Treatment of cancer using GFR α-4 chimeric antigen receptor
The present invention relates to compositions and methods for treating diseases, disorders or conditions associated with the expression of the Glycosyl-phosphatidylinositol (GPI)-linked GDNF family α-receptor 4 (GFRα4).
1. A nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the antigen binding domain comprises:
a heavy chain variable region having heavy chain complementarity determining regions (HC CDRs) 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 63, 65, and 67; and
a light chain variable region having light chain complementarity determining regions (LC CDRs) 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 71, 73, and 75,
wherein the antigen binding domain binds to isoform a or isoform b of human Glycosyl-phosphatidylinositol (GPI)-linked GDNF family α-receptor 4 (GFRα4) cell-surface receptor.
2. The nucleic acid molecule of claim 1 , wherein the antigen binding domain comprises an antibody or an antigen-binding fragment thereof.
3. The nucleic acid molecule of claim 2 , wherein the antigen-binding fragment is selected from the group consisting of a Fab or a single-chain variable fragment (scFv).
4. The nucleic acid molecule of claim 2 , wherein the antibody or antigen-binding fragment is a humanized antibody or a fragment thereof.
5. The nucleic acid molecule of claim 1 , wherein the antigen binding domain comprises:
(i) the amino acid sequence set forth in SEQ ID NO: 69;
(ii) an amino acid sequence having at least 1, 2, or 3 modifications but not more than 20 modifications of the amino acid sequence set forth in SEQ ID NO: 69; or
(iii) an amino acid sequence with 95-99% identity to the amino acid sequence set forth in SEQ ID NO: 69.
6. The nucleic acid molecule of claim 1 , wherein the antigen binding domain comprises:
(i) the amino acid sequence set forth in SEQ ID NO: 61;
(ii) an amino acid sequence having at least 1, 2, or 3 modifications but not more than 20 modifications of the amino acid sequence set forth in SEQ ID NO: 61; or
(iii) an amino acid sequence with 95-99% identity to the amino acid sequence set forth in SEQ ID NO: 61.
7. The nucleic acid molecule of claim 1 , wherein the antigen binding domain comprises the amino acid sequence of the light chain variable region set forth in SEQ ID NO: 69, and the amino acid sequence of the heavy chain variable region set forth in SEQ ID NO: 61.
8. The nucleic acid molecule of claim 1 , wherein the antigen binding domain comprises:
(i) an amino acid sequence selected from SEQ ID NO: 78 or SEQ ID NO: 79;
(ii) an amino acid sequence having at least 1, 2, or 3 modifications but not more than 30 modifications to SEQ ID NO: 78 or SEQ ID NO: 79; or
(iii) an amino acid sequence with 95-99% identity to SEQ ID NO: 78 or SEQ ID NO: 79.
9. The nucleic acid molecule of claim 1 , wherein the nucleic acid sequence of the antigen binding domain comprises:
(i) a nucleotide sequence selected form SEQ ID NO: 76 or SEQ ID NO: 77, or
(ii) a nucleotide sequence with 95-99% identity to a nucleotide sequence selected from SEQ ID NO: 76 or SEQ ID NO: 77.
10. The nucleic acid molecule of claim 1 , wherein the transmembrane domain is selected from the group consisting of the alpha, beta, or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.
11. The nucleic acid molecule of claim 1 , wherein the transmembrane domain comprises:
(i) the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence that comprises at least 1, 2, or 3 but not more than 5 modifications of the amino acid sequence of SEQ ID NO: 6, or a sequence with 95-99% identity to the amino acid sequence of SEQ ID NO: 6; or
(ii) is encoded by the nucleotide sequence of SEQ ID NO: 17, or a sequence with 95-99% identity to the nucleotide sequence of SEQ ID NO: 17.
12. The nucleic acid molecule of claim 1 , wherein the CAR further comprises a hinge region.
13. The nucleic acid molecule of claim 12 , wherein the hinge region comprises:
(i) the amino acid sequence of SEQ ID NO: 2, or a sequence with 95-99% identity to the amino acid sequence of SEQ ID NO: 2; or
(ii) is encoded by a nucleic acid sequence comprising the nucleotide sequence of SEQ ID NO: 13, or a sequence with 95-99% identity to the nucleotide sequence of SEQ ID NO: 13.
14. The nucleic acid molecule of claim 1 , wherein the intracellular signaling domain comprises a functional signaling domain of a protein selected from the group consisting of an MHC class I molecule, a TNF receptor protein, an Immunoglobulin-like protein, a cytokine receptor, an integrin, a signaling lymphocytic activation molecule (SLAM protein), an activating NK cell receptor, BTLA, a Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a/CD18), 4-1BB(CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, 5 LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, and a ligand that specifically binds with CD83.
15. The nucleic acid molecule of claim 1 , wherein the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having at least 1, 2, or 3 modifications but not more than 10 modifications of the amino acid sequence of SEQ ID NO:7, or an amino acid sequence with 95-99% identity to the amino acid sequence of SEQ ID NO:7.
16. The nucleic acid molecule of claim 15 , wherein the intracellular signaling domain is encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO:18, or a sequence with 95-99% identity to the nucleotide sequence of SEQ ID NO:18.
17. The nucleic acid molecule of claim 1 , wherein the intracellular signaling domain comprises at least one of: a functional signaling domain of 4-1BB and a functional signaling domain of CD3 zeta.
18. The nucleic acid molecule of claim 1 , wherein the intracellular signaling domain comprises:
(i) at least one of: the amino acid sequence of SEQ ID NO: 7 and the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10; or
(ii) at least one of: an amino acid sequence having at least 1, 2, or 3 modifications but not more than 10 modifications of the amino acid sequence of SEQ ID NO:7 and the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10; or
(iii) at least one of: an amino acid sequence with 95-99% identity to the amino acid sequence of SEQ ID NO:7 and the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10.
19. The nucleic acid molecule of claim 1 , wherein the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:7 and the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10.
20. The nucleic acid molecule of claim 1 , wherein the nucleic acid sequence encoding the intracellular signaling domain comprises at least one of:
(i) the nucleotide sequence of SEQ ID NO: 18, or a sequence with 95-99% identity to SEQ ID NO: 18, and
(ii) the nucleotide sequence of SEQ ID NO: 20 or SEQ ID NO: 21, or a sequence with 95-99% identity to SEQ ID NO: 20 or SEQ ID NO: 21.
21. The nucleic acid molecule of claim 1 , further comprising a leader sequence encoding the amino acid sequence of SEQ ID NO: 1.
22. The nucleic acid molecule of claim 1 , wherein the CAR comprises:
(i) the amino acid sequence of any of SEQ ID NOs: 86, 92, 96, 100, or 104;
(ii) an amino acid sequence having at least 1, 2, or 3 modifications but not more than 30 modifications of any of SEQ ID NOs: 86, 92, 96, 100, or 104; or
(iii) an amino acid sequence with 95-99% identity to any of SEQ ID NOs: 86, 92, 96, 100, or 104.
23. The nucleic acid molecule of claim 1 , comprising the nucleotide sequence of any of SEQ ID NOs: 91, 95, 99, or 103, or a nucleotide sequence with 95-99% identity to any of SEQ ID NOs: 91, 95, 99, or 103.
24. A nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the antigen binding domain comprises:
a heavy chain variable region having heavy chain complementarity determining regions (HC CDRs) 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 63, 65, and 67; and
a light chain variable region having light chain complementarity determining regions (LC CDRs) 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 71, 73, and 75,
wherein the antigen binding domain binds to isoform a or isoform b of human Glycosyl-phosphatidylinositol (GPI)-linked GDNF family a-receptor 4 (GFRa4) cell-surface receptor.
25. The nucleic acid molecule of claim 24 , wherein the intracellular signaling domain comprises at least one of: a functional signaling domain of 4-1BB and a functional signaling domain of CD3 zeta.
26. A vector comprising the nucleic acid molecule of claim 1 , wherein the vector is selected from the group consisting of a DNA vector, an RNA vector, a plasmid, a lentiviral vector, an adenoviral vector, and a retroviral vector.
27. The vector of claim 26 , further comprising an EF-1 promoter comprising the sequence of SEQ ID NO: 11.
28. A cell comprising the nucleic acid molecule of claim 1 .
29. The cell of claim 28 , wherein the cell is selected from the group consisting of a T cell, a natural killer (NK) cell, a cytotoxic T lymphocyte (CTL), and a regulatory T cell.
30. The cell of claim 28 , for use in the treatment of a disease associated with expression of a thyroid cell antigen.
31. The cell of claim 28 , further comprising a fusion protein comprising a first domain comprising at least a portion of an inhibitory molecule and a second domain associated with a positive signal.
32. The cell of claim 31 , wherein the first domain comprises at least a portion of PD-1 and the second domain is selected from the group consisting of a costimulatory domain, a primary signaling domain, and an intracellular signaling domain.