Methods of making chimeric antigen receptor-expressing cells
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), and compositions and reaction mixtures comprising the same.
1. A method of making a population of chimeric antigen receptor (CAR)-expressing immune effector cells, the method comprising:
(a) providing a population of cells comprising immune effector cells from an apheresis sample from a patient with chronic lymphocyticleukemia;
(b) depleting CD25-expressing cells from the population of cells of step (a) to provide a population of CD25-depleted cells, wherein the population of CD25-depleted cells is a population that contains less than 50% CD25+ cells in the population of cells of step (a) when assessed by flow cytometry;
(c) contacting the population of CD25-depleted cells with anti-CD3 and anti-CD28 coated beads; and
(d) transducing the population of CD25-depleted cells with a lentivirus vector encoding a chimeric antigen receptor;
to thereby provide a population of CAR-expressing immune effector cells.
2. The method of claim 1 , wherein the population of CD25-depleted cells is a population that contains less than 30% CD25+ cells.
3. The method of claim 1 , wherein the population of CD25-depleted cells is a population that contains less than 20% CD25+ cells.
4. The method of claim 1 , wherein the population of CD25-depleted cells is a population that contains less than 10% CD25+ cells.
5. The method of claim 1 , further comprising a step of depleting cells that express a tumor antigen prior to the contacting step.
6. The method of claim 1 , further comprising a step of depleting cells that express a check point inhibitor prior to the contacting step.
7. The method of claim 1 , wherein the CD25-expressing cells are depleted by contacting the population of cells from the apheresis sample with anti-CD25 antibody coated beads.
8. A population of chimeric antigen receptor expressing immune effector cells produced by the method of claim 1 .
9. A population of chimeric antigen receptor expressing immune effector cells produced by the method of claim 5 .
10. A population of chimeric antigen receptor expressing immune effector cells produced by the method of claim 6 .
11. A population of chimeric antigen receptor expressing immune effector cells produced by the method of claim 7 .
12. A method of making an expanded population of chimeric antigen receptor (CAR)-expressing immune effector cells, the method comprising:
(a) providing a population of cells comprising immune effector cells from an apheresis sample from a patient with chronic lymphocytic leukemia;
(b) depleting CD25-expressing cells from the population of cells of step (a) to provide a population of CD25-depleted cells, wherein the population of CD25-depleted cells is a population that contains less than 50% CD25+ cells in the population of cells of step (a) when assessed by flow cytometry;
(c) contacting the population of CD25-depleted cells with anti-CD3 and anti-CD28 coated beads;
(d) transducing the population of CD25-depleted cells with a lentivirus vector encoding a chimeric antigen receptor; and
(e) culturing the chimeric antigen receptor expressing immune effector cells in culture media supplemented with IL-15 or a combination of IL-15 and IL-7 for a period of between 3 and 9 days;
to thereby provide an expanded population of CAR-expressing immune effector cells.
13. The method of claim 12 , wherein the culturing period is less than 6 days.
14. The method of claim 12 , wherein the culturing period is less than 5 days.
15. The method of claim 12 , wherein the culturing period is less than 4 days.
16. An expanded population of chimeric antigen receptor expressing immune effector cells produced by the method of claim 12 .
17. An expanded population of chimeric antigen receptor expressing immune effector cells produced by the method of claim 15 .
18. A method of making an expanded population of CD19 chimeric antigen receptor (CD19 CAR)-expressing immune effector cells, the method comprising:
(a) providing a population of cells comprising immune effector cells from an apheresis sample from a patient with chronic lymphocytic leukemia;
(b) depleting CD25-expressing cells from the population of cells to provide a population of CD25-depleted cells, wherein the population of CD25-depleted cells is a population that contains less than 50% CD25+ cells in the population of cells of step (a) when assessed by flow cytometry;
(c) contacting the population of CD25-depleted cells with anti-CD3 and anti-CD28 coated beads;
(d) transducing the population of CD25-depleted cells with a lentivirus vector encoding a CD19 CAR; and,
(e) culturing the chimeric antigen receptor expressing immune effector cells in culture media supplemented with IL-15 or a combination of IL-15 and IL-7 for a period of between 3 and 9 days;
to thereby provide an expanded population of CD19 CAR-expressing immune effector cells.
19. The method of claim 18 , wherein the culturing period is less than 4 days.
20. An expanded population of CD19 chimeric antigen receptor expressing immune effector cells produced by the method of claim 18 .
21. The method of claim 1 , further comprising expanding the population of CD25-depleted cells.
22. The method of claim 21 , wherein the population of CD25-depleted cells is expanded in the presence of a cytokine for a period of 8 days or less.
23. The method of claim 1 , wherein the population of CD25-depleted cells is a population that contains less than 50%, 30%, 20%, or 10% of CD25-depleted tumor cells.
24. The method of claim 1 , wherein the population of CAR-expressing immune effector cells expresses a CAR that specifically binds to CD19.
25. The method of claim 24 , wherein the population of CAR-expressing immune effector cells expresses a CAR comprising an antigen binding domain comprising a light chain variable domain (VL) and a heavy chain variable domain (VH) having the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 51.
26. The method of claim 12 , wherein the population of CAR-expressing immune effector cells expresses a CAR that specifically binds to CD19.
27. The method of claim 26 , wherein the population of CAR-expressing immune effector cells expresses a CAR comprising an antigen binding domain comprising a light chain variable domain (VL) and a heavy chain variable domain (VH) having the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 51.
28. The method of claim 18 , wherein the population of CAR-expressing immune effector cells expresses a CAR comprising an antigen binding domain comprising a light chain variable domain (VL) and a heavy chain variable domain (VH) having the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 51.