gene expression” refers to a convenient method for the rapid production of small amounts of protein. COS cells are usually used for characterization of transient expression.

  The term “stable gene expression” refers to the preparation of a stable cell line that permanently expresses the gene of interest by stable integration of the plasmid into the host chromosome.

  Tumor necrosis factor alpha receptor (TNFR) -a novel eukaryotic that contains DNA encoding human IgG Fc fusion protein activity and promotes expression of TNFR: Fc activity when transfected into an appropriate cell line Biological expression vectors have been constructed. This novel expression vector can be used to produce soluble TNFR: Fc. Recombinantly produced TNFR: Fc activity is useful in the treatment and prevention of various disorders, including rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, and psoriasis .

  The present invention relates to a novel eukaryotic expression vector used to produce soluble TNFR: Fc in increased amounts.

  Prokaryotic expression systems were part of the early repertoire of research tools in molecular biology. De novo synthesis of recombinant eukaryotic proteins in prokaryotic systems has caused several problems for eukaryotic gene products. The two most important of them were inappropriate protein folding and assembly and post-translational modifications, mainly lack of glycosylation and phosphorylation. Prokaryotic systems do not have all the appropriate protein synthesis mechanisms to produce eukaryotic structural and / or catalytic function proteins. Therefore, mammalian expression systems are usually preferred for the production of therapeutic proteins, simply because the required post-translational modifications are addressed in situ by the cell line. Currently, various mammalian expression systems are available for either transient or stable expression of recombinant genes. Usually, a Chinese hamster ovary (CHO) cell stable expression system (CHO SES) is used. Furthermore, baby hamster kidney (BHK) cells, human fetal kidney (HEK) 293 cells, mouse L-cells, and myeloma cell lines such as J558L and Sp2 / 0 are also used for the establishment of stable transfectants. It is used as a host.

  However, the integration of foreign DNA into the host cell genome is an unpredictable process. It has been well documented that transgene expression varies greatly from cell line to cell line and that integration can cause unexpected changes in phenotype. Reasons behind the large variation in clonal expression levels include the difference in plasmid copy number and the phenomenon known as the position effect initially described as a spotted position effect in Drosophila melanogaster. The location of integration can influence transgene expression by at least three mechanisms: local regulatory element activity, local chromatin structure, and local DNA methylation status. Two general approaches can be used to protect DNA from negative position effects or integration-dependent suppression. One approach involves incorporating the transgene directly into a predetermined site that is transcriptionally active using site-specific recombination methods. Another method is to incorporate DNA sequence elements found in the chromatin border region into the expression vector so that the gene is protected from the effects of surrounding chromatin regardless of the integration site. For recombinant protein expression, sequences that act as chromatin boundaries and protect the transfected gene from the effects of surrounding chromatin include insulator sequences and scaffold / matrix binding regions (S / MAR).

  S / MAR is a DNA sequence that binds with high affinity in vitro to an isolated nuclear scaffold or nuclear matrix. Expression studies suggest that placing insulators on both sides of the transgene reduces the positional effect and, therefore, suppresses clonal expression fluctuations. S / MAR is a relatively short (100-1000 bp long) sequence that connects the chromatin loop to the nuclear matrix. S / MAR has been observed to be on either side of the end of the domain encompassing the various transcription units. It has also been shown that S / MAR brings together transcriptionally active chromatin regions so that transcription is initiated in a chromosomal region occupying the same position as the surface of the nuclear matrix.

  Similarly, S / MAR can define the boundaries of independent chromatin domains so that only the surrounding cis-regulatory elements control the expression of genes within the domain. Several possible functions have been previously discussed for S / MAR, including the boundaries of chromatin domains, altering chromatin conformation, participating in the initiation of DNA replication, and chromosomes Organizing the chromatin structure. S / MAR is often found in centromere-related DNA and telomere sequences, and appears to be important for chromosome assembly during mitosis and maintenance of chromosome shape during metaphase. Thus, S / MAR is involved in multiple independent processes at various stages of the cell cycle.

  Experimentally identified S / MAR analysis revealed that typical elements are on the order of 300 base pairs (bp) and up to several kilobases (kb) long. These S / MARs can contain several sequence motifs, including an AT-rich nucleotide motif (> 70% AT). Most MARs appear to contain a MAR-specific sequence called the “MAR recognition signature”, which is a binary sequence consisting of two individual sequences AATAYAAA and AWWRTAANNWWGNNNNC within 200 bp. Other sequences that have been proposed to represent MAR sequences include DNA unwinding motifs (AATATATAATTATT), replication initiation protein sites (ATTA and ATTTA), homooligonucleotide repeats (eg, A-box AATAAAYAAA and T- Box TTTWTTTWTT), DNase I hypersensitive site, potential nucleosome-free region, polypurine-polypyrimidine sequence, and sequences that can assume non-B-type DNA or triple helix conformation under negative superhelical conditions It is.

  S / MAR can form gene boundaries between chromosomal domains that are independently organized into a structure that allows gene expression or a structure that does not allow gene expression, called euchromatin domain and heterochromatin domain, respectively. Was guessed. Therefore, transgenes flanked by S / MAR elements may constitute an autonomous chromatin domain whose expression remains independent of the adjacent chromosomal environment. Recently, S / MAR has been shown to increase the expression of flanking transgenes when inserted together in a chromosomal environment. Alternatively, S / MAR can actively reconstitute chromatin near its chromosomal integration site, thereby preventing transgene silencing, for example, by mediating histone modifications or nuclear localization changes .

  The well-characterized 3 kb MAR element, the Drosophila Scs border element, the hspSAP MAR, the mouse T cell receptor TCRa, and the rat LAP locus control region bordering the 5 'end of the chicken lysozyme (cLys) locus It has been reported in the system to produce an acceptable chromatin structure. These elements were introduced into expression vectors and the resulting transgene expression was assayed by stable transfection of CHO cells. Most of these elements showed a modest effect on the level of transgene expression in the CHO cell pool. In contrast, cLysMAR was 6 times more effective than the second best of these elements. Moreover, when the two cLysMAR elements were on both sides of the expression cassette, an additional 4-fold increase in expression level was seen.

  Multiple copies of cLysMAR are large (6 kb), which limits the general use of such chromatin elements given the size of the expression vector. One approach to improving the versatility of MAR consists of co-transfecting the transgene expression cassette with various amounts of cLysMAR in trans on another plasmid. This has been shown to increase expression to a level higher than that obtained with a plasmid having only one cLysMAR element. Thus, expression levels can be significantly increased by simply adding a plasmid with MAR to the current expression vector at the time of cotransfection. In another approach, short functional elements of cLysMAR were defined by deletion mutagenesis. These parts of the MAR when multimerized were found to have the same activity as the full-length element, despite being much smaller in size (P.-A. Girod and N. Mermod, unpublished data) ).

  Production of multimeric proteins faces one major problem. That is, in order to obtain efficient production, it is necessary to synthesize all subunits cooperatively at a stoichiometric level. Therefore, the same expression signal, such as promoter and 3 / − region, was used in different expression cassettes. Two linearized plasmids encoding heavy and light chain expression cassettes and having various types of adjacent chromatin elements were co-transfected into CRO cells in the presence or absence of MAR elements. Again, addition of the cLysMAR element increased the average and maximum productivity of isolated CRO cell clones by 5-10 fold.

  One of the optimal settings is to add MAR elements on the heavy and light chain expression vectors, in cis, and in trans by adding additional MAR-containing plasmids to the transfection mix. It was made up of. Co-transfection of cis and trans cLysMAR increased the average expression level in the pool of stably cotransfected cells. In addition, it reduced expression variability in different clones, thus making it possible to isolate clones that show high secretion levels more frequently.

  Chicken lysozyme 5'MAR was identified as one of the most active sequences in studies comparing the effects of various chromatin structural regulatory elements on transgene expression. It has also been shown to increase the level of regulated or constitutive transgene expression in various mammalian cell lines. Inclusion of the cLysMAR sequence increased the overall expression of the transgene when transfected into the CHO cell line.

  As noted above, mammalian expression systems are usually preferred for their production since therapeutic proteins require post-translational modifications. Currently, various mammalian cell expression systems are available for protein expression. However, the expression levels of recombinant proteins obtained from these expression vectors / expression systems in mammalian cells are not commercially feasible.

  ENBREL is a dimeric fusion protein consisting of the extracellular ligand binding portion of human 75 kilodalton (p75) tumor necrosis factor receptor (TNFR) linked to the Fc portion of human IgG1. The Fc component of Enbrel contains the CH2 domain, CH3 domain, and hinge region of IgG1, but does not contain the CH1 domain. ENBREL is produced by recombinant DNA technology in a Chinese hamster ovary (CHO) mammalian cell expression system. It consists of 934 amino acids and has an apparent molecular weight of about 150 kilodaltons.

  ENBREL is a significant scientific advance that has been shown to reduce the signs and symptoms of rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, and psoriasis.

  TNF is a natural cytokine involved in normal inflammatory and immune responses. TNF plays an important role in the inflammatory process of rheumatoid arthritis (RA), polyarticular juvenile rheumatoid arthritis (JRA), and the resulting joint lesions. Elevated TNF levels are seen in RA patient synovial fluid. Two different receptors for TNF (TNFR), 55 kilodalton protein (p55) and 75 kilodalton protein (p75), originally exist as monomeric molecules on the cell surface and in soluble form.

  The biological activity of TNF depends on binding to either cell surface TNF receptor. A protein such as ENBREL would inhibit the action of TNF by competitive inhibition, and thus render TNF biologically inactive by preventing TNF from binding to its cellular receptor.

  Fusion of the extracellular domain of TNFR to the Fc portion of human IgG1 would allow for molecular dimerization, resulting in optimal pharmacokinetics of the protein by increasing serum residence time. Since both domains of the fusion protein are of human origin, this chimeric protein would ideally not induce an immune response and would therefore be a suitable molecule for human use.

  The present invention relates to a novel expression vector that uses S / MAR as described above to produce Enbrel in larger quantities. When isolated from the culture medium, the expression product of this DNA sequence exhibits the biological activity of TNFR: Fc. Vector development, cloning, and subcloning, transfection, fermentation, and purification strategies have been disclosed.

Method
Expression Vector Cloning and Construction In the pcDNA3.1 vector, the gene of interest is regulated by the human cytomegalovirus (CMV) immediate early promoter / enhancer. The human cytomegalovirus (CMV) immediate early promoter / enhancer allows efficient, high level expression of recombinant proteins. The gene of interest, TNFR: Fc, was cloned into the pCDNA3.1 vector using a directional TOPO expression kit. Positive transformants were confirmed by colony PCR first and then using appropriate restriction enzymes. This transformant was double digested with BamHI and XhoI giving the expected pattern. This transformant was also confirmed by using the restriction enzymes ApaI and SacII, which clearly showed the expected pattern. Thereafter, the sequence of this insert was confirmed.

  The isolated scaffold / matrix binding region (S / MAR) (shown here as the S / MAR sequence) was first cloned into the pGEMTeasy vector by PCR and confirmed by appropriate restriction enzyme analysis and sequencing. The cloned S / MAR was then inserted upstream of the CMV promoter in the pCDNA3.1 vector using restriction sites. This insert was confirmed by restriction analysis.

Expression of recombinant TNFR Using a recombinant expression vector (in this case, pCDNA3.1), the gene encoding the TNFR: Fc fusion protein was expressed. A recombinant expression vector is a replicable DNA construct in which DNA encoding the protein of interest is linked to specific genetic elements that facilitate its expression. Such a collection of transcription units is usually transfected with a transcriptional promoter or enhancer, appropriate transcriptional and translational initiation and termination sites, coding sequences encoding the protein of interest, and transfected mammalian cell line clones. Consists of selectable markers that can help differentiate unrecognized mammalian cell line clones.

  Recombinant protein expression in mammalian cells is particularly preferred as part of the present invention, since it is known that such proteins fold correctly, thereby resulting in a fully functional conformation. Because. The cell line used for recombinant gene expression is the CHO-K1 cell line, which results in a uniform cell population. A CHO-K1 transfected colony containing a stably integrated transcription unit encoding a recombinant protein is a single culture, ie the cell is a progeny of one ancestral transformant. .

  Transformed host cells are transfected with an expression vector containing the complete transcription unit. The expressed TNFR: Fc fusion protein is secreted into the culture supernatant. Enhancement of the level of expression product is achieved by selecting cell lines with a selectable marker such as a gene encoding antibiotic (neomycin) resistance.

  Many variations of the present invention will occur to those skilled in the art in view of the above description. Such obvious variations are within the full intended scope of the appended claims.

Claims (51)

  SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, their complements, mutants, and functional fragments, and the group consisting of sequences that are at least 70% homologous thereto An isolated nucleic acid comprising one or more selected sequences.   2. The isolated nucleic acid of claim 1, wherein the one or more sequences comprise an S / MAR sequence.   3. The isolated nucleic acid of claim 2, wherein the one or more S / MAR sequences increase biomolecule expression when the sequence is used in an expression system.   4. The isolated nucleic acid of claim 3, wherein the one or more S / MAR sequences increase the expression of the biomolecule regardless of the orientation of the sequence in the expression system.   4. The isolated nucleic acid of claim 3, wherein the biomolecule is a protein.   4. The isolated nucleic acid of claim 3, wherein the biomolecule is a fusion protein.   7. The isolated nucleic acid of claim 6, wherein the fusion protein is a recombinant soluble tumor necrosis factor alpha receptor (TNFR) -human IgG Fc fusion protein.   The isolated nucleic acid of claim 2, wherein the one or more S / MAR sequences comprise one or more nucleotide sequence motifs.   9. The isolated nucleic acid of claim 8, wherein the one or more nucleotide sequence motifs comprise at least one AT rich nucleotide motif.   A method of constructing an expression vector having increased expression efficiency, comprising inserting the isolated nucleic acid of claim 1 into an expression vector.   The method of claim 10, wherein the expression vector is a mammalian expression vector.   A vector comprising the isolated nucleic acid of claim 1.   The vector according to claim 12, wherein the vector is a bacterial plasmid, a bacteriophage vector, a yeast episomal vector, an artificial chromosome vector, or a viral vector.   The vector according to claim 12, wherein the vector is a mammalian expression vector.   A method for producing a recombinant host cell comprising introducing the isolated nucleic acid of claim 1 or the expression vector of claim 12 into a host cell.   16. The method of claim 15, wherein the isolated nucleic acid or the expression vector is introduced by transfection.   17. The method of claim 16, wherein the isolated nucleic acid becomes integrated with the genome of the recombinant host cell by transfection.   A host cell produced by the method of claim 15.   A host cell comprising the vector of claim 12.   20. A host cell according to claim 18 or 19, wherein the host cell is a eukaryotic cell.   20. A host cell according to claim 18 or 19, wherein the host cell is a mammalian cell.   (A) a sequence encoding a protein operably linked to one or more expression control elements; and (b) SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6. An expression vector comprising a nucleic acid molecule comprising their complements, variants, and functional fragments, and one or more S / MAR sequences selected from the group consisting of sequences at least 70% homologous thereto.   The expression vector according to claim 22, wherein the protein is a fusion protein.   23. The expression vector of claim 22, wherein the protein is a recombinant soluble tumor necrosis factor alpha receptor (TNFR) -human IgG Fc fusion protein.   A host cell comprising the expression vector according to claim 22.   26. The host cell of claim 25, wherein the host cell is a eukaryotic cell.   26. The host cell of claim 25, wherein the host cell is a mammalian cell.   (A) a sequence encoding a tumor necrosis factor alpha receptor (TNFR) -human IgG Fc fusion protein operably linked to one or more expression control elements; (b) SEQ ID NO: 1, SEQ ID NO: 2, sequence One or more S / s selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, their complements, variants, and functional fragments, and sequences at least 70% homologous thereto. An expression vector comprising a nucleic acid molecule comprising a MAR sequence.   A host cell comprising the expression vector according to claim 28.   30. The host cell of claim 29, wherein the host cell is a eukaryotic cell.   30. The host cell of claim 29, wherein the host cell is a mammalian cell.   The one or more expression control elements comprise at least one of a transcription promoter, transcription enhancer, transcription repressor, polyadenylation site, origin of replication site, translation initiation signal, and translation termination signal. The expression vector described.   33. The expression vector of claim 32, wherein the one or more S / MAR sequences are upstream of the transcription promoter.   33. The expression vector of claim 32, wherein the one or more S / MAR sequences are downstream of the transcription promoter.   33. The expression vector of claim 32, wherein the one or more S / MAR sequences are downstream of the translation termination signal.   33. The expression vector of claim 32, wherein the one or more S / MAR sequences are upstream of the transcription promoter and downstream of the translation termination signal.   33. The expression vector of claim 32, wherein the one or more S / MAR sequences are downstream of the transcription promoter and downstream of the translation termination signal.   30. The expression vector of claim 28, wherein the one or more S / MAR sequences are 0-10 KB away from a sequence encoding a tumor necrosis factor alpha receptor (TNFR) -human IgG Fc fusion protein.   29. The expression vector of claim 28, wherein the one or more S / MAR sequences are 0-10 KB away from the origin of the replication site.   30. The expression vector of claim 28, wherein the expression vector is used for recombinant expression of a tumor necrosis factor alpha receptor (TNFR) -human IgG Fc fusion protein.   A method for producing a protein comprising the steps of: (a) transforming a mammalian cell into (I) a sequence encoding the protein and (II) SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, And an expression vector comprising SEQ ID NO: 6, their complements, variants, and functional fragments, and one or more S / MAR sequences selected from the group consisting of sequences at least 70% homologous thereto. A method comprising: (b) culturing the transfected mammalian cells under conditions suitable for protein expression; and (c) isolating the expressed protein.   42. The method of claim 41, wherein the protein is a fusion protein.   42. The method of claim 41, wherein the protein is a recombinant soluble tumor necrosis factor alpha receptor (TNFR) -human IgG Fc fusion protein.   A method for producing a tumor necrosis factor alpha receptor (TNFR) -human IgG Fc fusion protein comprising: (a) transforming a mammalian cell into (I) a tumor necrosis factor alpha receptor (TNFR) -human IgG Fc fusion protein (II) SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, their complements, variants, and functional fragments, and at least 70 thereof Transfecting with an expression vector comprising one or more S / MAR sequences selected from the group consisting of% homologous sequences; and (b) suitable for expression of the fusion protein of the transfected mammalian cells. Culturing under conditions, and (c) isolating the expressed fusion protein.   45. The method of claim 44, further comprising purifying the isolated fusion protein.   45. Tumor necrosis factor alpha receptor (TNFR) -human IgG Fc fusion protein produced by the method of claim 44.   A method for producing a tumor necrosis factor alpha receptor (TNFR) -human IgG Fc fusion protein comprising: (a) encoding a mammalian cell with a tumor necrosis factor alpha receptor (TNFR) -human IgG Fc fusion protein And (b) transfecting said mammalian cell with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, complements thereof. Co-transfecting with a plasmid comprising one or more S / MAR sequences selected from the group consisting of somatic, mutant, and functional fragments, and sequences at least 70% homologous thereto; (c) transfected Culturing the cultured mammalian cells under conditions suitable for the expression of the fusion protein, and (d) simply expressing the expressed fusion protein. And a step of separating.   48. The method of claim 47, further comprising purifying the isolated fusion protein.   48. Tumor necrosis factor alpha receptor (TNFR) -human IgG Fc fusion protein produced by the method of claim 47.   The one or more S / MAR sequences are SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, their complements, variants, and functional fragments, and A factor that affects the activity of one or more S / MAR sequences, comprising one or more sequences selected from the group consisting of sequences that are at least 70% homologous to.   49. The factor of claim 48, wherein the factor is at least one of a genetic factor or an epigenetic factor.

Images