Compositions and methods for treating cancer with anti-ROR1 immunotherapy
Chimeric antigen receptors containing ROR1 antigen binding domains are disclosed. Nucleic acids, recombinant expression vectors, host cells, antigen binding fragments, and pharmaceutical compositions, relating to the chimeric antigen receptors are also disclosed. Methods of treating or preventing cancer in a subject, and methods of making chimeric antigen receptor T cells are also disclosed.
1. An isolated chimeric antigen receptor (CAR) comprising at least one extracellular antigen binding domain comprising a scFv ROR1 antigen binding domain comprising the amino acid sequence of SEQ ID NO: 2 or 8, a transmembrane domain comprising a CD8 transmembrane domain, an intracellular signaling domain comprising a functional signaling domain of 4-1BB and a CD3 zeta intracellular domain.
2. The isolated CAR of claim 1 , wherein the at least one extracellular antigen binding domain comprising the scFv ROR1 antigen binding domain is encoded by the nucleotide sequence comprising SEQ ID NO: 1 or SEQ ID NO: 7.
3. The isolated CAR of claim 2 , wherein the CD8 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 22, or an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 22.
4. The isolated CAR of claim 2 , wherein the at least one extracellular antigen binding domain and the intracellular signaling domain, or both are connected to the transmembrane domain by a linker or a spacer domain.
5. The isolated CAR of claim 4 , wherein the linker or the spacer domain is obtained from the extracellular domain of IgG4, CD8 or CD28, and is linked to the transmembrane domain.
6. The isolated CAR of claim 1 , wherein the CAR comprises the amino acid sequence of SEQ ID NO: 4, 6, 10, or 12.
7. A method of making a cell comprising transducing a T cell with a vector comprising a nucleic acid molecule encoding a CAR comprising at least one extracellular antigen binding domain comprising a scFv ROR1 antigen binding domain comprising the amino acid sequence of SEQ ID NO: 2, or 8, a transmembrane domain comprising a CD8 transmembrane domain, an intracellular signaling domain comprising a functional signaling domain of 4-1BB and a CD3 zeta intracellular domain.
8. The method of claim 7 , wherein the at least one extracellular antigen binding domain comprising the scFv ROR1 antigen binding domain is encoded by the nucleotide sequence comprising SEQ ID NO: 1 or SEQ ID NO: 7.
9. The method of claim 7 , wherein the CD8 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 22, or an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 22.
10. The method of claim 7 , wherein the at least one extracellular antigen binding domain and the intracellular signaling domain, or both are connected to the transmembrane domain by a linker or spacer domain.
11. The method of claim 10 , wherein the linker or the spacer domain is obtained from the extracellular domain of IgG4, CD8 or CD28, and is linked to the transmembrane domain.
12. The method of claim 7 , wherein the CAR comprises the amino acid sequence of SEQ ID NO: 4, 6, 10, or 12.
13. A method of generating a population of RNA-engineered cells comprising introducing an in vitro transcribed RNA or synthetic RNA into a cell, wherein the RNA comprises a nucleic acid molecule encoding a CAR comprising at least one extracellular antigen binding domain comprising a scFv ROR1 antigen binding domain comprising the amino acid sequence of SEQ ID NO: 2, or 8, a transmembrane domain comprising a CD8 transmembrane domain, an intracellular signaling domain comprising a functional signaling domain of 4-1BB and a CD3 zeta intracellular domain.
14. The method of claim 13 , wherein the at least one extracellular antigen binding domain comprising the scFv ROR1 antigen binding domain is encoded by a nucleotide sequence comprising the RNA complement of SEQ ID NO: 1 or SEQ ID NO: 7.
15. The method of claim 13 , wherein the CD8 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 22, or an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 22.
16. The method of claim 13 , wherein the at least one extracellular antigen binding domain and the at least one intracellular signaling domain, or both are connected to the transmembrane domain by a linker or a spacer domain.
17. The method of claim 16 , wherein the linker or the spacer domain is obtained from the extracellular domain of IgG4, CD8 or CD28, and is linked to the transmembrane domain.
18. The method of claim 13 , wherein the CAR comprises the amino acid sequence of SEQ ID NO: 4, 6, 10, or 12.
19. An isolated CAR comprising the amino acid sequence of SEQ ID NO: 4, 6, 10, or 12.