Functional Study Of Anti-human B7-H4Monoclonal Antibodies And Optimization Of An ELISA Kit For SB7-H4

As a negative costimulatory molecules, B7-H4is a recently identifed B7familymember that regulate the adaptive immune response by inhibiting T cellproliferation,activation and cytokine production. Human B7-H4belongs to the type Ⅰtransmembrane protein. Comparing with other B7family members, It has a20–30%amino acid homology in the extracellular portion. Though B7-H4mRNA is widelyexpressed in human lymphoid and non-lymphoid tissues, B7-H4protein seems to belimited in normal tissues. Dendritic cells (DCs), monocytes and T, B lymphocytes whichisolated from the fresh blood did not express B7-H4molecule. However, after theactivation in vitro B7-H4would be expressed in all above cells. Recent studies showthat human tumor cells such as colon, prostate, ovarian cancer cells, and lung tissueindicated the expression of B7-H4abnormally. These findings prompted that in thetumor microenvironment B7-H4plays an important role in the process of tumor cellsevade immune surveillance and maintain immune tolerance. Thus, B7-H4is expected tobe one of the diagnosis, prognosis and treatment of target molecules in cancer patients.This study was designed by the method of Jurkat cells co-cultured with SPCA-1cells, Using the mouse anti-human monoclonal antibody5G3(prepared by ourlaboratory) to block the B7-H4molecules on SPCA-1cells. By co-culture, we willcheck the biological character changes of Jurkat cells.The sB7-H4ELISA kit was optimized and used to detect the sB7-H4expression inthe serum of patients with non-small cell lung cancer.PartⅠ The change of biological characteristics of Jurkat cells after coculturedwith SPCA-1cellsObjective: To identify the characterization of Jurkat cells by establishing an in vitrococulture system.Methods: B7-H4expression on SPCA-1cells was analysised by RT-PCR and FCMmethods. After SPCA-1cocultured with Jurkat for72h, The expression of CD3and CD69, Apoptosis of Jurkat cells all were detected by FCM, Cell proliferation wasdetected by CCK8method.Results: SPCA-1cells dramatically inhibited the CD3and CD69expressions oncocultured Jurkat cells, Jurkat cell apoptosis and cell proliferation decreased.Conclusion: In non-small cell lung cancer cell lines，B7-H4negatively regulates Tcell-mediated antitumour immunity.This conclusion would be helpful to theinvestigation of B7-H4in future.Part Ⅱ Optimization of sB7-H4enzyme-linked assay kit and detection ofserum with non-small cell lung cancerObjective: To optimalize sB7-H4enzyme-linked assay kit and detect sB7-H4levelsin patients with non-small cell lung cancer.Methods: On the basis of sB7-H4ELISA kit which was generated by our lab, weused1F10as coating antibody, biotinylated8F10and5G3as detecting antibody. AnELISA kit for human sB7-H4was optimalized and applied for the examination of serumlevels of sB7-H4in non-small cell lung cancers.Results: An ELISA kit for human sB7-H4was optimalize successfully with0.25ng/mL～16ng/ml of lower limitation measurement.The highest sensitivity of this ELISA kit was determined by4μg/ml of8F10and0.1μg/ml of5G3. The serum sB7-H4levels in NSCLC and healthy control groups were(10.53±2.96) and (9.0882.43)μg/ml, rspectively．The levels of NSCLC group weresignificantly higher than those of healthy group(p<0.05).The serum sB7-H4levels ofⅢ/Ⅳstage NSCLC patients (10.803.45)μg/ml were relatively higher than those in theⅠ/Ⅱstage (10.332.67) μg/ml(p<0.05). In NSCLC, Men(10.612.61)μg/ml andwomen（10.38±2.48）μg/ml, Older than50years old (10.262.54)μg/ml and youngerthan50years old (10.412.65)μg/ml, All of them are nearly the same levels ofsB7-H4.The serum sB7-H4level in lung cancer patients before treatment was obviouslyhigher than that after treatment([10.75±2.72μg/ml]vs.[9.93±2.88μg/ml])withstatistically significant difference((p<0.05).Conclusion: A sensitive and specific ELISA kit for detecting human sB7-H4wasoptimalized, When this ELISA kit was applied, We found serum levels of sB7-H4innon-small cell lung cancers was higher than healthy controls. And it related to clinical staging, regardless of age and gender, sB7-H4is expected to become a target moleculelung cancer diagnosis and treatment.In summary, The biological characteristics of Jurkat cells after cocultured withSPCA-1cells; Optimization of sB7-H4enzyme-linked assay kit and detection of serumwith non-small cell lung cancer, All were studied successfully. So these results wouldlay foundation for further study in clinical applications.