Methods of making T cell compositions

• US 10,479,975 B2
• Filed: 06/05/2015
• Issued: 11/19/2019
• Est. Priority Date: 06/06/2014
• Status: Active Grant

• Current Assignee: 2seventy Bio, Inc. (bluebird bio, Inc.)
• Original Assignee: bluebird bio, Inc.
• Inventors: Friedman, Kevin
• Primary Examiner(s): Kelly, Robert M
• Application Number:
  US15/316,792
• Publication Number:
  US 20170218337A1
• Time in Patent Office: 1,628 Days
• Field of Search: None
• CPC Class Codes: A61K 2239/38 characterised by the dose, timing or administration schedule
• CPC Class Codes: A61K 2239/46 characterised by the cancer treated
• CPC Class Codes: A61K 2239/48 Blood cells, e.g. leukemia or lymphoma
• 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 39/464417 Receptors for tumor necrosis factors [TNF], e.g. lymphotoxin receptor [LTR], CD30
• CPC Class Codes: A61P 1/18 for pancreatic disorders, e.g. pancreatic enzymes
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 13/10 of the bladder
• CPC Class Codes: A61P 15/00 Drugs for genital or sexual disorders for disorders of sex hormones A61P5/24; Contraceptives
• CPC Class Codes: A61P 17/00 Drugs for dermatological disorders
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/02 specific for leukemia
• CPC Class Codes: C07K 14/7051 T-cell receptor (TcR)-CD3 complex
• CPC Class Codes: C07K 16/2878 against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
• CPC Class Codes: C07K 2319/02 containing a signal sequence
• CPC Class Codes: C07K 2319/03 containing a transmembrane segment
• 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 2501/51 : 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 2501/599 : with CD designations not provided for elsewhere
• CPC Class Codes: C12N 2501/727 : Kinases (EC 2.7.)
• CPC Class Codes: C12N 2501/999 : Small molecules not provided for elsewhere
• CPC Class Codes: C12N 2510/00 : Genetically modified cells
• CPC Class Codes: C12N 5/0636 : T lymphocytes

Abstract

The invention provides improved T cell compositions and methods for manufacturing T cells. More particularly, the invention provides methods of T cell manufacturing that result in adoptive T cell immunotherapies with improved survival, expansion, and persistence in vivo.

Claims

1. A method for manufacturing T cells comprising:

a) activating a population of T cells and stimulating the population of T cells to proliferate;

b) transducing the T cells with a viral vector comprising a polynucleotide encoding an engineered T cell receptor (TCR) or a chimeric antigen receptor (CAR); and

c) culturing the transduced T cells to proliferate;

wherein steps a)-c) are performed in the presence of a phosphatidylinositol-3 kinase (PI3K) inhibitor, and

wherein T cell differentiation is decreased in the T cells manufactured according to steps a)-c) performed in the presence of the PI3K inhibitor compared to T cell differentiation in T cells manufactured according to steps a)-c) performed in the absence of the PI3K inhibitor.

2. The method of claim 1, wherein the method comprises isolating peripheral blood mononuclear cells as the source of T cells.

3. The method of claim 1, wherein activation of the T cells comprises contacting the T cells with an anti-CD3 antibody or CD3-binding fragment thereof.

4. The method of claim 1, wherein stimulation of the T cells comprises contacting the T cells with an anti-CD28 antibody or a CD28-binding fragment thereof, B7-1 or a CD28-binding fragment thereof, or B7-2 or a CD28-binding fragment thereof.

5. The method of claim 1, wherein the viral vector is a retroviral vector.

6. The method of claim 1, wherein the viral vector is a lentiviral vector.

7. The method of claim 1, wherein the viral vector comprises the polynucleotide encoding the CAR.

8. The method of claim 7, wherein the CAR comprises

a) an extracellular domain that binds an antigen selected from the group consisting of:

alpha folate receptor, 5T4, α

vβ

6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7/8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetal AchR, GD2, GD3, Glypican-3 (GPC3), HLA-A1+MAGE1, HLA-A2+MAGE1, HLA-A3+MAGE1, HLA-A1+NY-ESO-1, HLA-A2+NY-ESO-1, HLA-A3+NY-ESO-1, IL-11Rα

, IL-13Rα

2, Lambda, Lewis-Y, Kappa, Mesothelin, Muc1, Muc16, NCAM, NKG2D Ligands, NY-ESO-1, PRAME, PSCA, PSMA, ROR1, SSX, Survivin, TAG72, TEMs, and VEGFR2;

b) a transmembrane domain derived from a polypeptide selected from the group consisting of;

CD8α

;

CD4, CD28, CD45, PD-1, and CD152;

c) one or more intracellular costimulatory signaling domains selected from the group consisting of;

CD28, CD54 (ICAM), CD134 (OX40), CD137 (41BB), CD152 (CTLA4), CD273 (PD-L2), CD274 (PD-L1), and CD278 (ICOS); and

d) a CD3ζ

signaling domain.

9. The method of claim 8, wherein the extracellular domain comprises an antibody or antigen binding fragment that binds the antigen.

10. The method of claim 8, wherein the transmembrane domain is derived from CD8α

or CD28.

11. The method of claim 8, wherein the one or more costimulatory signaling domains selected from the group consisting of:

CD28, CD134, and CD137.

12. The method of claim 8, further comprising a hinge region polypeptide.

13. The method of claim 12, wherein the hinge region polypeptide comprises a hinge region of IgG1 or CD8α

.

14. The method of claim 8, further comprising a signal peptide.

15. The method of claim 14, wherein the signal peptide comprises an IgG1 heavy chain signal polypeptide or a CD8α

signal polypeptide.

16. The method of claim 1, wherein the PI3K inhibitor is selected from the group consisting of:

BEZ235, LY294002, GDC-0941, BYL719, GSK2636771, TGX-221, AS25242, CAL-101, IPI-145, and ZSTK474.

17. The method of claim 16, wherein the PI3K inhibitor is a pan-PI3K inhibitor selected from the group consisting of:

BEZ235, LY294002, and GDC-0941.

18. The method of claim 16, wherein the PI3K inhibitor is a selective PI3K inhibitor selected from the group consisting of:

BYL719, GSK2636771, TGX-221, AS25242, CAL-101, and IPI-145.

19. The method of claim 16, wherein the PI3K inhibitor is the PI3-K inhibitor ZSTK474.

20. The method of claim 1, wherein manufacturing the T cells in the presence of the PI3K inhibitor in steps a)-c) increases the number of T cells expressing one or more markers selected from the group consisting of:

CD62L, CCR7, CD28, CD27, CD122, and CD127 compared to T cells manufactured according to steps a)-c) performed in the absence of the PI3K inhibitor.

21. The method of claim 1, wherein manufacturing the T cells in the presence of the PI3K inhibitor in steps a)-c) decreases CD57 or KLRG1 expression in the T cells compared to CD57 or KLRG1 expression in T cells manufactured according to steps a)-c) performed in the absence of the PI3K inhibitor.