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 anti-B cell maturation antigen (BCMA) 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 anti-BCMA CAR comprises an extracellular antigen binding domain comprising an antibody or antigen binding fragment thereof that binds BCMA.
8. The method of claim 1 , wherein the anti-BCMA CAR comprises a transmembrane domain obtained from CD8a or CD28.
9. The method of claim 1 , wherein the anti-BCMA CAR comprises one or more costimulatory signaling domains obtained from a polypeptide selected from the group consisting of: CD28, CD134, and CD137.
10. The method of claim 1 , wherein the anti-BCMA CAR further comprises a hinge region polypeptide.
11. The method of claim 10 , wherein the hinge region polypeptide comprises a hinge region of IgG1 or CD8a.
12. The method of claim 1 , wherein the anti-BCMA CAR further comprises a signal peptide.
13. The method of claim 12 , wherein the signal peptide comprises an IgG1 heavy chain signal polypeptide or a CD8α signal polypeptide.
14. 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.
15. The method of claim 1 , wherein the PI3K inhibitor is ZSTK474.
16. 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.
17. 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.
18. 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 lentiviral vector comprising a polynucleotide encoding an anti-BCMA CAR, wherein the CAR comprises a signal peptide, an extracellular antigen binding domain that binds BCMA, a hinge region polypeptide, a transmembrane domain, a costimulatory domain, and a primary signaling domain; and
c) culturing the transduced T cells to proliferate;
wherein steps a)-c) are performed in the presence of a 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.
19. The method of claim 18 , wherein the method comprises isolating peripheral blood mononuclear cells as the source of T cells.
20. The method of claim 18 , wherein activation of the T cells comprises contacting the T cells with an anti-CD3 antibody or CD3-binding fragment thereof.
21. The method of claim 18 , 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.
22. The method of claim 18 , wherein the extracellular antigen binding domain comprises an antibody or antigen binding fragment thereof that binds BCMA.
23. The method of claim 18 , wherein the transmembrane domain is obtained from CD8α or CD28.
24. The method of claim 18 , wherein the costimulatory signaling domain is obtained from a polypeptide selected from the group consisting of: CD28, CD134, and CD137.
25. The method of claim 18 , wherein the hinge region polypeptide comprises a hinge region of IgG1 or CD8α.
26. The method of claim 18 , wherein the signal peptide comprises an IgG1 heavy chain signal polypeptide or a CD8α signal polypeptide.
27. The method of claim 18 , 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.
28. The method of claim 18 , wherein the PI3K inhibitor is ZSTK474.
29. 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 lentiviral vector comprising a polynucleotide encoding an anti-BCMA CAR, wherein the CAR comprises a CD8α signal peptide, an antibody fragment that binds BCMA, a CD8α hinge region polypeptide, a CD8α transmembrane domain, a CD137 costimulatory domain, and a CD3t primary signaling domain; and
c) culturing the transduced T cells to proliferate;
wherein steps a)-c) are performed in the presence of a 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.
30. The method of claim 29 , wherein the method comprises isolating peripheral blood mononuclear cells as the source of T cells.
31. The method of claim 29 , wherein activation of the T cells comprises contacting the T cells with an anti-CD3 antibody or CD3-binding fragment thereof.
32. The method of claim 29 , 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.
33. The method of claim 29 , 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.
34. The method of claim 29 , wherein the PI3K inhibitor is ZSTK474.
35. 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 lentiviral vector comprising a polynucleotide encoding an anti-BCMA CAR, wherein the CAR comprises a CD8α signal peptide, an anti-BCMA single chain variable fragment (scFv), a CD8α hinge region polypeptide, a CD8α transmembrane domain, a CD13 ζ costimulatory domain, and a CD3t primary signaling domain; and
c) culturing the transduced T cells to proliferate;
wherein steps a)-c) are performed in the presence of ZSTK474, and wherein T cell differentiation is decreased in the T cells manufactured according to steps a)-c) performed in the presence of ZSTK474 compared to T cell differentiation in T cells manufactured according to steps a)-c) performed in the absence of ZSTK474.
36. The method of claim 35 , wherein the method comprises isolating peripheral blood mononuclear cells as the source of T cells.
37. The method of claim 35 , wherein activation of the T cells comprises contacting the T cells with an anti-CD3 antibody or CD3-binding fragment thereof.
38. The method of claim 35 , 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.