Cloning And Expression Of Human B7-H4 And Preparation Of Mouse Monoclonal Antibodies Against Human B7-H4

Human B7-H4 (also called B7S1 and B7x) , a member of B7 superfamily, is a type I membrane protein encoding 183 aa. B7-H4, cloned by Chen et al in 2003, is located on chromosome 1p11.1. Genomic DNA of B7-H4 consists of six exons and five introns and spans 66Kb, while the most prominent transcript for B7-H4 is approximately 1.8 Kb. Despite mRNA expression in several human tissues including both lymphoid and non-lymphoid, immunohistochemistry analysis does not reveal positive staining in any organs from healthy individuals. However, B7-H4 can be induced on T cells, B cells, monocytes and dendritic cells after in vitro stimulation. In contrast, immunohistochemical analysis demonstrated that B7-H4 was constitutively expressed in freshly isolated cancer tissues such as breast cancer, ovarian cancer and lung cancer. Probing of a mouse tissue Northern blot indicated that mouse B7-H4 has up to four different mRNA transcripts, depending on the tissue type. Sensitive RT-PCR showed that human tissue contained at least two different transcripts of B7-H4. These results suggest that B7-H4 expression is tightly controlled in the translational level in peripheral tissues. As one part of the important costimulatory molecules, B7-H4 pathway provides the important costimulatory signal to activate T and B cells in the process of immune response. Engagement of B7-H4 can inhibit CD4~+T cell proliferation in vivo by cell cycle arrest at G0/G1 phase. And programmed cell death is not a major mechanism responsible for inhibited T cell proliferation. Recent research showed B7-H4 signal also plays a vital role in the development of anti-tumor immunity and autoimmune diseases. B7-H4 is an ideal targetfor immunotherapy because its expression is mostly restricted to some tumor tissues. Therefore, the construction of B7-H4 transfected cells, the obtaining of B7-H4 antagonistic monoclonal antibody and investigation of their effects on B7-H4 mediated signal transduction may have significant theoretic and clinical values in tumor, transplant rejection and autoimmune diseases.1 Cloning of human B7-H4 gene and efficient expression ofrecombinant protein in E. ColiTo explore the biofunctions of human B7-H4, a new member of B7 superfamily, the fragment of human B7-H4 encoding the extracellular region (IgV-like and IgC-like domains) was obtained by PCR (Polymerase Chain Reaction) from human cDNA FLJ22418 and then cloned into the prokaryotic expression vector pGEX-5X-3 expressing glutathione S-transferase (GST) fusion protein as inclusion bodies. The insoluble protein following washing, denaturation, renaturation, dialysis was purified through GST affinity chromatography column. The elution profile was monitored and the analysis by SDS-PAGE showed that the purity reached to 90 %.2 Positive responses of T cells costimulated by GST/hB7-H4 in vitroIn the presence of the first signal imitated by anti-CD3 monoclonal antibody, T lymphocyte proliferation was observed by incubating purified T cells with immobilized GST/hB7-H4 fusion protein by 3H thymidine assay. The concentrations of IL-2 and TNF-y in the supernatant of T cells were determined by ELISA. The results showed that the GST/hB7-H4 fusion protein produced in bacteria had obvious biological activity to inhibit the T lymphocyte and inhibit IL-2 secretion. It is indicated that hB7-H4 may involve with the inhibitory effect of T cells.3 Construction of B7-H4 transfected cellscDNA fragment encoding human B7-H4 was inserted into retrovirus expression vector pEGZ-HA-Term with gene cloning techniques. Then the derived recombinant expression vector together with helper virus expression vectors were transfected intopackage cell 293T by liposome to produce recombinant retrovirus, which was used to infect L929 cells for 72h. The transfected L929 cells expressing B7-H4 stably were obtained through Zeocin selection and grew well after long time passaging in vitro and storage in liquid nitrogen. The expression of B7-H4 on cell surface stably maintains above 95%.4 The costimulation of L929/B7-H4 cell line to T cellsThe transfected cells L929/B7-H4 or L929/mock were added to anti-CD3 activated T cells and cocultured for 4 days. 3H-TdR incorporation assay as well as the analysis of cell apoptosis showed that L929/B7-H4 could inhibit proliferation of T cells. And the transfected cells could also inhibit CD4+ T cells evidenced by phenotype analysis. In addition, the transfected cells could inhibit T cells to secrete IL-2, IL-10 and IFN-y by ELISA.5 Establishment of hybridoma cell line specifically secretingmonoclonal antibody (mAb) against B7-H4BALB/c mice were immunized with human B7-H4 transfected L929 cells as an immune antigen and the splenocytes were fused with murine myeloma SP2/0 cells by the cell fusion hybridoma technique. By means of multiple cell subcloning and repeated screening with L929/B7-H4 as antibody screening positive cell while L929/mock as negative control, a hybridoma cell line (named as 1G7) stably secreting anti-B7-H4 monoclonal antibody was selected out Then the Ig isotypes were identified with test paper. The isotope of 1G7 was mouse IgGl while the isotope of light chain is k. The hybridoma cells grew well after long-term culture in vitro (40 passages) and storage in liquid nitrogen.The ascites were produced according to the ascites-inducing procedure in mouse abdominal cavity. The ratio of ascites formation was above 95 percent and the average yield is about 5 milliliter (ml) per mouse. After the ascites were purified by protein G affinity chromatography, the protein content was between 0.8 and 10 milligram (mg)/ml with 1:1000dilution by FACS. And the usuage of doing FACS was 0.2~2.0 microgram (ng)/l * 106 cells.6 The biological characteristics of anti-B7-H4 mAb in vitroThe study of antibody's biological function showed that mAb 1G7 could induce T cells activation and proliferation in a dose-dependent way with pre-coating agonist anti-CD3 mAb (0.3 ug/ml) (PO.05). Above all indicated that the anti-B7-H4 mAb was functional antibody and it might have potential values in clinical application of organ-transplant rejection, autoimmune diseases and tumor.7 Cloning of mouse anti-hB7-H4 mAb variable regionAmplification of murine variable regions (mVH & mVL) was achieved by RT-PCR using a pair of specific consensus primers from hybridoma 1G7. And the sequence of variable regions was analysis by bioinformatic methods. It made a solid foundation for humanization of mAb 1G7.In conclusion, human B7-H4 gene was cloned, fusion protein B7-H4/GST was successfully expressed, B7-H4 transfected cell line was constructed and a hybridoma continuously and steadily secreting specific anti-B7-H4 mAb was also successfully obtained. In China, we firstly obtained an antagonistic anti-B7-H4 mAb (1G7). And it is verified that blocking B7-H4 on cell surface could strength T cell response. These results were original. In addition, obtain of antagonistic anti-B7-H4 mAb and cloning of its variable regions laid material basis for further studying B7-H4 pathway and antibody humanization.