FIELD OF THE INVENTION

The present invention relate to vectors and compounds of expression for Recombinant Monoclonal antibody to CD20.

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.

Lymphomas are cancers of the lymphatic system - the body's blood-filtering tissues that help to fight infection and disease. Like other cancers, lymphomas occur when cells divide in uncontrolled manner in which growth control is lost. Consecutively, lymphatic cells may overcrowd, invade, destroy lymphoid tissues, and metastasize (spread) to other organs. There are two types of lymphomas: "Hodgkin's Disease" (HD) and Non-Hodgkin's Lymphoma (NHL). NHL is a heterogeneous disease, which causes approximately 50,000 new cases and almost 25,000 deaths every year, in North America alone. The incidence of these malignancies has been increasing steadily over the past several decades in North American and Western European countries at an annual rate of 4%. NHLs are among the leading types of cancers and causes of cancer- related mortality, accounting for -3% of cancer deaths in the US and UK in both men and women.

NHL occurs more often in patients between the ages of 40 and 70. A number of factors, including congenital and acquired immunodeficiency states, and infectious, physical, and chemical agents, have been associated with an increased risk for NHL. Infectious agents, such as viral infections (e.g., Epstein-Barr virus, HIV, human T-cell leukemia virus), and bacterial infections (e.g., Helicobacter pylori) may be associated with the development of NHL. NHLs are much less predictable than HD, and they are more likely to spread to areas beyond the lymph nodes.

Histologically, ~90% of NHLs are of B-cell (10% of T-cell) origin, the most prevalent worldwide being diffuse large (33%) and follicular (22%) types. Most aggressive form of NHL is diffuse large B-cell type, 50% of which present in an advanced stage. Majority (67%) of patients who present with a disseminated disease (i.e. advanced stage) at the time of diagnosis, require systemic therapy.

Thus far, the standard chemotherapy, including single-agent chlorambucil, cyclophosphamide or fludarabine, or combination regimens (e.g. cyclophosphamide, vincristine and prednisone [CVP] or cyclophosphamide, doxorubicin, vincristine and prednisone [CHOP]), have not proven curative. Up to 75%) of patients achieve remission (i.e. complete response [CR]), but most relapse after a median of 2 years and only about 20% remain disease free for >10 years. In patients with early or non bulky localized disease stage, the 5-year failure-free and overall survival (OS) rates of 80-90% and 60-70%, respectively, have been reported with 3-4 cycles of combination chemotherapy followed by involved-field radiotherapy. However, for those with disease in advanced stages, the standard first line treatment, which over the past two decades comprised CHOP chemotherapy regimen, yielded limited success; CR rates have been reported in 50-60% and 5-year OS in only 35-45% of patients.

Monoclonal antibody-based therapies gained importance to overcome the pit fall in the chemotherapy. Monoclonal antibodies were first described in 1975. Developments in the field of recombinant DNA led to advances in the generation of chimeric antibodies and humanized antibodies.

The Rituximab is a genetically engineered chimeric murine/human monoclonal antibody directed against the CD20 antigen found on the surface of normal and malignant B lymphocytes. Rituximab has a binding affinity for the CD20 antigen of approximately 8.0 nM. Rituximab action appears to be mediated via three potential humoral and cell-mediated effector mechanisms, including complement-dependent cytotoxicity (CDC), antibody- dependent cellular cytotoxicity (ADCC) and complement-dependent cellular cytotoxicity. Recent studies have shown that CDC appears to be the predominant mechanism by which Rituximab exerts its therapeutic effect.

In 1997, the US Food and Drug Administration (FDA) approved rituximab, a monoclonal antibody for the treatment of low grade non- Hodgkin's B cell lymphomas. Since then, it has been used in over 500 000 patients with generally excellent tolerability.

In order to facilitate production of large quantities of Monoclonal antibody to CD20 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 CD20 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 CD20.

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 CD20 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 CD20 expressed by the expression vector carrying Scaffold/Matrix Attachment Region(s) (S/MAR).

DESCRIPTION OF FIGURES

Figure 1: Construct map of pCDNA3.1/anti-CD20 He Figure 2: Construct map of pCDNA3.1/ anti-CD20 Lc Figure 3: Construct map of pCDNA3.1/MAR1/ anti-CD20-Hc Figure 4: Construct map of pCDNA3.1/MARl/anti-CD20-Lc

DETAILED DESCRIPTION OF THE INVENTION

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 trasnfef. 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 ■ ■ info 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.

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 CD20.

In still another embodiment of the present invention, the Monoclonal antibody to CD20 protein is a recombinant Monoclonal-antibody to CD20 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 CD20 protein expressed by the expression vector carrying Scaffold/Matrix Attachment Region(s) (S/MAR) (Fig. 1-4).

The present invention comprises novel DNA compounds which encode Monoclonal antibody to CD20 activity. A novel eukaryoticexpression vector has been constructed that comprise the novel Monoclonal antibody to CD20 protein activity-encoding DNA and drive expression of Monoclonal antibody to CD20 activity when transfected into .an appropriate cell line. The novel expression vector can be used to produce soluble Monoclonal antibody to CD20. The recombinant-produced Monoclonal antibody to CD20 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 CD20 in increased quantity.

Prokaryotic expression systems were part of the early repertoire of research tools in molecular biology. The de nova 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 describe'd 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 30 000^0 000 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 as-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.

The Rituximab is a genetically engineered chimeric murine/human monoclonal antibody directed against the CD20 antigen found on the surface of normal and malignant B lymphocytes. Rituximab has a binding affinity for the CD20 antigen of approximately 8.0 nM. Rituximab action appears to be mediated via three potential humoral and cell-mediated effector mechanisms, including complement-dependent cytotoxicity (CDC), antibody- dependent cellular cytotoxicity (ADCC) and complement-dependent cellular cytotoxicity. Recent studies have shown that CDC appears to be the predominant mechanism by which Rituximab exerts its therapeutic effect the standard chemotherapy, including single-agent chlorambucil, cyclophosphamide or fludarabine, or combination regimens (e.g. cyclophosphamide, vincristine and prednisone [CVP] or cyclophosphamide, doxorubicin, vincristine and prednisone [CHOP]), have not proven curative. Up to 75% of patients achieve remission (i.e. complete response [CR]), but most relapse after a median of 2 years and only about 20% remain disease free for >10 years. In patients with early or non bulky localized disease stage, the 5-year failure-free and overall survival (OS) rates of 80-90% and 60-70%, respectively, have been reported with 3^1 cycles of combination chemotherapy followed by involved-field radiotherapy. However, for those with disease in advanced stages, the standard first line treatment, which over the past two decades comprised CHOP chemotherapy regimen, yielded limited success; CR rates have been reported in 50-60% and 5-year OS in only 35-45% of patients.

Largely through a series of investigator sponsored trials supported in part by Genentech (South San Francisco, CA), the use of rituximab has been extended to a much wider spectrum of B cell malignancies, and strikingly, to a variety of autoimmune diseases in which B cells have been thought to play a role.

Several maintenance therapy regimens with rituximab in follicular lymphoma have been shown to improve treatment outcomes. Rituximab has also been evaluated in combination with cytotoxic chemotherapeutic regimens, including fiudarabine- and cyclophosphamide- based regimens, for the treatment of follicular lymphoma. The addition of rituximab to other chemotherapy regimens such as cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP) and fludarabine-based regimens has also improved treatment outcomes.

Rituximab is also implicated in the other autoimmune diseases In all, anecdotal information is available on some 30 autoimmune conditions. Following the pioneering work by Jo Edwards in a small-uncontrolled series of patients who had been resistant to conventional therapy, and randomised controlled trials (RCT) showed efficacy similar to what has been seen in the antitumour necrosis factor (anti-TNF) trials.

Experience from lymphoma, with 960,000 patients treated, showed that B cell depletion using rituximab was well-tolerated, which paved the way for the evaluation of rituximab in RA. Although not approved by FDA, It has recently been shown in two other recent publications demonstrating striking anecdotal data suggesting that rituximab may work in Wegener's granulomatosis and dermatomyositis.

Present invention relates to a novel expression vector using the above-mentioned S/MAR to produce Rituximab in larger quantity. Upon isolation from culture media, products of expression of the DNA sequence display the biological activities of Monoclonal antibody to CD20.

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 Fournier, 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. Kasper DL, Braunwald E, Fauci AS, et al. Harrison's principles of internal medicine, 16th Ed.; Chapter 97: Malignancies of lymphoid cells [online]. Available from URL: www.harisons online.com

15. Jemal A, Murray T, Ward E, et al. Cancer statistics, 2005. Cancer J Clin Jan/Feb 2005; 55(1): 10-30. ~" *—

16. Evans LS, Hancock BW. Non-Hodgkin lymphoma. Lancet. 362 2003 Jul 12; 362: 139-46.

17. National Cancer Institute. Adult non-Hodgkin's lymphoma (PDQU): treatment (health professional version) [online]. http://www.cancer.gov/cancerinfo/pdq/treatment/aduIt-non-hodgkins/ health professional.

18. Zinzani PL. Lymphoma: diagnosis, staging, natural history, and treatment strategies. Semin Oncol 2005 Feb; 32 (1 Pt 2): 4-10

19. Miller TP, Leblanc M, Spier C, et al. CHOP alone compared to CHOP plus radiotherapy for early stage aggressive non-Hodgkin's lymphomas: update of the Southwest Oncology Group (SWOG) randomized trial [abstract no. 3024]. Blood 2001 Nov 16; 98 (11 Part 1 of 2): 724-5a.

20. Risto S. Cvetkovi'c and Caroline M. Perry 2006 Drugs 66(6) 791-820.

21. Winter G, Harris WJ. Humanized antibodies. Immunol Todayl993; 14 (6): 243-6.

22. Levene AP, Singh G, Palmieri C. Therapeutic monoclonal antibodies in oncology. J R Soc Med 2005; 98 (4): 146-52.

23. Harjunpaa A. Junnikkala S, Meri S. Rituximab (anti-CD20) therapy of B-cell lymphomas: direct complement killing is superior to cellular effector mechanisms. Scand J Immunol2000; 51 (6): 634-41.

24. Golay J, Manganini M, Facchinetti V, et al. Rituximab-mediated antibody-dependent cellular cytotoxicity against neoplastic B cells is stimulated strongly by interIeukin-2. Haematologica2003; 88 (9): 1002-12.

We Claim

1) A matrix attachment region sequence[s] (SEQ ID 1) or its complementary sequencejs], variants] and fragments] 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], variants] and fragments] thereof said comprising step of amplifying S/MAR into the expression vector.

5) An expression vector carrying a matrix attachment region sequence[s] or its complementary sequence[s], variants] and fragments] thereof as claimed in Claim 1.

6) The expression vector as claimed in claim 5 wherein said expression vector is not limited to mammalian expression vector.

7) A eukaryotic cell with a matrix attachment region sequence[s] or its complementary sequencefs], variants] and fragments] thereof as claimed in claim 1.

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) The sequence as claimed in claim 1, wherein transcription factors can bind to those said sequences.

10) Factors (Proteins/Nucleic acids etc), which influence the biological activity of, said sequences as mentioned in claim 1.

11) Epigenetic factors, such as but not limited to, methylation, acetylation, phosphorylation which influence the biological activity of, said sequences as mentioned in claim 1.

12) Combination of factors as mentioned in Claim 10 and 11, which may act in concert which influence the biological activity of said sequences as mentioned in Claim 1.

13) Position of these said sequences as mentioned in Claim 1 in genome.

14) Pattern of motifs present in sequences as claimed in Claim 1.

15) Position of the sequences, as claimed in Claim 1, at upstream of promoter in vector backbone.

16) Position of the sequences, as claimed in Claim 1, at downstream of termination signal in vector backbone.

17) Position of the sequences, as claimed in Claim 1, at either upstream of promoter or downstream of termination signal in vector backbone or both.

18) Distance of the sequences, as claimed in Claim 1, from start site

19) Distance of the sequences, as claimed in Claim 1, from eukaryotic origin of replication.

20) The vector as claimed in claim 5, wherein the expression vector is used for production of anti-CD20 monoclonal antibody.

21) 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.

22) An anti-CD20 monoclonal antibody protein expressed by the expression vector carrying Scaffold/Matrix Attachment Region(s) as claimed in Claim 1.