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WO2011015917A2 - Vecteurs et composés pour expression de tnk-tpa recombinant (tenecteplase) - Google Patents

Vecteurs et composés pour expression de tnk-tpa recombinant (tenecteplase) Download PDF

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Publication number
WO2011015917A2
WO2011015917A2 PCT/IB2010/001894 IB2010001894W WO2011015917A2 WO 2011015917 A2 WO2011015917 A2 WO 2011015917A2 IB 2010001894 W IB2010001894 W IB 2010001894W WO 2011015917 A2 WO2011015917 A2 WO 2011015917A2
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WO
WIPO (PCT)
Prior art keywords
sequence
expression
tpa
expression vector
tnk
Prior art date
Application number
PCT/IB2010/001894
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English (en)
Other versions
WO2011015917A3 (fr
Inventor
Willoo Morawala Patell
Sami N. Guzder
Sunit Maity
Sunil Shekar
Original Assignee
Avesthagen Limited
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Avesthagen Limited filed Critical Avesthagen Limited
Publication of WO2011015917A2 publication Critical patent/WO2011015917A2/fr
Publication of WO2011015917A3 publication Critical patent/WO2011015917A3/fr

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Classifications

    • 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
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6456—Plasminogen activators
    • C12N9/6459—Plasminogen activators t-plasminogen activator (3.4.21.68), i.e. tPA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21069—Protein C activated (3.4.21.69)

Definitions

  • the present invention relate to vectors and compounds of expression for Recombinant Monoclonal antibody to human epidermal growth factor receptor-2 (HER-2).
  • HER-2 human epidermal growth factor receptor-2
  • tPA human tissue plasminogen activator
  • mammalian expression system is generally preferred for manufacturing most of therapeutic proteins, as they require post-translational modifications.
  • 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.
  • the level of expression of a recombinant protein achieved from these expression vectors/systems in mammalian cells is not commercially viable.
  • Plasminogen activators are enzymes that activate the zymogen, plasminogen to generate the serine proteinase plasmin, which in turn degrades fibrin.
  • plasminogen activators include streptokinase, urokinase and human tissue plasminogen activator (t- PA). The mechanism of action of each of these plasminogen activators is different from each other. Streptokinase forms a complex with plasminogen generating plasmin activity, urokinase cleaves plasminogen directly and t-PA forms a ternary complex with fibrin and plasminogen, leading to plasminogen activation in the locality of the clot.
  • Tissue type plasminogen activator a multidomain, glycosylated, serine protease is a fibrin specific activator of plasminogen and a very effective thrombolytic agent.
  • t-PA is a recombinant protein whose primary application is in the treatment of heart attack and stroke patients.
  • Natural t-PA has a plasma half-life of about six minutes or less. Due to its rapid clearance from the circulation, t-PA has to be infused to achieve thrombolysis. Front loaded dosing with increased concentrations of t-PA has shown more rapid and complete lysis compared to the standard infusion protocol and early potency is correlated with improved survival rate. Bolus administration could further improve the lytic rate by quickly exposing the target clot to a higher concentration of the enzyme, but single bolus administration of natural or wild type (wt) t-PA cannot be generally used, due its clearance rate.
  • wt wild type
  • TNK-tPA Tenecteplase
  • 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.
  • 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).
  • TNK-tPA In order to facilitate production of large quantities of TNK-tPA from cell culture, a novel expression vector has been developed with genetic compounds. Use of this expression vector has been shown to increase the expression of therapeutic protein.
  • the cloning, sub-cloning and expression of TNK-tPA have been mentioned in this application.
  • the main objective of the present invention is to obtain 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 soluble TNK- tPA (Tenecteplase).
  • 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 soluble TNK-tPA (Tenecteplase) protein expressed by the expression vector carrying Scaffold/Matrix Attachment Region(s) (S/MAR).
  • the present invention relates to the construction of an eukaryotic expression vector carrying Scaffold/Matrix Attachment Region(s) (S/MAR) used for production of Recombinant Monoclonal antibody to human epidermal growth factor receptor-2 (HER-2).
  • S/MAR Scaffold/Matrix Attachment Region(s)
  • FIG. 1 Construct map of pCDNA3.1/Tpa: TNK-tPA 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.
  • FIG. 2 Construct map of pCDNA3.1/MARl/tPA: TNK-tPA segment was cloned in Notl and CIaI site of the vector containing S/MAR sequence upstream of the CMVand the presence of other component of the vector are depicted in the legend in the figure.
  • Figure 3 Comparative Protein Expression: 7 fold increase in expression of a therapeutic protein was observed using MARl as regulatory element in the vector backbone at Cell line development stage.
  • the present invention relates to a host cell comprising an expression vector carrying Scaffold/Matrix Attachment Region(s) (S/MAR) and expressing soluble TNK-tPA (Tenecteplase)(Fig. 1-3).
  • the soluble TNK-tPA (Tenecteplase) protein is a recombinant Monoclonal antibody to human TNF- alpha protein.
  • the vector is a eukaryotic vector.
  • 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 cw-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 inventors overcame problems arising from the site-specific effect when genes are expressed in prokaryotic systems, and designed an optimal expression vector that increases the expressed amount of the proteins.
  • the present invention comprises novel DNA compounds which encode soluble TNK-tPA (Tenecteplase) activity.
  • a novel eukaryotic expression vector has been constructed that comprise the novel soluble TNK-tPA (Tenecteplase) activity-encoding DNA and drive expression of soluble TNK-tPA (Tenecteplase) activity when transfected into an appropriate cell line.
  • the novel expression vector can be used to produce soluble TNK- tPA (Tenecteplase)
  • the recombinant-produced soluble TNK-tPA (Tenecteplase) activity is useful in the treatment and prevention of stroke.
  • the present invention also relates to use of novel eukaryotic expression vector used for producing soluble TNK-tPA (Tenecteplase) in increased quantity.
  • the expression vector contains ORF of the TNK-tPA.
  • the ORFs are flanked by the CMV promoter at the upstream and SV40 poly A signal at the down stream.
  • a human gastrin terminator is inserted in front of the SV40 polyA signal.
  • the vector also contains the bacterial beta-lactamase gene from Transposon Tn3 (AmpR), conferring ampicillin resistance, and the bacterial CoIEl origin of replication (Fig 1).
  • the vector (vector containing TNK-tPA with out S/MAR sequence) is transfected and the expression of the TNK-tPA compared with that of the vector which is detailed below.
  • the expression vector contains ORF of the TNK-tPA.
  • the ORFs are flanked by the CMV promoter at the upstream and SV40 poly A signal at the down stream.
  • a human gastrin terminator Is 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 contains the bacterial beta-lactamase gene from Transposon Tn3 (AmpR), conferring ampicillin resistance, and the bacterial CoIEl origin of replication (Fig 2).
  • the vector (vector containing TNK-tPA) with S/MAR sequence was transfected and the expression of the TNK-tPA was compared with that of the vector which is detailed above (Fig 3).
  • the comparative study showed that using MARl as regulatory element in the vector back bone led to 7 fold increase in the expression of the therapeutic protein.
  • the ORF of the TNK-tPA was amplified with the primers containing Notl and Clal respectively and cloned with the same in to the vector explained above.

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  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne des vecteurs et des composés d’expression et leur construction pour l’expression à un niveau élevé de protéine TNK-tPA (Tenecteplase) soluble qui peut être utilisée en tant que médicament.
PCT/IB2010/001894 2009-08-03 2010-08-02 Vecteurs et composés pour expression de tnk-tpa recombinant (tenecteplase) WO2011015917A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IN1846CH2009 2009-08-03
IN1846/CHE/2009 2009-08-03

Publications (2)

Publication Number Publication Date
WO2011015917A2 true WO2011015917A2 (fr) 2011-02-10
WO2011015917A3 WO2011015917A3 (fr) 2011-04-21

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130244280A1 (en) * 2010-10-08 2013-09-19 Cadila Healthcare Limited Expression vector for high level expression of recombinant proteins

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DK1395669T3 (da) * 2001-01-26 2009-11-16 Selexis Sa Matriks bindingsregioner og fremgangsmåder til anvendelse af disse
WO2003024199A2 (fr) * 2001-09-21 2003-03-27 Avigenics, Inc. Production d'oiseaux transgeniques par transfection induite par le sperme
CN1938428A (zh) * 2003-11-12 2007-03-28 先灵公司 多基因表达的质粒系统
AU2009280913A1 (en) * 2008-08-12 2010-02-18 Avesthagen Limited An expression vector and a method thereof
AU2009309387A1 (en) * 2008-10-28 2010-05-06 Avesthagen Limited An expression vector and processes thereof

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130244280A1 (en) * 2010-10-08 2013-09-19 Cadila Healthcare Limited Expression vector for high level expression of recombinant proteins

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