The Preparation Of Mouse Anti-human B7-H4 Monoclonal Antibody And The Expression Characteristics Of B7-H4 In Tumor Cells

B7-H4 is another negative costimulatory molecule that was newly discovered in B7 family. Although its mRNA shows an extensive expression pattern in lymphoid and non-lymphoid tissues, the protein of B7-H4 only presents weak expression in normal tissues because of the stringent regulation at the post-transcriptional level. Accumulated studies have found that the tumor cells and tumor-associated macrophages in tumor tissues such as ovarian cancer, lung cancer, breast cancer, renal cell carcinoma and adenoid cystic carcinoma overexpress B7-H4 molecules. Under the stimulation of B7-H4 signal, the number of CD3+T cells around the tumors was reduced significantly and the proliferation of activated CTL was inhibited. As a result, the host immune responses against the tumors were greatly weakened. B7-H4 signal could also inhibits the tumor apoptosis and promotes malignant transformation of endothelial cells,which provides all the necessary conditions for tumor immune escape. Clinical studies have found that the expression of B7-H4 in tumor cells is closely related with adverse clinical and pathological features,such as the prevalence of lymph node metastases and poorer prognosis. Moreover, high level of soluble B7-H4 molecules in the blood serum of cancer patients provides a new biomarker and evidence for clinical diagnosis, especially in the detection of ovarian cancer at early stage. As the great attention had paid to the negative co-stimulatory molecule B7-H1, B7-H4 becomes a new hotspot in the research of tumor immune escape and clinical diagnosis.This study aims at the preparation of mouse anti-human B7-H4 monoclonal antibodies by using transfected cells expressing high level of B7-H4 as the antigens and cell fusion technology and try to provide effective research tools for further exploration of the biological significance of B7-H4 molecule in the tumor immune escape and new ways for clinical diagnosis, prognosis and treatment.PartⅠThe preparation of mouse anti-human B7-H4 monoclonal antibody and the analysis of its biological characteristics[Objective] The follow experiments were carried out to prepare novel mouse anti-human B7-H4 monoclonal antibodies for illuminating the regulatory expression and function of human B7-H4.[Methods] Routine immunization of BALB/ c mice by a tansfected cell line 293T/ B7-H4 as antigens.Then the immunized spleen was fused with SP2/0,a kind of plasmocytoma cells, and screen the positive clones by FCS with the 293T/B7-H4 as a positive control while 293T/ mock as the negative control.After acquisition of the hybidoma secreting anti-B7-H4 mAb,the investigation of its biological activities by Western blot,rapid isotyping analysis, karyotype analysis,competitive inhibition test, indirect immunofluorescence and 3H-TdR incorporation assay were followed.[Results] After screening and subcloning, a monoclonal antibody, named 3C8 was obtained. Immunophenotyping showed that the mAb could bind to 293T/B7-H4 transfected cells, but not to 293T/mock cells, indicating that the antibody is specific for human B7-H4. The isotype of the mAb was proved to be IgG1 withκlight chain. Western blot showed that mAb 3C8 could specifically bind to the B7-H4 molecule extracted from 293T/B7-H4 cells. The competition test conducted among mAbs evidenced that the mAb was completely different from the antigen binding sites of B7-H4 with another mAb 1G7, while it shared much more similar binding sites with the commercial antibody H74. It was revealed that the mAbs could detect the expression of B7-H4 on the CD14+ monocyte from healthy volunteers. The expression changed when inducing dendritic cells from monocytes with different factors.It showed that the expression of B7-H4 was downregulated by IL-4, while it was upregulated by LPS.The result of 3H-TdR incorporation assay revealed that the mAb 3C8 could block the suppression of PHA activated T cell proliferation effectively. [Conclusion] A novel anti-human B7-H4 monoclonal antibody was generated, which could recognize different epitopes from 1G7 and successfully block the signal from B7-H4. The results would help to further prepare ELISA kit for the detetion of soluble B7-H4 molecules and find the new way of investigation of the immune homeostasis and regulation.PartⅡThe preliminary study of the expression characteristics of B7-H4 in tumor cells[Objective] To explore the expression characteristics of B7-H4 in tumor cells and tumor microenvironment and to open up new ways for further study of the relationship between B7-H4 and tumor immune escape,the experiments were done as follows.[Methods] The analysis of the expression of B7-H4 on CD14+monocyte from different human tumor cell lines, pleural fluid and ascites of cancer patients were carried out by indirect immunofluorescence and FCS, while the distribution of B7-H4 in cancer (lung cancer) cell lines was detected by means of immunocytochemical technology. The analysis of the expression pattern of B7-H4 protein and mRNA were revealed by Western blot and RT-PCR respectively.[Results] B7-H4 were detected on many tumor cell lines, especially on the epithelial tumor cell lines such as lung and stomach cancer. The monocytes in patients'pleural fluid and ascites were different from the single B7-H4+ group of the monocytes in the peripheral blood of healthy people.There were two groups: CD14+B7-H4+ and CD14+B7-H4-. Immunocytochemical experiments showed that there were still a lot of B7-H4 molecules lying in the cytoplasm of the tumor cell lines. Western blot results revealed that the molecular weight of B7-H4 in the tumor cell lines, tumor-infiltrating mononuclear cells and healthy human peripheral blood mononuclear cells is about 34KD, a bit smaller than the normal ones.But RT-PCR results didn't show the existence of B7-H4 alternative splicing forms in these tumors and tumor-associated cells.[Conclusion] Though the main transcription of B7-H4 cDNA is in the normal size in the tumor cells and CD14+monocytes in patients'pleural fluid and ascites, the expression of B7-H4 protein's molecular weighs less than normal ones and glycosylation level is not high. The regulatory role and mechanism of B7-H4 protein with low glycosylation level in tumor and tumor micro-environment is worth of concern and further in-depth study. In addition, whether CD14+B7-H4+ and CD14+B7-H4- in patients'pleural fluid and ascites represented monocyte-macrophage cell subsets of different immune regulation function is yet to be proved.PartⅢThe cloning of antibody 3C8 variable region gene and recombination of the chimeric light and heavy chain gene[Objective] On the basis of the functional antibody 3C8, cloning and recombination of the chimeric heavy and light chain genes will pave the road for human-mouse chimeric antibodies'construction.[Methods] The degenerate primers were designed according to the conservative framework of the antibody and leader sequence. The variable regions of heavy and light chain genes were then cloned by RT-PCR; Then following bioinformatics analysis were applied to confirm the CDR sequence, predict the natural signal peptide, analyze second topology of antibody variable region and 3D stucture. The recombination of the variable region of mouse antibody to the constant region gene of human IgG1 andκchain was carried out by TP-PCR.[Results] The variable region genes in the heavy and light chain with natural signal peptide sequence were obtained successfully; By bioinformatics analysis, the antibody variable region of the light chain was formed by gene rearrangements of V1-117*01 and J1*01 subgroups. And the antibody variable region of the heavy chain was formed by gene rearrangements of V1-14*01 and J4*01 and D2-2*01. Its light and heavy chain signal peptide were cleavaged at the SSS-DS and VHS-EV between the19 and 20 amino acid respectively. The second topology and the three-dimensional simulation showed that the structure of the cloned sequence was mainly formed byβ-sheet except the signal peptide shaped asα-helix. The CDR regions of the antibody was located just between theβ-fold in reverse parallel and exposed on the entire protein surface where it can bind to the antigen easily. The chimeric heavy and light chain genes recombined by PCR was about 750 bp and 1500 bp and the following experiments is under way.[Conclusion] The success of cloning and reforming of the antibody variable region gene laid a solid foundation for the further research in chimeric antibody 3C8 and antibody affinity change that based on the DNA point mutation technology.In summary, this study successfully obtained a novel functional anti-human B7-H4 antibody. It can block B7-H4 signal in vitro effectively; The cloning of antibody variable region genes and recombination of the chimeric light and heavy chain genes will provide necessary material basis for continuing research in reconstruction of humanized antibody and tumor immunotherapy targeting B7-H4. In addition, our study also revealed that low-molecular-weight expression of B7-H4 in tumor and normal tissues was not due to the different shear at the mRNA level, but closely related with the protein post-translational modification.The different expression level of B7-H4 on CD14+ monocyte membrane in peripheral blood of healthy people and patients'pleural fluid, ascites will provide new clues for exploring the immune homeostasis and tumor immune escape.