ENHANCED ANTIGEN PRESENTING ABILITY OF RNA CAR T CELLS BY CO-INTRODUCTION OF COSTIMULATORY MOLECULES

• US 20160340406A1
• Filed: 01/21/2015
• Published: 11/24/2016
• Est. Priority Date: 01/21/2014
• Status: Active Grant

• Current Assignee: Novartis Ag, Trustees Of The University Of Pennsylvania (University of Pennsylvania)
• Original Assignee: Novartis Ag, Trustees Of The University Of Pennsylvania (University of Pennsylvania)
• Inventors: Zhao, Yangbing, Shedlock, Devon J., June, Carl H., Liu, Xiaojun
• Granted Patent: US 11,028,143 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: A61K 2039/5158 Antigen-pulsed cells, e.g. T-cells
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 39/0011 Cancer antigens
• CPC Class Codes: A61K 39/02 Bacterial antigens
• CPC Class Codes: A61K 39/12 Viral antigens
• CPC Class Codes: A61K 39/39 characterised by the immunostimulating additives, e.g. chemical adjuvants
• CPC Class Codes: A61K 39/3955 against proteinaceous materials, e.g. enzymes, hormones, lymphokines
• 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 39/4631 Chimeric Antigen Receptors [CAR]
• CPC Class Codes: A61K 39/464429 Molecules with a "CD" designation not provided for elsewhere
• CPC Class Codes: A61K 39/464468 Mesothelin [MSLN]
• CPC Class Codes: A61K 39/464491 Melan-A/MART
• CPC Class Codes: C07K 14/54 Interleukins [IL]
• CPC Class Codes: C07K 14/5418 IL-7
• CPC Class Codes: C07K 14/5443 IL-15
• CPC Class Codes: C07K 14/55 IL-2
• CPC Class Codes: C07K 14/7051 T-cell receptor (TcR)-CD3 complex
• CPC Class Codes: C07K 14/70517 CD8
• CPC Class Codes: C07K 14/70521 CD28, CD152
• CPC Class Codes: C07K 14/70532 B7 molecules, e.g. CD80, CD86
• CPC Class Codes: C07K 14/70575 : NGF/TNF-superfamily, e.g. CD70, CD95L, CD153, CD154 NGF C07K14/48, TNF C07K14/525
• CPC Class Codes: C07K 14/70596 : Molecules with a "CD"-designation not provided for elsewhere
• CPC Class Codes: C07K 16/28 : against receptors, cell surface antigens or cell surface determinants
• CPC Class Codes: C07K 2317/53 : Hinge
• CPC Class Codes: C07K 2317/622 : Single chain antibody (scFv)
• CPC Class Codes: C07K 2319/30 : Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
• CPC Class Codes: C12N 15/86 : Viral vectors
• 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 2510/00 : Genetically modified cells
• CPC Class Codes: C12N 2760/16034 : Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
• CPC Class Codes: C12N 5/0638 : Cytotoxic T lymphocytes [CTL] or lymphokine activated killer cells [LAK]
• CPC Class Codes: C12N 7/00 : Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00
• CPC Class Codes: G01N 33/505 : involving T-cells
• CPC Class Codes: Y02A 50/30 : Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention provides T cells comprising nucleic acid sequence encoding a chimeric antigen receptor and a nucleic acid sequence encoding an enhancer of T cell priming, compositions including the T cells, and methods of using the T cells to treat diseases associated with the expression of disease-associated antigens.

Claims

1. A T cell comprising nucleic acid, wherein:

(a) the nucleic acid comprises a first nucleic acid sequence encoding a chimeric antigen receptor (CAR) comprising an extracellular domain, a transmembrane domain, and an intracellular signaling domain, and

(b) the nucleic acid comprises a second nucleic acid sequence encoding a polypeptide which enhances T cell priming, or a functional fragment or variant thereof, provided that

(i) the first and/or second nucleic acid sequence comprises an RNA;

or

(ii) the CAR further comprises a second intracellular signaling domain.

2. The T cell of claim 1, wherein the first and second nucleic acid sequences are disposed on a single nucleic acid molecule.

3. The T cell of claim 2, wherein the nucleic acid molecule comprises RNA.

4. The T cell of claim 2, wherein the nucleic acid molecule comprises DNA.

5. The T cell of claim 1, wherein the first and second nucleic acid sequences are disposed on two or more distinct nucleic acid molecules.

6. The T cell of claim 5, wherein one or both nucleic acid molecules comprise RNA molecules.

7. The T cell of claim 5, wherein one or both nucleic acid molecules comprise DNA molecules.

8. The T cell of claim 5, wherein one nucleic acid molecule comprises an RNA molecule and the other nucleic acid comprises a DNA molecule.

9. The T cell of claim 1, wherein the second intracellular signaling domain comprises a co stimulatory signaling domain.

10. The T cell of claim 1, wherein the CAR comprises one or more costimulatory signaling domains.

11. The T cell of claim 1, wherein the intracellular signaling domain comprises a CD3zeta domain and the second intracellular signaling domain comprises a 4-1BB domain.

12. The T cell of claim 1, wherein the first nucleic acid sequence comprises an RNA.

13. The T cell of claim 1, wherein the second nucleic acid sequence comprises an RNA.

14. The T cell of claim 1, wherein the first and the second nucleic acid sequence each comprise RNA.

15. The T cell of any of claims 12-14, wherein the T cell is transfected to transiently express the first and/or second RNAs.

16. The T cell of any of claims 12-14, wherein the cell does not comprise an exogenous DNA encoding the first or second RNA.

17. The T cell of any of claims 12-14, wherein the first and/or second RNAs are generated by in vitro transcription.

18. The T cell of any of claims 12-14, wherein the first and/or second RNAs are synthetic RNAs.

19. The T cell of any of claims 12-14, wherein the first and/or second RNAs are introduced into the T cell by electroporation.

20. The T cell of claim 1, wherein the CAR further comprises one or more costimulatory signaling domains, and wherein the first and/or second nucleic acid sequence comprises DNA or cDNA.

21. The T cell of claim 20, wherein the first and/or second nucleic acid sequence comprises a vector.

22. The T cell of claim 21, wherein the vector is a viral vector.

23. The T cell of claim 22, wherein the viral vector is a retroviral vector or a lentiviral vector.

24. The T cell of claim 22 or 23, wherein the T cell is virally transduced to express the first and/or second nucleic acid sequence.

25. The T cell of any of the preceding claims, wherein the extracellular domain of the CAR comprises an antigen-binding domain.

26. The T cell of any of the preceding claims, wherein the antigen-binding domain is a scFv domain.

27. The T cell of any one of the preceding claims, wherein the transmembrane domain comprises a transmembrane protein of a protein selected from the group consisting of the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154.

28. The T cell of any one of the preceding claims, wherein the intracellular signaling domain comprises a functional signaling domain of CD3 zeta, common FcR gamma (FCER1G), Fc gamma RIIa, FcR beta (Fc Epsilon R1b), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP10, and DAP12.

29. The T cell of any one of the preceding claims, wherein the extracellular domain is connected to the transmembrane domain by a hinge region.

30. The T cell of any one of the preceding claims, wherein the CAR further comprises one or more costimulatory signaling domains.

31. The T cell of any one of the preceding claims, wherein the costimulatory signaling domain is a functional signaling domain from a protein selected from the group consisting of OX40, CD27, CD28, CD30, CD40, PD-1, CD2, CD7, CD258, NKG2C, B7-H3, a ligand that binds to CD83, ICAM-1, LFA-1 (CD11a/CD18), ICOS and 4-1BB (CD137), or any combination thereof.

32. The T cell of any one of the preceding claims, wherein the polypeptide which enhances T cell priming is selected from the group consisting of a costimulatory molecule, a soluble cytokine, a polypeptide involved in antigen presentation, a polypeptide involved in trafficking and/or migration, or a polypeptide involved in dendritic cell targeting, or a functional fragment or variant thereof.

33. The T cell of claim 32, wherein the costimulatory molecule is selected from the group consisting of CD70, CD83, CD80, CD86, CD40, CD154, CD137L (4-1BBL), CD252 (OX40L), CD275 (ICOS-L), CD54 (ICAM-1), CD49a, CD43, CD48, CD112 (PVRL2), CD150 (SLAM), CD155 (PVR), CD265 (RANK), CD270 (HVEM), TL1A, CD127, IL-4R, GITR-L, CD160, CD258, TIM-4, CD153 (CD30L), CD200R (OX2R), CD44, ligands thereof, and functional fragments and variants thereof.

34. The T cell of claim 32, wherein the soluble cytokine is selected from the group consisting of:

IL-2, IL-12, IL-6, IL-7, IL-15, IL-18, IL-21, GM-CSF, IL-18, IL-21, IL-27, and functional fragments and variants thereof.

35. The T cell of claim 32, wherein the polypeptide involved in antigen presentation is selected from the group consisting of CD64, MHC I, MHC II, and functional fragments and variants thereof.

36. The T cell of claim 32, wherein the polypeptide involved in trafficking and/or migration is selected from the group consisting of CD183, CCR2, CCR6, CD50, CD197, CD58, CD62L, and functional fragments and variants thereof.

37. The T cell of claim 32, wherein the polypeptide involved in DC targeting is selected from the group consisting of TLR ligands, anti-DEC-205 antibody, an anti-DC-SIGN antibody, and functional fragments and variants thereof.

38. The T cell of any one of the preceding claims, wherein the antigen-binding domain binds to an antigen associated with a disease state.

39. The T cell of claim 38, wherein the disease state is selected from the group consisting of a proliferative disease, a precancerous condition, a non-cancer indication, a viral infection, and a bacterial infection.

40. The T cell of claim 39, wherein the antigen-binding domain binds to a tumor antigen, a viral antigen, or a bacterial antigen.

41. The T cell of claim 40, wherein the tumor antigen is an antigen associated with a cancer selected from the group consisting of brain cancer, bladder cancer, breast cancer, cervical cancer, colorectal cancer, liver cancer, kidney cancer, lymphoma, leukemia, lung cancer, melanoma, metastatic melanoma, mesothelioma, neuroblastoma, ovarian cancer, prostate cancer, pancreatic cancer, renal cancer, skin cancer, thymoma, sarcoma, non-Hodgkin'"'"'s lymphoma, Hodgkin'"'"'s lymphoma, uterine cancer, and combinations thereof.

42. The T cell of any one of the preceding claims, which has enhanced antigen presentation ability relative to a T cell which lacks the second nucleic acid sequence.

43. The T cell of any one of the preceding claims, which has enhanced T cell priming ability relative to a T cell which lacks the second nucleic acid sequence.

44. The T cell of any one of the preceding claims, wherein the T cell is transfected to transiently express a nucleic acid comprising a third nucleic acid sequence encoding a polypeptide which enhances T cell priming, or a functional fragment or variant thereof, which differs from the polypeptide encoded by the second nucleic acid sequence.

45. The T cell of claim 44, wherein the T cell has increased T cell priming ability relative to a T cell comprising the first nucleic acid sequence and second nucleic acid sequence, but not the third nucleic acid sequence.

46. The T cell of claim 44 or 45, wherein the third nucleic acid sequence comprises an RNA.

47. The T cell of claim 46, wherein the T cell is transfected to transiently express the third RNA.

48. The T cell of claim 46, wherein the cell does not comprise an exogenous DNA encoding the third RNA.

49. The T cell of claim 44 or 45, wherein the CAR comprises one or more costimulatory signaling domains, and wherein the third nucleic acid sequence comprises DNA.

50. The T cell of any of claims 44-49, wherein the T cell further comprises one or more additional distinct nucleic acid sequences encoding a polypeptide which enhances T cell priming, or a functional fragment or variant thereof, which differ from the polypeptides encoded by the second and third nucleic acid sequences.

51. The T cell of claim 50, wherein the one or more additional nucleic acid sequences comprises RNA.

52. The T cell of claim 50, wherein the CAR comprises one or more costimulatory signaling domains, and wherein the one or more additional nucleic acids comprise DNA

53. The T cell of any one of the preceding claims, wherein expression of the polypeptide encoded by the second and/or additional nucleic acid sequences does not substantially affect the cell-killing function of the CAR encoded by the first nucleic acid sequence.

54. The T cell of any one of the preceding claims, wherein the T cell has increased efficacy in killing tumor cells or reducing tumor size in a subject with a tumor relative to a T cell comprising only the first nucleic acid sequence.

55. The T cell of any one of the preceding claims, wherein the T cell enhances the priming of T cells with a tumor antigen, a viral antigen, a bacterial antigen.

56. The T cell of any one of the preceding claims, wherein the first, second, and/or additional nucleic acid sequences are transcribed from an in vitro transcription vector.

57. The cell of any of one of the preceding claims, made by introducing a nucleic acid wherein

(a) the nucleic acid comprises a first nucleic acid sequence encoding a chimeric antigen receptor (CAR) comprising an extracellular domain, a transmembrane domain, and an intracellular signaling domain, and

(b) the nucleic acid comprises a second nucleic acid sequence encoding a polypeptide which enhances T cell priming, or a functional fragment or variant thereof;

provided that

(i) the first and/or second nucleic acid sequence comprises an RNA;

or

(ii) the CAR further comprises a second intracellular signaling domain.

58. A composition comprising the T cell of any one of the preceding claims.

59. The composition of claim 53, further comprising a second therapeutic agent.

61. A method of providing anti-tumor immunity in a subject comprising administering to the subject an effective amount of a T cell of any one of claims 1-57.

62. A method of treating a subject having a disease associated with the expression of a disease-associated antigen, comprising administering to the subject an effective amount of the T cell of any of claims 1-57.

63. The method of claim 62, wherein the disease is selected from the group consisting of a proliferative disease, a precancerous condition, a non-cancer related indication, a viral infection, and a bacterial infection.

64. A method of enhancing epitope spreading in a subject with cancer comprising administering to the subject the T cell of any one of claims 1-57.

65. A method of vaccinating a subject comprising administering to the subject the T cell of any one of claims 1-57.

66. The method of claim 65, further comprising administering to the subject an antigen.

67. The method of claim 66, wherein the antigen is a tumor antigen, a viral antigen, or a bacterial antigen.

68. The method of any one of claims 61-67, wherein the subject is a human.

60. A method of generating a T cell having enhanced anti-tumor activity, the method comprising introducing a nucleic acid, wherein:

(a) the nucleic acid comprises a first nucleic acid sequence encoding a chimeric antigen receptor (CAR) comprising an extracellular domain, a transmembrane domain, and an intracellular signaling domain, and

(b) the nucleic acid comprises a second nucleic acid sequence encoding a polypeptide which enhances T cell priming, or a functional fragment or variant thereof;

provided that

(i) the first and/or second nucleic acid sequence comprises an RNA;

or

(ii) the CAR further comprises a second intracellular signaling domain.