Polynucleotides encoding rituximab-resistant chimeric antigen receptors
Provided herein are polynucleotides encoding chimeric antigen receptors (CARs) comprising a CD19 antigen binding domain that specifically binds to CD19 and is resistant to rituximab binding; and immune cells comprising these CD19-specific CARs, e.g., CAR-T cells. Also provided are methods of making and using these CD19-specific CARs, and immune cells comprising these CD19-specific CARs.
1. An isolated polynucleotide encoding a polypeptide comprising an anti-CD19 chimeric antigen receptor (CAR) that comprises the amino acid sequence of SEQ ID NO: 9, wherein the polypeptide does not comprise a rituximab binding site, wherein the polynucleotide comprises a nucleic acid sequence having at least 90% identity to SEQ ID NO: 3 and a short EF1a promoter that is capable of expressing the anti-CD19 chimeric antigen receptor (CAR) in a mammalian T cell, and wherein the short EF1a promoter comprises the nucleic acid sequence of SEQ ID NO:16 and does not comprise the nucleic acid sequence of SEQ ID NO:39.
2. The isolated polynucleotide of claim 1 , wherein the polypeptide further comprises a safety switch.
3. The isolated polynucleotide of claim 2 , wherein the safety switch is linked to the CD19 CAR using a linker peptide.
4. The isolated polynucleotide of claim 2 , wherein the safety switch is linked to the anti-CD19 CAR using a T2A linker.
5. The isolated polynucleotide of claim 2 , wherein the safety switch comprises an antibody binding site.
6. The isolated polynucleotide of claim 2 , wherein the polypeptide comprises a CD34 epitope.
7. The isolated polynucleotide of claim 6 , wherein the CD34 epitope is a QBEND-10 epitope.
8. The isolated polynucleotide of claim 1 , wherein the polypeptide further comprises a CD8 hinge/transmembrane domain.
9. The isolated polynucleotide of claim 1 , wherein the polynucleotide encodes a polypeptide that comprises any one of the amino acid sequences of SEQ ID NOs: 11-14.
10. A vector comprising the isolated polynucleotide of claim 1 .
11. The vector of claim 10 , wherein the vector is a retroviral vector, a DNA vector, a plasmid, an RNA vector, an adenoviral vector, an adenovirus associated vector, a lentiviral vector, or any combination thereof.
12. An engineered immune cell comprising the isolated polynucleotide of claim 1 , wherein the engineered immune cell does not express a rituximab binding site.
13. An engineered immune cell comprising the vector of claim 10 , wherein the engineered immune cell does not express a rituximab binding site.
14. The engineered immune cell of claim 12 , wherein the immune cell is a T cell, tumor infiltrating lymphocyte (TIL), NK cell, TCR-expressing cell, dendritic cell, or NK-T cell.
15. The engineered immune cell of claim 14 , wherein the cell is an autologous T cell.
16. The engineered immune cell of claim 14 , wherein the cell is an allogeneic T cell.
17. The engineered immune cell of claim 12 , wherein the cell is resistant to rituximab.
18. A pharmaceutical composition comprising the engineered immune cell of claim 12 and a pharmaceutically acceptable excipient.
19. The isolated polynucleotide of claim 1 , wherein the polynucleotide comprises a nucleic acid sequence having at least 95% identity to SEQ ID NO: 3.
20. The isolated polynucleotide of claim 1 , wherein the polynucleotide comprises a nucleic acid sequence having at least 96% identity to SEQ ID NO: 3.
21. The isolated polynucleotide of claim 1 , wherein the polynucleotide comprises a nucleic acid sequence having at least 98% identity to SEQ ID NO: 3.
22. The isolated polynucleotide of claim 1 , wherein the polynucleotide comprises a nucleic acid sequence having at least 99% identity to SEQ ID NO: 3.
23. The isolated polynucleotide of claim 1 , wherein the polynucleotide comprises the nucleic acid sequence shown in SEQ ID NO: 3.