Canine autologous immunotherapy using dendritic cell induced cancer killing immunocytes
Described are compositions of matter, protocols, and treatment means for induction of immune mediated killing in dogs suffering from cancer. The invention provides means of extracting peripheral blood from a canine patient, expanding immunocytes capable of killing cancer cells in vitro, and re-administering said immunocytes into a patient in need of therapy. In one embodiment, immunocytes expanded are T cells possessing tumor cytotoxic activity induced by stimulation of NKG2D.
1. A method of inducing cytotoxicity against cancer cells in CD355+ lymphocytes comprising: a) obtaining canine blood; b) isolating peripheral blood mononuclear cells from the canine blood; c) isolating monocytic cells from said peripheral blood mononuclear cells; d) isolating CD355+ lymphocyte cells from the peripheral blood mononuclear cells; e) inducing said monocytic cells to differentiate into dendritic cells; f) co-culturing said dendritic cells with said CD355+ expressing lymphocyte cells such that said CD355+ lymphocyte cells possess cytotoxicity against cancer cells.
2. The method of claim 1 , wherein said peripheral blood mononuclear cells are isolated from said canine blood by a density gradient.
3. The method of claim 2 , wherein said density gradient is percoll.
4. The method of claim 2 , wherein said density gradient is ficoll.
5. The method of claim 1 , wherein said monocytic cells are isolated by plastic adherence.
6. The method of claim 1 , wherein said monocytic cells are isolated by magnetic activated cell separation for CD14.
7. The method of claim 1 , wherein said monocytic cells are isolated by fluorescent activated cell separation for CD14.
8. The method of claim 1 , wherein said lymphocyte cells expressing CD355 are isolated by a method selected from the group consisting of: a) magnetic activated cell separation, and b) fluorescent activated cell separation.
9. The method of claim 1 , wherein said monocytic cells are differentiated into dendritic cells by culture in GM-CSF and IL-4.
10. The method of claim 9 , wherein said culture with GM-CSF and IL-4 is for a period of 4-8 days.
11. The method of claim 10 , wherein said culture with GM-CSF and IL-4 is for a period of 7 days.
12. The method of claim 11 , wherein said culture with GM-CSF and IL-4 contains 100 IU of GM-CSF and 100 IU of IL-4.
13. The method of claim 1 , wherein said CD355 positive lymphocyte cells are cultured in IL-2 at 20 ng/ml for approximately 7 days.
14. The method of claim 13 , wherein said canine derived dendritic cells are co-cultured with canine derived CD355 positive lymphocyte cells for a period of 3 days to 30 days.
15. The method of claim 14 , wherein said co-culture is performed in a mixture of cytokines containing 20 ng of IL-2 per ml, and 100 IU of GM-CSF per ml.
16. The method of claim 1 , wherein canine peripheral blood mononuclear cells are cultured in a combination of 20 ng of IL-2 per ml, and 100 IU of GM-CSF per ml for a period of 7 days.