Construction And Biological Function Of Humanized Anti-CD20 Antibody With High Affinity

Non Hodgkin's lymphoma(NHL)is a malignant tumor that is predominantly in lymphoid tissue.It is a common type of B cell lymphoma.The radiotherapy and chemotherapy technology has been unable to treat the diseases,and targeted therapy mechanism of monoclonal antibody technology began to be widely studied and applied.The study found that NHL cells express CD20 antigen,then this phenotype molecules can be used as a target for targeted therapy.Rituximab is the first monoclonal drug to be used to treat the NHL by the US Food and Drug Administration(FDA).However,Rituximab contain 30 % of the mouse sequence,the patient after long-term medication,easy to cause human anti-mouse antibody immune response(HAMA)and produce serious adverse reactions.At the same time,Rituximab's clinical response rate of only 50 %,and only 10 % of the complete rate of remission.Therefore,seriously restricting the long-term application of Rituximab.Aiming at this phenomenon,people start to modification of Rituximab,some with the human skeleton area,which was similar to the mouse source sequence,to replace the mouse framework area,and some use to improve the affinity of Rituximab to enhance the anti-tumor activity.Objective: The transformation of novel antibodies can be carried out from both affinity and immunogenicity,which can increase the binding of antigens and antibodies by increasing the affinity.The immunogenicity is mainly by reducing the murine source sequence of antibody to reduce the resistance in the human body.The aim of this study was to construct a CD20 antibody with a higher affinity and lower immunogenicity and to verify whether it has a corresponding biological activity.Methods: 1.The light chain and heavy chain gene of CD20 antibody obtained by the patent analysis,and the design of high affinity and humanized transformation.After that,they were cloned to pc DNA3.1/ZEO(+)? pc DNA3.1(+)eukaryotic expression vector,and then to transfection of CHO-K1 cell,and screening of monoclonal cell lines by limiting dilution.Transcription of m RNA was tested by reverse transcription-polymerase chain reaction(RT-PCR)and the expression of antibodywas tested by sandwich enzyme-linked immuno sorbent assay(ELISA),and the suspension was done in the serum-free medium.2.The binding activity of cell supernatant to CD20-positive cells was detected by flow cytometry,and its biological function of cytotoxic activity,ADCC and CDC effect.was tested.Results: 1.The light and heavy chain gene of the high affinity humanized anti-CD20 antibodies were designed,and light chain gene segment and heavy chain gene segment were constructed recombination eukaryotic expression vectors of pc DNA3.1/ZEO(+)-CD20 L ?pc DNA3.1(+)-CD20 H who could express instantaneously in CHO-K1 cell and 17 monoclonal cell lines were screened.RT-PCR revealed antibody gene?s transcription in cells was successful.Through ELISA determination,quantity of antibody expression was between0.45-4.72 ?g/m L.And monoclonal cell lines can be suspended in serum-free medium.2.Flow cytometry confirmed that anti-CD20 antibody from secretion of supernatant could bind with Raji cell strain.And culture supernatant had better ADCC and CDC killing activity,but the result of direct cytotoxicity was not obvious.