Anti-leukemia Effects Of Modified GM3 Dendritic Cell Vaccine

TACAs are the most exposed, and often the most abundant as well, TAAs on the surface of cancer cells. Therefore, they are ideal targets for the immunotherapy of cancer. GM3 is a sialylated trisaccharide TACA abundantly expressed by leukemia and other tumors in the form of glycolipids or glycoproteins. Since naturally occurring TACAs are rarely immunogenic, we have designed a modified GM3 derivative (GM3NPhAc) of which the N-acetyl of sialic acid is instead of the N-phenylactyl, being linked to a carrier molecule - keyhole limpet hemocyanin (KLH) as a TACA-based cancer vaccine (GM3NPhAc-KLH). We have found that it markedly induces many types of antibodies such as IgM, IgG1, IgG2a and IgG3 as an unnatural antigen. Cell-mediated immune response is dominant in the anti-tumor immune response, but GM3NPhAc–KLH mainly induces humoral immune. To overcome this problem, we have explored a new strategy based on joint application of the APC function of dendritic cells (DCs) and the high immunogenicity of GM3NPhAc–KLH. DCs are pulsed with GM3NPhAc–KLH as a vaccine; meanwhile, the animals or the tumur cells are forced to express the arti?cial GM3NPhAc, in place of the native GM3 on tumor cell surfaces, through treatment with N-phenylacetyl-D-mannosamine (ManNPhAc), an unnatural monosaccharide that can serve as a biosynthetic precursor of arti?cial oligosaccharides, in terms of that biosynthesis of carbohydrates has no rigid templates and is controlled by a series of specific enzymes including sialic acid transferase highly expressed in tumors. It is expected to induce a specific anti-tumor immune response easily to clear tumor cells and provide a new efficient, low toxicity approach. This study is designed to investigate use of modified monosaccharides(ManNPhAc) as precursors to affect the biosynthesis of GM3NPhAc antigen on tumor cell surfaces and explore the specific anti-leukemia effect of the dendritic cell vaccine pulsed with GM3NPhAc–KLH.The results are shown as follows:1. FBL3 tumor cells were treated with ManNPhAc(0 - 2 mM)for 24 h, 48 h, 72 h, ELISA results indicated that treated with ManNPhAc(0 - 2 mM)for 48 - 72 h significantly improved GM3NPhAc exprssion on FBL3 cell surface; GM3NPhAc specific staining on FBL3 cell surface after 72h ManNPhAc treatment was conformed by immunohistochemistry; Additionally, immunohistochemistry results showed high GM3NPhAc exprssion on tumor cell surface of FBL3 bearing mice i.p ManNPhAc 2 mg/mouse/day for a week and no GM3NPhAc exprssion without ManNPhAc treatment, which were challenged with 5×105 FBL3 cells, indicating murine leukemia cell could be e?ectively glycoengineered to express GM3NPhAc new antigen.2. ADCC study showed that specific GM3NPhAc-antibody-dependent peritoneal macrophage cytotoxicity to the glycoengineered FBL3 cell was significant increased and the cytotoxicity reached 65%-80% with 0.05 - 0.35 mM GM3NPhAc-antibody incubation of FBL3 cells. Meanwhile, highly cytotoxicity to the glycoengineered FBL3 cell in the presence of complements was evaluated by CDC study, the cytotoxicity of FBL3 cells incubating with 0.15 mM ManNPhAC for 72h was 100%, suggesting that a small amount of modified precursors are able to partially replace the original TACAs, then these glycoengineered cells can be recognized by mAb of new antigen and killed by the immune system.3. DCs were generated from murine bone marrow cells and identified by FCAS;On day5 DCs(2×10~6)were cultured with GM3NPhAc-KLH (20μg/ml) overnight to prepare DC vaccine.4. Mice were immunized and reboosted seven days later.On the day after reboost, serum was collected and preparation of splenocytes was stimulated with GM3NPhAc-KLH to induce CTL. ELISA detected that Igκin murine serum of GM3NPhAc-KLH-DC group increased significantly (P<0.01), showed vaccine activity. CTL-induced cytotoxicity to the target FBL3 cell which expressed the modified GM3 was compared by LDH release, FBL3 expressed the natural antigen as a control. The cytotoxicity of glycoengineered FBL3 cell mediated by CTL of GM3NPhAc-KLH-DC group was significantly higher than that of KLH-DC group, and the CTL mediated cytotoxicity was more potent to the glycoengineered FBL3 cell than to the unmodified FBL3 cell. It is indicated that GM3NPhAc-KLH-DC is a specific DC vaccine.5. Mice were immunized and reboosted seven days later and were challenged by FBL3 cells (5×10~5) on d17, then i.p ManNPhAc 1 mg/mouse/day for a week, tumor growth and survival were measured. We observed the tumor volume was smaller in mice treated with GM3NPhAc-KLH-DC group than the DC and KLH-DC group, in addition, the survival prolonged. The results suggests GM3NPhAc-KLH-DC vaccine possesses an anti-tumor activity. Conclusion: GM3NPhAc-KLH-DC vaccine can induce specific anti-tumor immune response and anti-tumor activity. To combine DC vaccine with glycoengineering, it is provided a new approach to high selective or targeted tumor immunotherapy.