FIELD OF THE INVENTION
The present invention relate to vectors and compounds of expression for Recombinant Monoclonal antibody to VEGF.
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 hundreds 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.
Cancer of the colon and rectum (CRC) constitutes a major public health problem and is more prevalent in Western countries where the incidence is nearly double that of developing countries. There, it affects about one of twenty humans and ranks second amongst the most common malignancies in both men and women, with about 334,000 new cases diagnosed every year, distributed almost evenly between the sexes. Deaths from cancers of the colon and rectum rank second (189,000) after lung cancer.

Approximately 30% of all patients with CRC have metastatic disease at diagnosis, and 50% of early-stage patients will eventually develop metastatic or advanced disease. The prognosis for this patient population is poor. In Europe, 5-year relative survival for patients diagnosed with cancer of the colon or rectum during 1985-1989 was 48% for patients with colon cancer and 44% for patients with cancer of the rectum. Despite the recent addition of new therapeutic agents, efficacy remains unsatisfactory. Until the mid-1990s the only available drug, with limited activity in mCRC, was 5-fluorouracil (5FU). The recent introduction of two new cytotoxic drugs, irinotecan and oxaliplatin, in addition to 5FU resulted in significant progress in the treatment of mCRC. In 1997, Scientists at Genentech reported the humanization of the mouse anti-VEGF monoclonal antibody (Mab A.4.6.n22). By site-directed mutagenesis of a human antibody framework, the residues involved in the six complementarity- determining regions, and also several framework residues, were changed to murine counterparts. The humanized anti-VEGF monoclonal antibody (rhuMab VEGF; bevacizumab; Avastin) bound VEGF with affinity very similar to that of the original antibody (^d -0.5 nM).
In order to facilitate production of large quantities of Monoclonal antibody to VEGF 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, expression and purification of Monoclonal antibody to VEGF 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 VEGF.
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 VEGF 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 VEGF expressed by the expression vector carrying Scaffold/Matrix Attachment Region(s) (S/MAR).
LIST OF FIGURES
Figure 1: Construct map of pCDNA3.1/anti-VEGF He Figure 2: Construct map of pCDNAS.l/ MARl/ anti-VEGF Lc
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 VEGF.
In still another embodiment of the present invention, the Monoclonal antibody to VEGF protein is a recombinant Monoclonal antibody to VEGF protein.
In another embodiment of the present invention, the 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).
The present invention also relates to Monoclonal antibody to VEGF protein expressed by the expression vector carrying Scaffold/Matrix Attachment Region(s) (S/MAR) (Figure 1 and Figure 2).
The present invention comprises novel DNA compounds which encode Monoclonal antibody to VEGF activity. A novel eukaryotic expression vector has been constructed that comprise the novel Monoclonal antibody to VEGF protein activity-encoding DNA and drive expression of Monoclonal antibody to VEGF activity when transfected into an appropriate cell line. The novel expression vector can be used to produce soluble Monoclonal antibody to VEGF. The recombinant-produced Monoclonal antibody to VEGF 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 VEGF 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.

Bevacizumab is a recombinant humanized IgGl monoclonal antibody (93% human, 7% murine sequences) that binds with high affinity to human vascular endothelial growth factor (VEGF). It was generated by humanization of the murine parent antibody (A4.6.1). It neutralizes VEGF's biologic activity through a steric blocking of binding of VEGF to its receptors Flt-1 (VEGFR-1) and KDR (VEGFR-2) on the surface of endothelial cells. For the first-line treatment of metastatic colorectal cancer, the addition of bevacizumab to bolus-IFL chemotherapy conferred a clinically meaningful and statistically significant benefit for all study endpoints, including overall survival, progression-free survival and responserate, and was associated with an acceptable side-effect profile and later it was approved by both FDA and EMEA for this condition. Aterwards, it was also approved for in combination with carboplatin and paclitaxel for the first-line treatment of patients with unresectable, locally advanced, recurrent or metastatic non-squamous non-small cell lung cancer (NSCLC). Currently, several additional late-stage clinical studies are underway to determine its safety and effectiveness for patients with brain cancer, non-metastatic colon cancer, metastatic breast cancer, metastatic renal cell carcinoma, metastatic ovarian cancer, prostate and pancreatic cancer.
Present invention relates to a novel expression vector using the above-mentioned S/MAR to produce Bevacizumab in larger quantity. Upon isolation from culture media, products of expression of the DNA sequence display the biological activities of Monoclonal antibody to VEGF.
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, contain 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" are 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 inheritence. Host organisms containingthe 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 line that permanently express the gene of interest depending on the stable integration of plasmid into the host chromosome. REFERENCES
1. Yin, J. et. al. Journal of Biotech. 127, 335-347 (2007)
2. Geisse, S. et. al. Protein Express. Purif. 8, 271 -282 (1996)
3. Henikoff, S. Curr. Opin. Genet Dev. 2, 907-912(1992)
4. Kleinjan, DJ. et. al. Hum. Mol. Genet. 7,1611-1618(1998)
5. Allen, GC. et. al. Plant Mol. Biol. 43, 361-76 (2000)
6. Girod, PA. and Mermod, N. Gene Transfer and Expression in Mammalian Cells, Elsevier Sciences, 359-379 (2003)
7. Boulikas, T. Int. Rev. Cytol. 162A, 279-388 (1995)
8. kalos, M. and Foumier, REK. Mol.Cell.Biol. 15, 198-207 (1995)
9. Phi-Val, L. et. al. Mol. Cell. Biol. 10,2302-2307 (1990)
10. Zahn-Zabal, M. et. al. Journal of Biotech. 87, 29-42 (2001)
11. Girod, PA. et. al. Biotechnol. Bioeng. 91,1-11 (2005)
12. Singh, GB.et. al. NAR. 25,1419-1425 (1997)
13. Frish,, M. et. al. Genom. Biol. 12, 349-354 (2002)
14. Ferrara, N. et. al. Nature Rev Drug Discover. 2004; 3: 391 -400.
15. Presta, L. G. et al. Cancer Res. 1997; 57: 4593-4599.
16. Midgley, R. and D. Kerr. Lancet 1999; 353: 391-9.
17. Ferrara, N. Nature Rev. Cancer. 2002; 2: 795-803.

We Claim
1) An isolated nucleic acid comprising one or more sequences selected from the group consisting of SEQ ID NO: 1, complements, variants, and functional fragments thereof and sequences being at least 70% homologous thereto.
2) The isolated nucleic acid of claim 1, wherein the one or more sequences comprise S/MAR sequences.
3) The isolated nucleic acid of claim 2, wherein the one or more S/MAR sequences increase expression of a biomolecule when said sequences are used in an expression system.
4) The isolated nucleic acid of claim 3, wherein the one or more S/MAR sequences increase expression of the biomolecule independently of the orientation of said sequences in the expression system.
5) The isolated nucleic acid of claim 3, wherein the biomolecule is a protein.
6) The isolated nucleic acid of claim 6, wherein the protein is recombinant monoclonal antibody to VEGF
7) The isolated nucleic acid of claim 2, wherein the one or more S/MAR sequences contain one or more nucleotide sequence motifs.
8) The isolated nucleic acid of claim 7, wherein the one or more nucleotide sequence motifs includes at least one AT-rich nucleotide motif.
9) A method for constructing an expression vector having increased expression efficiency, the method comprising inserting the isolated nucleic acid of claim 1 into an expression vector.
10) The method according to claim 9, wherein the expression vector is a mammalian expression vector.
11) A vector comprising the isolated nucleic acid of claim 1.
12) The vector of claim 11, wherein said vector is a bacterial plasmid, a bacteriophage vector, a yeast episomal vector, an artificial chromosomal vector, or a viral vector.
13) The vector of claim 11, wherein said vector is a mammalian expression vector.
14) A method for producing a recombinant host cell, the method comprising introducing the isolated nucleic acid of claim 1 or the expression vector of claim 11 into a host cell.

15) The method according to claim 14, wherein the isolated nucleic acid or the expression vector is introduced by way of transfection.
16) The method according to claim 15, wherein the isolated nucleic acid gets integrated with the genome of the recombinant host cell upon transfection.
17) A host cell produced according to the method of claim 14.
18) A host cell comprising the vector of claim 11.
19) The host cell according to claims 17 or 18, wherein said host cell is a eukaryotic cell.
20) The host cell according to claims 17 or 18, wherein said host cell is a mammalian cell.
21) An expression vector comprising a nucleic acid molecule that comprises (a) a sequence encoding a protein operably linked to one or more expression control elements and (b) and one or more S/MAR sequences selected from the group consisting of SEQ ID NO: 1, complements, variants, and functional fragments thereof and sequences being at least 70% homologous thereto.
22) The expression vector of claim 21, wherein the protein is recombinant recombinant monoclonal antibody to VEGF
23) A host cell comprising the expression vector of claim 22.
24) The host cell of claim 23, wherein said host cell is a eukaryotic cell.
25) The host cell of claim 23, wherein said host cell is a mammalian cell.
26) The expression vector of claim 21, wherein the one or more expression control elements comprise at least one of transcriptional promoter, transcriptional enhancer, transcriptional repressor, polyadenylation site, origin of replication site, translation initiation signal and translation termination signal.
27) The expression vector of claim 26, wherein the one or more S/MAR sequences are located upstream of the transcriptional promoter.
28) The expression vector of claim 26, wherein the one or more S/MAR sequences are located downstream of the transcriptional promoter.
29) The expression vector of claim 26, wherein the one or more S/MAR sequences are located downstream of the translation termination signal.

30) The vector of claim 26, wherein the one or more S/MAR sequences are located upstream of the transcriptional promoter and downstream of the translation termination signal.
31) The vector of claim 26, wherein the one or more S/MAR sequences are located downstream of the transcriptional promoter and of the translation termination signal.
32) The expression vector of claim 26, wherein the one or more S/MAR sequences are located at a distance of 0 to 10 KB from the sequence encoding recombinant monoclonal antibody to VEGF protein.
33) The expression vector of claim 26, wherein the one or more S/MAR sequences are located at a distance of 0 to 10 KB from the origin of replication site.
34) The expression vector of claim 26, wherein said expression vector is used for recombinant expression of monoclonal antibody to VEGF protein.
35) A factor which influences the activity of one or more S/MAR sequences, wherein the one or more S/MAR sequences comprises one or more sequences selected from the group consisting of SEQ ID NO: 1, complements, variants, and functional fragments thereof and sequences being at least 70% homologous thereto.
36) The factor of claim 35, wherein said factor is at least one of a genetic factor, or an
epigenetic factor.