Regulating chimeric antigen receptors
This invention is in the area of compositions and methods for regulating chimeric antigen receptor immune effector cell, for example T-cell (CAR-T), therapy to modulate associated adverse inflammatory responses, for example, cytokine release syndrome and tumor lysis syndrome, using targeted protein degradation.
1. A T cell or natural killer (NK) cell comprising:
a. a chimeric antigen receptor polypeptide, wherein the chimeric antigen receptor polypeptide comprises:
i. an extracellular ligand binding domain comprising a single chain variable fragment (scFv) antibody that binds human CD19, BCMA, or CD38;
ii. a CD8, CD28, or CD137 transmembrane domain; and
iii. a cytoplasmic domain comprising at least one intracellular signaling domain, wherein the at least one intracellular signaling domain comprises CD3; and
b. a cytoplasmic costimulatory polypeptide, wherein the cytoplasmic costimulatory polypeptide comprises:
i. at least one intracellular signaling domain selected from a CD28, 4-1BB, OX40, CD27, ICOS, DAP-10, and DAP-12 signaling domain; and
ii. a heterobifunctional compound targeting protein, wherein the heterobifunctional compound targeting protein comprises the amino acid sequence selected from SEQ ID NO: 1 and 2 and wherein the heterobifunctional compound targeting protein binds to a heterobifunctional compound selected from the group consisting of dFKBP-1 to dFKBP-13;
wherein the binding of the heterobifunctional compound to the heterobifunctional compound targeting protein brings the cytoplasmic costimulatory polypeptide into proximity of a ubiquitin ligase;
and wherein the cytoplasmic costimulatory polypeptide is ubiquitinated and then degraded by a proteasome.
2. The T cell or natural killer (NK) cell of claim 1 , wherein the heterobifunctional compound targeting protein comprises the amino acid sequence of SEQ ID NO: 2.
3. The T cell or natural killer (NK) cell of claim 1 , wherein the T cell or natural killer (NK) cell is a T cell.
4. The T cell or natural killer (NK) cell of claim 1 , wherein the chimeric antigen receptor polypeptide further comprises a CD8α hinge domain.
5. The T cell or natural killer (NK) cell of claim 1 , wherein the at least one intracellular signaling domain of the cytoplasmic costimulatory polypeptide is CD28.
6. A therapeutic system, comprising:
a. the T cell or natural killer (NK) cell of claim 1 ; and
b. a heterobifunctional compound comprising any one of dFKBP-1 to dFKBP-13.
7. A method of reducing an adverse immune response in a subject caused by an activated T cell or natural killer (NK) cell that expresses a chimeric antigen receptor polypeptide and a cytoplasmic costimulatory polypeptide of claim 1 comprising:
administering to the subject experiencing an adverse immune response an effective amount of a heterobifunctional compound comprising any one of dFKBP-1 to dFKBP-13;
wherein the subject had previously been administered the T cell or natural killer (NK) cell of claim 1 .
8. A T cell or natural killer (NK) cell comprising a chimeric antigen receptor polypeptide, wherein the chimeric antigen receptor polypeptide comprises:
a. an extracellular ligand binding domain comprising a single chain variable fragment (scFv) antibody that binds human CD19, BCMA, or CD38;
b. a CD8, CD28, or CD137 transmembrane domain; and
c. a cytoplasmic domain, wherein the cytoplasmic domain comprises:
i. at least one intracellular signaling domain, wherein the at least one intracellular signaling domain comprises CD3; and
ii. a heterobifunctional compound targeting protein, wherein the heterobifunctional compound targeting protein comprises an amino acid sequence selected from SEQ ID NO: 1 and 2 and wherein the heterobifunctional compound targeting protein binds to a heterobifunctional compound;
wherein the binding of the heterobifunctional compound to the heterobifunctional compound targeting protein brings the chimeric antigen receptor polypeptide into proximity of a ubiquitin ligase;
and wherein the chimeric antigen receptor polypeptide is ubiquitinated and then degraded by a proteasome.
9. The T cell or natural killer (NK) cell of claim 8 , wherein the T cell or natural killer (NK) cell is a T cell.
10. The T cell or natural killer (NK) cell of claim 1 , wherein the chimeric antigen receptor polypeptide further comprises a CD8α hinge domain.
11. A therapeutic system for downregulating activation of a T cell or natural killer (NK) cell expressing a chimeric antigen receptor polypeptide, the system comprising:
a. the T cell or natural killer (NK) cell of claim 8 ; and
b. a heterobifunctional compound comprising any one of dFKBP-1 to dFKBP-13.
12. A method of reducing an adverse immune response in a subject caused by an activated T cell or natural killer (NK) cell that expresses a chimeric antigen receptor polypeptide of claim 8 comprising:
administering to the subject experiencing an adverse immune response an effective amount of a heterobifunctional compound comprising any one of dFKBP-1 to dFKBP-13;
wherein the subject had previously been administered the T cell or natural killer (NK) cell of claim 8 .
13. The T cell or natural killer (NK) cell of claim 1 , wherein the extracellular ligand binding domain binds CD19.
14. The T cell or natural killer (NK) cell of claim 8 , wherein the extracellular ligand binding domain binds CD19.
15. The T cell or natural killer (NK) cell of claim 1 , wherein the extracellular ligand binding domain that binds CD19 comprises scFv comprising the amino acid sequence of SEQ ID NO: 10 or 89.
16. The T cell or natural killer (NK) cell of claim 1 , wherein the extracellular ligand binding domain that binds BCMA comprises a variable light chain (VL) and a variable heavy chain (VH) wherein the VL comprises the amino acid sequence of SEQ ID NO: 68 or SEQ ID NO: 78 and wherein the VH comprises the amino acid sequence of SEQ ID NO: 70 or SEQ ID NO: 79.
17. The T cell or natural killer (NK) cell of claim 1 , wherein the extracellular ligand binding domain that binds CD38 comprises a variable light chain (VL) and a variable heavy chain (VH) wherein the VL comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 86 and the VH comprises the amino acid sequence of SEQ ID NO: 82 or SEQ ID NO: 87.
18. The T cell or natural killer (NK) cell of claim 1 , wherein the CD28 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 16.
19. The T cell or natural killer (NK) cell of claim 1 , wherein the CD8 transmembrane domain comprises a CD8 alpha hinge comprising the amino acid sequence of SEQ ID NO: 15.
20. The T cell or natural killer (NK) cell of claim 1 , wherein the CD3 intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 17.
21. The T cell or natural killer (NK) cell of claim 8 , wherein the extracellular ligand binding domain that binds CD19 comprises scFv comprising the amino acid sequence of SEQ ID NO: 10.
22. The T cell or natural killer (NK) cell of claim 8 , wherein the extracellular ligand binding domain that binds BCMA comprises a variable light chain (VL) and a variable heavy chain (VH) wherein the VL comprises the amino acid sequence of SEQ ID NO: 68 or SEQ ID NO: 78 and the VH comprises the amino acid sequence of SEQ ID NO: 70 or SEQ ID NO: 79.
23. The T cell or natural killer (NK) cell of claim 8 , wherein the extracellular ligand binding domain that binds CD38 comprises a variable light chain (VL) and a variable heavy chain (VH) wherein the VL comprises the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 86 and the VH comprises the amino acid sequence of SEQ ID NO: 82 or SEQ ID NO: 87.
24. The T cell or natural killer (NK) cell of claim 8 , wherein the CD28 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 16.
25. The T cell or natural killer (NK) cell of claim 8 , wherein the CD8 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 15.
26. The T cell or natural killer (NK) cell of claim 8 , wherein the CD3 intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 17.
27. The T cell or natural killer (NK) cell of claim 8 , wherein the chimeric antigen receptor polypeptide further comprises a CD8α hinge domain.
28. The T cell or natural killer (NK) cell of claim 1 , wherein the extracellular ligand binding domain that binds CD19 comprises a variable light chain (VL) and a variable heavy chain (VH) wherein the VL comprises the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 89 and the VH comprises the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 90.
29. The T cell or natural killer (NK) cell of claim 8 , wherein the extracellular ligand binding domain that binds CD19 comprises a variable light chain (VL) and a variable heavy chain (VH) wherein the VL comprises the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 89 and the VH comprises the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 90.