WO2011015917A2

WO2011015917A2 - Vectors and compounds for expression of recombinant tnk-tpa (tenecteplase)

Info

Publication number: WO2011015917A2
Application number: PCT/IB2010/001894
Authority: WO
Inventors: Willoo Morawala Patell; Sami N. Guzder; Sunit Maity; Sunil Shekar
Original assignee: Avesthagen Limited
Priority date: 2009-08-03
Filing date: 2010-08-02
Publication date: 2011-02-10
Also published as: WO2011015917A3

Abstract

The present application relates to a scaffold/matrix attachment region (S/MAR) sequence. In particular, the application relates to the use of expression comprising at least one S/MAR sequence for the high level recombinant expression of soluble TNK-tPA (Tenecteplase) protein.

Description

Vectors and Compounds of Expression For Expression of Recombinant Tnk TPA (Tenecteplase)

FIELD OF THE INVENTION

The present invention relate to vectors and compounds of expression for Recombinant Monoclonal antibody to human epidermal growth factor receptor-2 (HER-2).

BACKGROUND OF THE INVENTION

In 1986, 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. Moreover, several hundred are in pipeline. Like tPA, 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, industry continues to look at new technologies and process development strategies that will reduce timelines and also will help in reducing the cost.

As 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. 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 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. Among the plasminogen activators studied are 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.

First characterized in 1979, as an important and potent biological pharmaceutical agent in the treatment of various vascular diseases due to its high fibrin specificity and potent ability to dissolve blood clots in vivo, the protein was ultimately developed into a commercial product by Genentech Inc. with clinical trials initiated in 1984. Tissue type plasminogen activator (t-PA) 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.

Many investigators have produced longer half-life versions of t-PA that could be administered as a bolus, but almost all of the variants turned out to have significantly decreased fibrinolytic activities. To develop a molecule with reduced clearance rate while retaining full fibrinolytic activity, systematic mutagenesis studies were applied to t-PA on its various domains. This research led to the discovery and development of TNK-tPA (Tenecteplase), a genetically modified form of t-PA with enhanced pharmacologic and pharmacokinetic characteristics. 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. 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).

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.

OBJECTIVES OF THE PRESENT INVENTION

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).

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). SUMMARY OF THE INVENTION

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).

DETAILED DESCRIPTION OF THE DRAWINGS

Figure 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.

Figure 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.

DETAILED DESCRIPTION OF THE INVENTION

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). In another embodiment of the present invention, the soluble TNK-tPA (Tenecteplase) protein is a recombinant Monoclonal antibody to human TNF- alpha protein. In another embodiment of the present invention, 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.

As such, they may define boundaries of independent chromatin domains, such that only the encompassing cw-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.

Hereinafter, the present invention will be explained in detail. 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.

Claims

We Claim

1) A matrix attachment region sequence[s] (SEQ 1) or its complementary sequence[s], variantfs] 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], variant[s] and fragmentfs] thereof.

5) An expression vector carrying a matrix attachment region sequence[s] or its complementary sequence[s], variant[s] and fragment[s] thereof.

6) The expression vector as claimed in claim 5 wherein said expression vector is mammalian expression vector.

7) A eukaryotic cell with a matrix attachment region sequence [s] or its complementary sequence[s], variant[s] and fragment[s] 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) Position of these said sequences as mentioned in claim 1 in genome.

10) Position of said sequence in vector backbone, could be upstream of promoter.

11) Position of said sequence in vector backbone, could be downstream of termination signal.

12) Combination of above claim 10 and 11.

13) The vector as claimed in claim 5, wherein the expression vector is used for production of soluble TNK-tPA (Tenecteplase)

14) 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.

15) Soluble TNK-tPA (Tenecteplase) protein expressed by the expression vector carrying Scaffold/Matrix Attachment Region(s). 6) An expression vector, a method for construction, a host cell and soluble TNK-tPA (Tenecteplase) protein as substantially herein described with accompanying examples and figures.