Method for treating colon cancer with tumour-reactive T-lymphocytes

1. A method for treating a patient suffering from colon cancer, the method comprising:

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

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 autologous denatured colon tumour homogenate together with IL-2 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 by adding autologous denatured colon tumour homogenate when the CD25 cell surface marker is down-regulated on T-lymphocytes; and

v) administering tumour-reactive T-lymphocytes obtained in step (iv) to the patient;

wherein the T-lymphocytes are CD4+ helper or CD8+ T-lymphocytes and are not CD4+ CD25+ Hi lymphocytes.

2. The method according to claim 1 , wherein the second phase is initiated when 5% or less of the T-lymphocyte population expresses CD25.

3. The method according to either of claims 1 or 2 , wherein the first phase is initiated by adding IL-2.

4. The method according to claim 1 , wherein the second phase further comprises addition of antigen presenting cells to the T-lymphocytes.

5. The method according to claim 4 , wherein the antigen presenting cells are irradiated peripheral blood leukocytes containing antigen-presenting B-cells and/or monocytes.

6. The method according to claim 1 , wherein the second phase further comprises adding at least one substance capable of up-regulating IL-12R on the T-lymphocytes.

7. The method according to claim 6 , wherein the substance capable of up-regulating IL-12R on the T-lymphocytes is interferon-α.

8. The method according to claim 1 , wherein the second phase further comprises adding one or more substances capable of antagonizing development of Th2 type T-lymphocytes.

9. The method according to claim 8 , 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.

10. The method according to claim 9 , 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.

11. The method according to claim 9 or 10 , wherein a further amount of the one or more substances capable of antagonizing development of Th2 type T-lymphocytes is added regularly throughout the second phase.

12. The method according to claim 1 , wherein the second phase further comprises adding one or more substances promoting the development of Th1 type T-lymphocytes and the one or more substances are selected from the group consisting of IL-7, IL-12, IL-15 and IL-21.

13. The method according to claim 9 or 12 for the preparation of Th1 type T-lymphocytes of the memory or effector type.

14. The method according to claim 1 , further comprising monitoring the expression of CD25 cell surface marker on the T-lymphocytes continuously during the first phase and second phase and wherein the T-lymphocytes are harvested from a second phase culture medium when CD25 on T-lymphocytes in the second phase is down-regulated.

15. The method according to claim 1 , further comprising monitoring the expression of CD25 cell surface marker on the T-lymphocytes continuously during the first phase and second phase and wherein, when CD25 on T-lymphocytes in the second phase is down-regulated, the second phase of step iv) is repeated.

16. A method for treating a patient suffering from colon cancer, the method comprising administering tumour-reactive T-lymphocytes to the patient, wherein the T-lymphocytes are CD4+ helper or CD8+ T-lymphocytes and are not CD4+ CD25+ Hi lymphocytes and have been obtained by:

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

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 autologous denatured colon tumour homogenate together with IL-2 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 by adding autologous denatured colon tumour homogenate when the CD25 cell surface marker is down-regulated on T-lymphocytes.

17. The method according to claim 16 , wherein the second phase is initiated when 5% or less of the T-lymphocyte population expresses CD25.

18. The method according to claim 16 , wherein the second phase further comprises addition of antigen presenting cells to the T-lymphocytes.

19. The method according to claim 16 , wherein the second phase further comprises adding one or more substances promoting the development of Th1 type T-lymphocytes and the one or more substances are selected from the group consisting of IL-7, IL-12, IL-15 and IL-21.

20. A method for treating a patient suffering from colon cancer, the method comprising:

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

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

iii) isolating T-lymphocytes from said lymph nodes;

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

a) a first phase of stimulating CD4+ helper or CD8+ T-lymphocytes with autologous denatured colon tumour homogenate together with IL 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 by adding autologous denatured colon tumour homogenate when the CD25 cell surface marker is down-regulated on T-lymphocytes; and

v) administering tumour-reactive T-lymphocytes obtained in step (iv) to the patient;

wherein the T-lymphocytes are CD4+ helper or CD8+ T-lymphocytes and are not CD4+ CD25+ Hi lymphocytes.

21. A method for treating a patient suffering from colon cancer, the method comprising administering tumour-reactive T-lymphocytes to the patient, wherein the T-lymphocytes are CD4+ helper or CD8+ T-lymphocytes and are not CD4+ CD25+ Hi lymphocytes and have been obtained by:

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

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

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

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

a) a first phase of stimulating CD4+ helper or CD8+ T-lymphocytes with autologous denatured colon tumour homogenate together with IL-2 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 by adding autologous denatured colon tumour homogenate when the CD25 cell surface marker is down-regulated on T-lymphocytes.