Treatment of cancer using a CD123 chimeric antigen receptor
The invention provides compositions and methods for treating diseases associated with expression of CD123. The invention also relates to chimeric antigen receptor (CAR) specific to CD123, vectors encoding the same, and recombinant cells comprising the CD123 CAR. The invention also includes methods of administering a genetically modified cell expressing a CAR that comprises a CD123 binding domain.
1. An isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises a CD123 binding domain, a transmembrane domain, and an intracellular signaling domain, and wherein said CD123 binding domain comprises:
(a) a heavy chain variable domain comprising three complementary determining regions of heavy chain complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) present in order of HC CDR1, HC CDR2, and HC CDR3, wherein HC CDR1 comprises the amino acid sequence of SEQ ID NO: 487, HC CDR2 comprises the amino acid sequence of SEQ ID NO: 492, and HC CDR3 comprises the amino acid sequence of SEQ ID NO: 497; and
a light chain variable domain comprising three complementary determining regions of light chain complementary determining region 1 (LC CDR1), light chain complementary determining region 2 (LC CDR2), and light chain complementary determining region 3 (LC CDR3) present in order of LC CDR1, LC CDR2, and LC CDR3, wherein LC CDR1 comprises the amino acid sequence of SEQ ID NO: 502, LC CDR2 comprises the amino acid sequence of SEQ ID NO: 507, and LC CDR3 comprises the amino acid sequence of SEQ ID NO: 512; or
(b) a heavy chain variable domain comprising three complementary determining regions of heavy chain complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) present in order of HC CDR1, HC CDR2, and HC CDR3, wherein HC CDR1 comprises the amino acid sequence of SEQ ID NO: 517, HC CDR2 comprises the amino acid sequence of SEQ ID NO: 522, and HC CDR3 comprises the amino acid sequence of SEQ ID NO: 527; and
a light chain variable domain comprising three complementary determining regions of light chain complementary determining region 1 (LC CDR1), light chain complementary determining region 2 (LC CDR2), and light chain complementary determining region 3 (LC CDR3) present in order of LC CDR1, LC CDR2, and LC CDR3, wherein LC CDR1 comprises the amino acid sequence of SEQ ID NO: 532, LC CDR2 comprises the amino acid sequence of SEQ ID NO: 537, and LC CDR3 comprises the amino acid sequence of SEQ ID NO: 542; or
(c) a heavy chain variable domain comprising three complementary determining regions of heavy chain complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) present in order of HC CDR1, HC CDR2, and HC CDR3, wherein HC CDR1 comprises the amino acid sequence of SEQ ID NO: 335, HC CDR2 comprises the amino acid sequence of SEQ ID NO: 363, and HC CDR3 comprises the amino acid sequence of SEQ ID NO: 391; and
a light chain variable domain comprising three complementary determining regions of light chain complementary determining region 1 (LC CDR1), light chain complementary determining region 2 (LC CDR2), and light chain complementary determining region 3 (LC CDR3) present in order of LC CDR1, LC CDR2, and LC CDR3, wherein LC CDR1 comprises the amino acid sequence of SEQ ID NO: 419, LC CDR2 comprises the amino acid sequence of SEQ ID NO: 447, and LC CDR3 comprises the amino acid sequence of SEQ ID NO: 475.
2. The isolated nucleic acid molecule of claim 1 , which encodes a CAR comprising:
(i) the amino acid sequence of the light chain variable region of SEQ ID NO: 276; or
(ii) an amino acid sequence with 95-99% identity to the amino acid sequence of the light chain variable region of SEQ ID NO: 276.
3. The isolated nucleic acid molecule of claim 1 , which encodes a CAR comprising:
(i) the amino acid sequence of the heavy chain variable region of SEQ ID NO: 217; or
(ii) an amino acid sequence with 95-99% identity to the amino acid sequence of the heavy chain variable region of SEQ ID NO: 217.
4. The isolated nucleic acid molecule of claim 1 , which encodes a CAR comprising the amino acid sequence of the light chain variable region of SEQ ID NO: 276, and the amino acid sequence of the heavy chain variable region of SEQ ID NO: 217.
5. The isolated nucleic acid molecule of claim 1 , wherein the encoded CD123 binding domain comprises:
(i) the amino acid sequence of SEQ ID NO:480;
(ii) an amino acid sequence having at least one, two or three modifications but not more than 10 modifications to SEQ ID NO: 480; or
(iii) an amino acid sequence with 95-99% identity to SEQ ID NO: 480.
6. The isolated nucleic acid molecule of claim 1 , wherein the CD123 binding domain comprises a nucleotide sequence chosen from SEQ ID NO: 479 and 481, or a nucleotide sequence with 95-99% identity thereof.
7. The isolated nucleic acid molecule of claim 1 , wherein:
(i) the encoded CAR comprises a transmembrane domain that comprises a transmembrane domain of a protein 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; or
(ii) the encoded transmembrane domain comprises the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence with 95-99% identity to the amino acid sequence of SEQ ID NO:6; or
(iii) the nucleic acid sequence encoding the transmembrane domain comprises nucleotides 928-999 of sequence of SEQ ID NO: 40, or a nucleotide sequence with 95-99% identity thereof.
8. The isolated nucleic acid molecule of claim 1 , wherein the encoded CD123 binding domain is connected to the transmembrane domain by a hinge region, wherein
(i) the encoded hinge region comprises the amino acid sequence of SEQ ID NO:2, or an amino acid sequence with 95-99% identity to SEQ ID NO: 2; or
(ii) the nucleic acid sequence encoding the hinge region comprises nucleotides 793-927 of sequence of SEQ ID NO: 40, or a nucleotide sequence with 95-99% identity thereof.
9. The isolated nucleic acid molecule of claim 1 , wherein the encoded intracellular domain comprises a costimulatory domain, wherein the costimulatory domain comprises a functional signaling domain derived from a protein selected from the group consisting of a 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, 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, and PAG/Cbp.
10. The isolated nucleic acid molecule of claim 9 , wherein the costimulatory domain comprises 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.
11. The isolated nucleic acid molecule of claim 9 , wherein the nucleic acid sequence encoding the costimulatory domain comprises nucleotides 1000-1125 of sequence of SEQ ID NO: 40, or a nucleotide sequence with 95-99% identity thereof.
12. The isolated nucleic acid molecule of claim 1 , wherein the encoded intracellular signaling domain comprises a functional signaling domain of 4-1BB and a functional signaling domain of CD3 zeta.
13. The isolated nucleic acid molecule of claim 1 , wherein the encoded 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; or 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.
14. The isolated nucleic acid molecule of claim 1 , wherein the encoded 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, wherein the sequences comprising the intracellular signaling domain are expressed in the same frame and as a single polypeptide chain.
15. The isolated nucleic acid molecule of claim 1 , wherein the nucleic acid sequence encoding the intracellular signaling domain comprises nucleotides 1000-1125 of sequence of SEQ ID NO: 40, or a nucleotide sequence with 95-99% identity thereof, and/or nucleotides 1126-1461 of SEQ ID NO: 40 or SEQ ID NO:21, or a nucleotide sequence with 95-99% identity thereof.
16. The isolated nucleic acid molecule of claim 1 , further comprising a leader sequence which encodes the amino acid sequence of SEQ ID NO:1.
17. The isolated nucleic acid molecule of claim 1 , which encodes a CAR comprising:
(i) the amino acid sequence of SEQ ID NO: 99;
(ii) an amino acid sequence having at least one, two or three modifications but not more than 10 modifications to SEQ ID NO: 99; or
(iii) an amino acid sequence with 95-99% identity to SEQ ID NO: 99.
18. The isolated nucleic acid molecule of claim 1 , comprising the nucleotide sequence of SEQ ID NO: 40, or a nucleotide sequence with 95-99% identity to SEQ ID NO: 40.
19. An isolated chimeric antigen receptor (CAR) polypeptide, wherein the CAR comprises a CD123 binding domain, a transmembrane domain, and an intracellular signaling domain, and wherein said CD123 binding domain comprises:
(a) a heavy chain variable domain comprising three complementary determining regions of heavy chain complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) present in order of HC CDR1, HC CDR2, and HC CDR3, wherein HC CDR1 comprises the amino acid sequence of SEQ ID NO: 487, HC CDR2 comprises the amino acid sequence of SEQ ID NO: 492, and HC CDR3 comprises the amino acid sequence of SEQ ID NO: 497; and
a light chain variable domain comprising three complementary determining regions of light chain complementary determining region 1 (LC CDR1), light chain complementary determining region 2 (LC CDR2), and light chain complementary determining region 3 (LC CDR3) present in order of LC CDR1, LC CDR2, and LC CDR3, wherein LC CDR1 comprises the amino acid sequence of SEQ ID NO: 502, LC CDR2 comprises the amino acid sequence of SEQ ID NO: 507, and LC CDR3 comprises the amino acid sequence of SEQ ID NO: 512; or
(b) a heavy chain variable domain comprising three complementary determining regions of heavy chain complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) present in order of HC CDR1, HC CDR2, and HC CDR3, wherein HC CDR1 comprises the amino acid sequence of SEQ ID NO: 517, HC CDR2 comprises the amino acid sequence of SEQ ID NO: 522, and HC CDR3 comprises the amino acid sequence of SEQ ID NO: 527; and
a light chain variable domain comprising three complementary determining regions of light chain complementary determining region 1 (LC CDR1), light chain complementary determining region 2 (LC CDR2), and light chain complementary determining region 3 (LC CDR3) present in order of LC CDR1, LC CDR2, and LC CDR3, wherein LC CDR1 comprises the amino acid sequence of SEQ ID NO: 532, LC CDR2comprises the amino acid sequence of SEQ ID NO: 537, and LC CDR3 comprises the amino acid sequence of SEQ ID NO: 542; or
(c) a heavy chain variable domain comprising three complementary determining regions of heavy chain complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) present in order of HC CDR1, HC CDR2, and HC CDR3, wherein HC CDR1 comprises
the amino acid sequence of SEQ ID NO: 335, HC CDR2 comprises the amino acid sequence of SEQ ID NO: 363, and HC CDR3 comprises the amino acid sequence of SEQ ID NO: 391; and
a light chain variable domain comprising three complementary determining regions of light chain complementary determining region 1 (LC CDR1), light chain complementary determining region 2 (LC CDR2), and light chain complementary determining region 3 (LC CDR3) present in order of LC CDR1, LC CDR2, and LC CDR3, wherein LC CDR1 comprises the amino acid sequence of SEQ ID NO: 419, LC CDR2 comprises the amino acid sequence of SEQ ID NO: 447, and LC CDR3 comprises the amino acid sequence of SEQ ID NO: 475.
20. The isolated CAR polypeptide of claim 19 , comprising:
(i) the amino acid sequence of the light chain variable region of SEQ ID NO: 276; or
(ii) an amino acid sequence with 95-99% identity to the amino acid sequence of the light chain variable region of SEQ ID NO: 276.
21. The isolated CAR polypeptide of claim 19 , comprising:
(i) the amino acid sequence of the heavy chain variable region of SEQ ID NO: 217; or
(ii) an amino acid sequence with 95-99% identity to the amino acid sequence of the heavy chain variable region of SEQ ID NO: 217.
22. The isolated CAR polypeptide of claim 19 , comprising the amino acid sequence of the light chain variable region of SEQ ID NO: 276, and the amino acid sequence of the heavy chain variable region of SEQ ID NO: 217.
23. The isolated CAR polypeptide of claim 19 , comprising:
(i) the amino acid sequence of SEQ ID NO:480;
(ii) an amino acid sequence having at least one, two or three modifications but not more than 10 modifications to SEQ ID NO: 480; or (iii) an amino acid sequence with 95-99% identity to SEQ ID NO: 480.
24. The isolated CAR polypeptide of claim 19 , wherein the transmembrane domain comprises a transmembrane domain from a protein 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.
25. The isolated CAR polypeptide of claim 19 , wherein the transmembrane domain comprises:
(i) the amino acid sequence of SEQ ID NO: 6, or
(ii) a sequence with 95-99% identity to the amino acid sequence of SEQ ID NO:6.
26. The isolated CAR polypeptide of claim 19 , wherein the CD123 binding domain is connected to the transmembrane domain by a hinge region, wherein the hinge region comprises SEQ ID NO:2, or a sequence with 95-99% identity thereof.
27. The isolated CAR polypeptide of claim 19 , wherein the encoded intracellular domain comprises a costimulatory domain, wherein the costimulatory domain comprises a functional signaling domain derived from a protein selected from the group consisting of a 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, 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, and PAG/Cbp.
28. The isolated CAR polypeptide of claim 19 , wherein the costimulatory domain comprises the amino acid sequence of SEQ ID NO:7, or a sequence with 95-99% identity to the amino acid sequence of SEQ ID NO:7.
29. The isolated CAR polypeptide of claim 19 , wherein the intracellular signaling domain comprises a functional signaling domain of 4-1BB and a functional signaling domain of CD3 zeta.
30. The isolated CAR polypeptide of claim 19 , 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; or 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.
31. The isolated CAR polypeptide of claim 19 , comprising:
(i) the amino acid sequence of SEQ ID NO: 99;
(ii) an amino acid sequence having at least one, two or three modifications but not more than 10 modifications to SEQ ID NO: 99; or
(iii) an amino acid sequence with 95-99% identity to SEQ ID NO: 99.
32. A vector comprising the nucleic acid molecule of claim 1 , wherein the vector is a DNA vector, an RNA vector, a plasmid, a lentivirus vector, adenoviral vector, or a retrovirus vector.
33. An isolated immune effector cell comprising the nucleic acid molecule of claim 1 .
34. A method of making an immune effector cell, comprising transducing the immune effector cell with the vector of claim 32 .
35. 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 the nucleic acid molecule of claim 1 .
36. The isolated cell of claim 33 , further expressing a chimeric molecule that comprises a first polypeptide that comprises at least a portion of an inhibitory molecule, associated with a second polypeptide that comprises a positive signal from an intracellular signaling domain.
37. The isolated nucleic acid molecule of claim 1 , wherein the encoded intracellular signaling domain comprises a primary signaling domain comprising a functional signaling domain derived from CD3 zeta, TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta , CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278, FcεRI, DAP10, DAP12, or CD66d.
38. The isolated nucleic acid molecule of claim 37 , wherein the primary signaling domain comprises a functional signaling domain of CD3 zeta, wherein the CD3 zeta comprises the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10, or a sequence with 95-99% identity to the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10.
39. The isolated CAR polypeptide of claim 19 , wherein the intracellular signaling domain comprises a primary signaling domain comprising a functional signaling domain derived from CD3 zeta, TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta , CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278, FcεRI, DAP10, DAP12, or CD66d.
40. The isolated CAR polypeptide of claim 39 , wherein the primary signaling domain comprises a functional signaling domain of CD3 zeta, wherein the CD3 zeta comprises the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10, or a sequence with 95-99% identity to the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10.