Vectors and compounds for expression of recombinant infliximab

Mammalian protein expression system has the function of protein folding and post-translational modification which let the protein closer to the natural protein, so that to obtain the same biological activity with natural protein. Therefore, the Mammalian Cell Expression System is the most widely used in the development and production of recombinant protein drugs, particularly in the therapeutic monoclonal antibodies. Email:marketing@medicilon.com.cn web:www.medicilon.com

The present invention relates to vectors and in particular to compounds of expression for expression of recombinant anti-human TNF alpha monoclonal antibody comprising at least ORF of one of the two polypeptide (heavy and light) chain of the Infliximab, at least one scaffold/matrix attached region (S/MAR), processes for their construction, and their use, in particular for the high level expression of proteins which can be used as medicaments.

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.

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.