The Design And Biological Function Study Of Novel Humanized CD20Monoclonal Antibody

Non-Hodgkin’s lymphoma （NHL） is one of the most common cancer in the bloodsystem. The most of NHL is associated with B cell and the therapeutic efficiency using of many traditional chemotherapeutics for NHL is not very well. Use of monoclonal antibody to treat cancer targetly is the dream of human beings for a long time.In1997, the first anti-CD20monoclonal antibody, named Rituximab, was appliedto the treatment of NHL in the world. With the using widely of Rituximab, the mortality rate of NHL was decreased gradually. In the following10years, many therapeuticregimens were developed based on Rituximab, to aim at an increasing on the effect ofRituximab and to obtain an obvious improvement. At the same time, the remission rate and complete remission rate of NHL patients were increased, and the prognosis iswell. The most important factor in the Rituximab is the choice of the proper target-CD20. CD20is the transmembrane protein on the B leukomonocyte and it contain279amino acids. As a non-glycosylation, the molecular weight is reported to be33-37kDdepending on the different levels of phosphorylation. The CD20has four transmembrane domains and both of the N-terminal and C-terminal are located on the inner side of cytoplasm, which is between the third and fourth transmembrane domain. It has a large loop, which contains43amino acids to form prime epitope. The CD20antigen is relatively easy to expose and also to approach. The physiological effect of CD20remains to be clarified, based on a series of biological response on B cell after the combination of the CD20antigen with CD20antibody, also infering that the CD20would join in the proliferation, differentiation, signal transduction and the transmembrane deliverof Ca²⁺ on B cell.Rituximab belongs to one of the human-mouse chimeric antibodies, and30%of its sequence comes from the original murine. During the clinical application, immunogenicity was reported in the17-20%patients. So it is unable to be used for a long time.The application of Rituximab was restrict especially for the treatment of autoimmunedisease. Keeping this in mind, to carry out the humanization of CD20, to find novel antibody with a better effect and a lower immunogenicity is the hot issue on the resear ch for anti-CD20antibody.In this study, we use unique antibody humanization technology, based on the sequence of the murine monoclonal antibody2B8（the original murine monoclonal antibodyof Rituximab from Roche）, designed and selected with germline antibody frame sequence which has less binding sites to the MHC II （HLA-DR）, and the immunogenicityhad been decreased obviously. At the same time, we regulated to the sequence structure of CDR region through molecular dock, which can keep and increase the activity ofantigen recognition and anti-tumor effect in both vitro and vivo.Screening the novel humanized anti-CD20antibody through full humanization, comparedwith Rituximab, the murine sequence of the novel humanized anti-CD20antibody wasdecreased by23%. The novel humanized anti-CD20antibody retained the same antigenbinding sites on the large loop of Rituximab. The mechanism of action is same to Rituximaband the activity is clear in vitro and in vivo, while the immunogenicity is decreased obviously.On these grounds, compared to Rituximab, the novel humanized anti-CD20antibody has thesimilar effect on anti-tumor and lower toxicity. On the other hand, it also has an extensiveapplication prospect on the treatment of autoimmune diseases, which involves many kinds ofB cell abnormal.In this study, we designed ten kinds of formulations for the process of humanizedanti-CD20antibody and investigated the stability of the antibody at different temperature（-80℃，-20℃，4℃，25℃and40℃） for30days、60days、90days and120days. The third one wasplaned to be using as the formulation process of the anti-CD20antibody. We also analyzed thephysicochemical property （such as purity and isoelectric point） of the novel humanizedanti-CD20antibody through capillary electrophoresis, which presented that the samples withdifferent lots had different percentage of basic and acid components. The different influenceon the biological activity of the antibody.