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 polynucleotide encoding a CD19-directed chimeric antigen receptor, the chimeric antigen receptor comprising:
an extracellular anti-CD19 binding moiety comprising a single chain Fragment variable (scFv), wherein the scFv comprises:
a variable heavy (VH) domain, wherein the encoded VH domain comprises the amino acid sequence of SEQ ID NO: 120, and
a variable light (VL) domain;
a hinge, wherein the hinge is a CD8 alpha hinge;
a transmembrane domain, wherein the transmembrane domain comprises a CD8 alpha transmembrane domain; and
an intracellular signaling domain, wherein the intracellular signaling domain comprises:
an OX40 subdomain, wherein the OX40 subdomain is encoded by a sequence having at least 95% sequence identity to SEQ ID NO. 5,
a CD3 zeta subdomain, wherein the CD3 zeta subdomain is encoded by a sequence having at least 95% sequence identity to SEQ ID NO. 7, and
wherein the polynucleotide also encodes membrane-bound interleukin-15 (mbIL15), wherein the mbIL15 comprises a sequence having at least 95% sequence identity to SEQ ID NO. 12.
2. The polynucleotide of claim 1 , wherein the OX40 subdomain is encoded by SEQ ID NO. 5, and wherein the CD3 zeta subdomain is encoded by SEQ ID NO. 7.
3. The polynucleotide of claim 1 , wherein the encoded OX40 subdomain comprises the amino acid sequence of SEQ ID NO: 6.
4. The polynucleotide of claim 1 , wherein the encoded CD3 zeta subdomain comprises the amino acid sequence of SEQ ID NO: 8.
5. A polynucleotide encoding a CD19-directed chimeric
antigen receptor, the chimeric antigen receptor comprising:
an extracellular anti-CD19 binding moiety comprising a single chain Fragment variable (scFv), wherein the scFv comprises:
a variable heavy (VH) domain,
wherein the encoded VH domain comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 120; and
a variable light (VL) domain,
wherein the encoded VL domain comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 118;
a hinge;
a transmembrane domain; and
an intracellular signaling domain, wherein the intracellular signaling domain comprises:
an OX40 subdomain,
a CD3 zeta subdomain; and
wherein the polynucleotide also encodes membrane-bound interleukin-15 (mbIL15), wherein the mbIL15 comprises a sequence having at least 95% sequence identity to SEQ ID NO. 12.
6. The polynucleotide of claim 5 , wherein the OX40 subdomain is encoded by a sequence having at least 95% sequence identity to SEQ ID NO. 5.
7. The polynucleotide of claim 5 , wherein the CD3 zeta subdomain is encoded by a sequence having at least 95% sequence identity to SEQ ID NO. 7.
8. The polynucleotide of claim 5 , wherein the polynucleotide does not encode SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, or SEQ ID NO: 116.
9. The polynucleotide of claim 5 , wherein the encoded VH domain comprises the amino acid sequence of SEQ ID NO: 120.
10. The polynucleotide of claim 5 , wherein the encoded VL domain comprises the amino acid sequence of SEQ ID NO: 118.
11. A polynucleotide encoding a CD 19 -directed chimeric antigen receptor, the chimeric antigen receptor comprising:
an extracellular anti-CD19 binding moiety comprising a single chain Fragment variable (scFv), wherein the scFv comprises:
a variable heavy (VH) domain, and
a variable light (VL) domain, wherein the encoded VL domain comprises
the amino acid sequence of SEQ ID NO: 118;
a hinge, wherein the hinge is a CD8 alpha hinge;
a transmembrane domain, wherein the transmembrane domain comprises a CD8 alpha transmembrane domain; and
an intracellular signaling domain, wherein the intracellular signaling domain comprises:
an OX40 subdomain, wherein the OX 40 subdomain is encoded by a sequence having at least 95% sequence identity to SEQ ID NO. 5,
a CD3 zeta subdomain, wherein the CD3 zeta subdomain is encoded by a sequence having at least 95% sequence identity to SEQ ID NO. 7, and
wherein the polynucleotide also encodes membrane-bound interleukin-15 (mbIL15), wherein the mbIL15 comprises a sequence having at least 95% sequence identity to SEQ ID NO. 12.
12. The polynucleotide of claim 11 , wherein the OX40 subdomain is encoded by SEQ ID NO. 5, and wherein the CD3 zeta subdomain is encoded by SEQ ID NO. 7.
13. The polynucleotide of claim 11 , wherein the encoded OX40 subdomain comprises the amino acid sequence of SEQ ID NO: 6 and wherein the encoded CD3 zeta subdomain comprises the amino acid sequence of SEQ ID NO: 8.
14. A polynucleotide encoding a CD19-directed chimeric antigen receptor, the chimeric antigen receptor comprising:
an extracellular anti-CD19 binding moiety comprising a single chain Fragment variable (scFv), wherein the scFv comprises:
a variable heavy (VH) domain, and
a variable light (VL) domain;
a hinge, wherein the hinge is a CD8 alpha hinge;
a transmembrane domain, wherein the transmembrane domain comprises a CD8 alpha transmembrane domain; and
an intracellular signaling domain, wherein the intracellular signaling domain comprises:
an OX40 subdomain, wherein the OX40 subdomain is encoded by a sequence having at least 95% sequence identity to SEQ ID NO. 5,
a CD3 zeta subdomain, wherein the CD3 zeta subdomain is encoded by a sequence having at least 95% sequence identity to SEQ ID NO. 7, and
wherein the polynucleotide also encodes membrane-bound interleukin-15 (mbIL15), wherein the comprises a sequence having at least 95% sequence identity to SEQ ID NO. 12, and
wherein the encoded CD19-directed chimeric antigen receptor has at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 187.
15. The polynucleotide of claim 14 , wherein the OX40 subdomain is encoded by SEQ ID NO. 5, and wherein the CD3 zeta subdomain is encoded by SEQ ID NO. 7.
16. The polynucleotide of claim 14 , wherein the encoded OX40 subdomain comprises the amino acid sequence of SEQ ID NO: 6 and wherein the encoded CD 3 zeta subdomain comprises the amino acid sequence of SEQ ID NO: 8.
17. A polynucleotide encoding a CD19-directed chimeric antigen receptor, the chimeric antigen receptor comprising:
an extracellular anti-CD19 binding moiety comprising a single chain Fragment variable (scFv), wherein the scFv comprises:
a variable heavy (VH) domain, and
a variable light (VL) domain;
a hinge, wherein the hinge is a CD8 alpha hinge;
a transmembrane domain, wherein the transmembrane domain comprises a CD8 alpha transmembrane domain; and
an intracellular signaling domain, wherein the intracellular signaling domain comprises:
an OX40 subdomain, wherein the OX40 subdomain is encoded by a sequence having at least 95% sequence identity to SEQ ID NO. 5,
a CD3 zeta subdomain, wherein the CD3 zeta subdomain is encoded by a sequence having at least 95% sequence identity to SEQ ID NO. 7, and
wherein the polynucleotide also encodes membrane-bound interleukin-15 (mbIL15), wherein the mbIL15 comprises a sequence having at least 95% sequence identity to SEQ ID NO. 12, and
wherein the encoded CD19-directed chimeric antigen receptor comprises the amino acid sequence set forth in SEQ ID NO: 187.
18. The polynucleotide of claim 17 , wherein the OX40 subdomain is encoded by SEQ ID NO. 5, and wherein the CD3 zeta subdomain is encoded by SEQ ID NO. 7.
19. The polynucleotide of claim 17 , wherein the encoded OX40 subdomain comprises the amino acid sequence of SEQ ID NO: 6 and wherein the encoded CD3 zeta subdomain comprises the amino acid sequence of SEQ ID NO: 8.