WO2011015924A2 - Vectors and compounds for expression of recombinant trastuzumab - Google Patents

Vectors and compounds for expression of recombinant trastuzumab Download PDF

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WO2011015924A2
WO2011015924A2 PCT/IB2010/001906 IB2010001906W WO2011015924A2 WO 2011015924 A2 WO2011015924 A2 WO 2011015924A2 IB 2010001906 W IB2010001906 W IB 2010001906W WO 2011015924 A2 WO2011015924 A2 WO 2011015924A2
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sequence
expression
attachment region
expression vector
matrix attachment
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WO2011015924A3 (en
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Villoo Morawala Patell
Sami N. Guzder
Sunit Maity
Sunil Shekar
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Avesthagen Limited
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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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • 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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/46—Vector systems having a special element relevant for transcription elements influencing chromatin structure, e.g. scaffold/matrix attachment region, methylation free island

Definitions

  • the present invention relates to use of novel expression vectors and compounds of expression used for production of Recombinant Monoclonal antibody to human epidermal growth factor receptor-2 (HER-2) in increased quantity.
  • the present invention further realtes to method of construction of the expression vector and to obtain monoclonal antibody to human epidermal growth factor receptor-2 (HER-2) protein expressed by the expression vector carrying Scaffold/Matrix Attachment Region (S/MAR).
  • S/MAR Scaffold/Matrix Attachment Region
  • mammalian expression system is generally preferred for manufacturing most of therapeutic proteins, because they require post-translational modifications.
  • mammalian cell expression systems are now available for expression of proteins.
  • expression vectors use a strong viral or cellular promoter/enhancer to drive the expression of recombinant gene,
  • the level of expression of a recombinant protein attained from these expression vectors/systems in mammalian cells is not commercially viable.
  • Trastuzumab (Herceptin) is a recombinant humanized IgGIk monoclonal antibody glycoprotein that binds specifically to human epidermal growth factor receptor-2 (HER-2).
  • Herceptin is designed to target and block the function of HER-2 protein over expression.
  • Trastuzumab contains primarily human constant framework regions with the Complementarity- determining regions (CDR) from a murine antibody with specificity for human HER-2.
  • CDR Complementarity- determining regions
  • the humanized antibody against HER-2, Trastuzumab having an approximate molecular weight of 155 kilo-daltons is produced by a mammalian cell (Chinese Hamster Ovary) [CHO] suspension culture in a nutrient medium containing the antibiotic gentamicin. Gentamicin is not detectable in the final product.
  • Trastuzumab is packaged as a sterile, white to pale yellow, preservative-free lyophilized powder for intravenous (IV) administration.
  • Cancerous breast tissue cells that over-express the HER-2 gene produce extra protein receptors on the cell surface.
  • the higher density of receptors triggers the cell to divide and multiply at an accelerated rate, thus contributing to tumour growth.
  • Trastuzumab binds to numerous HER-2 receptor sites found on the cell surface, blocking the receptor sites and possibly preventing further growth by interrupting the growth signal.
  • Trastuzumab as a agent is indicated for the treatment of patients with metastatic breast cancer whose tumors over-express the HER-2 protein and who have received one or more chemotherapy regimens for their metastatic disease.
  • Trastuzumab in combination with paclitaxel is indicated for treatment of patients with metastatic breast cancer whose tumors over-express the HER-2 protein and who have not received chemotherapy for their metastatic disease. This antibody should only be used in patients whose tumors have HER-2 protein over-expression.
  • the foremost objective of the present invention is to construct an expression vector carrying Scaffold/Matrix Attachment Region(s) (SMAR).
  • Another main objective of the present invention is to obtain an expression vector carrying Scaffold/Matrix Attachment Region(s) (S/MAR) used for production of Monoclonal antibody to human epidermal growth factor receptor-2 (HER-2).
  • S/MAR Scaffold/Matrix Attachment Region(s)
  • Yet another objective of the present invention is to develop a method for construction of an expression vector carrying Scaffold/Matrix Attachment Region(s) (S/MAR).
  • Still another objective of the present invention is to obtain a host cell comprising an expression vector carrying Scaffold/Matrix Attachment Region(s) (S/MAR).
  • Still another objective of the present invention is to obtain Monoclonal antibody to human epidermal growth factor receptor-2 (HER-2) protein expressed by the expression vector carrying Scaffold/Matrix Attachment Region(s) (S/MAR).
  • HER-2 human epidermal growth factor receptor-2
  • S/MAR Scaffold/Matrix Attachment Region(s)
  • the present invention relates to an expression vector carrying Scaffold/Matrix Attachment Region(s) (S/MAR); a method for construction of an expression vector carrying Scaffold/Matrix Attachment Region(s) (S/MAR), said method comprising step of inserting S/MAR into the expression vector; a host cell comprising an expression vector carrying Scaffold/Matrix Attachment Region(s) (S/MAR); Monoclonal antibody to human epidermal growth factor receptor-2 (HER-2) expressed by the expression vector carrying Scaffold/Matrix Attachment Region(s) (S/MAR). DETAILED DESCRIPTION OF THE DRAWINGS
  • Figure 1 Construct map of pCDNA3.1/anti-HER2 Hc-: antiHER-2 Hc segment was cloned in Notl and CIaI site of the vector containing NO S/MAR sequence and the presence of other component of the vector are depicted in the legend in the figure and also explained in detail in the section 'the detail description of the invention'.
  • Figure 2 Construct map of pCDNA3.1/anti-HER2 Lc-: antiHER-2 Lc segment was cloned in Notl and CIaI site of the vector containing NO S/MAR sequence and the presence of other component of the vector are depicted in the legend in the figure and also explained in detail in the section 'the detail description of the invention'.
  • FIG. 3 Construct map of pCDNA3.1/MARl/anti HER2-Hc-: antiHER-2 Hc segment was cloned in Notl and CIaI site of the vector containing S/MAR sequence upstream of the CMV and the presence of other component of the vector are depicted in the legend in the figure and also explained in detail in the section 'the detail description of the invention'.
  • FIG. 4 Construct map of pCDNA3.1/MARl/anti HER2-Lc: - antiHER2 Lc segment was cloned in Notl and CIaI site of the vector containing S/MAR sequence upstream of the CMV and the presence of other component of the vector are depicted in the legend in the figure and also explained in detail in the section 'the detail description of the invention'.
  • the present invention relates to an expression vector carrying Scaffold/Matrix Attachment Region(s) (S/MAR).
  • Present invention also relates to the expression vector carrying Scaffold/Matrix Attachment Region(s) (S/MAR) sequences.
  • the vector is a Eukaryotic vector.
  • the vector is used for production of Monoclonal antibody to human epidermal growth factor receptor-2 (HER-2).
  • the Monoclonal antibody to human epidermal growth factor receptor-2 (HER-2) protein is a recombinant Monoclonal antibody to human epidermal growth factor receptor-2 (HER-2) protein.
  • the above-mentioned vector is a Eukaryotic vector.
  • the present invention also relates to a host cell comprising an expression vector carrying Scaffold/Matrix Attachment Region(s) (S/MAR).
  • S/MAR Scaffold/Matrix Attachment Region
  • Present invention encompasses a novel expression vector using the above said S/MAR to produce larger quantity of recombinant Trastuzumab.
  • the expression vector is constructed by using ORF (Open Reading Frame) of one of the two polypeptide (heavy) chain of the Trastuzumab (anti HER-2 antibody).
  • ORF' s are flanked by the CMV promoter at the upstream and SV40 poly A signal at the down stream.
  • a human gastrin terminator was inserted in front of the SV40 polyA signal.
  • the vector also contained the bacterial beta-lactamase gene from Transposon Tn3 (AmpR), conferring ampicillin resistance, and the bacterial CoIEl origin of replication (Fig 1).
  • the expression vector is constructed by using ORF of one of the two polypeptide (light) chain of the Trastuzumab (anti HER-2 antibody).
  • the ORF' s are flanked by the CMV promoter at the upstream and SV40 poly A signal at the down stream.
  • a human gastrin terminator was inserted in front of the SV40 polyA signal.
  • the vector also contained the bacterial beta-lactamase gene from Transposon Tn3 (AmpR), conferring ampicillin resistance, and the bacterial CoIEl origin of replication (Fig 2).
  • vector vector containing Hc chain and the other vector containing Lc chain of Tratuzumab with out S/MAR sequence were co-transfected and the expression of the monoclonal antibody against the HER-2 (anti HER-2 antibody) were compared with that of the vector which is detailed below.
  • the expression vector contained ORF of one of the two polypeptide (heavy) chains of the Trastuzumab (and HER-2 antibody).
  • the ORF' s are flanked by the CMV promoter at the upstream and SV40 poly A signal at the down stream.
  • a human gastrin terminator was inserted in front of the SV40 polyA signal.
  • the whole Expression cassette is flanked by human S/MAR (Scaffold/Matrix Attachment Regions) elements at the upstream of the promoter.
  • the vector also contained the bacterial beta-lactamase gene from Transposon Tn3 (AmpR), conferring ampicillin resistance, and the bacterial CoIEl origin of replication (Fig3).
  • the expression vector contained ORF of one of the two polypeptide (light) chains of the Trastuzumab (and HER-2 antibody).
  • the ORF's are flanked by the CMV promoter at the upstream and SV40 poly A signal at the down stream.
  • a human gastrin terminator was inserted in front of the SV40 polyA signal.
  • the whole Expression cassette is flanked by human S/MAR (Scaffold/Matrix Attachment Regions) elements at the upstream of the promoter.
  • the vector also contained the bacterial beta-lactamase gene from Transposon Tn3 (AmpR), conferring ampicillin resistance, and the bacterial CoIEl origin of replication (Fig 4)
  • vector vector containing Hc chain and the other vector containing Lc chain of Tratuzumab with S/MAR sequence were co transfected and the expression of the monoclonal antibody against the HER-2 (anti HER2 antibody) were compared with that of the vector which is detailed above (Figl&2).
  • the ORF of the heavy chain and the light chain of Trastuzumab was amplified with the primers comprising of Notl and Clal respectively and cloned with the same in to the vector explained above.
  • the present invention also relates to Monoclonal antibody to human epidermal growth factor receptor-2 (HER-2) protein expressed by the expression vector carrying Scaffold/Matrix Attachment Region(s) (S/MAR) (Fig. 1-4).
  • HER-2 human epidermal growth factor receptor-2
  • S/MAR Scaffold/Matrix Attachment Region(s)
  • the present invention comprises novel DNA compounds that encode Monoclonal antibody to human epidermal growth factor receptor-2 (HER-2) activity.
  • a novel Eukaryotic expression vector has been constructed that comprise the novel Monoclonal antibody to human epidermal growth factor receptor-2 (HER-2) protein activity-encoding DNA and drive expression of Monoclonal antibody to human epidermal growth factor receptor-2 (HER-2) activity when transfected into an appropriate cell line.
  • the novel expression vector can be used to produce soluble Monoclonal antibody to human epidermal growth factor receptor-2 (HER-2).
  • the recombinant-produced Monoclonal antibody to human epidermal growth factor receptor-2 (HER-2) activity is useful in the treatment and prevention of varieties of cancer.
  • the present invention relates to use of novel Eukaryotic expression vector used for producing soluble Monoclonal antibody to human epidermal growth factor receptor-2 (HER-2) in increased quantity.
  • novel Eukaryotic expression vector used for producing soluble Monoclonal antibody to human epidermal growth factor receptor-2 (HER-2) in increased quantity.
  • Prokaryotic expression systems were part of the early repertoire of research tools and techniques 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.
  • problems Among the two most critical were improper protein folding and assembly, and the lack of posttranslational 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 system is generally preferred for manufacturing of therapeutic proteins, for simple reason that as post-translational modifications required will be addressed by the system.
  • CHO SES Chinese Hamster Ovary (CHO) cell stable expression systems
  • BHK baby hamster kidney
  • HEK Human Embryonic Kidney
  • mouse L-cells mouse L-cells
  • myeloma cell lines like J558L and Sp2/0, etc.
  • the integration of foreign DNA into the genome of a host cell is a chaotic and typically random process. It has been well documented that the transgene expression is highly variable among cell lines and its integration may cause unexpected changes in the phenotype. Reasons underlying the large variability in clonal expression levels include differing plasmid copy numbers and a phenomenon known as the position effect, which was initially described in Drosophila melanogaster as position-effect variegation.
  • the position of integration can influence transgene expression through at least three mechanisms: the activity of local regulatory elements, the local chromatin structure and the local state of DNA methylation. Two common approaches can be used to protect DNA from negative position effects or integration-dependent repression.
  • One approach will be to direct transgene integration into a predetermined site that is transcriptionally active using site-specific recombination methods. Another method is to simply incorporate into the expression vector DNA sequence elements found in chromatin border regions, such that regardless of the integration site the gene will be protected from surrounding chromatin influences. For recombinant protein expression, 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 suggested that flanking transgene with 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. MARs often include the origins of replication (ORI) and can possess a concentrated area of transcription factor binding sites. Approximately 100 000 matrix attachment sites are believed to exist in the mammalian nucleus of which 30 000-40 000 serve as ORIs. MARs have been observed to flank the ends of domains encompassing various transcriptional units. It has also been shown that MARs bring together the transcriptionally active regions of chromatin such that the transcription is initiated in the region of the chromosome that coincides with the surface of nuclear matrix.
  • ORI origins of replication
  • S/MARs may define boundaries of independent chromatin domains, such that only the encompassing ds-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.
  • S/MARs 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.
  • 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 had also shown to increase the levels of regulated or constitutive transgene expression in various mammalian cell lines. Recently, inclusion of this MAR sequence increased overall expression of transgene when transfected into CHO cell line.
  • mammalian expression system is generally preferred for manufacturing most of therapeutic proteins, as they require post-translational modifications.
  • a variety of mammalian cell expression systems are now available for expression of proteins.
  • the level of expression of a recombinant protein achieved from these expression vectors/systems in mammalian cells is not commercially viable.
  • the present invention relates to a construction of the novel expression vector by using the above- mentioned S/MAR to produce Trastuzumab in larger quantity.
  • products of expression of the DNA sequence display the biological activities of Monoclonal antibody to human epidermal growth factor receptor-2 (HER-2).
  • Chrosome is 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 informations. It is made up of different nucleotides A, G, T or C.
  • a “gene” is a deoxyribonucleotide (DNA) sequence coding for a given mature protein. “Gene” shall not include untranslated flanking regions such as RNA transcription initiation signals, polyadenylation addition sites, promoters or enhancers. “Promoter” is a nucleic acid sequence that controls 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.
  • 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.
  • 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 precedign the secreted mature protein. In mature protein it is not present as it is cleaved.
  • vector referes herein is a nucleic acid molecule capable of transporitgn 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.
  • Plasmid is 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 the synthesis of RNA from a DNA template.
  • Translation means the synthesis of a polypeptide from messenger RNA.
  • Cross refers to the placement of two or more DNA elements linked on the same plasmid.
  • 'Trans refers to the placement of two or more elements on two or more different plasmids.
  • Orientation refers to the order of nucleotides in the DNA sequence.
  • an "isolated nucleic acid fragment” is 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 organism. Gene amplification can be classified in two categories (i) developmentally regulated gene expression as seen in Xenopus oocytes and (ii) spontaneously occuring 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 nucelic 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, e.g., 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.
  • Stem cells e.g., 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.
  • Transfection means the introduction of a foreign material like DNA into eukaryotic cells by any means of trasnfer. Different method of transfection includes Calcium phosphate, electroporation, lipofectamine and DEAE-Dextran transfection etc.
  • '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.
  • Transient gene expression refers to a convenient method for the rapid production of small quantities of proetin. Generally COS cells are mostly used for transient expression characterization. "Stable gene expression” means preparation of stable cell lines that permanently express the gene of interest depending on the stable integration of plasmid into the host chromosome.

Abstract

The present invention relates to obtain expression vectors and compounds of expression of recombinant anti-Human Epidermal Growth Factor Receptor-2 (HER-2) monoclonal antibody protein. The present invention further relates to use of novel expression vectors carrying Scaffold/Matrix Attachment Region (S/MAR) for production of Recombinant Monoclonal antibody to human epidermal growth factor receptor-2 (HER-2) in increased quantity.

Description

Vectors And Compounds For Expression Of Recombinant Trastuzumab
FIELD OF THE INVENTION
The present invention relates to use of novel expression vectors and compounds of expression used for production of Recombinant Monoclonal antibody to human epidermal growth factor receptor-2 (HER-2) in increased quantity. The present invention further realtes to method of construction of the expression vector and to obtain monoclonal antibody to human epidermal growth factor receptor-2 (HER-2) protein expressed by the expression vector carrying Scaffold/Matrix Attachment Region (S/MAR).
BACKGROUND OF THE INVENTION
In 1986, Food and Drug Administration (FDA) approved human Tissue Plasminogen Activator (tPA; Genentech, CA, USA) protein from mammalian cells to be used for therapeutic purpose. It was the beginning, currently there are many more monoclonal antibodies, which got the regulatory approval from FDA. Moreover, several hundreds are in pipeline. Like t-PA, most of these proteins are expressed immortalized Chinese Hamster Ovary (CHO) cells, but other cell lines, such as mouse myeloma (NSO), Baby Hamster Kidney (BHK), Human Embryo Kidney (HEK-293) are approved for recombinant protein production. There are two critical issues during the production of therapeutics (a) time taken to provide the material (b) lowering the price of the material to the common user. Therefore, biotechnology and pharmaceutical industry continues to look at new technologies and process development strategies that will decrease timelines and also will help in reducing the price.
As mentioned, mammalian expression system is generally preferred for manufacturing most of therapeutic proteins, because they require post-translational modifications. 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 recombinant gene, However, the level of expression of a recombinant protein attained from these expression vectors/systems in mammalian cells is not commercially viable. Trastuzumab (Herceptin) is a recombinant humanized IgGIk monoclonal antibody glycoprotein that binds specifically to human epidermal growth factor receptor-2 (HER-2). Herceptin is designed to target and block the function of HER-2 protein over expression. The antibody, Trastuzumab contains primarily human constant framework regions with the Complementarity- determining regions (CDR) from a murine antibody with specificity for human HER-2. The humanized antibody against HER-2, Trastuzumab having an approximate molecular weight of 155 kilo-daltons is produced by a mammalian cell (Chinese Hamster Ovary) [CHO] suspension culture in a nutrient medium containing the antibiotic gentamicin. Gentamicin is not detectable in the final product. Trastuzumab is packaged as a sterile, white to pale yellow, preservative-free lyophilized powder for intravenous (IV) administration.
Cancerous breast tissue cells that over-express the HER-2 gene produce extra protein receptors on the cell surface. The higher density of receptors triggers the cell to divide and multiply at an accelerated rate, thus contributing to tumour growth. Trastuzumab binds to numerous HER-2 receptor sites found on the cell surface, blocking the receptor sites and possibly preventing further growth by interrupting the growth signal.
US Patent Application number- 11/400,638 provides Trastuzumab as a agent is indicated for the treatment of patients with metastatic breast cancer whose tumors over-express the HER-2 protein and who have received one or more chemotherapy regimens for their metastatic disease. Trastuzumab in combination with paclitaxel is indicated for treatment of patients with metastatic breast cancer whose tumors over-express the HER-2 protein and who have not received chemotherapy for their metastatic disease. This antibody should only be used in patients whose tumors have HER-2 protein over-expression.
In order to facilitate production of large quantities of Monoclonal antibody to human epidermal growth factor receptor-2 (HER-2) from cell culture, a novel expression vector has been design and developed with genetic compounds. One of the uses of this expression vector has been shown to increase and amplify the expression of therapeutic protein. The cloning, expression and purification of Monoclonal antibody to human epidermal growth factor receptor-2 (HER-2) have been mentioned in this present application. OBJECTIVES OF THE PRESENT INVENTION
The foremost objective of the present invention is to construct an expression vector carrying Scaffold/Matrix Attachment Region(s) (SMAR).
Another main objective of the present invention is to obtain an expression vector carrying Scaffold/Matrix Attachment Region(s) (S/MAR) used for production of Monoclonal antibody to human epidermal growth factor receptor-2 (HER-2).
Yet another objective of the present invention is to develop a method for construction of an expression vector carrying Scaffold/Matrix Attachment Region(s) (S/MAR).
Still another objective of the present invention is to obtain a host cell comprising an expression vector carrying Scaffold/Matrix Attachment Region(s) (S/MAR).
Still another objective of the present invention is to obtain Monoclonal antibody to human epidermal growth factor receptor-2 (HER-2) protein expressed by the expression vector carrying Scaffold/Matrix Attachment Region(s) (S/MAR).
STATEMENT OF THE PRESENT INVENTION
Accordingly, the present invention relates to an expression vector carrying Scaffold/Matrix Attachment Region(s) (S/MAR); a method for construction of an expression vector carrying Scaffold/Matrix Attachment Region(s) (S/MAR), said method comprising step of inserting S/MAR into the expression vector; a host cell comprising an expression vector carrying Scaffold/Matrix Attachment Region(s) (S/MAR); Monoclonal antibody to human epidermal growth factor receptor-2 (HER-2) expressed by the expression vector carrying Scaffold/Matrix Attachment Region(s) (S/MAR). DETAILED DESCRIPTION OF THE DRAWINGS
Figure 1: Construct map of pCDNA3.1/anti-HER2 Hc-: antiHER-2 Hc segment was cloned in Notl and CIaI site of the vector containing NO S/MAR sequence and the presence of other component of the vector are depicted in the legend in the figure and also explained in detail in the section 'the detail description of the invention'.
Figure 2: Construct map of pCDNA3.1/anti-HER2 Lc-: antiHER-2 Lc segment was cloned in Notl and CIaI site of the vector containing NO S/MAR sequence and the presence of other component of the vector are depicted in the legend in the figure and also explained in detail in the section 'the detail description of the invention'.
Figure 3: Construct map of pCDNA3.1/MARl/anti HER2-Hc-: antiHER-2 Hc segment was cloned in Notl and CIaI site of the vector containing S/MAR sequence upstream of the CMV and the presence of other component of the vector are depicted in the legend in the figure and also explained in detail in the section 'the detail description of the invention'.
Figure 4: Construct map of pCDNA3.1/MARl/anti HER2-Lc: - antiHER2 Lc segment was cloned in Notl and CIaI site of the vector containing S/MAR sequence upstream of the CMV and the presence of other component of the vector are depicted in the legend in the figure and also explained in detail in the section 'the detail description of the invention'.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to an expression vector carrying Scaffold/Matrix Attachment Region(s) (S/MAR).
Present invention also relates to the expression vector carrying Scaffold/Matrix Attachment Region(s) (S/MAR) sequences.
In another embodiment of the present invention, the vector is a Eukaryotic vector.
In yet another embodiment of the present invention, the vector is used for production of Monoclonal antibody to human epidermal growth factor receptor-2 (HER-2). In still another embodiment of the present invention, the Monoclonal antibody to human epidermal growth factor receptor-2 (HER-2) protein is a recombinant Monoclonal antibody to human epidermal growth factor receptor-2 (HER-2) protein.
In another embodiment of the present invention, the above-mentioned vector is a Eukaryotic vector.
The present invention also relates to a host cell comprising an expression vector carrying Scaffold/Matrix Attachment Region(s) (S/MAR).
Present invention encompasses a novel expression vector using the above said S/MAR to produce larger quantity of recombinant Trastuzumab.
Foremost, the expression vector is constructed by using ORF (Open Reading Frame) of one of the two polypeptide (heavy) chain of the Trastuzumab (anti HER-2 antibody). The ORF' s are flanked by the CMV promoter at the upstream and SV40 poly A signal at the down stream. A human gastrin terminator was inserted in front of the SV40 polyA signal. The vector also contained the bacterial beta-lactamase gene from Transposon Tn3 (AmpR), conferring ampicillin resistance, and the bacterial CoIEl origin of replication (Fig 1).
The expression vector is constructed by using ORF of one of the two polypeptide (light) chain of the Trastuzumab (anti HER-2 antibody). The ORF' s are flanked by the CMV promoter at the upstream and SV40 poly A signal at the down stream. A human gastrin terminator was inserted in front of the SV40 polyA signal. The vector also contained the bacterial beta-lactamase gene from Transposon Tn3 (AmpR), conferring ampicillin resistance, and the bacterial CoIEl origin of replication (Fig 2).
These vector (vector containing Hc chain and the other vector containing Lc chain of Tratuzumab with out S/MAR sequence were co-transfected and the expression of the monoclonal antibody against the HER-2 (anti HER-2 antibody) were compared with that of the vector which is detailed below. The expression vector contained ORF of one of the two polypeptide (heavy) chains of the Trastuzumab (and HER-2 antibody). The ORF' s are flanked by the CMV promoter at the upstream and SV40 poly A signal at the down stream. A human gastrin terminator was inserted in front of the SV40 polyA signal. The whole Expression cassette is flanked by human S/MAR (Scaffold/Matrix Attachment Regions) elements at the upstream of the promoter. The vector also contained the bacterial beta-lactamase gene from Transposon Tn3 (AmpR), conferring ampicillin resistance, and the bacterial CoIEl origin of replication (Fig3).
The expression vector contained ORF of one of the two polypeptide (light) chains of the Trastuzumab (and HER-2 antibody). The ORF's are flanked by the CMV promoter at the upstream and SV40 poly A signal at the down stream. A human gastrin terminator was inserted in front of the SV40 polyA signal. The whole Expression cassette is flanked by human S/MAR (Scaffold/Matrix Attachment Regions) elements at the upstream of the promoter. The vector also contained the bacterial beta-lactamase gene from Transposon Tn3 (AmpR), conferring ampicillin resistance, and the bacterial CoIEl origin of replication (Fig 4)
These vector (vector containing Hc chain and the other vector containing Lc chain of Tratuzumab with S/MAR sequence were co transfected and the expression of the monoclonal antibody against the HER-2 (anti HER2 antibody) were compared with that of the vector which is detailed above (Figl&2).
The ORF of the heavy chain and the light chain of Trastuzumab was amplified with the primers comprising of Notl and Clal respectively and cloned with the same in to the vector explained above.
The present invention also relates to Monoclonal antibody to human epidermal growth factor receptor-2 (HER-2) protein expressed by the expression vector carrying Scaffold/Matrix Attachment Region(s) (S/MAR) (Fig. 1-4).
The present invention comprises novel DNA compounds that encode Monoclonal antibody to human epidermal growth factor receptor-2 (HER-2) activity. A novel Eukaryotic expression vector has been constructed that comprise the novel Monoclonal antibody to human epidermal growth factor receptor-2 (HER-2) protein activity-encoding DNA and drive expression of Monoclonal antibody to human epidermal growth factor receptor-2 (HER-2) activity when transfected into an appropriate cell line. The novel expression vector can be used to produce soluble Monoclonal antibody to human epidermal growth factor receptor-2 (HER-2). The recombinant-produced Monoclonal antibody to human epidermal growth factor receptor-2 (HER-2) activity is useful in the treatment and prevention of varieties of cancer.
The present invention relates to use of novel Eukaryotic expression vector used for producing soluble Monoclonal antibody to human epidermal growth factor receptor-2 (HER-2) in increased quantity.
Prokaryotic expression systems were part of the early repertoire of research tools and techniques 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. Among the two most critical were improper protein folding and assembly, and the lack of posttranslational 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 system is generally preferred for manufacturing of therapeutic proteins, for simple reason that as post-translational modifications required will be addressed by the system. A variety of mammalian cell expression systems are now available for either the transient expression of recombinant genes or stably transfected ones. Generally, Chinese Hamster Ovary (CHO) cell stable expression systems (CHO SES) are used for this purpose to express recombinant genes. Moreover, baby hamster kidney (BHK) cells, Human Embryonic Kidney (HEK) 293 cells, mouse L-cells, and myeloma cell lines like J558L and Sp2/0, etc., are also employed as hosts for the establishment of stable transfectants.
However, the integration of foreign DNA into the genome of a host cell is a chaotic and typically random process. It has been well documented that the transgene expression is highly variable among cell lines and its integration may cause unexpected changes in the phenotype. Reasons underlying the large variability in clonal expression levels include differing plasmid copy numbers and a phenomenon known as the position effect, which was initially described in Drosophila melanogaster as position-effect variegation. The position of integration can influence transgene expression through at least three mechanisms: the activity of local regulatory elements, the local chromatin structure and the local state of DNA methylation. Two common approaches can be used to protect DNA from negative position effects or integration-dependent repression. One approach will be to direct transgene integration into a predetermined site that is transcriptionally active using site-specific recombination methods. Another method is to simply incorporate into the expression vector DNA sequence elements found in chromatin border regions, such that regardless of the integration site the gene will be protected from surrounding chromatin influences. For recombinant protein expression, 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 suggested that flanking transgene with 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. MARs often include the origins of replication (ORI) and can possess a concentrated area of transcription factor binding sites. Approximately 100 000 matrix attachment sites are believed to exist in the mammalian nucleus of which 30 000-40 000 serve as ORIs. MARs have been observed to flank the ends of domains encompassing various transcriptional units. It has also been shown that MARs bring together the transcriptionally active regions of chromatin such that the transcription is initiated in the region of the chromosome that coincides with the surface of nuclear matrix.
As such, they may define boundaries of independent chromatin domains, such that only the encompassing ds-regulatory elements control the expression of the genes within the domain. A number of possible functions have been discussed earlier for 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. S/MARs 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. 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 had also shown to increase the levels of regulated or constitutive transgene expression in various mammalian cell lines. Recently, inclusion of this MAR sequence increased overall expression of transgene when transfected into CHO cell line.
As previously mentioned, mammalian expression system is generally preferred for manufacturing most of 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 a recombinant protein achieved from these expression vectors/systems in mammalian cells is not commercially viable.
The present invention relates to a construction of the novel expression vector by using the above- mentioned S/MAR to produce Trastuzumab in larger quantity. Upon isolation from culture media, products of expression of the DNA sequence display the biological activities of Monoclonal antibody to human epidermal growth factor receptor-2 (HER-2).
The following definitions are used in order to help in understanding the invention. "Chromosome" is 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 informations. It is made up of different nucleotides A, G, T or C.
A "gene" is a deoxyribonucleotide (DNA) sequence coding for a given mature protein. "Gene" shall not include untranslated flanking regions such as RNA transcription initiation signals, polyadenylation addition sites, promoters or enhancers. "Promoter" is a nucleic acid sequence that controls 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.
"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.
"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 precedign the secreted mature protein. In mature protein it is not present as it is cleaved.
The definition of "vector" referes herein is a nucleic acid molecule capable of transporitgn 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.
"Plasmid" is 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 the synthesis of RNA from a DNA template.
"Translation" means the synthesis of a polypeptide from messenger RNA. "Cis" refers to the placement of two or more DNA elements linked on the same plasmid. 'Trans" refers to the placement of two or more elements on two or more different plasmids. "Orientation" refers to the order of nucleotides in the DNA sequence.
As used herein, an "isolated nucleic acid fragment" is 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 organism. Gene amplification can be classified in two categories (i) developmentally regulated gene expression as seen in Xenopus oocytes and (ii) spontaneously occuring gene expression as amplification of the lac region reported in Escherichia coli. The best-known gene amplification in mammalian cells is dihydrofolate reductase (DHFR).
'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 nucelic 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, e.g., 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.
"Transfection" means the introduction of a foreign material like DNA into eukaryotic cells by any means of trasnfer. Different method of transfection includes Calcium phosphate, electroporation, lipofectamine and DEAE-Dextran transfection etc.
'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.
"Cotransfection" is the method of simultaneous transfection.
'Transient gene expression" refers to a convenient method for the rapid production of small quantities of proetin. Generally COS cells are mostly used for transient expression characterization. "Stable gene expression" means preparation of stable cell lines that permanently express the gene of interest depending on the stable integration of plasmid into the host chromosome.

Claims

We claim
1) A matrix attachment region sequence[s] (SEQ 1) or its complementary sequence[s], variants] and fragment[s] thereof.
2) The sequence as claimed in claim 1, wherein said sequence increases protein production by modulating transcription efficiency.
3) The sequence as claimed in claim 1, wherein said sequence promotes transient and stable transfection to enhance expression of recombinant proteins.
4) A process to obtain a matrix attachment region sequence[s] or its complementary sequence[s], variants] and fragments] thereof.
5) An expression vectors carrying a matrix attachment region sequence[s] or its complementary sequencefs], variants] and fragment[s] thereof.
Urhe expression vectors as claimed in claim 5 wherein said expression vector is mammalian expression vector.
7) A eukaryotic cell with a matrix attachment region sequencefs] or its complementary sequence[s], variants] and fragments] thereof.
8) The sequence as claimed in claim 1, wherein said sequence promotes transient and stable transfection to enhance expression of recombinant proteins orientation independently.
9) The sequence as claimed in claim 1, wherein transcription factors can bind to those said sequences.
10) Position of these said sequences as mentioned in claim 1 in genome.
11 ) Position of said sequence in vector backbone, could be upstream of promoter.
12) Position of said sequence in vector backbone, could be downstream of termination signal.
13) Combination of above claim 11 and 12.
14) The vectors as claimed in claim 5, wherein the expression vector is used for production of Trastuzumab (HER2) monoclonal antibody.
15) A method for construction of an expression vector carrying Scaffold/Matrix Attachment Region(s) (S/MAR), said method comprising step of inserting S/MAR into the expression vector.
16) HER2 monoclonal antibody protein expressed by the expression vector carrying Scaffold/Matrix Attachment Region(s).
PCT/IB2010/001906 2009-08-03 2010-08-03 Vectors and compounds for expression of recombinant trastuzumab WO2011015924A2 (en)

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002074969A2 (en) * 2001-01-26 2002-09-26 University Of Lausanne Matrix attachment regions and methods for use thereof
WO2003024199A2 (en) * 2001-09-21 2003-03-27 Avigenics, Inc. Production of transgenic avians using sperm-mediated transfection
WO2005047512A2 (en) * 2003-11-12 2005-05-26 Shering Corporation Plasmid system for multigene expression
WO2008142124A1 (en) * 2007-05-21 2008-11-27 Vivalis Recombinant protein production in avian ebx® cells
WO2010018444A2 (en) * 2008-08-12 2010-02-18 Avesthagen Limited An expression vector and a method thereof
WO2010049777A1 (en) * 2008-10-28 2010-05-06 Avesthagen Limited An expression vector and processes thereof
WO2010118360A1 (en) * 2009-04-09 2010-10-14 The Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College Production of proteins using transposon-based vectors

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002074969A2 (en) * 2001-01-26 2002-09-26 University Of Lausanne Matrix attachment regions and methods for use thereof
WO2003024199A2 (en) * 2001-09-21 2003-03-27 Avigenics, Inc. Production of transgenic avians using sperm-mediated transfection
WO2005047512A2 (en) * 2003-11-12 2005-05-26 Shering Corporation Plasmid system for multigene expression
WO2008142124A1 (en) * 2007-05-21 2008-11-27 Vivalis Recombinant protein production in avian ebx® cells
WO2010018444A2 (en) * 2008-08-12 2010-02-18 Avesthagen Limited An expression vector and a method thereof
WO2010049777A1 (en) * 2008-10-28 2010-05-06 Avesthagen Limited An expression vector and processes thereof
WO2010118360A1 (en) * 2009-04-09 2010-10-14 The Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College Production of proteins using transposon-based vectors

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CARTER, P. ET AL.: 'Humanization of an anti-p185HER2 antibody for human cancer therapy' PNAS vol. 89, 1992, pages 4285 - 4289 *
GIROD, P-A. ET AL.: 'Use of the chicken lysozyme 5' matrix attachment region to generate high producer CHO cell lines' BIOTECHNOLOGY AND BIOENGINEERING vol. 91, no. 1, 2005, pages 1 - 11 *
ZAHN-ZABAL, M. ET AL.: 'Development of stable cell lines for production or regulated expression using matrix attachment regions' JOURNAL OF BIOTECHNOLOGY vol. 87, 2001, pages 29 - 42 *

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