Compositions and methods for cancer therapy with dengue virus and dendritic cells
A technology of dendritic cells and dengue virus, applied in biochemical equipment and methods, medical raw materials derived from viruses/bacteriophages, viruses, etc., can solve problems such as unsatisfactory results, low ability of complete response, and limited success
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Embodiment 1
[0155] Example 1. Generation and pulse treatment of murine dendritic cells (DC)
[0156] Mature DCs were generated from mouse bone marrow using the method as described by Lutz M. et al. (J. Immunol. Methods 223:77-92, 1999). The bone marrow suspension was incubated in culture dishes for 10 days in medium supplemented with recombinant murine GM-CSF. Non-adherent cells were collected, centrifuged, and resuspended in medium containing GM-CSF and lipopolysaccharide. Two days later, DCs were harvested and their viability was determined by trypan blue exclusion. Purity of DCs was determined by flow cytometry analysis. DCs were pulsed with 10 μg / ml of synthetic peptide for 18 hours. After 18 hours of incubation, DCs were harvested, washed twice in HBSS, and resuspended in HESS for additional analysis (see Examples 2 and 3).
Embodiment 2
[0157] Example 2. Dengue virus and dendritic cells for the treatment of melanoma in a first mouse model
[0158] Mouse model experiments using dengue virus (DV) strains and tumor antigen-primed dendritic cells (DCs) were performed to observe the results of combined targeting of cancer cells. DV C57BL / 6 mice were inoculated with 0.05 ml of 1x10 via tail base injection 6 or 1x10 7 Dengue virus (DEN-2 strain #1710) in pfu / ml. 2,000 ( rIL-2) and 500 IU (rIFN-γ) administered recombinant murine IL-2 (Genzyme) and IFN-γ (Sigma Pharmaceuticals). Seven days after dengue virus administration, C57BL / 6 mice were immunized intravenously with mouse DCs incubated separately with the two peptides. Synthetic peptides. An H-2b-restricted peptide from ovalbumin (OVA-8), SIINFEKL (SEQ ID NO: 7), was used as a control. Using B16 melanoma-associated H-2b-restricted peptide (EGSRNQDWL (SEQ ID NO: 1 )) derived from antigen gp100 / pme117 and B16 melanoma-associated H-2b-restricted peptide derived...
Embodiment 3
[0162] Example 3. Dengue virus and dendritic cells for the treatment of melanoma in a second mouse model
[0163] Mouse model experiments were performed using DCs primed with dengue virus (DV) strains and tumor antigens to observe the results of combined targeting of cancer cells. Cytokines were administered to mice to parallel the response to DV observed in humans.
[0164] Tumors were established in mice using the H-2b-restricted B16 mouse melanoma cell line (ATCC #CRL-6322). Peptides (derived from antigen gp100 / pme117 and B16 melanoma-associated H-2b-restricted peptide derived from TRP-1 / gp75) for pulsed dendritic cells were synthesized. Dendritic cells were generated from mouse bone marrow according to the method described by Lutz et al. (J. Immunol. Methods 223:77-92, 1999).
[0165] On day 0, mice received 5x10 4live B16 melanoma cells to establish lung metastases. On day 7, mice were inoculated with 0.05 ml of 1x10 by injection at the base of the tail 6 or 1x10 7 ...
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