Method of identifying tumor associated antigens
Compositions and methods of producing improved cancer vaccines are described. In addition, methods of identifying tumor associated antigens are also described.
1. A method of identifying one or more tumor associated antigens of a target tumor comprising the steps of:
(a) transfecting genomic or cDNA from cells of a target tumor into recipient cells expressing at least one MHC determinant that is allogeneic to a plurality of recipient animals;
(b) diluting a suspension of the transfected recipient cells into a plurality of cell pools;
(c) expanding the cells of the plurality of cell pools of step (b);
(d) immunizing each of the recipient animals with a portion of each of the expanded cell pools of step (c);
(e) isolating spleen cells from the recipient animals immunized according to step (d);
(f) incubating the isolated spleen cells with the target tumor cells;
(g) detecting an immune response of the spleen cells to the target tumor cells and identifying the cell pool that generates the highest level of immune response (the immuno high pool) and the cell pool that generates the lowest level of immune response (the immuno low pool);
(h) optionally enriching the immuno high pool by repeating step (b) to step (g) with the immuno high pool;
(i) isolating RNA from the cells of the immuno high pool and from control cells;
(j) performing microarray analysis on RNA isolated from the cells of the immuno high pool and RNA isolated from the control cells; and
(k) identifying one or more genes expressed at increased levels in the cells from the immuno high pool as compared to the control cells,
wherein the one or more genes identified are tumor associated antigens of the target tumor.
2. The method of claim 1 , wherein the control cells are cells of the immuno low pool.
3. The method of claim 1 , wherein the recipient cells further express a cytokine.
4. The method of claim 3 , wherein the cytokine is IL-2.
5. The method of claim 1 , wherein the recipient cells are fibroblasts.
6. The method of claim 1 , wherein the target tumor is a solid tumor or a hematological tumor.
7. The method of claim 6 , wherein the target tumor is melanoma, lymphoma, plasmacytoma, sarcoma, glioma, thymoma, leukemia, breast cancer, squamous carcinoma, prostate cancer, colon cancer, esophageal cancer, brain cancer, lung cancer, ovarian cancer, cervical cancer, or hepatoma.
8. The method of claim 7 , wherein the target tumor is breast cancer, lung cancer or squamous carcinoma.
9. The method of claim 1 , wherein the immune response is detected in step (g) by Elispot IFN-gamma assay or 51 Cr-release assay.
10. The method of claim 1 , wherein the suspension of transfected recipient cells are diluted in step (b) to a cell density from about 1×10 3 to about 1×10 5 cells in each cell pool.
11. The method of claim 10 , wherein the suspension of transfected recipient cells are diluted in step (b) to a cell density of about 1×10 3 cells in each cell pool.