Method for expansion of tumour-reactive T-lymphocytes for immunotherapy of patients with cancer
The present invention discloses an improved method for expansion and activation of tumor-reactive lymphocytes, in particular CD4+ helper and/or CD8+ T-lymphocytes, which may be used for treating and/or preventing cancer. The method provides high numbers of tumor-reactive T-lymphocytes within a short time span and the possibility of directing development of tumor-reactive CD4+ helper and/or CD8+ T-lymphocytes towards specific subpopulations. The method comprises a first phase of stimulating tumor-reactive CD4+ T helper and/or CD8+ T-lymphocytes with tumor-derived antigen together with at least one substance having agonistic activity towards the IL-2 receptor to promote survival of tumor-reactive CD4+ T helper and/or CD8+ T-lymphocytes; and a second phase of activating and promoting growth of tumor-reactive CD4+ T helper and/or CD8+ T-lymphocytes, wherein the second phase is initiated when the CD25 cell surface marker (or IL-2R marker) is down-regulated on CD4+ T helper and/or CD8+ T-lymphocytes.
1. A method for the expansion of tumour-reactive CD4+ T helper and/or CD8+ T-lymphocytes, the method comprising:
i) a first phase of stimulating tumour-reactive CD4+ T helper or CD8+ T-lymphocytes with a tumour-derived antigen together with at least one substance having agonistic activity towards an IL-2 receptor to promote survival of tumour-reactive CD4+ T helper or CD8+ T-lymphocytes; and
ii) a second phase of activating and promoting growth of tumour-reactive CD4+ T helper or CD8+ T-lymphocytes, wherein the second phase is initiated when the CD25 cell surface marker is down-regulated on CD4+ T helper or CD8+ T-lymphocytes, wherein down-regulation is defined as that 5% or less of the T-lymphocyte population expresses CD25 and wherein the second phase is initiated by the addition of tumour-derived antigen to the T-lymphocytes for activating tumour-reactive CD25-negative T-lymphocytes.
2. A method according to claim 1 , wherein the first phase is initiated by adding the at least one substance having agonistic activity towards the IL-2 receptor.
3. A method according to claim 2 , wherein the substance having agonistic activity towards the IL-2 receptor is IL-2.
4. A method according to claim 1 , wherein the tumour-derived antigen is a denatured homogenate of a tumour.
5. A method according to claim 4 , wherein the tumour-derived antigen is autologous.
6. A method according to claims 1 , which further comprises addition to the T-lymphocytes of antigen presenting cells together with the tumour-derived antigen.
7. A method according to claim 6 , wherein the antigen presenting cells are irradiated peripheral blood leucocytes containing antigen-presenting B-cells or monocytes.
8. A method according to claim 1 , wherein the second phase comprises adding at least one substance capable of up-regulating IL-12R on the T-lymphocytes.
9. A method according to claim 1 , wherein the second phase comprises adding one or more substances capable of antagonizing development of Th2 type T-lymphocytes.
10. A method according to claim 9 , wherein the one or more substances capable of antagonizing development of Th2 type T-lymphocytes are one or more substances capable of neutralizing IL-4, IL-5, IL-10, and/or TGF-beta.
11. A method according to claim 10 , wherein the one or more substances capable of neutralizing IL-4, IL-5, IL-10, or TGF-beta are anti IL-4 antibody, anti IL-5 antibody or anti IL-10 antibody.
12. A method according to any of claim 1 , 10 or 11 , wherein a further amount of the one or more substance capable of antagonizing development of Th2 type T-lymphocytes is added regularly throughout the second phase.
13. A method according to claim 1 , wherein the second phase comprises adding one or more substances promoting the development of Th1 type T-lymphocytes.
14. A method according to claim 13 , wherein the one or more substances promoting the development of Th1 type T-lymphocytes is a substance having agonistic activity towards the IL-7, IL-12, IL-15 and/or IL-21 receptor.
15. A method according to claim 14 , wherein the one or more substances is selected from IL-7, IL-12, IL-15 and IL-21.
16. A method according to claim 1 for the preparation of Th1-lymphocytes of the memory or effector type.
17. A method according to claim 1 , which further comprises monitoring the expression of cell surface markers continuously during the first phase and second phase, and wherein the T-lymphocytes are harvested when CD25 on T-lymphocytes in the second phase is down-regulated.
18. A method according to claim 17 , wherein the T-lymphocytes are subjected to at least one additional round of the second phase, when CD25 on T-lymphocytes is down-regulated.
19. A method according to claim 1 , wherein the T-lymphocytes are derived from lymph nodes draining a primary tumour or a metastasis, or they are derived from blood.