Feature Analysis Of Binding Of Anti-CD28 Antibody With Antigen And Studying Of Antibody-mediated Effect

CD28 gene is located at human chromosome 2 q33-34 region, and the gene coding region consists of 1,104 bp. It express on the most of the resting and activated T cells, approximately 95% CD4+ and 50% CD8+T cells express CD28 molecule. In addition, some NK cells and malignant plasma cells also express CD28 molecule. Its natural ligands are B7-1 (CD80) and B7-2 (CD86).The interaction between CD28 and ligands expressed on the surface of antigen-presenting cells would mediate costimulatory signal which is important to T cell responses. Recently research shows that B7-CD28 signal is not limited between antigen-presenting cells and T cells, there are abnormal B7-CD28 signal pathway in some tumors such as multiple myeloma cells, T lymphoma cell, and the pathway may be involved in tumor metastasis, lead to disease progression. The purpose of our study is to prepare the monoclonal antibody against human CD28 and to identify its biological function; further we select mouse T lymphoma cell lines transfected human CD28 gene(CD28-T) and human T lymphoma cell lines natural express human CD28 molecules(Jurkat) as the research object ,analyze the effect of antibodies on tumor cells growth and proliferation in vitro, then we establish tumor model to analyze the effect of antibody in the process of tumorigenicity oand tumor growth and decline, in order to find effective anti-tumor biological agents.Objective : To prepare the anti-human CD28 monoclonal antibody and to study its biological function.Methods: Ascite is induced in BALB/c mice by intraperitoneal injection of well-grown hybridoma. Protein G affinity chromatography is used to purify mAb. Indirect immunoflurescence assay is used to analyze the mAb titer. We analyze the monoclonal antibodies recognized antigenic sites by FCM.FCM is used to analyze the recognition of CD28 mAb to different cell lines which differently expressed CD28 molecule.Results: The average output of ascite is to 5 ml each mouse., and the concentration of protein is 3mg/ml.Indirect immunoflurescence assay suggested that the titer of purified CD28 mAb to cells is 0.5μg/5×10~5cells. Competition experiment indicated that SQA-11 recognized a different antigen epitope from that of commercial mAb. Phenotypic analysis suggested that SQA-11 could well recognize CD28 molecule expressed on U266,CD28-T and Jurkat.Conclusion: We prepared anti-human CD28 monoclonal antibody and it laid material basis for the further study of CD28 signaling pathway in the tumor growth and metastasis.Objective : To establish tumor model to analyze the effect of antibody in the process of tumorigencity and tumor growth and decline, in order to find effective anti-tumor biological agents.Methods: The ability of proliferation of CD28-T and Jurkat under the circumstance of SQA-11 is analyzed via MTT assay. CD28-T and Jurkat cells are subcutaneous injected into the BALB/c nude mouse respectively and investigate the average days of tumor formation and their oncogenic capabilities. 40 days later the mice were killed,and the expression of CD28 molecule on cells from mice tumor tissues were analyzed by FCM. In order to analyze the effect of SQA-11 in the process of tumor growth and decline,we coculture tumor cell with SQA-11 before injection and intratumoral inject SQA-11 after tumor have formed respectively.Results: The result of MTT assay showed that the proliferation of CD28-T and Jurkat cells were inhibited when the cells were cocultured with SQA-11 after 72h. Subcutaneous inoculation experiments demonstrated that the CD28-T tumor formed on day 3 and the formation rate reached 100%(40/40);Jurkat tumor formed on day 14 and the formation rate was 80%(32/40).The result of FCM analysis showed that the positive expression ratio of CD28 molecule on CD28-T tumor tissue and Jurkat tumor tissue were 90.4% and 74.3% respectively, and there were no statistically significant difference between tumor tissue cells and tumor cell lines. CD28-T and Jurkat were cocultured with SQA-11 before injecting ,the CD28-T tumor formation was delayed two days that tumor formed on day 5,and the tumor formation rate was also 100%(20/20)and the Jurkat tumor formation was delayed five days that there were tumor formation on day 19,and the tumor formation rate was 50%(10/20).SQA-11was injected into tumor tissue after the tumor formation, the result showed that there are no significant effect on the growth of CD28-T tumor, but the growth of Jurkat tumor was inhibited obviously.Conclusion:SQA-11 could inhibit the growth of human tumor which naturally expressed human CD28 molecule, but there are no significant effect on mouse tumor which expressed transfected human CD28 molecule.