METHODS FOR INCREASING T-CELL FUNCTION

Abstract

Provided herein are methods of increasing T-cell function and T-cells produced by these methods. Also provided herein are methods of treating a subject using T-cells produced by these methods.

Claims

1. A method for increasing T-cell function, wherein the method comprises introducing into a T-cell: (i) a first nucleic acid sequence encoding a FOXP3 polypeptide; and (ii) a second nucleic acid sequence encoding a binding agent.

2. The method of claim 1, wherein the first nucleic acid sequence encoding a FOXP3 polypeptide comprises a mutation in exon 2, wherein the FOXP3 polypeptide comprising a mutation in exon 2 results in increased nuclear localization of the FOXP3 polypeptide as compared to a FOXP3 polypeptide comprising an exon 2 that does not include a mutation.

3. The method of claim 2, wherein the mutation comprises deletion of exon 2.

4. The method of any one of claims 1-3, wherein the introducing step further comprises introducing a nucleic acid construct, wherein the nucleic acid construct comprises the first nucleic acid sequence and the second nucleic acid sequence, wherein the first nucleic acid sequence or the second nucleic acid sequence are operably linked to a promoter, and wherein the first nucleic acid sequence and the second nucleic acid sequence are operably linked, into the T-cell.

5. The method of any one of claims 1-4, wherein the introducing step further comprises introducing a third nucleic acid sequence encoding a receptor polypeptide into the T-cell.

6. The method of claim 4 or 5, wherein the nucleic acid construct further includes the third nucleic acid sequence, wherein the third nucleic acid sequence is operably linked to a promoter.

7. The method of claim 6, wherein the third nucleic acid sequence is operably linked to the first nucleic acid sequence and/or the second nucleic acid sequence.

8. The method of any one of claims 5-7, wherein the receptor polypeptide is a chemokine receptor polypeptide.

9. The method of claim 8, wherein the chemokine receptor polypeptide is, CCR6, CCR9 or GRP15.

10. The method of any one of claims 1-9, wherein the binding agent is an antibody or antigen-binding fragment.

11. The method of claim 10, wherein the antigen-binding domain is an antigen-binding fragment selected from the group consisting of a Fab, a F(ab).sub.2 fragment, a scFV, a scAb, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody.

12. The method of claim 11, wherein the antigen-binding fragment is a scFv that is capable of binding to a cell adhesion molecule.

13. The method of claim 12, wherein the cell adhesion molecule is MADCAM-1.

14. The method of claim 13, wherein the antigen-binding fragment comprises an amino acid sequence at least 80% identical to a sequence selected from the group consisting of SEQ ID NOs: 6-29.

15. The method of claim 13, wherein the antigen-binding fragment comprises: (i) the three heavy chain CDR sequences present in SEQ ID NO: 30 and the three light chain CDR sequences present in SEQ ID NO: 31; (ii) the three heavy chain CDR sequences present in SEQ ID NO: 32 and the three light chain CDR sequences present in SEQ ID NO: 33; (iii) the three heavy chain CDR sequences present in SEQ ID NO: 34 and the three light chain CDR sequences present in SEQ ID NO: 35; (iv) the three heavy chain CDR sequences present in SEQ ID NO: 36 and the three light chain CDR sequences present in SEQ ID NO: 37; (v) the three heavy chain CDR sequences present in SEQ ID NO: 38 and the three light chain CDR sequences present in SEQ ID NO: 39; (vi) the three heavy chain CDR sequences present in SEQ ID NO: 40 and the three light chain CDR sequences present in SEQ ID NO: 41; (vii) the three heavy chain CDR sequences present in SEQ ID NO: 42 and the three light chain CDR sequences present in SEQ ID NO: 43; (viii) the three heavy chain CDR sequences present in SEQ ID NO: 44 and the three light chain CDR sequences present in SEQ ID NO: 45; (ix) the three heavy chain CDR sequences present in SEQ ID NO: 46 and the three light chain CDR sequences present in SEQ ID NO: 47; (x) the three heavy chain CDR sequences present in SEQ ID NO: 48 and the three light chain CDR sequences present in SEQ ID NO: 49; (xi) the three heavy chain CDR sequences present in SEQ ID NO: 50 and the three light chain CDR sequences present in SEQ ID NO: 51; (xii) the three heavy chain CDR sequences present in SEQ ID NO: 52 and the three light chain CDR sequences present in SEQ ID NO: 53; (xiii) the three heavy chain CDR sequences present in SEQ ID NO: 54 and the three light chain CDR sequences present in SEQ ID NO: 55; (xiv) the three heavy chain CDR sequences present in SEQ ID NO: 56 and the three light chain CDR sequences present in SEQ ID NO: 57; (xv) the three heavy chain CDR sequences present in SEQ ID NO: 58 and the three light chain CDR sequences present in SEQ ID NO: 59; (xvi) the three heavy chain CDR sequences present in SEQ ID NO: 60 and the three light chain CDR sequences present in SEQ ID NO: 61; (xvii) the three heavy chain CDR sequences present in SEQ ID NO: 62 and the three light chain CDR sequences present in SEQ ID NO: 63; (xviii) the three heavy chain CDR sequences present in SEQ ID NO: 64 and the three light chain CDR sequences present in SEQ ID NO: 65; (xix) the three heavy chain CDR sequences present in SEQ ID NO: 66 and the three light chain CDR sequences present in SEQ ID NO: 67; (xx) the three heavy chain CDR sequences present in SEQ ID NO: 68 and the three light chain CDR sequences present in SEQ ID NO: 69; (xxi) the three heavy chain CDR sequences present in SEQ ID NO: 70 and the three light chain CDR sequences present in SEQ ID NO: 71; (xxii) the three heavy chain CDR sequences present in SEQ ID NO: 72 and the three light chain CDR sequences present in SEQ ID NO: 73; (xxiii) the three heavy chain CDR sequences present in SEQ ID NO: 74 and the three light chain CDR sequences present in SEQ ID NO: 75; or (xxiv) the three heavy chain CDR sequences present in SEQ ID NO: 76 and the three light chain CDR sequences present in SEQ ID NO: 77.

16. The method of any one of claims 1-9, wherein the binding agent is a chimeric antigen receptor, wherein the chimeric antigen receptor comprises an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain comprises an antibody or an antigen-binding domain capable of binding to an antigen on an autoimmune cell, and wherein the intracellular domain comprises a cytoplasmic signaling domain and one or more co-stimulatory domains.

17. The method of claim 16, wherein the antigen-binding domain is an antigen-binding fragment selected from the group consisting of a Fab, a F(ab).sub.2 fragment, a scFV, a scAb, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody.

18. The method of claim 17, wherein the antigen-binding fragment is a scFv that is capable of binding to a cell adhesion molecule.

19. The method of claim 18, wherein the cell adhesion molecule is MADCAM-1.

20. The method of claim 18, wherein the antigen-binding fragment comprises an amino acid sequence at least 80% identical to a sequence selected from the group consisting of SEQ ID NOs: 6-29.

21. The method of claim 16, wherein the cytoplasmic signaling domain is a CD3 zeta domain.

22. The method of claim 16, wherein the co-stimulatory domain comprises at least one of a CD48, 4-1BB, ICOS, X-40, and CD27 domain.

23. The method of any one of claims 1-22, wherein the introducing step further comprises introducing an additional nucleic acid sequence encoding a therapeutic gene product into the T-cell.

24. The method of claim 4 or 6, wherein the nucleic acid construct further comprises an additional sequence encoding the therapeutic gene product.

25. The method of claim 24, wherein the additional nucleic acid sequence is operably linked a promoter or is operably linked to the first nucleic acid sequence, the second nucleic acid sequence and/or the third nucleic acid sequence.

26. The method of any one of claims 4, 12, and 24, wherein the nucleic acid construct comprises a viral vector selected from the group consisting of a lentiviral vector, a retroviral vector, an adenoviral vector, and an adeno-associated viral (AAV) vector.

27. The method of claim 26, wherein the viral vector is a lentiviral vector.

28. The method of claim 27, wherein the introducing step comprises viral transduction.

29. The method of any one of claims 1-28, wherein the T-cell is a CD4.sup.+ T-cell or a CD4.sup.+/CD45RA.sup.+ T-cell.

30. The method of any one of claims 1-29, wherein the method further comprises: obtaining a T-cell from a patient or obtaining T-cells allogenic to the patient.

31. The method of claim 30, wherein the method further comprises: treating the obtained T-cells to isolate a population of cells enriched for CD4.sup.+ T-cells or CD4.sup.+/CD45RA.sup.+ T-cells.

32. A T-cell produced by the method of any one of claims 1-31.

33. A composition comprising the T-cell of claim 32.

34. A T-cell comprising: a first nucleic acid sequence encoding a FOXP3 polypeptide; and a second nucleic acid sequence encoding a binding agent.

35. The T-cell of claim 34, wherein the first nucleic acid sequence encoding a FOXP3 polypeptide comprises a mutation in exon 2, wherein the FOXP3 polypeptide comprising a mutation in exon 2 results in increased nuclear localization of the FOXP3 polypeptide as compared to a FOXP3 polypeptide comprising an exon 2 that does not include a mutation.

36. The T-cell of claim 35, wherein the mutation comprises deletion of exon 2.

37. The T-cell of any one of claims 34-36, wherein the first nucleic acid sequence is operably linked to a promoter.

38. The T-cell of any one of claims 34-37, wherein the second nucleic acid sequence is operably linked to a promoter.

39. The T-cell of any one of claims 34-38, wherein the T-cell further comprises a third nucleic acid sequence encoding a receptor polypeptide.

40. The T-cell of claim 39, wherein the third nucleic acid sequence is operably linked to a promoter.

41. The T-cell of claim 39 or 40, wherein the receptor polypeptide is a chemokine receptor polypeptide.

42. The T-cell of claim 41, wherein the chemokine receptor polypeptide is CCR6, CCR9, or GRP15.

43. The T-cell of any one of claims 34-42, wherein the binding agent is an antibody or antigen-binding domain.

44. The T-cell of claim 43, wherein the antigen-binding domain is an antigen-binding fragment selected from the group consisting of a Fab, a F(ab).sub.2 fragment, a scFV, a scAb, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody.

45. The T-cell of claim 44, wherein the antigen-binding fragment is a scFv that is capable of binding to a cell adhesion molecule.

46. The T-cell of claim 45, wherein the cell adhesion molecule is MADCAM-1.

47. The T-cell of claim 46, wherein the antigen-binding fragment comprises an amino acid sequence at least 80% identical to a sequence selected from the group consisting of SEQ ID NOs: 6-29.

48. The method of claim 46, wherein the antigen-binding fragment comprises: (i) the three heavy chain CDR sequences present in SEQ ID NO: 30 and the three light chain CDR sequences present in SEQ ID NO: 31; (ii) the three heavy chain CDR sequences present in SEQ ID NO: 32 and the three light chain CDR sequences present in SEQ ID NO: 33; (iii) the three heavy chain CDR sequences present in SEQ ID NO: 34 and the three light chain CDR sequences present in SEQ ID NO: 35; (iv) the three heavy chain CDR sequences present in SEQ ID NO: 36 and the three light chain CDR sequences present in SEQ ID NO: 37; (v) the three heavy chain CDR sequences present in SEQ ID NO: 38 and the three light chain CDR sequences present in SEQ ID NO: 39; (vi) the three heavy chain CDR sequences present in SEQ ID NO: 40 and the three light chain CDR sequences present in SEQ ID NO: 41; (vii) the three heavy chain CDR sequences present in SEQ ID NO: 42 and the three light chain CDR sequences present in SEQ ID NO: 43; (viii) the three heavy chain CDR sequences present in SEQ ID NO: 44 and the three light chain CDR sequences present in SEQ ID NO: 45; (ix) the three heavy chain CDR sequences present in SEQ ID NO: 46 and the three light chain CDR sequences present in SEQ ID NO: 47; (x) the three heavy chain CDR sequences present in SEQ ID NO: 48 and the three light chain CDR sequences present in SEQ ID NO: 49; (xi) the three heavy chain CDR sequences present in SEQ ID NO: 50 and the three light chain CDR sequences present in SEQ ID NO: 51; (xii) the three heavy chain CDR sequences present in SEQ ID NO: 52 and the three light chain CDR sequences present in SEQ ID NO: 53; (xiii) the three heavy chain CDR sequences present in SEQ ID NO: 54 and the three light chain CDR sequences present in SEQ ID NO: 55; (xiv) the three heavy chain CDR sequences present in SEQ ID NO: 56 and the three light chain CDR sequences present in SEQ ID NO: 57; (xv) the three heavy chain CDR sequences present in SEQ ID NO: 58 and the three light chain CDR sequences present in SEQ ID NO: 59; (xvi) the three heavy chain CDR sequences present in SEQ ID NO: 60 and the three light chain CDR sequences present in SEQ ID NO: 61; (xvii) the three heavy chain CDR sequences present in SEQ ID NO: 62 and the three light chain CDR sequences present in SEQ ID NO: 63; (xviii) the three heavy chain CDR sequences present in SEQ ID NO: 64 and the three light chain CDR sequences present in SEQ ID NO: 65; (xix) the three heavy chain CDR sequences present in SEQ ID NO: 66 and the three light chain CDR sequences present in SEQ ID NO: 67; (xx) the three heavy chain CDR sequences present in SEQ ID NO: 68 and the three light chain CDR sequences present in SEQ ID NO: 69; (xxi) the three heavy chain CDR sequences present in SEQ ID NO: 70 and the three light chain CDR sequences present in SEQ ID NO: 71; (xxii) the three heavy chain CDR sequences present in SEQ ID NO: 72 and the three light chain CDR sequences present in SEQ ID NO: 73; (xxiii) the three heavy chain CDR sequences present in SEQ ID NO: 74 and the three light chain CDR sequences present in SEQ ID NO: 75; or (xxiv) the three heavy chain CDR sequences present in SEQ ID NO: 76 and the three light chain CDR sequences present in SEQ ID NO: 77.

49. The T-cell of any one of claims 34-42, wherein the binding agent is a chimeric antigen receptor, wherein the chimeric antigen receptor comprises an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain comprises an antibody or antigen-binding domain capable of binding to an antigen on an autoimmune cell, and wherein the intracellular domain comprises a cytoplasmic signaling domain and one or more co-stimulatory domains.

50. The T-cell of claim 49, wherein the antigen-binding domain is an antigen-binding fragment selected from the group consisting of a Fab, a F(ab).sub.2 fragment, a scFV, a scAb, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody.

51. The T-cell of claim 50, wherein the antigen-binding fragment is a scFv that is capable of binding to a cell adhesion molecule.

52. The T-cell of claim 51, wherein the cell adhesion molecule is MADCAM-1.

53. The T-cell of claim 52, wherein the antigen-binding fragment comprises an amino acid sequence at least 80% identical to a sequence selected from the group consisting of SEQ ID NOs: 6-29.

54. The T-cell of claim 49, wherein the cytoplasmic signaling domain is a CD3 zeta domain.

55. The T-cell of claim 49, wherein the co-stimulatory domain comprises at least one of a CD48, 4-1BB, ICOS, X-40, and CD27 domain.

56. The T-cell of any one of claims 42-68, wherein the T-cell further comprises an additional nucleic acid sequence encoding a therapeutic gene product, wherein the additional nucleic acid sequence is operably linked to a promoter.

57. A composition comprising a T-cell of any one of claims 34-56.

58. A method of producing a T-cell population expressing an exogenous FOXP3 polypeptide and a receptor polypeptide, the method comprising culturing a T-cell of any one of claims 32 and 34-56 in growth media under conditions sufficient to expand the population of T-cells.

59. A population of T-cells prepared by the method of claim 58.

60. A composition comprising the population of T-cells of claim 59.

61. A vector comprising a first nucleic acid sequence encoding a FOXP3 polypeptide and a second nucleic acid sequence encoding a binding agent.

62. The vector of claim 61, wherein the first nucleic acid sequence comprises a mutation that results in nuclear localization of the FOXP3 polypeptide.

63. The vector of claim 61, wherein the first nucleic acid sequence encoding a FOXP3 polypeptide comprises a mutation in exon 2, wherein the FOXP3 polypeptide comprising a mutation in exon 2 results in increased nuclear localization of the FOXP3 polypeptide as compared to a FOXP3 polypeptide comprising an exon 2 that does not include a mutation.

64. The vector of claim 61, wherein the mutation comprises deletion of exon 2.

65. The vector of any one of claims 61-64, wherein the first nucleic acid sequence is 5 positioned relative to the second nucleic acid in the vector.

66. The vector of claim 65, wherein the first nucleic acid sequence is operably linked to a promoter.

67. The vector of claim 66, wherein the vector further comprises an additional nucleic acid sequence between the first nucleic acid sequence and the second nucleic acid sequence, wherein the additional nucleic acid sequence operably links the second nucleic acid sequence to the first nucleic acid sequence.

68. The vector of any one of claims 61-64, wherein the second nucleic acid sequence is 5 positioned relative to the first nucleic acid in the vector.

69. The vector of claim 68, wherein the second nucleic acid sequence is operably linked to a promoter.

70. The vector of any one of claims 61-69, wherein the vector further comprises a third nucleic acid sequence encoding a receptor polypeptide.

71. The vector of claim 70, wherein the third sequence is operably linked to the first sequence and/or the second sequence.

72. The vector of claim 70 or 71, wherein the third sequence is operably linked to a promoter.

73. The vector of any one of claims 70-72, wherein the receptor polypeptide is a chemokine receptor polypeptide.

74. The vector of claim 73, wherein the chemokine receptor polypeptide is CCR6, CCR9, or GRP15.

75. The vector of any one of claims 61-74, wherein the binding agent is an antibody or antigen-binding domain.

76. The vector of claim 75, wherein the antigen-binding domain is an antigen-binding fragment selected from the group consisting of a Fab, a F(ab).sub.2 fragment, a scFV, a scAb, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody.

77. The vector of claim 75, wherein the antigen-binding fragment is a scFv that is capable of binding to an antigen on an autoimmune cell or a cell adhesion molecule.

78. The vector of claim 77, wherein the cell adhesion molecule is MADCAM-1.

79. The vector of claim 78, wherein the antigen-binding fragment comprises an amino acid sequence at least 80% identical to a sequence selected from the group consisting of SEQ ID NOs: 6-29.

80. The vector of claim 78, wherein the antigen-binding fragment comprises: (i) the three heavy chain CDR sequences present in SEQ ID NO: 30 and the three light chain CDR sequences present in SEQ ID NO: 31; (ii) the three heavy chain CDR sequences present in SEQ ID NO: 32 and the three light chain CDR sequences present in SEQ ID NO: 33; (iii) the three heavy chain CDR sequences present in SEQ ID NO: 34 and the three light chain CDR sequences present in SEQ ID NO: 35; (iv) the three heavy chain CDR sequences present in SEQ ID NO: 36 and the three light chain CDR sequences present in SEQ ID NO: 37; (v) the three heavy chain CDR sequences present in SEQ ID NO: 38 and the three light chain CDR sequences present in SEQ ID NO: 39; (vi) the three heavy chain CDR sequences present in SEQ ID NO: 40 and the three light chain CDR sequences present in SEQ ID NO: 41; (vii) the three heavy chain CDR sequences present in SEQ ID NO: 42 and the three light chain CDR sequences present in SEQ ID NO: 43; (viii) the three heavy chain CDR sequences present in SEQ ID NO: 44 and the three light chain CDR sequences present in SEQ ID NO: 45; (ix) the three heavy chain CDR sequences present in SEQ ID NO: 46 and the three light chain CDR sequences present in SEQ ID NO: 47; (x) the three heavy chain CDR sequences present in SEQ ID NO: 48 and the three light chain CDR sequences present in SEQ ID NO: 49; (xi) the three heavy chain CDR sequences present in SEQ ID NO: 50 and the three light chain CDR sequences present in SEQ ID NO: 51; (xii) the three heavy chain CDR sequences present in SEQ ID NO: 52 and the three light chain CDR sequences present in SEQ ID NO: 53; (xiii) the three heavy chain CDR sequences present in SEQ ID NO: 54 and the three light chain CDR sequences present in SEQ ID NO: 55; (xiv) the three heavy chain CDR sequences present in SEQ ID NO: 56 and the three light chain CDR sequences present in SEQ ID NO: 57; (xv) the three heavy chain CDR sequences present in SEQ ID NO: 58 and the three light chain CDR sequences present in SEQ ID NO: 59; (xvi) the three heavy chain CDR sequences present in SEQ ID NO: 60 and the three light chain CDR sequences present in SEQ ID NO: 61; (xvii) the three heavy chain CDR sequences present in SEQ ID NO: 62 and the three light chain CDR sequences present in SEQ ID NO: 63; (xviii) the three heavy chain CDR sequences present in SEQ ID NO: 64 and the three light chain CDR sequences present in SEQ ID NO: 65; (xix) the three heavy chain CDR sequences present in SEQ ID NO: 66 and the three light chain CDR sequences present in SEQ ID NO: 67; (xx) the three heavy chain CDR sequences present in SEQ ID NO: 68 and the three light chain CDR sequences present in SEQ ID NO: 69; (xxi) the three heavy chain CDR sequences present in SEQ ID NO: 70 and the three light chain CDR sequences present in SEQ ID NO: 71; (xxii) the three heavy chain CDR sequences present in SEQ ID NO: 72 and the three light chain CDR sequences present in SEQ ID NO: 73; (xxiii) the three heavy chain CDR sequences present in SEQ ID NO: 74 and the three light chain CDR sequences present in SEQ ID NO: 75; or (xxiv) the three heavy chain CDR sequences present in SEQ ID NO: 76 and the three light chain CDR sequences present in SEQ ID NO: 77.

81. The vector of any one of claims 61-74, wherein the binding agent is a chimeric antigen receptor, wherein the chimeric antigen receptor comprises an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain comprises an antibody or antigen-binding domain capable of binding to an antigen on an autoimmune cell, and wherein the intracellular domain comprises a cytoplasmic signaling domain and one or more co-stimulatory domains.

82. The vector of claim 81, wherein the antigen-binding domain is an antigen-binding fragment selected from the group consisting of a Fab, a F(ab).sub.2 fragment, a scFV, a scAb, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody.

83. The vector of claim 82, wherein the antigen-binding fragment is a scFv that is capable of binding to an antigen on an autoimmune cell or a cell adhesion molecule.

84. The vector of claim 83, wherein the cell adhesion molecule is MADCAM-1.

85. The vector of claim 83, wherein the antigen-binding fragment comprises an amino acid sequence at least 80% identical to a sequence selected from the group consisting of SEQ ID NOs: 6-29.

86. The vector of claim 81, wherein the cytoplasmic signaling domain is a CD3 zeta domain.

87. The vector of claim 81, wherein the co-stimulatory domain comprises at least one of a CD48, 4-1BB, ICOS, X-40, and CD27 domain.

88. The vector of any one of claims 61-87, wherein the vector further comprises an additional nucleic acid sequence encoding a therapeutic gene product.

89. The vector of claim 88, wherein the additional nucleic sequence is operably linked a promoter.

90. The vector of any one of claims 88-89, wherein the additional sequence is operably linked to the first nucleic acid sequence, or the second nucleic acid sequence.

91. The vector of any one of claims 88-90, wherein the additional nucleic acid sequence is operably linked to the first nucleic acid sequence, the second nucleic acid sequence or the third nucleic acid sequence.

92. The vector of any one of claims 61-91, wherein the vector comprises a viral vector selected from the group consisting of a lentiviral vector, a retroviral vector, an adenoviral vector, or an adeno-associated viral (AAV) vector.

93. The vector of claim 92, wherein the viral vector is a lentiviral vector.

94. A composition comprising the vector of any one of claims 61-93.

95. A kit comprising the composition of any one of claims 33, 57, 60, and 94.

96. A method of treating an autoimmune disease or disorder in a patient comprising administering a T-cell of any one of claims 32 and 34-56, or a composition of any one of claims 33, 57, 60, and 94.

97. The method of claim 96, wherein the subject is previously diagnosed or identified as having an autoimmune disease or disorder.

98. The method of claim 97, wherein the autoimmune disease or disorder is lupus, rheumatoid arthritis, multiple sclerosis, insulin dependent diabetes mellitis, myasthenia gravis, Graves disease, autoimmune hemolytic anemia, autoimmune thrombocytopenia purpura, Goodpasture's syndrome, pemphigus vulgaris, acute rheumatic fever, post-streptococcal glomerulonephritis, Crohn's disease, Celiac disease, or polyarteritis nodosa.

99. The method of claim 96, wherein administering the autologous or allogenic T-cell population comprises intravenous injection or intravenous infusion.

100. The method of claim 96, wherein the administering results in amelioration of one or more symptoms of the autoimmune disease or disorder.

101. A polypeptide comprising an antigen-binding domain comprising: an amino acid sequence at least 80% identical to a sequence selected from the group consisting of SEQ ID NOs: 6-29.

102. A polypeptide comprising an antigen-binding domain comprising: (i) the three heavy chain CDR sequences present in SEQ ID NO: 30 and the three light chain CDR sequences present in SEQ ID NO: 31; (ii) the three heavy chain CDR sequences present in SEQ ID NO: 32 and the three light chain CDR sequences present in SEQ ID NO: 33; (iii) the three heavy chain CDR sequences present in SEQ ID NO: 34 and the three light chain CDR sequences present in SEQ ID NO: 35; (iv) the three heavy chain CDR sequences present in SEQ ID NO: 36 and the three light chain CDR sequences present in SEQ ID NO: 37; (v) the three heavy chain CDR sequences present in SEQ ID NO: 38 and the three light chain CDR sequences present in SEQ ID NO: 39; (vi) the three heavy chain CDR sequences present in SEQ ID NO: 40 and the three light chain CDR sequences present in SEQ ID NO: 41; (vii) the three heavy chain CDR sequences present in SEQ ID NO: 42 and the three light chain CDR sequences present in SEQ ID NO: 43; (viii) the three heavy chain CDR sequences present in SEQ ID NO: 44 and the three light chain CDR sequences present in SEQ ID NO: 45; (ix) the three heavy chain CDR sequences present in SEQ ID NO: 46 and the three light chain CDR sequences present in SEQ ID NO: 47; (x) the three heavy chain CDR sequences present in SEQ ID NO: 48 and the three light chain CDR sequences present in SEQ ID NO: 49; (xi) the three heavy chain CDR sequences present in SEQ ID NO: 50 and the three light chain CDR sequences present in SEQ ID NO: 51; (xii) the three heavy chain CDR sequences present in SEQ ID NO: 52 and the three light chain CDR sequences present in SEQ ID NO: 53; (xiii) the three heavy chain CDR sequences present in SEQ ID NO: 54 and the three light chain CDR sequences present in SEQ ID NO: 55; (xiv) the three heavy chain CDR sequences present in SEQ ID NO: 56 and the three light chain CDR sequences present in SEQ ID NO: 57; (xv) the three heavy chain CDR sequences present in SEQ ID NO: 58 and the three light chain CDR sequences present in SEQ ID NO: 59; (xvi) the three heavy chain CDR sequences present in SEQ ID NO: 60 and the three light chain CDR sequences present in SEQ ID NO: 61; (xvii) the three heavy chain CDR sequences present in SEQ ID NO: 62 and the three light chain CDR sequences present in SEQ ID NO: 63; (xviii) the three heavy chain CDR sequences present in SEQ ID NO: 64 and the three light chain CDR sequences present in SEQ ID NO: 65; (xix) the three heavy chain CDR sequences present in SEQ ID NO: 66 and the three light chain CDR sequences present in SEQ ID NO: 67; (xx) the three heavy chain CDR sequences present in SEQ ID NO: 68 and the three light chain CDR sequences present in SEQ ID NO: 69; (xxi) the three heavy chain CDR sequences present in SEQ ID NO: 70 and the three light chain CDR sequences present in SEQ ID NO: 71; (xxii) the three heavy chain CDR sequences present in SEQ ID NO: 72 and the three light chain CDR sequences present in SEQ ID NO: 73; (xxiii) the three heavy chain CDR sequences present in SEQ ID NO: 74 and the three light chain CDR sequences present in SEQ ID NO: 75; or (xxiv) the three heavy chain CDR sequences present in SEQ ID NO: 76 and the three light chain CDR sequences present in SEQ ID NO: 77.

Description

DESCRIPTION OF THE DRAWINGS

[0041] FIG. 1 is a diagram showing an exemplary T-cell with enforced expression of a FOXP3 polypeptide. Enforced expression of a FOXP3 polypeptide results in a core Treg suppressive program (e.g., IL-2 consumption and increase in CD25 expression, an increase in adenosine, an increase in CD39 expression, and expression of CTLA-4).

[0042] FIG. 2 is a diagram showing an exemplary T-cell with enforced expression of a FOXP3 polypeptide and a therapeutic gene product. Expression of a therapeutic gene product in addition to a FOXP3 polypeptide can result in enhancement of a core Treg program. Examples of suitable therapeutic gene products include, without limitation, IL6R scFv, IFN?R scFv, IL-10, IL-4, IL-13, and any anti-fibrotic protein.

[0043] FIG. 3 is a diagram showing an exemplary T-cell with enforced expression of a FOXP3 polypeptide and a receptor polypeptide (e.g., a chemokine receptor polypeptide).

[0044] FIG. 4 is a diagram showing an exemplary T-cell with enforced expression of a FOXP3 polypeptide and a receptor polypeptide (e.g., a chimeric antigen receptor polypeptide).

[0045] FIG. 5 is a schematic showing an exemplary workflow for identifying anti-MADCAM-1 scFvs.

[0046] FIG. 6 is a plot showing the binding affinities (K.sub.D)(M) for a set of anti-MADCAM-1 scFvs. Each circle represents a different anti-MADCAM-1 scFv.

DETAILED DESCRIPTION

[0047] Provided herein are methods and materials that can be used to treat mammals identified as having an autoimmune disease. Provided herein are methods for increasing T-cell function that include introducing into a T-cell a first nucleic acid sequence encoding a FOXP3 polypeptide and a second nucleic acid sequence encoding a binding agent. Also provided are T-cells produced by any of the methods described herein. Also provided herein are T-cells that include: a first nucleic acid sequence encoding a FOXP3 polypeptide and a second nucleic acid sequence encoding a binding agent. Also provided herein are vectors that include a first nucleic acid sequence encoding a FOXP3 polypeptide and a second nucleic acid sequence encoding a binding agent. Also provided herein are methods and materials for treating a mammal having an autoimmune disease, where the methods include administering to the mammal an effective amount of a T-cell (e.g., any of the T-cells described herein).

FOXP3 Polypeptides

[0048] In some embodiments, a first nucleic acid sequence encoding a FOXP3 polypeptide having one or more mutations is introduced into a T-cell (e.g., CD4.sup.+ T-cell, CD4.sup.+CD45RA.sup.+ T-cell, CD4.sup.+CD62L.sup.+ T-cell, or central memory T-cell). For example, a mutation in the first nucleic acid sequence encoding a FOXP3 polypeptide can include, without limitation, mutations that result in an amino acid substitution that changes the sub-cellular localization (e.g., increased nuclear localization) of the encoded FOXP3 polypeptide.

[0049] In some cases, a cell (e.g., a CD4.sup.+ T-cell) with a FOXP3 polypeptide-dependent expression profile can have increased immunosuppressive function. For example, a cell transduced with a FOXP3 polypeptide having one or more amino acid substitutions, amino acid insertions, and/or amino acid substitutions as described herein can have increased expression of genes that are transcriptional targets of a FOXP3. Increased expression of these genes (e.g., Il-2, Ctla-4, and Tnfrsf18) can result in increased Treg cell function (e.g., inhibition of responder cell proliferation).

[0050] In some embodiments, a FOXP3 polypeptide can have one or more amino acid substitutions, amino acid insertions, and/or amino acid deletions within an amino acid sequence of exon 2. In some embodiments, the amino acid deletion includes deletion of all or part of the amino acid sequence corresponding to exon 2. In some embodiments, a FOXP3 polypeptide having the amino acid sequence corresponding to an exon 2-deleted from the FOXP3 polypeptide (FOXP3d2) can result in an increased nuclear localization of the FOXP3 polypeptide. FOXP3 polypeptides harboring any one or more amino acid deletions in the amino acid sequence exon 2 can sequestered to the nucleus (or show increased nuclear localization as compared to a FOXP3 polypeptide not including one or more amino acid deletions in the amino acid sequence of exon 2).

[0051] As used herein, FOXP3 refers to the FOXP3 gene or protein that is a transcription factor in the Forkhead box (Fox) family of transcription factors (Sakaguchi et al., Int'l Immun. 21(10):1105-1111, 2009; Pandiyan, et al., Cytokine 76(1):13-24, 2015), or a variant thereof (e.g., a FOXP3 protein having one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty) amino acid substitutions, amino acid deletions, or amino acid insertions as compared to a wildtype FOXP3 protein). In some embodiments, when preparing a T-cell to be used in the treatment of a mammal having an autoimmune disease by administering to the mammal the T-cell, FOXP3 refers to human FOXP3 or a variant thereof. An example of a wildtype human FOXP3 polypeptide includes, without limitation, NCBI reference sequence: NP 001107849.1 or a functional fragment thereof.

[0052] In some embodiments referring to a FOXP3 polypeptide, the amino acid sequence of the FOXP3 polypeptide is at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to:

TABLE-US-00001 (SEQIDNO:1) MPNPRPGKPSAPSLALGPSPGASPSWRAAPKASDL LGARGPGGTFQGRDLRGGAHASSSSLNPMPPSQLQ LPTLPLVMVAPSGARLGPLPHLQALLQDRPHFMHQ LSTVDAHARTPVLQVHPLESPAMISLTPPTTATGV FSLKARPGLPPGINVASLEWVSREPALLCTFPNPS APRKDSTLSAVPQSSYPLLANGVCKWPGCEKVFEE PEDFLKHCQADHLLDEKGRAQCLLQREMVQSLEQQ LVLEKEKLSAMQAHLAGKMALTKASSVASSDKGSC CIVAAGSQGPVVPAWSGPREAPDSLFAVRRHLWGS HGNSTFPEFLHNMDYFKFHNMRPPFTYATLIRWAI LEAPEKQRTLNEIYHWFTRMFAFFRNHPATWKNAI RHNLSLHKCFVRVESEKGAVWTVDELEFRKKRSQR PSRCSNPTPGP.

[0053] In some embodiments referring to a first nucleic acid sequence encoding a FOXP3 (e.g., full length FOXP3) polypeptide, the nucleic acid sequence is at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to:

TABLE-US-00002 (SEQIDNO:2) ATGCCTAACCCCCGCCCTGGAAAACCATCTGCCCC TTCACTGGCCCTGGGACCTTCACCCGGAGCCTCAC CATCTTGGAGAGCCGCCCCCAAGGCCAGCGACCTG CTGGGAGCCAGAGGCCCCGGCGGCACCTTCCAGGG CAGGGATCTGCGCGGCGGCGCCCACGCCAGCTCCT CTAGCCTGAACCCCATGCCCCCTTCTCAGCTCCAG CTGCCCACACTGCCCCTGGTCATGGTGGCACCTAG CGGAGCAAGGCTGGGACCACTGCCACACCTCCAGG CCCTGCTCCAGGACAGACCTCACTTTATGCACCAG CTGTCCACCGTGGATGCACACGCAAGGACACCCGT GCTCCAGGTGCACCCTCTGGAGTCTCCAGCCATGA TCAGCCTGACCCCACCAACCACAGCAACAGGCGTG TTCTCCCTGAAGGCCAGACCTGGCCTGCCTCCAGG CATCAACGTGGCCTCCCTGGAGTGGGTGTCTAGGG AGCCAGCCCTGCTGTGCACCTTTCCTAATCCATCT GCCCCCCGCAAGGACTCCACACTGTCTGCCGTGCC ACAGTCCTCTTACCCCCTGCTGGCCAACGGCGTGT GCAAGTGGCCTGGCTGTGAGAAGGTGTTCGAGGAG CCAGAGGATTTTCTGAAGCACTGCCAGGCCGACCA CCTGCTGGATGAGAAGGGAAGGGCACAGTGTCTGC TCCAGAGGGAGATGGTGCAGAGCCTGGAGCAGCAG CTGGTGCTGGAGAAGGAGAAGCTGTCCGCCATGCA GGCACACCTGGCAGGCAAGATGGCACTGACCAAGG CCAGCTCCGTGGCCTCTAGCGACAAGGGCAGCTGC TGTATCGTGGCCGCCGGCTCCCAGGGACCAGTGGT GCCCGCCTGGTCTGGACCCAGGGAGGCACCTGACA GCCTGTTCGCCGTGCGGAGACACCTGTGGGGCAGC CACGGCAATTCCACCTTCCCCGAGTTTCTGCACAA CATGGATTACTTCAAGTTTCACAATATGCGGCCCC CTTTTACCTATGCCACACTGATCAGATGGGCCATC CTGGAGGCCCCAGAGAAGCAGCGCACCCTGAACGA AATCTACCACTGGTTCACACGGATGTTTGCCTTCT TTAGAAATCACCCCGCCACCTGGAAGAACGCCATC AGGCACAATCTGTCCCTGCACAAGTGTTTCGTGCG CGTGGAGTCTGAGAAGGGCGCCGTGTGGACAGTGG ATGAGCTGGAGTTCAGAAAGAAGAGAAGCCAGAGA CCATCCAGGTGTTCAAACCCTACCCCAGGACCC.

[0054] In some embodiments, referring to a nucleic acid sequence encoding a FOXP3 polypeptide having a mutation in exon 2, the nucleic acid sequence corresponding to FOXP3 exon 2 is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to: CCTGCCCTTGGACAAGGACCCGATGCCCAACCCCAGGCCTGGCAAGCCCTCGGCCC CTTCCTTGGCCCTTGGCCCATCCCCAGGAGCCTCGCCCAGCTGGAGGGCTGCACCCA AAGCCTCAGACCTGCTGGGGGCCCGGGGCCCAGGGGGAACCTTCCAGGGCCGAGAT CTTCGAGGCGGGGCCCATGCCTCCTCTTCTTCCTTGAACCCCATGCCACCATCGCAG CTGCAG (SEQ ID NO: 3). In some embodiments referring to a nucleic acid sequence encoding a FOXP3 polypeptide having a deleted exon 2, the nucleic acid sequence that is deleted from full length FOXP3 polypeptide (SEQ ID NO: 2) is SEQ ID NO: 3 or a fragment of SEQ ID NO: 3.

Binding Agents

[0055] This document provides methods and materials for introducing into a T-cell (e.g., CD4.sup.+ T-cell, CD4.sup.+CD45RA.sup.+ T-cell, CD4.sup.+CD62L.sup.+ T-cell, or central memory T-cell) a first nucleic acid sequence encoding a FOXP3 polypeptide (e.g., any of the exemplary FOXP3 polypeptides described herein) and a second nucleic acid sequence encoding a binding agent (e.g., any of the exemplary binding agents described herein). This document also provides methods and materials for introducing into a T-cell (e.g., CD4.sup.+ T-cell, CD4.sup.+CD45RA.sup.+ T-cell, CD4.sup.+CD62L.sup.+ T-cell, or central memory T-cell) a first nucleic acid sequence encoding a FOXP3 polypeptide (e.g., any of the exemplary FOXP3 polypeptides described herein), a second nucleic acid sequence encoding a binding agent (e.g., any of the binding agents described herein), and a third nucleic acid sequence encoding a receptor polypeptide (e.g., any of the exemplary receptor polypeptides described herein). Also provided herein are methods and materials for introducing into a T-cell (e.g., CD4.sup.+ T-cells, CD4.sup.+CD45RA.sup.+ T-cells, CD4.sup.+CD62L.sup.+ T-cell, or central memory T-cells) a first nucleic acid sequence encoding a FOXP3 polypeptide (e.g., any of the exemplary FOXP3 polypeptides described herein), a second nucleic acid sequence encoding a binding agent (e.g., any of the exemplary anti-MADCAM-1 antigen binding fragments), a third nucleic acid sequence encoding a receptor polypeptide (e.g., any of the exemplary polypeptides described herein), and a fourth nucleic acid sequence encoding a therapeutic gene product (e.g., any of the exemplary therapeutic gene products described herein).

[0056] In some embodiments, upon administering a T-cell that includes a binding agent (e.g., any of the binding agents described herein) to a subject, the binding agent enables the T-cell to home to a particular location or tissue in the subject. In some embodiments, upon administering a T-cell that includes a binding agent to a subject, the binding agent increases the ability of the T-cell to home to a particular location or tissue in the subject as compared to a T-cell that does not include the binding agent (i.e., a T-cell where the binding agent was not introduced into the cell). In some embodiments, the T-cell that includes the binding agent has increased (e.g., at least a 5% increase, at least a 10% increase, at least a 20% increase, at least a 30% increase, at least a 40% increase, at least a 50% increase, at least a 60% increase, at least a 70% increase, at least a 80% increase, at least a 90% increase, at least a 100% increase, at least a 120% increase, at least a 140% increase, at least a 160% increase, at least a 180% increase, or at least a 200% increase) homing to a particular location or tissue in the subject as compared to a T-cell that does not include the binding agent.

[0057] As used herein, binding agent refers to any of a variety of polypeptides that bind with specificity to its binding partner. In some embodiments, a T-cell (e.g., a CD4.sup.+ T-cell, a CD4.sup.+CD45RA.sup.+ T-cell, a CD4.sup.+CD62L.sup.+ T-cell, or central memory T-cell) can be transduced with a nucleic acid sequence encoding a mutated FOXP3 polypeptide and a nucleic acid sequence encoding a binding agent as described herein and optionally, nucleic acid sequence(s) encoding a receptor polypeptide and/or a therapeutic gene product. In some embodiments, a binding agent can be any polypeptide that enhances the immunosuppressive effect of a T-cell (e.g., a CD4.sup.+ T-cell, a CD4.sup.+CD45RA.sup.+ T-cell, a CD4.sup.+CD62L.sup.+ T-cell, or central memory T-cell). In some embodiments, a binding agent can be a polypeptide that binds to molecules found specifically on autoimmune cells or tissues. In some embodiments, a binding agent can be a polypeptide that binds to molecules found specifically on endothelial cells. In some embodiments, a binding agent can be a polypeptide that binds to MADCAM-1. In some embodiments, MADCAM-1 is expressed on endothelial cells.

[0058] In some embodiments, a binding agent can be a chimeric antigen receptor (CAR) as described herein, where the CAR has an extracellular domain, a transmembrane domain, and an intracellular domain. In cases where the binding agent is a CAR, the extracellular domain includes a polypeptide capable of binding to a molecule found specifically on autoimmune cells or tissues, and the intracellular domain includes, e.g., a cytoplasmic signaling domain and one or more co-stimulatory domains. For example, the extracellular domain can include an scFv capable of binding to antigen on an autoimmune cell. In some embodiments, the scFv can bind to antigens found specifically on endothelial cells. In some embodiments, the scFv can bind to a polypeptide that binds to MADCAM-1.

[0059] As used herein, the term chimeric antigen receptor or CAR refers to a chimeric antigen receptor comprising an extraceullar domain, a transmembrane domain, and an intracellular domain. In some cases, the extracellular domain can comprise an antigen-binding domain as described herein. In some cases, the transmembrane domain can comprise a transmembrane domain derived from a natural polypeptide obtained from a membrane-binding or transmembrane protein. For example, a transmembrane domain can include, without limitation, a transmembrane domain from a T-cell receptor alpha or beta chain, a CD3 zeta chain, a CD28 polypeptide, or a CD8 polypeptide. In some cases, the intracellular domain can comprise a cytoplasmic signaling domain as described herein. In some cases, the intracellular domain can comprise a co-stimulatory domain as described herein.

[0060] In some embodiments, the CAR includes an extracellular domain having a scFv domain directed to MADCAM-1, a transmembrane domain, and an intracellular signaling domain. MADCAM-1 is expressed on endothelial cells and is responsive to numerous inflammatory mediators (see, e.g., Ando et al., BMC Physiol. 7:6331, 2007; Oshima et al., Cell Physiol. 281:C1096-C1105, 2001). MADCAM-1 can mediate both leukocyte adhesion and migration through the endothelium into tissues.

[0061] In some embodiments, the affinity of the anti-MADCAM-1 scFv is tuned to bind at high antigen density that exists in diseased tissues but avoid targeting healthy tissues with basal MADCAM-1 expression. In some embodiments, the MADCAM-1 scFv is coupled to a transmembrane domain which spans the membrane and is derived from a CD8 transmembrane domain (e.g., CD8a stalk and CD8a hinge). The intracellular signaling domain is comprised of the CD3zeta domain and an additional co-stimulatory domain such as CD28, 4-1BB, ICOS, OX-40, and/or CD27 to transmit a proliferative/survival signal upon antigen recognition.

[0062] In some embodiments, the antigen-binding domain is an antigen-binding fragment selected from the group consisting of a Fab, a F(ab).sub.2 fragment, a scFv, a scAb, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody. In some embodiments, the antigen-binding fragment is a scFv that is capable of binding to an antigen on an autoimmune cell. In some embodiments, the scFv is capable of binding to a cell adhesion molecule. In some embodiments, the cell adhesion molecule is MADCAM-1. In some embodiments, the cytoplasmic signaling domain is a CD3 zeta domain. In some embodiments, the co-stimulatory domain comprises at least one of a CD28, 4-1BB, ICOS, X-40, or CD27 domain.

[0063] In some embodiments where the chimeric antigen receptor polypeptide includes a CD3 zeta cytoplasmic signaling domain, the CD3 zeta cytoplasmic signaling domain has an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to: MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADA PAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDK MAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (NCBI Reference No.: NP_932170) (SEQ ID NO: 4) or a functional fragment thereof that has activating or stimulatory activity.

[0064] In some embodiments where the chimeric antigen receptor polypeptide includes a CD28 co-stimulatory domain, the CD28 co-stimulatory domain is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to:

TABLE-US-00003 (SEQIDNO:5) IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPG PSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKR SRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAY.

[0065] In some embodiments, a binding agent refers to an antibody, an antigen-binding domain, or an antigen-binding fragment thereof. As used herein, the term antibody, antigen-binding domain, or antigen-binding fragment refers to an intact immunoglobulin or to an antigen-binding portion thereof. In some embodiments, a binding agent refers to an intact immunoglobulin or to an antigen-binding portion thereof. Antigen-binding portions may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Examples of antigen-binding portions include Fab, Fab, F(ab).sub.2, Fv, domain antibodies (dAbs), and complementarity determining region (CDR) fragments, single-chain antibodies (scFv), chimeric antibodies, diabodies, triabodies, tetrabodies, and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen-binding to the polypeptide.

[0066] As used herein, the term scFv antibody fragments comprise the V.sub.H and V.sub.L domains of an antibody, wherein these domains are present in a single polypeptide chain. Included in the definition are single domain antibodies, including camelids. In some cases, the antibody or antigen-binding domain thereof is human or humanized.

[0067] As used herein, T-cell function refers to a T-cell's (e.g., any of the exemplary T-cells described herein) survival, stability, and/or ability to execute its intended function. For example, a CD4.sup.+ T-cell can have an immunosuppressive function. A CD4.sup.+ T-cell including a nucleic acid encoding a FOXP3 polypeptide can have a FOXP3-dependent expression profile that increases the immunosuppressive function of the T-cell. For example, a cell transduced with a mutated FOXP3 polypeptide as described herein can have increased expression of genes that are transcriptional targets of a FOXP3 that can result in increased Treg cell function.

[0068] As used herein, the term activation refers to induction of a signal on an immune cell (e.g., a B-cell or T-cell) that to results in initiation of the immune response (e.g., T-cell activation). In some cases, upon binding of an antigen to a T-cell receptor (TCR) or an exogenous chimeric antigen receptor (CAR), the immune cell can undergo changes in protein expression that result in the activation of the immune response. In some embodiments, upon activation of the CAR in T-cell (e.g., a T-cell that includes an exogenous FOXP3 polypeptide), the T-cell proliferates. In some cases, the proliferation of the T-cell following CAR activation is related to the signaling pathways activated CAR activation. In some cases, a TCR or CAR includes a cytoplasmic signaling sequence that can drive T-cell activation. For example, upon binding of the antigen, a chimeric antigen receptor comprising an intracellular domain that includes a cytoplasmic signaling sequence (e.g., an immune-receptor tyrosine-based inhibition motifs (ITAM)) that can be phosphorylated. A phosphorylated ITAM results in the induction of a T-cell activation pathway that ultimately results in a T-cell immune response. Examples of ITAMs include, without limitation, CD3 gamma, CD3 delta, CD3 epsilon, TCR zeta, FcR gamma, FcR beta, CD5, CD22, CD79a, and CD66d.

[0069] As used herein, the term stimulation refers to stage of TCR or CAR signaling where a co-stimulatory signal can be used to achieve a robust and sustained TCR or CAR signaling response. As described herein, a co-stimulatory domain can be referred to as a signaling domain. In some cases, a signaling domain (e.g., co-stimulatory domain) can be a CD27, CD28, OX40, CD30, CD40, B7-H3, NKG2C, LIGHT, CD7, CD2, 4-1BB, or PD-1.

[0070] As used herein, the terms binding, binds, or specifically binds in the context of the binding of an antibody or antigen binding fragment to a target antigen typically is a binding with an affinity corresponding to a K.sub.D of about 10.sup.?5 M or less (e.g., about 10.sup.?6 M or less, about 10.sup.?7 M or less, about 10.sup.?8 M or less, or about 10.sup.?9 M or less) when determined by surface plasmon resonance (SPR). As used herein, the term K.sub.D (M) refers to the dissociation equilibrium constant of a particular antibody-antigen interaction.

[0071] In some embodiments, any of the antigen-binding domains, antibodies, or binding agents further include a secretion signal peptide. For example, a nucleic acid sequence encoding a binding agent further includes a nucleic acid sequence encoding a secretion signal peptide. In some embodiments, a binding agent includes, without limitation, an antibody or antigen-binding fragment (e.g., a scFv) capable of binding to a cell adhesion molecule (e.g., without limitation, MADCAM-1).

[0072] As used herein, MADCAM-1 refers to mucosal addressin cell adhesion molecule 1 polypeptide. When preparing the T-cell or treating a mammal with the T-cell, MADCAM-1 refers to human MADCAM-1. An example of a human MADCAM-1 polypeptide includes, without limitation, NCBI reference sequence: NP_570116.2, or NP_570118.1, or a fragment thereof. In some embodiments, an anti-MADCAM-1 antibody or antigen-binding domain or an antigen-binding fragment thereof exhibits a binding affinity to MADCAM-1 that enables binding to and extravasation of the T-cell from the circulation into a tissue. The affinity of the anti-MADCAM-1 antibody or antibody binding fragment scFv is selected to bind to cells that express at high MADCAM-1 density that exists in diseased tissues but avoid targeting healthy tissues with basal MADCAM-1 expression.

[0073] In some embodiments, the antigen-binding domain or antigen-binding fragment comprises an amino acid sequence at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to a sequence selected from the group of SEQ ID NOs: 6-29. In some embodiments, the antigen-binding fragment comprising a sequence selected from the group SEQ ID NOs: 6-29 is altered to introduce one or more amino acid substitutions, insertions, or deletions. For example, the antigen-binding fragment selected from the group of SEQ ID NOs: 6-29 is mutated in such a manner as to enable increased binding affinity to a target antigen as compared to the binding affinity to an antigen that is not the target antigen. In another example, the antigen-binding fragment selected from the group of SEQ ID NOs: 6-29 is mutated in such a manner as to decrease binding affinity to a target antigen as compared to the binding affinity to an antigen that is not the target antigen. In some cases, the one or more amino acid substitutions, insertions, or deletions are located within a CDR region of the antigen-binding fragment. In some cases, one or more, two or more, three or more, four or more, five or more or six CDR regions include one or more amino acid substitutions, insertions, or deletions, as compared to the non-altered amino acid sequence (e.g., amino acid sequences as shown in SEQ ID NOs: 6-29).

[0074] As used herein, the term variable region or variable domain can refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable regions of the heavy chain and light chain of an antibody may be further subdivided into complementarity-determining regions (CDRs) and interspersed with regions that are more conserved, termed framework regions (FRs). The terms complementarity determining regions and CDRs, are known in the art to refer to non-contiguous sequences of amino acids within antibody variable regions, which confer antigen specificity and/or binding affinity.

[0075] In some embodiments, the antigen-binding domain includes a heavy chain variable domain that includes an amino acid sequence at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to SEQ ID NOs: 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, or 76. In some embodiments, the antigen-binding domain includes a light chain variable domain that includes an amino acid sequence at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to SEQ ID NOs: 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, or 77.

[0076] In some embodiments, the antigen-binding domain or antigen-binding fragment includes: [0077] a heavy chain variable domain comprising a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to SEQ ID NO: 30 and a light chain variable domain comprising a sequence that is at least 80% (e.g., at least 85%, at least 90, at least 95%, at least 99%, or 100%) identical to SEQ ID NO: 31; [0078] a heavy chain variable domain comprising a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to SEQ ID NO: 32 and a light chain variable domain comprising a sequence that is at least 80% (e.g., at least 85%, at least 90, at least 95%, at least 99%, or 100%) identical to SEQ ID NO: 33; [0079] a heavy chain variable domain comprising a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to SEQ ID NO: 34 and a light chain variable domain comprising a sequence that is at least 80% (e.g., at least 85%, at least 90, at least 95%, at least 99%, or 100%) identical to SEQ ID NO: 35; [0080] a heavy chain variable domain comprising a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to SEQ ID NO: 36 and a light chain variable domain comprising a sequence that is at least 80% (e.g., at least 85%, at least 90, at least 95%, at least 99%, or 100%) identical to SEQ ID NO: 37; [0081] a heavy chain variable domain comprising a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to SEQ ID NO: 38 and a light chain variable domain comprising a sequence that is at least 80% (e.g., at least 85%, at least 90, at least 95%, at least 99%, or 100%) identical to SEQ ID NO: 39; [0082] a heavy chain variable domain comprising a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to SEQ ID NO: 40 and a light chain variable domain comprising a sequence that is at least 80% (e.g., at least 85%, at least 90, at least 95%, at least 99%, or 100%) identical to SEQ ID NO: 41; [0083] a heavy chain variable domain comprising a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to SEQ ID NO: 42 and a light chain variable domain comprising a sequence that is at least 80% (e.g., at least 85%, at least 90, at least 95%, at least 99%, or 100%) identical to SEQ ID NO: 43; [0084] a heavy chain variable domain comprising a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to SEQ ID NO: 44 and a light chain variable domain comprising a sequence that is at least 80% (e.g., at least 85%, at least 90, at least 95%, at least 99%, or 100%) identical to SEQ ID NO: 45; [0085] a heavy chain variable domain comprising a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to SEQ ID NO: 46 and a light chain variable domain comprising a sequence that is at least 80% (e.g., at least 85%, at least 90, at least 95%, at least 99%, or 100%) identical to SEQ ID NO: 47; [0086] a heavy chain variable domain comprising a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to SEQ ID NO: 48 and a light chain variable domain comprising a sequence that is at least 80% (e.g., at least 85%, at least 90, at least 95%, at least 99%, or 100%) identical to SEQ ID NO: 49; [0087] a heavy chain variable domain comprising a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to SEQ ID NO: 50 and a light chain variable domain comprising a sequence that is at least 80% (e.g., at least 85%, at least 90, at least 95%, at least 99%, or 100%) identical to SEQ ID NO: 51; [0088] a heavy chain variable domain comprising a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to SEQ ID NO: 52 and a light chain variable domain comprising a sequence that is at least 80% (e.g., at least 85%, at least 90, at least 95%, at least 99%, or 100%) identical to SEQ ID NO: 53; [0089] a heavy chain variable domain comprising a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to SEQ ID NO: 54 and a light chain variable domain comprising a sequence that is at least 80% (e.g., at least 85%, at least 90, at least 95%, at least 99%, or 100%) identical to SEQ ID NO: 55; [0090] a heavy chain variable domain comprising a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to SEQ ID NO: 56 and a light chain variable domain comprising a sequence that is at least 80% (e.g., at least 85%, at least 90, at least 95%, at least 99%, or 100%) identical to SEQ ID NO: 57; [0091] a heavy chain variable domain comprising a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to SEQ ID NO: 58 and a light chain variable domain comprising a sequence that is at least 80% (e.g., at least 85%, at least 90, at least 95%, at least 99%, or 100%) identical to SEQ ID NO: 59; [0092] a heavy chain variable domain comprising a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to SEQ ID NO: 60 and a light chain variable domain comprising a sequence that is at least 80% (e.g., at least 85%, at least 90, at least 95%, at least 99%, or 100%) identical to SEQ ID NO: 61; [0093] a heavy chain variable domain comprising a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to SEQ ID NO: 62 and a light chain variable domain comprising a sequence that is at least 80% (e.g., at least 85%, at least 90, at least 95%, at least 99%, or 100%) identical to SEQ ID NO: 63; [0094] a heavy chain variable domain comprising a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to SEQ ID NO: 64 and a light chain variable domain comprising a sequence that is at least 80% (e.g., at least 85%, at least 90, at least 95%, at least 99%, or 100%) identical to SEQ ID NO: 65; [0095] a heavy chain variable domain comprising a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to SEQ ID NO: 66 and a light chain variable domain comprising a sequence that is at least 80% (e.g., at least 85%, at least 90, at least 95%, at least 99%, or 100%) identical to SEQ ID NO: 67; [0096] a heavy chain variable domain comprising a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to SEQ ID NO: 68 and a light chain variable domain comprising a sequence that is at least 80% (e.g., at least 85%, at least 90, at least 95%, at least 99%, or 100%) identical to SEQ ID NO: 69; [0097] a heavy chain variable domain comprising a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to SEQ ID NO: 70 and a light chain variable domain comprising a sequence that is at least 80% (e.g., at least 85%, at least 90, at least 95%, at least 99%, or 100%) identical to SEQ ID NO: 71; [0098] a heavy chain variable domain comprising a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to SEQ ID NO: 72 and a light chain variable domain comprising a sequence that is at least 80% (e.g., at least 85%, at least 90, at least 95%, at least 99%, or 100%) identical to SEQ ID NO: 73; [0099] a heavy chain variable domain comprising a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to SEQ ID NO: 74 and a light chain variable domain comprising a sequence that is at least 80% (e.g., at least 85%, at least 90, at least 95%, at least 99%, or 100%) identical to SEQ ID NO: 75; or [0100] a heavy chain variable domain comprising a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to SEQ ID NO: 76 and a light chain variable domain comprising a sequence that is at least 80% (e.g., at least 85%, at least 90, at least 95%, at least 99%, or 100%) identical to SEQ ID NO: 77.

[0101] In some embodiments, the antigen-binding domain or antigen-binding fragment includes an (i) the three heavy chain CDR sequences present in SEQ ID NO: 30 and the three light chain CDR sequences present in SEQ ID NO: 31; (ii) the three heavy chain CDR sequences present in SEQ ID NO: 32 and the three light chain CDR sequences present in SEQ ID NO: 33; (iii) the three heavy chain CDR sequences present in SEQ ID NO: 34 and the three light chain CDR sequences present in SEQ ID NO: 35; (iv) the three heavy chain CDR sequences present in SEQ ID NO: 36 and the three light chain CDR sequences present in SEQ ID NO: 37; (v) the three heavy chain CDR sequences present in SEQ ID NO: 38 and the three light chain CDR sequences present in SEQ ID NO: 39; (vi) the three heavy chain CDR sequences present in SEQ ID NO: 40 and the three light chain CDR sequences present in SEQ ID NO: 41; (vii) the three heavy chain CDR sequences present in SEQ ID NO: 42 and the three light chain CDR sequences present in SEQ ID NO: 43; (viii) the three heavy chain CDR sequences present in SEQ ID NO: 44 and the three light chain CDR sequences present in SEQ ID NO: 45; (ix) the three heavy chain CDR sequences present in SEQ ID NO: 46 and the three light chain CDR sequences present in SEQ ID NO: 47; (x) the three heavy chain CDR sequences present in SEQ ID NO: 48 and the three light chain CDR sequences present in SEQ ID NO: 49; (xi) the three heavy chain CDR sequences present in SEQ ID NO: 50 and the three light chain CDR sequences present in SEQ ID NO: 51; (xii) the three heavy chain CDR sequences present in SEQ ID NO: 52 and the three light chain CDR sequences present in SEQ ID NO: 53; (xiii) the three heavy chain CDR sequences present in SEQ ID NO: 54 and the three light chain CDR sequences present in SEQ ID NO: 55; (xiv) the three heavy chain CDR sequences present in SEQ ID NO: 56 and the three light chain CDR sequences present in SEQ ID NO: 57; (xv) the three heavy chain CDR sequences present in SEQ ID NO: 58 and the three light chain CDR sequences present in SEQ ID NO: 59; (xvi) the three heavy chain CDR sequences present in SEQ ID NO: 60 and the three light chain CDR sequences present in SEQ ID NO: 61; (xvii) the three heavy chain CDR sequences present in SEQ ID NO: 62 and the three light chain CDR sequences present in SEQ ID NO: 63; (xviii) the three heavy chain CDR sequences present in SEQ ID NO: 64 and the three light chain CDR sequences present in SEQ ID NO: 65; (xix) the three heavy chain CDR sequences present in SEQ ID NO: 66 and the three light chain CDR sequences present in SEQ ID NO: 67; (xx) the three heavy chain CDR sequences present in SEQ ID NO: 68 and the three light chain CDR sequences present in SEQ ID NO: 69; (xxi) the three heavy chain CDR sequences present in SEQ ID NO: 70 and the three light chain CDR sequences present in SEQ ID NO: 71; (xxii) the three heavy chain CDR sequences present in SEQ ID NO: 72 and the three light chain CDR sequences present in SEQ ID NO: 73; (xxiii) the three heavy chain CDR sequences present in SEQ ID NO: 74 and the three light chain CDR sequences present in SEQ ID NO: 75; or (xxiv) the three heavy chain CDR sequences present in SEQ ID NO: 76 and the three light chain CDR sequences present in SEQ ID NO: 77.

[0102] In some embodiments, an anti-MADCAM-1 antibody or antigen-binding domain or antigen-binding fragment thereof may also be described or specified in terms of their binding affinity to MADCAM-1 (e.g., human MADCAM-1). In some embodiments, preferred binding affinities to MADCAM-1 include those with a dissociation constant or K.sub.D of no greater than 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, 10 nM, 15 nM, 20 nM, 25 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 110 nM, 120 nM, 130 nM, 140 nM, 150 nM, 200 nM, 250 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1000 nM, 1100 nM, 1200 nM, 1300 nM, 1400 nM, 1500 nM, 1600 nM, 1700 nM, 1800 nM, 1900 nM, or 2000 nM. In some embodiments, an anti-MADCAM-1 antibody or antigen-binding domain or antigen-binding fragment thereof exhibits a binding affinity (K.sub.D) to MADCAM-1 that is between about 10 nM to about 1000 nM (e.g., about 10 nM to about 950 nM, about 10 nM to about 900 nM, about 10 nM to about 850 nM, about 10 nM to about 800 nM, about 10 nM to about 750 nM, about 10 nM to about 700 nM, about 10 nM to about 650 nM, about 10 nM to about 600 nM, about 10 nM to about 550 nM, about 10 nM to about 500 nM, about 10 nM to about 450 nM, about 10 nM to about 400 nM, about 10 nM to about 350 nM, about 10 nM to about 300 nM, about 10 nM to about 250 nM, about 10 nM to about 200 nM, about 10 nM to about 200 nM, about 10 nM to about 150 nM, about 10 nm to about 100 nM, about 10 nM to about 50 nm, about 50 nM to about 950 nM, about 50 nM to about 900 nM, about 50 nM to about 850 nM, about 50 nM to about 800 nM, about 50 nM to about 750 nM, about 50 nM to about 700 nM, about 50 nM to about 650 nM, about 50 nM to about 600 nM, about 50 nM to about 550 nM, about 50 nM to about 500 nM, about 50 nM to about 450 nM, about 50 nM to about 400 nM, about 50 nM to about 350 nM, about 50 nM to about 300 nM, about 50 nM to about 250 nM, about 50 nM to about 200 nM, about 50 nM to about 200 nM, about 50 nM to about 150 nM, about 50 nm to about 100 nM, about 100 nM to about 1000 nM, about 100 nM to about 950 nM, about 100 nM to about 900 nM, about 100 nM to about 850 nM, about 100 nM to about 800 nM, about 100 nM to about 750 nM, about 100 nM to about 700 nM, about 100 nM to about 650 nM, about 100 nM to about 600 nM, about 100 nM to about 550 nM, about 100 nM to about 500 nM, about 100 nM to about 450 nM, about 100 nM to about 400 nM, about 100 nM to about 350 nM, about 100 nM to about 300 nM, about 100 nM to about 250 nM, about 100 nM to about 200 nM, about 100 nM to about 200 nM, about 100 nM to about 150 nM, about 150 nM to about 1000 nM, about 150 nM to about 950 nM, about 150 nM to about 900 nM, about 150 nM to about 850 nM, about 150 nM to about 800 nM, about 150 nM to about 750 nM, about 150 nM to about 700 nM, about 150 nM to about 650 nM, about 150 nM to about 600 nM, about 150 nM to about 550 nM, about 150 nM to about 500 nM, about 150 nM to about 450 nM, about 150 nM to about 400 nM, about 150 nM to about 350 nM, about 150 nM to about 300 nM, about 150 nM to about 250 nM, about 150 nM to about 200 nM, about 200 nM to about 1000 nM, about 200 nM to about 950 nM, about 200 nM to about 900 nM, about 200 nM to about 850 nM, about 200 nM to about 800 nM, about 200 nM to about 750 nM, about 200 nM to about 700 nM, about 200 nM to about 650 nM, about 200 nM to about 600 nM, about 200 nM to about 550 nM, about 200 nM to about 500 nM, about 200 nM to about 450 nM, about 200 nM to about 400 nM, about 200 nM to about 350 nM, about 200 nM to about 300 nM, about 200 nM to about 250 nM, about 250 nM to about 1000 nM, about 250 nM to about 950 nM, about 250 nM to about 900 nM, about 250 nM to about 850 nM, about 250 nM to about 800 nM, about 250 nM to about 750 nM, about 250 nM to about 700 nM, about 250 nM to about 650 nM, about 250 nM to about 600 nM, about 250 nM to about 550 nM, about 250 nM to about 500 nM, about 250 nM to about 450 nM, about 250 nM to about 400 nM, about 250 nM to about 350 nM, about 250 nM to about 300 nM, about 300 nM to about 1000 nM, about 300 nM to about 950 nM, about 300 nM to about 900 nM, about 300 nM to about 850 nM, about 300 nM to about 800 nM, about 300 nM to about 750 nM, about 300 nM to about 700 nM, about 300 nM to about 650 nM, about 300 nM to about 600 nM, about 300 nM to about 550 nM, about 300 nM to about 500 nM, about 300 nM to about 450 nM, about 300 nM to about 400 nM, about 300 nM to about 350 nM, about 350 nM to about 1000 nM, about 350 nM to about 950 nM, about 350 nM to about 900 nM, about 350 nM to about 850 nM, about 350 nM to about 800 nM, about 350 nM to about 750 nM, about 350 nM to about 700 nM, about 350 nM to about 650 nM, about 350 nM to about 600 nM, about 350 nM to about 550 nM, about 350 nM to about 500 nM, about 350 nM to about 450 nM, about 350 nM to about 400 nM, about 400 nM to about 1000 nM, about 400 nM to about 950 nM, about 400 nM to about 900 nM, about 400 nM to about 850 nM, about 400 nM to about 800 nM, about 400 nM to about 750 nM, about 400 nM to about 700 nM, about 400 nM to about 650 nM, about 400 nM to about 600 nM, about 400 nM to about 550 nM, about 400 nM to about 500 nM, about 400 nM to about 450 nM, about 450 nM to about 1000 nM, about 450 nM to about 950 nM, about 450 nM to about 900 nM, about 450 nM to about 850 nM, about 450 nM to about 800 nM, about 450 nM to about 750 nM, about 450 nM to about 700 nM, about 450 nM to about 650 nM, about 450 nM to about 600 nM, about 450 nM to about 550 nM, about 450 nM to about 500 nM, about 500 nM to about 1000 nM, about 500 nM to about 950 nM, about 500 nM to about 900 nM, about 500 nM to about 850 nM, about 500 nM to about 800 nM, about 500 nM to about 750 nM, about 500 nM to about 700 nM, about 500 nM to about 650 nM, about 500 nM to about 600 nM, about 500 nM to about 550 nM, about 550 nM to about 1000 nM, about 550 nM to about 950 nM, about 550 nM to about 900 nM, about 550 nM to about 850 nM, about 550 nM to about 800 nM, about 550 nM to about 750 nM, about 550 nM to about 700 nM, about 550 nM to about 650 nM, about 550 nM to about 600 nM, about 600 nM to about 1000 nM, about 600 nM to about 950 nM, about 600 nM to about 900 nM, about 600 nM to about 850 nM, about 600 nM to about 800 nM, about 600 nM to about 750 nM, about 600 nM to about 700 nM, about 600 nM to about 650 nM, about 650 nM to about 1000 nM, about 650 nM to about 950 nM, about 650 nM to about 900 nM, about 650 nM to about 850 nM, about 650 nM to about 800 nM, about 650 nM to about 750 nM, about 650 nM to about 700 nM, about 700 nM to about 1000 nM, about 700 nM to about 950 nM, about 700 nM to about 900 nM, about 700 nM to about 850 nM, about 700 nM to about 800 nM, about 700 nM to about 750 nM, about 750 nM to about 1000 nM, about 750 nM to about 950 nM, about 750 nM to about 900 nM, about 750 nM to about 850 nM, about 750 nM to about 800 nM, about 800 nM to about 1000 nM, about 800 nM to about 950 nM, about 800 nM to about 900 nM, about 800 nM to about 850 nM, about 850 nM to about 1000 nM, about 850 nM to about 950 nM, about 850 nM to about 900 nM, or about 900 to about 950 nM) (e.g., as measured by SPR).

[0103] In some embodiments, an anti-MADCAM-1 antibody or antigen-binding domain or antigen-binding fragment thereof exhibits a binding affinity (K.sub.D) to MADCAM-1 that is between about 0.1 nM to about 10 nM (e.g., about 0.1 nM to about 9 nM, about 0.1 nM to about 8 nM, about 0.1 nM to about 7 nM, about 0.1 nM to about 6 nM, about 0.1 nM to about 5 nM, about 0.1 nM to about 4 nM, about 0.1 nM to about 3 nM, about 0.1 nM to about 2 nM, about 0.1 nM to about 1 nM, about 0.1 nM to about 0.9 nM, about 0.1 nM to about 0.8 nM, about 0.1 nM to about 0.7 nM, about 0.1 nM to about 0.6 nM, about 0.1 nM to about 0.5 nM, about 0.1 nM to about 0.4 nM, about 0.1 nM to about 0.3 nM, about 0.1 nM to about 0.2 nM, about 0.2 nM to about 10 nM, about 0.2 nM to about 9 nM, about 0.2 nM to about 8 nM, about 0.2 nM to about 7 nM, about 0.2 nM to about 6 nM, about 0.2 nM to about 5 nM, about 0.2 nM to about 4 nM, about 0.2 nM to about 3 nM, about 0.2 nM to about 2 nM, about 0.2 nM to about 1 nM, about 0.2 nM to about 0.9 nM, about 0.2 nM to about 0.8 nM, about 0.2 nM to about 0.7 nM, about 0.2 nM to about 0.6 nM, about 0.2 nM to about 0.5 nM, about 0.2 nM to about 0.4 nM, about 0.2 nM to about 0.3 nM, about 0.3 nM to about 10 nM, about 0.3 nM to about 9 nM, about 0.3 nM to about 8 nM, about 0.3 nM to about 7 nM, about 0.3 nM to about 6 nM, about 0.3 nM to about 5 nM, about 0.3 nM to about 4 nM, about 0.3 nM to about 3 nM, about 0.3 nM to about 2 nM, about 0.3 nM to about 1 nM, about 0.3 nM to about 0.9 nM, about 0.3 nM to about 0.8 nM, about 0.3 nM to about 0.7 nM, about 0.3 nM to about 0.6 nM, about 0.3 nM to about 0.5 nM, about 0.3 nM to about 0.4 nM, about 0.4 nM to about 10 nM, about 0.4 nM to about 9 nM, about 0.4 nM to about 8 nM, about 0.4 nM to about 7 nM, about 0.4 nM to about 6 nM, about 0.4 nM to about 5 nM, about 0.4 nM to about 4 nM, about 0.4 nM to about 3 nM, about 0.4 nM to about 2 nM, about 0.4 nM to about 1 nM, about 0.4 nM to about 0.9 nM, about 0.4 nM to about 0.8 nM, about 0.4 nM to about 0.7 nM, about 0.4 nM to about 0.6 nM, about 0.4 nM to about 0.5 nM, about 0.5 nM to about 10 nM, about 0.5 nM to about 9 nM, about 0.5 nM to about 8 nM, about 0.5 nM to about 7 nM, about 0.5 nM to about 6 nM, about 0.5 nM to about 5 nM, about 0.5 nM to about 4 nM, about 0.5 nM to about 3 nM, about 0.5 nM to about 2 nM, about 0.5 nM to about 1 nM, about 0.5 nM to about 0.9 nM, about 0.5 nM to about 0.8 nM, about 0.5 nM to about 0.7 nM, about 0.5 nM to about 0.6 nM, about 0.6 nM to about 10 nM, about 0.6 nM to about 9 nM, about 0.6 nM to about 8 nM, about 0.6 nM to about 7 nM, about 0.6 nM to about 6 nM, about 0.6 nM to about 5 nM, about 0.6 nM to about 4 nM, about 0.6 nM to about 3 nM, about 0.6 nM to about 2 nM, about 0.6 nM to about 1 nM, about 0.6 nM to about 0.9 nM, about 0.6 nM to about 0.8 nM, about 0.6 nM to about 0.7 nM, about 0.7 nM to about 10 nM, about 0.7 nM to about 9 nM, about 0.7 nM to about 8 nM, about 0.7 nM to about 7 nM, about 0.7 nM to about 6 nM, about 0.7 nM to about 5 nM, about 0.7 nM to about 4 nM, about 0.7 nM to about 3 nM, about 0.7 nM to about 2 nM, about 0.7 nM to about 1 nM, about 0.7 nM to about 0.9 nM, about 0.7 nM to about 0.8 nM, about 0.8 nM to about 10 nM, about 0.8 nM to about 9 nM, about 0.8 nM to about 8 nM, about 0.8 nM to about 7 nM, about 0.8 nM to about 6 nM, about 0.8 nM to about 5 nM, about 0.8 nM to about 4 nM, about 0.8 nM to about 3 nM, about 0.8 nM to about 2 nM, about 0.8 nM to about 1 nM, about 0.8 nM to about 0.9 nM, about 0.9 nM to about 10 nM, about 0.9 nM to about 9 nM, about 0.9 nM to about 8 nM, about 0.9 nM to about 7 nM, about 0.9 nM to about 6 nM, about 0.9 nM to about 5 nM, about 0.9 nM to about 4 nM, about 0.9 nM to about 3 nM, about 0.9 nM to about 2 nM, about 0.9 nM to about 1 nM, about 1 nM to about 10 nM, about 1 nM to about 9 nM, about 1 nM to about 8 nM, about 1 nM to about 7 nM, about 1 nM to about 6 nM, about 1 nM to about 5 nM, about 1 nM to about 4 nM, about 1 nM to about 3 nM, about 1 nM to about 2 nM, about 2 nM to about 10 nM, about 2 nM to about 9 nM, about 2 nM to about 8 nM, about 2 nM to about 7 nM, about 2 nM to about 6 nM, about 2 nM to about 5 nM, about 2 nM to about 4 nM, about 2 nM to about 3 nM, about 3 nM to about 10 nM, about 3 nM to about 9 nM, about 3 nM to about 8 nM, about 3 nM to about 7 nM, about 3 nM to about 6 nM, about 3 nM to about 5 nM, about 3 nM to about 4 nM, about 4 nM to about 10 nM, about 4 nM to about 9 nM, about 4 nM to about 8 nM, about 4 nM to about 7 nM, about 4 nM to about 6 nM, about 4 nM to about 5 nM, about 5 nM to about 10 nM, about 5 nM to about 9 nM, about 5 nM to about 8 nM, about 5 nM to about 7 nM, about 5 nM to about 6 nM, about 6 nM to about 10 nM, about 6 nM to about 9 nM, about 6 nM to about 8 nM, about 6 nM to about 7 nM, about 7 nM to about 10 nM, about 7 nM to about 9 nM, about 7 nM to about 8 nM, about 8 nM to about 10 nM, about 8 nM to about 9 nM, or about 9 nM to about 10 nM) (e.g., as measured by SPR).

[0104] In some embodiments, an anti-MADCAM-1 antibody or antigen-binding domain or antigen-binding fragment thereof exhibits a binding affinity (K.sub.D) to MADCAM-1 that is between about 1000 nM to about 2000 nM (e.g., about 1000 nM to about 1900 nM, about 1000 nM to about 1800 nM, about 1000 nM to about 1700 nM, about 1000 nM to about 1600 nM, about 1000 nM to about 1500 nM, about 1000 nM to about 1400 nM, about 1000 nM to about 1300 nM, about 1000 nM to about 1200 nM, about 1000 nM to about 1100 nM, about 1100 nM to about 2000 nM, about 1100 nM to about 1900 nM, about 1100 nM to about 1800 nM, about 1100 nM to about 1700 nM, about 1100 nM to about 1600 nM, about 1100 nM to about 1500 nM, about 1100 nM to about 1400 nM, about 1100 nM to about 1300 nM, about 1100 nM to about 1200 nM, about 1200 nM to about 2000 nM, about 1200 nM to about 1900 nM, about 1200 nM to about 1800 nM, about 1200 nM to about 1700 nM, about 1200 nM to about 1600 nM, about 1200 nM to about 1500 nM, about 1200 nM to about 1400 nM, about 1200 nM to about 1300 nM, about 1300 nM to about 2000 nM, about 1300 nM to about 1900 nM, about 1300 nM to about 1800 nM, about 1300 nM to about 1700 nM, about 1300 nM to about 1600 nM, about 1300 nM to about 1500 nM, about 1300 nM to about 1400 nM, about 1400 nM to about 2000 nM, about 1400 nM to about 1900 nM, about 1400 nM to about 1800 nM, about 1400 nM to about 1700 nM, about 1400 nM to about 1600 nM, about 1400 nM to about 1500 nM, about 1500 nM to about 2000 nM, about 1500 nM to about 1900 nM, about 1500 nM to about 1800 nM, about 1500 nM to about 1700 nM, about 1500 nM to about 1600 nM, about 1600 nM to about 2000 nM, 1600 nM to about 1900 nM, about 1600 nM to about 1800 nM, about 1600 nM to about 1700 nM, about 1700 nM to about 2000 nM, about 1700 nM to about 1900 nM, about 1700 nM to about 1800 nM, about 1800 nM to about 2000 nM, about 1800 nM to about 1900 nM, or about 1900 nM to about 2000 nM) (e.g., as measured by SPR).

[0105] In some embodiments, preferred binding affinities to MADCAM-1 include those with a dissociation constant or K.sub.D of no greater than 1 ?M, 2 ?M, 3 ?M, 4 ?M, 5 ?M, 6 ?M, 7 ?M, 8 ?M, 9 ?M, 10 ?M, 15 ?M, 20 ?M, 25 ?M, 30 ?M, 40 ?M, 50 ?M, 60 ?M, 70 ?M, 80 ?M, 90 ?M, 100 ?M, 110 ?M, 120 ?M, 130 ?M, 140 ?M, 150 ?M, 200 ?M, 250 ?M, 300 ?M, 400 ?M, 500 ?M, 600 ?M, 700 ?M, 800 ?M, 900 ?M, or 1000 ?M.

[0106] In some embodiments, an anti-MADCAM-1 antibody or antigen-binding domain or antigen-binding fragment thereof exhibits a binding affinity (K.sub.D) to MADCAM-1 that is between about 1 TM to about 100 TM (e.g., about 1 ?M to about 90 ?M, about 1 ?M to about 80 PM, about 1 ?M to about 70 ?M, about 1 ?M to about 60 ?M, about 1 ?M to about 50 ?M, about 1 ?M to about 40 ?M, about 1 ?M to about 30 ?M, about 1 ?M to about 20 ?M, about 1 ?M to about 10 ?M, about 10 ?M to about 100 ?M, about 10 ?M to about 90 ?M, about 10 ?M to about 80 ?M, about 10 ?M to about 70 ?M, about 10 ?M to about 60 ?M, about 10 ?M to about 50 ?M, about 10 ?M to about 40 ?M, about 10 ?M to about 30 ?M, about 10 ?M to about 20 ?M, about 20 ?M to about 100 ?M, about 20 ?M to about 90 ?M, about 20 ?M to about 80 ?M, about 20 ?M to about 70 ?M, about 20 ?M to about 60 ?M, about 20 ?M to about 50 ?M, about 20 ?M to about 40 ?M, about 20 ?M to about 30 ?M, about 30 ?M to about 100 PM, about 30 ?M to about 90 ?M, about 30 ?M to about 80 ?M, about 30 ?M to about 70 PM, about 30 ?M to about 60 ?M, about 30 ?M to about 50 ?M, about 30 ?M to about 40 ?M, about 40 ?M to about 100 ?M, about 40 ?M to about 90 ?M, about 40 ?M to about 80 ?M, about 40 ?M to about 70 ?M, about 40 ?M to about 60 ?M, about 40 ?M to about 50 ?M, about 50 ?M to about 100 ?M, about 50 ?M to about 90 ?M, about 50 ?M to about 80 ?M, about 50 ?M to about 70 PM, about 50 ?M to about 60 ?M, about 60 ?M to about 100 ?M, about 60 ?M to about 90 ?M, about 60 ?M to about 80 ?M, about 60 ?M to about 70 ?M, about 70 ?M to about 100 PM, about 70 ?M to about 90 ?M, about 70 ?M to about 80 ?M, about 80 ?M to about 100 ?M, about 80 ?M to about 90 ?M, or about 90 ?M to about 100 ?M (e.g., as measured by SPR).

Receptor Polypeptides

[0107] This document provides methods and materials for introducing into a T-cell (e.g., CD4.sup.+ T-cell, CD4.sup.+CD45RA.sup.+ T-cell, CD4.sup.+CD62L.sup.+ T-cell, or central memory T-cell) a nucleic acid sequence encoding a receptor or a homing receptor polypeptide (e.g., any of the exemplary receptor polypeptides described herein). In some embodiments, upon administering a T-cell that includes a receptor polypeptide to a subject, the receptor polypeptide increases the ability of the T-cell to home to a particular location or tissue in the subject as compared to a T-cell that does not include the receptor polypeptide (i.e., a T-cell where the receptor polypeptide was not introduced into the cell). For example, a receptor polypeptide can direct a CD4.sup.+/CD45RA.sup.+ T-cell to a particular tissue or area within a human where cells responsible for causing symptoms of an autoimmune disease are located. In some embodiments, the receptor polypeptide can be a chemokine receptor polypeptide, and a cell expressing the chemokine receptor can be chemotactically directed to a particular location (e.g., lymphoid tissue) within a mammal (e.g., a human).

[0108] In some embodiments, the T-cell that includes the receptor polypeptide has increased (e.g., at least a 5% increase, at least a 10% increase, at least a 20% increase, at least a 30% increase, at least a 40% increase, at least a 50% increase, at least a 60% increase, at least a 70% increase, at least a 80% increase, at least a 90% increase, at least a 100% increase, at least a 120% increase, at least a 140% increase, at least a 160% increase, at least a 180% increase, or at least a 200% increase) homing to a particular location or tissue in the subject as compared to a T-cell that does not include the receptor polypeptide.

[0109] Examples of chemokine receptors that can be transduced into a cell along with a FOXP3 polypeptide (e.g., any of the exemplary FOXP3 polypeptides described herein) include, without limitation, CCR6, CCR9, and GPR15. In some embodiments, a ligand for a chemokine receptor polypeptide can be present on the surface of a cell residing in a lymphoid organ (e.g., spleen, lymph nodes, bone marrow, and/or tonsils). In some embodiments, a ligand for a chemokine receptor polypeptide can be present on the surface of a cell residing in an inflamed peripheral tissue (e.g., lung) in a mammal (e.g., a human). For example, a T-cell (e.g., a CD4.sup.+ T-cell, a CD4.sup.+CD45RA.sup.+ T-cell, a CD4.sup.+CD62L.sup.+ T-cell, or central memory T-cell) transduced with a FOXP3 polypeptide (e.g., any of the exemplary FOXP3 polypeptides described herein) and a CXCR4 chemokine receptor polypeptide can be chemotactically directed to a CXCL12 ligand associated with inflammation in the bone marrow of a mammal. In another example, a T-cell (e.g., a CD4.sup.+ T-cell, a CD4.sup.+CD45RA.sup.+ T-cell, a CD4.sup.+CD62L.sup.+ T-cell, or central memory T-cell) transduced with a FOXP3 polypeptide (e.g., any of the exemplary FOXP3 polypeptides described herein) and a CCR6 chemokine receptor polypeptide can be directed to a particular tissue or area within a mammal where a CCL20 polypeptide is located (e.g., a ligand for the CCR6 chemokine receptor polypeptide). A CCL20 polypeptide can be associated with chronic inflammatory conditions (e.g., inflammatory bowel disease). In yet another example, a T-cell (e.g., a CD4.sup.+ T-cell, a CD4.sup.+CD45RA.sup.+ T-cell, a CD4.sup.+CD62L.sup.+ T-cell, or central memory T-cell) transduced with a FOXP3 polypeptide (e.g., any of the exemplary FOXP3 polypeptides described herein) and a CCR7 chemokine receptor polypeptide can be directed to a particular tissue or area within a mammal where a CCL19 and/or a CCL21 polypeptide are located, as CCL19 and CCL21 are ligands for the CCR7 chemokine receptor polypeptide. CCL19 and CCL20 polypeptides can be associated with chronic inflammatory conditions in the thymus and lymph nodes.

[0110] In some embodiments, a receptor polypeptide includes, without limitation, CCR6 polypeptide or a functional fragment thereof, a CCR9 polypeptide or a functional fragment thereof, and a GPR15 polypeptide or a functional fragment thereof.

[0111] As used herein, CCR6 refers to C-C motif chemokine receptor 6 polypeptide. When preparing the T-cell or treating a mammal with the T-cell, CCR6 refers to human CCR6. Non-limiting examples of a human CCR6 polypeptide include, without limitation, NCBI reference sequence: NP_004358.2 or a functional fragment thereof or NCBI reference sequence: NP_113597.2 or a functional fragment thereof.

[0112] As used herein, CCR9 refers to C-C motif chemokine receptor 9 polypeptide. When preparing the T-cell or treating a mammal with the T-cell, CCR9 refers to human CCR9. Non-limiting examples of a human CCR9 polypeptide include, without limitation, NCBI reference sequence: NP_001243298.1 or a functional fragment thereof; NCBI reference sequence: NP_001373376.1 or a functional fragment thereof; NCBI reference sequence: NP_001373377.1 or a functional fragment thereof; NCBI reference sequence: NP_006632.2 or a functional fragment thereof; or NCBI reference sequence: NP_112477.1 or a functional fragment thereof.

[0113] As used herein, GPR15 refers to G protein-coupled receptor 15 polypeptide. When preparing the T-cell or treating a mammal with the T-cell, GPR15 refers to human GPR15. An example of a human GPR15 polypeptide includes, without limitation, NCBI reference sequence: NP_005281.1 or a functional fragment thereof.

[0114] In some embodiments, the receptor polypeptide can be a GPR15 receptor polypeptide (e.g., a G protein-coupled receptor 15). For example, a cell (e.g., a CD4.sup.+ T-cells, a CD4.sup.+CD45RA.sup.+ T-cell, a CD4.sup.+CD62L.sup.+ T-cell, or central memory T-cell) transduced with a FOXP3 polypeptide (e.g., any of the exemplary FOXP3 polypeptides described herein) and a GPR15 receptor polypeptide can be directed to a particular tissues or area within a mammal where a GPR15L polypeptide is located (e.g., the colon).

Therapeutic Gene Products

[0115] This document provides methods and materials for introducing into a T-cell (e.g., CD4.sup.+ T-cell, CD4.sup.+CD45RA.sup.+ T-cell, CD4.sup.+CD62L.sup.+ T-cell, or central memory T-cell) a nucleic acid sequence encoding a therapeutic gene product (e.g., a nucleic acid sequence encoding a therapeutic gene product). Any appropriate therapeutic gene product that enhances the immunosuppressive effects of a T-cell (e.g., a CD4.sup.+ T-cell, a CD4.sup.+CD45RA.sup.+ T-cell, a CD4.sup.+CD62L.sup.+ T-cell, or central memory T-cell) can be used.

Tissue Targeting

[0116] Also described herein are T-cells that include a first nucleic acid sequence encoding a FOXP3 polypeptide and a second nucleic acid sequence encoding a binding agent (e.g., any of the exemplary binding agents described herein), and optionally, a nucleic acid encoding a receptor polypeptide (e.g., any of the exemplary receptor polypeptides described herein), where the T-cell homes to a specific location or specific tissue when administered to a subject. As used herein, the term homing refers the ability of a T-cell to locate and enter an environmental niche (e.g., a particular location, organ, or tissue). The migration can result from, without limitation, receptor-ligand interactions, cytokine release, tissue damage, inflammatory signals, or other chemotactic signals. The terms home, homes or homed can be used interchangeably with homing.

[0117] In some embodiments, a T-cell that includes a combination of binding agent (e.g., any of the exemplary binding agents described herein) and optionally, a further a receptor polypeptide (e.g., any of the exemplary receptor polypeptides described herein) that determines, at least in part, the specific location in the body of the subject to which the T-cell is homed after being administered to the subject. Non-limiting examples of combinations of receptor polypeptides and binding agents are listed in Table 1. Table 1 also lists non-limiting homing locations for the various combinations of receptor polypeptides and binding agents.

TABLE-US-00004 TABLE 1 Combinations of receptor polypeptides, binding agents, and homing locations. Receptor polypeptide Binding agent (scFv) Homing location CCR6 MADCAM-1 Intestine CCR9 MADCAM-1 Intestine GPR15 MADCAM-1 Colon

[0118] In some embodiments, a T-cell includes a first nucleic acid sequence encoding a FOXP3 polypeptide, a second nucleic acid sequence encoding a CXCR6 polypeptide, and a third nucleic acid encoding an anti-MADCAM-1 scFv, where the T-cell homes to any inflammatory site or inflamed tissue when administered to a subject.

[0119] In some embodiments, a T-cell includes a first nucleic acid sequence encoding a FOXP3 polypeptide, a second nucleic acid sequence encoding a CCR6 polypeptide, and a third nucleic acid encoding an anti-MADCAM-1 scFv, where the T-cell homes to the intestine when administered to a subject.

[0120] In some embodiments, a T-cell includes a first nucleic acid sequence encoding a FOXP3 polypeptide, a second nucleic acid sequence encoding a CCR9 polypeptide, and a third nucleic acid encoding an anti-MADCAM-1 scFv, where the T-cell homes to the intestine when administered to a subject.

[0121] In some embodiments, a T-cell includes a first nucleic acid sequence encoding a FOXP3 polypeptide, a second nucleic acid sequence encoding a GPR15 polypeptide, and a third nucleic acid encoding an anti-MADCAM-1 scFv, where the T-cell homes to the colon when administered to a subject.

[0122] In some embodiments, a T-cell includes a first nucleic acid sequence encoding a FOXP3 polypeptide, a second nucleic acid sequence encoding a CXCR5 polypeptide, and a third nucleic acid encoding an anti-MADCAM-1 scFv, where the T-cell homes to the germinal center when administered to a subject.

[0123] In some embodiments, a T-cell that includes a FOXP3 polypeptide and a receptor polypeptide (e.g., any of the exemplary receptor polypeptides described herein), where the receptor polypeptide determines, at least in part, the specific location in the body of the subject to which the T-cell is homed after being administered to the subject. In some embodiments, a T-cell that includes a FOXP3 polypeptide and a binding agent (e.g., any of the exemplary binding agents described herein), where the binding agent determines, at least in part, the specific location in the body of the subject to which the T-cell is homed after being administered to the subject.

Methods of Producing T-Cells

[0124] As described herein, any appropriate method of producing cells (e.g., T-cells) comprising a FOXP3 polypeptide and a binding agent can be used to generate the T-cells as described herein. In some embodiments, a cell (e.g., a T-cell) that is transduced with the nucleic acid sequences described herein is isolated from a mammal (e.g., a human) using any appropriate method (e.g., magnetic activated sorting or flow cytometry-mediated sorting). In some cases, nucleic acid sequences encoding a FOXP3 polypeptide and a binding agent can be transformed into a cell (e.g., a T-cell) along with nucleic acid sequences encoding a receptor polypeptide and/or a therapeutic gene product. For example, a T-cell can be made by transducing nucleic acid sequences encoding a FOXP3 polypeptide and a binding agent into a cell (e.g., a T-cell) using a lentivirus.

[0125] In another example, a T-cell can be made by transducing nucleic acid sequences encoding a FOXP3 polypeptide, a binding agent, and a receptor polypeptide into a cell (e.g., a T-cell) using a lentivirus. In yet another example, a T-cell can be made by co-transducing nucleic acid sequences encoding a FOXP3 polypeptide, a binding agent, a receptor polypeptide, and a therapeutic gene product into a cell (e.g., a T-cell) using a lentivirus. In all cases described herein, the nucleic acid sequences are operably linked to a promoter or are operably linked to other nucleic acid sequences using a self-cleaving 2A polypeptide or IRES sequence.

[0126] Methods of introducing nucleic acids and expression vectors into a cell (e.g., an eukaryotic cell) are known in the art. Non-limiting examples of methods that can be used to introduce a nucleic acid into a cell include lipofection, transfection, electroporation, microinjection, calcium phosphate transfection, dendrimer-based transfection, cationic polymer transfection, cell squeezing, sonoporation, optical transfection, impalefection, hydrodynamic delivery, magnetofection, viral transduction (e.g., adenoviral and lentiviral transduction), and nanoparticle transfection. As used herein, transformed and transduced are used interchangeably.

[0127] In some embodiments, the transformed cell can be an immune cell, an epithelial cell, an endothelial cell, or a stem cell. In some examples, an immune cell is a T-cell, and the detection of memory T-cells can include, e.g., the detection of the level of expression of one or more of CD45RO, CCR7, L-selectin (CD62L), CD44, CD45RA, integrin ?e?7, CD43, CD4, CD8, CD27, CD28, IL-7R?, CD95, IL-2R?, and LFA-1. Additional examples of T-cells that can be transduced are described herein.

Nucleic Acids/Vectors

[0128] Also provided herein are nucleic acids sequences that encode any of the polypeptides described herein. For example, nucleic acid sequences are included that encode for a FOXP3 polypeptide, a binding agent, a receptor polypeptide, and a therapeutic gene product. Also provided herein are vectors that include any of the nucleic acids encoding any of the polypeptides described herein (e.g., the polypeptides include, without limitation, a FOXP3 polypeptide, a binding agent, a receptor polypeptide, and a therapeutic gene product). Also provided herein is a set of vectors that include any of the nucleic acid sequences encoding any of the polypeptides described herein. For example, the set of vectors includes two vectors, three vectors, or four vectors where each vector includes one or more nucleic acids encoding any of the polypeptides described herein.

[0129] In some embodiments, the vector includes: a first nucleic acid sequence encoding a FOXP3 polypeptide; and a second nucleic acid sequence encoding a binding agent. In some embodiments, the vector includes: a first nucleic acid sequence encoding a FOXP3 polypeptide, a second nucleic acid sequence encoding a binding agent, and a nucleic acid sequence encoding a receptor polypeptide, where each nucleic acid sequence is operably linked to a promoter or operably linked to one of the other nucleic acid sequences in the vector.

[0130] In some embodiments, the vector includes: a first nucleic acid sequence encoding a FOXP3 polypeptide, a second nucleic acid sequence encoding a binding agent, a nucleic acid sequence encoding a receptor polypeptide, and a nucleic acid encoding a therapeutic gene product, where each nucleic acid sequence is operably linked to a promoter or operably linked to one of the other nucleic acid sequences in the vector.

[0131] Any of the vectors described herein can be an expression vector. For example, an expression vector can include a promoter sequence operably linked to the sequence encoding any of the polypeptides as described herein. Non-limiting examples of vectors include plasmids, transposons, cosmids, and viral vectors (e.g., any adenoviral vectors (e.g., pSV or pCMV vectors), adeno-associated virus (AAV) vectors, lentivirus vectors, and retroviral vectors), and any Gateway? vectors. In some cases, a vector can include sufficient cis-acting elements that supplement expression where the remaining elements needed for expression can be supplied by the host mammalian cell or in an in vitro expression system. Skilled practitioners will be capable of selecting suitable vectors and mammalian cells for making any of the T-cells as described herein. Any appropriate promoter (e.g., EF1 alpha) can be operably linked to any of the nucleic acid sequences described herein. Non-limiting examples of promoters to be used in any of the vectors or constructs described herein include EF1a, SFFV, PGK, CMV, CAG, UbC, MSCV, MND, EF1a hybrid, and/or CAG hybrid. As used herein, the term operably linked is well known in the art and refers to genetic components that are combined such that they carry out their normal functions. For example, a nucleic acid sequence is operably linked to a promoter when its transcription is under the control of the promoter. In another example, a nucleic acid sequence can be operably linked to other nucleic acid sequence by a self-cleaving 2A polypeptide or an internal ribosome entry site (IRES). In such cases, the self-cleaving 2A polypeptide allows the second nucleic acid sequence to be under the control of the promoter operably linked to the first nucleic acid sequence. The nucleic acid sequences described herein can be operably linked to a promoter. In some cases, the nucleic acid sequences described herein can be operably linked to any other nucleic acid sequence described herein using a self-cleaving 2A polypeptide or IRES. In some cases, the nucleic acid sequences are all included on one vector and operably linked either to a promoter upstream of the nucleic acid sequences or operably linked to the other nucleic acid sequences through a self-cleaving 2A polypeptide or an IRES.

Polypeptides

[0132] Also provided are polypeptides that include an antigen-binding domain comprising: an amino acid sequence at least 80% identical (e.g., at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to a sequence selected from the group of SEQ ID NOs: 6-29. In some embodiments, the polypeptide includes an antigen-binding domain that includes a heavy chain variable domain including an amino acid sequence at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to SEQ ID NOs: 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, or 76. In some embodiments, the polypeptide includes an antigen-binding domain that includes a light chain variable domain including an amino acid sequence at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to SEQ ID NOs: 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, or 77.

[0133] Also provided are polypeptides that include an antigen-binding domain comprising: (i) the three heavy chain CDR sequences present in SEQ ID NO: 30 and the three light chain CDR sequences present in SEQ ID NO: 31; (ii) the three heavy chain CDR sequences present in SEQ ID NO: 32 and the three light chain CDR sequences present in SEQ ID NO: 33; (iii) the three heavy chain CDR sequences present in SEQ ID NO: 34 and the three light chain CDR sequences present in SEQ ID NO: 35; (iv) the three heavy chain CDR sequences present in SEQ ID NO: 36 and the three light chain CDR sequences present in SEQ ID NO: 37; (v) the three heavy chain CDR sequences present in SEQ ID NO: 38 and the three light chain CDR sequences present in SEQ ID NO: 39; (vi) the three heavy chain CDR sequences present in SEQ ID NO: 40 and the three light chain CDR sequences present in SEQ ID NO: 41; (vii) the three heavy chain CDR sequences present in SEQ ID NO: 42 and the three light chain CDR sequences present in SEQ ID NO: 43; (viii) the three heavy chain CDR sequences present in SEQ ID NO: 44 and the three light chain CDR sequences present in SEQ ID NO: 45; (ix) the three heavy chain CDR sequences present in SEQ ID NO: 46 and the three light chain CDR sequences present in SEQ ID NO: 47; (x) the three heavy chain CDR sequences present in SEQ ID NO: 48 and the three light chain CDR sequences present in SEQ ID NO: 49; (xi) the three heavy chain CDR sequences present in SEQ ID NO: 50 and the three light chain CDR sequences present in SEQ ID NO: 51; (xii) the three heavy chain CDR sequences present in SEQ ID NO: 52 and the three light chain CDR sequences present in SEQ ID NO: 53; (xiii) the three heavy chain CDR sequences present in SEQ ID NO: 54 and the three light chain CDR sequences present in SEQ ID NO: 55; (xiv) the three heavy chain CDR sequences present in SEQ ID NO: 56 and the three light chain CDR sequences present in SEQ ID NO: 57; (xv) the three heavy chain CDR sequences present in SEQ ID NO: 58 and the three light chain CDR sequences present in SEQ ID NO: 59; (xvi) the three heavy chain CDR sequences present in SEQ ID NO: 60 and the three light chain CDR sequences present in SEQ ID NO: 61; (xvii) the three heavy chain CDR sequences present in SEQ ID NO: 62 and the three light chain CDR sequences present in SEQ ID NO: 63; (xviii) the three heavy chain CDR sequences present in SEQ ID NO: 64 and the three light chain CDR sequences present in SEQ ID NO: 65; (xix) the three heavy chain CDR sequences present in SEQ ID NO: 66 and the three light chain CDR sequences present in SEQ ID NO: 67; (xx) the three heavy chain CDR sequences present in SEQ ID NO: 68 and the three light chain CDR sequences present in SEQ ID NO: 69; (xxi) the three heavy chain CDR sequences present in SEQ ID NO: 70 and the three light chain CDR sequences present in SEQ ID NO: 71; (xxii) the three heavy chain CDR sequences present in SEQ ID NO: 72 and the three light chain CDR sequences present in SEQ ID NO: 73; (xxiii) the three heavy chain CDR sequences present in SEQ ID NO: 74 and the three light chain CDR sequences present in SEQ ID NO: 75; or (xxiv) the three heavy chain CDR sequences present in SEQ ID NO: 76 and the three light chain CDR sequences present in SEQ ID NO: 77.

[0134] In some embodiments, a polypeptide described herein can be an antibody (e.g., a human or humanized antibody). In some embodiments, the antibody can be a human or humanized IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, a polypeptide described herein can be an antigen-binding antibody fragment (e.g., an scFv).

Compositions and Kits

[0135] Also provided herein are compositions (e.g., pharmaceutical compositions) that include any of the T-cells described herein, a population of any of the T-cells described herein, any of the antibodies or antigen-binding fragments described herein, or any of the nucleic acids or vectors described herein. In some embodiments, the compositions include any of the T-cells (e.g., any of the T-cells described herein, including any of the T-cells produced using any of the methods described herein). In some embodiments, the compositions are pharmaceutical compositions (e.g., pharmaceutical compositions formulated for different routes of administration (e.g., intravenous or subcutaneous)). In some embodiments, the pharmaceutical compositions can include a pharmaceutically acceptable carrier (e.g., phosphate buffered saline).

[0136] Also provided herein are compositions that include a binding agent comprising an antigen-binding domain or antigen-binding fragment comprising an amino acid sequence at least 80% (e.g., at least 85%, 90%, 95%, 99% and 100%) identical to a sequence selected from SEQ ID NOs: 6-29.

[0137] Also provided are kits that include any of the compositions (e.g., pharmaceutical compositions) described herein that include any of the nucleic acids, any of the T-cells, or any of the vectors described herein. In some embodiments, a kit can include a solid composition (e.g., a lyophilized composition including any of the vectors or nucleic acids described herein) and a liquid for solubilizing the lyophilized composition.

Cells

[0138] Also provided herein are cells (e.g., any of the exemplary cells described herein or known in the art) comprising one or more of any of the nucleic acids described herein (e.g., a first nucleic acid encoding a FOXP3 polypeptide, a second nucleic acid encoding a binding agent, and optionally, one or both of a nucleic acid encoding a receptor polypeptide and a nucleic acid that encodes a therapeutic gene product). Also provided herein are cells (e.g., any of the exemplary cells described herein or known in the art) that include one or more of any of the vectors described herein. In some embodiments, the cells are any of the exemplary types of T-cells described herein or known in the art. In some embodiments, a T-cell includes, without limitation, a CD4.sup.+ T-cell, a CD4.sup.+CD45RA.sup.+ T-cell, a CD4.sup.+CD62L.sup.+ T-cell, or a central memory T-cell).

[0139] In some embodiments of any of the methods described herein, the cell can be a eukaryotic cell. As used herein, the term eukaryotic cell refers to a cell having a distinct, membrane-bound nucleus. Such cells may include, for example, mammalian (e.g., rodent, non-human primate, or human) cells. Non-limiting examples of mammalian cells include Chinese hamster ovary cells and human embryonic kidney cells (e.g., HEK293 cells).

Methods of Treatment

[0140] Also provided herein are methods of treating a mammal (e.g., a human) having an autoimmune disease that includes administering to the mammal (e.g., human) a therapeutically effective amount of a cell (e.g., any of the exemplary T-cells described herein) or any of the compositions (e.g., pharmaceutical compositions) described herein.

[0141] In some embodiments, these methods can result in a reduction in the number, severity, or frequency of one or more symptoms of the autoimmune diseases in the mammal (e.g., as compared to the number, severity, or frequency of the one or more symptoms of the autoimmune disease in the mammal prior to treatment). For example, a mammal having an autoimmune disease having been administered a T-cell as described here can experience a reduction in inflammation or autoantibody production.

[0142] Also provided herein are methods of treating a mammal (e.g., a human) having an autoimmune disease that includes administering to the mammal (e.g., a human) a therapeutically effective amount of a T-cell that includes a first nucleic acid sequence encoding a FOXP3 polypeptide, a second nucleic acid sequence encoding a CXCR3 polypeptide, and a third nucleic acid encoding an anti-MADCAM-1 scFv, where the T-cell homes to any inflammatory site or inflamed tissue when administered to the mammal (e.g., a human).

[0143] Also provided herein are methods of treating a mammal (e.g., a human) having an autoimmune disease that includes administering to the mammal (e.g., a human) a therapeutically effective amount of a T-cell that includes a first nucleic acid sequence encoding a FOXP3 polypeptide, a second nucleic acid sequence encoding a CCR6 polypeptide, and a third nucleic acid encoding an anti-MADCAM-1 scFv, where the T-cell homes to the intestine when administered to the mammal (e.g., a human).

[0144] Also provided herein are methods of treating a mammal (e.g., a human) having an autoimmune disease that includes administering to the mammal (e.g., a human) a therapeutically effective amount of a T-cell that includes a first nucleic acid sequence encoding a FOXP3 polypeptide, a second nucleic acid sequence encoding a CCR9 polypeptide, and a third nucleic acid encoding an anti-MADCAM-1 scFv, where the T-cell homes to the intestine when administered to the mammal (e.g., a human).

[0145] Also provided herein are methods of treating a mammal (e.g., a human) having an autoimmune disease that includes administering to the mammal (e.g., a human) a therapeutically effective amount of a T-cell that includes a first nucleic acid sequence encoding a FOXP3 polypeptide, a second nucleic acid sequence encoding a GPR15 polypeptide, and a third nucleic acid encoding an anti-MADCAM-1 scFv, where the T-cell homes to the colon when administered to the mammal (e.g., a human).

[0146] Also provided herein are methods of treating a mammal (e.g., a human) having an autoimmune disease that includes administering to the mammal (e.g., a human) a therapeutically effective amount of a T-cell that includes a first nucleic acid sequence encoding a FOXP3 polypeptide, a second nucleic acid sequence encoding a CXCR5 polypeptide, and a third nucleic acid encoding an anti-MADCAM-1 scFv, where the T-cell homes to the germinal center when administered to the mammal (e.g., a human).

[0147] Any appropriate method of administration can be used to administer the T-cells to a mammal (e.g. a human) having an autoimmune disease. Examples of methods of administration include, without limitation, parenteral administration and intravenous injection.

[0148] A pharmaceutical composition containing the T-cells and a pharmaceutically acceptable carrier or buffer can be administered to a mammal (e.g., a human) having an autoimmune disease. For example, a pharmaceutical composition (e.g., a T-cell along with a pharmaceutically acceptable carrier) to be administered to a mammal having an autoimmune disease can be formulated in an injectable form (e.g., emulsion, solution and/or suspension). In some embodiments, a pharmaceutical composition containing the T-cells can include phosphate buffered saline.

[0149] Pharmaceutically acceptable carriers, fillers, and vehicles that can be used in a pharmaceutical composition described herein can include, without limitation, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.

[0150] Effective dosage can vary depending on the severity of the autoimmune disease, the route of administration, the age and general health condition of the subject, excipient usage, the possibility of co-usage with other therapeutic treatments, and the judgment of the treating physician. An effective amount of a T-cell can be any amount that reduces inflammation and autoantibody production within a mammal having an autoimmune disease without producing significant toxicity to the mammal. For example, an effective amount of T-cells administered to a mammal having an autoimmune disease can be from about 1?10.sup.6 cells to about 1?10.sup.10 (e.g., from about 1?10.sup.6 to about 1?10.sup.9, from about 1?10.sup.6 to about 1?10.sup.8, from about 1?10.sup.6 to about 1?10.sup.7, from about 1?10.sup.7 to about 1?10.sup.10, from about 1?10.sup.7 to about 1?10.sup.9, from about 1?10.sup.7 to about 1?10.sup.8, from about 1?10.sup.8 to about 1?10.sup.10, from about 1?10.sup.8 to about 1?10.sup.9, or form about 1?10.sup.9 to about 1?10.sup.10) cells. In some cases, the T-cells can be a purified population of immune cells generated as described herein. In some cases, the purity of the population of T-cells can be assessed using any appropriate method, including, without limitation, flow cytometry. In some cases, the population of T-cells to be administered can include a range of purities from about 70% to about 100%, from about 70% to about 90%, from about 70% to about 80%, from about 80% to about 90%, from about 90% to about 100%, from about 80% to about 100%, from about 80% to about 90%, or from about 90% to 100%. In some cases, the dosage (e.g., number of T-cells to be administered) can adjusted based on the level of purity of the T-cells.

[0151] The frequency of administration of a T-cell can be any frequency that reduces inflammation or autoantibody production within a mammal having an autoimmune disease without producing toxicity to the mammal. In some cases, the actual frequency of administration can vary depending on various factors including, without limitation, the effective amount, duration of treatment, use of multiple treatment agents, route of administration, and severity of the condition may require an increase or decrease in frequency of administration.

[0152] An effective duration for administering a composition containing a T-cell can be any duration that reduces inflammation or autoantibody production within a mammal having an autoimmune disease without producing toxicity to the mammal. In some cases, the effective duration can vary from several days to several months. In general, the effective treatment duration for administering a composition containing a T-cell to treat an autoimmune disease can range in duration from about one month to about five years (e.g., from about two months to about five years, from about three months to about five years, from about six months to about five years, from about eight months to about five years, from about one year to about five years, from about one month to about four years, from about one month to about three years, from about one month to about two years, from about six months to about four years, from about six months to about three years, or from about six months to about two years). In some cases, the effective treatment duration for administering a composition containing a T-cell can be for the remainder of the life of the mammal.

[0153] In some cases, a course of treatment and/or the severity of one or more symptoms related to autoimmune disease can be monitored. Any appropriate method can be used to determine whether the autoimmune disease is being treated. For example, immunological techniques (e.g., ELISA) can be performed to determine if the level of autoantibodies present within a mammal being treated as described herein is reduced following the administration of the T-cells. Remission and relapse of the disease can be monitored by testing for one or more markers of autoimmune disease.

[0154] Any appropriate autoimmune disease can be treated with a T-cell as described herein. In some cases, an autoimmune disease caused by the accumulation of autoantibodies can be treated with a T-cell as described herein. Examples of autoimmune diseases include, without limitation, lupus, rheumatoid arthritis, multiple sclerosis, insulin dependent diabetes mellitis, myasthenia gravis, Grave's disease, autoimmune hemolytic anemia, autoimmune thrombocytopenia purpura, Goodpasture's syndrome, pemphigus vulgaris, acute rheumatic fever, post-streptococcal glomerulonephritis, Crohn's disease, Celiac disease, and polyarteritis nodosa.

[0155] The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

EXAMPLES

Example 1. Enforced Expression of FOXP3 in CD4.SUP.+ T-Cells

[0156] A nucleic acid sequence encoding a FOXP3 polypeptide is transformed into CD4.sup.+/CD45RA.sup.+ T-cells to generate enforced expression of FOXP3 (eFOXP3) (FIG. 1). The nucleotide sequence encoding the FOXP3 polypeptide (e.g., a wild-type FOXP3 polypeptide or a FOXP3 polypeptide and is operably linked to an SSFV promoter. The nucleic acid sequence encoding the FOXP3 polypeptide is transformed into primary CD4.sup.+/CD45RA.sup.+ T-cells along with a nucleic acid sequence encoding a chimeric antigen receptor polypeptide comprising an intracellular domain made up of a CD3zeta domain and a CD28 domain. Following transformation, the cells are expanded in the presence of rIL-2 and optionally purified, e.g., based on low expression of CD127 (IL7 receptor). Markers of the suppressive phenotype of the transduced cells (e.g., CD4.sup.+/CD45RA.sup.+ transformed with an exogenous FOXP3 polypeptide) is determined by expression of Treg-associated molecules (e.g., CD25, CTLA-4, and GITR) and/or a cytokine profile upon re-stimulation of the transduced cell (e.g., low production of IL-2, IFNgamma, and IL-17).

Example 2. Expression of Therapeutic Outputs

[0157] CD4.sup.+/CD45RA.sup.+ T-cells are transduced with a lentivirus having nucleic acid sequences encoding a FOXP3 polypeptide having mutations as described herein, a receptor polypeptide, and a therapeutic gene product (FIG. 2). Here, a CD4.sup.+/CD45RA.sup.+ T-cell is transformed with a nucleic acid sequence encoding a FOXP3 polypeptide, a nucleic acid sequence encoding a CXCR3 chemokine receptor polypeptide, and is also transformed with a nucleic acid sequence encoding a scFv antigen-binding fragment that is capable of binding to an IL-6R antigen expressed on a cell associated with an autoimmune disease. The binding of the scFV to an epitope of IL-6R blocks the binding of IL-6R to IL-6. An antibody used in this example includes Tocilizumab, which is a humanized anti-IL-6R antibody. The variable light and heavy chain domains of Tocilizumab (See, U.S. Pat. No. 5,795,965) are provided to the cells using nucleic acid sequence encoding a scFv linked to a secretion signal and operably linked by a constitutive promoter such as EF-1?. Mutations are introduced into the amino acid sequence of Tocilizumab that render the heavy and light chains more favorable binding properties to the IL-6R (See, U.S. Pat. No. 8,562,991). Tregs are not known to naturally produce IL-6 blocking mediators (e.g., antibody or antigen-binding fragments to IL-6R). Therefore, expression of such blockers transformed into a CD4.sup.+/CD45RA.sup.+ T-cell along with an a nucleic acid sequence encoding a FOXP3 polypeptide will render the T-cells more effective in inflammatory environments than T-cells not transformed with the nucleic acid sequences described herein. The binding of the scFv to IL-6R.sup.+ is confirmed by flow cytometry. Secretion of the scFv is verified by ELISA, and the biological activity is confirmed by inhibition of IL-6 signaling in a reporter cell assay (e.g., IL-6 Luciferase stable reporter cell line from Novus Biologicals).

Example 3. Enforced Expression of Chemokine Receptors and Tissue Targeting of CD4.SUP.+.FOXP3.SUP.+ T-Cells

[0158] CD4.sup.+CD45RA.sup.+ T-cells are transduced with a lentivirus having nucleic acid sequences encoding a CXCR3 polypeptide and a nucleic acid sequence encoding a FOXP3 polypeptide (FIG. 3). Following transformation, the cells are expanded in the presence of high dose rIL-2 and purified based on low expression of CD127 (IL7 receptor). Other means of purifying the cells as described herein or known in the art can also be used to purify the T-cell. CXCR3 expression is confirmed by flow cytometric analysis using an antibody against the CXCR3 polypeptide (e.g., anti-human CD183 (CXCR3) Biolegend Cat. No. RUO353707). CXCR3 function is also confirmed by an in vitro chemotaxis assay (e.g., transwell migration assay). Briefly, transduced cells are placed in a transwell system with a CXCR3 ligand (e.g., 50-300 ng/mL CXCL10 (human rCXCL10 from R&D Systems Cat No. 266-IP-010)) or a control chemokine present on the side of the membrane opposite the transduced cells. Migration of cells across the membrane is evaluated by flow cytometry using an antibody against CXCR3. Specificity is further confirmed by blocking migration with anti-CXCR3 blocking antibody.

Example 4. Attachment with Cell Surface Binding Agent

[0159] CD4.sup.+/CD45RA.sup.+ T-cells are transduced with a lentivirus having nucleic acid sequences encoding a FOXP3 polypeptide and a chimeric antigen receptor polypeptide including an scFv capable of binding to an intercellular adhesion molecule-1 (MADCAM-1) (FIG. 4). MADCAM-1 is expressed on endothelial cells and is responsive to numerous inflammatory mediators (see, e.g., Xu et al., Immunol. Cell Biol. 85(8):633-639, 2008).

[0160] The chimeric antigen receptor includes an extracellular domain having a scFv domain directed to MADCAM-1, a transmembrane domain and an intracellular signaling domain. The affinity of the scFv is tuned to bind at high antigen density that exists in diseased tissues but avoid targeting healthy tissues with basal MADCAM-1 expression. The scFv is coupled to a transmembrane domain which spans the membrane and is derived from a CD8 transmembrane domain (e.g., CD8? stalk and CD8? hinge). The intracellular signaling domain is comprised of the CD3zeta domain and an additional co-stimulatory domain such as CD28, 4-1BB, ICOS, OX-40 and/or CD27 to transmit a proliferative/survival signal upon antigen recognition.

Example 5. Antibody GenerationHumanization and Binding Affinity for MADCAM-1 scFv

[0161] FIG. 5 shows the exemplary workflow used to identify candidate anti-MADCAM-1 scFVs. Humanized MADCAM-1 single-chain variable fragment (scFv) antibodies originated from rabbit immunoglobulin G antibodies with kappa light chains from K1 or K2 subclasses, and were generated by grafting of rabbit complementarity-determining region (CDR) onto human antibody framework regions. Monoclonal antibodies were first generated by immunization of O. cuniculus with recombinantly expressed human MADCAM-1 extracellular domains. Clones were selected by antigen ELISA and binding to an antigen expressing cell line. CDRs for sequenced antibodies were defined as the union (Zhang et al., MAbs 9(3):419-429, 2017) of sequences identified by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, Diane Publishing Company, 1992) and IMGT (Lefranc et al., Dev. Comp. Immunol. 27:55-77, 2003) methods. Antibodies were humanized by CDR grafting onto human framework candidates identified by sequence similarity and critical framework amino acids back mutated as experimentally determined. Given the unique disulfide bonding patterns in rabbit IgG antibodies both in the framework and CDR regions; consideration was taken to ensure proper pairing of disulfide bonds. scFvs were subsequently generated by fusing humanized variable heavy (VH) and variable light (VL) regions in the VH-VL orientation with a standard (G.sub.4S).sub.3 linker. Mutations (e.g., amino acid substitutions) were introduced into CDR regions to modulate (e.g., increase) KD.

[0162] Binding affinity (KD) of MADCAM-1 scFv antibodies from Table 2 was measured using biolayer interferometry (See also FIG. 6). Streptavidin biosensors were loaded for 5 seconds with 10 Tg/mL biotinylated-MADCAM-1 antigen followed by acquisition of the baseline signal in phosphate buffer saline supplemented with bovine serum albumin and sodium azide. For the association step loaded probes were incubated for 250 seconds with a titration of anti-MADCAM-1 scFv antibodies at concentrations generally between 0 nM and 2 TM. This was followed by a dissociation step in buffer lacking anti-MADCAM-1 scFv for 250 seconds. Experimental data was normalized to a reference probe and high-frequency noise was removed by Savitzky-Golay filtering. On and off-rate constants were determined using a globally fitted 1:1 binding model.

TABLE-US-00005 TABLE 2 K.sub.D for MADCAM-1 scFv Clones Clone kon (1/Ms) koff (1/s) Kd (nm) MAdCAM1 scFv 32.3 4.07E+05 4.91E?03 12.1 MAdCAM1 scFv 32.4 5.77E+05 1.08E?02 18.7 MAdCAM1 scFv 32.5 2.72E+05 1.46E?03 5.4 MAdCAM1 scFv 32.6 3.27E+05 2.19E?03 6.7 MAdCAM1 scFv 32.7 4.16E+05 9.91E?03 23.8 MAdCAM1 scFv 32.8 5.34E+05 8.30E?03 15.5 MAdCAM1 scFv 8.1 2.07E+04 1.86E?03 89.9 MAdCAM1 scFv 8.2 3.78E+04 2.95E?03 78 MAdCAM1 scFv 8.4 1.27E+04 3.93E?04 30.9 MAdCAM1 scFv 8.5 1.38E+04 1.58E?03 114.5 MAdCAM1 scFv 8.6 2.57E+04 2.18E?03 84.8 MAdCAM1 scFv 8.7 1.66E+04 4.28E?03 257.8 MAdCAM1 scFv 8.8 2.24E+04 6.96E?03 310.7 MAdCAM1 scFv 17.1 2.47E+05 5.68E?04 2.3 MAdCAM1 scFv 17.3 3.04E+05 8.68E?04 2.9 MAdCAM1 scFv 17.5 2.58E+05 9.67E?04 3.7 MAdCAM1 scFv 17.7 3.00E+05 5.37E?04 1.8 MAdCAM1 scFv 2.1 7.08E+04 6.02E?03 85 MAdCAM1 scFv 2.2 5.08E+04 1.67E?02 328.7 MAdCAM1 scFv 10.1 1.22E+04 1.25E?03 102.5 MAdCAM1 scFv 10.2 5.13E+03 6.46E?03 1259.3 MAdCAM1 scFv 10.3 1.16E+04 1.14E?03 98.3 MAdCAM1 scFv 10.7 2.38E+04 1.08E?03 45.4 MAdCAM1 scFv 10.8 2.79E+03 2.59E?03 928.3

Example 6. Transmigration of T-Cells

[0163] Engineered T-cells as described in Example 4 (e.g., CD4.sup.+ T-cells having nucleic acid sequences encoding a FOXP3 polypeptide and a chimeric antigen receptor polypeptide including an scFv capable of binding to MADCAM-1) require selective binding to endothelial cells present in inflamed tissues which have high levels of MADCAM-1 expression. In order to enter the inflamed tissue and mediate their therapeutic effect, the engineered T-cells undergo transendothelial migration (TEM) or diapedesis. Here, T-cells including a FOXP3 polypeptide and a chimeric antigen receptor (CAR) having an anti-MADCAM-1 scFv extracellular domain were guided to locations within the subject (e.g., inflamed tissue, intestine, or colon) by the MADCAM-1 scFv. The engineered T-cell's ability to transmigrate once reaching the location in the subject depends on, but not limited to, binding affinity (K.sub.D) of the scFv.

[0164] Engineered T-cells expressing a particular MADCAM-1-scFv-CAR can be screened for their ability to bind to endothelial cells using binding assays conducted under flow conditions similar to that of the hemodynamic shear stress of blood flow. An exemplary assay is described in Brown et al., BMC Immunol. 2:9, 2001, which is herein incorporated by reference in its entirety. Briefly, engineered T-cells are exposed to human endothelial cells under conditions of physiological shear stress, and the number of T-cells bound are measured under flow forces in the range of 0.5-8 dynes/cm.sup.2. Optimally, the engineered T-cell will bind minimally to normal endothelial cells in their basal state and will bind to a greater extent to inflammatory cytokine-activated endothelial cells. Examples of inflammatory cytokines are IL-1, TNF?, and IFN?. Endothelial cells treated with such cytokines dramatically upregulate MADCAM-1 and other cell adhesion molecules, thus mimicking the vasculature in inflamed tissues, as described in Dustin et al., J. Cell Biol. 107(1):321-331, 1988, which is herein incorporated by reference in its entirety.

[0165] To measure TEM, an in vitro two chamber migration assay was developed to screen engineered T-cells (e.g., CD4.sup.+ T-cells having nucleic acid sequences encoding a FOXP3 polypeptide and a chimeric antigen receptor polypeptide including an scFV capable of binding to MADCAM-1). Non-limiting examples of assays of measuring TEM are as described in Muller and Luscinskas, Methods Enzymol. 443:155-176, 2009, which is herein incorporated by reference in its entirety. Human umbilical endothelial cells, human microvascular endothelial cells, or intestinal endothelial cells were grown in an inner chamber as a monolayer on extracellular matrix (ECM) based hydrogels. The ECM support was pre-coated with fibronectin or human serum without growth factors to enhance plating efficiency. Established monolayers were then treated with TNF? to mimic the inflamed state and to increase the levels of MADCAM-1. TNF? was titrated to determine concentrations necessary to recapitulate MADCAM-1 expression in diseased tissues. Engineered T-cells (e.g., CD4.sup.+ T-cells having nucleic acid sequences encoding a FOXP3 polypeptide and a chimeric antigen receptor polypeptide including a scFv capable of binding to MADCAM-1) were then added to the inner chamber and actively monitored by live-cell fluorescence microscopy for activation and diapedesis. Through the course of monolayer establishment and transmigration assays, the outer chamber was supplemented with media in the presence or absence of chemoattractants, such as selected chemokines that are present in disease tissue and ligands of chemokine receptors expressed on the T-cells. Only engineered T-cells that included an scFv capable of binding MADCAM-1 with a particular binding affinity enable transmigration from the inner chamber to the outer chamber, as measured by fluorescence microscopy.

Other Embodiments

[0166] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

TABLE-US-00006 SequenceAppendix SEQ IDNO: Name Sequence SEQID FOXP3 MPNPRPGKPSAPSLALGPSPGASPSWRAAPKASDLLGARGPGG NO:1 polypeptide TFQGRDLRGGAHASSSSLNPMPPSQLQLPTLPLVMVAPSGARL GPLPHLQALLQDRPHFMHQLSTVDAHARTPVLQVHPLESPAMI SLTPPTTATGVFSLKARPGLPPGINVASLEWVSREPALLCTFPNP SAPRKDSTLSAVPQSSYPLLANGVCKWPGCEKVFEEPEDFLKH CQADHLLDEKGRAQCLLQREMVQSLEQQLVLEKEKLSAMQAH LAGKMALTKASSVASSDKGSCCIVAAGSQGPVVPAWSGPREAP DSLFAVRRHLWGSHGNSTFPEFLHNMDYFKFHNMRPPFTYATL IRWAILEAPEKQRTLNEIYHWFTRMFAFFRNHPATWKNAIRHN LSLHKCFVRVESEKGAVWTVDELEFRKKRSQRPSRCSNPTPGP SEQID FOXP3full atgcctaacccccgccctggaaaaccatctgcccc NO:2 length ttcactggccctgggaccttcacccggagcctcac nucleic catcttggagagccgcccccaaggccagcgacctg acid ctgggagccagaggccccggcggcaccttccaggg cagggatctgcgcggcggcgcccacgccagctcct ctagcctgaaccccatgcccccttctcagctccag ctgcccacactgcccctggtcatggtggcacctag cggagcaaggctgggaccactgccacacctccagg ccctgctccaggacagacctcactttatgcaccag ctgtccaccgtggatgcacacgcaaggacacccgt gctccaggtgcaccctctggagtctccagccatga tcagcctgaccccaccaaccacagcaacaggcgtg ttctccctgaaggccagacctggcctgcctccagg catcaacgtggcctccctggagtgggtgtctaggg agccagccctgctgtgcacctttcctaatccatct gccccccgcaaggactccacactgtctgccgtgcc acagtcctcttaccccctgctggccaacggcgtgt gcaagtggcctggctgtgagaaggtgttcgaggag ccagaggattttctgaagcactgccaggccgacca cctgctggatgagaagggaagggcacagtgtctgc tccagagggagatggtgcagagcctggagcagcag ctggtgctggagaaggagaagctgtccgccatgca ggcacacctggcaggcaagatggcactgaccaagg ccagctccgtggcctctagcgacaagggcagctgc tgtatcgtggccgccggctcccagggaccagtggt gcccgcctggtctggacccagggaggcacctgaca gcctgttcgccgtgcggagacacctgtggggcagc cacggcaattccaccttccccgagtttctgcacaa catggattacttcaagtttcacaatatgcggcccc cttttacctatgccacactgatcagatgggccatc ctggaggccccagagaagcagcgcaccctgaacga aatctaccactggttcacacggatgtttgccttct ttagaaatcaccccgccacctggaagaacgccatc aggcacaatctgtccctgcacaagtgtttcgtgcg cgtggagtctgagaagggcgccgtgtggacagtgg atgagctggagttcagaaagaagagaagccagaga ccatccaggtgttcaaaccctaccccaggaccc SEQID FOXP3 CCTGCCCTTGGACAAGGACCCGATGCCCAACCCCAGGCCTG NO:3 exon2 GCAAGCCCTCGGCCCCTTCCTTGGCCCTTGGCCCATCCCCAG GAGCCTCGCCCAGCTGGAGGGCTGCACCCAAAGCCTCAGAC CTGCTGGGGGCCCGGGGCCCAGGGGGAACCTTCCAGGGCCG AGATCTTCGAGGCGGGGCCCATGCCTCCTCTTCTTCCTTGAA CCCCATGCCACCATCGCAGCTGCAG SEQID CD3zeta mkwkalftaailqaqlpiteaqsfglldpklcylldgilfiy NO:4 cytoplasmic gviltalflrvkfsrsadapayqqgqnqlynelnlgrreeyd signaling vldkrrgrdpemggkpqrrknpqeglynelqkdkmaeaysei domain gmkgerrrgkghdglyqglstatkdtydalhmqalppr SEQID CD28co- IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLV NO:5 stimulatory VVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPG domai PTRKHYQPYAPPRDFAAY SEQID MAdCA EVQLLESGGGLVQPGGSLRLSCAASGFSFSSSYWICWVRQAPG NO:6 M1scFv KGLEWVSCIYGGSSGATYYANWAKGRFTISRDNSKNTLYLQM 32.3 NSLRAEDTAVYYCARSGSTTSGGVYRWYFNLWGQGTLVTVSS GGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCQASENI YNLLAWYQQKPGKVPKLLIYDASTLQSGVPSRFSGSGSGTDFT LTISSLQPEDVATYYCQFTYYGGTYESAFGGGTKVEIK SEQID MAdCA EVQLLESGGGLVQPGGSLRLSCAASGFSFSSSYWICWVRQAPG NO:7 M1scFv KGLEWVSCIYGGSSGATYYANWAKGRFTISRDNSKNTLYLQM 32.4 NSLRAEDTAVYYCARSGSTTSGGVYRWYFNLWGQGTLVTVSS GGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCQASENI YNLLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFT FTISSLQPEDIATYYCQFTYYGGTYESAFGGGTKVEIK SEQID MAdCA QVQLVESGGGVVQPGRSLRLSCAASGFSFSSSYWICWVRQAPG NO:8 M1scFv KGLEWVACIYGGSSGATYYANWAKGRFTISRDNSTNTLFLQM 32.5 NSLRAEDTAVYYCARSGSTTSGGVYRWYFNLWGQGTLVTVSS GGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCQASENI YNLLAWYQQKPGKVPKLLIYDASTLQSGVPSRFSGSGSGTDFT LTISSLQPEDVATYYCQFTYYGGTYESAFGGGTKVEIK SEQID MAdCA QVQLVESGGGVVQPGRSLRLSCAASGFSFSSSYWICWVRQAPG NO:9 M1scFv KGLEWVACIYGGSSGATYYANWAKGRFTISRDNSTNTLFLQM 32.6 NSLRAEDTAVYYCARSGSTTSGGVYRWYFNLWGQGTLVTVSS GGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCQASENI YNLLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFT FTISSLQPEDIATYYCQFTYYGGTYESAFGGGTKVEIK SEQID MAdCA EVQLVESGGGLVQPGGSLRLSCAASGFSFSSSYWICWVRQAPG NO:10 M1scFv KGLEWVSCIYGGSSGATYYANWAKGRFTISRHNSKNTLYLQM 32.7 NSLRAEDTAVYYCARSGSTTSGGVYRWYFNLWGQGTLVTVSS GGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCQASENI YNLLAWYQQKPGKVPKLLIYDASTLQSGVPSRFSGSGSGTDFT LTISSLQPEDVATYYCQFTYYGGTYESAFGGGTKVEIK SEQID MAdCA EVQLVESGGGLVQPGGSLRLSCAASGFSFSSSYWICWVRQAPG NO:11 M1scFv KGLEWVSCIYGGSSGATYYANWAKGRFTISRHNSKNTLYLQM 32.8 NSLRAEDTAVYYCARSGSTTSGGVYRWYFNLWGQGTLVTVSS GGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCQASENI YNLLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFT FTISSLQPEDIATYYCQFTYYGGTYESAFGGGTKVEIK SEQID MAdCA EVQLVESGGGLVQPGGSLRLSCAASGFTLSSSYYMYWVRQAP NO:12 M1scFv GKGLEWVSVIYAGDGNTYYASWAKGRFTISRDNSKNTLYLQM 8.1 NSLRAEDTAVYYCARGYVGYGYAIDLWGQGTLVTVSSGGGGS GGGGSGGGGSDIQMTQSPSTLSASVGDRVTITCQASQSIYSSLA WYQQKPGKAPKLLIYYASILPSGVPSRFSGSGSGTEFTLTISSLQ PDDFATYYCQATTYDSTYPNAFGGGTKVEIK SEQID MAdCA EVQLVESGGGLVQPGGSLRLSCAASGFTLSSSYYMYWVRQAP NO:13 M1scFv GKGLEWVSVIYAGDGNTYYASWAKGRFTISRDNSKNTLYLQM 8.2 NSLRAEDTAVYYCARGYVGYGYAIDLWGQGTLVTVSSGGGGS GGGGSGGGGSAIQLTQSPSSLSASVGDRVTITCQASQSIYSSLA WYQQKPGKAPKLLIYYASILPSGVPSRFSGSGSGTDFTLTISSLQ PEDFATYYCQATTYDSTYPNAFGGGTKVEIK SEQID MAdCA EVQLLESGGGLVQPGGSLRLSCAASGFTLSSSYYMYWVRQAPG NO:14 M1scFv KGLEWVSAIYAGDGNTYYASWAKGRFTISRDNSKNTLYLQMN 8.4 SLRAEDTAVYYCARGYVGYGYAIDLWGQGTLVTVSSGGGGSG GGGSGGGGSAIQLTQSPSSLSASVGDRVTITCQASQSIYSSLAW YQQKPGKAPKLLIYYASILPSGVPSRFSGSGSGTDFTLTISSLQ PEDFATYYCQATTYDSTYPNAFGGGTKVEIK SEQID MAdCA QVQLVESGGGVVQPGRSLRLSCAASGFTLSSSYYMYWVRQAP NO:15 M1scFv GKGLEWVAVIYAGDGNTYYASWAKGRFTISRDNSKNTLYLQM 8.5 NSLRAEDTAVYYCARGYVGYGYAIDLWGQGTLVTVSSGGGGS GGGGSGGGGSDIQMTQSPSTLSASVGDRVTITCQASQSIYSSLA WYQQKPGKAPKLLIYYASILPSGVPSRFSGSGSGTEFTLTISSLQ PDDFATYYCQATTYDSTYPNAFGGGTKVEIK SEQID MAdCA QVQLVESGGGVVQPGRSLRLSCAASGFTLSSSYYMYWVRQAP NO:16 M1scFv GKGLEWVAVIYAGDGNTYYASWAKGRFTISRDNSKNTLYLQM 8.6 NSLRAEDTAVYYCARGYVGYGYAIDLWGQGTLVTVSSGGGGS GGGGSGGGGSAIQLTQSPSSLSASVGDRVTITCQASQSIYSSLA WYQQKPGKAPKLLIYYASILPSGVPSRFSGSGSGTDFTLTISSLQ PEDFATYYCQATTYDSTYPNAFGGGTKVEIK SEQID MAdCA EVQLVETGGGLIQPGGSLRLSCAASGFTLSSSYYMYWVRQAPG NO:17 M1scFv KGLEWVSVIYAGDGNTYYASWAKGRFTISRDNSKNTLYLQMN 8.7 SLRAEDTAVYYCARGYVGYGYAIDLWGQGTLVTVSSGGGGSG GGGSGGGGSDIQMTQSPSTLSASVGDRVTITCQASQSIYSSLAW YQQKPGKAPKLLIYYASILPSGVPSRFSGSGSGTEFTLTISSLQ PDDFATYYCQATTYDSTYPNAFGGGTKVEIK SEQID MAdCA EVQLVETGGGLIQPGGSLRLSCAASGFTLSSSYYMYWVRQAPG NO:18 M1scFv KGLEWVSVIYAGDGNTYYASWAKGRFTISRDNSKNTLYLQMN 8.8 SLRAEDTAVYYCARGYVGYGYAIDLWGQGTLVTVSSGGGGSG GGGSGGGGSAIQLTQSPSSLSASVGDRVTITCQASQSIYSSLAW YQQKPGKAPKLLIYYASILPSGVPSRFSGSGSGTDFTLTISSLQ PEDFATYYCQATTYDSTYPNAFGGGTKVEIK SEQID MAdCA EVQLVESGGGLVQPGGSLRLSCAASGFSFSSTYYMCWVRQAPG NO:19 M1scFv KGLEWVSCIYTGSGNTDYASWAKGRFTISRHNSKNTLYLQMNS 17.1 LRAEDTAVYYCARGGIVDSYFTYFDLWGQGTLVTVSSGGGGS GGGGSGGGGSDIQMTQSPSTLSASVGDRVTITCQASESIFSNLA WYQQKPGKAPKLLIYWASTLASGVPSRFSGSGSGTEFTLTISSL QPDDFATYYCQSYVYSSSSSNDFGGGTKVEIK SEQID MAdCA QVQLLESGGGLVKPGGSLRLSCAASGFSFSSTYYMCWIRQAPG NO:20 M1scFv KGLEWVSCIYTGSGNTDYASWAKGRFTISRDNAKNSLYLQMN 17.3 SLRAEDTAVYYCARGGIVDSYFTYFDLWGQGTLVTVSSGGGGS GGGGSGGGGSDIQMTQSPSTLSASVGDRVTITCQASESIFSNLA WYQQKPGKAPKLLIYWASTLASGVPSRFSGSGSGTEFTLTISSL QPDDFATYYCQSYVYSSSSSNDFGGGTKVEIK SEQID MAdCA QVQLVESGGGVVQPGRSLRLSCAASGFSFSSTYYMCWVRQAP NO:21 M1scFv GKGLEWVACIYTGSGNTDYASWAKGRFTISRDNSTNTLFLQM 17.5 NSLRAEDTAVYYCARGGIVDSYFTYFDLWGQGTLVTVSSGGG GSGGGGSGGGGSDIQMTQSPSTLSASVGDRVTITCQASESIFSNL AWYQQKPGKAPKLLIYWASTLASGVPSRFSGSGSGTEFTLTISS LQPDDFATYYCQSYVYSSSSSNDFGGGTKVEIK SEQID MAdCA EVQLVESGGGLVKPGGSLRLSCAASGFSFSSTYYMCWVRQAPG NO:22 M1scFv KGLEWVSCIYTGSGNTDYASWAKGRFTISRDNAKNSLYLQMN 17.7 SLRAEDTAVYYCARGGIVDSYFTYFDLWGQGTLVTVSSGGGGS GGGGSGGGGSDIQMTQSPSTLSASVGDRVTITCQASESIFSNLA WYQQKPGKAPKLLIYWASTLASGVPSRFSGSGSGTEFTLTISSL QPDDFATYYCQSYVYSSSSSNDFGGGTKVEIK SEQID MAdCA QVQLLESGGGLVKPGGSLRLSCAASGFSFSSGYYMCWIRQAPG NO:23 M1scFv KGLEWVSCIYADSSYTYYASWAKGRFTISRDNAKNSLYLQMN 2.1 SLRAEDTAVYYCARSNGDYFYGMDLWGQGTLVTVSSGGGGS GGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCQASQSINSWLS WYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGTDFTFTISSLQ PEDIATYYCQSNYYSTSTAFGGGTKVEIK SEQID MAdCA QVQLLESGGGLVKPGGSLRLSCAASGFSFSSGYYMCWIRQAPG NO:24 M1scFv KGLEWVSCIYADSSYTYYASWAKGRFTISRDNAKNSLYLQMN 2.2 SLRAEDTAVYYCARSNGDYFYGMDLWGQGTLVTVSSGGGGS GGGGSGGGGSAIQMTQSPSSLSASVGDRVTITCQASQSINSWLS WYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGTDFTLTISSLQ PEDFATYYCQSNYYSTSTAFGGGTKVEIK SEQID MAdCA QVQLVESGGGVVQPGRSLRLSCAASGIDFSGYHYICWVRQAPG NO:25 M1scFv KGLEWVACIYTVSSGIWYATWAKGRFTISRDNSTNTLFLQMNS 10.1 LRAEDTAVYYCARDDADVSGYWFDLWGQGTLVTVSSGGGGS GGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCQASQSISNLLA WYQQKPGKAPKLLIYKASTLASGVPSRFSGSGSGTDFTFTISSLQ PEDIATYYCQQGITNNGIDHGFGGGTKVEIK SEQID MAdCA QVQLVESGGGVVQPGRSLRLSCAASGIDFSGYHYICWVRQAPG NO:26 M1scFv KGLEWVACIYTVSSGIWYATWAKGRFTISRDNSTNTLFLQMNS 10.2 LRAEDTAVYYCARDDADVSGYWFDLWGQGTLVTVSSGGGGS GGGGSGGGGSAIQLTQSPSSLSASVGDRVTITCQASQSISNLLA WYQQKPGKAPKLLIYKASTLASGVPSRFSGSGSGTDFTLTISSL QPEDFATYYCQQGITNNGIDHGFGGGTKVEIK SEQID MAdCA EVQLVESGGGLVKPGGSLRLSCAASGIDFSGYHYICWVRQAPG NO:27 M1scFv KGLEWVSCIYTVSSGIWYATWAKGRFTISRDNAKNSLYLQMNS 10.3 LRAEDTAVYYCARDDADVSGYWFDLWGQGTLVTVSSGGGGS GGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCQASQSISNLLA WYQQKPGKAPKLLIYKASTLASGVPSRFSGSGSGTDFTFTISSLQ PEDIATYYCQQGITNNGIDHGFGGGTKVEIK SEQID MAdCA EVQLLESGGGLVQPGGSLRLSCAASGIDFSGYHYICWVRQAPG NO:28 M1scFv KGLEWVSCIYTVSSGIWYATWAKGRFTISRDNSKNTLYLQMNS 10.7 LRAEDTAVYYCARDDADVSGYWFDLWGQGTLVTVSSGGGGS GGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCQASQSISNLLA WYQQKPGKAPKLLIYKASTLASGVPSRFSGSGSGTDFTFTISSLQ PEDIATYYCQQGITNNGIDHGFGGGTKVEIK SEQID MAdCA EVQLLESGGGLVQPGGSLRLSCAASGIDFSGYHYICWVRQAPG NO:29 M1scFv KGLEWVSCIYTVSSGIWYATWAKGRFTISRDNSKNTLYLQMNS 10.8 LRAEDTAVYYCARDDADVSGYWFDLWGQGTLVTVSSGGGGS GGGGSGGGGSAIQLTQSPSSLSASVGDRVTITCQASQSISNLLA WYQQKPGKAPKLLIYKASTLASGVPSRFSGSGSGTDFTLTISSL QPEDFATYYCQQGITNNGIDHGFGGGTKVEIK SEQID MAdCA EVQLLESGGGLVQPGGSLRLSCAASGFSFSSSYWICWVRQAPG NO:30 M1scFv KGLEWVSCIYGGSSGATYYANWAKGRFTISRDNSKNTLYLQM 32.3HC NSLRAEDTAVYYCARSGSTTSGGVYRWYFNLWGQGTLVTVSS SEQID MAdCA DIQMTQSPSSLSASVGDRVTITCQASENIYNLLAWYQQKPGKVP NO:31 M1scFv KLLIYDASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQ 32.3LC FTYYGGTYESAFGGGTKVEIK SEQID MAdCA EVQLLESGGGLVQPGGSLRLSCAASGFSFSSSYWICWVRQAPG NO:32 M1scFv KGLEWVSCIYGGSSGATYYANWAKGRFTISRDNSKNTLYLQM 32.4HC NSLRAEDTAVYYCARSGSTTSGGVYRWYFNLWGQGTLVTVSS SEQID MAdCA DIQMTQSPSSLSASVGDRVTITCQASENIYNLLAWYQQKPGKAP NO:33 M1scFv KLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQF 32.4LC TYYGGTYESAFGGGTKVEIK SEQID MAdCA QVQLVESGGGVVQPGRSLRLSCAASGFSFSSSYWICWVRQAPG NO:34 M1scFv KGLEWVACIYGGSSGATYYANWAKGRFTISRDNSTNTLFLQM 32.5HC NSLRAEDTAVYYCARSGSTTSGGVYRWYFNLWGQGTLVTVSS SEQID MAdCA DIQMTQSPSSLSASVGDRVTITCQASENIYNLLAWYQQKPGKVP NO:35 M1scFv KLLIYDASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQ 32.5LC FTYYGGTYESAFGGGTKVEIK SEQID MAdCA QVQLVESGGGVVQPGRSLRLSCAASGFSFSSSYWICWVRQAPG NO:36 M1scFv KGLEWVACIYGGSSGATYYANWAKGRFTISRDNSTNTLFLQM 32.6HC NSLRAEDTAVYYCARSGSTTSGGVYRWYFNLWGQGTLVTVSS SEQID MAdCA DIQMTQSPSSLSASVGDRVTITCQASENIYNLLAWYQQKPGKAP NO:37 M1scFv KLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYC 32.6LC QFTYYGGTYESAFGGGTKVEIK SEQID MAdCA EVQLVESGGGLVQPGGSLRLSCAASGFSFSSSYWICWVRQAPG NO:38 M1scFv KGLEWVSCIYGGSSGATYYANWAKGRFTISRHNSKNTLYLQM 32.7HC NSLRAEDTAVYYCARSGSTTSGGVYRWYFNLWGQGTLVTVSS SEQID MAdCA DIQMTQSPSSLSASVGDRVTITCQASENIYNLLAWYQQKPGKVP NO:39 M1scFv KLLIYDASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQ 32.7LC FTYYGGTYESAFGGGTKVEIK SEQID MAdCA EVQLVESGGGLVQPGGSLRLSCAASGFSFSSSYWICWVRQAPG NO:40 M1scFv KGLEWVSCIYGGSSGATYYANWAKGRFTISRHNSKNTLYLQM 32.8HC NSLRAEDTAVYYCARSGSTTSGGVYRWYFNLWGQGTLVTVSS SEQID MAdCA DIQMTQSPSSLSASVGDRVTITCQASENIYNLLAWYQQKPGKAP NO:41 M1scFv KLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQF 32.8LC TYYGGTYESAFGGGTKVEIK SEQID MAdCA EVQLVESGGGLVQPGGSLRLSCAASGFTLSSSYYMYWVRQAP NO:42 M1scFv GKGLEWVSVIYAGDGNTYYASWAKGRFTISRDNSKNTLYLQM 8.1HC NSLRAEDTAVYYCARGYVGYGYAIDLWGQGTLVTVSS SEQID MAdCA DIQMTQSPSTLSASVGDRVTITCQASQSIYSSLAWYQQKPGKAP NO:43 M1scFv KLLIYYASILPSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQA 8.1LC TTYDSTYPNAFGGGTKVEIK SEQID MAdCA EVQLVESGGGLVQPGGSLRLSCAASGFTLSSSYYMYWVRQAP NO:44 M1scFv GKGLEWVSVIYAGDGNTYYASWAKGRFTISRDNSKNTLYLQM 8.2HC NSLRAEDTAVYYCARGYVGYGYAIDLWGQGTLVTVSS SEQID MAdCA AIQLTQSPSSLSASVGDRVTITCQASQSIYSSLAWYQQKPGKAP NO:45 M1scFv KLLIYYASILPSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQA 8.2LC TTYDSTYPNAFGGGTKVEIK SEQID MAdCA EVQLLESGGGLVQPGGSLRLSCAASGFTLSSSYYMYWVRQAPG NO:46 M1scFv KGLEWVSAIYAGDGNTYYASWAKGRFTISRDNSKNTLYLQMN 8.4HC SLRAEDTAVYYCARGYVGYGYAIDLWGQGTLVTVSS SEQID MAdCA AIQLTQSPSSLSASVGDRVTITCQASQSIYSSLAWYQQKPGKAP NO:47 M1scFv KLLIYYASILPSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQA 8.4LC TTYDSTYPNAFGGGTKVEIK SEQID MAdCA QVQLVESGGGVVQPGRSLRLSCAASGFTLSSSYYMYWVRQAP NO:48 M1scFv GKGLEWVAVIYAGDGNTYYASWAKGRFTISRDNSKNTLYLQM 8.5HC NSLRAEDTAVYYCARGYVGYGYAIDLWGQGTLVTVSS SEQID MAdCA DIQMTQSPSTLSASVGDRVTITCQASQSIYSSLAWYQQKPGKAP NO:49 M1scFv KLLIYYASILPSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQA 8.5LC TTYDSTYPNAFGGGTKVEIK SEQID MAdCA QVQLVESGGGVVQPGRSLRLSCAASGFTLSSSYYMYWVRQAP NO:50 M1scFv GKGLEWVAVIYAGDGNTYYASWAKGRFTISRDNSKNTLYLQM 8.6HC NSLRAEDTAVYYCARGYVGYGYAIDLWGQGTLVTVSS SEQID MAdCA AIQLTQSPSSLSASVGDRVTITCQASQSIYSSLAWYQQKPGKAP NO:51 M1scFv KLLIYYASILPSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQA 8.6LC TTYDSTYPNAFGGGTKVEIK SEQID MAdCA EVQLVETGGGLIQPGGSLRLSCAASGFTLSSSYYMYWVRQAPG NO:52 M1scFv KGLEWVSVIYAGDGNTYYASWAKGRFTISRDNSKNTLYLQMN 8.7HC SLRAEDTAVYYCARGYVGYGYAIDLWGQGTLVTVSS SEQID MAdCA DIQMTQSPSTLSASVGDRVTITCQASQSIYSSLAWYQQKPGKAP NO:53 M1scFv KLLIYYASILPSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQA 8.7LC TTYDSTYPNAFGGGTKVEIK SEQID MAdCA EVQLVETGGGLIQPGGSLRLSCAASGFTLSSSYYMYWVRQAPG NO:54 M1scFv KGLEWVSVIYAGDGNTYYASWAKGRFTISRDNSKNTLYLQMN 8.8HC SLRAEDTAVYYCARGYVGYGYAIDLWGQGTLVTVSS SEQID MAdCA AIQLTQSPSSLSASVGDRVTITCQASQSIYSSLAWYQQKPGKAP NO:55 M1scFv KLLIYYASILPSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQA 8.8LC TTYDSTYPNAFGGGTKVEIK SEQID MAdCA EVQLVESGGGLVQPGGSLRLSCAASGFSFSSTYYMCWVRQAPG NO:56 M1scFv KGLEWVSCIYTGSGNTDYASWAKGRFTISRHNSKNTLYLQMNS 17.1HC LRAEDTAVYYCARGGIVDSYFTYFDLWGQGTLVTVSS SEQID MAdCA DIQMTQSPSTLSASVGDRVTITCQASESIFSNLAWYQQKPGKAP NO:57 M1scFv KLLIYWASTLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQ 17.1LC SYVYSSSSSNDFGGGTKVEIK SEQID MAdCA QVQLLESGGGLVKPGGSLRLSCAASGFSFSSTYYMCWIRQAPG NO:58 M1scFv KGLEWVSCIYTGSGNTDYASWAKGRFTISRDNAKNSLYLQMN 17.3HC SLRAEDTAVYYCARGGIVDSYFTYFDLWGQGTLVTVSS SEQID MAdCA DIQMTQSPSTLSASVGDRVTITCQASESIFSNLAWYQQKPGKAP NO:59 M1scFv KLLIYWASTLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQ 17.3LC SYVYSSSSSNDFGGGTKVEIK SEQID MAdCA QVQLVESGGGVVQPGRSLRLSCAASGFSFSSTYYMCWVRQAP NO:60 M1scFv GKGLEWVACIYTGSGNTDYASWAKGRFTISRDNSTNTLFLQM 17.5HC NSLRAEDTAVYYCARGGIVDSYFTYFDLWGQGTLVTVSS SEQID MAdCA DIQMTQSPSTLSASVGDRVTITCQASESIFSNLAWYQQKPGKAP NO:61 M1scFv KLLIYWASTLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQ 17.5LC SYVYSSSSSNDFGGGTKVEIK SEQID MAdCA EVQLVESGGGLVKPGGSLRLSCAASGFSFSSTYYMCWVRQAPG NO:62 M1scFv KGLEWVSCIYTGSGNTDYASWAKGRFTISRDNAKNSLYLQMN 17.7HC SLRAEDTAVYYCARGGIVDSYFTYFDLWGQGTLVTVSS SEQID MAdCA DIQMTQSPSTLSASVGDRVTITCQASESIFSNLAWYQQKPGKAP NO:63 M1scFv KLLIYWASTLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQ 17.7LC SYVYSSSSSNDFGGGTKVEIK SEQID MAdCA QVQLLESGGGLVKPGGSLRLSCAASGFSFSSGYYMCWIRQAPG NO:64 M1scFv KGLEWVSCIYADSSYTYYASWAKGRFTISRDNAKNSLYLQMN 2.1HC SLRAEDTAVYYCARSNGDYFYGMDLWGQGTLVTVSS SEQID MAdCA DIQMTQSPSSLSASVGDRVTITCQASQSINSWLSWYQQKPGKAP NO:65 M1scFv KLLIYRASTLASGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQS 2.1LC NYYSTSTAFGGGTKVEIK SEQID MAdCA QVQLLESGGGLVKPGGSLRLSCAASGFSFSSGYYMCWIRQAPG NO:66 M1scFv KGLEWVSCIYADSSYTYYASWAKGRFTISRDNAKNSLYLQMN 2.2HC SLRAEDTAVYYCARSNGDYFYGMDLWGQGTLVTVSS SEQID MAdCA AIQMTQSPSSLSASVGDRVTITCQASQSINSWLSWYQQKPGKAP NO:67 M1scFv KLLIYRASTLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ 2.2LC SNYYSTSTAFGGGTKVEIK SEQID MAdCA QVQLVESGGGVVQPGRSLRLSCAASGIDFSGYHYICWVRQAPG NO:68 M1scFv KGLEWVACIYTVSSGIWYATWAKGRFTISRDNSTNTLFLQMNS 10.1HC LRAEDTAVYYCARDDADVSGYWFDLWGQGTLVTVSS SEQID MAdCA DIQMTQSPSSLSASVGDRVTITCQASQSISNLLAWYQQKPGKAP NO:69 M1scFv KLLIYKASTLASGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQ 10.1LC GITNNGIDHGFGGGTKVEIK SEQID MAdCA QVQLVESGGGVVQPGRSLRLSCAASGIDFSGYHYICWVRQAPG NO:70 M1scFv KGLEWVACIYTVSSGIWYATWAKGRFTISRDNSTNTLFLQMNS 10.2HC LRAEDTAVYYCARDDADVSGYWFDLWGQGTLVTVSS SEQID MAdCA AIQLTQSPSSLSASVGDRVTITCQASQSISNLLAWYQQKPGKAP NO:71 M1scFv KLLIYKASTLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ 10.2LC QGITNNGIDHGFGGGTKVEIK SEQID MAdCA EVQLVESGGGLVKPGGSLRLSCAASGIDFSGYHYICWVRQAPG NO:72 M1scFv KGLEWVSCIYTVSSGIWYATWAKGRFTISRDNAKNSLYLQMNS 10.3HC LRAEDTAVYYCARDDADVSGYWFDLWGQGTLVTVSS SEQID MAdCA DIQMTQSPSSLSASVGDRVTITCQASQSISNLLAWYQQKPGKAP NO:73 M1scFv KLLIYKASTLASGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQ 10.3LC GITNNGIDHGFGGGTKVEIK SEQID MAdCA EVQLLESGGGLVQPGGSLRLSCAASGIDFSGYHYICWVRQAPG NO:74 M1scFv KGLEWVSCIYTVSSGIWYATWAKGRFTISRDNSKNTLYLQMNS 10.7HC LRAEDTAVYYCARDDADVSGYWFDLWGQGTLVTVSS SEQID MAdCA DIQMTQSPSSLSASVGDRVTITCQASQSISNLLAWYQQKPGKAP NO:75 M1scFv KLLIYKASTLASGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQ 10.7LC GITNNGIDHGFGGGTKVEIK SEQID MAdCA EVQLLESGGGLVQPGGSLRLSCAASGIDFSGYHYICWVRQAPG NO:76 M1scFv KGLEWVSCIYTVSSGIWYATWAKGRFTISRDNSKNTLYLQMNS 10.8HC LRAEDTAVYYCARDDADVSGYWFDLWGQGTLVTVSS SEQID MAdCA AIQLTQSPSSLSASVGDRVTITCQASQSISNLLAWYQQKPGKAP NO:77 M1scFv KLLIYKASTLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ 10.8LC QGITNNGIDHGFGGGTKVEIK