MX2011001644A - An expression vector and a method thereof. - Google Patents
An expression vector and a method thereof.Info
- Publication number
- MX2011001644A MX2011001644A MX2011001644A MX2011001644A MX2011001644A MX 2011001644 A MX2011001644 A MX 2011001644A MX 2011001644 A MX2011001644 A MX 2011001644A MX 2011001644 A MX2011001644 A MX 2011001644A MX 2011001644 A MX2011001644 A MX 2011001644A
- Authority
- MX
- Mexico
- Prior art keywords
- seq
- expression
- sequences
- cell
- fusion protein
- Prior art date
Links
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
- C07K14/7151—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for tumor necrosis factor [TNF], for lymphotoxin [LT]
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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- C—CHEMISTRY; METALLURGY
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Abstract
The present invention relates to vectors and compounds for the expression of recombinant soluble proteins. More particularly, the present invention relates to nucleic acid molecules, expression vectors, and host cells for the expression of recombinant soluble Tumour Necrosis Factor Alfa receptor (TNFR) - Human IgG Fc fusion protein. The invention further relates to methods for preparing recombinant soluble Tumour Necrosis Factor Alfa receptor (TNFR) - Human IgG Fc fusion protein using the host cells transfected with the expression vectors.
Description
A VECTOR OF EXPRESSION AND A METHOD OF THE SAME
FIELD OF THE INVENTION
The present invention relates to vectors and compounds for the expression of. recombinant soluble proteins. More particularly, the present invention relates to nucleic acid molecules, expression vectors, and host cells for the expression of recombinant soluble Alpha Tumor Necrosis Factor (TNFR) receptor-human IgG Fe fusion protein. The invention further relates to a method for preparing the receptof the recombinant soluble Alpha Tumor Necrosis Factor (TNFR) -Fig protein of human IgG using the host cells transfected with the expression vectors,
BACKGROUND OF THE INVENTION
In 1986, the US FDA approved the human tissue plasminogen activator protein (tPA; Genentech, CA, USA) of mammalian cells that is used for therapeutic purposes. Advances in cell culture and recombinant DNA technologies have facilitated the expression of a variety of similar proteins of therapeutic value or other economic value using genetically engineered cells. Currently there are several monoclonal antibodies,
which have regulatory approval for therapeutic use and several hundred are in various stages of development and approval. Like tPA, most of these proteins are expressed in immortalized Chinese hamster ovary (CHO) cells, but other cell lines, such as mouse myeloma (NSO), baby hamster kidney (BHK), kidney of human embryo (HEK-293) are also approved | for the production of recombinant proteins.
The expression of many biologically active therapeutics, which are derived from higher eukaryotic sources, often requires posttranslational modifications which occur naturally in lower eukaryotic or prokaryotic cells, necessitating this. Accordingly, a mammalian expression system is preferred, generally for the manufacture of most therapeutic proteins, since the post-translational modifications required are also carried out on the lines. ace of cells. A variety of mammalian cell expression systems are now available for expression. of proteins. Generally the expression vectors use a strong viral or cellular promoter / enhancer to drive the expression of the recombinant gene. . However, the level of
expression of a recombinant protein achieved from
these vectors / expression systems in mammalian cells
It is not commercially viable. There are two critical problems
during the production of therapeutic products that need
be treated (a) the time taken to provide the material (b) decrease the price of material for access to the masses, especially in developing or underdeveloped countries. Therefore, the industry gives
Biotechnology continues to analyze new technologies and process development strategies that will reduce the deadlines and costs. |
ENBREL (Etanercept), also known as TNFR fusion protein: Fc, is a recombinant fusion protein comprising the extracellular domain of the superfamily of the
human tumor necrosis factor receptor, member IB t
I
I
(p75) and the Fe domain of human IgGl. It is an important scientific breakthrough for the treatment of rheumatoid arthritis, which in many patients has been shown to reduce the signs and symptoms of rheumatoid arthritis, juvenile rheumatoid arthritis of polyarticular course, ankylosing spondylitis, psoriatic arthritis and psoriasis. Tumor Necrosis Factor (TNF) is a cytokine of origin
natural that is involved in inflammatory responses and
normal immune It plays an important role in the
inflammatory processes of rheumatoid arthritis (RA), juvenile rheumatoid arthritis of polyarticular course (JRA) and the resulting joint pathology. Elevated levels of TNF are found in the synovial fluid of patients with RA.
The production of recombinant proteins such as TNFR: Fc within mammalian cells can be difficult due to the low genetic stability of the recombinant gene and / or the silencing of the recombinant gene. It has been reported that several molecular mechanisms can
| Lead to gene silencing that includes metilacipn
DNA, effect of negative position, integration dependent repression and so on, to name a few.
Thus, there is a need in the field to overcome the shortcomings of known methods for producing recombinant fusion proteins, by providing expression systems with superior genetic stability during large-scale production of these proteins. In order to facilitate the production of large amounts of TNFR: Fc from cell culture, a novel expression vector has been developed. The use of this expression vector has been shown to increase the expression of therapeutic prpthein. The cloning, expression, and purification .del
TNFRrFc has been mentioned in this application.
SUMMARY OF THE INVENTION
The present description includes one or more of the
features cited in the appended claims and / or
the following characteristics which, alone io in
any combination, may comprise a content
patentable
The present invention is based on the discovery
of the novel sequences of the Fija-ion Region at i
Scaffolding / Matrix (S / MAR), which can be used to
increase and stabilize the expression performance of the
recombinant proteins in mammals and other eukaryotic cells. S / MAR sequences increase stability
genetics near. the transcription cassettes and inhibit the
genetic silencing by interfering with such mechanisms and DNA methylation. Additionally, the presence of
S / MAR sequences are thought to decrease the variability of
clone to clone through the effects of decreasing position.
i
In this way, in one modality, the present
1 invention relates to an isolated nucleic acid that! has
one or more nucleotide sequences selected from the] group
which consists of 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 of them and sequences that are by
at least 70% homologous to them or sequences that I hybridize
to the isolated nucleic acid under conditions of severity.
In another embodiment, the present invention relates to an expression vector carrying sequence (s) of Region of
Fixation to Scaffolding / Matrix (S / MAR) and any
combination (is) of it. S / MAR sequences can be
I
select from the group consisting of SEQ ID NO: 1, S; ÉQ ID
NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SED. ID NO: 6, complements, variants and functional fragments thereof and sequences that are at least 70% homologous to the
same as determined by the sequence alignment of
DNA by pairs using matching methods similar to BLAST algorithm (Alignment Research Tool)
. i
Basic Local).
In another embodiment, the present invention relates to an expression vector that carries sequence (s) of Fixation Region to the Scaffold to the Matrix (S / MAR) or any
combination (s) thereof, and a sequence encoding the Alpha Tumor Necrosis Factor (TNFR) receptor - Fe protein of IgG linked operably to one or more
Expression control elements.
The S / MAR sequence (s) can be located upstream or downstream of the transcriptional promoter
inside the expression vector. In addition, the sequence (s;
S / MAR can be located at a distance of 0 to 10 | kb: from the
sequence (s) of Fixation Region to the Scaffolding / to the Matrix or any combination (s) thereof.
In yet another embodiment, the present invention relates to a method for producing the Alpha Tumor Necrosis Factor (TNFR) receptor-IgG Fe fusion protein by transfecting a mammalian cell with an expression control carrying the gene encoding the Alpha Tumor Necrosis Factor (TNFR) receptor - IgG Fe fusion protein, and co-transfect the same mammalian cell using a mid carrying the Region (s) sequence (s)
Fixation to Scaffolding / Matrix (S / MAR) any combination (s) of it.
In still a further embodiment, the present invention relates to epigenetic and genetic factors that affect the biological activity of the sequence (s) of the Fixation Region to the Scaffolding / to the Matrix (S / MAR)
BRIEF DESCRIPTION OF THE FIGURES
The invention is further explained with reference to the drawings in which:
Figure 1 illustrates the construct of the plasmid vector pCDNA3.1V TNFR: Fe;
Figure 2 illustrates the plasmid vector construct pCDNA3.1 / MARI / T FR: Fe;
Figure 3 illustrates the construct of | plasmid vector pCDNA3.1 / MAR2 / TNFR: Fe;
Figure 4 illustrates the construct of | Plasmid vector pCDNA3.1 / MAR3 / TNFR: Fe;
Figure 5 illustrates the construct of | Plasmid vector pCDNA3.1 / MAR4 / TNFR: Fe;
Figure 6 illustrates the construct of | Plasmid vector pCDNA3.1 / MAR5 / TNFR: Fe; Y
Figure 7 illustrates the construct of | Plasmid vector pCDNA3.1 / MAR6 / TNFR: Fe
DETAILED DESCRIPTION OF THE INVENTION
The term "Fixation Region to Scaffolding / Matrix (S / MAR)" as used herein refers to DNA elements rich in AT similar to those of non-consensus of several hundred base pairs (bp) in length , which organize the nuclear DNA of the eukaryotic genome within some 60,000 chromatin domains, by the periodic fixation to scaffolding or. matrix of the cell nucleus protein. They are typically found in non-coding regions such as flanking regions, border regions of chromatin and introns.
With "for at least 70% homology" DNA is proposed where the nucleotide sequence is at least 70%
homologous to a defined sequence measured by the DNA sequence alignment by pairs using matching methods such as the BLAST algorithm (Basic Local Alignment Investigation Tool).
The term "functional fragments of SEQ ID NO: 1, 2, 3, 4, 5 and 6" as used in this document
fragments of the sequences of a suffi- cletely large size to have the desired effect on expression performances
The term "flanking" as used herein proposes that the sequences in question connect either directly to the expression vector or are linked by the binding DNA sequences which may be up to 10 kb or more in length as long as These link sequences do not interfere with the desired effect of the sequences. The expression vector comprises at a minimum a gene of interest and expression control elements operably linked thereto.
The expression control elements comprise the usual regulatory elements such as transcriptional promoters, enhancers, repressors, RNA polymerase binding sites, polyadenylation sites, translation initiation signals, and translation termination signals and can be easily accomplished by a person
ordinarily expert in the field.
The term "complement" refers to a sequence of nucleic acids having a complementary nucleotide sequence and a reverse orientation as compared to a reference nucleotide sequence. For example, the 5 'sequence ATGCACGGG 3' is a sequence complementary to 5 'CCCGTGCAT 3'.
As used herein, the term "variant" refers to. sequences of polynucleotides or polypeptides different from the specifically identified sequences, wherein one or more nucleotides or amino acid residues are deleted, substituted, or added. The variants can be allelic variants of natural origin, or variants not of natural origin. The variants may be of the same or of other species and may encompass homologs, paralogs and orthologs. In certain embodiments, the variants have biological activities that are the same or similar to those of the sequences in question.
Variant polynucleotide sequences preferably exhibit 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 at least 55%, more preferably at least 56%, more preferably at least
minus 57%, more preferably at least 58%, more
preferably at least 59%, more preferably at least
less 60%, more preferably at least 61%, more preferably at least 62%, more preferably at least
less 63%, more preferably at least 64%, more preferably at least 65%, more preferably at least
less 66%, more preferably at least 67%, more preferably at least 68%, more preferably at least
less 69%, more preferably at least 70%, j more preferably at least 71%, more preferably at least
minus 72%, more preferably at least 73%, more preferably at least 74%, more preferably at least
minus 75%, more preferably at least 76%; more preferably at least 77%, more preferably at least
minus 78%, more preferably at least 79% more
preferably at least 80%, more preferably at least
less 81%, more preferably at least 82%, more
preferably at least 83%, more preferably at least
less 84%, more preferably at least 85%, more
preferably at least 86%, more preferably at least
minus 87%, more preferably at least 88%; more preferably at least 89%, more preferably at least
minus 90%, more preferably at least 91%, more
preferably at least 92%, more preferably at least
minus 93%, more preferably at least 94%,; more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and most preferably at least 99% identity to a sequence of the present invention .
The term "sequence portion", as used herein, refers to a certain nucleotide sequence of at least 2 nucleotides comprised in a larger oligonucleotide sequence. A sequence portion may be presented once in an oligonucleotide sequence, or any variety of
I
times. For example, the 5 'oligonucleotide -AUCAUCAUG-3' comprises three occurrences of the sequence portion 5'j-AU-3 '
i two occurrences of the sequence portions 5-UC-3 'i and 5'- Í
CA-3 ', and an occurrence of the sequence portion 5'-UG-3.
I
The term "epigenetic factors" as used herein refers to any external process or factor that, in operation, affects the expression of a gene or a set of genes, and contrasts with the "genetic factors" which refer to any internal process or factor that includes internal factors such as proteins], 'nucleic acids, and so on.
As used herein, the term protein
of the TNFR receptor: Fe "or" TNFR: Fe "refers to a protein having the amino acid sequence similar to the extracellular domain of the human TNFRII protein (p75) and which can bind to its native TNF-alpha ligand in turn inhibiting TNF-alpha from the binding to the cell membrane bound to TNFRI or TNFRII Of the two distinct forms of TNFR known to exist, the preferred TNFR of the present invention is TNFRII (p75). Soluble TNFRs lack the transmembrane region to facilitate secretion out of the cell.The soluble part of the TNFRII which is the extracellular domain is fused in the structure to the Fe region of human IgG.The fusion protein of the present invention is biologically active, ie it can bind to TNF in solution.
The term "biomolecule" as used e: this document refers to a substance, a compound or a component associated with a biological environment including, but not limited to, sugars, amino acids, peptides, proteins, oligonucleotides, polynucleotides, polypeptides , organic molecules, haptens, epitopes, biological cells, parts of biological cells, vitamins, hormones and the like.
The term "expression system" refers to any living or in vitro biological system that is used to
producing one or more proteins encoded by a polynucleotide.
The term "recombinant", as used herein, proposes that a protein be derived from recombinant expression systems, which in this specification is an expression system based on mammalian cells.
The term "isolated DNA sequence", as used herein, refers to a DNA polymer in the form of a separate fragment or as a part of a larger DNA construct. These sequences would be cloned in the expression vectors and would enable the isolation of the secretion in large quantities for identification, manipulation and recovery of the DNA fragment. These sequences will be provided in an open reading form with no interruptions by regions of non-translated DNA or introns
As used herein, the term "nucleotide sequence" refers to a deoxyribonucleotide heteropolymer. The DNA sequences encoding the proteins provided by this invention can be assembled from cDNA fragments and short oligonucleotide linkers, or from a series of oligonucleotides, to provide a synthetic gene which is capable of being expressed in a recombinant transcriptional unit.
'Chromosome' is an organized structure of DNA and proteins found within the cell.
Chromatin "is the complex of DNA and pjrotein, found within the nuclei of eukaryotic cells, which constitutes the chromosome.
they contain the genetic information. It is made up of different nucleotides A, G, T or C.
A "gene", as used herein, refers to a sequence of deoxyribonucleotides (DNA) that encode a mature protein provided. It does not include untranslated flanking regions such as RNA transcription initiation signals, polyadenylation addition sites, promoters or enhancers.
As used herein, the term "transcriptional promoter" refers to a sequence of > nucleic acid that controls the expression of a functional RNA or coding sequence. The promoters can be derived from a native gene, or they are composed of different elements derived from different promoters found in nature. The promoter can be any nucleic acid sequence which shows transcriptional activity in the host cell and can be derived from genes encoding proteins already homologous or heterologous to the! cell
host Examples of suitable promoters are the SV40 promoter, the MT-1 promoter (metallothionein gene), the immediate early promoter of human cytomegalovirus and so on.
"Transcriptional enhancer" refers to the gene sequence that acts to initiate transcription of the gene independent of the position or orientation of the gene.
"Transcriptional repressor" refers to the sequence of the gene that acts to inhibit the transcription of the gene independent of the position or orientation of the gene.
The term "signal peptide" refers to an amino-terminal polypeptide that precedes the secreted mature protein. In the mature protein is not present, as it is chalked
As used herein, the term "protein" refers to any polymer of two or more individual amino acids (either naturally occurring or not) linked via the peptide bonds, as occurs when the carboxyl carbon atom of the carboxylic acid group bonded to the alpha-carbon of an amino acid (or amino acid residue) is covalently linked to the amino-nitrogen atom of the amino group bonded to the alpha-carbon of an adjacent amino acid. These peptide bonds and the atoms that comprise them (ie, alpha-carbon atoms, carbon-carboxyl atoms (and their oxygen atom substitute), and
amino nitrogen atoms (and their substituted hydrogen atoms)) form the "polypeptide backbone" of the protein. In addition, as used herein, the term "protein" is understood to include the terms "polypeptide" and "peptide" (which, sometimes, may be used interchangeably herein).
The term "vector" as used herein refers to a molecule. of nucleic acid capable of transporting another nucleic acid to which it has been linked. The vectors, usually derived from. plasmids, function1 as a "molecular carrier", which will carry DNA fragments inside a host cell.
The vector can be any vector which can be conveniently subjected to recombinant DNA procedures, and the selection of the vector will often depend on the host cell into which it is to be introduced. In this way, the vector can be an autonomously replicating vector, that is, a vector which exists as an extra chromosomal entity, the replication of which is independent of chromosomal replication, for example, a plasmid. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the genome of the host cell and replicated together with the chromosome (s)
within which it has been integrated. The vector is preferably an expression vector in which a coding DNA sequence is operably linked to additional segments required for DNA transcription. In general, the expression vector is derived from plasmid or viral DNA, or may contain elements of both. The term, "operably linked" indicates that the segments are arranged so that they function smoothly for their intended purposes, for example - initiates transcription in a promoter and proceeds through the DNA sequence encoding the polypeptide.
The term "plasmids," as used herein, refers to small circular double-stranded polynucleotide structures of DNA found in bacteria and in some other organisms. Plasmids can be replicated independently of the chromosome of the host cell
The term "replication" refers to the synthesis of DNA from its DNA template strand.
The term "transcription" refers to the synthesis of RNA from an RNA template.
The term "translation" is. refers to the syn- thesis of a messenger RNA polypeptide.
The term "cis" refers to the placement of two or more DNA elements linked to the same plasmid.
The term "trans" refers to the collation of two or more elements in two or more different plasmids.
The term "Orientation" refers to the order of nucleotides in the DNA sequence.
As used in this document, the term
"isolated nucleic acid fragment" refers to a DNA or RNA polymer that is single-stranded or double-stranded. An isolated nucleic acid fragment in the form of a DNA polymer can be comprised of one or more segments of cDNA, genomic DNA or synthetic DNA.
The term "gene amplification" as used herein refers to the repeated, selective replication of a certain gene or genes without the proportional increase in the number of copies of other genes. It is a process of development and evolution that is important in many organisms. Gene amplification it can be classified into two categories (i) gene expression that is developmentally regulated as observed in Xenopus oocytes and (ii) gene expression that occurs spontaneously as the amplification of the lac region reported in Escherichia coli. The best-known gene amplification in mammalian cells is dihydrofolate reductase (DHFR).
The term "transformation" refers to the transfer of a nucleic acid fragment within the
genome of a host organism, resulting in a genetically stable inheritance. The host organisms that contain the transformed nucleic acid fragments are referred to as "transformed" organisms.
The term "eukaryotic cell" refers to any cell of a eukaryotic organism whose cells are organized within complex structures by the inner membrane and the cytoskeleton. Any eukaryotic cell that can be used for gene-protein manipulation and can also be maintained under cell culture conditions and subsequently transfected would be included in. this invention. Especially preferred cell types include stem cells, embryonic stem cells, Chinese hamster ovary cells (CHO), COS, BHK21, NIH3T3, HeLa, C2C12, HEK, MDCK, cancer cells, and differentiated or undifferentiated primary cells. Mammalian cells can include CHO cells, HeLa cells, baby hamster kidney cells (BHK), COS cells, HEK 293 cells |; 'other immortalized cell lines.
As used herein, term "transfection" refers to the introduction of foreign material such as DNA within, from eukaryotic cells by any means of transfer.The different methods of transfection include, but are not limited to, transfection of
Calcium phosphate, electroporation, transieccióri lipofectamina and transfection of DEAE-Dextran.
The term "transfected cell" refers to a eukaryotic cell in which foreign DNA has been introduced into eukaryotic cells. This DNA can be part of the host chromosome or it can be replicated | as an extrachromosomal element.
The term "co-transfection" refers to the method of simultaneously transfecting a eukaryotic cell with more than one foreign exogenous gene to the cell.
The term "transient gene expression" refers to a convenient method for rapid production of small amounts of proteins. COS cells I know 'use
I
generally for the characterization of transient expression.
The term "stable gene expression" refers to the preparation of stable cell lines that permanently express the gene of interest depending on the stable integration of the plasmid within the host chromosome.
A novel eukaryotic expression vector has been constructed which comprises the DNA encoding the activity of the Alpha Tumor Necrosis Factor (TN; FR) receptor, Fe-Human IgG fusion protein, and drives the expression
TNFR activity: Fc when transfected into an appropriate cell line. The novel expression vector can be used to produce the TNFR: soluble Fc. The activity of TNFR: recombinantly produced Fc is useful in the treatment and prevention of varieties of disorders including rheumatoid arthritis, juvenile rheumatoid arthritis of polyarticular course, ankylosing spondylitis, psoriatic arthritis, and psoriasis.
The present invention relates to a novel eukaryotic expression véctpr used to produce TNFR: soluble Fe in increased amount.
The prokaryotic expression systems were part of the previous repertoire of research tools in molecular biology. The de novo synthesis of recombinant eukaryotic proteins in a prokaryotic system imposed a variety of problems on the eukaryotic gene product. The two most critical among them were the folding and assembly of inappropriate proteins, and 'to the
t lack of post-translational modification, mainly glycosylation and phosphorylation. Prokaryotic systems do not possess all the appropriate protein synthesizing machinery to reduce a structural and / or catalytically functional eukaryotic protein. Therefore, mammalian expression systems are generally preferred for the
manufacture of therapeutic proteins, for the simple reason that the post-translational modifications required will be addressed by the cellular system (s) in situ. A variety of mammalian expression systems are now available for either transient or stable expression of recombinant genes. Generally, the stable expression systems of hamster ovary cells are used: Chinese (CHO) and (CHO SES). On the other hand, baby hamster kidney cells (BHK), human embryonic kidney cells; (HEK) 293, L-mouse cells, and myeloma cell lines such as J558L and Sp2 / 0, etc., are also used as hosts for the establishment of stable transients.
However, the integration of foreign DNA into the genome of a host cell is an unpredictable process. It has been well documented that transgene expression is highly variable among cell lines, and that integration may cause unexpected changes in the phenotype. The reason is that they base the great variability on the clonal expression levels include different plasmid copy numbers and a phenomenon known as position effect, which was initially described in the Drosophila melanogaster as the position-effect variegation. The integration position can affect transgenic expression through p'or
minus three mechanisms: the activity of local regulatory elements, the local chromatin structure, and the local state of DNA methylation. Common procedures can be used to protect DNA from the effects of negative position or integration-dependent repression. One method involves direct integration of the transgene into a predetermined site that is transcriptionally active using site-specific recombination methods.
Another method is to incorporate DNA sequence elements found in the border regions of chromatin within the expression vector, such that without considering integration site, the gene will be protected from influences of the surrounding chromatin. For the expression of the recombinant protein, the sequences that behave as chromatin boundaries and protect the transfected genes and the influences of the surrounding chromatin include isolating sequences and scaffolding / matrix binding regions (S / MARs).
S / MARs are DNA sequences that link isolated nuclear scaffolds or nuclear and in matrixes; vitro with high affinity. Expression studies suggest that flanking a transgene with an isolator could reduce the position effect, thereby suppressing the variability of clonal expression. The S / MARs are sequences
relatively short (100-100O bp long) that anchor the chromatin loops to the nuclear matrix. The S / MARs have been observed flanking the ends of the domains spanning several transcriptional units. It has also been observed that the S / MARs carry together the transcriptionally active regions of. chromatin such that transcription starts in the region of the chromosome that matches the surface of the nuclear matrix.
As such, they can define boundaries of independent chromatin domains, such that only integral cis-regulatory elements control the expression of genes within the chromatin. domain. A variety of possible functions have been raised previously for the S / MARs, which include forming limits of the chromatin domains, changing chromatin conformations, participating in the initiation of DNA replication and organizing the structure of chromatin of a chromosome. S / MARs are common in the associated centromere DNA and telomeric arrays, and appear to be important in the assembly of mitotic chromosomes: and the maintenance of chromosome shape during metaphase. In this way, S / MARs are involved in multiple independent processes during the different stages of the cell cycle.
Analyzes of S / MARs experimentally
identified have revealed a typical element that is as short as 300 base pairs (bp) and up to several kilobases (kb) long. These S / MARs. they may contain several sequence portions, which include AT-rich nucleotide portions (> 70% A-T). Most MARs seem to contain a specific MAR sequence called "recognition signature"
i
MAR ", which a bipartite sequence consisting of two individual sequences AATAAYAA and AWWVRTAANNWWGNNNC within 200 bp. Other sequences, proposed by indicative of the MAR sequence, are the portion that uncoils the DNA
(??????????????), sites of replication-initiating proteins (ATTA and ATTTA), repetitions of 'homo-? oligonucleotides (for example, the A-box AATAAAYAAA and the T-box TTWTWTTWTT), I-hypersensitive DNase sites, stretches without potential nucleosome, polypurine-polypyrimidine clues and sequences that can adopt non-B-DNA or helical tiples conformations under Negative super-winding conditions. |,.
It was speculated that S / MAR could form genetic boundaries between chromosomal domains that are organized independently within permissive or non-permissive structures for gene expression, referred to as eukomatrin and heterochromatin domains, respectively .. A transgene flanked by S / elements MAR can constitute 'by
thus an autonomous chromatin domain whose expression would remain independent of the adjacent chromosomal environment. Recently, S / MARs have been shown to increase the expression of adjacent transgenes when they are co-inserted in a chromosomal environment. Alternatively, S / MARs can actively reconfigure chromatin around their chromosomal integration site and consequently prevent transgenic silencing, for example by modifying histone modifications or changes in the sub-nuclear location.
A well characterized 3-kb MAR element that limits the 5 'end of the chicken lysosome site (cLys), the Drosophila Scs boundary element, MAR hspSAP, the TCRa mouse T cell receptor, and the control region of Rat LAP site, all have been reported to mediate; permissive chromatin structures in a variety of systems. These elements were introduced into the expression vectors and the resulting transgenic expression was assayed by stable transfection of CHO cells. Most of these elements showed modest effects on the expression levels of the transgene in clusters of CHO cells. In contrast, cLysMARs were six times more effective than the second best of these elements. On the other hand1, an additional four times increase in the level of expression
it was observed when two cLysMAR elements flanked the expression cassette.
Multiple copies of cLysMAR are large (6kb);, which consequently restrict the general use of these chromatin elements taking into account the size of expression vectors. A method for praising the versatility of MAR consists of co-transfecting the transgenic expression cassette, and various amounts of cLysMAR in trans onto a separate plasmid. This showed that it improves expression at higher levels than those obtained with plasmids carrying only one cLysMAR element. In this way, the plasmids carrying MAR can be simply added to the current expression vectors, in cotransfections, to significantly implement the expression levels. In another procedure, short functional elements of cLysMAR were defined by deletion mutagenesis. These portions of MAR, when multimerized, discovered that they are equally active as the full-length element, although of much smaller size (P.-A. Girod and N. Mermod, unpublished data) :.
The production of multimeric proteins faces a major problem: it has efficient production, all the subunits need to be synthesized in stoichiometric levels. Therefore, the signals of
Identical expressions, such as promoters and 3 / -regions were used in the different expression cassettes. Two linearized plasmids encoding the heavy and light chain expression cassette and which house adjacent chromatin elements of various classes were transfected simultaneously into the CRO cells, in the presence or absence of MAR elements. Again, the addition of cLysMAR elements increased the average and maximum productivities of clones of isolated CRO cells by 5 to 10 times.
One of the optimal settings consisted in aggregating the MAR elements both in cis, on each side of the heavy and light chain expression vectors, and in trans, by additionally adding a plasmid containing additional MAR to the transfection mixture. Co-transfection of cLysMAR into cis and trans increased the level of expression medium in a pool of stably co-transfected cells. On the other hand, it reduced the variability of expression in different clones, thus allowing the isolation of clones that exhibit high levels of secretion at a higher frequency.
The 5 'MAR of chicken lysozyme was identified as one of the most active sequences in a study that compared the effect of several regulatory elements of chromatin structure on transgenic expression. He also showed the
increase in levels of transgenic expression
Constitutive regulated in several mammalian cell lines. The inclusion of the cLysMAR sequence increased the
Total expression of the transgenes when co-transfected
within the CHO cell line.
As previously mentioned, mammalian expression systems are generally preferred 1 for manufacturing therapeutic proteins, since they require post-translational modifications. A variety of mammalian cell expression systems are now available for the expression of proteins. However, the level of expression of the recombinant protein achieved from these vectors / expression systems in mammalian cells
It is not commercially viable.
The ENBREL is a dimeric fusion protein that consists of the extracellular ligand binding portion of the human tumor (TNF) 75 kilodaltons (p75) receptor bound to the Fe portion of human IgGl. The Fe component of the Enbrel contains the CH2 domain, the CH3 domain and the hinge region, but not the CH1 domain of
the IgGl. ENBREL is produced by recombinant DNA technology in a cell expression system of
Chinese hamster ovary mammal (CHO). It consists of the
934 amino acids and has an obvious molecular weight of
approximately 150 kilodaltons.
The ENBREL is an important scientific breakthrough that has been shown to reduce the signs and symptoms of rheumatoid arthritis, juvenile rheumatoid arthritis of polyarticular course, ankylosing spondylitis, psoriatic arthritis, and psoriasis.
TNF is a cytokine of natural origin that is involved in normal inflammatory and immune responses. It plays an important role in the inflammatory processes of rheumatoid arthritis (RA), juvenile rheumatoid arthritis of polyarticular course (JRA) and the resulting joint pathology. Elevated levels of TNF were found in the synovial fluid of patients with RA. Two distinct receptors for TNF (TNFRs), a protein of 55 kilodaltons (p55) and a protein of at 75 kilodaltons (p75) exist naturally as monomeric molecules squared on cell surfaces and in soluble forms
The biological activity of TNF is dependent on the binding to any of the cell surface TNF receptors. A protein like ENBREL could inhibit the action of TNF by the. competitive inhibition returning | from: this way biologically inactive TNF by preventing the binding of TNF to its cellular receptors
The fusion of the extracellular domain of TNFR to the
Fe part of the human IgGl would allow the dimerization of the molecule leading to optimal pharmacokinetics of the protein by increasing the time resident in the serum. As both of the domains of the fusion protein are of human origin, the chimeric protein would not ideally induce an immune response, thus making it a suitable molecule for human use.
The present invention relates to a novel expression vetch using the S / MAR mentioned above to produce Enbrel in a larger amount. In the isolation of the culture media, the expression products of the DNA sequence show the biological activities of TNFR.Fc. The vector development, cloning and subcloning, transfection, fermentation and purification strategies are disclosed.
METHODOLOGY
Cloning and Construction of the Expression Vector
In the pCDNA3.1 vector, the gene of interest is regulated by the human cytomegalovirus (CMV) immediate promoter / early enhancer. It allows the high-level, efficient expression of the recombinant protein. The gene of interest, TNFR: Fe was cloned into the pCDNA3.1 vector using the address TOPO expression kit. The positive transformants were initially verified by the PCR of colonies and then
using appropriate restriction enzymes. It was digested twice using BamHI and XhoI that provided the expected pattern. It was also confirmed when using the restriction enzymes Apal and Sac II, the. which clearly demonstrated the expected pattern. The inserts - were then verified sequences.
The isolated scaffold / matrix binding regions (S / MARs), marked here as S / AR sequences were initially cloned into the pGEMTeasy vector by PCR and confirmed by analysis and sequencing of appropriate restriction enzymes. Subsequently, the cloned S / MARs were inserted upstream of the CMV promoter in the pCDNA3.1 vector using the restriction sites. The insertion was confirmed by the restriction analysis.
Expression of recombinant TNFR
The recombinant expression vectors, in this case, pCDNA3.1, were used to express the gene encoding TNFR fusion protein: .Fc. Recombinant expression vectors are replicable DNA constructs in which the DNA encoding the protein of interest binds to certain elements of genes that drive its expression. An assembly of that transcription unit generally comprises de-promoters or transcriptional enhancers, appropriate transcription and translational initiation and sites of
termination, a coding sequence that encodes the
protein of interest and a selection marker that can
help to strengthen among cell lines clones of
transiected and non-transfected mammals.
The expression of recombinant proteins in the
mammalian cells are particularly preferred as part
I
of this invention since these proteins are known
that fold correctly resulting in
consequence a fully functional conformation. The line
of cells that will be used for recombinant gene expression
is the cell line CHO-K1, and it will be a homogeneous population
of cells. The transfected colony of CHO-K1 containing the
stably integrated transcriptional unit that encodes the
recombinant protein will be a monoculture, that is to say
cells will be the progeny of a single ancestral transformant.
The transformed host cells; HE
transience with the expression vectors that contain the
complete transcriptional unit. The TNFR: Fe fusion protein expressed will be secreted into the supernatant of
culture. The elevated levels of the expression product are
achieved by selecting the line of cells that use a
selection marker such as a gene encoding the
antibiotic resistance (neomycin).
Many variations of the present invention are
They will suggest to those experts in the field in view of the above description. These obvious variations are within the full proposed scope of the appended claims. ,
Claims (51)
1. An isolated nucleic acid, characterized i because it comprises one or more sequences selected from the group consisting of 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, variants, and functional fragments of the mmesms and sequences that are at least 70% homologous to them.
2. The nucleic acid isolated according to claim 1 ,. characterized in that the one or more sequences comprise S / MAR sequences.
3. The isolated nucleic acid according to claim 2, characterized in that the one or more S / MAR sequences increase the expression of a biomolecule when the sequences are used in an expression system.
4. The isolated nucleic acid according to claim 3, characterized in that the one; or more sequences S / MAR increase the expression of the bicjmolécula independently of the orientation of the sequences in the expression system.
5. The isolated nucleic acid according to claim 3, characterized in that the biomolecule is a protein.
6. The isolated nucleic acid according to claim 3, characterized in that the biomolecule is a fusion protein.
7. The isolated nucleic acid according to claim 6, characterized in that the fusion protein is a recombinant soluble recombinant Tumor Necrosis Factor Alpha (TNFR) receptor - IgG Fe fusion protein. Human
The isolated nucleic acid according to claim 2, characterized in that the one or more S / MAR sequences contain one or more portions of nucleotide sequences.
9. The isolated nucleic acid according to claim 8, characterized in that the one or more nucleotide sequence portions includes at least one portion of nucleotides rich in AT.
10. A method for constructing an expression vector having increased expression efficiency, the method characterized in that it comprises inserting the isolated nucleic acid according to claim 1 into a vector of. expression.
11. The method according to claim 10, characterized in that the expression vector is a mammalian expression vector,
12. A vector, characterized in that it comprises the isolated nucleic acid according to claim 1.
13. The vector according to claim 12, characterized in that the vector is a bacterial plasmid, a bacteriophage vector, an episomal yeast vector, an artificial chromosomal vector or a viral vector.
14. The vector according to claim 12, characterized in that the vector is a vector of 'mammalian expression,
15. A method for producing a recombinant cell, the method characterized in that it comprises introducing the isolated nucleic acid according to claim 1 or the expression vector | of claim 12 within a cell.
16. The method according to claim 15, characterized in that the isolated nucleic acid or the expression vector is introduced by means of transference.
17. The method in accordance with the claim 16, characterized in that the isolated nucleic acid is integrated with the genome of the recombinant cell in the transience
18. A cell, characterized pc > rque produced according to the method according to the claim 15.
19. A cell, characterized because comprises the vector according to the claim; 12
20. The cell in accordance with claim 18 or 19, characterized in that the cell is a eukaryotic cell.
21. The cell in accordance with claim 18 or 19, characterized in that the cell Host is a mammalian cell.
22. An expression vector, characterized in that comprises a nucleic acid molecule comprising (a) a sequence that encodes a bound protein operably to one or more | expression control elements and (b) and one or more S / MAR sequences selected from the group consisting of 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, supplements, variants, and functional fragments thereof and sequences which are at least 70% homologous thereto.
23. The expression vector in accordance with claim 22, characterized in that the protein is a fusion protein.
24. The expression vector according to claim 22, characterized in that the receptor protein of the recombinant soluble Alpha Tumor Necrosis Factor (TNFR) - Fe protein of human IgG.
25. A host cell, characterized in that it comprises the expression vector according to claim 22.
26. The host cell according to claim 25, characterized in that the host cell is a eukaryotic cell.
27. The host cell according to claim 25, characterized in that the host cell is a mammalian cell.
28. An expression vector, characterized in that it comprises a nucleic acid molecule comprising (a) a sequence encoding the Alpha Tumor Necrosis Factor (TNFR) receptor - Fe IgG human fusion protein linked operably to one or more control elements of expression and (b) one or more. S / MAR sequences selected from the group consisting of 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, variants , and functional fragments thereof and sequences that are at least 70% homologous thereto.
29. A host cell, characterized in that it comprises the expression vector according to claim 28.
30. The host cell according to claim 29, characterized in that the host cell is a eukaryotic cell. ,
31. The host cell according to claim 29, characterized in that the host cell is a mammalian cell.
32. The conformity expression vector according to claim 28, characterized in that the | or more expression control elements comprise at least one of transcriptional promoter, transcriptional enhancer, transcriptional repressor, polyadenylation site, origin of the replication site, translation initiation signal and translation termination signal.
33. The expression vector according to claim 32, characterized in that the one or more S / MAR sequences are located upstream of the transcriptional promoter.
34. The expression vector according to claim 32, characterized in that the one | or more S / MAR sequences are located downstream of the transcriptional prómotor.
35. The conformity expression vector! with claim 32, characterized in that the one or more S / MAR sequences are located downstream of the translation completion signal.
36. The vector according to claim 32, characterized in that the one or more S / MAR sequences are located upstream of the transcriptional promoter and downstream of the translation termination signal.
37. The vector according to claim 32, characterized in that the one or more S / ÁR sequences are located downstream of the transcriptional promoter and the translation termination signal.
38. The expression vector according to claim 28, characterized in that the | or more S / MAR sequences are located at a distance of 0 to 10 KB from the sequence encoding the Alpha Tumor Necrosis Factor (TNFR) receptor - Fe IgG human fusion protein.
39. The expression vector according to claim 28, characterized in that the | or more S / MAR sequences are located a. a distance of 0 to 10 KB from the origin of the replication site.
40. The expression vector according to claim 28, characterized in that the expression vector is used for the recombinant expression of the receptor of the Alpha Tumor Necrosis Factor (TNFR) - Human IgG Fe fusion protein.
41. A method for producing a protein, the method characterized in that it comprises the steps | transfecting a mammalian cell with an expression vector comprising (I) a sequence encoding the protein and (II) one or more S / MAR sequences selected from the group consisting of 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, variants and functional fragments thereof and sequences that are at least 70% homologous to them; (b) culturing the transfected mammalian cell under conditions suitable for the expression of the protein; and (c) isolating the expressed protein.
42. The method in accordance with the claim 41, characterized in that the protein is a fusion protein.
43. The method according to claim 41, characterized in that the protein is the receptor for the Alpha Tumor Necrosis Factor (TNFR) - soluble recombinant - fusion protein. Human IgG Fe.
44. A method for producing the Alpha Tumor Necrosis Factor (TNFR) receptor - Fe IgG human fusion protein, the method characterized in that I understood the stages of (a) transfecting a mammalian cell | with an expression vector comprising (I) a secuericia coding for the receptor of the Factor of Tumor Necrosis i Alfa (TNFR) - Fe protein fusion of Human IgG and (II) one or more S / MAR sequences selected from the group consisting of of 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, variants and functional fragments thereof and sequences that they are at least 70% homologous to them; (b) culturing the transfected mammalian cell under conditions suitable for expression of the fusion protein; and (c) isolating the expressed fusion protein.
45. The method according to claim 44, characterized in that it further comprises the step of purifying the isolated fusion protein.
46. The Alpha Tumor Necrosis Factor (TNFR) receptor - Human IgG Fe fusion protein, characterized in that they are produced according to the method according to claim 44.
47. A method for producing the Alpha Tumor Necrosis Factor (TNFR) receptor-fusion protein: Human IgG Fc, the method characterized in that it comprises the steps of (a) transfecting a mammalian cell | with an expression vector comprising a sequence encoding the receptor of the Alpha Tumor Necrosis Factor (TNFR) fusion protein Fe of Human IgG; (b) co-transfecting the mammalian cell with a plasmid comprising one or more S / MAR sequences selected from the group consisting of 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, variants and functional fragments thereof and sequences that are at least 70% homologous thereto; (c) culturing the transfected mammalian cell under conditions suitable for expression of the fusion protein; and (d) isolating the expressed fusion protein.
48. The method according to claim 47, characterized in that it further comprises the step of purifying the isolated fusion protein.
49. The Alpha Tumor Necrosis Factor (TNFR) receptor - Human IgG Fe fusion protein, characterized in that it is produced according to the method according to claim 47.
50. A. factor, characterized in that it affects the activity of one or more of the S / MAR sequences, where the one or more S / MAR sequences comprises one or more sequences ii selected from the group consisting of 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, variants, and functional fragments of the same and sequences that are at least 70% homologous to them.
51. The factor of compliance with the claim 48, characterized in that the factor is at least one of a genetic factor, or an epigenetic factor.
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IN1941CH2008 | 2008-08-12 | ||
PCT/IB2009/006517 WO2010018444A2 (en) | 2008-08-12 | 2009-08-12 | An expression vector and a method thereof |
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JP (1) | JP2012506694A (en) |
KR (1) | KR20110044769A (en) |
CN (1) | CN102177240A (en) |
AU (1) | AU2009280913A1 (en) |
BR (1) | BRPI0918008A2 (en) |
CA (1) | CA2736580A1 (en) |
MX (1) | MX2011001644A (en) |
WO (1) | WO2010018444A2 (en) |
ZA (1) | ZA201101882B (en) |
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WO2010049777A1 (en) * | 2008-10-28 | 2010-05-06 | Avesthagen Limited | An expression vector and processes thereof |
WO2011015924A2 (en) * | 2009-08-03 | 2011-02-10 | Avesthagen Limited | Vectors and compounds for expression of recombinant trastuzumab |
WO2011015916A2 (en) * | 2009-08-03 | 2011-02-10 | Avesthagen Limited | Vectors and compounds for expression of recombinant infliximab |
WO2011015918A2 (en) * | 2009-08-03 | 2011-02-10 | Avesthagen Limited | Vectors and compounds for expression of recombinant cetuximab |
WO2011015917A2 (en) * | 2009-08-03 | 2011-02-10 | Avesthagen Limited | Vectors and compounds for expression of recombinant tnk-tpa (tenecteplase) |
GB201213117D0 (en) * | 2012-07-24 | 2012-09-05 | Ucl Business Plc | Transgene expression |
CN103740756B (en) * | 2013-12-25 | 2015-08-05 | 中国农业大学 | The non-viral free carrier that a kind of controllable is deleted and construction process thereof |
WO2019057774A1 (en) * | 2017-09-19 | 2019-03-28 | Deutsches Krebsforschungszentrum | Non-integrating dna vectors for the genetic modification of cells |
ES2821655T3 (en) * | 2017-09-19 | 2021-04-27 | Deutsches Krebsforsch | Non-integrating DNA vectors for genetic modification of cells |
WO2019088257A1 (en) * | 2017-11-02 | 2019-05-09 | 国立大学法人鳥取大学 | High-yield production method for protein by using mammalian artificial chromosome vector |
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ATE514783T1 (en) * | 2003-11-12 | 2011-07-15 | Schering Corp | PLASMID SYSTEM FOR EXPRESSING MULTIPLE GENES |
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BRPI0918008A2 (en) | 2018-07-17 |
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ZA201101882B (en) | 2012-07-25 |
WO2010018444A2 (en) | 2010-02-18 |
KR20110044769A (en) | 2011-04-29 |
CN102177240A (en) | 2011-09-07 |
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CA2736580A1 (en) | 2010-02-18 |
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