Study On The Preparation And Biological Function Of Humanized Anti-CD38 Antibody

Multiple myeloma(MM)is a progressive and fatal disease,which is mainly characterized plasma cell malignant proliferation and antibody or light chain over expression in the bone marrow.These diseases seriously affect the quality of human life and expected life.At present,the treatment for this disease mainly in the small chemical molecular drug based chemotherapy.The side effects of these drugs of chemotherapy and radiotherapy are significant.Although the drugs for MM have greatly improved the overall survival(OS)in recent years,MM is still incurable,and the needs of MM patients are far from being satisfied.The white cell differentiation group 38(cluster of differentiation 38,CD38)is highly expressed in lymphoid,bone marrow cells and some tissues derived from non hematopoietic cells,and is highly expressed in multiple myeloma malignant tumor cells.The role played by CD38 in cellular signaling suggests that CD38 has the potential as a target for monoclonal antibody drugs in the treatment of MM.In this paper,CD38 ECD(extracellular domain)protein and CHO-CD38FL(full-length)stable cell line were prepared by eukaryotic expression system.Immuned BALB/c mice with protein/cell lines alternately,when the serum titer of tail vein is required,using ECD immune spleen,killing mice and isolating spleen B cells.Fusion with myeloma cells(Sp2/0-Ag14),in the presence of PEG.Hybridoma supernatant were screened by ELISA and flow cytometry with protein,cell(CHO-CD38FL,Daudi),respectively.Positive hybridoma are subcloned by 2rounds of limited dilution.The hybridoma cells were injected into the abdominal cavity of mice,and ascites was prepared to obtain mouse antibodies.The affinity of antibodies with Daudi(CD38~+tumor cells)and CD38 ECD was determined by flow cytometry,Fortebio respectively.In vitro,evaluating the biologic effect of antibody by ADCC(antibody dependent cellular cytotoxicity)and complement effect(CDC)assay.The extraction total RNA of hybridoma cell and reversed transcription to cDNA,respectively.Desiging degenerate primers in the light chain,heavy chain constant region and leader peptide,then sequenced and amplified,cloned into T vector,transformed and sequenced.by blasting mouse sequence database(Abysis,Ig blast),to determine the family of light chain and heavy chain CDR region,FR region and variable region types.In IMGT database,searching the highest homology of mouse to human FR region.Then retained the murine CDR,with human FR instead of mouse FR to realize the humanized antibody.Also comparing humanized antibody in affinity,ADCC,CDCwith wild type.The result for molecular weight of the prepared CD38 ECD was consistent with the expected result(45kD),and CHO-CD38FL could bind to anti CD38 antibody.After 4 rounds of immunization,the serum titers of all 5 mice reached 1:10~4,which met the requirements of fusion.After fusion,screening and subcloning,2 cell lines were obtained,namely 38A3 and38C5.Ascites was prepared from 2 hybridoma cells.After purification,15mg and 18mg antibodies were obtained,and the purity was 95.31%and 94.92%respectively.The affinity of38A3,38C5 and positive control to Daudi cells was 3.09nM,13.72nM,4.46nM,respectively.and the affinity of antibody to CD38 ECD was 4.12nM,6.63nM,4.36nM,respectively.In ADCC effect,the maximal killing effect of 38A3,38C5 and positive antibody were 38.567%,34.499%and 37.771%,respectively,and the IC50 values were 1.464ng/ml,0.1661ng/ml,0.1473ng/ml,respectively.while in CDC,the IC50 of the antibody were close to0.01792μg/ml,0.01429μg/ml and 0.02267μg/ml respectively.The mouse antibody 38C5 was humanized by CDR grafting method.After humanized,the affinity of antibody,ADCC and CDC were 11.2nM,0.5708ng/ml,0.04829μg/ml,respectively.