Preparation And Biological Characterization Of Mouse Anti-human 4-1BBL Monoclonal Antibodies

Human 4-1BBL is a type II transmembrane glycoprotein,belongs to tumor necrosis factor(TNF) superfamily. 4-1BBL is widely expressed on many types of cells, including B cells, macrophages, dendritic cells,activated T cell, et al, but the unactivated T cell. It is reported that 4-1BBL is relevant to human hematological malignancies. It is reported that a soluble form of 4-1BBL(s4-1BBL) was present at high levels in the sera of some patients with various hematological diseases, but only at low levels in healthy donors. 4-1BBL/4-1BB expressed on some B or T lymphoma cell lines, in spite of low expression level, involves in the proliferation of lymphocyte or resistance to drug. Recent studies revealed that bi-directional transduction of signals existed for the 4-1BB receptor/ligand system. While cross-linking of 4-1BB activates T lymphocytes, cross-linking of the 4-1BB ligand has the opposite effect. This reverse signalling through the 4-1BB ligand inhibits proliferation of T lymphocytes and induces apoptosis. Reverse signalling exists also for monocytes and cross-linking of 4-1BB ligand on monocytes can induce the cell proliferaion, the production of M-CSF, IL-6, IL-8 and TNF-αand prolong the survival of monocytes. 4-1BBL reverse signalling also can promote the maturation of DCs from human CD34+ hematopoietic stem cells. For example, 4-1BBL reverse signalling can upregulate the expression of CD86, CD83 and production of IL-12 on DCs generated from CD34+ progenitor cells. Therefore, 4-1BBL reverse signalling play critical roles in immune response mediated by T cells, monocytes, DCs. However, immunoregulatory roles of the 4-1BB/4-1BBL bi-directional signalling and the associated mechanisms still need further and detailed investigation. Our study established the anti-human 4-1BBL mAbs, one of them(39A8)is a functional 4-1BBL mAb and provided a useful tool for studying the biological function of 4-1BBL.1. Preparation of anti-4-1BBL mAb4-1BBL transfected L929 cells(L929/4-1BBL), after pretreatment with mitomycin, were used as immunogen to immunize Balb/c mice to prepare anti-4-1BBL mAb. According to the B lymphocyte hybridoma technique, splenocytes harvested from the immunized mouse were fused with SP2/0, murine multiple myeloma cells. After repeated screening by L929/4-1BBL(as positive screening) and L929/mock cell line(as negative control), and multiple cloning in HAT conditioned culture, a hybridomas(39A8) secreting anti-4-1BBL mAb continuously and steadily were obtained. Fast-strip analysis showed that the isotopes of mAb 39A8 belonged to mouse IgG2a. The hybridoma grew well after long-term storage in the liquid nitrogen and in vitro culture.2. Identification of 4-1BBL mAb characteristicsTo further elucidate the recognition ability of the mAb against 4-1BBL molecule, the phenotype analysis was utilized by flow cytometry assay. The results indicated that the anti-4-1BBL mAb could recognize 4-1BBL molecule expressed on U937, THP-1, Hela, PC-3, Daudi, M231, SHG44, et al. The results suggested that the mAb can recognize the 4-1BBL molecules on different cells.The 4-1BBL mAb with different concentrations was added to the culture system of U937, THP-1 and 675321 cells constantly expressing 4-1BBL molecules. We observed cell growth, counted cell number and described growth at 24h, 48h, 72h and 96h respectively. The result showed that these mAbs caused the proliferation suppression of tumor cells. After 24h hours of treatment, trigger action was detected by using microexamination and cell counting. The results indicated that the 4-1BBL expressed on some tumor cells could also mediate reverse signals, which provided the proliferating effects similar to the growth cytokines on some tumor cells.In conclusion, this study has prepared one specific mouse anti-human 4-1BBL monoclonal antibody successfully, then has studied its biological characterization. We studied the 4-1BBL reversion signal to tumour cell initially by using the anti-4-1BBL mAb. Therefore, the successful preparation of this functional anti-4-1BBL mAb has provided the effective material base for the research of 4-1BBL/4-1BB later biology function and its signal way as well as the development of the clinical diagnosis tools.