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Project
In vitro cell-free protein synthesis and purification platform. (HERC16)
The LMCT is involved in immunotherapy with autologous dendritic cells electroporated with mRNA encoding tumor antigens. A platform for the in vitro synthesis of mRNA has been established. A major effort is the detection of immunologic biomarkers associated with clinical response after immune therapy. So far, most of the efforts are focused on the detection of a cellular response (1-3).
The immunosuppressive environment of the tumor does not support cross-priming, which is necessary for tumor immune recognition (Disis 2010). TriMix-DC immunotherapy will induce tumor-antigen specific T cells. Trafficking of these T cells secreting IFN-g could activate local tumor APC, restore key functions and upregulate immune accessory molecules, which would enhance cross-priming. The measurement of the restoration of effective cross-priming may be a biomarker more closely related to the antitumor response. Effective cross-priming could be assessed by the development of epitope or antigen spreading. The development of antigen spreading after a cancer vaccine, is measured by the generation of new immunity to antigens not present in the vaccine (Fig. 1).
Our expertise concerning the in vitro synthesis of mRNA will be used to extend our analyses towards antigens highly expressed by the tumor but not present in our DC vaccine (Mage-A3, -C2, tyrosinase, gp100). mRNA's encoding antigens such as NY-ESO, Mage-A1, -A4, -A10, -C1 will be synthesized. These antigens can then be used in our immune monitoring assays to investigate antigen spreading. We will develop a novel assay to detect humoral responses towards these antigens released upon tumor destruction. Such a response should be easier to detect in peripheral blood. A tag sequence will be cloned in frame with the tumor encoding sequence. The tag sequence will be recognized by BirA for biotinylation. Upon in vitro synthesis of this mRNA, an in vitro wheat germ cell-free translation reaction will be performed for protein synthesis. The protein antigens can then be coupled to differentially colored beads coated with streptavidine (Fig.2).
This assay to detect the antibody responses to the released antigens will be used as a surrogate for CD8+ T cell mediated tumor destruction. Such an assay can be expanded easily to large panels of target proteins. The assay will be validated using sera containing MAGE-A3 specific antibodies obtained from patients immunized with the MAGE-A3 protein or from patients with neutralizing antibodies against flu or other vaccine antigens.
The immunosuppressive environment of the tumor does not support cross-priming, which is necessary for tumor immune recognition (Disis 2010). TriMix-DC immunotherapy will induce tumor-antigen specific T cells. Trafficking of these T cells secreting IFN-g could activate local tumor APC, restore key functions and upregulate immune accessory molecules, which would enhance cross-priming. The measurement of the restoration of effective cross-priming may be a biomarker more closely related to the antitumor response. Effective cross-priming could be assessed by the development of epitope or antigen spreading. The development of antigen spreading after a cancer vaccine, is measured by the generation of new immunity to antigens not present in the vaccine (Fig. 1).
Our expertise concerning the in vitro synthesis of mRNA will be used to extend our analyses towards antigens highly expressed by the tumor but not present in our DC vaccine (Mage-A3, -C2, tyrosinase, gp100). mRNA's encoding antigens such as NY-ESO, Mage-A1, -A4, -A10, -C1 will be synthesized. These antigens can then be used in our immune monitoring assays to investigate antigen spreading. We will develop a novel assay to detect humoral responses towards these antigens released upon tumor destruction. Such a response should be easier to detect in peripheral blood. A tag sequence will be cloned in frame with the tumor encoding sequence. The tag sequence will be recognized by BirA for biotinylation. Upon in vitro synthesis of this mRNA, an in vitro wheat germ cell-free translation reaction will be performed for protein synthesis. The protein antigens can then be coupled to differentially colored beads coated with streptavidine (Fig.2).
This assay to detect the antibody responses to the released antigens will be used as a surrogate for CD8+ T cell mediated tumor destruction. Such an assay can be expanded easily to large panels of target proteins. The assay will be validated using sera containing MAGE-A3 specific antibodies obtained from patients immunized with the MAGE-A3 protein or from patients with neutralizing antibodies against flu or other vaccine antigens.
Date:26 Apr 2012 → 25 Apr 2017
Keywords:Chemometrics, Drug Analysis, Analytical Chemistry, Artificial Intelligence, Chromatography, Food Analysis
Disciplines:Nutrition and dietetics, Drug discovery and development, Analytical chemistry