Expression And Activity Detection Of Novel Humanized Anti-CD20Monoclonal Antibody

B cell lymphoma is a kind of solid tumor in B cells which includes hodgkin lymphoma(HL) and Non-Hodgkin’s lymphoma (NHL). Non-Hodgkin’s lymphoma is one of the most common cancer in the blood system which seriously threaten human life and health. So far, the therapeutic efficiency using of many traditional clinical chemotherapeutics for NHL has not been very well. Therefore, scientists develop all kinds of new drugs to treat non-hodgkin’s lymphoma. Monoclonal antibody targeting therapy have developed quickly for its high specificity and efficiency. CD20is a specific antigens on the surface of B lymphocyte which is a kind of non-glycosylation protein with279amino acids. Depending on the different levels of phosphorylation the weight of CD20molecular is reported to be33-37kD. CD20is a transmembrane protein on the B lymphocyte with four transmembrane domains. Both of the C-terminal and N-terminal are located on the inner side of cytoplasm. There are a large loop contained43amino acids which is between the third and fourth transmembrane domain form the prime epitope of CD20molecular. CD20expressed on the surface of B cells widely, from early pre-B cells to later in differentiation, but it is absent on terminally differentiated plasma cells. CD20is expressed on all stages of B cell development except the first and last. It is found on B-cell lymphomas, hairy cell leukemia, B-cell chronic lymphocytic leukemia, and melanoma cancer stem cells. The CD20antigen exposes on membrane surface and is easy to approach for antibody relatively, so, CD20antigen is the ideal target for the treatment of B cell lymphoma. Although the function of CD20is unknown, it may play a role in Ca2+influx across plasma membranes, maintaining intracellular Ca2+concentration and allowing activation of B cells.Rituximab is a genetically engineered chimeric murine and human monoclonal antibody directed against the CD20antigen found on the surface of normal and malignant B lymphocytes. Rituximab is a glycosylated IgGl kappa immunoglobulin containing murine light-chain and heavy-chain variable region sequences (Fab domain) and human constant region sequences (Fc domain). Rituximab is composed of1328amino acids and has an approximate molecular weight of144kD. Rituximab was developed by IDEC Pharmaceuticals named IDEC-C2B8. Rituximab destroys B cells and is therefore used to treat diseases which are characterized by excessive numbers of B cells, overactive B cells or dysfunctional B cells and the diseases include many lymphomas, leukemias, transplant rejection, and autoimmune disorders. Based on its safety and effectiveness in clinical trials, rituximab was approved by the U.S. Food and Drug Administration in1997to treat B-cell non-Hodgkin lymphomas resistant to other chemotherapy regimens. In2010it was approved by the European Commission for maintenance treatment after initial treatment of follicular lymphoma. Rituximab has been shown to be an effective rheumatoid arthritis treatment in three randomised controlled trials and is now licensed for use in refractory rheumatoid disease. In the United States, it has been FDA-approved for use in combination with methotrexate (MTX) for reducing signs and symptoms in adult patients with moderately to severely active rheumatoid arthritis (RA) who have had an inadequate response to one or more anti-TNF-alpha therapy. In Europe, the license is slightly more restrictive:it is licensed for use in combination with MTX in patients with severe active RA who have had an inadequate response to one or more anti-TNF therapy. Other autoimmune diseases that have been treated with rituximab include autoimmune hemolytic anemia, pure red cell aplasia, idiopathic thrombocytopenic purpura(ITP), Evans syndrome, vasculitis, bullous skin disorders, type1diabetes mellitus,Sjogren’s syndrome, and Devic’s disease, and Graves’ophthalmopathy. Rituximab belongs to one of the human-mouse chimeric antibodies, and30%of its sequence comes from the original murine. It is unable to be used for a long time with a possibility of immunogenicity. Therefore, it is particularly urgent to seek a more effective anti-CD20antibody for the treatment of B lymphoma.We designed the nucleotide sequence and found out the human IgG1which were similar to the Rituximab on the crystal structure in the Protein Data Bank (PDB) as candidates through Computer simulation software. Based on the Rituximab, we transformed the CDR of candidates and obtained the target gene fragments by overlapping PCR. Then the sequences of light chains and high chains were inserted into pcDNA3.3and pOptiVEC vectors. Plasmids were transfected into293F cells through transient transfection. After a large scale culture of the cell line, mAb was purified by affinity chromatography rProtein column A. Expression level of the humanized antibody was detected by SDS-PAGE. The affinity of the humanized antibodies to CD20was assessed by Fortebio assay. At last, the ability to kill tumour cells of antibodies was detected by experiments on the growth inhibition of nude mice transplantation tumor.Results data show that thirteen humanized monoclonal antibodies against CD20were expressed and purified successfully. In reducing SDS-PAGE, the antibodies exhibited two bands of approximately25KD and55KD, respectively. The band size of the antibodies matched the expected value. Fortebio assay revealed that the humanized antibodies could bind to CD20with high affinity (L4H7:1.91×10-9mol/L, L5H5:7.35x10-10mol/L, L5H7:1.91x10-9mol/L). The tumor growth inhibition experiment showed that the anti-tumor activity of L5H7mAb was better than the one of Rituximab. Next, we will increase the number of experimental animals to verify the antitumor activity and safety of antibody L5H7. We aim to get a safe and effective humanized anti-CD20monoclonal antibody with stronger affinity, higher antitumor activity and less side effects and provide a new method for effective treatment of non-hodgkin’s lymphoma.