Construction Of Recombinant Anti-CD20 Monoclonal Antibody Engineered Cell Lines

Malignant lymphoma is the lymph nodes and external immune cells of lymphoid tissue tumor, lymph cell or tissue derived from malignant change in the cell, including non-hodgkin’s lymphoma Non- Hodgkin ’s lymphoma(NHL) more difficult to cure.After radiation and chemotherapy is the cure rate is only 25% of the disease, so for the treatment of recurrent, refractory low-grade or follicles are in urgent need of new treatments for the treatment of non-hodgkin’s lymphoma.In the treatment of B cell lymphoma, CD20 molecule is an ideal target antigen, expressed in more than 95% of the normal or deterioration of the surface of B cells.Before starting to express it in B lymphocyte(pre- B) phase, the terminal end to differentiate into plasma cells, B cells has long been regarded as B cell surface characteristic of the logo.Resistance against CD20 single combined with CD20 on B cells, and lead to dissolve the immune response of B cells, cells dissolved possible mechanisms including complement dependent cytotoxic effect(CDC) and antibody dependent cell mediated cytotoxic effect(ADCC) [[I]].Used for B cell lymphoma antigen CD20 molecule monoclonal antibodies are important in the treatment of non-hodgkin’slymphoma.After a global multicenter clinical trial demonstrated significantly improve cancer survival rate and improve patients’ life quality, prolong the life cycle of curative effect, used in the current international standard of tumor treatment, has the characteristics of the small of adverse reaction.So far at least 500000 non-hodgkin’s lymphoma patients accepted global anti CD20 monoclonal antibody therapy, and as one of the world’s most successful antibody drug manufactured by roche’s used in the treatment of non-hodgkin’s lymphoma of rituxan injection(China import registered brand name "mabthera") [[I]], the global sales of about $6 billion in 2011, thus, to develop such drugs will bring huge benefits for tumor patients, and for the development of such drugs biotechnology company bring huge economic and social benefits, has broad market prospects.survival rate and improve patients’ life quality, prolong the life cycle of curative effect, used in the current international standard of this project is to build a kind of engineering cells bead, the expression of the active ingredient: restructuring anti CD20 monoclonal antibody has the same amino acid sequence as rituxan,.According to the literature reference mabthera including signal peptide was synthesized by the restructuring of the anti CD20 light chain of monoclonal antibody and heavy chain gene fragments, and insert it into p REA202 mammalian cell expression vector, build antibodies light chain and heavy chain expression vector p REA202 / REA202 L and p REA202 / REA202 H.Will build the expression of the antibody light chain and heavy chain carrier by liposome transfection CHO- DG44 cells, by G418 resistance screening positive clones, antibody by ELISA screening high expression of cloning, after screening, cloning, pressurized with MTX gradually won a plant height cell lines CHO- REA202 antibodies.Preliminary evaluation showed that the expression of cell line of the project product for complete resistance against CD20 single, antibody expression ability can reach 20 to 23 PCD(pg/cell/day).In the cell lines to establish cell bank, library work cells after recovery, continuous subculture, examine the stability of the engineered cells.According to the results, will be handed a seed cell culture stage as generation, production training stage every 48 hours to cultivate a generation.Cells extend the trial batches, 60 batches were taken 0, 10, 20, 40, 60 should investigate the generation of cells.The results show that: in the process of batches of CHO- REA202 REA202 the DNA sequence of a protein and heavy chain gene not hair change;REA202 protein and heavy chain gene copy number level not obvious change;Cell growth characteristics, doubling time and form was not obvious change;REA202 protein expression levels did not significantly change.The results showed that the engineered cells CHO-REA202 has good stability batches, in line with the "Chinese pharmacopoeia" to the requirement of the extend the stability of the cell.