Method for the production of activated marked tumor-specific T cells and use thereof in treatment of tumors

• US 6,399,054 B1
• Filed: 09/29/1998
• Issued: 06/04/2002
• Est. Priority Date: 03/30/1996
• Status: Expired due to Fees

• Current Assignee: Science Park Raf S.p.A. (Finraf SpA)
• Original Assignee: Science Park Raf S.p.A. (Finraf SpA)
• Inventors: Casorati, Giulia, Dellabona, Paolo
• Primary Examiner(s): Crouch, Deborah
• Assistant Examiner(s): Beckerleg, Anne Marie S.
• Application Number:
  US09/161,998
• Time in Patent Office: 1,344 Days
• Field of Search: 424/93.21, 530/387.3, 530/387.9, 530/388.8, 530/388.85, 530/391.1, 530/350, 435/320.1, 435/325
• US Class Current: 424/93.21
• CPC Class Codes: A61K 2039/5158 Antigen-pulsed cells, e.g. T-cells
• CPC Class Codes: A61K 2239/31 characterized by the route of administration
• CPC Class Codes: A61K 2239/38 characterised by the dose, timing or administration schedule
• CPC Class Codes: A61K 39/0011 Cancer antigens
• CPC Class Codes: A61K 39/4611 T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
• CPC Class Codes: A61K 39/464499 Undefined tumor antigens, e.g. tumor lysate or antigens targeted by cells isolated from tumor
• CPC Class Codes: C12N 2501/23 Interleukins [IL]
• CPC Class Codes: C12N 2501/51 B7 molecules, e.g. CD80, CD86, CD28 (ligand), CD152 (ligand)
• CPC Class Codes: C12N 2501/515 CD3, T-cell receptor complex
• CPC Class Codes: C12N 5/0636 T lymphocytes

Abstract

A method for the production of activated tumor-specific T cells by co-cultivating, ex vivo, tumor cells from a patient with T cells from that patient, comprising the steps of: i) incubating the tumor cells with a first fusion protein obtained from a B7 protein and one partner of a biological binding pair and a second fusion protein obtained from an antibody against a cell surface antigen and the other partner of the biological binding pair, ii) inhibiting the proliferation of the tumor cells prior to or after that incubation; iii) co-cultivating the tumor cells with the T cells to be activated, until activation of the T cells is attained; iv) separating the activated T cells from the tumor cells, is highly efficient and can be carried out in a simple manner.

Claims

1. A method for producing MHC-restricted, activated tumor-specific T cells, comprising the following steps:

(1) obtaining tumor cells from a patient;

(2) inhibiting the proliferation of the tumor cells and incubating said tumor cells with reagents comprising a ternary complex consisting essentially of

(i) a biotinylated antibody against a cell surface antigen of said tumor cells from said patient

or at least two Fab fragments of said antibody,

(ii) a ligand which is avidin, streptavidin or neutravidin, and

(iii) biotinylated B7-Ig,

to generate targeted tumor cells;

(3) obtaining T cells from said patient;

thereafter

(4) co-cultivating said targeted tumor cells with said T cells to activate said T cells in the presence of at least interleukin 2; and

thereafter

(5) isolating said activated T cells from the targeted tumor cells to obtain activated tumor-specific T cells.

2. The method of claim 1, further comprising marking said T cells prior to or after step (4).

3. The method of claim 1, wherein said T cells are obtained from peripheral blood mononuclear cells with the exclusion of B cells and monocytes.

4. The method of claim 1, wherein said T cells are CD4⁺ and/or CD8⁺ lymphocytes.

5. The method of claim 1, wherein said T cells are purified CD8⁺ lymphocytes and tumor-infiltrating T cells.

6. The method of claim 2, wherein said T cells are marked by transfecting said T cells with a gene which codes for a marker protein expressed on the surface of said T cells.

7. The method of claim 6, wherein said marker protein is CD24.

8. The method of claim 1, further comprising transfecting said T cells with a suicide gene, which gene directly or indirectly kills said T cells in order to reduce the number of activated tumor-specific T cells when needed.

9. The method of claim 8, wherein said suicide gene is a thymidine kinase gene.

10. The method of claim 1, wherein the proliferation of the tumor cells is inhibited by exposing said tumor cells to an effective amount of ionizing irradiation and/or mitomycin C.

11. The method of claim 1, wherein the inhibition of the proliferation of said tumor cells is conducted before the incubating step.

12. The method of claim 1, wherein said T cells and said tumor cells are co-cultivated at a ratio of about 100:

1 to about 5;

1.

13. The method of claim 1, wherein said tumor cells are taken from a surgical specimen of said patient.

14. The method of claim 1, wherein the inhibition of the proliferation of said tumor cells is conducted after the incubating step.

15. The method of claim 1, wherein said B7 protein is B7-1 or B7-2.

16. The method of claim 1, wherein said cell surface antigen is ERB B2 or a transferrin receptor.

17. The method of claim 1, wherein step (4) is carried out by co-cultivating said targeted tumor cells with said T cells in the presence of at least one other lymphokine to stimulate said T cells.

18. The method of claim 17, wherein said lymphokine is IL-6, IL-7 or a combination thereof.

19. The method of claim 17, further comprising, between steps (4) and (5), restimulating said T cells at least once by isolating said T cells and co-cultivating said isolated T cells with said pre-targeted tumor cells and said at least one other lymphokine in order to increase the number of activated tumor-specific T cells.

50. The method of claim 1, further comprising forming said ternary complex at the end of step 2).

20. A method of treating a tumor in a patient, comprising the steps of:

(A) obtaining tumor cells from said patient,

(B) inhibiting proliferation of said tumor cells and incubating said tumor cells with reagents comprising a ternary complex consisting essentially of

i) a biotinylated antibody against a cell surface antigen of said tumor cells from said patient or at least two Fab fragments of said antibody,

ii) a ligand which is avidin, streptavidin or neutravidin, and

iii) biotinylated B7-Ig,

(C) obtaining T cells from said patient, and thereafter

(D) co-cultivating said tumor cells of step (B) with said T cells to activate said T cells in the presence of at least interleukin 2, and thereafter

(E) isolating T cells from said tumor cells to obtain MHC-restricted, activated tumor-specific T cells, and thereafter

(F) administering an effective amount of said MHC-restricted, activated tumor-specific T cells to said patient.

21. The method of claim 20, wherein about 10⁷ to about 10⁹ activated tumor-specific T cells per square meter of body surface area are infused intravenously.

22. The method of claim 21, wherein the intravenous infusion is repeated once every four weeks.

23. The method of claim 20, further comprising marking said T cells prior to or after step (4) in order to monitor the persistence of said activated tumor-specific T cells in said patient.

24. The method of claim 20, further comprising transfecting said T cells with a suicide gene, which gene directly or indirectly kills said T cells to reduce the number of activated tumor-specific T cells in order to terminate the treatment when needed.

25. A method for producing MHC-restricted, activated tumor-specific T cells, comprising the following steps:

(1) obtaining tumor cells from a patient;

(2) inhibiting the proliferation of the tumor cells and conducting a three-step procedure;

I. incubating said tumor cells with a biotinylated antibody against a cell surface antigen of said tumor cells from said patient or at least two Fab fragments of said antibody to obtain a product of step I;

II. incubating the product of step I with a ligand which is avidin, streptavidin or neutravidin to obtain a product of step II; and

III. incubating the product of step II with biotinylated B7-Ig to generate targeted tumor cells;

(3) obtaining T cells from said patient;

(4) co-cultivating said targeted tumor cells with said T cells to activate said T cells in the presence of at least interleukin 2; and

thereafter

(5) isolating said T cells from the targeted tumor cells to obtain activated tumor-specific T cells.

26. The method of claim 25, further comprising marking said T cells prior to or after step (4).

27. The method of claim 25, wherein said T cells are obtained from peripheral blood mononuclear cells with the exclusion of B cells and monocytes.

28. The method of claim 25, wherein said T cells are CD4⁺ and/or CD8⁺ lymphocytes.

29. The method of claim 25, wherein said T cells are purified CD8⁺ lymphocytes and tumor-infiltrating T cells.

30. The method of claim 26, wherein said T cells are marked by transfecting said T cells with a gene which codes for a marker protein expressed on the surface of said T cells.

31. The method of claim 30, wherein said marker protein is CD24.

32. The method of claim 25, further comprising transfecting said T cells with a suicide gene, which gene directly or indirectly kills said T cells in order to reduce the number of activated tumor-specific T cells when needed.

33. The method of claim 32, wherein said suicide gene is a thymidine kinase gene.

34. The method of claim 25, wherein the proliferation of the tumor cells is inhibited by exposing said tumor cells to an effective amount of ionizing irradiation and/or mitomycin C.

35. The method of claim 25, wherein the inhibition of the proliferation of said tumor cells is conducted before step I.

36. The method of claim 25, wherein said T cells and said tumor cells are co-cultivated at a ratio of about 10:

1 to about 5;

1.

37. The method of claim 25, wherein said tumor cells are taken from a surgical specimen of said patient.

38. The method of claim 25, wherein the inhibition of the proliferation of said tumor cells is conducted after step III.

39. The method of claim 25, wherein said B7 protein is B7-1 or B7-2.

40. The method of claim 25, wherein said cell surface antigen is ERB B2 or a transferrin receptor.

41. The method of claim 25, wherein step (4) is carried out by co-cultivating said targeted tumor cells with said T cells in the presence of at least one other lymphokine to stimulate said T cells.

42. The method of claim 41, wherein said lymphokine is IL-6, IL-7 or a combination thereof.

43. The method of claim 41, further comprising, between steps (4) and (5), restimulating said T cells at least once by isolating said T cells and co-cultivating said isolated T cells with said pre-targeted tumor cells and said at least one other lymphokine in order to increase the number of activated tumor-specific T cells.

44. A method of treating tumors in a patient, comprising the following steps:

(A) obtaining tumor cells from said patient;

(B) inhibiting the proliferation of said tumor cells and conducting a three-step procedure;

I. incubating said tumor cells with a biotinylated antibody against a cell surface antigen of said tumor cells from said patient or at least two Fab fragments of said antibody to obtain a product of step I;

II. incubating the product of step I with a ligand which is avidin, streptavidin or neutravidin to obtain a product of step II; and

III. incubating the product of step II with biotinylated B7-Ig to generate targeted tumor cells;

(C) obtaining T cells from said patient;

(D) co-cultivating said targeted tumor cells with said T cells to activate said T cells in the presence of at least interleukin 2; and

thereafter

(E) isolating said T cells from said targeted tumor cells to obtain MHC-restricted, activated tumor-specific T cells, and thereafter

(F) administering an effective amount of said MHC-restricted, activated tumor-specific T cells to said patient.

45. The method of claim 44, wherein the activated tumor-specific T cells are administered intravenously to said patient.

46. The method of claim 45, wherein about 10⁷ to about 10⁹ activated tumor-specific T cells per square meter of body surface area are infused intravenously.

47. The method of claim 45, wherein the intravenous infusion is repeated once every four weeks.

48. The method of claim 44, further comprising marking said T cells prior to or after step (4) in order to monitor the persistence of said activated tumor-specific T cells in said patient.

49. The method of claim 44, further comprising transfecting said T cells with a suicide gene, which gene directly or indirectly kills said T cells to reduce the number of activated tumor-specific T cells in order to terminate the treatment when needed.