Ex vivo, fast and efficient process to obtain activated antigen-presenting cells that are useful for therapies against cancer and immune system-related diseases

Patent metadata

Field	Value
Publication number	US-9694059-B2
Application number	US-201313875119-A
Country	US
Kind code	B2
Filing date	May 1, 2013
Priority date	Sep 28, 2007
Publication date	Jul 4, 2017
Grant date	Jul 4, 2017

Abstract

The present invention relates to an ex vivo, fast and efficient process to obtain activated antigen-presenting cells that are useful for therapies against cancer and immune system-related diseases. At the same time, it is related to a cellular composition that contributes to stimulate the activated antigen-presenting cells to induce a specific immune response against tumors in patients with cancer or other pathologies involving immune responses.

First claim

The invention claimed is:

1. An in-vitro method to obtain activated antigen-presenting cells (APCs) that are dendritic cells (DCs), and that are useful in the preparation of vaccines for the treatment of cancer, the method comprising:
a) obtaining monocytes from peripheral blood cells (PBMC);
b) pre-activating monocytes obtained from step a) together with granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) for at least 10 hours;
c) incubating activated monocytes obtained from step b) for an additional 24 hours with a lysate obtained from three melanoma cell lines wherein the three melanoma cell lines are Mel 1 (IDAC accession number 260916-01), Mel 2 (IDAC accession number 260916-02), and Mel 3 (IDAC accession number 260916-03) and the three melanoma cell lines have been thermally pre-treated, occurring in a single step, differentiation, maturation and loading of DCs; and
d) harvesting and washing the APCs obtained in step c), wherein, in step c), thermally pre-treating comprises incubation of the melanoma cell lines at a temperature between 39-44° C. for 15 minutes to 4 hours in a serum-free culture medium, and said thermally pre-treating is followed by incubation of said melanoma cell lines at 37° C. for 1 to 6 hours.

2. The method of claim 1 , wherein said temperature of thermal pre-treatment is between 40 and 43° C.

3. The method of claim 2 , wherein said temperature of thermal pre-treatment is 42° C.

4. The method of claim 1 , wherein thermal pre-treatment is for between 1 to 3 hours.

5. The method of claim 4 , wherein thermal pre-treatment is for 2 hours.

6. The method of claim 1 , wherein the thermally pre-treated melanoma cell lines have been incubated at a temperature between 39 and 44° C., and then have been incubated at 37° C. from 2 to 4 hours.

7. The method of claim 6 , wherein incubation at 37° C. is for 3 hours.

8. The method of claim 1 , wherein, in step b), said monocytes are provided as PBMC, which are incubated with GM-CSF and said IL-4 at a concentration of 10-40×10 6 cells/ml in serum-free culture medium.

9. The method of claim 8 , wherein said concentration is between 20-30×10 6 cells/ml.

10. The method of claim 9 , wherein said concentration is 25×10 6 cells/ml.

11. The method of claim 1 , wherein, in step b), a concentration of said IL-4 is between 100-800 U/ml.

12. The method of claim 11 , wherein said concentration is between 400-600 U/ml.

13. The method of claim 12 , wherein said concentration is 500 U/ml.

14. The method of claim 1 , wherein, in step b), a concentration of said GM-CSF is between 500-1100 U/ml.

15. The method of claim 14 , wherein said concentration is between 700-900 U/ml.

16. The method of claim 15 , wherein said concentration is 800 U/ml.

17. The method of claim 1 wherein step c) further comprises incubating said monocytes with one or more pro-inflammatory factors selected from IFN-.gamma, IL-6, IL-1.Beta, prostaglandin E2, CpG, heat shock proteins, ligands of Toll-like receptors (TLR) and mixtures thereof.

18. The method of claim 1 , wherein, in step c), a concentration of TNF-alpha is between 100 pg/ml-100 ng/ml.

19. The method of claim 18 , wherein said concentration is between 1 ng/ml-50 ng/ml.

20. The method of claim 19 , wherein said concentration is between 2 ng/ml-20 ng/ml.

21. The method of claim 20 , wherein said concentration is 10 ng/ml.

22. The method of claim 1 , wherein a concentration of said lysate is between 1 μg/ml to 10 mg/ml.

23. The method of claim 22 , wherein said concentration of said lysate is between 10 μg/ml and 1 mg/ml.

24. The method of claim 23 , wherein said concentration of said lysate is 100 pg/ml.

25. The method of claim 1 , wherein, in step b), concentrations of IL-4 and GM-CSF are 500 U/ml and 800 U/ml, respectively.

26. The method of claim 1 , wherein said method further comprises step e) freezing APCs from step d).

Assignees

• Univ Chile
• Oncobiomed

Inventors

• Salazar Onfray Flavio Andres
• Lopez Nitsche Mercedes Natalia
• Pereda Ramos Cristian Javier
• Aguilera Insunza Raquel Elvira
• Escobar Alvarez Alejandro Felipe

Classifications

• A61P37/04

  Immunostimulants · CPC title

• C12N2501/02

  Compounds of the arachidonic acid pathway, e.g. prostaglandins, leukotrienes · CPC title

• C12N2501/25

  Tumour necrosing factors [TNF] · CPC title

• C12N2506/11

  from blood or immune system cells · CPC title

• C12N2500/84

  from mammals · CPC title