CD33 specific chimeric antigen receptors
Provided herein are chimeric antigen receptors (CARs) for cancer therapy, and more particularly, CARs containing a scFv from a CD33 monoclonal antibody. Provided are immune effector cells containing such CARs, and methods of treating proliferative disorders such as acute myeloid leukemia (AML), and relapsed or refractory AML.
1. A vector comprising a backbone and a nucleic acid sequence encoding:
(A) a cell tag comprising an amino acid sequence having at least 95% identity with the full length of SEQ ID NO: 54; and
(B) a chimeric antigen receptor (CAR) comprising:
(1) a CD33 antigen binding domain comprising:
(a) the amino acid sequence of SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 3;
(b) the amino acid sequence of SEQ ID NO: 9 and the amino acid sequence of SEQ ID NO: 10;
(c) amino acid sequence of SEQ ID NO: 11 and the amino acid sequence of SEQ ID NO: 12; or
(d) the amino acid sequence of the full length of SEQ ID NO: 13 and the amino acid sequence of SEQ ID NO: 14;
(2) a stalk domain comprising: (a) a hinge region from IgG1; (b) a hinge region from IgG4; (c) the CH2CH3 region of immunoglobulin; or (d) the CD8alpha hinge domain;
(3) a transmembrane domain that: (a) comprises the transmembrane region of: the alpha chain of a T-cell receptor, the beta chain of a T-cell receptor, the zeta chain of a T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8alpha, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154; or (b) is synthetic and comprises predominantly hydrophobic residues;
(4) a costimulatory signaling domain comprising 4-1BB, CD28, or both; and
(5) a CD3 zeta signaling domain.
2. The vector of claim 1 , wherein the vector is a lentivirus vector, a retroviral vector, or a non-viral vector.
3. The vector of claim 1 , wherein the CD33 antigen binding domain comprises the amino acid sequence of the full length of SEQ ID NO: 8.
4. The vector of claim 1 , wherein the costimulatory signaling domain comprises 4-1BB.
5. The vector of claim 1 , wherein the costimulatory signaling domain comprises CD28.
6. An immune effector cell comprising the vector of claim 1 .
7. The immune effector cell of claim 6 , wherein the immune effector cell is a T cell or a Natural Killer (NK) cell.
8. A method for stimulating a T cell-mediated immune response to a target cell population or tissue in a human subject in need thereof, the method comprising administering to the human subject an effective amount of the cell of claim 6 .
9. The method of claim 8 , wherein the human subject has been diagnosed with acute myeloid leukemia (AML).
10. The method of claim 9 , wherein the acute myeloid leukemia is relapsed or refractory AML.
11. The vector of claim 1 , wherein:
the CD33 antigen binding domain comprises the amino acid sequence of SEQ ID NO: 8;
the stalk domain comprises the amino acid sequence of SEQ ID NO: 22;
the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 18;
the costimulatory signaling domain comprises the amino acid sequence of SEQ ID NO: 28; and
the CD3 zeta signaling domain comprises the amino acid sequence of SEQ ID NO: 26.
12. The vector of claim 1 , wherein:
the CD33 antigen binding domain comprises the amino acid sequence of SEQ ID NO: 8;
the stalk domain comprises the amino acid sequence of SEQ ID NO: 22;
the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 18;
the costimulatory signaling domain comprises the amino acid sequence of SEQ ID NO: 24; and
the CD3 zeta signaling domain comprises the amino acid sequence of SEQ ID NO: 26.
13. A system for expressing a CAR in an immune effector cell, the system comprising the vector of claim 1 , wherein:
the CD33 antigen binding domain comprises the amino acid sequence of SEQ ID NO: 8;
the stalk domain comprises the amino acid sequence of SEQ ID NO: 22;
the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 18;
the costimulatory signaling domain comprises the amino acid sequence of SEQ ID NO: 24 or 28; and
the CD3 zeta signaling domain comprises the amino acid sequence of SEQ ID NO: 26.
14. The system of claim 13 , wherein the immune effector cell is a T cell or NK cell.
15. The system of claim 13 , further comprising a nucleic acid encoding at least one additional gene.
16. The system of claim 15 , wherein the additional gene encodes a cytokine.
17. The system of claim 16 , wherein the cytokine is IL-2, IL-15, IL-12, or IL-21.
18. A method of stimulating the proliferation and/or survival of engineered T-cells, the method comprising:
(a) obtaining a sample of cells from a subject, the sample comprising T-cells or T-cell progenitors;
(b) transfecting the cells with the vector of claim 1 and a vector encoding a transposase, to provide a population of engineered CD33 CAR-expressing T-cells; and
(c) optionally, culturing the population of CD33 CAR T-cells ex vivo for 2 days or less.
19. The immune effector cell of claim 7 , wherein the T cell is a cytotoxic T lymphocyte (CTL) or a regulatory T cell.
20. The vector of claim 1 , wherein the cell tag comprises the amino acid sequence of SEQ ID NO: 54.
21. A method for stimulating a T cell-mediated immune response to a target cell population or tissue in a human subject in need thereof, the method comprising administering to the human subject an effective amount of the cell of claim 6 .
22. A system for expressing a CAR in an immune effector cell, the system comprising the vector of claim 20 , wherein:
the CD33 antigen binding domain comprises the amino acid sequence of SEQ ID NO: 8;
the stalk domain comprises the amino acid sequence of SEQ ID NO: 22;
the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 18;
the costimulatory signaling domain comprises the amino acid sequence of SEQ ID NO: 24 or 28; and
the CD3 zeta signaling domain comprises the amino acid sequence of SEQ ID NO: 26.
23. A method of stimulating the proliferation and/or survival of engineered T-cells, the method comprising:
(a) obtaining a sample of cells from a subject, the sample comprising T-cells or T-cell progenitors;
(b) transfecting the cells with the vector of claim 20 and a vector encoding a transposase, to provide a population of engineered CD33 CAR-expressing T-cells; and
(c) optionally, culturing the population of CD33 CAR T-cells ex vivo for 2 days or less.
24. The vector of claim 20 , wherein:
the CD33 antigen binding domain comprises the amino acid sequence of SEQ ID NO: 8;
the stalk domain comprises the amino acid sequence of SEQ ID NO: 22;
the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 18;
the costimulatory signaling domain comprises the amino acid sequence of SEQ ID NO: 24 or 28; and
the CD3 zeta signaling domain comprises the amino acid sequence of SEQ ID NO: 26.
25. The vector of claim 24 , wherein the costimulatory signaling domain comprises the amino acid sequence of SEQ ID NO: 28.
26. The vector of claim 1 , further comprising a nucleic acid encoding a cytokine.
27. The vector of claim 26 , wherein the cytokine is IL-2, IL-15, IL-12, or IL-21.
28. The vector of claim 1 , expressing a fusion protein comprising IL-15 and IL-15Rα.
29. The vector of claim 28 , wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 37.
30. The immune effector cell of claim 6 , expressing a cytokine.
31. The immune effector cell of claim 30 , wherein the cytokine is IL-2, IL-15, IL-12, or IL-21.
32. The immune effector cell of claim 6 , expressing a fusion protein comprising IL-15 and IL-15Rα.
33. The immune effector cell of claim 32 , expressing a fusion protein comprising the amino acid sequence of SEQ ID NO: 37.
34. An immune effector cell comprising the vector of claim 29 .
35. A method for treating a cancer in a subject in need thereof, the method comprising administering the immune effector cell of claim 6 to the subject.
36. The method of claim 35 , wherein the cancer is acute myeloid leukemia.
37. The method of claim 36 , wherein the cancer is relapsed or refractory acute myeloid leukemia.
38. The method of claim 35 , wherein the immune effector cell is administered to the subject in an amount of 10 4 to 10 9 cells per kilogram body weight of the subject.
39. The method of claim 35 , wherein the immune effector cell is administered to the subject in an amount of 10 5 to 10 8 cells per kilogram body weight of the subject.
40. The method of claim 35 , wherein the immune effector cell is administered to the subject in an amount of 10 5 to 10 7 cells per kilogram body weight of the subject.
41. The method of claim 35 , wherein the immune effector cell is administered to the subject in an amount of 10 6 to 10 9 cells/kg body weight of the subject.
42. The method of claim 35 , wherein the immune effector cell is administered to the subject in an amount of 10 6 to 10 7 cells per kilogram body weight of the subject.
43. The method of claim 35 , wherein the immune effector cell is produced by introducing the vector into a cell and the immune effector cell is administered to the subject within 1 day of such introduction.
44. The method of claim 35 , wherein the immune effector cell is administered following lymphodepletion of the subject.
45. The system of claim 15 , wherein the additional gene encodes a fusion protein comprising IL-15 and IL-15Rα.