PHOSPHOGLYCERATE KINASE 1 (PGK) PROMOTERS AND METHODS OF USE FOR EXPRESSING CHIMERIC ANTIGEN RECEPTOR

• US 20180118834A1
• Filed: 01/15/2016
• Published: 05/03/2018
• Est. Priority Date: 01/16/2015
• Status: Active Grant

• Current Assignee: Novartis Ag, Trustees Of The University Of Pennsylvania (University of Pennsylvania)
• Original Assignee: China Novartis Institutes For Biomedical Research Co., Ltd. (Novartis Ag), Novartis Institutes for BioMedical Research, Inc. (Novartis Ag), Novartis Pharma AG (Novartis Ag), Trustees Of The University Of Pennsylvania (University of Pennsylvania)
• Inventors: Brogdon, Jennifer, Ebersbach, Hilmar Erhard, Glass, David, Huber, Thomas, Jascur, Julia, June, Carl H., Lee, Jihyun, Mannick, Joan, Milone, Michael C., Murphy, Leon, Posey, Avery D., Song, Huijuan, Wang, Yongqiang, Wei, Lai, Wu, Qilong, Yang, Qiumei, Zhang, Jiquan
• Granted Patent: US 11,459,390 B2
• Time in Patent Office: Days
• CPC Class Codes: A61K 2035/124 the cells being hematopoietic, bone marrow derived or blood cells
• CPC Class Codes: A61K 35/17 Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes when activated by a specific antigen A61K39/00
• 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 39/464412 CD19 or B4
• CPC Class Codes: A61K 48/0058 Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/02 specific for leukemia
• CPC Class Codes: C07K 14/705 Receptors; Cell surface antigens; Cell surface determinants tumour specific antigens C07K14/4748
• CPC Class Codes: C07K 14/7051 T-cell receptor (TcR)-CD3 complex
• CPC Class Codes: C07K 14/70521 CD28, CD152
• CPC Class Codes: C07K 16/28 against receptors, cell surface antigens or cell surface determinants
• CPC Class Codes: C07K 16/2863 against receptors for growth factors, growth regulators
• CPC Class Codes: C07K 16/30 from tumour cells
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/02 containing a signal sequence
• CPC Class Codes: C07K 2319/03 containing a transmembrane segment
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 2740/10043 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2740/15043 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2800/60 : Vectors containing traps for, e.g. exons, promoters

Abstract

The invention provides compositions and methods for treating diseases associated with expression of a tumor antigen as described herein. The invention also relates to nucleic acids comprising a truncated PGK promoter operably linked to a chimeric antigen receptor (CAR) specific to a tumor antigen as described herein, vectors encoding the same, and recombinant T cells comprising the CARs of the present invention. The invention also includes methods of administering a genetically modified T cell expressing a CAR that comprises an antigen binding domain that binds to a tumor antigen as described herein.

Claims

1. A nucleic acid molecule comprising a truncated PGK promoter operably linked to a nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising a costimulatory domain, a primary signalling domain, or both of a costimulatory domain and a primary signalling domain.

2. The nucleic acid molecule of claim 1, wherein said antigen binding domain binds to a tumor antigen selected from a group consisting of:

mesothelin, c-Met, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, Mesothelin, IL-11Ra, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-beta, SSEA-4, CD20, Folate receptor alpha, ERBB2 (Her2/neu), MUC1, EGFR, NCAM, Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gp100, bcr-abl, tyrosinase, EphA2, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor beta, TEM1/CD248, TEM7R, CLDN6, TSHR, GPRC5D, CXORF61, CD97, CD179a, ALK, Plysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, legumain, HPV E6,E7, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin and telomerase, PCTA-1/Galectin 8, MelanA/MART1, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, Androgen receptor, Cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxyl esterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, c-met, and IGLL1.

3. The nucleic acid molecule of claim 1 or 2, wherein the truncated PGK promoter comprises or consists of a nucleic acid sequence of a WT PGK promoter (wherein the WT PGK promoter is optionally the sequence of SEQ ID NO:

1) with a partial deletion in the WT sequence (wherein the deletion is optionally a deletion of at least 20, 25, 50, 100, 150, 200, 250, 300, 350, 400, or 450 nucleic acids).

4. The nucleic acid molecule of claim 2, wherein the partial deletion is from the 3′

end of the WT PGK promoter or from the 5′

end of the WT PGK promoter.

5. The nucleic acid molecule of any of the preceding claims, wherein the truncated PGK promoter comprises or consists of a sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to a PGK promoter selected from the group consisting of PGK100 (SEQ ID NO:

2);

PGK200 (SEQ ID NO;

     3);

PGK300 (SEQ ID NO;

     4); and

PGK400 (SEQ ID NO;

     5).

6. The nucleic acid molecule of any of the preceding claims, wherein the truncated PGK promoter comprises or consists of a nucleic acid sequence of a WT PGK promoter (wherein the WT PGK promoter is optionally the sequence of SEQ ID NO:

1) with a fragment of the WT sequence deleted.

7. The nucleic acid molecule of claim 6, wherein the deleted fragment has the sequence located at positions −

521 to −

422 of SEQ ID NO;

1, positions −

521 to −

324 of SEQ ID NO;

1, positions −

521 to −

221 of SEQ ID NO;

1, positions −

521 to −

118 of SEQ ID NO;

1, or the truncated PGK promoter comprises or consists of nucleic acids located at positions −

1 to −

422 of SEQ ID NO;

1, positions −

1 to −

324 of SEQ ID NO;

1, positions −

1 to −

221 of SEQ ID NO;

1, or positions −

1 to −

118 of SEQ ID NO;

1.

8. The nucleic acid molecule of any of the preceding claims, wherein the truncated PGK promoter is modified (wherein the modification optionally comprises nucleic acid substitutions, additions, deletions, or mutations) but retains the ability to direct expression of a CAR.

9. The nucleic acid molecule of any of the preceding claims, wherein the truncated PGK promoter comprises or consists of a sequence of at least 25, 50, 75, or 100 contiguous nucleic acids identical or substantially identical (optionally, 75%, 80%, 85%, 90%, or 95% identical) to a sequence of a WT PGK promoter (wherein the WT PGK promoter is optionally the sequence of SEQ ID NO:

1).

10. The nucleic acid molecule of any of the preceding claims, wherein the costimulatory domain is a 41BB, CD27 or CD28 costimulatory domain.

11. The nucleic acid molecule of any of the preceding claims, wherein the costimulatory domain comprises a CD28 costimulatory domain.

12. The nucleic acid molecule of any of the preceding claims, wherein the primary signaling domain is a CD3 zeta signaling domain.

13. The nucleic acid molecule of any of the preceding claims, wherein the CAR comprises a leader sequence, a tumor antigen binding domain, a hinge region, a transmembrane domain, and an intracellular signaling domain.

15. The vector of claim 14, wherein the nucleic acid comprising a truncated PGK promoter operably linked to a nucleic acid sequence encoding a CAR comprises or consists of the nucleic acid of any of claims 1-13.

21. A cell, which is optionally an immune effector cell, comprising a nucleic acid molecule of any of claims 1-13, or a vector of any of claims 14-20.

22. The cell of claim 21, wherein the cell is a human T cell or a human NK cell.

23. The cell of claim 22, wherein the human T cell is a CD8+ T cell.

29. The method of any of claims 26-28, wherein the cell is the cell described in any one of claims 21-23.

14. A vector comprising a nucleic acid, said nucleic acid comprising a truncated PGK promoter operably linked to a nucleic acid sequence encoding a CAR.

16. The vector of claim 14 or 15, wherein the vector is selected from the group consisting of a DNA, a RNA, a plasmid, a lentivirus vector, adenoviral vector, or a retrovirus vector.

17. The vector of any of claims 14-16, comprising a truncated PGK promoter operably linked to a nucleic acid sequence encoding a CAR so as to direct transcription of the nucleic acid sequence encoding the CAR at a desired level.

18. The vector of any of claims 14-17, wherein the vector is an in vitro transcribed vector, e.g., a vector that transcribes RNA of a nucleic acid molecule described herein.

19. The vector of any of claims 14-18, wherein the vector further comprises a poly(A) tail sequence, a 3′

UTR, a peptide cleavage site, which is optionally a T2A site, or any combination thereof.

20. The vector of any of claims 14-19, wherein the vector is a retroviral vector, and is optionally a lentiviral vector, and is optionally a self-inactivating lentiviral vector.

25. The method of claim 24, wherein the cell is the cell of any one of claims 21-23, and the vector is the vector in any one of claims 14-20.

24. A method of making a cell comprising transducing a cell, which cell is optionally an immune effector cell, which is optionally a T cell or a NK cell, with a vector of comprising a truncated PGK promoter operably linked to a nucleic acid sequence encoding a CAR.

26. A method of treating a subject, comprising administering to a subject a cell comprising a nucleic acid molecule comprising a truncated PGK promoter operably linked to a nucleic acid encoding a CAR.

27. The method of claim 26, wherein the subject has a cancer.

28. The method of claim 26 or 27, wherein the cell is autologous or allogeneic to the subject to be administered the cell.

30. A method of generating a population of RNA-engineered cells transiently expressing exogenous RNA, comprising contacting a population of cells with RNA transcribed from a nucleic acid, which nucleic acid comprises a truncated PGK1 promoter and a nucleic acid encoding a CAR, and wherein the cells are optionally immune effector cells (which are optionally T cells or NK cells).

31. A CAR-expressing cell therapy, for use in providing an anti-tumor immunity in a subject, wherein the CAR-expressing cell comprises a cell comprising a nucleic acid molecule comprising a truncated PGK promoter operably linked to a nucleic acid sequence encoding a CAR.

32. The use of claim 31, wherein the cell is an autologous T cell or NK cell, or an allogeneic T cell or NK cell.

33. The use of claim 31 or 32, wherein the subject is a human.

34. A CAR-expressing cell therapy, for use in the treatment of a subject having a disease associated with expression of a tumor antigen, wherein the CAR-expressing cell therapy comprises a cell comprising a truncated PGK promoter operably linked to a nucleic acid sequence encoding a CAR.

36. The use or method of claim 34 or 35, wherein the disease associated with a tumor antigen is a proliferative disease, (which proliferative disease is optionally a cancer or malignancy or a precancerous condition such as a myelodysplasia, a myelodysplastic syndrome or a preleukemia), or a non-cancer related indication associated with expression of a tumor antigen described herein.

37. The use or method of claim 36, wherein the cancer is a hematologic cancer, leukemia, or lymphoma.

38. The use or method of claim 36, wherein the cancer is selected from the group consisting of one or more acute leukemias including but not limited to B-cell acute lymphoid leukemia (“

BALL”

), T-cell acute lymphoid leukemia (“

TALL”

), acute lymphoid leukemia (ALL);

one or more chronic leukemias including but not limited to chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL);

additional hematologic cancers or hematologic conditions including, but not limited to B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt'"'"'s lymphoma, diffuse large B cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell- or a large cell-follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, Marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin lymphoma, Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom macroglobulinemia, and “

preleukemia”

which are a diverse collection of hematological conditions united by ineffective production (or dysplasia) of myeloid blood cells, and to disease associated with expression of a tumor antigen described herein include, but not limited to, atypical and/or non-classical cancers, malignancies, precancerous conditions or proliferative diseases expressing a tumor antigen as described herein; and

any combination thereof. In another embodiment, the disease associated with a tumor antigen described herein is a solid tumor.

39. The use or method of any of claims 34-38, wherein lymphocyte infusion, (optionally, allogeneic lymphocyte infusion), is used in the treatment of the cancer, wherein the lymphocyte infusion comprises at least one CAR-expressing cell, wherein the lymphocyte infusion is optionally an autologous lymphocyte infusion.

40. The use or method of any of claims 34-39, wherein the cell is an immune effector cell (optionally, an immune effector cell described herein, optionally a human T cell or a human NK cell).

41. The use or method of any of claims 34-40, wherein the subject has received a previous stem cell transplantation, which is optionally an autologous stem cell transplantation.

42. The use or method of any of claims 34-41, wherein the immune effector cell is administered in combination with an agent that increases the efficacy of a cell expressing a CAR molecule, an agent that ameliorates one or more side effect associated with administration of a cell expressing a CAR molecule, or an agent that treats the disease associated with a tumor antigen as.

43. The use or method of any of claims 34-42, wherein the cell expresses two or more CAR molecules.

44. The use or method of any of claims 34-43, wherein the therapy comprises a population of cells.

35. A method of treating a subject having a disease associated with expression of a tumor antigen comprising administering to the subject an effective amount of a cell comprising a truncated PGK promoter operably linked to a nucleic acid sequence encoding a CAR.

45. An isolated nucleic acid molecule comprising a truncated PGK promoter operably linked to a nucleic acid encoding a CAR, a vector comprising the isolated nucleic acid molecule, or the cell comprising the isolated nucleic acid molecule, for use as a medicament.

46. An isolated nucleic acid molecule comprising a truncated PGK promoter operably linked to a nucleic acid encoding a CAR, a vector comprising the isolated nucleic acid molecule, or the cell comprising the isolated nucleic acid molecule, for use in the treatment of a disease expressing a tumor antigen.

47. A population of autologous or allogeneic cells that are transfected or transduced with a vector comprising a nucleic acid molecule comprising a truncated PGK promoter operably linked to a nucleic acid encoding a CAR.

48. A method comprising, delivering a vector (optionally, by transfecting or electroporating) to a cell (optionally, a T cell or a NK cell), wherein the vector comprises a nucleic acid molecule comprising a truncated PGK promoter operably linked to a nucleic acid molecule encoding a CAR, which is transcribed as an mRNA molecule, and the CAR is translated from the RNA molecule and expressed on the surface of the cell.