Chimeric antigen receptors containing a chlorotoxin domain
Chimeric transmembrane immunoreceptors (CAR) which include an extracellular domain that includes chlorotoxin or a related toxin, or a variant of chlorotoxin or a related toxin, that binds to human glioma or other human tumor cells, a transmembrane region, a costimulatory domain and an intracellular signaling domain are described.
1. A nucleic acid molecule encoding a chimeric antigen receptor, wherein the chimeric antigen receptor comprises:
(i) chlorotoxin;
(ii) a transmembrane domain selected from the group consisting of:
a CD4 transmembrane domain,
a CD8 transmembrane domain,
a CD28 transmembrane domain, and
a CD3ζ transmembrane domain;
(iii) a costimulatory domain; and
(iv) CD3ζ signaling domain.
2. The nucleic acid molecule of claim 1 further comprising a spacer region located between the chlorotoxin and the transmembrane domain.
3. The nucleic acid molecule of claim 2 , wherein the spacer region comprises 5-300 amino acids.
4. The nucleic acid molecule of claim 2 , wherein the spacer region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-12.
5. The nucleic acid molecule of claim 2 , wherein the spacer comprises an IgG hinge region.
6. A nucleic acid molecule encoding a chimeric antigen receptor comprising an amino acid sequence selected from the group consisting of: an amino acid sequence that is identical to SEQ ID NO: 26 or differs from SEQ ID NO: 26 by no more than 5 single amino acid substitutions; an amino acid sequence that is identical to SEQ ID NO: 27 or differs from SEQ ID NO: 27 by no more than 5 single amino acid substitutions; an amino acid sequence that is identical to SEQ ID NO: 28 or differs from SEQ ID NO: 28 by no more than 5 single amino acid substitutions; an amino acid sequence that is identical to SEQ ID NO: 29 or differs from SEQ ID NO: 29 no more than 5 single amino acid substitutions; an amino acid sequence that is identical to SEQ ID NO: 30 or differs from SEQ ID NO: 30 by no more than 5 single amino acid substitutions; an amino acid sequence that is identical to SEQ ID NO: 31 or differs from SEQ ID NO: 31 by no more than 5 single amino acid substitutions; an amino acid sequence that is identical to SEQ ID NO: 32 or differs from SEQ ID NO: 32 by no more than 5 single amino acid substitutions; an amino acid sequence that is identical to SEQ ID NO: 33 or differs from SEQ ID NO: 33 by no more than 5 single amino acid substitutions; an amino acid sequence that is identical to SEQ ID NO: 34 or differs from SEQ ID NO: 34 by no more than 5 single amino acid substitutions; an amino acid sequence that is identical to SEQ ID NO: 35 or differs from SEQ ID NO: 35 by no more than 5 single amino acid substitutions; an amino acid sequence that is identical to SEQ ID NO: 36 or differs from SEQ ID NO: 36 by no more than 5 single amino acid substitutions; an amino acid sequence that is identical to SEQ ID NO: 37 or differs from SEQ ID NO: 37 by no more than 5 single amino acid substitutions; an amino acid sequence that is identical to SEQ ID NO: 38 or differs from SEQ ID NO: 38 by no more than 5 single amino acid substitutions; an amino acid sequence that is identical to SEQ ID NO: 39 or differs from SEQ ID NO: 39 by no more than 5 single amino acid substitutions; and an amino acid sequence that is identical to SEQ ID NO: 40 or differs from SEQ ID NO: 40 by no more than 5 single amino acid substitutions.
7. The nucleic acid molecule of claim 6 , wherein the chimeric antigen receptor comprises an amino acid sequence that is identical to SEQ ID NO: 27 or differs from SEQ ID NO: 27 by no more than 5 single amino acid substitutions.
8. The nucleic acid molecule of claim 6 , wherein the chimeric antigen receptor comprises an amino acid sequence that is identical to SEQ ID NO: 28 or differs from SEQ ID NO: 28 by no more than 5 single amino acid substitutions.
9. The nucleic acid molecule of claim 6 , wherein the chimeric antigen receptor comprises an amino acid sequence that is identical to SEQ ID NO: 29 or differs from SEQ ID NO: 29 by no more than 5 single amino acid substitutions.
10. The nucleic acid molecule of claim 6 , wherein the chimeric antigen receptor comprises an amino acid sequence that is identical to SEQ ID NO: 30 or differs from SEQ ID NO: 30 by no more than 5 single amino acid substitutions.
11. The nucleic acid molecule of claim 6 , wherein the chimeric antigen receptor comprises an amino acid sequence that is identical to SEQ ID NO: 31 or differs from SEQ ID NO: 31 by no more than 5 single amino acid substitutions.
12. The nucleic acid molecule of claim 6 , wherein the chimeric antigen receptor comprises an amino acid sequence that is identical to SEQ ID NO: 32 or differs from SEQ ID NO: 32 by no more than 5 single amino acid substitutions.
13. The nucleic acid molecule of claim 6 , wherein the chimeric antigen receptor comprises an amino acid sequence that is identical to SEQ ID NO: 33 or differs from SEQ ID NO: 33 by no more than 5 single amino acid substitutions.
14. The nucleic acid molecule of claim 6 , wherein the chimeric antigen receptor comprises an amino acid sequence that is identical to SEQ ID NO: 34 or differs from SEQ ID NO: 34 by no more than 5 single amino acid substitutions.
15. The nucleic acid molecule of claim 6 , wherein the chimeric antigen receptor comprises an amino acid sequence that is identical to SEQ ID NO: 35 or differs from SEQ ID NO: 35 by no more than 5 single amino acid substitutions.
16. The nucleic acid molecule of claim 6 , wherein the chimeric antigen receptor comprises an amino acid sequence that is identical to SEQ ID NO: 36 or differs from SEQ ID NO: 36 by no more than 5 single amino acid substitutions.
17. The nucleic acid molecule of claim 6 , wherein the chimeric antigen receptor comprises an amino acid sequence that is identical to SEQ ID NO: 37 or differs from SEQ ID NO: 37 by no more than 5 single amino acid substitutions.
18. The nucleic acid molecule of claim 6 , wherein the chimeric antigen receptor comprises an amino acid sequence that is identical to SEQ ID NO: 38 or differs from SEQ ID NO: 38 by no more than 5 single amino acid substitutions.
19. The nucleic acid molecule of claim 6 , wherein the chimeric antigen receptor comprises an amino acid sequence that is identical to SEQ ID NO: 39 or differs from SEQ ID NO: 39 by no more than 5 single amino acid substitutions.
20. The nucleic acid molecule of claim 6 , wherein the chimeric antigen receptor comprises an amino acid sequence that is identical to SEQ ID NO: 40 or differs from SEQ ID NO: 40 by no more than 5 single amino acid substitutions.
21. The nucleic acid molecule of claim 6 , wherein the chimeric antigen receptor comprises an amino acid sequence that is identical to SEQ ID NO: 26 or differs from SEQ ID NO:26 by no more than 5 single amino acid substitutions.
22. The nucleic acid molecule of claim 6 , wherein the single amino acid substitutions are conservative substitutions.
23. The nucleic acid molecule of claim 1 , wherein the chimeric antigen receptor comprises an amino acid sequence selected from SEQ ID NOs: 26-55.
24. The nucleic acid molecule of claim 1 , wherein the chimeric antigen receptor consists of an amino acid sequence selected from SEQ ID NOs: 26-55.
25. A vector comprising the nucleic acid molecule of claim 6 .
26. A vector comprising an expression cassette comprising the nucleic acid molecule of claim 6 .
27. A population of T cells or NK cells comprising the nucleic acid molecule claim 6 .
28. A population of T cells or NK cells comprising the vector of claim 25 .
29. A nucleic acid molecule encoding a chimeric antigen receptor, wherein the chimeric antigen receptor comprises: chlorotoxin comprising SEQ ID NO: 1; a spacer comprising a sequence selected from the group consisting of: SEQ ID NOs: 2-12; a transmembrane domain comprising a sequence selected from the group consisting of SEQ ID NOs: 13-20; a costimulatory domain comprising a sequence selected from the group consisting of SEQ ID NOs: 21-25, and a CD3ζ signaling domain comprising SEQ ID NO: 21.
30. The nucleic acid molecule of claim 29 , comprising a spacer comprising a sequence selected from the group consisting of: SEQ ID NOs: 2, 3, 5, 8, and 9-12; a transmembrane domain comprising a sequence selected from the group consisting of SEQ ID NOs: 15 and 16; a costimulatory domain comprising a sequence selected from the group consisting of SEQ ID NOs: 22-24, and a CD3ζ signaling domain comprising SEQ ID NO: 21.
31. The nucleic acid molecule of claim 29 , comprising a first costimulatory domain comprising SEQ ID NO: 22 or 23, and further comprising a second costimulatory domain comprising SEQ ID NO: 24.
32. The nucleic acid molecule of any of claims 29 - 31 , further comprising the sequence GGG between the costimulatory domain and the CD3ζ signaling domain.
33. The nucleic acid molecule of claim 31 , further comprising the sequence GGG between the first and second costimulatory domains.
34. A vector comprising the nucleic acid molecule of any of claims 29 - 31 and 33 .
35. A vector comprising the nucleic acid molecule of claim 32 .
36. A vector comprising an expression cassette comprising the nucleic acid molecule of any of claims 29 - 31 and 33 .
37. A vector comprising an expression cassette comprising the nucleic acid molecule of claim 32 .
38. A population of T cells or NK cells comprising the nucleic acid molecule of any of claims 29 - 31 and 33 .
39. A population of T cells or NK cells comprising the nucleic acid molecule of claim 32 .
40. A population of T cells or NK cells comprising the vector of claim 34 .
41. A population of T cells or NK cells comprising the vector of claim 35 .