| CD40, a type I transmembrane glycoprotein with molecular weight of 48000, belongs to nerve growth factor receptor (NGFR) and tumor necrosis factor receptor (TNFR) superfamily. CD40 is widely expressed on B lymphocytes, monocytes/ macrophages, dendritic cells, hematopoietic cells, epithelial cells, vascular endothelial cells as well as carcinoma cells. Interaction between CD40 and its ligand CD40L plays a key role in lymphocyte activation and differentiation, cytokine secretion and antibody production and class switching. Apart from direct effects on tumor cells, CD40 could mediate addition anti-tumor effects on CD40 highly expressed malignant cells through regulate innate and adaptive immune responses. Although three anti-CD40 monoclonal antibodies have entered preclinical trials, further studies are needed to evaluate their safety due to wide expression of CD40 in normal tissues. CD40 mutant (CD40mu), a C-to-A transition (H78Q), is discovered in some cell lines and freshly isolated tumor cells. It was confirmed that this mutant has altered partial epitope of CD40. This paper described three new antibodies against CD40mu 3E4,6B4 and 10C5, whose recognition specificity are quite different from antibody against normal human CD40. They can recognize CD40mu expressed on tumor cell lines (HO8910, H460) and fresh tumor tissues. 10C5 do not recognize CD40 expressed by vascular endothelial cell line (Eahy 926) and normal tissues indicating potential application value.1. Establishment of the hybridoma cell lines specifically secreting monoclonal antibody (mAb) against CD40muBALB/c mice were immunized with L929/CD40mu 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/CD40mu as antibody screening positive cell while L929/CD40wt as negative control, one hybridoma cell line, 3E4,6B4and10C5, stably secreting anti-CD40mu monoclonal antibody were obtained. The Ig isotype were IgG2b, IgG1 and IgG3 with test paper. The hybridoma cells grew well after long-term culture in vitro and were storaged 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 contents were about 1.0, 2.2,1.1 milligram(mg)/ml with 1:10000 dilution by FCM.2. The biological characteristics of anti-hCD40mu mAb in vitroIt was found that CD40mu was expressed on some malignant B cells and epithelial tumor cell line express as well as fresh tumor tissues. Epitopes recognized by these mAbs are quite different from 5C11 and other commercial anti-human CD40 monoclonal antibodies. They can identify a variety of tumor cell lines (HO8910, H460, etc.). 10C5 can not identify normal CD40 on vascular endothelial cells Eahy926 and some normal tissue. Preliminary experiments in vitro showed that 3E4 could induce the growth of human ovarian cancer cell line HO8910 and 10C5 could inhibit the growth of HO8910 cells in vitro and promote their apoptosis.To sum up, this study successfully prepared three specific mouse anti-human CD40mu monoclonal antibodies, discussed the expression of CD40mu on tumor cells and their biological functions; the mAbs caused biological effects by stimulating tumor cells expressing CD40mu: induce/inhibit growth of tumor cells in vitro and promote their apoptosis. The mAbs provided a material basis for further discussion of tumor cells biological functions expressing CD40mu. They also provided new ideas for the targeting biological therapy of tumor expressing CD40mu.
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