Method for treating malignant melanoma

1. A method for treating a patient suffering from malignant melanoma, the method comprising:

i) identifying in a patient one or more sentinel or metinel lymph nodes draining a malignant melanoma tumour or a metastasis from a malignant melanoma tumour;

ii) resecting the one or more lymph nodes and, optionally all or part of the tumour or metastasis;

iii) isolating tumour-reactive T-lymphocytes from said lymph nodes;

iv) in vitro expanding said tumour-reactive T-lymphocytes, wherein the in vitro expansion comprises

a) a first phase of stimulating tumour-reactive CD4+ helper or CD8+ T-lymphocytes with tumour-derived antigen together with at least one substance having agonistic activity towards the IL-2 receptor to promote survival of tumour-reactive CD4+ helper or CD8+ T-lymphocytes, and

b) a second phase of activating and promoting growth of tumour-reactive CD4+ helper or CD8+ T-lymphocytes, wherein the second phase is initiated when a CD25 cell surface marker or IL-2R marker is down-regulated on T-lymphocytes; and

v) administering the thus obtained tumour-reactive T-lymphocytes to the patient;

wherein the T-lymphocytes are CD4+ helper or CD8+ T-lymphocytes.

2. A method according to claim 1 , wherein the down-regulation is defined as that 5% or less of the T-lymphocyte population expresses CD25.

3. A method according to either of claim 1 or 2 , wherein the first phase is initiated by adding the at least one substance having agonistic activity towards the IL-2 receptor.

4. A method according to claim 1 , wherein the tumour-derived antigen is a denatured homogenate of a tumour.

5. A method according to either of claim 1 or 4 , wherein the second phase is initiated by the addition of malignant melanoma-derived antigen to the T-lymphocytes for activating tumour-reactive CD25-negative T-lymphocytes.

6. A method according to claim 5 , wherein the malignant melanoma-derived antigen is a denatured homogenate of a tumour.

7. A method according to claim 1 , which further comprises addition of antigen presenting cells to the T-lymphocytes together with the tumour-derived antigen.

8. A method according to claim 7 , wherein the antigen presenting cells are irradiated peripheral blood leucocytes containing antigen-presenting B-cells and/or monocytes.

9. 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.

10. 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.

11. A method according to claim 10 , 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.

12. A method according to claim 11 , wherein the one or more substances capable of neutralizing IL-4, IL-5, IL-10, and/or TGF-beta are anti IL-4 antibody, anti IL-5 antibody and/or anti IL-10 antibody.

13. A method according to any of claims 1 , 11 or 12 , 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.

14. A method according to claim 1 , wherein the second phase comprises adding one or more substances promoting the development of Th1 type T-lymphocytes.

15. A method according to claim 14 , 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 or IL-21 receptor.

16. A method according to claim 15 , wherein the one or more substances is selected from IL-7, IL-12, IL-15 and IL-21.

17. A method according to any of claims 11 , 14 or 15 for the preparation of Th1 type T-lymphocytes of the memory or effector type.

18. A method according to claim 1 , further comprising monitoring the expression of cell surface markers, on the T-lymphocytes 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.

19. A method according to claim 18 , wherein the T-lymphocytes are subjected to at least one additional round of the second phase when CD25 on T-lymphocytes is down-regulated.

20. A method according to claim 1 , wherein the T-lymphocytes are derived from lymph nodes draining the malignant melanoma tumour and/or a metastasis thereof, or they are derived from blood.

21. A method according to claim 1 , wherein the tumour-derived antigen is autologous tumour-derived antigen.

22. A method according to claim 1 , wherein the tumour-derived antigen is autologous denatured tumour extract.

23. A method for treating a patient suffering from malignant melanoma, the method comprising administering tumour-reactive T-lymphocytes to the patient, wherein the T-lymphocytes are CD4+ helper or CD8+ T-lymphocytes and are obtained by:

i) identifying in a patient one or more sentinel or metinel lymph nodes draining a malignant melanoma tumour or a metastasis from a malignant melanoma tumour;

ii) resecting the one or more lymph nodes and, optionally all or part of the tumour or metastasis;

iii) isolating tumour-reactive T-lymphocytes from said lymph nodes; and

iv) in vitro expanding said tumour-reactive T-lymphocytes, wherein the in vitro expansion comprises

a) a first phase of stimulating tumour-reactive CD4+ helper or CD8+ T-lymphocytes with tumour-derived antigen together with at least one substance having agonistic activity towards the IL-2 receptor to promote survival of tumour-reactive CD4+ helper or CD8+ T-lymphocytes, and

b) a second phase of activating and promoting growth of tumour-reactive CD4+ helper or CD8+ T-lymphocytes, wherein the second phase is initiated when a CD25 cell surface marker or IL-2R marker is down-regulated on T-lymphocytes.