EP2331693A2 - Vecteur d'expression et son procédé - Google Patents

Vecteur d'expression et son procédé

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Publication number
EP2331693A2
EP2331693A2 EP09806504A EP09806504A EP2331693A2 EP 2331693 A2 EP2331693 A2 EP 2331693A2 EP 09806504 A EP09806504 A EP 09806504A EP 09806504 A EP09806504 A EP 09806504A EP 2331693 A2 EP2331693 A2 EP 2331693A2
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EP
European Patent Office
Prior art keywords
seq
expression
fusion protein
expression vector
sequences
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP09806504A
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German (de)
English (en)
Inventor
Villoo Morawala Patell
Sunit Maity
Ashutosh Vyas
Gopalakrishnan Chellappa
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Avesthagen Ltd
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Avesthagen Ltd
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Publication date
Application filed by Avesthagen Ltd filed Critical Avesthagen Ltd
Publication of EP2331693A2 publication Critical patent/EP2331693A2/fr
Withdrawn legal-status Critical Current

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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]
    • 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
    • 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
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • 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 a method for preparing recombinant soluble Tumour Necrosis Factor Alfa receptor (TNFR) - Human IgG Fc fusion protein using the host cells transfected with the expression vectors.
  • TNFR Tumour Necrosis Factor Alfa receptor
  • tPA human tissue plasminogen activator
  • a mammalian expression system is generally preferred for manufacturing most of therapeutic proteins, since the post-translational modifications required are carried out in the cell line as well.
  • a variety of mammalian cell expression systems are now available for expression of proteins.
  • Generally expression vectors use a strong viral or cellular promoter/enhancer to drive the expression of the recombinant gene.
  • ENBREL also known as TNFR:Fc fusion protein
  • 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 IgGL
  • p75 the extracellular domain of the human tumor necrosis factor receptor superfamily
  • IgGL human IgGL
  • RA rheumatoid arthritis
  • JRA polyarticular-course juvenile rheumatoid arthritis
  • TNF TNF
  • the present disclosure includes one or more of the features recited in the appended claims and/or the following features which, alone or in any combination, may comprise patentable subject matter.
  • the present invention is based on the discovery of novel Scaffold/Matrix Attachment Region (S/MAR) sequences, which may be used for increasing and stabilizing the expression yield of recombinant proteins in mammalian and other eukaryotic cells.
  • S/MAR sequences increase genetic stability of nearby transcription cassettes and inhibit gene silencing by interfering with mechanisms such as DNA methylation. Furthermore, the presence of S/MAR sequences is thought to decrease clone-to-clone variability through decreasing position effects.
  • the present invention relates to an isolated nucleic acid having one or more nucleotide 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, variants and functional fragments thereof and sequences being at least 70% homologous thereto or sequences that hybridize to the isolated nucleic acid under stringent conditions.
  • the present invention relates to an expression vector carrying Scaffold/Matrix Attachment Region (S/MAR) sequence(s) and any combination(s) thereof.
  • S/MAR sequences may be 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 being at least 70% homologous thereto as determined by pair wise DNA sequence alignment using matching methods like the BLAST (Basic Local Alignment Search Tool) algorithm.
  • BLAST Basic Local Alignment Search Tool
  • the present invention relates to an expression vector carrying Scaffold/Matrix Attachment Region (S/MAR) sequence(s) or any combination(s) thereof, and a sequence encoding Tumor Necrosis Factor Alfa Receptor (TNFR) - IgG Fc fusion protein operably linked to one or more expression control elements.
  • S/MAR Scaffold/Matrix Attachment Region
  • TNFR Tumor Necrosis Factor Alfa Receptor
  • the S/MAR sequence(s) may be located upstream or downstream of the transcriptional promoter within the expression vector. Further, the S/MAR sequence(s) may be located at a distance of from 0 to 10 kb from the sequence encoding Tumour Necrosis Factor Alfa receptor (TNFR) - Human IgG Fc fusion protein.
  • TNFR Tumour Necrosis Factor Alfa receptor
  • the present invention relates to a method for construction of an expression vector carrying Scaffold/Matrix Attachment Region (S/MAR) sequence(s) or any combination(s) thereof.
  • the present invention relates to a host cell comprising an expression vector carrying Scaffold/Matrix Attachment Region (S/MAR) sequence(s) or any combination(s) thereof.
  • the present invention relates to a method for producing Tumor Necrosis Factor Alfa Receptor (TNFR) - IgG Fc fusion protein through recombinant expression using an expression vector carrying Scaffold/Matrix Attachment Region (S/MAR) sequence(s) or any combination(s) thereof.
  • TNFR Tumor Necrosis Factor Alfa Receptor
  • S/MAR Scaffold/Matrix Attachment Region
  • the present invention relates to Tumor Necrosis Factor Alfa Receptor (TNFR) - IgG Fc fusion protein expressed by the expression vector carrying Scaffold/Matrix Attachment Region (S/MAR) sequence(s) or any combination(s) thereof.
  • TNFR Tumor Necrosis Factor Alfa Receptor
  • S/MAR Scaffold/Matrix Attachment Region
  • the present invention relates to a method for producing Tumor Necrosis Factor Alfa Receptor (TNFR) - IgG Fc fusion protein by transfecting a mammalian cell with an expression construct carrying the gene encoding Tumor Necrosis Factor Alfa Receptor (TNFR) - IgG Fc fusion protein, and co-transfecting the same mammalian cell using a plasmid carrying Scaffold/Matrix Attachment Region (S/MAR) sequence(s) or any combination(s) thereof.
  • S/MAR Scaffold/Matrix Attachment Region
  • the present invention relates to epigenetic and genetic factors that influence the biological activity of Scaffold/Matrix Attachment Region (S/MAR) sequence(s).
  • S/MAR Scaffold/Matrix Attachment Region
  • Fig. 1 illustrates the construct of plasmid vector pCDNA3.1/ TNFR:Fc
  • Fig. 2 illustrates the construct of plasmid vector pCDNA3.1/MAR1/TNFR:Fc
  • Fig. 3 illustrates the construct of plasmid vector pCDNA3.1/MAR2/TNFR:Fc
  • Fig. 4 illustrates the construct of plasmid vector pCDNA3.1/MAR3/TNFR:Fc
  • Fig. 5 illustrates the construct of plasmid vector pCDNA3.1/MAR4/TNFR:Fc
  • Fig. 6 illustrates the construct of plasmid vector pCDNA3.1/MAR5/TNFR:Fc;
  • Fig. 7 illustrates the construct of plasmid vector pCDNA3.1/MAR6/TNFR:Fc
  • S/MAR Single/Matrix Attachment Region
  • At least 70% homology is meant DNA wherein the nucleotide sequence is least 70% homologous to a defined sequence measured by pair wise DNA sequence alignment using matching methods like the BLAST (Basic Local Alignment Search Tool) algorithm.
  • BLAST Basic Local Alignment Search Tool
  • linking means that the sequences in question are either directly connected to the expression vector or are connected by linking DNA sequences which may be up to 10 kb or more in length as long as such linking 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 operatively linked thereto.
  • the expression control elements comprise the usual regulator elements such as transcriptional promoters, enhancers, repressors, RNA polymerase binding sites, polyadenylation sites, translation initiation signals, and translation termination signals and may be readily accomplished by one ordinarily skilled in the art.
  • complement refers to a nucleic acid sequence having a complementary nucleotide sequence and reverse orientation as compared to a reference nucleotide sequence.
  • sequence 5" ATGCACGGG 3' is complementary to 5' CCCGTGCAT 3 1 .
  • variant refers to polynucleotide or polypeptide sequences different from the specifically identified sequences, wherein one or more nucleotides or amino acid residues is deleted, substituted, or added. Variants may be naturally occurring allelic variants, or non-naturally occurring variants. Variants may be from the same or from other species and may encompass homologues, paralogues and orthologues. In certain embodiments, variants possess 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 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
  • sequence motif refers to a certain nucleotide sequence of at least 2 nucleotides comprised in a larger oligonucleotide sequence.
  • a sequence motif may occur once in an oligonucleotide sequence, or it may occur any number of times.
  • the oligonucleotide 5'-AUCAUCAUG-3' comprises three occurrences of the sequence motif 5'-AU-3 ⁇ two occurrences of the sequence motifs 5-UC-3 1 and 5-CA-3', and one occurrence of the sequence motif 5-UG-3'
  • epigenetic factors refers to any external process or factor that, in operation, affects the expression of a gene or a set of genes, and stands in contrast to the "genetic factors” which refers to any internal process or factor that includes internal factors such as proteins, nucleic acids etc.
  • TNFR:Fc receptor protein refers to a protein having amino acid sequence similar to the extracellular domain of the human TNFRII (p75) protein and which can bind to its native ligand TNF-alpha in turn inhibiting the TNF-alpha from binding to the cell membrane bound TNFRI or TNFRII.
  • TNFRII the preferred TNFR of the present invention is the TNFRII (p75).
  • Soluble TNFR constructs are devoid of the transmembrane region for facilitating secretion out of the cell.
  • the soluble part of the TNFRII which is the extracellular domain is fused in frame to the Fc region of the human IgGL
  • the fusion protein of the present invention is biologically active, i.e. it can bind TNF in solution.
  • biomolecule 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.
  • expression system refers to any in vivo or in vitro biological system that is used to produce one or more protein encoded by a polynucleotide
  • recombinant means that a protein is derived from recombinant expression systems, which in this specification is a mammalian cell based expression system.
  • isolated DNA sequence refers to a DNA polymer in the form of a separate fragment or as a part of a larger DNA construct. Such sequences would be cloned in expression vectors and would enable isolation of the sequence in large amounts for identification, manipulation and recovery of the DNA fragment. Such sequences will be provided in an open reading form without any interruptions by non-translated DNA regions or by introns.
  • nucleotide sequence refers to a heteropolymer of deoxyribonucleotides.
  • 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
  • Chrosome is an organized structure of DNA and proteins found inside the cell.
  • Chromatin is the complex of DNA and protein, found inside the nuclei of eukaryotic cells, which makes up the chromosome.
  • DNA or "Deoxyribonucleic Acid” contains genetic information. It is made up of different nucleotides A, G, T or C.
  • a “gene”, as used herein refers to a deoxyribonucleotide (DNA) sequence coding for a given mature protein. It does not include untranslated flanking regions such as RNA transcription initiation signals, polyadenylation addition sites, promoters or enhancers.
  • transcriptional promoter refers to a nucleic acid sequence that controls the expression of a coding sequence or functional RNA. Promoters may be derived from a native gene, or be composed of different elements derived from different promoters found in nature. The promoter may be any nucleic acid sequence which shows transcriptional activity in the host cell and may be derived from genes encoding proteins either homologous or heterologous to the host cell. Examples of suitable promoters are the SV40 promoter, the MT-1 (metallothionein gene) promoter, the human cytomegalovirus immediate-early promoter etc.
  • Transcriptional enhancer refers to the sequence of gene that acts to initiate the 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.
  • signal peptide refers to an amino terminal polypeptide preceding the secreted mature protein. In mature protein it is not present, as it is cleaved.
  • the term “protein” refers to any polymer of two or more individual amino acids (whether or not naturally occurring) linked via peptide bonds, as occur when the carboxyl carbon atom of the carboxylic acid group bonded to the alpha-carbon of one amino acid (or amino acid residue) becomes covalently bound to the amino nitrogen atom of the amino group bonded to the alpha-carbon of an adjacent amino acid.
  • These peptide bond linkages, and the atoms comprising them i.e., alpha-carbon atoms, carboxyl carbon atoms (and their substituent oxygen atoms), and amino nitrogen atoms (and their substituent hydrogen atoms)
  • the term “protein” is understood to include the terms “polypeptide” and “peptide” (which, at times, may be used interchangeably herein).
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • Vectors usually derived from plasmids, functions like a "molecular carrier", which will carry fragments of DNA into a host cell.
  • the vector may be any vector which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced.
  • the vector may be an autonomously replicating vector, i.e., a vector which exists as an extra chromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid.
  • the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome (s) into which it has been integrated.
  • the vector is preferably an expression vector in which an encoding DNA sequence is operably linked to additional segments required for transcription of the DNA.
  • the expression vector is derived from plasmid or viral DNA, or may contain elements of both.
  • operably linked indicates that the segments are arranged so that they function in concert for their intended purposes, e. g. transcription initiates in a promoter and proceeds through the DNA sequence coding for the polypeptide
  • Plasmids refers to small circular double stranded polynucleotide structures of DNA found in bacteria and some other organisms. Plasmids can replicate independently of the host cell chromosome.
  • replication refers to the synthesis of DNA from its template DNA strand.
  • transcription refers to the synthesis of RNA from a DNA template.
  • translation refers to the synthesis of a polypeptide from messenger RNA.
  • trans refers to the placement of two or more DNA elements linked on the same plasmid.
  • Orientation refers to the order of nucleotides in the DNA sequence.
  • isolated nucleic acid fragment refers to a polymer of DNA or RNA that is single or double stranded.
  • An isolated nucleic acid fragment in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA or synthetic DNA.
  • gene amplification refers to the selective, repeated replication of a certain gene or genes without proportional increase in the copy number of other genes. It is an important widespread developmental and evolutionary process in many organisms. Gene amplification can be classified in two categories (i) developmentally regulated gene expression as seen in Xenopus oocytes and (ii) spontaneously occurring gene expression as amplification of the lac region reported in Escherichia coli. The best known gene amplification in mammalian cells is dihydrofolate reductase (DHFR).
  • DHFR dihydrofolate reductase
  • transformation refers to the transfer of a nucleic acid fragment into the genome of a host organism, resulting in genetically stable inheritance.
  • Host organisms containing the transformed nucleic acid fragments are referred to as "transformed" organisms.
  • eukaryotic cell refers to any cell from a eukaryotic organism whose cells are organized into complex structures by internal membrane and cytoskeleton. Any eukaryotic cell that can be used for gene/protein manipulation and also can be maintained under cell culture conditions and subsequently transfected would be included in this invention.
  • Especially preferable cell types include stem cells, embryonic stem cells, Chinese hamster ovary cells (CHO), COS, BHK21, NIH3T3, HeLa, C2C12, HEK, MDCK, cancer cells, and primary differentiated or undifferentiated cells.
  • the mammalian cells may include CHO cells, HeLa cells, baby hamster kidney (BHK) cells, COS cells, HEK 293 cells or other immortalized cell lines.
  • transfection refers to the introduction of a foreign material like DNA into eukaryotic cells by any means of transfer. Different methods of transfection include, but are not limited to, Calcium phosphate transfection, electroporation, lipofectamine transfection and DEAE-Dextran transfection.
  • transfected cell refers to the eukaryotic cell in which the foreign DNA has been introduced into the eukaryotic cells. This DNA can be part of the host chromosome or replicate as an extra chromosomal element.
  • co-transfection refers to the method of simultaneously transfecting a eukaryotic cell with more than one exogenous gene foreign to the cell.
  • transient gene expression refers to a convenient method for the rapid production of small quantities of protein. COS cells are generally used for the characterization of transient expression
  • stable gene expression refers to the preparation of stable cell lines that permanently express the gene of interest depending on the stable integration of plasmid into the host chromosome
  • a novel eukaryotic expression vector has been constructed that comprises the Tumor Necrosis Factor Alfa receptor (TNFR) - Human IgG Fc fusion protein activity-encoding DNA, and drives the expression of TNFR:Fc activity when transfected into an appropriate cell line.
  • the novel expression vector can be used to produce soluble TNFR:Fc.
  • the recombinantly produced TNFR:Fc activity is useful in the treatment and prevention of varieties of disorders including rheumatoid arthritis, polyarticular-course juvenile rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, and psoriasis.
  • the present invention relates to a novel eukaryotic expression vector used for producing soluble TNFR:Fc in increased quantity.
  • Prokaryotic expression systems were part of the early repertoire of research tools in molecular biology. The de novo synthesis of recombinant eukaryotic proteins in a prokaryotic system imposed a number of problems on the eukaryotic gene product. The two most critical among them were improper protein folding and assembly, and the lack of post-translational modification, principally glycosylation and phosphorylation. Prokaryotic systems do not possess all the appropriate protein synthesizing machinery to produce a structural and/or catalytically functional eukaryotic protein. Therefore, mammalian expression systems are generally preferred for manufacturing therapeutic proteins, for the simple reason that the post-translational modifications required will be addressed by the cellular system(s) in situ.
  • CHO SES Chinese hamster ovary
  • BHK baby hamster kidney
  • HEK human embryonic kidney
  • mouse L-cells mouse L-cells
  • myeloma cell lines like J558L and Sp2/0, etc.
  • One approach involves the 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 chromatin border regions into the expression vector, such that regardless of the integration site, the gene will be protected from surrounding chromatin influences.
  • sequences that behave as chromatin borders and protect transfected genes from surrounding chromatin influences include insulator sequences and scaffold/matrix-attachment regions (S/MARs).
  • S/MARs are DNA sequences that bind isolated nuclear scaffolds or nuclear matrices in vitro with high affinity. Expression studies suggest that flanking a transgene with an insulator could reduce the position effect, thus suppressing clonal expression variability. S/MARs are relatively short (100-1000 bp long) sequences that anchor the chromatin loops to the nuclear matrix. S/MARs have been observed to flank the ends of domains encompassing various transcriptional units. It has also been shown that S/MARs bring together the transcriptionally active regions of chromatin such that transcription is initiated in the region of the chromosome that coincides with the surface of the nuclear matrix.
  • S/MARs may define boundaries of independent chromatin domains, such that only the encompassing cis-regulatory elements control the expression of the genes within the domain.
  • S/MARs which include forming boundaries of chromatin domains, changing of chromatin conformations, participating in initiation of DNA replication and organizing the chromatin structure of a chromosome.
  • SMARs are common in centromere-associated DNA and telomeric arrays, and appear to be important in mitotic chromosome assembly and maintenance of chromosome shape during metaphase. Thus, S/MARs are involved in multiple independent processes during different stages of the cell cycle.
  • S/MARs have revealed a typical element to be as short as 300 base pairs (bp) and up to several kilobases (kb) long. These S/MARs may contain several sequence motifs, including AT-rich nucleotide motifs (> 70% A-T). Most MARs appear to contain a MAR-specific sequence called "MAR recognition signature," which is a bipartite sequence that consists of two individual sequences AATAAYAA and AVWVRTAANNWWGNNNC within 200 bp.
  • MAR recognition signature is a bipartite sequence that consists of two individual sequences AATAAYAA and AVWVRTAANNWWGNNNC within 200 bp.
  • sequences proposed to be indicative of MAR sequences, are the DNA- unwinding motif (AATATATTAATATT), replication initiator protein sites (ATTA and, ATTTA), homo-oligonucleotide repeats (e.g., the A-box AATAAAYAAA and the T-box TTWTWTTWTT), DNase l-hypersensitive sites, potential nucleosome-free stretches, polypurine-polypyrimidine tracks, and sequences that may adopt non-B-DNA or triple-helical conformations under conditions of negative supercoiling.
  • AATATATTAATATT DNA- unwinding motif
  • ATTA and, ATTTA replication initiator protein sites
  • homo-oligonucleotide repeats e.g., the A-box AATAAAYAAA and the T-box TTWTWTTWTT
  • DNase l-hypersensitive sites e.g., the A-box AATAAAYAAA and the T-box TTWTWTTWTT
  • S/MAR could form genetic boundaries between chromosomal domains that independently organize into structures permissive or non-permissive for gene expression, referred to as euchromatin and heterochromatin domains, respectively.
  • a transgene flanked by S/MAR elements may therefore constitute an autonomous chromatin domain whose expression would remain independent of the adjacent chromosomal environment.
  • S/MARs have been shown to increase the expression of adjacent transgenes when co-inserted into a chromosomal environment.
  • S/MARs may actively reconfigure chromatin around its chromosomal integration site and thereby prevent transgene silencing, for instance by mediating histone modifications or changes in sub-nuclear localization.
  • cLysMAR was six-fold more effective than the second best of these elements. Moreover, a further four-fold increase in expression level was seen when two cLysMAR elements flanked the expression cassette.
  • cLysMAR Multiple copies of cLysMAR are large (6kb), which thereby restricts the general use of such chromatin elements taking into account the size of the expression vectors.
  • One approach to improve MAR versatility consists of co-transfecting the transgene expression cassette, and various amounts of cLysMAR in trans on a separate plasmid. This was shown to enhance expression to levels higher than those obtained with plasmids bearing just one cLysMAR element. Thus, MAR-bearing plasmids can be simply added to current expression vectors, in co- transfections, to significantly increase expression levels.
  • short functional elements of cLysMAR were defined by deletion mutagenesis. These portions of MAR, when multimerized, were found to be equally active as the full-length element, although of much smaller size (P.-A. Girod and N. Mermod, unpublished data).
  • Multimeric protein production faces one major problem: to have efficient production, all subunits need to be coordinately synthesized at stoichiometric levels. Therefore, identical expression signals, 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 harbouring adjacent chromatin elements of various kinds were transfected simultaneously into CRO cells, in the presence or absence of MAR elements. Once again, addition of cLysMAR elements increased the average and maximal productivities of isolated CRO cell clones by 5- to 10-fold.
  • One of the optimal settings consisted of adding the MAR elements both in cis, on each of the heavy and light chain expression vectors, and in trans, by further adding to the transfection mix an additional MAR-containing plasmid.
  • the co-transfection of cLysMAR in cis and trans increased the mean expression level in a pool of stably co-transfected cells. Moreover, it reduced the expression variability in different clones, thus allowing the isolation of clones exhibiting high secretion levels at a higher frequency.
  • the chicken lysozyme 5' MAR was identified as one of the most active sequence in a study that compared the effect of various chromatin structure regulatory elements on transgene expression. It was also shown to increase the levels of regulated or constitutive transgene expression in various mammalian cell lines. Inclusion of cLysMAR sequence increased overall expression of transgenes when transfected into CHO cell line. As previously mentioned, mammalian expression systems are generally preferred for manufacturing therapeutic proteins, as they require post-translational modifications. A variety of mammalian cell expression systems are now available for expression of proteins. However, the level of expression of the recombinant protein achieved from these expression vectors/systems in mammalian cells is not commercially viable.
  • ENBREL is a dimeric fusion protein consisting of the extracellular ligand-binding portion of the human-75 kilodalton (p75) tumor necrosis factor receptor (TNFR) linked to the Fc portion of human IgGl
  • the Fc component of Enbrel contains the CH2 domain, the CH3 domain and hinge region, but not the CH1 domain of IgGL 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 approximately 150 kilodaltons.
  • ENBREL is an important scientific advance that has shown to reduce the signs and symptoms of rheumatoid arthritis, polyarticular-course juvenile rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, and psoriasis.
  • TNF is a naturally occurring cytokine that is involved in normal inflammatory and immune responses. It plays an important role in the inflammatory processes of rheumatoid arthritis (RA), polyarticular-course juvenile rheumatoid arthritis (JRA) and the resulting joint pathology. Elevated levels of TNF are found in the synovial fluid of RA patients. Two distinct receptors for TNF (TNFRs), a 55 kilodalton protein (p55) and a 75 kilodalton protein (p75) exist naturally as monomeric molecules on cell surfaces and in soluble forms.
  • TNFRs Two distinct receptors for TNF
  • p55 55 kilodalton protein
  • p75 75 kilodalton protein
  • TNF Biological activity of TNF is dependent upon binding to either cell surface TNF receptors.
  • a protein like ENBREL would inhibit the action of TNF by competitive inhibition thus rendering TNF biologically inactive by preventing the binding of TNF to its cellular receptors.
  • the fusion of the extracellular domain of the TNFR to the Fc part of the human IgGI would allow the dimerization of the molecule leading to optimal pharmacokinetics of the protein by increasing the serum resident time.
  • 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 vector using the above-mentioned S/MAR to produce Enbrel in larger quantity.
  • products of expression of the DNA sequence display the biological activities of TNFR.Fc.
  • Vector development, cloning and sub-cloning, transfection, fermentation and purification strategies are disclosed.
  • telomere In pCDNA3.1 vector, the gene of interest is regulated by Human cytomegalovirus (CMV) immediate-early promoter/enhancer. It permits efficient, high level expression of the recombinant protein.
  • CMV Human cytomegalovirus
  • the gene of interest, TNFR:Fc was cloned into pCDNA3.1 vector using direction TOPO expression kit. The positive transformants were initially verified by colony PCR and later using appropriate restriction enzymes. It was double digested using BamHI and Xhol that gave the expected pattern. It was also confirmed by using the restriction enzymes Apal and Sac II, which clearly demonstrated the expected pattern. The inserts were later sequence verified.
  • S/MARs Isolated scaffold/matrix-attachment regions
  • Recombinant expression vectors in this case pCDNA3.1 , were used to express the gene encoding TNFR:Fc fusion protein.
  • Recombinant expression vectors are replicable DNA constructs in which the DNA encoding for the protein of interest is linked to certain gene elements that drive its expression.
  • An assembly of such a transcription unit generally comprises of - transcriptional promoters or enhancers, appropriate transcription and translational initiation and termination sites, a coding sequence that encodes for the protein of interest and a selection marker that can help to differentiate between the transfected and the non-transfected mammalian cell line clones.
  • the expression of recombinant proteins in mammalian cells is particularly preferred as part of this invention since such proteins are known to be correctly folded thereby resulting in a fully functional conformation.
  • the cell line that will be used for recombinant gene expression is the CHO-K1 cell line, and will be a homogenous population of cells.
  • the transfected colony of CHO-K1 containing the stably integrated transcriptional unit encoding for the recombinant protein will be a monoculture, i.e. the cells will be the progeny of a single ancestral transformant.
  • the transformed host cells will be transfected with expression vectors containing the complete transcriptional unit.
  • the expressed TNFR:Fc fusion protein will be secreted into the culture supernatant. Elevated levels of expression product are achieved by selecting for cell lines using a selection marker such as a gene coding for antibiotic (neomycin) resistance.

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Abstract

La présente invention concerne des vecteurs et des composés pour l'expression de protéines solubles recombinantes. Plus particulièrement, la présente invention concerne des molécules d'acide nucléique, des vecteurs d'expression et des cellules hôtes pour l'expression de la protéine de fusion récepteur du facteur de nécrose tumorale alfa (TNFR) – Fc de IgG humaine, soluble, recombinante. L'invention concerne en outre des procédés de préparation de la protéine de fusion récepteur du facteur de nécrose tumorale alfa (TNFR) – Fc de IgG humaine, soluble, recombinante, à l'aide des cellules hôtes transfectées par les vecteurs d'expression.
EP09806504A 2008-08-12 2009-08-12 Vecteur d'expression et son procédé Withdrawn EP2331693A2 (fr)

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JP (1) JP2012506694A (fr)
KR (1) KR20110044769A (fr)
CN (1) CN102177240A (fr)
AU (1) AU2009280913A1 (fr)
BR (1) BRPI0918008A2 (fr)
CA (1) CA2736580A1 (fr)
MX (1) MX2011001644A (fr)
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CN102257141A (zh) * 2008-10-28 2011-11-23 阿维斯塔根有限公司 表达载体和其加工方法
WO2011015918A2 (fr) * 2009-08-03 2011-02-10 Avesthagen Limited Vecteurs et composés pour l'expression de cetuximab de recombinaison
WO2011015916A2 (fr) * 2009-08-03 2011-02-10 Avesthagen Limited Vecteurs et composés pour expression d’infliximab recombinant
WO2011015924A2 (fr) * 2009-08-03 2011-02-10 Avesthagen Limited Vecteurs et composés pour expression du trastuzumab de recombinaison
WO2011015917A2 (fr) * 2009-08-03 2011-02-10 Avesthagen Limited Vecteurs et composés pour expression de tnk-tpa recombinant (tenecteplase)
GB201213117D0 (en) * 2012-07-24 2012-09-05 Ucl Business Plc Transgene expression
CN103740756B (zh) * 2013-12-25 2015-08-05 中国农业大学 一种可调控删除的非病毒游离载体及其构建方法
WO2019057774A1 (fr) * 2017-09-19 2019-03-28 Deutsches Krebsforschungszentrum Vecteurs d'adn non intégrants pour la modification génétique de cellules
EP3456821B2 (fr) * 2017-09-19 2024-01-24 Deutsches Krebsforschungszentrum Vecteurs d'adn non intégrants destinés à la modification génétique de cellules
WO2019088257A1 (fr) * 2017-11-02 2019-05-09 国立大学法人鳥取大学 Procédé de production à haut rendement d'une protéine à l'aide d'un vecteur de chromosome artificiel de mammifère

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CN1938428A (zh) * 2003-11-12 2007-03-28 先灵公司 多基因表达的质粒系统

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WO2010018444A3 (fr) 2010-05-14
KR20110044769A (ko) 2011-04-29
ZA201101882B (en) 2012-07-25
MX2011001644A (es) 2011-10-06
BRPI0918008A2 (pt) 2018-07-17
WO2010018444A2 (fr) 2010-02-18
JP2012506694A (ja) 2012-03-22
CA2736580A1 (fr) 2010-02-18
AU2009280913A1 (en) 2010-02-18
CN102177240A (zh) 2011-09-07

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