Methods of making T cell compositions
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.
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, αvβ6 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-13Rα2, 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 CD3ζsignaling 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.