METHODS FOR IMPROVING THE EFFICACY AND EXPANSION OF IMMUNE CELLS

• US 20170137783A1
• Filed: 07/21/2016
• Published: 05/18/2017
• Est. Priority Date: 07/21/2015
• 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: June, Carl H., Levine, Bruce L., Bedoya, Felipe, Ghassemi, Saba, Kawalekar, Omkar U., Melenhorst, Jan J., Milone, Michael, Powell, JR., Daniel J., Zheng, Zoe
• Granted Patent: US 10,829,735 B2
• Time in Patent Office: Days
• 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 2239/59 Reproductive system, e.g. uterus, ovaries, cervix or testes
• 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 39/464468 Mesothelin [MSLN]
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/02 specific for leukemia
• CPC Class Codes: A61P 37/04 Immunostimulants
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• 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 14/70517 CD8
• CPC Class Codes: C07K 14/70578 NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95 NGF-receptor C07K14/71, TNF-receptor C07K14/7151
• CPC Class Codes: C07K 16/18 against material from animals or humans
• CPC Class Codes: C07K 16/28 against receptors, cell surface antigens or cell surface determinants
• CPC Class Codes: C07K 16/2803 against the immunoglobulin superfamily
• CPC Class Codes: C07K 16/30 from tumour cells
• CPC Class Codes: C07K 2317/622 Single chain antibody (scFv)
• CPC Class Codes: C07K 2319/03 : containing a transmembrane segment
• CPC Class Codes: C07K 2319/33 : fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
• CPC Class Codes: C07K 2319/74 : containing a fusion for binding to a cell surface receptor
• CPC Class Codes: C12N 2501/2302 : Interleukin-2 (IL-2)
• CPC Class Codes: C12N 2501/2307 : Interleukin-7 (IL-7)
• CPC Class Codes: C12N 2501/2315 : Interleukin-15 (IL-15)
• CPC Class Codes: C12N 2501/50 : Cell markers; Cell surface determinants
• CPC Class Codes: C12N 2510/00 : Genetically modified cells
• CPC Class Codes: C12N 2740/16043 : viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 5/0087 : Purging against subsets of blood cells, e.g. purging alloreactive T cells
• CPC Class Codes: C12N 5/0636 : T lymphocytes

Abstract

The invention provides methods of making immune effector cells (e.g., T cells, NK cells) that can be engineered to express a chimeric antigen receptor (CAR), compositions and reaction mixtures comprising the same, and methods of treatment using the same.

Claims

1. A method of expanding and/or activating a population of immune cells, comprising:

introducing a nucleic acid encoding a first Chimeric Antigen Receptor (CAR) molecule into the immune cell population, under conditions suitable for transient expression of the CAR molecule, thereby producing a first CAR-expressing cell population, wherein the CAR molecule comprises an antigen binding domain of an antibody molecule; and

contacting the first CAR-expressing cell population with a ligand of the CAR antigen binding domain chosen from a cognate antigen molecule, or an anti-antigen idiotypic antibody molecule, under conditions such that immune cell expansion and/or activation occurs, thereby producing an expanded and/or activated immune cell population.

2. A method of expanding and/or activating a population of immune cells, comprising:

providing a first CAR-expressing cell population, said first CAR-expressing cell population comprising a transiently expressed first CAR molecule, and said CAR molecule comprises an antigen binding domain of an antibody molecule; and

contacting said CAR-expressing cell population with a ligand of the CAR molecule chosen from a cognate antigen molecule, or an anti-antigen idiotypic antibody molecule, under conditions such that immune cell expansion and/or activation occurs, thereby producing an expanded and/or activated immune cell population.

3. The method of claim 2, wherein the expansion and/or activation of the population of immune cells is carried out in vitro, ex vivo or in vivo.

4. The method of claim 2, wherein:

the population of immune cells is acquired, from a blood sample from a subject;

the population of immune cells comprises immune effector cells chosen from T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, myeloid-derived phagocytes, or a combination thereof;

the population of immune cells comprises primary T cells or a subset of lymphocytes chosen from anergized T cells, naï

ve T cells, T-regulatory cells, Th-17 cells, stem T cells, or a combination thereof;

the population of immune cells comprises peripheral blood mononucleated cells (PBMCs), cord blood cells, or a combination thereof;

or

the population of immune cells comprises cells that express a low level of, substantially impaired, or do not have, a functional T cell receptor.

9. The method of claim 2, wherein the ligand is:

a cognate antigen molecule;

an anti-antigen idiotypic antibody molecule;

immobilized or attached to a non-naturally occurring substrate;

present on a surface of a cell.

14. The method of claim 2, wherein the T cells are expanded in vivo by lymph node injection, or by injection of the tumor-infiltrating lymphocytes (TIL) into a tumor.

15. The method of claim 2, wherein the nucleic acid encoding the first CAR molecule is an RNA molecule.

16. The method of claim 2, wherein:

the first CAR molecule is transiently expressed in the immune cell population for a finite period of time or number of cell replications;

the first CAR molecule is internalized post a single ligand stimulation;

the immune cell does not receive repeated ligand stimulation;

the strength of the immune cell stimulation is customized to a desired level by adjusting one or both of;

the first CAR-surface density, or the affinity of the CAR antigen binding domain to the ligand;

the first CAR-expressing immune cells are cultured in the presence of the ligand of the CAR molecule for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 18, 21, 22, 23 or 24 hours, or about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 days;

the first CAR-expressing cells are cultured for a period of 8 days or less;

the CAR-expressing cells shows at least 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 or higher population doublings;

wherein the first CAR-expressing immune cell population expands to a total of 400-600, or about 500 cells, wherein the cell expansion is measured between 10 and 25 days after stimulation with the ligand;

the expanded and/or activated immune cell population comprises immune effector cells having a less differentiated phenotype;

or

wherein the younger T cell is chosen from a naï

ve T cell (T_N), a memory stem cell (T_SCM), a central memory T cell (T_CM), or a combination thereof;

26. The method of claim 2, further comprising contacting the expanded and/or activated immune cell population with a nucleic acid encoding a second CAR molecule, thereby producing a second CAR-expressing cell population.

27. The method of claim 26, wherein the nucleic acid encoding the second CAR molecule is selected from the group consisting of a DNA, an RNA, a plasmid, a lentivirus vector, adenoviral vector, or a retrovirus vector.

28. The method of claim 26, wherein the first and second CAR molecules are directed to the same antigen or different antigens.

29. The method of claim 28, wherein the cancer associated antigen is chosen from CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1 (CLECL1), CD33, EGFRvIII, GD2, GD3, BCMA, Tn Ag, PSMA, ROR1, FLT3, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, Mesothelin, IL-11Rα

, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-beta, SSEA-4, CD20, Folate receptor alpha, ERBB2 (Her2/neu), MUC1, EGFR, NCAM, Prostase, PAP, ELF2M, Ephrin B2, FAP, 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, GPRCSD, CXORF61, CD97, CD179a, ALK, Polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, 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, legumain, HPV E6, E7, intestinal carboxyl esterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, or IGLL1.

31. The method of claim 26, wherein the first and second CAR molecules are the same or different CAR molecules.

32. The method of claim 26, wherein the first and second CAR molecules are each independently chosen from a CD19 CAR, a BCMA CAR, a CD33 CAR, a CLL-1 CAR, EGFRvIII CAR, a GFR alpha 4 CAR, an ROR1 CAR, a CD20 CAR, a CD22 CAR, a CD123 CAR, a CD10 CAR, a CD34 CAR, a FLT-3 CAR, a CD79b CAR, a CD179b CAR, a mesothelin CAR or a CD79a CAR, or any combination thereof.

34. The method of claim 2, wherein the immune cell population transiently expressing the first CAR is expanded and/or activated in vitro or ex vivo by contacting said immune cell population with a CD19-antigen or anti-CD19 idiotypic antibody immobilized onto a non-cellular or cellular substrate.

36. A method of treating a cancer, or providing anti-tumor immunity, in a subject, comprising administering to the subject an expanded and/or activated immune cell population made according to claim 2, alone or in combination with an additional therapy, thereby treating or providing anti-tumor immunity to the subject.

47. The method of claim 26, wherein the first and second CAR molecules are mesothelin CAR and CD19 CAR molecules, respectively.

55. The method of claim 36, wherein the cancer is a hematological cancer chosen from one or more of:

a B-cell acute lymphocytic leukemia (B-ALL), T-cell acute lymphocytic leukemia (T-ALL), acute lymphocytic leukemia (ALL), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), B cell promyelocytic 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 (MCL), marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin'"'"'s lymphoma (NHL), Hodgkin'"'"'s lymphoma (HL), plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, and Waldenstrom macroglobulinemia.

57. The method of claim 2, wherein the population of cells is expanded in the presence of a cytokine chosen from IL2 or IL-15 and IL-7.

58. The method of claim 2, further comprising removing T regulatory cells from the immune cell population, to thereby provide a population of T regulatory-depleted cells.

63. The method claim 2, further comprising removing cells from the acquired immune effector cell population which express a check point inhibitor chosen from one or more of PD1+ cells, LAG3+ cells, and TIM3+ cells, to thereby provide a population of T regulatory-depleted cells, and check point inhibitor depleted cells.

71. An immune cell preparation or reaction mixture, comprising a population of immune effector cells made according to the method of claim 2.

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37. (canceled)

38. A method of treating, or providing anti-tumor immunity to, a subject having a cancer, comprising:

administering to the subject an effective amount of an immune cell population that expresses a first CAR molecule and a second CAR molecule, simultaneously or sequentially, wherein the first CAR molecule is transiently expressed, and the second CAR molecule is stably expressed.

39. A method of treating, or providing anti-tumor immunity to, a subject having a cancer, comprising:

administering to the subject an effective amount of an immune cell population expressing a second CAR molecule, wherein the immune cell population was previously obtained by expanding and/or activating in vitro or ex vivo an immune cell population transiently expressing a first CAR molecule, said first CAR molecule comprising an antigen binding domain of an antibody molecule.

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72. An immune effector cell preparation or reaction mixture that expresses a first and a second CAR molecule, simultaneously or sequentially, wherein the first CAR molecule is transiently expressed, and the second CAR molecule is stably expressed.