FIELD OF THE INVENTION

The present invention relates to an expression vector including a scaffold matrix attachment region element (hereinafter referred to as "S/MAR region") and more particularly, to an expression vector for expressing Recombinant Infliximab.

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 in 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.

The present invention comprises novel DNA compounds, which encode Monoclonal antibody to human TNF-alpha activity. A novel eukaryotic expression vector has been constructed that comprise the novel Monoclonal antibody to human TNF-alpha protein activity-encoding DNA and drive expression of Monoclonal antibody to human TNF- alpha activity when transfected into an appropriate cell line. The novel expression vector can- be used to produce soluble Monoclonal antibody to human TNF-alpha. The recombinant-produced Monoclonal antibody to human TNF-alpha 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 TNF-alpha 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. 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 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.

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.

Infliximab is a recombinant chimeric (partially humanized) murine monoclonal antibody glycoprotein that binds specifically to human tumor necrosis factor alpha (TNF-alpha) with an association constant of 10 ¹⁰ M ^"1 . TNF-alpha is a naturally occurring cytokine that is involved in normal inflammatory and immune responses. The antibody, Infliximab has the antigen-binding region of a murine immunoglobulin Gl (IgGl) and the constant (Fc) or framework regions of human IgGl kappa immunoglobulin. The antibody, Infliximab, has an approximate molecular weight of 149,100 daltons and is produced by a transformed murine SP2/O cell line cultured by continuous perfusion. Infliximab is supplied as a sterile, white, lyophilized powder for intravenous infusion.

Infliximab neutralizes the biological activity of TNF-alpha and inhibits binding of TNF- alpha with its receptors. Cells expressing transmembrane TNF-alpha bound by Infliximab, can be lysed in vitro by complement or effector cells. Infliximab does not neutralize TNF-beta a related cytokine that utilizes the same receptors as TNF-alpha. Elevated concentrations of TNF-alpha are known to exist in the joints of RA (Rheumatoid Arthritis) patients and the stools of Crohn's disease patients. In RA, Infliximab reduces the infiltration of inflammatory cells into inflamed areas of the joints and also reduces expression of molecules mediating cellular adhesion, chemo attraction and tissue degradation e.g. ICAM-I, IL-8, MCP-I, MMP-I & 3 etc. In Crohn's disease also, Infliximab reduces infiltration of inflammatory cells and decreases TNF-alpha production in inflamed areas of the intestine.

Infliximab, in combination with methotrexate, is indicated for the reduction in signs and symptoms of RA in patients who have had an inadequate response to methotrexate. Infliximab is also indicated for the reduction in signs and symptoms of Crohn's disease in patients with moderately to severely active Crohn's disease who have had an inadequate response to conventional therapy.

In order to facilitate production of large quantities of Infliximab 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 Infliximab 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) (S/MAR).

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 TNF-alpha.

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 TNF-alpha 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 Monoclonal antibody to human TNF-alpha.

DETAILED DESCRIPTION OF THE DRAWINGS

Figure 1: Construct map of pCDNA3.1/anti-TNF alpha -Hc-: anti-TNF alpha Hc segment was cloned in Notl and CIaI site of the vector containing NO S/MAR sequence.

The presence of other component of the vector are depicted in the legend in the figure and also explained in the detailed description of the invention.

Figure 2: Construct map of pCDNA3.1/anti-TNF alpha -Lc-: anti-TNF alpha 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 the detailed description of the invention.

Figure 3: Construct map of pCDNA3.1/MARl/ anti-TNF-alpha-Hc-: anti-TNF alpha

Hc segment was cloned in Notl and CM site of the vector containing S/MAR sequence upstream of CMV promoter and the presence of other component of the vector are depicted in the legend in the figure and also explained in the detailed description of the invention.

Figure 4: Construct map of pCDNA3.1/MARl/ anti-TNF-alpha-Lc-: anti-TNF alpha

Lc segment was cloned in Notl and CIaI site of the vector containing S/MAR sequence upstream of CMV promoter and the presence of other component of the vector are depicted in the legend in the figure and also explained in the detail description of the invention. 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 Monoclonal antibody to human TNF-alpha protein (Fig. 1-4).

Present invention relates to a novel expression vector using the above-mentioned S/MAR to produce Infliximab in larger quantity. Upon isolation from culture media, products of expression of the DNA sequence display the biological activities of Monoclonal antibody to human TNF-alpha.

S/MARs are DNA sequences that bind isolated nuclear scaffolds or nuclear matrices in vitro with high affinity. Analyses of experimentally identified S/MARs have revealed a typical element to be as short as 300 base pairs and up to several kbs 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 AWWRTAANNWWGNNNC within 200 bp. Other 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 AAT AAA YAAA and the T-box TTWTWTTWTT), DNase I-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.

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 genes.

An expression vector for animal cells of the present invention comprises suitable base sequences, which are further added to conventional expression vectors. The suitable base sequences include a nuclear matrix attachment region (hereinafter referred to as "MAR") and a scaffold attachment region (hereinafter referred to as "SAR"), which stimulate foreign gene expression in a host cells from position effects at the insertion site, and increase the expressed amount of the foreign genes.

The first expression vector is constructed using ORF of the heavy chain of recombinant Infliximab, which is flanked by CMV promoter upstream and SV40 poly A signal down stream. The vector also contains the bacterial beta-lactamase gene from Transposon Tn3 (AmpR), conferring ampicillin resistance, the bacterial CoIEl origin of replication and specifically a human gastrin terminator inserted in front of the SV40 polyA signal. The second expression vector is constructed similarly using ORF of the light chain of recombinant Infliximab. These vectors (vector containing Hc chain and the other vector containing Lc chain of Infliximab) with out S/MAR sequence are co transfected in an established cell line. The ORF of the heavy chain and the light chain of Infliximab are amplified with the primers containing Notl and Clal respectively. The whole Expression cassette is flanked by human S/MAR (Scaffold/Matrix Attachment Regions) elements at the upstream of the promoter.

The first expression vector contains ORF of one of the two polypeptide (heavy) chain of the Infliximab (anti TNF alpha antibody). The ORF is 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 second expression vector contains ORF of one of the two polypeptide (light) chain of the Infliximab (anti TNF alpha antibody). The ORF is 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 contains the bacterial beta-lactamase gene from Transposon Tn3 (AmpR), conferring ampicillin resistance, and the bacterial CoIEl origin of replication (Fig 2). These vectors (vector containing Hc chain and the other vector containing Lc chain of Infliximab with out S/MAR sequence are co transfected and the expression of the monoclonal antibody against the TNF alpha (anti TNF alpha 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 Infliximab (anti TNF alpha 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 Infliximab (anti TNF alpha 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 vectors (vector containing Hc chain and the other vector containing Lc chain of Infliximab with S/MAR sequence are co transfected and the expression of the monoclonal antibody against the TNF alpha (anti TNF alpha 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 Infliximab was amplified with the primers containing Notl and Clal respectively and cloned with the same in to the vector explained above. Initially pCDNA3.1/tPA was cloned using BamHI/XhoI restriction sites. Afterwards, the cloned fragment was confirmed sequentially and further confirmed by restriction digestion with BgIII. Later, MARl sequence was inserted into this vector to synthesize pCDNA3.1/MARl/tPA.