Methods of making chimeric antigen receptor-expressing cells

Patent metadata

Field	Value
Publication number	US-10273300-B2
Application number	US-201514981142-A
Country	US
Kind code	B2
Filing date	Dec 28, 2015
Priority date	Dec 29, 2014
Publication date	Apr 30, 2019
Grant date	Apr 30, 2019

Abstract

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.

First claim

What is claimed is:

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.

Assignees

• Univ Pennsylvania
• Novartis Ag

Inventors

• Bedoya Felipe
• Ghassemi Saba
• June Carl H
• Levine Bruce L
• Melenhorst Jan J
• Milone Michael C
• Powell Jr Daniel J
• Zheng Zoe

Classifications

• A61P35/00

  Antineoplastic agents · CPC title

• C07K14/7051 Primary

  T-cell receptor (TcR)-CD3 complex · CPC title

• C12N2501/2307

  Interleukin-7 (IL-7) · CPC title

• C12N2501/515

  CD3, T-cell receptor complex · CPC title

• C07K16/2803 Primary

  against the immunoglobulin superfamily · CPC title