Effects And Mechanisms Of Recombinant DsNKG2D-IL-15Fusion Protein Against Tumor

NKG2D is a key activating receptor expressed on NK and CD8+T cells. MHC class I chain-related protein A (MICA) as one of human NKG2D ligands is highly expressed on tumor cells. Retionic acid early inducible protein-1(RAE-1) is one of mouse NKG2D ligands. IL-15promotes proliferation, activation and survival of NK and CD8+T cells. Two new fusion proteins in which double soluble NKG2D extracellular domains were fused to N-terminus of IL-15, called human dsNKG2D-IL-15and mouse dsNKG2D-IL-15respectively, were generated based on genetic engineering technology in our lab. We expected that double soluble NKG2D domains bound to tumor cells and IL-15activated NK and CD8+T cells. Thus the fusion protein could be used for tumor therapy.Previous study demonstrates that the recombinant human dsNKG2D-IL-15protein promotes activities of NK cells, and the recombinant mouse dsNKG2D-IL-15protein not only binds to RAE-1positive tumor cells, but also stimulates activation, proliferation and cytotoxicity of NK cells. In this study we aimed to verify effects and mechanism of recombinant dsNKG2D-IL-15fusion proteins against tumor, and to provide necessary experimental data for successively clinical study.Our study could be divided into two parts.Part Ⅰ. Effects and mechanisms of recombinant human dsNKG2D-IL-15fusion protein against tumorAims:To observe whether the recombinant human dsNKG2D-IL-15protein binds to MICA positive tumor cells or not, and to explore effects of tumor cells anchored with dsNKG2D-IL-15on activation, proliferation and function of NK cells, and effects of human dsNKG2D-IL-15on growth of transplanted gastric tumor in nude mice.Methods:The binding of the recombinant human dsNKG2D-IL-15protein to MICA positive tumor cells was analyzed by flow cytometry. After incubation of soluble dsNKG2D-IL-15or plate-bound dsNKG2D-IL-15fusion protein with human peripheral blood mononuclear cells (PBMC) overnight, expression of CD69, NKp46, NKG2D, NKG2A, CXCR3, DNAM-1receptors on NK cells was checked by flow cytometry. IFN-γ production was analyzed by a flow cytometric-intracellular staining assay. Proliferation was detected by the MTS/PMS method. NK cell killing activity was measured by both a CD107a degranulation assay and a LDH releasing kit. Next expression of CD69, NKG2D, CD107a on NK cells and IFN-γ production of NK cells stimulated with K562-MICA cells anchored with dsNKG2D-IL-15were analyzed similarly. A gastric cell line, SGC-7901, was injected subcutaneously in the back of Balb/c nude mice. Five days later, recombinant human dsNKG2D-IL-15, mouse dsNKG2D-IL-15, or PBS was injected peritoneally each day for21days. Volumes of tumors and mouse survival situations were documented every day. On day21tumor mice were sacrificed to isolate spleens and tumor tissues. Frequencies of NK cells and NKG2D positive NK cells of spleens and tumor tissues were examined in all mice.Results:Recombinant human dsNKG2D-IL15fusion protein bounds to MICA positive tumor cells. Soluble and immobilized dsNKG2D-IL-15both up-regulated NK cell CD69expression on NK cells and enhanced cytotoxicity, IFN-y secretion, proliferation of NK cells. With stimulations of human dsNKG2D-IL-15CD16expression level on NK cells decreased, and NKp46, CXCR3, NKG2A expressions on NK cells were up-regulated, while there was no significant change of DNAM-1expression on NK cells. Tumor cells anchored with human dsNKG2D-IL-15promoted activation, IFN-y secretion, and killing activity of NK cells. In tumor-bearing nude mice, human dsNKG2D-IL-15treatment inhibited gastric tumor growth and elongated life span of tumor mice significantly compared with PBS or mouse dsNKG2D-IL-15treatments. In addition, frequencies of NK cells and NKG2D+NK cells in spleens and tumor tissues were higher in human dsNKG2D-IL-15treatment groups than those in PBS or mouse dsNKG2D-IL-15treatment groups.Conclusions:The recombinant human dsNKG2D-IL-15fusion could bind to MICA positive tumor cells and promote activation, proliferation, and cytotoxicity of NK cells. Treatment with dsNKG2D-IL-15retards growth of MICA positive tumors and prolongs survival of tumor-bearing nude mice. The anti-tumor effect is associated with NK cell activation and NK cell migration into tumors. 2. Effects and mechanisms of recombinant mouse dsNKG2D-IL-15fusion protein against tumor in vivoAims:To observe effects and mechanisms of recombinant mouse dsNKG2D-IL-15fusion protein on growth of transplanted colon tumor in normal Balb/c mice.Methods:The binding of the recombinant mouse dsNKG2D-IL-15protein to Ba/f3-RAE-1cells was analyzed by flow cytometry. NK cell cytotoxicity against tumor cells anchored with mouse dsNKG2D-IL-15was detected by the LDH releasing assay. A mouse colon cancer cell line, CT-26, was inoculated into healthy Balb/c mice subcutaneously. At the same day or five days later, these mice were injected with mouse dsNKG2D-IL-15, or human dsNKG2D-IL-15, or PBS, peritoneally. Tumor volumes and mouse survivals were documented every day. Twenty-one days later, tumor mice were sacrificed to isolate spleens and tumor tissues. Frequencies of NK cells, CD8+T cells, NKG2D+NK cells, NKG2D+CD8+T cells in spleens and tumor tissues were analyzed.Results:Recombinant mouse dsNKG2D-IL-15fusion protein bound to RAE-1positive tumor cells. NK cell killing activity was enhanced as RAE-1positive tumor cells anchored with dsNKG2D-IL-15. Transplanted colon cancer in Balb/c mice study showed that mouse dsNKG2D-IL-15inhibited tumor growth and elongated life span, compared with human dsNKG2D-IL-15or PBS treatments. Frequencies of NK cells, NKG2D+NK cells, CD8+T cells, and NKG2D+CD8+T cells in spleens and tumor tissues were enhanced in the mouse dsNKG2D-IL-15treatment group.Conclusions:The recombinant mouse dsNKG2D-IL-15fusion protein not only binds to RAE-1positive tumor cells, but also enhances NK cell killing against RAE-1positive tumor cells. With the mouse dsNKG2D-IL-15treatment in vivo RAE-1positive tumor growth is retarded and life span is prolonged. The anti-tumor effect is associated with activation of NK and CD8+T cells and high densities of NK and CD8+T cells in tumors.