CD19-directed chimeric antigen receptors and uses thereof in immunotherapy
Provided for herein in several embodiments are immune cell-based (e.g., natural killer (NK) cell) compositions comprising CD19-directed chimeric antigen receptors. In some, embodiments the anti-CD19 binder portion of the CAR is humanized. In several embodiments, the humanized anti-CD19 CAR expressing cells exhibit enhanced expression of the CAR as well as enhanced cytotoxicity and/or persistence. Several embodiments include methods of using of the anti-CD19 CAR expressing immune cells in immunotherapy.
1. A polypeptide comprising a CD19-directed chimeric antigen receptor (CAR), the CAR comprising:
an extracellular anti-CD19 binding moiety comprising a single-chain variable fragment (scFv), wherein the scFv comprises a variable heavy (VH) domain and a variable light (VL) domain,
wherein the VH domain comprises the CDR-H1, CDR-H2, and CDR-H3 of the VH domain set forth in SEQ ID NO: 120, and
wherein the VL domain comprises the CDR-L1, CDR-L2, and CDR-L3 of the VL domain set forth in SEQ ID NO: 118;
a CD8 alpha hinge domain;
a CD8 alpha transmembrane domain; and
an intracellular signaling domain comprising an OX40 subdomain and a CD3zeta subdomain.
2. The polypeptide of claim 1 , wherein the OX40 subdomain comprises the amino acid sequence set forth in SEQ ID NO:6.
3. The polypeptide of claim 1 , wherein the CD3zeta subdomain comprises the amino acid sequence set forth in SEQ ID NO:8.
4. The polypeptide of claim 1 , wherein the polypeptide is encoded by a polynucleotide that also encodes a membrane-bound interleukin-15 (mbIL15).
5. The polypeptide of claim 4 , wherein the mbIL15 comprises the amino acid sequence set forth in SEQ ID NO: 12.
6. An immune cell that expresses the polypeptide of claim 1 .
7. The immune cell of claim 6 , wherein the immune cell also expresses a membrane-bound interleukin-15 (mbIL15).
8. The immune cell of claim 6 , wherein the immune cell is a natural killer (NK) or a T cell.
9. A method of obtaining an immune cell that expresses a CD19-directed chimeric antigen receptor, the method comprising:
transfecting an immune cell with a polynucleotide encoding a CD19-directed chimeric antigen receptor (CAR), the CAR comprising:
an extracellular anti-CD19 binding moiety comprising a variable heavy (VH) domain, and a variable light (VL) domain, wherein the VH domain comprises the CDR-H1, CDR-H2, and CDR-H3 of the VH domain set forth in SEQ ID NO: 120 and the VL domain comprises the CDR-L1, CDR-L2, and CDR-L3 of the VL domain set forth in SEQ ID NO: 118;
a hinge domain;
a CD8 alpha transmembrane domain; and
an intracellular signaling domain comprising an OX40 subdomain and a CD3 zeta subdomain.
10. The method of claim 9 , wherein the immune cell is a natural killer (NK) cell or a T cell.
11. The polypeptide of claim 1 , wherein the VH domain comprises the amino acid sequence set forth in SEQ ID NO: 120.
12. The polypeptide of claim 1 , wherein the VL domain comprises the amino acid sequence set forth in SEQ ID NO: 118.
13. The polypeptide of claim 1 , wherein the VH domain comprises the amino acid sequence set forth in SEQ ID NO: 120, and the VL domain comprises the amino acid sequence set forth in SEQ ID NO: 118.
14. The immune cell of claim 6 , wherein the immune cell is a natural killer (NK) cell.
15. The immune cell of claim 14 , wherein the immune cell also expresses a membrane-bound interleukin-15 (mbIL15).
16. The immune cell of claim 6 , wherein the immune cell is a T cell.
17. The immune cell of claim 16 , wherein the immune cell also expresses a membrane-bound interleukin-15 (mbIL15).
18. A polypeptide comprising a CD19-directed chimeric antigen receptor (CAR), the CAR comprising:
an extracellular anti-CD19 binding moiety comprising a single-chain variable fragment (scFv), wherein the scFv comprises a variable heavy (VH) domain and a variable light (VL) domain,
wherein the VH domain comprises the amino acid sequence set forth in SEQ ID NO: 120, and
wherein the VL domain comprises the amino acid sequence set forth in SEQ ID NO: 118;
a CD8alpha transmembrane domain; and
an intracellular signaling domain comprising an OX40 subdomain and a CD3zeta subdomain.
19. The polypeptide of claim 18 , wherein the OX40 subdomain comprises the amino acid sequence set forth in SEQ ID NO: 6.
20. An immune cell expressing the polypeptide of claim 18 .
21. The immune cell of claim 20 , wherein the immune cell is a natural killer (NK) cell.
22. The immune cell of claim 20 , wherein the immune cell also expresses a membrane-bound interleukin-15 (IL-15).
23. An immune cell expressing:
(a) a CD19-directed chimeric antigen receptor (CAR), the CAR comprising:
an extracellular anti-CD19 binding moiety comprising a single-chain variable fragment (scFv), wherein the scFv comprises a variable heavy (VH) domain and a variable light (VL) domain,
wherein the VH domain comprises the CDR-H1, CDR-H2, and CDR-H3 of the VH domain set forth in SEQ ID NO: 120, and
wherein the VL domain comprises the CDR-L1, CDR-L2, and CDR-L3 of the VL domain set forth in SEQ ID NO: 118;
a transmembrane domain; and
an intracellular signaling domain comprising an OX40 subdomain and a CD3zeta subdomain; and
(b) a membrane-bound interleukin-15 (mbIL15).
24. The immune cell of claim 23 , wherein the VH domain comprises the amino acid sequence set forth in SEQ ID NO: 120, and the VL domain comprises the amino acid sequence set forth in SEQ ID NO: 118.