Expression Of The Human CD38 Antigen And Preparation And Functional Study Of Its Monoclonal Antibodies

Lymphoma and multiple myeloma have become a critical threat against human's life, in which conventional chemotherapy, radiothrapy and immunothrapy have only limited value,but it may be ideal for treatment with passive antibody against a suitable cell surface antigen on the neoplastic plasma cell. At present , the monoclonal antibodies(McAb) against the molecules on the cell surface are being used in clinics for the diagnosis and treatment of various diseases and has become a promising approach. The CD38 antigen is a nonlineage-restricted type- Ⅱ transmembrane glycoprotein that has emerged as a multifunctional protein in recent year.It can serve as an ectoenzyme that catalyzes the synthesis and hydrolysis of cyclic ADP-ribose,a recently identified Ca mobilizing agent that acts independently of inositol triphosphate. Ligation of CD38 with agonistic antibodies induces diverse effects in hematopoeitic cells that range from growth stimulation to induction and prevention from apoptosis, induction of cytokines, activation of kinases, and phosphorylation of certain protein. The CD38 is known to be present on the majority of neoplastic plasma cells, It has been reported that CD38 can serve as a target antigen in immunotoxin thrapy of myeloma and lymphoma. Clinical studies of anti-CD38 McAb have demonstrated considerable anti-tumor activity and safety. So, it may be a very effective approach to lymphoma and multiple myeloma.Anti-CD38 McAbs which have been reported may be separated in two broad groups(IB4,AT2 and IB6 and OKT10,SUN-4B7and AT1) depending on the epitope bound. Notwithstanding a different location, IB4 and OKTIO may perform as agonistic reagents, inducing proliferation and cytokines release, however, IB6 and AT2 which react with epitopes partially shared with the IB4, do not induce measurable effects on PBMC proliferation or IFN-γ release. Consequentely,the agonistic features are not strictly dependent on the region of the molecule recognized.,they also relate to the characteristics of each monoclonal antibody.Although anti-CD38 McAb has been used in clinic, and it has been considered a promising approach, but has not been reported in China. Based on the research background and the applicational situation,our aim is to ① express and purify the human CD38 protein in prokaryotic and eukaryotic system; ② prepare monoclonal antibodies of CD38 molecule in order to study its biological effect and the effective mechanism; (3) To locate the region of antigenic epitopes recognized by anti-CD38 McAbs.In this study, four subjects were investigated:(1) Cloning and expression Of the cDNA fragment of the extracellular domain of ' human CD38 antigen.The cDNA sequences of human CD38 were amplified by RT-PCR techmique from human B lymphoma cell line Daudi, and were inserted into pGEMT to obtain recombinant vector pGEM-T /CD38,The validity of the sequences was confirmed by automatic DNA sequencing. The 770bp cDNA fragment of the extracellular domain of human CD38 was amplified from the recombinant vector pGEMT /CD38, then it was subcloned into expression vector pET28a(+) , E.coli BL21 was transfected with recombinant vector and induced with IPTG SDS-PAGE analysis demonstrated that there existed a recombinant protein, and its relative molecular weight was accordance with that of our expected. Western-blot analysis indicated that the recombinant protein can be recognized by standard anti-CD38 mAb. This work lay a fundation for the preparation of anti-CD38 McAbs.(2) Cloning and expression of human CD38 full length cDNA in COS7 cells.The full length cDNA sequence of human CD38 were amplified by PCR from the recombinant vector pGEMT /CD38, The validity of the sequences was confirmed by automatic DNA sequencing. Insert the valid CD38 gene into pcDNA3.1(+) plasmid to obtain recombinant mammalian expression vector pcDNA3.1(+)/CD38Z, Using lipofectin gene transfer technique system, recombinant expression vector containing CD38 gene was transfected into COS-7 cells. The expression of CD38 molecules on the surface of COS-7 cells was detected by FACS and immunohistochemical technique.(3) Preparation and function assay of monoclonal antibodies to human CD38. Daudi cell line which highly expresses CD38 molecule was used as antigen to immunize Balb/c mice. The spleen cells of the immunized mice were used to prepare the McAb by hybridoma techniques. Hybridoma cells was selected by indirect immunofluorescence experiments , A monoclonal antibody against CD38 was obtained by hybridoma techniques and named as Ic6. The results of immunoprecipitation showed that the antibody could recognize 45KDa molecules on the surface of Daudi cell. FCM analysis indicated that Ic6 accorded with anti-CD38 McAb in specific reaction with cell lines. Western-Blot analysis revealed that Ic6 could recognize the recombinant CD38 protein. These Functional assay by MTT revealed that Ic6 could markedly kill target cells by CDC(complement dependant cytotoxicity) and significantly inhibited the proliferation of several tumor cell lines, but could not induce apoptosis of these tumor cell lines. 3H-TdR incorporation of PBMC was decreased significantly after treated with varing concentration of 1C6.(4) A preliminary study on targeting antigenic epitopes of monoclonal antibody against human CD38 antigen.Several recombinant fusion espression vectors were constructed which contained human CD38 genes deleted different parts respectively. After induction with IPTG, the fusion proteins were expressed and analyzed by SDS-PAGE and Western blot. The result of Western blot showed that the region recognized by 1C6 spans between aa220-242 ,close to the carboxy terminus of the molecule. The determination of epitope recognized by 1C6 was not only helpful to acquire more about the interaction of antigen and antibody, but also facilitative to the works of engineered antibody and drug...