Antigen-specific immune effector cells
Provided herein are methods for the production of antigen-specific effector T cells and NK cells from pluripotent stem cells which express a chimeric antigen receptor (CAR). Further provided herein are methods for the adoptive cell therapy by administering the effector T cells and/or NK cells provided herein.
1. A method of producing antigen-specific effector T cells and/or NK cells comprising:
(a) engineering induced pluripotent stem cells (iPSCs) to express a chimeric antigen receptor (CAR), thereby producing CAR-iPSCs;
(b) priming CAR-iPSCs for mesoderm induction by directed differentiation of the CAR-iPSCs to CD34 + hematopoietic progenitor cells (HPCs) by generating embryoid bodies (EBs) in the presence of a ROCK inhibitor, a GSK-3 inhibitor, FGF2, and VEGF;
(c) contacting the EBs with BMP-4, VEGF, and FGF2 to induce mesoderm induction;
(d) differentiating the EBs in the presence of Flt-3 ligand, IL3, SCF, and TPO, thereby producing a population of HPCs comprising at least 40% CD34+CD43+ cells;
(e) further differentiating the HPCs in the population to T cells and/or NK cells comprising culturing the HPC population on a fibronectin or fragment thereof and Notch DLL-4 coated surface in the presence of ascorbic acid and nicotinamide under hypoxic conditions; and
(f) expanding the T cells and/or NK cells by co-culturing with antigen-specific target cells, thereby producing antigen-specific effector T cells and/or NK cells.
2. The method of claim 1 , wherein the iPSCs are reprogrammed from T cells.
3. The method of claim 1 , wherein differentiating of step (b) is essentially free of BMP4 and step (d) is essentially free of BMP4, VEGF and FGF2.
4. The method of claim 1 , wherein the GSK-3 inhibitor is CHIR99021.
5. The method of claim 1 , wherein the T cells are CD4 + T cells, CD8 + T cells, cytotoxic T cells, regulatory T cells, natural killer T cells, naïve T cells, memory T cells, or gamma delta T cells.
6. The method of claim 1 , wherein step (b) further comprises selecting for cells that express CD34 and CD43 prior to differentiating to antigen-specific T cells and/or NK cells.
7. The method of claim 6 , wherein the cells that express both CD34 and CD43 comprise at least 65 percent of the total cell population.
8. The method of claim 1 , wherein at least 5 percent of the HPCs express the CAR.
9. The method of claim 1 , wherein the culture in step (e) further comprises SCF, FLT-3 ligand, TPO, and IL7.
10. The method of claim 9 , wherein the culture of step (e) further comprises a GSK-3 inhibitor, IL-2, and/or IL-12.
11. The method of claim 1 , wherein expanding the T cells of step (f) further comprises culturing the antigen-specific T cells in the presence of anti-CD3 antibody, IL-2, and IL-15.
12. The method of claim 1 , wherein at least 1.5 percent of the differentiated CD34 + HPCs are CD3+CAR + T cells.
13. The method of claim 1 , wherein at least 5 percent of the antigen-specific effector T cells display cytotoxic activity against target cells.
14. The method of claim 1 , wherein the iPSCs are homozygous for one or more of the loci alleles HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP or HLA-DQ.
15. The method of claim 1 , wherein expanding comprises culturing the antigen-specific T cells in the presence of anti-CD3 antibody, FLT3-ligand, IL-7, IL-2, IL-15, and/or IL-21.
16. The method of claim 1 , wherein the method comprises culturing the cells under defined, feeder-free conditions.
17. The method of claim 11 , wherein the expanding the T cells produce a cell population comprising CD3 + T cells.
18. The method of claim 1 , wherein the fibronectin fragment is retronectin.
19. The method of claim 1 , wherein the population of HPCs comprise at least 45% CD34+CD43+ cells.
20. The method of claim 19 , wherein the population of HPCs comprise at least 50% CD34+CD43+ cells.
21. The method of claim 20 , wherein the population of HPCs comprise at least 60% CD34+CD43+ cells.
22. The method of claim 21 , wherein the population of HPCs comprise at least 70% CD34+CD43+ cells.