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1. WO2020223327 - ANTIGEN SPECIFIC CD19-TARGETED CAR-T CELLS

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[ EN ]

WHAT IS CLAIMED IS:

1. A chimeric antigen receptor (CAR) polypeptide, comprising a B lymphocyte antigen-binding domain, a transmembrane domain, and an intracellular signaling domain.

2. The CAR polypeptide of claim 1, wherein the B-lymphocyte antigen is a CD 19 antigen, a CD20 antigen, or a CD22 antigen.

3. The CAR polypeptide of claim 1 or 2, wherein the B-lymphocyte antigen-binding domain is an anti-CD 19 functional antibody fragment.

4. The CAR polypeptide of any one of claims 1 to 3, wherein the B-lymphocyte antigen-binding domain is an anti-CD 19 single-chain variable fragment (scFv).

5. The CAR polypeptide of any one of claims 1 to 4, wherein the B-lymphocyte antigen-binding domain comprises the amino acid sequence set forth in SEQ ID NO. 9.

6. The CAR polypeptide of any one of claims 1 to 5, wherein the transmembrane domain is derived from a transmembrane or membrane-bound polypeptide.

7. The CAR polypeptide of any one of claims 1 to 6, wherein the transmembrane domain comprises at least one transmembrane domain of any one of the polypeptides CD28, NKp30, CDS, DAPIO, 41BB, DAP 12, CD3C, CD3e, CD45, CD4, CD5, CD8, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, 0X40, CD2, CD27, LFA-1, ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFl), CD 160, CD 19, IL2R b, IL2Ry, IL7Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld, ITGAE, CD103, ITGAL, CDl la, LFA-1, ITGAM, CDl lb, ITGAX, CDl lc, ITGB1,

CD29, ITGB2, CD 18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244,

2B4) , CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD 160 (BY55), PSGL1, CD 100 (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMFl, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, PAG/Cbp, mutants thereof, or any combination thereof.

8. The CAR polypeptide of any one of claims 1 to 7, wherein the transmembrane domain comprises the transmembrane domain of CD28 and/or 4 IBB.

9. The CAR polypeptide of any one of claims 1 to 8, wherein the signaling domain comprises at least one signaling domain of any one of the polypeptides CD8, CD3^ CD35, CD3y, CD3e, FcyRI-y, FcyRIII-y, FceRIp, FceRIy, DAP10, DAP 12, CD32, CD79a, CD79b, CD28, CD3C, CD4, b2c, CD137 (41BB), ICOS, CD27, CD285, CD80, NKp30, 0X40, mutants thereof, or any combination thereof.

10. The CAR polypeptide of any one of claims 1 to 9, further comprising at least one co-stimulatory signaling region.

11. The CAR polypeptide of claim 10, wherein the co-stimulatory signaling region comprises a signaling domain of any one of the polypeptides CD8, CD3^ CD35, CD3y, CD3e, FcyRI-y, FcyRIII-y, FceRIp, FceRIy, DAP10, DAP 12, CD32, CD79a, CD79b, CD28, CD3C, CD4, b2c, CD137 (41BB), ICOS, CD27, CD285, CD80, NKp30, 0X40, mutants thereof, or any combination thereof.

12. The CAR polypeptide of claim 10, wherein the co-stimulatory signaling region comprises a signaling domain of CD28 or a mutant thereof.

13. The CAR polypeptide of claim 12, wherein the CD28 signaling domain comprises at least one or more mutations in any one of subdomains YMNM, PRRP, PYAP, or any combination thereof .

14. The CAR polypeptide of claim 12 or 13, wherein the CD28 signaling domain lacks any one of subdomains YMNM, PRRP, PYAP.

15. The CAR polypeptide of any one of claims 12 to 14, wherein the CD28 signaling domain lacks any two of the subdomains selected from YMNM, PRRP, or PYAP.

16. The CAR polypeptide of claim 10 or 11, wherein the co-stimulatory signaling region comprises a signaling domain of CD137 (41BB) or a mutant thereof.

17. The CAR polypeptide of any one of claims 1 to 16, wherein the at least one signaling domain comprises a native Oϋ3z or a mutant thereof.

18. The CAR polypeptide of claim 17, wherein the mutant CD3z lacks a C-terminal immunoreceptor tyrosine-based activation motif (IT AM).

19. The CAR polypeptide of claim 17, wherein the mutant CD3z lacks two C-terminal immunoreceptor tyrosine-based activation motifs (IT AMs).

20. The CAR polypeptide of claim 17, wherein the mutant CD3z comprises only one immunoreceptor tyrosine-based activation motif (IT AM).

21. The CAR polypeptide of any one of claims 17 to 20, wherein the mutant CD3z comprises the amino acid sequence set forth in SEQ ID NO. 11.

22. The CAR polypeptide of any one of claims 1 to 21, further comprising a hinge sequence.

23. The CAR polypeptide of claim 22, wherein the hinge sequence is derived from a CD8a molecule or a CD28 molecule.

24. The CAR polypeptide of any one of claims 1 to 23, further comprising a signal peptide.

25. The CAR polypeptide of claim 24, wherein the signal peptide is derived from CD8a leader sequence.

26. The CAR polypeptide of any one of claims 1 to 25, comprising any one of the amino acid sequences set forth in SEQ ID NOs. 6 to 11, any fragment thereof, or any combination thereof.

27. The CAR polypeptide of any one of claims 1 to 25, comprising the amino acid sequences set forth in SEQ ID NOs. 7.

28. A nucleic acid encoding the CAR polypeptide of any one of claims 1 to 27.

29. The nucleic acid of claim 28, comprising any one of the nucleotide sequences set forth in SEQ ID NOs. 1 to 5, a fragment thereof, or any combination thereof.

30. The nucleic acid of claim 28, comprising the nucleotide sequence set forth in SEQ ID NO. 1.

31. A vector comprising the nucleic acid of any one of claims 28 to 30.

32. An immune cell comprising the nucleic acid of claim 31.

33. An immune cell comprising the vector of claim 32.

34. An immune cell expressing the CAR polypeptide of any one of claims 1 to 27.

35. The immune cell of any one of claims 32 to 34, wherein the immune cell is a leukocyte.

36. The immune cell of any one of claims 32 to 35 wherein the immune cell is a lymphocyte, a monocyte, a macrophage, a dendritic cell, a mast cell, a neutrophil, a basophil, or an eosinophil.

37. The immune cell of any one of claims 32 to 36 wherein the immune cell is a lymphocyte selected from an abT cell, gdT cell, a Natural Killer (NK) cell, a Natural Killer T (NKT) cell, a B cell, an innate lymphoid cell (ILC), a cytokine induced killer (CIK) cell, a cytotoxic T lymphocyte (CTL), a lymphokine activated killer (LAK) cell, a regulatory T cell, or any combination thereof.

38. The immune cell of claim 37, wherein the immune cell is a cytotoxic T lymphocyte (CTL).

39. The immune cell of claim 37 or 38, wherein the immune cell is a viral antigen-sensitized CTL.

40. The immune cell of any one of claims 37 to 39, wherein the immune cell is a CTL sensitized to a viral antigen from any one of Epstein-Barr virus (EBV), cytomegalovirus (CMV), B.K. virus (BKV), John Cunningham virus (JCV), picomavirus (e.g., Hepatitis A

virus), hepadnavirus (e.g., Hepatitis B virus), hepacivirus (e.g., Hepatitis C virus), deltavirus (e.g., Hepatitis D virus), hepevirus (e.g., Hepatitis E virus), or any combination thereof.

41. The immune cell of any one of claims 37 to 40, wherein the immune cell is an EBV-sensitized CTL.

42. A bi-specific chimeric antigen receptor (CAR) T cell expressing

(a) a CAR polypeptide that selectively binds a B-lymphocyte antigen and

(b) a CAR polypeptide that selectively binds a tumor-associated antigen.

43. The bi-specific chimeric CAR T cell of claim 42, wherein the B-lymphocyte antigen is a CD 19 antigen, a CD20 antigen, or a CD22 antigen.

44. The bi-specific CAR T cell of claim 39 or 40, wherein the tumor-associated antigen is GPC3, MAGE-A1, MAGE-A2, MAGE-C2, SSX-2, Ny-ESO-1, hTERT, or a viral hepatitis antigen.

45. The bi-specific CAR T cell of any one of claims 42 to 44, wherein the antigen binding domain of the chimeric antigen receptors each comprise a functional antibody fragment.

46. The bi-specific CAR T cell of any one of claims 42 to 45, wherein the antigen binding domain of the chimeric antigen receptors each comprise a single-chain variable fragment (scFv).

47. The bi-specific CAR T cell of any one of claims 42 to 46, wherein the

transmembrane domain of the chimeric antigen receptors each comprise at least one transmembrane domain of any of CD28, 4 IBB, mutants thereof, or any combination thereof.

48. The bi-specific CAR T cell of any one of claims 42 to 47, wherein the signaling domain of at least one of the chimeric antigen receptors comprises at least one signaling domain of CD3^ mutants thereof, or any combination thereof.

49. The bi-specific CAR T cell of any one of claims 42 to 47, wherein the signaling domain of the chimeric antigen receptors each comprise at least one signaling domain of Oϋ3z, mutants thereof, or any combination thereof.

50. The bi-specific CAR T cell of claim 48 or 49, wherein the mutant CD3z lacks a C-terminal immunoreceptor tyrosine-based activation motif (IT AM).

51. The bi-specific CAR T cell of claim 48 or 49, wherein the mutant CD3z lacks two C-terminal immunoreceptor tyrosine-based activation motifs (IT AMs).

52. The bi-specific CAR T cell of claim 48 or 49, wherein the mutant CD3z comprises only one immunoreceptor tyrosine-based activation motif (IT AM).

53. The bi-specific CAR T cell of any one of claims 42 to 52, wherein at least one of the chimeric antigen receptors comprises at least one co-stimulatory signaling region.

54. The bi-specific CAR T cell of any one of claims 42 to 52, wherein each chimeric antigen receptor comprises at least one co-stimulatory signaling region.

55. The bi-specific CAR T cell of claim 53 or 54, wherein the co-stimulatory signaling region comprises a signaling domain of any one of CD28 or a mutant thereof, CD137

(4 IBB) or a mutant thereof, or any combination thereof.

56. The CAR polypeptide of claim 55, wherein the CD28 signaling domain comprises at least one or more mutations in any one of subdomains YMNM, PRRP, PYAP, or any combination thereof .

57. The CAR polypeptide of claim 55 or 56, wherein the CD28 signaling domain lacks function of at least one the subdomains selected from YMNM, PRRP, or PYAP.

58. The CAR polypeptide of any one of claims 55 to 57, wherein the CD28 signaling domain lacks function of any two of the subdomains selected from YMNM, PRRP, or PYAP.

59. The bi-specific CAR T cell of any one of claims 42 to 58, further comprising a hinge sequence derived from CD8a or CD28.

60. The CAR-expressing cell of any one of claims 34 to 59, wherein the CAR-expressing cell is genetically modified to no longer express one or more immune checkpoint molecules.

61. The CAR-expressing cell of any one of claims 34 to 59, further comprising expression of a dominant-negative form of one or more immune checkpoint molecules.

62. The CAR-expressing cell of any one of claims 34 to 59, further comprising expression of switch receptors specific for one or more immune checkpoint molecules.

63. The CAR-expressing cell of any one of claims 34 to 59, further comprising expression antibodies, or functional fragments thereof, capable of blocking signaling by one or more checkpoint molecules.

64. The CAR-expressing cell of any one of claims 60 to 63, wherein the immune checkpoint molecules are selected from programmed death 1 (PD-1), programmed death-ligand 1 (PD-L1), programmed death-ligand 2 (PD-L2), cytotoxic T lymphocyte antigen-4 (CTLA-4), B- and T-lymphocyte attenuator (BTLA), T cell immunoglobulin mucin-3 (TIM-3), lymphocyte-activation protein 3 (LAG-3), T cell immunoreceptor with Ig and ITIM domains (TIGIT), leukocyte-associated immunoglobulin- like receptor 1 (LAIRl), natural killer cell receptor 2B4 (2B4), CD 160, and transforming growth factor b (TGF-b) receptor.

65. The immune cell of any one of claims 60 to 64, wherein the immune checkpoint molecule is PD-1 and/or CTLA-4.

66. A method of treating a B-lymphocyte antigen-associated cancer in a subject, the method comprising administering an effective amount of an adoptive immunotherapy composition comprising CAR-expressing cells of any one of claims 32 to 65.

67. The method of claim 66, wherein the B-lymphocyte antigen-associated cancer is a hematologic cancer selected from: acute leukemia, chronic leukemia, lymphocytic leukemia, myelogenous leukemia, a pre-leukemic condition, Hodgkin lymphoma, Non-Hodgkin lymphoma, EBV-associated lymphoproliferative disease, mature B cell neoplasm,

mature T cell and/or natural killer (NK) cell neoplasm, precursor lymphoid neoplasm, and immunodeficiency-associated lymphoproliferative disorder.

68. The method of claim 66 or 67, wherein the B-lymphocyte antigen-associated cancer is EBV-associated lymphoproliferative disease.

69. The method of any one of claims 66 to 68, wherein the subject has received, is receiving, or will receive an additional anti-cancer therapy.

70. The method of claim 69, wherein the additional anti-cancer therapy comprises surgery, radiation, chemotherapy, immunotherapy, or hormone therapy.

71. The method of any one of claims 66 to 70, wherein the adoptive immunotherapy composition is administered intrapleurally, intravenously, subcutaneously, intranodally, intratumorally, intrathecally, intraperitoneally, intracranially, or by direct administration to an organ.

72. The method of any one of claims 66 to 71, wherein the subject is human.

73. The method of claim 72, wherein the CAR-expressing cells of the adoptive immunotherapy composition are derived from the subject.

74. The method of claim 73, wherein the CAR-expressing cells of the adoptive immunotherapy composition are derived from a donor sample, or from a bank or library of donor samples.

75. The method of any one of claims 66 to 74, further comprising administering at least one immune checkpoint inhibitor.

76. The method of claim 75, wherein the immune checkpoint inhibitor comprises an anti -PD- 1 antibody, anti-PD-Ll antibody, anti-PD-L2 antibody, anti-CTLA-4 antibody, or a combination thereof.

77. The method of claim 75, wherein the immune checkpoint inhibitor comprises an RNAi molecule such as an antisense RNA molecule (asRNA), micro RNA molecule

(miRNA), short hairpin RNA molecule (shRNA), or small interfering RNA molecule (siRNA).

78. The method of claim 75, wherein the immune checkpoint inhibitor comprises a CRISPR RNA (crRNA) molecule.

79. The method of claim 75, wherein the immune checkpoint inhibitor comprises a dominant-negative form of an immune checkpoint molecule.

80. The method of claim 75, wherein the immune checkpoint inhibitor comprises a recombinant switch receptor.

81. The method of any of claims 75to 80, wherein immune checkpoint inhibitor is expressed by a vector comprising a nucleic acid molecule encoding the immune checkpoint inhibitor, wherein the vector is selected from a DNA vector, an RNA vector, a plasmid, or a viral vector.

82. The method of claim 81, wherein the vector comprising a nucleic acid molecule encoding the immune checkpoint inhibitor is expressed in the CAR-expressing cells of the adoptive immunotherapy composition.