ROR-1 specific chimeric antigen receptors and uses thereof
Provided herein are chimeric antigen receptors (CARs) for cancer therapy, and more particularly, CARs containing a scFv from an anti-ROR-1 monoclonal antibody. Provided are immune effector cells containing such CARs, and methods of treating proliferative disorders.
1. A nucleic acid encoding a chimeric antigen receptor (CAR), comprising:
(a) a ROR-1 antigen binding domain comprising: (i) a first polypeptide comprising the an amino acid sequence of SEQ ID NO: 36; and (ii) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 17;
(b) a transmembrane domain; and
(c) a CD3 zeta signaling domain.
2. The nucleic acid of claim 1 , wherein the CD3 zeta signaling domain comprises a polypeptide having at least 90% identity with the amino acid sequence of SEQ ID NO: 93.
3. The nucleic acid of claim 1 , further encoding a polypeptide having at least 90% identity with an amino acid sequence of any one of SEQ ID NOs: 94-108.
4. The nucleic acid of claim 1 , further encoding a cell tag.
5. The nucleic acid claim 4 , wherein the cell tag is a truncated epidermal growth factor receptor.
6. The nucleic acid of claim 4 , wherein the cell tag comprises a polypeptide having at least 90% identity with the amino acid sequence of SEQ ID NO: 109 or SEQ ID NO: 110.
7. The nucleic acid of claim 1 , further encoding: (a) a first gene switch polypeptide comprising a DNA binding domain fused to a first nuclear receptor ligand binding domain; (b) a second gene switch polypeptide comprising a transactivation domain fused to a second nuclear receptor ligand binding domain; and (c) a linker connecting the first gene switch polypeptide and the second gene switch polypeptide.
8. A vector comprising the nucleic acid of claim 4 and a nucleic acid sequence encoding a cytokine.
9. The vector of claim 8 , wherein the cell tag comprises a polypeptide having at least 90% identity with the amino acid sequence of SEQ ID NO: 110.
10. The vector of claim 8 , wherein the cytokine is IL-15.
11. The vector of claim 8 , wherein the backbone is Tc1/mariner-type transposon DNA plasmid.
12. An immune effector cell comprising the nucleic acid of claim 1 .
13. The immune effector cell of claim 12 further comprising a cell tag and a cytokine.
14. The immune effector cell of claim 13 , wherein the cytokine is IL-15.
15. The immune effector cell of claim 13 , wherein the cell tag comprises a polypeptide having at least 90% identity with the amino acid sequence of SEQ ID NO: 110.
16. The immune effector cell of claim 13 , wherein the CAR comprises a polypeptide having the amino acid sequence of SEQ ID NO: 79.
17. 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 12 wherein the cell administered to the human subject also comprises a cell tag, and wherein the target cell population or tissue expresses ROR-1.
18. A method of treating cancer in a human subject in need thereof, the method comprising administering to the human subject one or more doses of an effective amount of engineered T-cells, wherein the engineered T-cells comprise a CAR encoded by the nucleic acid of claim 1 and a membrane bound IL-15, and wherein the cancer expresses ROR-1.
19. A vector comprising the nucleic acid of claim 4 and a nucleic acid sequence encoding a fusion protein comprising: IL-15, or a functional fragment or variant thereof; and IL-15RΔ, or a functional fragment or variant thereof.
20. The vector of claim 19 , wherein the fusion protein comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 113.
21. The vector of claim 19 , wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 113.
22. The nucleic acid of claim 1 , wherein the CAR further comprises a spacer.
23. The nucleic acid of claim 22 , wherein the spacer comprises a stalk region and at least one stalk extension region.
24. The nucleic acid of claim 23 , wherein each stalk extension region contains at least one fewer dimerization site as compared to the stalk region.
25. The nucleic acid of claim 24 , wherein the stalk region is proximal to the transmembrane domain.
26. The nucleic acid of claim 22 , wherein the spacer comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 85.
27. The nucleic acid of claim 1 , wherein the transmembrane domain comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 87.
28. The nucleic acid of claim 1 , wherein the CAR further comprises a 4-11313 co-stimulatory signaling domain and/or a CD28 co-stimulatory domain.
29. The nucleic acid of claim 28 , wherein the CAR comprises a CD28 co-stimulatory domain.
30. The nucleic acid of claim 29 , wherein the CD28 co-stimulatory domain comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 90.
31. The nucleic acid of claim 1 , wherein the CAR comprises:
(a) a spacer comprising a polypeptide comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 85;
(b) a transmembrane domain comprising a polypeptide comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 87;
(c) a CD28 costimulatory signaling domain comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 90; and
(d) a CD3 zeta signaling domain comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 93.
32. The nucleic acid of claim 1 , wherein the CAR comprises the amino acid sequence of SEQ ID NO: 79.
33. The nucleic acid of claim 1 , wherein the CD3 zeta signaling domain comprises a polypeptide having at least 95% identity with the amino acid sequence of SEQ ID NO: 93.
34. The nucleic acid of claim 1 , wherein the CD3 zeta signaling domain comprises a polypeptide having at least 98% identity with the amino acid sequence of SEQ ID NO: 93.
35. The nucleic acid of claim 1 , wherein the CD3 zeta signaling domain comprises the amino acid sequence of SEQ ID NO: 93.
36. The nucleic acid of claim 22 , wherein the spacer comprises an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 85.
37. The nucleic acid of claim 22 , wherein the spacer comprises an amino acid sequence having at least 98% identity with the amino acid sequence of SEQ ID NO: 85.
38. The nucleic acid of claim 22 , wherein the spacer comprises the amino acid sequence of SEQ ID NO: 85.
39. The nucleic acid of claim 1 , wherein the transmembrane domain comprises an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 87.
40. The nucleic acid of claim 1 , wherein the transmembrane domain comprises an amino acid sequence having at least 98% identity with the amino acid sequence of SEQ ID NO: 87.
41. The nucleic acid of claim 1 , wherein the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 87.
42. The nucleic acid of claim 29 , wherein the CD28 co-stimulatory domain comprises an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 90.
43. The nucleic acid of claim 29 , wherein the CD28 co-stimulatory domain comprises an amino acid sequence having at least 98% identity with the amino acid sequence of SEQ ID NO: 90.
44. The nucleic acid of claim 29 , wherein the CD28 co-stimulatory domain comprises the amino acid sequence of SEQ ID NO: 90.
45. The vector of claim 8 , wherein the cell tag comprises a polypeptide having at least 95% identity with the amino acid sequence of SEQ ID NO: 110.
46. The vector of claim 8 , wherein the cell tag comprises a polypeptide having at least 98% identity with the amino acid sequence of SEQ ID NO: 110.
47. The vector of claim 8 , wherein the cell tag comprises the amino acid sequence of SEQ ID NO: 110.
48. The vector of claim 19 , wherein the fusion protein comprises an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 113.
49. The vector of claim 19 , wherein the fusion protein comprises an amino acid sequence having at least 98% identity with the amino acid sequence of SEQ ID NO: 113.
50. The nucleic acid of claim 1 , wherein the CAR comprises:
(a) a spacer comprising a polypeptide comprising an amino acid sequence having at least 95% identity with SEQ ID NO: 85;
(b) a transmembrane domain comprising a polypeptide comprising an amino acid sequence having at least 95% identity with SEQ ID NO: 87;
(c) a CD28 costimulatory signaling domain comprising an amino acid sequence having at least 95% identity with SEQ ID NO: 90; and
(d) a CD3 zeta signaling domain comprising an amino acid sequence having at least 95% identity with SEQ ID NO: 93.
51. The nucleic acid of claim 1 , wherein the CAR comprises:
(a) a spacer comprising a polypeptide comprising an amino acid sequence having at least 98% identity with SEQ ID NO: 85;
(b) a transmembrane domain comprising a polypeptide comprising an amino acid sequence having at least 98% identity with SEQ ID NO: 87;
(c) a CD28 costimulatory signaling domain comprising an amino acid sequence having at least 98% identity with SEQ ID NO: 90; and
(d) a CD3 zeta signaling domain comprising an amino acid sequence having at least 98% identity with SEQ ID NO: 93.
52. The nucleic acid of claim 1 , wherein the CAR comprises:
(a) a spacer comprising the amino acid sequence of SEQ ID NO: 85;
(b) a transmembrane domain comprising the amino acid sequence of SEQ ID NO: 87;
(c) a CD28 costimulatory signaling domain comprising the amino acid sequence of SEQ ID NO: 90; and
(d) a CD3 zeta signaling domain comprising the amino acid sequence of SEQ ID NO: 93.
53. The nucleic acid of claim 1 , wherein the CAR comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 79.
54. The nucleic acid of claim 1 , wherein the CAR comprises an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 79.
55. The nucleic acid of claim 1 , wherein the CAR comprises an amino acid sequence having at least 98% identity with the amino acid sequence of SEQ ID NO: 79.
56. The vector of claim 8 encoding: (a) a CAR comprising an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 79; (b) a cell tag comprising an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 110; and (c) a cytokine comprising an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 113.
57. The vector of claim 8 encoding: (a) a CAR comprising an amino acid sequence having at least 98% identity with the amino acid sequence of SEQ ID NO: 79; (b) a cell tag comprising an amino acid sequence having at least 98% identity with the amino acid sequence of SEQ ID NO: 110; and (c) a cytokine comprising an amino acid sequence having at least 98% identity with the amino acid sequence of SEQ ID NO: 113.
58. The vector of claim 8 encoding: (a) a CAR comprising the amino acid sequence of SEQ ID NO: 79; (b) a cell tag comprising the amino acid sequence of SEQ ID NO: 110; and (c) a cytokine comprising the amino acid sequence of SEQ ID NO: 113.