Polypeptides comprising modified il-2 polypeptides and uses thereof

Abstract

To provide improved T cell modulating therapeutics.SOLUTION: Provided herein are polypeptides comprising a modified IL-2, where the modified IL-2 has reduced affinity for the IL-2 receptor relative to wild type IL-2. In some embodiments, polypeptides comprising a modified IL-2 that bind and agonize activated T cells are provided. Uses of the polypeptides comprising a modified IL-2 are also provided.SELECTED DRAWING: Figure 1A

Description

[Technical field]

[0001] [CROSS REFERENCE TO RELATED APPLICATIONS] This application claims the benefit of priority to U.S. Provisional Application No. 62 / 789,075, filed January 7, 2019, which is incorporated by reference in its entirety for all purposes.

[0002] The present invention relates to modified IL-2 that has reduced affinity for CD25 and CD122, and such modified IL-2 fused to a targeting moiety. The present invention also relates to methods of using the modified IL-2 and polypeptides comprising modified IL-2, including, but not limited to, methods of treating cancer. [Background technology]

[0003] IL-2 is a potent cytokine that stimulates the proliferation of T and NK cells through either the heterotrimeric IL-2 receptor (IL-2R) composed of CD25, CD122, and CD132, or the heterodimeric IL-2 receptor composed of only CD122 and CD132. Both forms of IL-2R are potent mediators of T cell survival, proliferation, and overall activation status. IL-2 is generally produced by T and NK cells after activation and mediates cis and trans signaling in the local microenvironment. IL-2R signaling can induce the differentiation of naive T cells into effector and memory T cells, and can also stimulate suppressive regulatory T cells. The trimeric form of IL-2R has a higher affinity for IL-2 than the dimeric form, but both are moderately high affinity, leading to rapid receptor-mediated internalization and degradation, resulting in a very short half-life. Recombinant human IL-2 (rhIL-2, Proleukin) is used clinically to treat renal cell carcinoma and malignant melanoma, but is associated with significant toxicity. Vascular leak syndrome is a major toxicity issue for cancer patients treated with Proleukin, due to the effects of IL-2 signaling on endothelial cells expressing the high affinity IL-2R.

[0004] T cells are activated by ligation of their TCR with adjacent cells presenting MHC bound with complementary peptides, which leads to clustering of the TCR complex and signaling through NFAT. T cell costimulation via CD28 is mediated by CD80 and CD86, enhancing T cell activation. After initial activation, T cells upregulate a variety of proteins, including cytokine receptors, as well as a number of costimulatory and checkpoint receptors that serve to regulate T cell responses.

[0005] Durable antitumor clinical responses have been reported in recent years with antagonistic antibodies against checkpoint receptors such as CTLA-4, PD-1, and PD-L1. However, even in the most responsive indications, response rates are limited to approximately 30% of patients. Thus, improved T cell modulating therapeutics are needed. Summary of the Invention

[0006] Provided herein are polypeptides comprising modified IL-2 comprising at least one substitution at at least one amino acid position. In some embodiments, the modified IL-2 has reduced binding affinity to CD25, CD122, and / or IL-2R compared to wild-type IL-2. In some embodiments, the modified IL-2 has reduced activity against resting or activated T cells compared to wild-type IL-2.

[0007] Embodiment 1. A polypeptide comprising a modified IL-2, said modified IL-2 comprising at least one substitution at at least one amino acid position selected from P65, D84, E95, M23, and H16.

[0008] Embodiment 2. 2. The polypeptide of embodiment 1, wherein said modified IL-2 is modified human IL-2.

[0009] Embodiment 3. 3. The polypeptide of embodiment 1 or 2, wherein said amino acid positions correspond to the amino acid positions in SEQ ID NO:1.

[0010] Embodiment 4. 23. The polypeptide of any one of the preceding embodiments, wherein said modified IL-2 comprises a substitution at amino acid position P65.

[0011] Embodiment 5. 5. The polypeptide of embodiment 4, wherein said substitutions are selected from P65R, P65E, P65K, P65H, P65Y, P65Q, P65D, and P65N.

[0012] Embodiment 6. 23. The polypeptide of any one of the preceding embodiments, wherein said modified IL-2 comprises a substitution at amino acid position H16.

[0013] Embodiment 7. 7. The polypeptide of embodiment 6, wherein said substitutions are selected from H16A, H16G, H16S, H16T, H16V, and H16P.

[0014] Embodiment 8. 23. The polypeptide of any one of the preceding embodiments, wherein said modified IL-2 comprises a substitution at amino acid position D84.

[0015] EMBODIMENT 9. 9. The polypeptide of embodiment 8, wherein said substitutions are selected from D84S, D84G, D84A, D84T, D84V, and D84P.

[0016] Embodiment 10. The polypeptide of any one of the preceding embodiments, wherein the modified IL-2 comprises substitutions at amino acid positions P65, H16, and D84.

[0017] Embodiment 11. 11. The polypeptide of embodiment 10, wherein said modified IL-2 comprises the substitutions P65R, H16A, and D84S.

[0018] Embodiment 12. 23. The polypeptide of any one of the preceding embodiments, wherein said modified IL-2 comprises a substitution at amino acid position M23.

[0019] Embodiment 13. 13. The polypeptide of embodiment 12, wherein said substitutions are selected from M23A, M23G, M23S, M23T, M23V, and M23P.

[0020] Embodiment 14. 14. The polypeptide of embodiment 13, wherein said modified IL-2 comprises the substitutions P65R, H16A, D84S, and M23A.

[0021] EMBODIMENT 15. 23. The polypeptide of any one of the preceding embodiments, wherein said modified IL-2 comprises a substitution at amino acid position E95.

[0022] Embodiment 16. 16. The polypeptide of embodiment 15, wherein said substitution is selected from E95Q, E95G, E95S, E95T, E95V, E95P, E95H, and E95N.

[0023] EMBODIMENT 17. 17. The polypeptide of embodiment 16, wherein said modified IL-2 comprises the substitutions P65R, H16A, D84S, and E95Q.

[0024] Embodiment 18. 18. The polypeptide of embodiment 17, wherein said modified IL-2 comprises the substitutions P65R, H16A, D84S, M23A, and E95Q.

[0025] EMBODIMENT 19. 23. The polypeptide of any one of the preceding embodiments, wherein said modified IL-2 comprises a substitution at amino acid position F42.

[0026] EMBODIMENT 20. 20. The polypeptide of embodiment 19, wherein said substitution at F42 is selected from F42K, F42A, F42R, F42A, F42G, F42S, and F42T.

[0027] EMBODIMENT 21. The polypeptide of any one of the preceding embodiments, wherein said modified IL-2 comprises at least one substitution at at least one amino acid position selected from Y45 and L72.

[0028] EMBODIMENT 22. 22. The polypeptide of embodiment 21, wherein said modified IL-2 comprises at least one substitution selected from Y45A and L72G.

[0029] EMBODIMENT 23. The polypeptide of any one of the preceding embodiments, wherein said modified IL-2 comprises at least one substitution at at least one amino acid position selected from T3 and C125.

[0030] EMBODIMENT 24. 24. The polypeptide of embodiment 23, wherein said modified IL-2 comprises at least one substitution selected from T3A and C125A.

[0031] 25. The modified IL-2 includes H16A-F42K, D84S-F42K, E15S-F42K, M23A-F42K, E95Q-F42K, P65R-H16A, P65R-D84S, P65R-E15S, P65R-M23A, P65R-E95Q, T3A-C125S, T3A-P65R-C125S, T3A-H16A-C125S, T3A-D84S- 5. The polypeptide of any one of the preceding embodiments, comprising a range of substitutions selected from: T3A-H16A-P65R-C125S, T3A-P65R-D84S-C125S, T3A-H16A-P65R-D84S-C125S, T3A-H16A-P65R-D84S-C125S, T3A-H16A-M23A-P65R-D84S-C125S, T3A-H16A-P65R-D84S-E95Q-C125S, and T3A-H16A-M23A-P65R-D84S-E95Q-C125S.

[0032] 26. 26. The polypeptide of embodiment 25, wherein said modified IL-2 comprises said set of substitutions and does not comprise any additional substitutions.

[0033] EMBODIMENT 27. The polypeptide of any one of the above embodiments, wherein the modified IL-2 comprises an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:84.

[0034] 28. The polypeptide of any one of the above embodiments, wherein the modified IL-2 comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from SEQ ID NO:3 to SEQ ID NO:9, SEQ ID NO:11 to SEQ ID NO:21, and SEQ ID NO:23 to SEQ ID NO:31.

[0035] 29. The polypeptide of any one of the above embodiments, wherein the modified IL-2 comprises an amino acid sequence selected from SEQ ID NO:3 to SEQ ID NO:9, SEQ ID NO:11 to SEQ ID NO:21, and SEQ ID NO:23 to SEQ ID NO:31.

[0036] EMBODIMENT 30. 2. The polypeptide of any one of the preceding embodiments, wherein the polypeptide comprises an Fc region.

[0037] EMBODIMENT 31. The polypeptide of embodiment 30, wherein the modified IL-2 is fused to the N-terminus or C-terminus of the Fc region.

[0038] EMBODIMENT 32. 32. The polypeptide of embodiment 30 or 31, wherein said Fc region comprises a substitution at Kabat amino acid position T366.

[0039] EMBODIMENT 33. 33. The polypeptide of embodiment 32, wherein the Fc region comprises a T366W substitution.

[0040] EMBODIMENT 34. 32. The polypeptide of embodiment 31, wherein said Fc region comprises at least one substitution at at least one Kabat amino acid position selected from T366, L368, and Y407.

[0041] EMBODIMENT 35. 35. The polypeptide of embodiment 34, wherein the Fc region comprises T366S, L368A, and Y407V mutations.

[0042] 36. 36. The polypeptide of any one of embodiments 30 to 35, wherein said Fc region comprises a substitution at a Kabat position selected from S354 and Y349.

[0043] 37. 37. The polypeptide of embodiment 36, wherein the Fc region comprises a S354C or a Y349C substitution.

[0044] 38. 38. The polypeptide of any one of embodiments 30-37, wherein said Fc region comprises a substitution at Kabat amino acid position H435.

[0045] 39. 39. The polypeptide of embodiment 38, wherein said Fc region comprises a substitution selected from H435R and H435K.

[0046] EMBODIMENT 40. 40. The polypeptide of any one of embodiments 30 to 39, wherein said Fc region comprises at least one substitution at at least one Kabat amino acid position selected from M252 and M428.

[0047] EMBODIMENT 41. 41. The polypeptide of embodiment 40, wherein the Fc region comprises M252Y and M428V substitutions.

[0048] EMBODIMENT 42. 42. The polypeptide of any one of embodiments 30-41, wherein said Fc region comprises a deletion of Kabat amino acids E233, L234, and L235.

[0049] EMBODIMENT 43. 42. The polypeptide of any one of embodiments 30-41, wherein said Fc region comprises at least one substitution at at least one amino acid position selected from L234, L235, and P329.

[0050] EMBODIMENT 44. 44. The polypeptide of embodiment 43, wherein the Fc region comprises L234A, L235A, and P329G substitutions.

[0051] EMBODIMENT 45. The polypeptide of any one of embodiments 30 to 44, wherein the Fc region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from SEQ ID NO: 47 to SEQ ID NO: 83.

[0052] EMBODIMENT 46. 45. The polypeptide of any one of embodiments 30 to 44, wherein said Fc region is a portion of a heavy chain constant region.

[0053] EMBODIMENT 47. 47. The polypeptide of embodiment 46, wherein said heavy chain constant region is an IgG constant region.

[0054] 48. 48. The polypeptide of embodiment 47, wherein said heavy chain constant region is an IgG1, IgG2, IgG3, or IgG4 constant region.

[0055] 49. The polypeptide of any one of embodiments 30 to 48, wherein the modified IL-2 is fused to the C-terminus of the Fc region or heavy chain constant region.

[0056] EMBODIMENT 50. 50. The polypeptide of embodiment 49, wherein the modified IL-2 is fused to the C-terminus of the Fc region or heavy chain constant region by a linker comprising 1 to 20 amino acids.

[0057] EMBODIMENT 51. 51. The polypeptide of embodiment 50, wherein the linker comprises a glycine amino acid.

[0058] EMBODIMENT 52. 52. The polypeptide of embodiment 51, wherein the linker comprises glycine and serine amino acids.

[0059] EMBODIMENT 53. 53. The polypeptide of any one of embodiments 50-52, wherein most or all of the amino acids in the linker are glycine and serine.

[0060] EMBODIMENT 54. The polypeptide of any one of embodiments 30 to 33, 42, and 49 to 53, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 102, SEQ ID NO: 103, or SEQ ID NO: 104.

[0061] EMBODIMENT 55. 2. The polypeptide of any one of the preceding embodiments, wherein said polypeptide comprises at least one antigen binding domain.

[0062] EMBODIMENT 56. 56. The polypeptide of embodiment 55, wherein the polypeptide comprises two, three, or four antigen-binding domains.

[0063] EMBODIMENT 57. 57. The polypeptide of embodiment 55 or 56, wherein at least one antigen-binding domain specifically binds to a T cell antigen or a natural killer cell antigen.

[0064] EMBODIMENT 58. At least one antigen-binding domain is a CD4 + T cell antigen or CD8 +58. The polypeptide of any one of embodiments 55-57, which specifically binds to a T cell antigen.

[0065] EMBODIMENT 59. The at least one antigen-binding domain is an antigen-binding domain that binds to activated CD4 + T cells or activated CD8 + 59. The polypeptide of embodiment 58, which specifically binds to an antigen on a T cell.

[0066] 60. 60. The polypeptide of any one of embodiments 55-59, wherein at least one antigen-binding domain is agonistic.

[0067] 61. 60. The polypeptide of any one of embodiments 55-59, wherein said antigen-binding domain is an antagonist.

[0068] 62. 62. The polypeptide of any one of embodiments 55-61, wherein at least one antigen-binding domain specifically binds to PD-1, CTLA-4, LAG3, TIM3, 4-1BB, OX40, GITR, CD8a, CD8b, CD4, NKp30, NKG2A, TIGIT, TGFβR1, TGFβR2, Fas, NKG2D, NKp46, PD-L1, CD107a, ICOS, TNFR2, or CD16a.

[0069] 63. 63. The polypeptide of any one of embodiments 55-62, wherein at least one antigen-binding domain specifically binds to PD-1.

[0070] 64. The polypeptide of any one of embodiments 55 to 63, wherein at least one antigen-binding domain is a human or humanized antigen-binding domain.

[0071] 65. 65. The polypeptide of embodiment 64, wherein each antigen-binding domain is independently a human or humanized antigen-binding domain.

[0072] 66. 66. The polypeptide of any one of embodiments 55 to 65, wherein at least one antigen-binding domain comprises a VHH domain.

[0073] 67. 67. The polypeptide of embodiment 66, wherein each antigen-binding domain comprises a VHH domain.

[0074] 68. 66. The polypeptide of any one of embodiments 55 to 65, wherein at least one antigen-binding domain comprises a VH domain and a VL domain.

[0075] 69. 69. The polypeptide of embodiment 68, wherein at least one antigen-binding domain comprises a VH domain and a VL domain of an antibody selected from pembrolizumab, nivolumab, AMP-514, TSR-042, STI-A1110, ipilimumab, tremelimumab, urelumab, utomilumab, atezolizumab, and durvalumab.

[0076] EMBODIMENT 70. 70. The polypeptide of embodiment 68 or 69, wherein said at least one antigen-binding domain comprises a single chain Fv (scFv).

[0077] EMBODIMENT 71. 70. The polypeptide of embodiment 68 or 69, wherein the polypeptide comprises a heavy chain constant region, the VH domain is fused to the heavy chain constant region, and the VL domain is associated with the VH domain.

[0078] EMBODIMENT 72. 72. The polypeptide of embodiment 71, wherein said VL domain is fused to a light chain constant region.

[0079] EMBODIMENT 73. 73. The polypeptide of embodiment 72, wherein said light chain constant region is selected from kappa and lambda.

[0080] 74. The polypeptide of any one of embodiments 55 to 73, wherein each of said antigen-binding domains is the same.

[0081] 75. The polypeptide of any one of embodiments 55 to 74, wherein each of the antigen-binding domains specifically binds to the same antigen.

[0082] EMBODIMENT 76. The polypeptide of any one of embodiments 55 to 73, wherein at least one of the antigen-binding domains specifically binds to a different antigen than at least one of the other antigen-binding domains.

[0083] 77. The polypeptide of any one of embodiments 55 to 73, wherein at least one antigen-binding domain specifically binds to PD-1, and at least one other antigen-binding domain specifically binds to a T cell antigen or a natural killer cell antigen other than PD-1.

[0084] 78. 78. The polypeptide of any one of embodiments 55-77, wherein at least one antigen-binding domain binds to PD-1, CTLA-4, LAG3, TIM3, 4-1BB, OX40, GITR, CD8a, CD8b, CD4, NKp30, NKG2A, TIGIT, TGFβR1, TGFβR2, Fas, NKG2D, NKp46, PD-L1, CD107a, ICOS, TNFR2, or CD16a.

[0085] 79. The polypeptide of any one of embodiments 31-78, wherein the polypeptide forms a homodimer under physiological conditions.

[0086] 80. 80. The polypeptide of any one of embodiments 1-79, wherein the modified IL-2 binds to human IL-2R with an affinity that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 50-fold, or at least 100-fold lower than the affinity of human wild-type IL-2 for IL-2R.

[0087] 81. A complex comprising a first polypeptide and a second polypeptide, wherein said first polypeptide is the polypeptide of any one of embodiments 1-79.

[0088] 82. 82. The conjugate of embodiment 81, wherein said first polypeptide comprises a first Fc region and said second polypeptide comprises a second Fc region.

[0089] 83. 83. The conjugate of embodiment 81 or 82, wherein each Fc region is an isotype selected from human IgG1, IgG2, IgG3, and IgG4.

[0090] 84. 84. The conjugate of embodiment 83, wherein each Fc region is human IgG1.

[0091] 85. The conjugate of any one of embodiments 81 to 84, wherein each Fc region comprises a deletion of amino acids E233, L234, and L235.

[0092] 86. The conjugate of any one of embodiments 81 to 85, wherein each Fc region comprises a H435R or H435K mutation.

[0093] 87. The conjugate of any one of embodiments 81 to 86, wherein said Fc region comprises the mutations M252Y and M428L or the mutations M252Y and M428V.

[0094] 88. The conjugate of any one of embodiments 81 to 87, wherein the first Fc region or the second Fc region comprises a T366W mutation and the other Fc region comprises the mutations T366S, L368A, and Y407V.

[0095] 89. 89. The conjugate of embodiment 88, wherein said first Fc region or said second Fc region comprises a S354C mutation.

[0096] EMBODIMENT 90. The conjugate of any one of embodiments 81 to 89, wherein each Fc region independently comprises an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from SEQ ID NO: 47 to SEQ ID NO: 83.

[0097] EMBODIMENT 91. 91. The conjugate of any one of embodiments 81-90, wherein said second polypeptide does not comprise modified IL2.

[0098] EMBODIMENT 92. 92. The conjugate of any one of embodiments 81-91, wherein said first polypeptide comprises at least one antigen-binding domain.

[0099] EMBODIMENT 93. 93. The conjugate of any one of embodiments 81-92, wherein said second polypeptide comprises at least one antigen-binding domain.

[0100] EMBODIMENT 94. 94. The conjugate of any one of embodiments 81-93, wherein the first polypeptide comprises a first antigen-binding domain, an Fc region, and a modified IL-2.

[0101] EMBODIMENT 95. 95. The conjugate of embodiment 94, wherein said first antigen-binding domain is fused to the N-terminus of said Fc region and said modified IL-2 is fused to the C-terminus of said Fc region.

[0102] EMBODIMENT 96. 96. The conjugate of embodiment 94 or 95, wherein said second polypeptide comprises a second antigen-binding domain and an Fc region.

[0103] EMBODIMENT 97. 97. The conjugate of embodiment 96, wherein said first antigen-binding domain and said second antigen-binding domain are the same or different.

[0104] EMBODIMENT 98. a) the first antigen-binding domain and the second antigen-binding domain both bind to PD-1; b) the first antigen-binding domain binds to PD-1 and the second antigen-binding domain binds to LAG3; c) the first antigen-binding domain binds to PD-1 and the second antigen-binding domain binds to CTLA-4; d) the first antigen-binding domain binds to PD-1 and the second antigen-binding domain binds to 4-1BB; e) the first antigen-binding domain binds to PD-1 and the second antigen-binding domain binds to OX40; f) the first antigen-binding domain binds to PD-1 and the second antigen-binding domain binds to GITR; g) the first antigen-binding domain binds to PD-1 and the second antigen-binding domain binds to CD8a; h) the first antigen-binding domain binds to PD-1 and the second antigen-binding domain binds to CD8b; i) the first antigen-binding domain binds to PD-1 and the second antigen-binding domain binds to CD4; j) the first antigen-binding domain binds to PD-1 and the second antigen-binding domain binds to NKp30; k) the first antigen-binding domain binds to PD-1 and the second antigen-binding domain binds to NKG2A; l) the first antigen-binding domain binds to PD-1 and the second antigen-binding domain binds to TIGIT; m) the first antigen-binding domain binds to PD-1 and the second antigen-binding domain binds to NKG2D; n) the first antigen-binding domain binds to PD-1 and the second antigen-binding domain binds to TGFBR2; o) the first antigen-binding domain binds to PD-1 and the second antigen-binding domain binds to Fas; p) the first antigen-binding domain binds to PD-1 and the second antigen-binding domain binds to CD107a; q) the first antigen-binding domain binds to PD-1 and the second antigen-binding domain binds to NKp46; r) the first antigen-binding domain binds to CD8a and the second antigen-binding domain binds to TGFRβR2; s) the first antigen-binding domain binds to CD8a and the second antigen-binding domain binds to Fas; t) the first antigen-binding domain binds to NKG2D and the second antigen-binding domain binds to TGFRβR2; u) the first antigen-binding domain binds to NKG2D and the second antigen-binding domain binds to Fas; v) the first antigen-binding domain binds to NKG2A and the second antigen-binding domain binds to TGFRβR2; w) the first antigen-binding domain binds to NKG2A and the second antigen-binding domain binds to Fas; x) the first antigen-binding domain binds to NKp46 and the second antigen-binding domain binds to TGFRβR2; y) the first antigen-binding domain binds to NKp46 and the second antigen-binding domain binds to Fas; z) the first antigen-binding domain binds to CTLA-4 and the second antigen-binding domain binds to LAG3; aa) the first antigen-binding domain binds to CTLA-4 and the second antigen-binding domain binds to Tim3; bb) the first antigen-binding domain binds to CTLA-4 and the second antigen-binding domain binds to OX40; cc) the first antigen-binding domain binds to CTLA-4 and the second antigen-binding domain binds to GITR; dd) the first antigen-binding domain binds to CTLA-4 and the second antigen-binding domain binds to CD107a; ee) the first antigen-binding domain binds to CTLA-4 and the second antigen-binding domain binds to NKp46; or ff) The conjugate of embodiment 97, wherein the first antigen-binding domain binds to ICOS and the second antigen-binding domain binds to TNFR2.

[0105] EMBODIMENT 99. The conjugate of any one of embodiments 81-98, wherein the modified IL-2 binds to human IL-2R with an affinity that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 50-fold, or at least 100-fold lower than the affinity of human wild-type IL-2 for IL-2R.

[0106] EMBODIMENT 100. A pharmaceutical composition comprising a polypeptide according to any one of embodiments 1 to 80 or a conjugate according to any one of embodiments 81 to 99 and a pharma- ceutically acceptable carrier.

[0107] EMBODIMENT 101. An isolated nucleic acid encoding the polypeptide of any one of embodiments 1 to 80 or the conjugate of any one of embodiments 81 to 99.

[0108] EMBODIMENT 102. An expression vector comprising the nucleic acid of embodiment 101.

[0109] EMBODIMENT 103. 102. An isolated host cell comprising the nucleic acid of embodiment 101 or the expression vector of embodiment 102.

[0110] EMBODIMENT 104. An isolated host cell expressing the polypeptide of any one of embodiments 1 to 80 or the conjugate of any one of embodiments 81 to 99.

[0111] EMBODIMENT 105. A method for producing a polypeptide of any one of embodiments 1 to 80 or a conjugate of any one of embodiments 81 to 99, comprising incubating a host cell of embodiment 103 or 104 under conditions suitable for expression of said polypeptide or conjugate.

[0112] EMBODIMENT 106. 106. The method of embodiment 105, further comprising isolating said polypeptide or complex.

[0113] EMBODIMENT 107. CD4 + T cells and / or CD8 + A method for increasing proliferation of T cells, comprising contacting T cells with a polypeptide of any one of embodiments 1-80 or a complex of any one of embodiments 81-99.

[0114] EMBODIMENT 108. CD4 + T cells and / or CD8 + The method of embodiment 107, wherein the T cells are present in vitro.

[0115] EMBODIMENT 109. CD4 + T cells and / or CD8 + The method of embodiment 107, wherein the T cells are present in vivo.

[0116] EMBODIMENT 110. The method of any one of embodiments 107-109, wherein the increase is at least 1.5-fold, at least 2-fold, at least 3-fold, or at least 5-fold.

[0117] EMBODIMENT 111. A method for increasing NK cell proliferation, comprising contacting NK cells with a polypeptide of any one of embodiments 1-80 or a complex of any one of embodiments 81-99.

[0118] EMBODIMENT 112. 112. The method of embodiment 111, wherein the increase is at least 1.5-fold, at least 2-fold, at least 3-fold, or at least 5-fold.

[0119] EMBODIMENT 113. A method for treating cancer, comprising administering to a subject with cancer a pharma- ceutical effective amount of a polypeptide of any one of embodiments 1-80, or a conjugate of any one of embodiments 81-99, or a pharmaceutical composition of embodiment 100.

[0120] EMBODIMENT 114. The cancers include basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and central nervous system cancer, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, colorectal cancer, connective tissue cancer, digestive system cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, glioblastoma, liver cancer, hepatocellular carcinoma, intraepithelial neoplasia, kidney or renal cancer, laryngeal cancer, liver cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, melanoma, myeloma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer. , prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, respiratory system cancer, salivary gland cancer, sarcoma, skin cancer, squamous cell carcinoma, stomach cancer, testicular cancer, thyroid cancer, uterine or endometrial cancer, urinary system cancer, vulvar cancer, lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, B-cell lymphoma, low-grade / follicular non-Hodgkin lymphoma (NHL), small lymphocytic (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic 114. The method of embodiment 113, wherein the patient is selected from: advanced non-small cell lung cancer (NHL), high-grade lymphoblastic NHL, high-grade small non-dividing cell NHL, bulky disease NHL, mantle cell lymphoma, AIDS-related lymphoma, Waldenstrom's macroglobulinemia, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, and chronic myeloblastic leukemia.

[0121] EMBODIMENT 115. 115. The method of embodiment 113 or 114, further comprising administering an additional therapeutic agent.

[0122] EMBODIMENT 116. The method of embodiment 115, wherein said additional therapeutic agent is an anti-cancer agent.

[0123] EMBODIMENT 117. The method of embodiment 116, wherein the anti-cancer agent is selected from a chemotherapeutic agent, an anti-cancer biologic, a radiation therapy agent, a CAR-T therapy agent, and an oncolytic virus.

[0124] EMBODIMENT 118. 118. The method of embodiment 116 or 117, wherein said additional therapeutic agent is an anti-cancer biologic.

[0125] EMBODIMENT 119. The method of embodiment 118, wherein said anti-cancer biologic is an agent that inhibits PD-1 and / or PD-L1.

[0126] EMBODIMENT 120. The method of embodiment 118, wherein said anti-cancer biologic is an agent that inhibits VISTA, gpNMB, B7H3, B7H4, HHLA2, CTLA4, or TIGIT.

[0127] EMBODIMENT 121. The method of any one of embodiments 116 to 120, wherein the anti-cancer agent is an antibody.

[0128] EMBODIMENT 122. The method of embodiment 118, wherein the anti-cancer biologic is a cytokine.

[0129] EMBODIMENT 123. The method of embodiment 116, wherein the anti-cancer agent is a CAR-T therapeutic agent.

[0130] EMBODIMENT 124. The method of embodiment 116, wherein the anticancer agent is an oncolytic virus.

[0131] EMBODIMENT 125. The method of any one of embodiments 113-124, further comprising tumor resection and / or radiation therapy. [Brief description of the drawings]

[0132] [Figure 1A-1H] Schematic diagrams of various IL-2 fusion protein formats. FIG. 1A shows IL-2 linked to the N-terminus of a heterodimeric knob-in-hole IgG1 Fc. FIG. 1B shows IL-2 linked to the C-terminus of a heterodimeric IgG1 Fc of a single domain antibody. FIG. 1C-FIG. 1E show IL-2 linked to one VHH (FIG. 1E), two identical VHH (FIG. 1C), or two different VHH (FIG. 1D). FIG. 1F shows IL-2 linked to the C-terminus of a homodimeric heavy chain constant region of a conventional antibody. FIG. 1G shows IL-2 linked to the C-terminus of a heterodimeric heavy chain constant region of a conventional antibody. FIG. 1H shows IL-2 fused to the C-terminus of a heterodimeric scFv antibody. [Figure 2A-2C] Figure 2 shows the binding of IL-2 fusion proteins containing wild-type IL-2 (Figure 2A) or modified IL-2 (Figures 2A-C) fused to the N-terminus of heterodimeric Fc as shown in Figure 1A to 293F cells transiently transfected with various combinations of IL-2 receptors (CD25, CD122, and CD132) as measured by flow cytometry. "UT 293F" indicates untransfected 293F cells. [Figure 3A-3B]FIG. 1B shows the binding of fusion proteins comprising wild-type IL-2 or modified IL-2 fused to the N-terminus of the heterodimeric Fc as shown in FIG. 1A to 293F cells transiently transfected with CD25 and CD122 as measured by flow cytometry. [Figure 4A-4B] FIG. 1B shows the binding of fusion proteins comprising wild-type IL-2 or modified IL-2 fused to the N-terminus of the heterodimeric Fc as shown in FIG. 1A to 293F cells transiently transfected with CD122 and CD132, or CD25, CD122, and CD132, as measured by flow cytometry. [Figure 5A-5B] Figure 1 shows the binding of fusion proteins comprising wild type or modified IL-2 fused to the C-terminus of a non-targeting VHH linked to a heterodimeric Fc as shown in Figure 1B to resting and activated CD4+ T cells as measured by flow cytometry. "Isotype control" indicates a control protein without IL-2. [Figure 6A-6B] Figure 6A shows binding of fusion proteins comprising wild-type IL-2 or modified IL-2 fused to the C-terminus of a non-targeting VHH linked to a heterodimeric Fc as shown in Figure 1B to enriched regulatory T cells (Treg, Figure 6A), induced regulatory T cells (induced Treg, Figure 6B), and enriched responder CD4+ T cells (Tresp, Figure 6C) as measured by flow cytometry. [Figure 7A-7D]Figure 7 shows the activity of fusion proteins comprising wild-type IL-2 or modified IL-2 fused to the C-terminus of a non-targeting VHH linked to a heterodimeric Fc as shown in Figure 1B on resting CD4+ and CD8+ T cells. Proliferation (Figures 7A and 7C) and CD71 levels (Figures 7B and 7D) were measured. Figures 7E and 7F show the activity of wild-type IL-2 or modified IL-2 fused to the C-terminus of a non-targeting VHH linked to a heterodimeric Fc as shown in Figure 1B on resting CD4+ and CD8+ T cells as measured by flow cytometric detection of intracellular phosphorylated STAT5 levels. "Isotype" indicates a control protein without IL-2. [Figure 8A-8B] FIG. 1B shows proliferation and CD25 levels as markers of activation of enriched Tregs after 7 days of treatment with fusion proteins containing wild-type IL-2 or modified IL-2 fused to the C-terminus of a non-targeting VHH linked to a heterodimeric Fc as shown in FIG. [Figure 9A-9D] Figures 9C and 9D show the activity and binding of pembrolizumab, an analog of pembrolizumab with IL-2-RAS fused to the heavy chain C-terminus as shown in Figure IF, and IL-2-RAS alone (Figures 9C and 9D) to CD8+ and CD4+ T cells. Activity on CD8+ (Figure 9A) and CD4+ (Figure 9B) T cells was measured by flow cytometric detection of CellTrace™ Violet. The extent of binding to CD8+ (Figure 9C) and CD4+ (Figure 9D) T cells was measured by flow cytometry. [Figure 10A-10D] 10A and 10B show the dependence of induction of proliferation of CD8+ and CD4+ T cells on IL-2. The effect of pembrolizumab, untargeted IL-2-RAS, and an analog of pembrolizumab in which IL-2-RAS is fused to the heavy chain C-terminus as shown in FIG. 1F on proliferation of CD8+ T cells (FIGS. 10A and 10C) or CD4+ T cells (FIGS. 10B and 10D) without pre-blocking (FIGS. 10A and 10B) or with pre-blocking with saturating concentrations of pembrolizumab (FIGS. 10C and 10D) is shown. [Figure 11] FIG. 1 shows the restoration of CD4+ T responder (Tresp) cell proliferation by pembrolizumab analogs with IL-2-RAS fused to the heavy chain C-terminus as shown in FIG. 1F, IL-2-RAS fused to the C-terminus of a non-targeting VHH as shown in FIG. 1B, and wild-type IL-2 fused to the C-terminus of a non-targeting VHH as shown in FIG. 1B. Tresp proliferation was induced by CD3 binding (Tresp+beads) and then suppressed with autologous regulatory T cells (Tregs). The "Tresp+beads" line shows baseline Tresp cell proliferation by CD3 binding in the absence of Treg cells. The "No Ab" line shows baseline Tresp cell proliferation by CD3 binding in the presence of Treg cells. [Figure 12A-12B] Figure 12 shows transactivation of T cells by plate-bound non-targeted wild-type IL-2 ("IL-2 WT") or IL-2-RAS fused to the C-terminus of non-targeted VHH as shown in Figure 1B. T cell activation was measured by flow cytometric detection of intracellular phosphorylated STAT5 levels. Responses of CD8+ T cells (Figure 12A) and CD4+ T cells (Figure 12B) are shown. [Figures 13A-13I] Figure 13 shows the activity and binding of IL-2-RAS fused to the C-terminus of a heterodimeric scFv antibody targeting NKp46 as shown in Figure 1H, a heterodimeric scFv antibody targeting NKp46 alone, and fusion proteins containing wild-type IL-2 or IL-2-RAS fused to the C-terminus of a non-targeting VHH linked to a heterodimeric Fc as shown in Figure 1B, on NK cells, CD8+ T cells, and CD4+ T cells. Proliferation of NK cells (Figure 13A), CD8+ T cells (Figure 13B), and CD4+ T cells (Figure 13C), and pSTAT levels of NK cells (Figure 13D), CD8+ T cells (Figure 13E), and CD4+ T cells (Figure 13F) were measured by flow cytometry. Binding of the indicated polypeptides to NK cells (FIG. 13G), CD8+ T cells (FIG. 13H), and CD4+ T cells (FIG. 13I) was also measured by flow cytometry. [Fig. 14A-14H]FIG. 1 shows the activity and binding of IL-2-RAS fused to the C-terminus of a conventional anti-LAG3 heterodimeric antibody (MAb) as shown in FIG. 1G, IL-2-RAS fused to the C-terminus of an anti-LAG3 VHH with a heterodimeric Fc as shown in FIG. 1B, IL-2-RAS fused to the C-terminus of a non-targeting VHH as shown in FIG. 1B, wild-type IL-2 fused to the C-terminus of a non-targeting heterodimeric Fc as shown in FIG. 1B, or a LAG3-targeting MAb or LAG3-targeting VHH-Fc molecule without IL-2, to CD8+ T cells or CD4+ T cells. Proliferation of CD8+ (FIG. 14A) and CD4+ (FIG. 14B) T cells and expression of activation markers CD25 (FIG. 14C) and CD71 (FIG. 14E) on CD8+ (FIG. 14C) and CD4+ (FIG. 14D) T cells were measured by flow cytometry. Figures 14G and 14H show binding to pre-activated CD8+ (FIG. 14G) and CD4+ (FIG. 14H) T cells. [Figure 15] Figure 1 shows the activity of fusion proteins containing the indicated modified IL-2 fused to the C-terminus of VHH with heterodimeric Fc as shown in Figure 1B on HEK-Blue IL-2 reporter cells that do not express the target antigen of the VHH and therefore rely solely on the binding of modified IL-2 to the overexpressed IL-2 receptor for reporter gene induction. The activity of secreted embryonic alkaline phosphatase expressed in reporter cells in response to IL-2 receptor-mediated induction of pSTAT5 signaling was measured. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0133] The embodiments presented herein relate to polypeptides, including modified IL-2, that modulate the activity of T cells, and their use in various methods of treating cancer.

[0134] Definitions and Various Embodiments The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0135] All references cited in this specification, including patent applications, patent publications, and Genbank accession numbers, are incorporated herein by reference to the same extent as if each individual reference was specifically and individually indicated to be incorporated by reference in its entirety.

[0136] The techniques and procedures described or referenced herein are generally well understood and generally described in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (FM AuSable, et al. eds., (2003)), the series METHODS IN ENZYMOLOGY (Academic Press, Inc.), PCR 2: A PRACTICAL APPROACH (MJ MacPherson, BD Hames and GR Taylor eds. (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMAL CELL CULTURE (RI Freshney, ed. (1987)), Oligonucleotide Synthesis (MJ Gait, ed., 1984), Methods in Molecular Biology, Humana Press, Cell Biology: A Laboratory Notebook (JE Cellis, ed., 1998) Academic Press, Animal Cell Culture (RI Freshney, ed., 1987), Introduction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press, Cell and Tissue Culture Laboratory Procedures (A. Doyle, JB Griffiths, and DG Newell eds., 1993-8) J. Wiley and Sons, Handbook of Experimental Immunology (DM Weir and C.C. Blackwell, eds.), Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987), PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994), Current Protocols in Immunology (JE Coligan et al. eds., 1991), Short Protocols in Molecular Biology (Wiley and Sons, 1999), Immunobiology (CA Janeway and P. Travers, 1997), Antibodies (P. Finch, 1997), Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988-1989), Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000), Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999), The Antibodies (M. Zanetti and JD Capra, eds., Harwood Academic Publishers, 1995), and Cancer: Principles and Practice of Oncology (VT DeVita et al., eds., JB Lippincott Company, 1993), and their latest editions. It is used with a methodology that is consistent with the

[0137] Unless otherwise specified, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context or expressly indicated otherwise, singular terms shall include the plural and plural terms shall include the singular. In the event of discrepancies in definitions among various sources or references, the definitions set forth herein shall prevail.

[0138] Generally, the numbering of residues in immunoglobulin heavy chains is based on the method described by Kabat et al., Sequences of The numbering of the EU index is as in Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). "EU index as in Kabat" refers to the residue numbering of the human IgG1 EU antibody.

[0139] It is understood that embodiments of the invention described herein include "consisting of" and / or "consisting essentially of" embodiments. As used herein, the singular forms "a," "an," and "the" are used unless otherwise indicated. Plural references are included unless otherwise noted. Use of the term "or" herein is not to be construed as implying that the alternatives are mutually exclusive.

[0140] In this application, the use of "or" means "and / or" unless expressly stated otherwise or understood by a person skilled in the art. In the context of a multiple dependent claim, the use of "or" refers to more than one preceding independent or dependent claim.

[0141] The phrases "reference sample", "reference cell", or "reference tissue" refer to a sample with at least one known characteristic that can be used as a comparison with a sample with at least one unknown characteristic. In some embodiments, a reference sample can be used as a positive or negative indicator. A reference sample can be used to establish, for example, the level of protein and / or mRNA present in a healthy tissue relative to the level of protein and / or mRNA present in a sample with an unknown characteristic. In some embodiments, the reference sample is a sample from the same subject, but from a part of the subject different from the part being tested. In some embodiments, the reference sample is a sample from a tissue area surrounding or adjacent to the cancer. In some embodiments, the reference sample is not from the subject being tested, but from a subject known to have or not have a disorder of interest (e.g., a particular cancer or T-cell related disorder). In some embodiments, the reference sample is from the same subject, but at a time before the subject developed cancer. In some embodiments, the reference sample is a sample from a benign cancer sample from the same subject or a different subject. When a negative reference sample is used for comparison, the expression level or amount of the molecule of interest in the negative reference sample indicates a level at which one skilled in the art would recognize, in view of the present disclosure, that the molecule is absent and / or present at a low level.When a positive reference sample is used for comparison, the expression level or amount of the molecule of interest in the positive reference sample indicates a level at which one skilled in the art would recognize, in view of the present disclosure, that the molecule is present at a certain level.

[0142] The terms "benefit", "clinical benefit", "responsiveness" and "therapeutic response" as used herein in the context of benefiting from or responding to the administration of a therapeutic agent can be measured by assessing various endpoints, such as some degree of inhibition of disease progression, including slowing and complete halt, reduction in the number of disease episodes and / or symptoms, reduction in lesion size, inhibition (i.e., reduction, slowing, or complete halt) of disease cell invasion into adjacent peripheral organs and / or tissues, inhibition (i.e., reduction, slowing, or complete halt) of disease spread, some degree of relief of one or more symptoms associated with the disorder, disease-free presentation after treatment, such as increased length of progression-free survival, increased overall survival, higher response rate, and / or reduced mortality at a given time point after treatment. A "non-responsive" or "non-responsive" subject or cancer is one that does not meet the above criteria of "responding".

[0143] The terms "nucleic acid molecule," "nucleic acid," and "polynucleotide" are used interchangeably and may refer to a polymer of nucleotides. Such polymers of nucleotides may contain natural and / or non-natural nucleotides, including, but not limited to, DNA, RNA, and PNA. A "nucleic acid sequence" refers to the linear sequence of nucleotides contained in a nucleic acid molecule or polynucleotide.

[0144] The terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues and are not limited to a minimum length. Such polymers of amino acid residues may contain natural or non-natural amino acid residues and may include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. Included in this definition are both full-length proteins and fragments thereof. These terms also include post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. Furthermore, for purposes of this disclosure, a "polypeptide" refers to a polymer of any naturally occurring amino acid residue, so long as the protein maintains the desired activity. "Amino acid sequence" refers to a protein containing modifications (generally conservative in nature) such as deletions, additions, and substitutions to the sequence. These modifications may be deliberate, as by site-directed mutagenesis, or may be accidental, such as by mutations of hosts which produce the protein or by errors in PCR amplification. "Amino acid sequence" refers to the linear sequence of amino acids contained in a polypeptide or protein.

[0145] As used herein, "IL-2" or "interleukin-2" refers to any naturally occurring mature IL-2 resulting from processing of an IL-2 precursor in a cell. The term includes IL-2 from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus or rhesus monkeys) and rodents (e.g., mice and rats), unless otherwise indicated. The term also includes naturally occurring variants of IL-2, such as splice variants or allelic variants. A non-limiting exemplary human IL-2 amino acid sequence is shown, for example, in GenBank Accession No. NP_000577.2. See SEQ ID NO: 1 (mature form).

[0146] As used herein, "modified IL-2" refers to a polypeptide that differs from the wild-type IL-2 amino acid sequence by a substitution at at least one amino acid position.

[0147] The term "specifically binds" to an antigen or epitope is a term well understood in the art, and methods for determining such specific binding are also well known in the art. A molecule is said to exhibit "specific binding" or "preferential binding" if it reacts or associates more frequently, more rapidly, with a longer duration and / or with a higher affinity with a particular cell or substance than it reacts or associates with another cell or substance. An antigen-binding domain "specifically binds" or "preferentially binds" to an antigen if it binds with higher affinity, avidity, more readily, and / or with a longer duration than it binds to other substances. For example, an sdAb or VHH-containing polypeptide that specifically or preferentially binds to an epitope is an sdAb or VHH-containing polypeptide that binds to this epitope with higher affinity, avidity, more readily, and / or with a longer duration than it binds to other epitopes on the same target antigen or epitopes on other target antigens. It is also understood by interpreting this definition that, for example, an antigen-binding domain that specifically or preferentially binds to a first antigen may or may not specifically or preferentially bind to a second antigen. Thus, "specific binding" or "preferential binding" does not necessarily require (although it may include) exclusive binding. Generally, but not necessarily, reference to binding means preferential binding. "Specificity" refers to the ability of a binding protein to selectively bind to an antigen.

[0148] As used herein, the term "modulate" with respect to the activity of IL-2 refers to a change in the activity of IL-2. In some embodiments, "modulate" refers to an increase in IL-2 activity.

[0149] As used herein, the term "epitope" refers to a site on a target molecule (e.g., an antigen, such as a protein, nucleic acid, carbohydrate, or lipid) to which an antigen-binding molecule (e.g., an antigen-binding domain-containing polypeptide) binds. Epitopes often comprise chemically active surface arrangements of molecules, such as amino acids, polypeptides, or sugar side chains, and have specific three-dimensional structural features and specific charge characteristics. Epitopes can be formed from both contiguous and / or juxtaposed non-contiguous residues (e.g., amino acids, nucleotides, sugars, lipid moieties) of a target molecule. Epitopes formed from contiguous residues (e.g., amino acids, nucleotides, sugars, lipid moieties) are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. Epitopes can be, but are not limited to, at least 3, at least 5, or 8-10 residues (e.g., In some embodiments, an epitope may comprise a residue sequence of less than 20 residues (e.g., amino acids or nucleotides), less than 15 residues, or less than 12 residues in length. Two antibodies may bind to the same epitope in an antigen if they exhibit competitive binding to the antigen. In some embodiments, an epitope may be specified by a certain minimum distance to the CDR residues on the antigen-binding molecule. In some embodiments, an epitope may be specified by the above distance and further limited to those residues involved in binding (e.g., hydrogen bonding) between the antigen-binding molecule residues and the antigen residues. Epitopes may also be specified by various scans. For example, an alanine scan or an arginine scan may indicate one or more residues with which an antigen-binding molecule may interact. Unless explicitly indicated, a set of residues as an epitope does not exclude other residues from being part of the epitope for a particular antigen-binding domain or molecule. Rather, the existence of such a set indicates a minimum epitope string (or set of types). Thus, in some embodiments, the set of residues identified as an epitope is not an exclusive list of residues for the epitope on the antigen, but rather represents a minimal epitope associated with the antigen.

[0150] A "non-linear epitope" or "conformational epitope" comprises non-contiguous polypeptides, amino acids and / or sugars within an antigenic protein to which an epitope-specific antigen-binding molecule (e.g., an antigen-binding domain-containing polypeptide) binds. In some embodiments, at least one residue is non-contiguous with other represented residues of the epitope, although one or more residues may be contiguous with other residues.

[0151] A "linear epitope" comprises a contiguous polypeptide, amino acid and / or sugar in an antigenic protein to which an epitope-specific antigen-binding molecule (e.g., an antigen-binding domain-containing polypeptide) binds. Note that in some embodiments, not all of the residues in a linear epitope need to be directly bound (or involved in binding) by an antigen-binding molecule. In some embodiments, a linear epitope can be derived from immunization with a peptide that essentially consists of the sequence of the linear epitope, or from a structural section of a protein that is relatively isolated from the rest of the protein (so that the antigen-binding molecule can interact, at least primarily, with just that sequence section).

[0152] The terms "antibody" and "antigen-binding molecule" are used in the broadest sense and include, but are not limited to, conventional antibodies (typically comprising at least one heavy chain and at least one light chain), single domain antibodies (sdAbs, typically comprising only one chain similar to a heavy chain), VHH-containing polypeptides (polypeptides comprising at least one antibody variable domain of only a heavy chain, i.e., VHH), and various polypeptides comprising an antigen-binding domain comprising any of the above-mentioned fragments, so long as they exhibit the desired antigen-binding activity. In some embodiments, the antibody comprises a dimerization domain. Such dimerization domains include, but are not limited to, a heavy chain constant domain (comprising CH1, hinge, CH2, and CH3, where CH1 is typically paired with a light chain constant domain CL, while the hinge mediates dimerization) and an Fc region (comprising hinge, CH2, and CH3, where the hinge mediates dimerization). The term antibody also includes, but is not limited to, chimeric antibodies, humanized antibodies, and antibodies of various species such as camel (including llama), shark, mouse, human, cynomolgus monkey, etc.

[0153] The terms "single domain antibody" and "sdAb" are used interchangeably herein to refer to an antibody having a single monomeric domain, typically a heavy chain (or VHH) without any light chain.

[0154] As used herein, the term "VHH" or "VHH domain" or "VHH antigen-binding domain" refers to the antigen-binding portion of a single domain antibody, such as a camelid antibody or a shark antibody. In some embodiments, a VHH comprises three CDRs and four framework regions, designated FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In some embodiments, a VHH may be truncated at the N-terminus or C-terminus to comprise only a partial FR1 and / or FR4, or to lack one or both of these framework regions, so long as the VHH substantially retains antigen binding and specificity.

[0155] The term "VHH-containing polypeptide" refers to a polypeptide that comprises at least one VHH domain. In some embodiments, a VHH polypeptide comprises two, three, or more than four VHH domains, where each VHH domain may be the same or different. In some embodiments, a VHH-containing polypeptide comprises an Fc region. In some such embodiments, a VHH polypeptide may form a dimer. Non-limiting structures of a VHH-containing polypeptide include a VHH 1 -Fc, VHH 1 -VHH 2 -Fc, and VHH 1 -VHH 2 -VHH 3 -Fc, where VHH 1 , V.H.H. 2 , and VHH 3 may be the same or different. In some embodiments of such structures, a VHH may be linked to another VHH by a linker, or a VHH may be linked to an Fc by a linker. In some such embodiments, the linker comprises 1-20 amino acids, preferably 1-20 amino acids consisting primarily of glycine and optionally serine. In some embodiments, when the VHH-containing polypeptide comprises an Fc, it forms a dimer. Thus, the structure VHH 1 -VHH 2 -Fc is considered to be tetravalent if it forms a dimer (i.e., the dimer has four VHH domains). Similarly, the structure VHH 1 -VHH 2 -VHH 3 -Fc is considered to be hexavalent if it forms a dimer (i.e., the dimer has six VHH domains).

[0156] The term "monoclonal antibody" refers to an antibody (including sdAb or VHH-containing polypeptide) from a substantially homogeneous antibody population. That is, the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. Thus, a sample of monoclonal antibodies is capable of binding to the same epitope on the antigen. The modifier "monoclonal" indicates the nature of the antibody being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies are the first described by Kohler and Milstein, 1975, Nature 256:495, The monoclonal antibodies may be made by the hybridoma method described in, or by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567. The monoclonal antibodies may also be made using the techniques described in, for example, McCafferty et al., 1990, Nature 348:552-554. The vector can be isolated from a phage library prepared by the method described above.

[0157] The term "CDR" refers to a complementarity determining region defined by at least one specific manner to one skilled in the art. In some embodiments, the CDRs may be defined according to any of the Chothia numbering scheme, the Kabat numbering scheme, a combination of Kabat and Chothia, the AbM definition, and / or the contact definition. A VHH comprises three CDRs designated CDR1, CDR2, and CDR3.

[0158] As used herein, the term "heavy chain constant region" refers to a region that comprises at least three heavy chain constant domains, i.e., H 1, hinge, C H 2, and C H3. Of course, deletions and modifications that do not alter function within the domain are included within the scope of the term "heavy chain constant region" unless otherwise specified. Non-limiting exemplary heavy chain constant regions include gamma, delta, and alpha. Non-limiting exemplary heavy chain constant regions also include epsilon and mu. Each heavy chain constant region corresponds to an antibody isotype. For example, an antibody containing a gamma constant region is an IgG antibody, an antibody containing a delta constant region is an IgD antibody, and an antibody containing an alpha constant region is an IgA antibody. Furthermore, an antibody containing a mu constant region is an IgM antibody, and an antibody containing an epsilon constant region is an IgE antibody. Certain isotypes can be further subdivided into subclasses. For example, IgG antibodies include, but are not limited to, IgG1 (gamma constant region), IgG2 (alpha constant region), IgG3 (alpha constant region), IgG4 (alpha constant region), IgG5 (alpha constant region), IgG6 (alpha constant region), IgG7 (alpha constant region), IgG8 (alpha constant region), IgG9 (alpha constant region), IgG10 (alpha constant region), IgG11 (alpha constant region), IgG12 (alpha constant region), IgG13 (alpha constant region), IgG14 (alpha constant region), IgG15 (alpha constant region), IgG16 (alpha constant region), IgG17 (alpha constant region), IgG18 (alpha constant region), IgG19 (alpha constant region), IgG110 (alpha constant region), IgG111 (alpha constant region), IgG121 (alpha constant region), IgG130 (alpha constant region), IgG140 (alpha constant region), IgG150 (alpha constant region), IgG160 (alpha constant region), IgG171 (alpha constant region), IgG181 (alpha constant region), IgG192 (alpha constant region), IgG193 (alpha constant region), IgG194 (alpha constant region), IgG195 (alpha constant region), IgG186 (alpha constant region), IgG196 (alpha constant region), IgG197 (alpha constant region), IgG198 (alpha constant region), IgG199 (alpha constant region), IgG111 (alpha constant region), IgG112 (alpha constant region), IgG113 (alpha constant region), IgG114 (alpha constant region), IgG115 1 (including constant region) antibody, IgG2(γ 2 (including constant region) antibody, IgG3(γ 3 (including the constant region) antibody, and IgG4 (γ 4 Examples of IgA antibodies include, but are not limited to, IgA1 (α 1 (including constant regions) antibody and IgA2 (α 2 Examples of IgM antibodies include, but are not limited to, IgM1 and IgM2.

[0159] As used herein, "Fc region" refers to a portion of a heavy chain constant region comprising CH2 and CH3. In some embodiments, the Fc region comprises a hinge, CH2, and CH3. In various embodiments, when the Fc region comprises a hinge, the hinge mediates dimerization between two Fc-containing polypeptides. The Fc region can be of any antibody heavy chain constant region isotype discussed herein. In some embodiments, the Fc region is IgG1, IgG2, IgG3, or IgG4.

[0160] As used herein, an "acceptor human framework" refers to a heavy chain variable domain (V) derived from a human immunoglobulin framework or a human consensus framework, as discussed herein. H) framework amino acid sequence. The acceptor human framework derived from a human immunoglobulin framework or a human consensus framework can comprise the same amino acid sequence or can comprise amino acid sequence changes. In some embodiments, the number of amino acid changes is less than 10, or less than 9, or less than 8, or less than 7, or less than 6, or less than 5, or less than 4, or less than 3 across all human frameworks within a single antigen-binding domain such as a VHH.

[0161] "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody or VHH-containing polypeptide) and its binding partner (e.g., an antigen). The affinity or apparent affinity of a molecule X for its partner Y is generally measured by the dissociation constant (K D ) or K D(見かけ) Affinity can be expressed by any of the conventional methods known in the art, including those described herein (e.g., ELISA K D , KinExA, flow cytometry, and / or surface plasmon resonance devices, etc. Such methods include, but are not limited to, BIAcore™, Octet™, or methods involving flow cytometry.

[0162] As used herein, "K D As used herein, the term "K" refers to the equilibrium dissociation constant of an antigen-binding molecule / antigen interaction. D When the term "K" is used, it includes D and K D(見かけ) Includes:

[0163] In some embodiments, the K D is measured by flow cytometry using antigen-expressing cell lines and fitting the mean fluorescence measured at each antibody concentration to a nonlinear one-site binding equation (Graphpad's Prism Software). In some such embodiments, K D is KD(見かけ) It is.

[0164] The term "biological activity" refers to any one or more biological properties of a molecule (whether naturally occurring as found in vivo or provided or made possible by recombinant means). Biological properties include, but are not limited to, binding of a ligand, induction or increase in cell proliferation (such as T cell proliferation), and induction or increase in the expression of cytokines. The increase is cited.

[0165] As used herein, the term "IL-2 activity" or "biological activity" of IL-2 includes at least one of any biological action or biologically relevant function of IL-2. In some embodiments, IL-2 activity includes the ability of IL-2 to induce T cell proliferation and / or activate natural killer (NK) cells. Non-limiting exemplary IL-2 activities include increased pSTAT5 expression, increased CD4+ expression, and increased IL-2 expression. + T cells and / or CD8 + Increased T cell proliferation, increased CD71 expression on T cells, and CD4 + T cells and CD8 + These include a decrease in the suppressive activity of Treg cells against T cell activation and proliferation.

[0166] An "agonist" or "activating" antibody (such as an sdAb or VHH-containing polypeptide) is an antibody that increases and / or activates the biological activity of a target antigen. In some embodiments, an agonist antibody binds to an antigen and increases its biological activity by at least about 20%, 40%, 60%, 80%, 85% or more.

[0167] An "antagonist," "blocking," or "neutralizing" antibody is an antibody that reduces and / or inactivates the biological activity of a target antigen. In some embodiments, a neutralizing antibody binds to an antigen and reduces its biological activity by at least about 20%, 40%, 60%, 80%, 85%, 90%, 95%, 99% or more.

[0168] An "affinity matured" VHH-containing polypeptide refers to a VHH-containing polypeptide which has one or more modifications in one or more CDRs which result in an improvement in the affinity of the VHH-containing polypeptide for an antigen compared to a parent VHH-containing polypeptide which does not have such modifications.

[0169] "Humanized VHH" as used herein refers to a VHH in which one or more framework regions have been substantially replaced with human framework regions. In some cases, certain framework region (FR) residues of a human immunoglobulin are replaced by corresponding non-human residues. Furthermore, a humanized VHH may contain residues that are not found in the original VHH or in the human framework sequence, but are included to further improve and optimize the performance of the VHH or VHH-containing polypeptide. In some embodiments, a humanized VHH-containing polypeptide comprises a human Fc region. As will be appreciated, a humanized sequence may be identified by its primary sequence and does not necessarily indicate the process by which the antibody was made.

[0170] A "functional Fc region" possesses an "effector function" of a native sequence Fc region. Exemplary "effector functions" include Fc receptor binding, Clq binding and complement dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation. Such effector functions generally require combining the Fc region with a binding domain (e.g., an antibody variable domain) and can be assessed using a variety of assays.

[0171] A "native sequence Fc region" comprises an amino acid sequence identical to that of an Fc region found in nature. Native sequence human Fc regions include native sequence human IgG1 Fc regions (non-A and A allotypes), native sequence human IgG2 Fc regions, native sequence human IgG3 Fc regions, and native sequence human IgG4 Fc regions, as well as naturally occurring variants thereof.

[0172] A "variant Fc region" comprises an amino acid sequence that differs from the amino acid sequence of a native sequence Fc region by at least one amino acid modification. In some embodiments, a "variant Fc region" comprises an amino acid sequence that differs from the amino acid sequence of a native sequence Fc region by at least one amino acid modification. but which retains at least one effector function of a native-sequence Fc region. In some embodiments, the variant Fc region has at least one amino acid substitution, e.g., about 1 to about 10 amino acid substitutions, preferably about 1 to about 5 amino acid substitutions, in the native-sequence Fc region or the Fc region of the parent polypeptide compared to the native-sequence Fc region or the Fc region of the parent polypeptide. In some embodiments, the variant Fc region herein has at least about 80% sequence identity, at least about 90% sequence identity, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a native-sequence Fc region and / or the Fc region of the parent polypeptide.

[0173] "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. In some embodiments, the FcγR is a native human FcR. In some embodiments, the FcR binds IgG antibodies (gamma receptors) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences but differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain (see, e.g., Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed, e.g., in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991), Capel et al., Immunomethods 4:25-34 (1994), and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those identified in the future, are encompassed by the term "FcR" herein. For example, the term "Fc receptor" or "FcR" also includes the fetal receptor FcRn, which mediates the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) and immunoglobulin It plays a role in regulating homeostasis. Methods for measuring binding to FcRn are known (see, for example, Ghetie and Ward, Immunol. Today 18(12):592-598 (1997); Ghetie et al., Nature Biotechnology, 15(7):637-640 (1997); Hinton et al., J. Biol. Chem. 279(8):6213-6216 (2004); WO 2004 / 92219 (Hinton et al.)).

[0174] As used herein, the terms "substantially similar" or "substantially the same" refer to a sufficiently high degree of similarity between two or more numerical values ​​such that one of skill in the art would consider the difference between the two or more values ​​to have little or no biological and / or statistical significance within the context of the biological characteristic measured by the values. In some embodiments, two or more substantially similar values ​​differ by no more than the approximate value of any one of 5%, 10%, 15%, 20%, 25%, or 50%.

[0175] A polypeptide "variant" refers to a biologically active polypeptide having at least about 80% amino acid sequence identity with a native sequence polypeptide, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Such variants include, for example, polypeptides in which one or more amino acid residues are added or deleted at the N-terminus or C-terminus of the polypeptide. In some embodiments, a variant has at least about 80% amino acid sequence identity. In some embodiments, a variant has at least about 90% amino acid sequence identity. In some embodiments, a variant has at least about 95% amino acid sequence identity with a native sequence polypeptide.

[0176] As used herein, the term "peptide sequence," "polypeptide sequence," or "antibody sequence" refers to a peptide sequence, polypeptide sequence, or antibody sequence. "Percent (%) amino acid sequence identity" and "homology" as used herein are defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in a particular peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment to determine percent amino acid sequence identity can be performed by any method known to those skilled in the art using publicly available computer software, such as, for example, BLAST, BLAST-2, ALIGN, or MEGALIGN™ (DNASTAR, Inc.) software. This can be achieved in a variety of ways that are within the skill of the art. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0177] Amino acid substitutions can include, but are not limited to, replacing one amino acid in a polypeptide with another amino acid. Exemplary substitutions are shown in Table 1. Amino acid substitutions can be introduced into an antibody of interest and the products screened for a desired activity, such as retained / improved antigen or receptor binding, reduced antigen or receptor binding, reduced immunogenicity, or improved ADCC or CDC.

[0178] [Table 1]

[0179] Amino acids can be grouped according to common side chain properties: (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile, (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln, (3) Acidic: Asp, Glu, (4) Basic: His, Lys, Arg, (5) Residues that affect chain orientation: Gly, Pro, (6) Aromatic: Trp, Tyr, Phe

[0180] Non-conservative substitutions involve exchanging a member of one of these classes for another class. .

[0181] The term "vector" is used to describe a polynucleotide that can be manipulated to contain a cloned polynucleotide or polynucleotides that can be propagated in a host cell. A vector can contain one or more of the following elements: an origin of replication, one or more regulatory sequences (e.g., promoters and / or enhancers, etc.) that control the expression of a polypeptide of interest, and / or one or more selectable marker genes (e.g., antibiotic resistance genes and genes that can be used in colorimetric assays, e.g., β-galactosidase, etc.). The term "expression vector" refers to a vector used to express a polypeptide of interest in a host cell.

[0182] "Host cell" refers to a cell that can be or has been the recipient of a vector or isolated polynucleotide. A host cell can be a prokaryotic or eukaryotic cell. Exemplary eukaryotic cells include mammalian cells, such as primate or non-primate cells, fungal cells, such as yeast, plant cells, and insect cells. Non-limiting exemplary mammalian cells include, but are not limited to, NSO cells, PER.C6™ cells (Crucell), and 293F cells and CHO cells, and derivatives thereof, such as 293- Host cells include CHO-6E cells, CHO-DG44 cells, CHO-K1 cells, CHO-S cells, and CHO-DS cells. A host cell includes the progeny of a single host cell, although the progeny may not necessarily be completely identical (in morphology or genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. A host cell also includes cells transfected in vivo with a polynucleotide(s) provided herein.

[0183] The term "isolated" as used herein refers to a molecule that is separated from at least some of the components that it is typically found or produced with in nature. For example, a polypeptide is referred to as "isolated" when it is separated from at least some of the components of the cell that produced it. If the polypeptide is secreted by the cell after expression, physically separating the supernatant containing the polypeptide from the cell that produced it is considered to "isolate" the polypeptide. Similarly, a polynucleotide is referred to as "isolated" when it is not part of a larger polynucleotide that it is typically found in nature (e.g., in the case of a DNA polynucleotide, genomic DNA or mitochondrial DNA, etc.) or when it is separated from at least some of the components of the cell that produced it, for example, in the case of an RNA polynucleotide. Thus, a DNA polynucleotide contained in a vector within a host cell can be referred to as "isolated".

[0184] The terms "individual" and "subject" are used interchangeably herein to refer to animals, e.g., mammals. In some embodiments, the present invention is directed to treating mammals, including, but not limited to, humans, rodents, monkeys, cats, dogs, horses, cows, pigs, sheep, goats, mammalian laboratory animals, mammalian farm animals, mammalian sport animals, and mammalian pets. In some instances, "individual" or "subject" refers to an individual or subject in need of treatment for a disease or disorder. In some embodiments, the subject receiving treatment may be a patient, which means the subject has been identified as having or at sufficient risk of suffering from a disorder relevant to the treatment.

[0185] As used herein, "disease" or "disorder" refers to a condition for which treatment is needed and / or desired.

[0186] The terms "tumor cell," "cancer cell," "cancer," "tumor," and / or "neoplasm" are used interchangeably herein, unless otherwise specified, and refer to a cell that is capable of inhibiting the normal functioning of bodily organs and systems. It refers to a cell (or cells) that exhibits uncontrolled proliferation and / or abnormal increased cell survival and / or inhibition of apoptosis that prevents or inhibits cell proliferation. This definition includes benign and malignant cancers, polyps, hyperplasias, and latent tumors or micrometastases.

[0187] The terms "cancer" and "tumor" encompass solid cancers and hematological / lymphatic cancers, as well as malignant tumors such as dysplasia, premalignant tumors, and benign tumors. Also included in this definition are cells with abnormal proliferation unchecked by the immune system (e.g., immune evasion and escape mechanisms) (e.g., virally infected cells). Exemplary cancers include, but are not limited to, basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and central nervous system cancer, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, colorectal cancer, connective tissue cancer, digestive system cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer (including gastrointestinal cancer), glioblastoma, liver cancer, hepatocellular carcinoma, intraepithelial neoplasia, renal or kidney cancer, laryngeal cancer, leukemia, liver cancer, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), melanoma, myeloma, neuroblastoma, oral cancer (lips, tongue, mouth, and pharynx), ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, respiratory system cancer, salivary gland cancer, sarcoma, skin cancer, squamous cell carcinoma, stomach cancer, testicular cancer, thyroid cancer, uterine or endometrial cancer, urinary system cancer, vulvar cancer, Hodgkin's lymphoma, and non-Hodgkin's lymphoma lymphomas, including non-Hodgkin's lymphoma, as well as B-cell lymphomas (including low-grade / follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-dividing cell NHL, bulky disease NHL), mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom's macroglobulinemia, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and other carcinomas and sarcomas, as well as post-transplant lymphoproliferative disorders (PTLD), and abnormal blood vessel growth associated with nematoses, edema (such as edema associated with brain tumors), and Meigs syndrome.

[0188] The term "non-tumor cells" as used herein refers to normal cells or tissues. Exemplary non-tumor cells include, but are not limited to, T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, dendritic cells, monocytes, macrophages, epithelial cells, fibroblasts, hepatocytes, stromal kidney cells, fibroblast-like synoviocytes, osteoblasts, and cells located in breast, skeletal muscle, pancreas, stomach, ovary, small intestine, placenta, uterus, testes, kidney, lung, heart, brain, liver, prostate, colon, lymphoid organs, bone, and bone-derived mesenchymal stem cells. The term "peripherally located cells or tissues" as used herein refers to non-tumor cells that are not located near tumor cells and / or within the tumor microenvironment.

[0189] The term "cells or tissues in the tumor microenvironment" as used herein refers to cells, molecules, extracellular matrix and / or blood vessels that surround and / or nourish tumor cells. Exemplary cells or tissues in the tumor microenvironment include, but are not limited to, tumor vasculature, tumor-infiltrating lymphocytes, fibroblastic reticular cells, endothelial progenitor cells (EPCs), cancer-associated fibroblasts, pericytes, other stromal cells, components of the extracellular matrix (ECM), dendritic cells, antigen-presenting cells, T cells, regulatory T cells (Treg cells), macrophages, neutrophils, myeloid-derived suppressor cells (MDSCs) and other immune cells located in the vicinity of the tumor. Methods for identifying tumor cells and / or cells / tissues located in the tumor microenvironment are well known in the art, as described herein below.

[0190] In some embodiments, "increase" or "decrease" refers to a statistically significant increase or decrease, respectively. As will be apparent to one of skill in the art, "modulation" also refers to causing a change (which may be either an increase or a decrease) in the affinity, avidity, specificity and / or selectivity of a target or antigen for one or more of its ligands, binding partners, associate partners into homomultimeric or heteromultimeric forms or substrates, compared to the same conditions except in the presence of a test agent, or to one or more conditions (pH, ionic strength, etc.) in the medium or environment in which the target or antigen is present. This may involve causing a change (which may be either an increase or a decrease) in the sensitivity of the target or antigen to the target (such as intensity, presence of cofactors, etc.) and / or cell proliferation or cytokine production. This may be determined in any suitable manner and / or using any suitable assay known per se or as described herein, depending on the target involved.

[0191] As used herein, an "immune response" is intended to encompass a cellular and / or humoral immune response that is sufficient to inhibit or forestall the onset of or ameliorate a symptom of a disease (e.g., cancer or cancer metastasis). An "immune response" can encompass aspects of both the innate and adaptive immune systems.

[0192] As used herein, "treatment" is an approach to obtain beneficial or desired clinical results. As used herein, "treatment" covers any administration or application of a therapeutic agent for a disease in a mammal, including a human. For purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, any one or more of the following: alleviation of one or more symptoms, reduction in the extent of the disease, prevention or delay of disease progression (e.g., metastasis, e.g., to the lungs or lymph nodes), prevention or delay of disease recurrence, delay or slowing of disease progression, amelioration of disease state, arrest of disease or disease progression, arrest or slowing of disease or its progression, arrest of its development, and remission (whether partial or total). "Treatment" also includes reduction of pathological consequences of proliferative diseases. The methods provided herein contemplate any one or more of these aspects of treatment. In accordance with the above, the term treatment does not require 100 percent elimination of all aspects of the disorder.

[0193] By "ameliorate" is meant that one or more symptoms are lessened or improved compared to when the therapeutic agent is not administered. "Amelioration" also includes a shortening or reduction in the duration of the symptoms.

[0194] The term "anti-cancer agent" is used herein in its broadest sense to refer to an agent used to treat one or more cancers. Exemplary classes of such agents include, but are not limited to, chemotherapeutic agents, anti-cancer biologics (such as cytokines, receptor extracellular domain-Fc fusions, and antibodies), radiotherapy agents, CAR-T therapy agents, therapeutic oligonucleotides (such as antisense oligonucleotides and siRNAs), and oncolytic viruses.

[0195] The term "biological sample" refers to a quantity of material from a living or formerly living organism, including, but not limited to, blood (e.g., whole blood), plasma, serum, urine, amniotic fluid, synovial fluid, endothelial cells, leukocytes, monocytes, other cells, organs, tissues, bone marrow, lymph nodes, and spleen.

[0196] The term "control" or "reference" refers to a composition known to be free of the analyte (a "negative control") or to a composition known to contain the analyte (a "positive control"). A positive control may contain a known concentration of the analyte.

[0197] The term "abrogate" or "abrogate" refers to the reduction or cessation of any phenotypic characteristic, or the reduction or cessation of the incidence, extent, or likelihood of that characteristic. "Reduce" or "abrogate" refers to the decrease, reduction, or cessation of an activity, function, and / or amount compared to a reference. In some embodiments, "reduce" or "abrogate" refers to the ability to cause an overall reduction of 10% or more. In some embodiments, "reduce" or "abrogate" refers to the ability to cause an overall reduction of 50% or more. In some embodiments, "reduce" or "abrogate" refers to the ability to cause an overall reduction of 75%, 85%, 90%, 95% or more. In some embodiments, the above is inhibited or reduced over a period of time relative to a control over the same period of time.

[0198] As used herein, "delaying the onset of disease" means delaying, preventing, slowing, retarding, stabilizing, inhibiting, and / or prolonging the onset of a disease (such as cancer). This delay can be of varying lengths of time, depending on the history of the disease and / or the individual being treated. As will be apparent to one of skill in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease. For example, late-stage cancer, such as the onset of metastases, can be delayed.

[0199] "Prevention," as used herein, includes providing prevention against the occurrence or recurrence of a disease in a subject who may have a predisposition to the disease, but has not yet been diagnosed with the disease. Unless otherwise indicated, the terms "reduce," "inhibit," or "prevent" do not indicate or require complete prevention over the entire period of time, but only prevention over the period being measured.

[0200] A "therapeutically effective amount" of a substance / molecule, agonist or antagonist may vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the substance / molecule, agonist or antagonist to elicit a desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or detrimental effects of the substance / molecule, agonist or antagonist are outweighed by the therapeutically beneficial effects. A therapeutically effective amount may be delivered in one or more administrations. A therapeutically effective amount refers to an amount effective to achieve a desired therapeutic and / or prophylactic result at the required dosages for the required time.

[0201] The terms "pharmaceutical formulation" and "pharmaceutical composition" refer to a preparation that is in a form that allows the biological activity of the active ingredient(s) to be effective and that does not contain additional components that are unacceptably toxic to the subject to which the formulation is administered. Such formulations may be sterile.

[0202] "Pharmaceutically acceptable carrier" refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material, formulation aid, or carrier conventional in the art used with therapeutic agents that together comprise a "pharmaceutical composition" for administration to a subject. A pharmaceutically acceptable carrier is non-toxic to a recipient at the dosage and concentration used and is compatible with other ingredients of the formulation. A pharmaceutically acceptable carrier is appropriate for the formulation in which it is used.

[0203] Administration "in combination with" one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.

[0204] The term "concurrently" is used herein to refer to the administration of two or more therapeutic agents where at least a portion of the administration overlaps in time, or where the administration of one therapeutic agent falls within a short period of time relative to the administration of the other therapeutic agent, or where the therapeutic effects of both therapeutic agents overlap for at least some period of time.

[0205] The term "sequentially" is used herein to refer to the administration of two or more therapeutic agents that do not overlap in time or where the therapeutic effects of the therapeutic agents do not overlap.

[0206] As used herein, "in combination with" refers to the administration of one therapy in addition to another. Thus, "in combination with" refers to the administration of one therapy before, during, or after the administration of another therapy to an individual.

[0207] The term "package insert" is used to refer to instructions typically included in commercial packaging for a therapeutic product, which contain information regarding directions, usage, dosage, administration, concomitant therapy, contraindications and / or warnings regarding the use of such therapeutic product.

[0208] An "article of manufacture" is any manufacture (e.g., package or container) or kit that contains at least one reagent, e.g., a pharmaceutical agent for treating a disease or disorder (e.g., cancer), or a probe that specifically detects a biomarker described herein. In some embodiments, the article of manufacture or kit is advertised, delivered, or sold as a unit for performing a method described herein.

[0209] The terms "label" and "detectable label" refer to a moiety that, for example, when attached to an antibody or antigen, renders the reaction (e.g., binding) between members of a specific binding pair detectable. A labeled member of a specific binding pair is said to be "detectably labeled." Thus, the term "labeled binding protein" refers to a protein that incorporates a label that provides for the identification of the binding protein. In some embodiments, the label is a detectable marker that can generate a signal that is detectable visually or by instrumental means, for example, the incorporation of a radiolabeled amino acid or the attachment of a biotinyl moiety to the polypeptide that can be detected by marked avidin (e.g., streptavidin that contains a fluorescent marker or an enzymatic activity that can be detected by optical or colorimetric methods). Examples of labels for polypeptides include, but are not limited to, radioisotopes or radionuclides (e.g., 3 H, 14 C. 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I, 177 Lu, 166 Ho, or 153Sm), chromogens, fluorescent labels (e.g., FITC, rhodamine, lanthanide fluorophores), enzyme labels (e.g., horseradish peroxidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags), and magnetic agents such as gadolinium chelates. Representative examples of labels commonly used in immunoassays include moieties that emit light, e.g., acridinium compounds, and moieties that emit fluorescence, e.g., fluorescein. In this regard, the moiety itself may not be detectably labeled, but may become detectable upon reaction with yet another moiety.

[0210] Exemplary Modified IL-2-Containing Polypeptides Provided herein are polypeptides comprising modified IL-2. In some embodiments, the modified IL-2 comprises at least one amino acid substitution that reduces the affinity of the modified IL-2 for the IL-2 receptor as compared to wild-type IL-2. In various embodiments, the polypeptides comprising modified IL-2 provided herein are agonists of IL-2R. In some embodiments, the modified IL-2 is modified human IL-2 and the IL-2R is human IL-2R. In some embodiments, the modified IL-2 binds to human IL-2R with an affinity that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 50-fold, or at least 100-fold lower than the affinity of human wild-type IL-2 for IL-2R.

[0211] In various embodiments, the modified IL-2-containing polypeptides comprise at least one antigen binding domain that binds to a T cell or natural killer (NK) cell antigen. In some embodiments, the modified IL-2-containing polypeptides provided herein comprise one, two, three, four, five, six, seven, or eight antigen binding domains, where at least one or all bind to a T cell or natural killer cell antigen. In some embodiments, the modified IL-2-containing polypeptides provided herein comprise one, two, three, or four antigen binding domains, where at least one or all bind to a T cell or natural killer cell antigen. In some embodiments, the modified IL-2-containing polypeptides comprise an anti-IL-2-containing polypeptide. does not bind to or activate IL-2R in the absence of the original binding domain, hi some embodiments, a modified IL-2-containing polypeptide binds to and / or activates IL-2R on a cell only if the polypeptide contains an antigen binding domain that binds to the same antigen on the cell as IL-2R.

[0212] In various embodiments, the modified IL-2 comprises at least one substitution at at least one amino acid position selected from P65, D84, E95, M23, and H16. In some embodiments, the modified IL-2 comprises substitutions at amino acid positions P65, H16, and D84. In some embodiments, the modified IL-2 comprises substitutions at amino acid positions P65, H16, D84, and M23. In some embodiments, the modified IL-2 comprises substitutions at amino acid positions P65, H16, D84, and E95. In some embodiments, the modified IL-2 comprises substitutions at amino acid positions P65, H16, D84, M23, and E95.

[0213] In some embodiments, the substitution at amino acid position P65 is selected from P65R, P65E, P65K, P65H, P65Y, P65Q, P65D, and P65N. In some embodiments, the substitution at amino acid position H16 is selected from H16A, H16G, H16S, H16T, H16V, and H16P. In some embodiments, the substitution at amino acid position D84 is selected from D84S, D84G, D84A, D84T, D84V, and D84P. In some embodiments, the substitution at amino acid position M23 is selected from M23A, M23G, M23S, M23T, M23V, and M23P. In some embodiments, the substitution at amino acid position E95 is selected from E95Q, E95G, E95S, E95T, E95V, E95P, E95H, and E95N.

[0214] In some embodiments, the modified IL-2 further comprises a substitution at amino acid position F42. In some such embodiments, the substitution at F42 is selected from F42K, F42A, F42R, F42A, F42G, F42S, and F42T.

[0215] In some embodiments, the modified IL-2 further comprises at least one substitution at at least one amino acid position selected from Y45 and L72. In some such embodiments, the modified IL-2 comprises at least one substitution selected from Y45A and L72G.

[0216] In some embodiments, the modified IL-2 further comprises at least one substitution at at least one amino acid position selected from T3 and C125. In some such embodiments, the modified IL-2 comprises at least one substitution selected from T3A and C125A.

[0217] In some embodiments, the modified IL-2 comprises the substitutions P65R, H16A, and D84S. In some embodiments, the modified IL-2 comprises the substitutions P65R, H16A, D84S, and M23A. In some embodiments, the modified IL-2 comprises the substitutions P65R, H16A, D84S, and E95Q. In some embodiments, the modified IL-2 comprises the substitutions P65R, H16A, D84S, M23A, and E95Q. In some embodiments, the modified IL-2 is selected from the group consisting of H16A-F42K, D84S-F42K, E15S-F42K, M23A-F42K, E95Q-F42K, P65R-H16A, P65R-D84S, P65R-E15S, P65R-M23A, P65R-E95Q, T3A-C125S, T3A-P65R-C125S, T3A-H16A-C125S, T3A-D84S -C125S, T3A-H16A-P65R-C125S, T3A-P65R-D84S-C125S, T3A-H16A-P65R-D84S-C125S, T3A-H16A-M23A-P65R-D84S-C125S, T3A-H16A-P65R-D84S-E95Q-C125S, and T3A-H16A-M23A-P65R-D84S-E95Q-C125S The compound includes a substitution selected from:

[0218] In any of the embodiments described herein, the modified IL-2 may be modified human IL-2. In various embodiments, the amino acid positions of the substitutions correspond to the amino acid positions in SEQ ID NO:1.

[0219] In some embodiments, the modified IL-2 comprises an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:84 and includes one or more of the substitutions discussed herein. In some embodiments, the modified IL-2 comprises an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from SEQ ID NO:3-9, SEQ ID NO:11-21, and SEQ ID NO:23-31 and includes one or more of the substitutions discussed herein. In some embodiments, the modified IL-2 comprises an amino acid sequence selected from SEQ ID NO:3-9, SEQ ID NO:11-21, and SEQ ID NO:23-31. In some embodiments, the modified IL-2 comprises an amino acid sequence selected from SEQ ID NO:3, SEQ ID NO:5-SEQ ID NO:9, SEQ ID NO:12-SEQ ID NO:21, and SEQ ID NO:23-SEQ ID NO:31.

[0220] In some embodiments, the modified IL-2-containing polypeptide comprises at least one antigen-binding domain that binds to a T cell or natural killer cell antigen and an Fc region. In some embodiments, the modified IL-2-containing polypeptide provided herein comprises one, two, three, or four antigen-binding domains and an Fc region. In some embodiments, the Fc region mediates dimerization of the modified IL-2-containing polypeptide under physiological conditions, such that dimer formation doubles the number of antigen-binding sites. For example, a modified IL-2-containing polypeptide comprising three antigen-binding domains and an Fc region is trivalent as a monomer, but under physiological conditions, the Fc region can mediate dimerization such that the modified IL-2-containing polypeptide exists as a hexavalent dimer under such conditions.

[0221] In various embodiments, the polypeptide comprising modified IL-2 comprises a sequence selected from SEQ ID NO:3-9, SEQ ID NO:11-21, and SEQ ID NO:23-31. In various embodiments, the polypeptide comprising modified IL-2 comprises a sequence selected from SEQ ID NO:3, SEQ ID NO:5-9, and SEQ ID NO:12-21, and SEQ ID NO:23-31. In various embodiments, the polypeptide comprising modified IL-2 comprises SEQ ID NO:21. In some embodiments, the polypeptide further comprises an antigen binding domain. In some embodiments, the antigen binding domain is humanized.

[0222] In some embodiments, at least one antigen binding domain is selected from the group consisting of natural or native cognate binding partners, Anticalin (engineered lipocalin), Darpin, Fynomer, Centyrin (engineered fibronectin type III domain), cystine knot domain, Affilin, Affibody, or engineered CH3 domain. In some embodiments, the natural cognate binding partner comprises a ligand or extracellular domain of a natural cognate binding partner of a tumor-associated antigen (TAA) or a variant thereof that exhibits binding activity to the TAA, or a binding fragment thereof.

[0223] In some embodiments, a polypeptide comprising modified IL-2 and at least one antigen binding domain enhances anti-tumor T cell or natural killer cell responses while avoiding Tregs, peripheral T cells, and endothelial cells. In some such embodiments, the at least one antigen binding domain targets the modified IL-2 to activated T cells. In some embodiments, the modified IL-2 binds to and modulates the IL-2R only if the IL-2R is on the same cell as the antigen to which the at least one antigen binding domain binds. In some embodiments, the modified IL-2 does not bind to or activate IL-2R when the IL-2R is on a cell that is different from a cell that expresses the antigen to which the at least one antigen binding domain binds.

[0224] In various embodiments, the antigen binding domain binds to a protein selected from PD-1, CTLA-4, LAG3, TIM3, 4-1BB, OX40, GITR, CD8a, CD8b, CD4, NKp30, NKG2A, TIGIT, TGFβR1, TGFβR2, Fas, NKG2D, NKp46, PD-L1, CD107a, ICOS, TNFR2, and CD16a. In some embodiments, the polypeptide comprising the modified IL-2 binds to a protein selected from nivolumab (BMS; PD-1), pembrolizumab (Merck; PD-1), AMP-514 (Amplimmune; PD-1), TSR-042 (Tesaro / AnaptysBio; ANB-011). ;PD-1), STI-A1110 (Sorrento Therapeutics;PD-1), ipilim Mab (BMS; CTLA-4), tremelimumab (AstraZeneca, CTLA-4), urelumab (BMS, 4-1BB), utomilumab (Pfizer, 4-1BB), atezolizumab (Roche, PD-L1), durvalumab (AstraZeneca, PD-L1), monalizumab (NKG2A, Innate Pharma and AstraZeneca), and BMS-986016 (Bristo-Meyers Squibb, LAG-3) antigen-binding domains.

[0225] In some embodiments, the polypeptide comprises at least one antigen binding domain that specifically binds to PD-1. In some embodiments, the polypeptide comprises at least one antigen binding domain that specifically binds to LAG3. In some embodiments, the polypeptide comprises at least one antigen binding domain that specifically binds to NKp46. In some embodiments, the polypeptide comprises at least one antigen binding domain that specifically binds to NKG2D. In some embodiments, the polypeptide comprises at least one antigen binding domain that specifically binds to CD8a.

[0226] In some embodiments, the antigen-binding domain may be humanized. Polypeptides comprising humanized antigen-binding domains (such as VHH-containing polypeptides) are useful as therapeutic molecules because humanized antigen-binding domains and humanized antibodies reduce or eliminate human immune responses to non-human antibodies that may result in an immune response to antibody therapeutics and reduce the efficacy of the therapeutics. Generally, a humanized antigen-binding domain or humanized antibody comprises one or more variable domains in which the CDRs (or portions thereof) are derived from a non-human antibody and the FRs (or portions thereof) are derived from human antibody sequences. The humanized antigen-binding domain or humanized antibody also optionally comprises at least a portion of a human constant region. In some embodiments, some FR residues in the humanized antigen-binding domain or humanized antibody are replaced with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve the specificity or affinity of the antibody.

[0227] Humanized antibodies and methods for making them are reviewed, e.g., in Almagro and Fransson, (2008) Front. Biosci. 13: 1619-1633, and described, e.g., in Riechmann et al., (1988) Nature 332:323-329, Queen et al., (1989) Proc. Natl Acad. Sci. USA 86: 10029-10033, U.S. Pat. No. 5,821,337, U.S. Pat. No. 7,527,791, U.S. Pat. No. 6,982,321, and U.S. Pat. No. 7,087,409, Kashmiri et al., (2005) Methods 36:25-34, Padlan, (1991) Mol. Immunol. 28:489-498 (describing "resurfacing"), Dall'Acqua, et al. et al., (2005) Methods 36:43-60 (describing "FR shuffling"), and Osbourn et al., (2005) Methods 36:61-68 and Klimka et al., (2000) Br. J. Cancer, 83:252-260 (describing a "guided selection" approach to FR shuffling).

[0228] Human framework regions that may be used for humanization include, but are not limited to, framework regions selected using the "best-fit" method (see, e.g., Sims et al. (1993) J. Immunol. 151:2296), framework regions derived from consensus sequences of human antibodies of particular subgroups of heavy chain variable regions (see, e.g., Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285, and Presta et al. (1993) J. Immunol, 151:2623), human mature (somatically mutated) framework regions, and / or human IgG1-specific framework regions. or human germline framework regions (see, e.g., Almagro and Fransson, (2008) Front. Biosci. 13:1619-1633), and framework regions obtained from screening of FR libraries (see, e.g., Baca et al., (1997) J. Biol. Chem. 272: 10678-10684, and Rosok et al., (1996) J. Biol. Chem. 271 :22611-22618). Typically, the FR region of a VHH is replaced with a human FR region to generate a humanized VHH. In some embodiments, certain FR residues of the human FR are replaced to improve one or more properties of the humanized VHH. A VHH domain having such replaced residues is also referred to herein as "humanized".

[0229] In various embodiments, the Fc region contained in the modified IL-2-containing polypeptide is a human Fc region or is derived from a human Fc region.

[0230] In some embodiments, the Fc region included in the modified IL-2-containing polypeptide is derived from a human Fc region and contains a three amino acid deletion in the lower hinge corresponding to IgG1 E233, L234, and L235, and is referred to herein as "Fc xELL." Fc xELL polypeptides do not bind to FcγR and are therefore referred to as "effector silent" or "effector null," although in some embodiments, the xELL Fc region binds to FcRn, thereby providing extended half-life and FcRn mediated recycling. ) with associated transcytosis.

[0231] In some embodiments, the Fc region included in the modified IL-2 containing polypeptide is derived from a human Fc region and includes the mutations M252Y and M428V, referred to herein as "Fc-YV." In some embodiments, the Fc region included in the modified IL-2 containing polypeptide is derived from a human Fc region and includes the mutations M252Y and M428L, referred to herein as "Fc-YL." In some embodiments, such mutations enhance binding to FcRn at the acidic pH of the endosome (near 6.5) while losing detectable binding at neutral pH (about 7.2), allowing for enhanced FcRn-mediated recycling and extended half-life.

[0232] In some embodiments, the Fc region included in the modified IL-2-containing polypeptides herein is derived from a human Fc region and includes mutations designed for heterodimerization, referred to herein as "knob" and "hole." In some embodiments, the "knob" Fc region includes the mutation T366W. In some embodiments, the "hole" Fc region includes the mutations T366S, L368A, and Y407V. In some embodiments, the Fc region used for heterodimerization includes an additional mutation, such as the mutation S354C on a first member of the heterodimeric Fc pair, which forms an asymmetric disulfide with the corresponding mutation Y349C on the second member of the heterodimeric Fc pair. In some embodiments, one member of the heterodimeric Fc pair includes the modification H435R or H435K to prevent Protein A binding while maintaining FcRn binding. In some embodiments, one member of the heterodimeric Fc pair comprises the modification H435R or H435K, while the second member of the heterodimeric Fc pair is not modified at H435. In various embodiments, the hole Fc region comprises the modification H435R or H435K (sometimes referred to as "hole-R" when the modification is H435R), while the knob Fc region does not. In some cases, the hole-R mutation is used to differentiate a heterodimeric Fc region from a possible homodimeric hole Fc region. Improve purification of.

[0233] Non-limiting exemplary Fc regions that can be used in modified IL-2-containing polypeptides include Fc regions comprising the amino acid sequences of SEQ ID NO:47-SEQ ID NO:83.

[0234] In some embodiments, the modified IL-2-containing polypeptide comprising at least one antigen-binding domain and an Fc region comprises an amino acid sequence selected from SEQ ID NO:3-9, SEQ ID NO:11-21, and SEQ ID NO:23-31, and an Fc region fused to the C-terminus of the amino acid sequence. In some embodiments, the modified IL-2-containing polypeptide comprising at least one antigen-binding domain and an Fc region comprises an amino acid sequence selected from SEQ ID NO:3, SEQ ID NO:5-9, SEQ ID NO:12-21, and SEQ ID NO:23-31, and an Fc region fused to the C-terminus of the amino acid sequence. In some embodiments, the modified IL-2-containing polypeptide comprising at least one antigen-binding domain and an Fc region comprises the amino acid sequence of SEQ ID NO:21, and an Fc region fused to the C-terminus of the amino acid sequence. In some embodiments, the modified IL-2-containing polypeptide comprising at least one antigen-binding domain and an Fc region comprises an amino acid sequence selected from SEQ ID NO:3-9, SEQ ID NO:11-21, and SEQ ID NO:23-31, and an Fc region fused to the N-terminus of the amino acid sequence. In some embodiments, the modified IL-2-containing polypeptide comprising at least one antigen-binding domain and an Fc region comprises an amino acid sequence selected from SEQ ID NO:3, SEQ ID NO:5-9, SEQ ID NO:12-21, and SEQ ID NO:23-31, and an Fc region fused to the N-terminus of the amino acid sequence. In some embodiments, the modified IL-2-containing polypeptide comprising at least one antigen-binding domain and an Fc region comprises the amino acid sequence of SEQ ID NO:21, and an Fc region fused to the N-terminus of the amino acid sequence. In some embodiments, the modified IL-2-containing polypeptide comprising at least one antigen-binding domain and an Fc region comprises an amino acid sequence selected from SEQ ID NO:34-43, and SEQ ID NO:46. In some embodiments, the polypeptide comprises SEQ ID NO: 43 and an antigen binding domain that binds to an antigen expressed on a T cell or a natural killer cell. In some embodiments, the polypeptide comprises SEQ ID NO: 46 and an antigen binding domain that binds to an antigen expressed on a T cell or a natural killer cell.

[0235] Exemplary Activities of Modified IL-2-Containing Polypeptides In various embodiments, the modified IL-2-containing polypeptides provided herein are agonists of IL-2R activity. Agonist activity can be determined in some embodiments using methods shown in the Examples herein, for example, using 293F cells or similar cells. In some embodiments, the modified IL-2-containing polypeptides provided herein are agonists of IL-2R activity when targeted to T cells, but exhibit little or no agonist activity in the absence of targeting. In some embodiments, the modified IL-2-containing polypeptides provided herein are agonists of IL-2R activity when targeted to NK cells and / or T cells, but exhibit little or no agonist activity in the absence of targeting. In some embodiments, the modified IL-2-containing polypeptides targeted to T cells or NK cells contain at least one antigen-binding domain that specifically binds to an antigen expressed on T cells or NK cells.

[0236] In some embodiments, the modified IL-2-containing polypeptides provided herein are CD4 in vitro and / or in vivo + T cells and / or CD8 + In some embodiments, the polypeptide increases the proliferation of CD4 T cells in the presence of Treg cells. + T cells and / or CD8 + In some such embodiments, CD4 + T cells and / or CD8 + T cells are activated CD4 + T cells and / or CD8 + In some embodiments, the modified IL-2-containing polypeptides provided herein are T cells. The peptide activates CD4 in vitro. + T cells and / or CD8 + In some embodiments, the modified IL-2-containing polypeptide increases the proliferation of activated CD4 T cells. + T cells and / or CD8 +T cell proliferation was measured using CD4 + T cells and / or CD8 + In some embodiments, the polypeptide increases T cell proliferation by at least 1.5-fold, at least 2-fold, at least 3-fold, or at least 5-fold. + T cells and / or CD8 + Increase T cell proliferation by at least 1.5-fold, at least 2-fold, at least 3-fold, or at least 5-fold, and inhibit resting CD4 + T cells and / or CD8 + The proliferation of T cells is not substantially increased relative to the proliferation observed in the absence of the polypeptide.

[0237] In some embodiments, the modified IL-2-containing polypeptides provided herein are Increases proliferation of NK cells in vitro and / or in vivo. In some such embodiments, the NK cells are activated NK cells. In some embodiments, the modified IL-2-containing polypeptides provided herein increase proliferation of activated NK cells in vitro. In some embodiments, the modified IL-2-containing polypeptides increase proliferation of activated NK cells at least 1.5-fold, at least 2-fold, at least 3-fold, or at least 5-fold compared to NK cell proliferation in the absence of the polypeptide. In some embodiments, the polypeptides increase proliferation of activated NK cells at least 1.5-fold, at least 2-fold, at least 3-fold, or at least 5-fold and do not substantially increase proliferation of resting NK cells compared to proliferation observed in the absence of the polypeptide.

[0238] activated CD4 + T cells and / or CD8 + Increased T cell proliferation can be determined by any method in the art, such as, for example, the methods shown in the Examples herein. Non-limiting exemplary assays are as follows: CD4 + T cells and / or CD8 +T cells can be isolated from one or more healthy human donors. The T cells are stained with CellTrace Violet (CTV), activated with anti-CD3 antibodies, contacted with modified IL-2-containing polypeptides, and analyzed by FACS. Loss of CTV staining indicates proliferation. In some embodiments, CD4 + T cells and / or CD8 + Increased proliferation of T cells was observed, for example, in CD4 + T cells and / or CD8 + By measuring the proliferation of T cells, the proliferation is determined as an average from a series of experiments or from pooled T cells. In some embodiments, CD4 + T cells and / or CD8 + The increase in T cell proliferation is determined as an average from experiments performed using T cells from at least 5 or at least 10 different healthy donors, or from a pool of T cells from at least 5 or at least 10 different healthy donors. In some embodiments, the modified IL-2-containing polypeptides provided herein inhibit CD4 T cell proliferation even in the presence of Treg cells. + T cells and / or CD8 + Increases T cell proliferation.

[0239] In some embodiments, the modified IL-2-containing polypeptides provided herein are CD4 in vitro and / or in vivo + T cells and / or CD8 + Increases CD71 expression on T cells. CD71 expression is indicative of T cell activation. In some embodiments, the modified IL-2-containing polypeptides provided herein increase CD4 + T cells and / or CD8 + In some embodiments, the modified IL-2-containing polypeptide increases CD4 + T cells and / or CD8 +In some embodiments, the polypeptide increases CD71 expression on T cells by at least 1.5-fold, at least 2-fold, at least 3-fold, or at least 5-fold compared to CD71 expression in the absence of the polypeptide. + T cells and / or CD8 + Increase CD71 expression on T cells by at least 1.5-fold, at least 2-fold, at least 3-fold, or at least 5-fold, and inhibit resting CD4 + T cells and / or CD8 + In some embodiments, the polypeptide does not substantially increase CD71 expression on T cells compared to CD71 expression observed in the absence of the polypeptide. + T cells and / or CD8 + Increases CD71 expression on T cells.

[0240] CD4 + T cells and / or CD8 + Increased CD71 expression on T cells can be determined by any method in the art, such as, for example, the methods shown in the Examples herein. Non-limiting exemplary assays are as follows: CD4 + T cells and / or CD8 + T cells can be isolated from one or more healthy human donors, stimulated with anti-CD3 antibodies, contacted with modified IL-2-containing polypeptides, and analyzed for CD71 expression by FACS. + T cells and / or CD8 + Increased CD71 expression on T cells was observed, for example, in CD4 T cells isolated from different healthy human donors. + T cells and / or CD8 + By measuring CD71 expression on T cells, it is determined as an average from a series of experiments or pooled T cells. In some embodiments, CD4 + T cells and / or CD8 +The increase in CD71 expression on T cells is determined as an average from experiments performed using T cells from at least 5 or at least 10 different healthy donors, or from a pool of T cells from at least 5 or at least 10 different healthy donors. In some embodiments, the modified IL-2-containing polypeptides provided herein increase CD4 expression even in the presence of Treg cells. + T cells and / or CD8 + Increases CD71 expression on T cells.

[0241] In some embodiments, the modified IL-2-containing polypeptides provided herein are CD4 in vitro and / or in vivo + T cells and / or CD8 + Increases pSTAT5 expression in T cells. pSTAT5 expression is indicative of T cell activation. In some embodiments, the modified IL-2-containing polypeptides provided herein increase CD4 + T cells and / or CD8 + In some embodiments, the modified IL-2-containing polypeptide increases pSTAT5 expression in T cells. + T cells and / or CD8 + In some embodiments, the polypeptide increases pSTAT5 expression on T cells by at least 1.5-fold, at least 2-fold, at least 3-fold, or at least 5-fold compared to pSTAT5 expression in the absence of the polypeptide. + T cells and / or CD8 + Increases pSTAT5 expression on T cells. CD4 + T cells and / or CD8 + Increased pSTAT5 expression in T cells can be determined by any method in the art, such as, for example, the methods set forth in the Examples herein. In some embodiments, the modified IL-2-containing polypeptides provided herein increase pSTAT5 expression in T cells by upregulating CD4 T cells even in the presence of Treg cells. + T cells and / or CD8 + Increases pSTAT5 expression in T cells.

[0242] In some embodiments, the modified IL-2-containing polypeptides provided herein are Increases pSTAT5 expression in NK cells in vitro and / or in vivo. pSTAT5 expression is indicative of NK cell activation. In some embodiments, the modified IL-2-containing polypeptides provided herein increase pSTAT5 expression in NK cells in vitro. In some embodiments, the modified IL-2-containing polypeptides increase pSTAT5 expression on NK cells by at least 1.5-fold, at least 2-fold, at least 3-fold, or at least 5-fold compared to pSTAT5 expression in the absence of the polypeptide. In some embodiments, the polypeptides increase pSTAT5 expression in NK cells in the presence of Treg cells. Increased pSTAT5 expression in NK cells can be determined by any method in the art, such as, for example, the methods shown in the Examples herein.

[0243] In some embodiments, the modified IL-2-containing polypeptides provided herein reduce or attenuate the suppressive activity of regulatory T cells (Tregs). + T cells and / or CD8 + Treg suppressive activity against T cells is reduced by at least 10%, at least 20%, at least 30%, or at least Reduce by 50%. Conventional CD4 + T cells and / or CD8 + The reduction in Treg suppressive activity on T cells can be determined by any method in the art, such as, for example, the methods shown in the Examples herein. Non-limiting exemplary assays are as follows: Treg and CD4 + T cells are isolated from healthy human donor PBMCs and then differentially labeled with proliferating cell fluorescent dyes. CD4 +T cells are stimulated with anti-CD3 antibodies, while Treg cells are incubated in the presence of modified IL-2-containing polypeptides provided herein. The two T cell populations are co-cultured for three days to stimulate CD4 + Proliferation and activation of T cells is monitored by flow cytometry. In some embodiments, the modified IL-2-containing polypeptides provided herein are capable of inhibiting, for example, CD4 T cell proliferation and activation in the presence of Treg cells but in the absence of the modified IL-2-containing polypeptides provided herein. + T cells and / or CD8 + Compared with T cell activation and proliferation, CD4 + T cells and / or CD8 + Increases T cell activation and proliferation.

[0244] Polypeptide Expression and Production Nucleic acid molecules are provided that include a polynucleotide encoding a modified IL-2-containing binding polypeptide. Thus, in various embodiments, nucleic acid molecules are provided that encode a polypeptide comprising modified IL-2. In some embodiments, the nucleic acid molecule encodes modified IL-2 and at least one antigen binding domain. In various embodiments, the nucleic acid molecule encodes modified IL-2, an Fc region, and optionally at least one antigen binding domain. In some embodiments, the Fc region comprises a mutation designed for heterodimerization, such as a "knob" or "hole" mutation. In some embodiments, nucleic acid molecules are provided that encode a modified IL-2-containing polypeptide comprising modified IL-2, at least one antigen binding domain, and an Fc region, where the Fc region is fused to the C-terminus of the at least one antigen binding domain and the modified IL-2 is fused to the C-terminus of the Fc region. In any of the above-described embodiments, the nucleic acid molecule can also encode a leader sequence that directs secretion of the modified IL-2-containing polypeptide, where the leader sequence is typically cleaved such that it is not present in the secreted polypeptide. The leader sequence may be the native heavy chain (or VHH) leader sequence or may be another heterologous leader sequence.

[0245] The nucleic acid molecule can be constructed using recombinant DNA techniques routine in the art. In some embodiments, the nucleic acid molecule is an expression vector suitable for expression in a selected host cell.

[0246] Vectors are provided that contain nucleic acids encoding the modified IL-2-containing polypeptides described herein. Such vectors include, but are not limited to, DNA vectors, phage vectors, viral vectors, retroviral vectors, and the like. In some embodiments, vectors are selected that are optimized for expression of the polypeptide in a desired cell type, such as 293F cells, CHO cells or CHO-derived cells, or NSO cells. Exemplary such vectors are described, for example, in Running Deer et al., Biotechnol. Prog. 20:880-889 (2004).

[0247] In some embodiments, modified IL-2-containing polypeptides may be expressed in prokaryotic cells, such as bacterial cells, or in eukaryotic cells, such as fungal cells (such as yeast), plant cells, insect cells, and mammalian cells. Such expression may be carried out, for example, according to procedures known in the art. Exemplary eukaryotic cells that may be used for expression of the polypeptide include, but are not limited to, COS cells, including COS7 cells, 293 cells, including 293F cells, CHO cells, including CHO-S, DG44, Lec13 CHO cells, and FUT8 CHO cells, PER.C6™ cells (Crucell), and NSO cells. In some embodiments, the polypeptides may be expressed in prokaryotic cells, such as bacterial ... In embodiments, modified IL-2-containing polypeptides can be expressed in yeast. See, e.g., U.S. Patent Application Publication No. 2006 / 0270045. In some embodiments, A particular eukaryotic host cell is selected based on its ability to make desired post-translational modifications to a polypeptide, for example, in some embodiments, CHO cells produce polypeptides with higher levels of sialylation than the same polypeptides produced in 293F cells.

[0248] Introduction of one or more nucleic acids (e.g., vectors) into a desired host cell can be accomplished by any method, including, but not limited to, calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid mediated transfection, electroporation, transduction, infection, etc. Non-limiting exemplary methods are described, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual, 3 rd ed. Cold Spring Harbor Laboratory Press (2001). Nucleic acids can be prepared according to any suitable method. , may be transiently or stably transfected into the desired host cell.

[0249] Also provided are host cells comprising any of the nucleic acids or vectors described herein. In some embodiments, host cells are provided that express the modified IL-2-containing polypeptides described herein. The modified IL-2-containing polypeptides expressed in the host cells can be purified by any suitable method. Such methods include, but are not limited to, the use of affinity matrix or hydrophobic interaction chromatography. Suitable affinity ligands include agents that bind to the ROR1 ECD and Fc region. For example, Protein A, Protein G, Protein A / G, or antibody affinity columns can be used to purify modified IL-2-containing polypeptides comprising an Fc region by binding to the Fc region. Hydrophobic interaction chromatography, e.g., butyl or phenyl columns, can also be suitable for purifying some polypeptides, such as antibodies. Ion exchange chromatography (e.g., anion exchange and / or cation exchange chromatography) can also be suitable for purifying some polypeptides, such as antibodies. Mixed mode chromatography (e.g., reversed phase / anion exchange, reversed phase / cation exchange, hydrophilic interaction / anion exchange, hydrophilic interaction / cation exchange, etc.) can also be suitable for purifying some polypeptides, such as antibodies. Many methods for purifying polypeptides are known in the art.

[0250] In some embodiments, the modified IL-2-containing polypeptides are produced in a cell-free system. Non-limiting exemplary cell-free systems are described, for example, in Sitaraman et al., Methods Mol. Biol. 498: 229-44 (2009), Spirin, Trends Biotechnol. 22: 538-45 (2004), and Endo et al., Biotechnol. Adv. 21: 695-713 (2003).

[0251] In some embodiments, a modified IL-2-containing polypeptide produced by the above method is provided. In some embodiments, the modified IL-2-containing polypeptide is produced in a host cell. In some embodiments, the modified IL-2-containing polypeptide is produced in a cell-free system. In some embodiments, the modified IL-2-containing polypeptide is purified. In some embodiments, a cell culture medium comprising the modified IL-2-containing polypeptide is provided.

[0252] In some embodiments, a composition is provided that comprises an antibody produced by the above method. In some embodiments, the composition comprises a modified IL-2-containing polypeptide produced in a host cell. In some embodiments, the composition comprises a modified IL-2-containing polypeptide produced in a cell-free system. In some embodiments, the composition comprises a purified modified IL-2-containing polypeptide.

[0253] Exemplary Methods of Treating Disease Using Modified IL-2-Containing Polypeptides In some embodiments, methods are provided for treating a disease in an individual comprising administering a modified IL-2-containing polypeptide. Such diseases include, but are not limited to, CD4 + T cells and / or CD8 +The present invention includes any disease that would benefit from increased proliferation and activation of T cells. In some embodiments, a method for treating cancer in an individual is provided. The method comprises administering to the individual an effective amount of a modified IL-2-containing polypeptide provided herein. Such a method of treatment can be a method of treatment in a human or an animal. In some embodiments, a method of treatment in a human is provided. Non-limiting exemplary cancers that can be treated using the modified IL-2 containing polypeptides provided herein include basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and central nervous system cancer, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, colorectal cancer, connective tissue cancer, digestive system cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, glioblastoma, hepatic cancer, hepatocellular carcinoma, intraepithelial neoplasia, renal or kidney cancer, laryngeal cancer, liver cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, melanoma, myeloma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, respiratory system cancer, salivary gland cancer, sarcoma, skin cancer, squamous cell carcinoma, gastric cancer, , testicular cancer, thyroid cancer, uterine or endometrial cancer, urinary system cancer, and vulvar cancer, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, B-cell lymphoma, low-grade / follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-dividing cell NHL, bulky disease NHL, mantle cell lymphoma, AIDS-related lymphoma, Waldenstrom's macroglobulinemia, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, and chronic myeloblastic leukemia.

[0254] The modified IL-2-containing polypeptide may be administered to the subject as needed. The frequency of administration may be determined by a person skilled in the art, such as the attending physician, based on considerations such as the condition being treated, the age of the subject being treated, the severity of the condition being treated, the general health of the subject being treated, and the like. In some embodiments, an effective dose of the modified IL-2-containing polypeptide is administered to the subject one or more times. In some embodiments, an effective dose of the modified IL-2-containing polypeptide is administered to the subject daily, twice weekly, weekly, biweekly, monthly, and the like. An effective dose of the modified IL-2-containing polypeptide is administered to the subject at least once. In some embodiments, an effective dose of the modified IL-2-containing polypeptide may be administered multiple times, including multiple times over at least one month, at least six months, or at least one year.

[0255] In some embodiments, the pharmaceutical composition comprising the modified IL-2-containing polypeptide is administered in an amount effective to treat cancer (including cancer prevention) and / or increase T-cell proliferation. The therapeutically effective amount typically depends on the weight of the subject being treated, the physical or health condition of the subject, the extent of the condition being treated, or the age of the subject being treated. In general, the polypeptide may be administered in an amount ranging from about 0.05 mg / kg body weight to about 100 mg / kg body weight per dose. In some embodiments, the polypeptide may be administered in an amount ranging from about 10 μg / kg body weight to about 100 mg / kg body weight per dose. In some embodiments, the polypeptide may be administered in an amount ranging from about 50 μg / kg body weight to about 5 mg / kg body weight per dose. In some embodiments, the polypeptide may be administered in an amount ranging from about 100 μg / kg body weight to about 10 mg / kg body weight per dose. In some embodiments, the polypeptide may be administered in an amount ranging from about 100 μg / kg body weight to about 20 mg / kg body weight per dose. In some embodiments, the polypeptide may be administered in an amount ranging from about 0.5 mg / kg body weight to about 20 mg / kg body weight per dose. In some embodiments, the polypeptide may be administered in an amount ranging from about 0.5 mg / kg body weight to about 10 mg / kg body weight per dose. In some embodiments, the polypeptide may be administered in an amount ranging from about 0.05 mg / kg body weight to about 20 mg / kg body weight per dose. In some embodiments, the polypeptide may be administered in an amount ranging from about 0.05 mg / kg body weight to about 10 mg / kg body weight per dose. In some embodiments, the polypeptide may be administered in an amount ranging from about 5 mg / kg body weight or less, e.g., less than 4 mg / kg, less than 3 mg / kg, less than 2 mg / kg, or less than 1 mg / kg of antibody. It may be administered at

[0256] In some embodiments, the modified IL-2-containing polypeptides may be administered in vivo by a variety of routes, including, but not limited to, intravenous, intraarterial, parenteral, intraperitoneal, or subcutaneous. Depending on the intended use, the appropriate formulation and route of administration may be selected.

[0257] In some embodiments, therapeutic treatment with modified IL-2-containing polypeptides is accomplished by increasing T cell proliferation and / or activation, hi some embodiments, increasing T cell proliferation and / or activation inhibits cancer growth.

[0258] Pharmaceutical Compositions In some embodiments, compositions comprising modified IL-2-containing polypeptides are provided in formulations that include a wide variety of pharma- ceutically acceptable carriers (see, e.g., Gennaro, Remington: The Science and Practice of Pharmacy with Facts and Comparisons: Drugfacts Plus, 20th ed. (2003); Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7 th ed., Lippencott Williams and Wilkins (2004), Kibbe et al., Handbook of Pharmaceutical Excipients, 3 rd ed., Pharmaceutical Press (2000). A variety of pharma- ceutically acceptable carriers, including agents, adjuvants, and diluents, can be used. In addition, a variety of pharma- ceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents, etc., can also be used. Non-limiting exemplary carriers include saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.

[0259] In some embodiments, the pharmaceutical composition comprises a modified IL-2-containing polypeptide at a concentration of at least 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, 125 mg / mL, 150 mg / mL, 175 mg / mL, 200 mg / mL, 225 mg / mL, or 250 mg / mL.

[0260] Combination therapy The modified IL-2-containing polypeptide may be administered alone or in combination with other therapeutic modalities, such as other anti-cancer agents. The modified IL-2-containing polypeptide may be provided prior to, substantially simultaneously with, or subsequent to (i.e., simultaneously or sequentially with) other therapeutic modalities. In some embodiments, the therapeutic methods described herein may further include administering a biologic, such as radiation therapy, chemotherapy, vaccination, targeted tumor therapy, CAR-T therapy, oncolytic virus therapy, cancer immunotherapy, cytokine therapy, surgical resection, chromatin modification, resection, cryotherapy, an antisense agent against a tumor target, an siRNA agent against a tumor target, a microRNA agent against a tumor target, or an anti-cancer / anti-tumor agent, or an antibody, cytokine, or receptor extracellular domain-Fc fusion.

[0261] In some embodiments, the modified IL-2-containing polypeptides provided herein are given simultaneously with a second therapeutic agent, such as a PD-1 antibody. Examples of PD-1 antibodies include nivolumab (BMS), pembrolizumab (Merck), AMP-514 (Amplimmune), TSR-042 (Tesaro / AnaptysBio, ANB-011), STI-A1110 (Sorrento Therapeutics), and other agents against programmed death-1 (PD-1).

[0262] In some embodiments, the modified IL-2-containing polypeptides provided herein are given simultaneously with a second therapeutic agent, e.g., a PD-L1 therapeutic agent. Examples of PD-L1 therapeutic agents include The studies included pidilizumab (CureTech, CT-011), durvalumab (Medimmune / AstraZeneca), atezolizumab (Genentech / Roche), avelumab (Pfizer), AMP-224 (Amplimmune), BMS-936559 (Bristol-Myers Squibb), STI-A1010 (Sorrento Therapeutics), and programmed death-1 ligand (PD-L1 ) and other agents acting against the

[0263] In some embodiments, the modified IL-2-containing polypeptides provided herein are given concomitantly with CAR-T (chimeric antigen receptor T cell) therapy, oncolytic virus therapy, cytokine therapy, and / or agents that target other checkpoint molecules, such as VISTA, gpNMB, B7H3, B7H4, HHLA2, CD73, CTLA4, TIGIT, etc.

[0264] Non-Limiting Exemplary Methods of Diagnosis and Treatment In some embodiments, the methods described herein are useful for evaluating a subject and / or a sample from a subject (e.g., a cancer patient). In some embodiments, the evaluation is one or more of diagnosis, prognosis, and / or response to treatment.

[0265] In some embodiments, the methods described herein include assessing the presence, absence, or level of a protein. In some embodiments, the methods described herein include assessing the presence, absence, or level of expression of a nucleic acid. The compositions described herein can be used for these measurements. For example, in some embodiments, the methods described herein include contacting a tumor specimen or cells cultured from the tumor with a therapeutic agent described herein.

[0266] In some embodiments, the evaluation may indicate treatment, including treatment with the polypeptides described herein. In some embodiments, the evaluation may indicate the use or withholding of adjuvant therapy after resection. Adjuvant therapy, also called adjuvant therapy, is a treatment given in addition to the primary, main, or initial treatment. As a non-limiting example, adjuvant therapy may be an additional treatment, usually given after surgery, when all detectable disease has been removed but there remains a statistical risk of relapse due to latent disease. In some embodiments, the polypeptide is used as an adjuvant therapy in the treatment of cancer. In some embodiments, the antibody is used as the sole adjuvant therapy in the treatment of cancer. In some embodiments, the antibody described herein is withheld as an adjuvant therapy in the treatment of cancer. For example, when a patient is unlikely to respond to the antibody described herein or has only a minimal response, treatment may not be administered for quality of life and to avoid unnecessary toxicity from ineffective chemotherapy. In such cases, palliative care may be used.

[0267] In some embodiments, the polypeptide is administered as a neoadjuvant therapy before resection. In some embodiments, neoadjuvant therapy refers to a therapy that shrinks and / or downgrades the tumor before any surgery. In some embodiments, neoadjuvant therapy refers to a chemotherapy drug administered to a cancer patient before surgery. In some embodiments, neoadjuvant therapy refers to a polypeptide administered to a cancer patient before surgery. Cancer types for which neoadjuvant chemotherapy is usually considered include, for example, breast cancer, colorectal cancer, ovarian cancer, cervical cancer, bladder cancer, and lung cancer. In some embodiments, the antibody is used as a neoadjuvant therapy in the treatment of cancer. In some embodiments, the use is before resection.

[0268] In some embodiments, the tumor microenvironment contemplated by the methods described herein includes tumor vasculature, tumor-infiltrating lymphocytes, fibroblastic reticular cells, endothelial progenitor cells (EPCs), cancer-associated fibroblasts, pericytes, other stromal cells, components of the extracellular matrix (ECM), dendritic cells, antigen-presenting cells, and the like. The immune cells may be one or more of: indicator cells, T cells, regulatory T cells, macrophages, neutrophils, and other immune cells located in the vicinity of the tumor.

[0269] kit Also provided are articles of manufacture and kits that include any of the modified IL-2-containing polypeptides described herein and suitable packaging. In some embodiments, the invention includes a kit comprising (i) a modified IL-2-containing polypeptide and (ii) instructions for using the kit to administer the modified IL-2-containing polypeptide to an individual.

[0270] Suitable packaging for the compositions described herein are known in the art and include, for example, vials (e.g., sealed vials), containers, ampoules, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. These articles of manufacture may be further sterilized and / or sealed. Unit dosage forms comprising the compositions described herein are also provided. These unit dosage forms may be stored in suitable packaging in single or multiple unit dosage forms, and may also be further sterilized and sealed. Instructions provided in the kits of the invention are typically written instructions on a label or insert (e.g., a paper sheet included in the kit), although machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable. Instructions for use of the antibody generally include information regarding dosage, administration schedule, and route of administration for the intended therapeutic or industrial use. The kit may further include instructions for selecting an appropriate individual treatment.

[0271] The containers may be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses. Kits may also be provided that contain a sufficient dose of the molecules disclosed herein to provide effective treatment to an individual for an extended period of time, such as any approximate period of 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months or more. Kits may also contain multiple unit doses of the molecules and instructions for use, and may be packaged in sufficient quantities for storage and use in pharmacies, such as hospital pharmacies and compounding pharmacies. In some embodiments, the kits include a dried (e.g., lyophilized) composition that can be reconstituted, resuspended, or rehydrated to generally form a stable aqueous suspension of the polypeptide. EXAMPLES

[0272] The examples discussed below are intended to be purely illustrative of the present invention and should not be considered as limiting the present invention in any way. These examples are not intended to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.), but some experimental error and deviation should be taken into account. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure.

[0273] Example 1: The P65R mutation of IL-2 essentially eliminates CD25 binding IL-2 mutants were designed to disrupt the CD25 interface by steric occlusion (P65R and P65E) and were tested for binding to 293F cells transiently transfected with one or more components of the IL-2 receptor (CD25, CD122, and / or CD132). The mutants were compared to IL-2-F42K, a mutant reported to have reduced affinity for CD25. Increasing concentrations of fusion proteins containing wild-type human IL-2 (SEQ ID NO: 32), IL-2-F42K (SEQ ID NO: 33), IL-2-P65R (SEQ ID NO: 35), or IL-2-P65E (SEQ ID NO: 34) fused to the N-terminus of the "knob" Fc and complexed with the "hole" Fc (SEQ ID NO: 44) were added to transfected 293F cells and incubated at 4° C. for 45 minutes.

[0274] Binding was analyzed by flow cytometry essentially as follows: Cells were washed once in 200 μL FACS buffer (PBS, 2% FBS, 0.05% sodium azide) and cell pellets were resuspended in 100 μl surface marker staining solution (containing A647-conjugated anti-human Fcg secondary antibody at a dilution of 1:300 in FACS buffer). Cells were incubated for 45 min at 4° C. before final washing and analysis on the flow cytometer. Cell debris was excluded by size exclusion with FSC / SSC and dead cells were excluded based on their positive propidium iodide signal. Single cells were selected using exclusion of doublets and aggregates with FSC-A / FSC-H. Transiently transfected cells also expressed cytoplasmic EGFP and cells that were FL1 positive were analyzed. An increase in the MFI level of the anti-human secondary antibody indicated IL-2 binding. FlowJo software was used to analyze cell populations. The raw mean fluorescence intensity ("MFI") for each marker was then exported and analyzed using Excel and GraphPad PRISM. Values ​​were graphed and titration curves were fitted to assess dose-response relationships using a nonlinear regression One-site -- Total curve fit.

[0275] As shown in Figures 2A-C, the fusion proteins containing the IL-2-P65E mutant showed slightly reduced affinity for IL-2R compared to the fusion proteins containing wild-type IL-2. The fusion proteins containing IL-2 F42K showed lower affinity than the fusion proteins containing IL-2-P65E, while the fusion proteins containing IL-2-P65R showed the lowest affinity for the heterotrimeric IL-2R (Figure 2A). Furthermore, the fusion proteins containing IL-2-P65R showed no detectable binding to CD25 / CD132 and only weakly bound to CD25 / CD122 (Figure 2C), whereas the fusion proteins containing IL-2-F42K retained some affinity for CD25 / CD132 (Figure 2B) and bound to CD25 / CD122 with higher affinity than the fusion proteins containing IL-2-P65R (Figures 2B and 2C). Thus, P65R-mutated IL-2 had significantly reduced binding to the CD25-containing IL-2 receptor.

[0276] Example 2: IL-2 modifications to reduce affinity for CD122 As described in Example 1, the P65R IL-2 mutation was designed to disrupt the CD25 interface by steric occlusion. In addition, IL-2 mutations were designed to reduce affinity for the CD122 interface by elimination of certain contact residue interactions (e.g., D84S, E95Q, M23A, H16A, and E15S). Single or double mutants were fused to the N-terminus of the "knob" portion of a heterodimeric Fc (the knob was stabilized by a disulfide to a hole containing the "hole" Fc SEQ ID NO: 44) for monovalent IL-2 binding to the IL-2R. Relative binding affinities were assessed by transiently transfecting 293F cells with CD25 and CD122 (co-transfection with CD132 showed similar results, although the additional binding avidity reduced the observed affinity differences). Bound IL-2-Fc fusion protein was detected with a fluorescent anti-human secondary antibody essentially as described in Example 1 and analyzed by flow cytometry.

[0277] As shown in Figures 3A and 3B, all fusion proteins containing double IL-2 mutants incorporating F42K with a mutation at the CD122 interface (SEQ ID NO: 36 to SEQ ID NO: 39), except for IL-2-F42K-E15S (SEQ ID NO: 85), showed reduced binding affinity to CD25 / CD122 compared to those containing the single mutant IL-2-F42K (SEQ ID NO: 33).

[0278] Example 3: IL-2-RAS (P65R, H16A, and D84S) has reduced affinity for CD122 in the context of trimeric and dimeric forms of IL-2R The CD122 affinity-reducing mutations described in Example 2 were combined with the P65R mutation to construct IL-2 double and triple mutants. The IL-2 mutants were then bound to the IL-2 receptor (IL-2 The IL-2R subunits were fused to the N-terminus of a heterodimeric Fc "knob" portion for monovalent IL-2 binding to the IL-2R subunits and paired with a "hole" Fc comprising SEQ ID NO: 44. The relative binding affinities of the resulting fusion proteins were assessed in 293F cells transiently transfected with IL-2R subunits, essentially as described in Example 1.

[0279] Compared to fusion proteins containing wild-type IL-2 (SEQ ID NO: 32), fusion proteins containing IL-2-P65R-H16A (SEQ ID NO: 41) and IL-2-P65R-D84S (SEQ ID NO: 42) had reduced affinity for both CD122 / CD132 (heterodimeric IL-2R) (Figure 4A) and heterotrimeric IL-2R (Figure 4B), whereas the affinity of the fusion protein containing the triple mutant IL-2 P65R-H16A-D84S ("IL-2-RAS", SEQ ID NO: 43) was even further attenuated (Figures 4A and 4B). Maximum binding and EC 50 The shift in binding observed for these IL-2 mutants in both the on-rate (EC 50 These results suggest that the antibody reduced the binding time (shift to the right) and off-rate (decrease in maximum binding).

[0280] Example 4. IL-2-RAS has reduced affinity for resting and pre-activated T cells To isolate T cells, non-T cell populations were labeled with biotinylated anti-lineage marker antibodies against CD14, CD16, CD19, CD20, CD36, CD56, CD123, and TCRγ / δ (BioLegend) for 20 min at room temperature. The non-T cell population was depleted by incubation with 100 × 10 IgG particles at room temperature for 20 min (500 μl of bead slurry plus 100 × 10 IgG). 6 (500 μl cell suspension per cell, incubate 2×8 min on magnet). The supernatant of unbound cells contained the isolated T cells.

[0281] A portion of isolated T cells (5.5 × 10 in 3 mL) 6 ) were activated by incubating in 6-well plates precoated with 1 μg / ml anti-CD3 OKT3 antibody (BD Biosciences) for 2 days, washed with PBS / 2% FBS, and then cultured at 2 × 10 6 The resting T cells or pre-activated T cells were used directly in the binding assay. Binding of a non-targeting VHH-Fc isotype control and a fusion protein comprising IL-2-RAS or wild type IL-2 fused to the C-terminus of a non-targeting VHH linked to a heterodimeric Fc to resting or pre-activated T cells was measured by flow cytometry essentially as described in Example 1, except that the following secondary antibodies were used: AF647 anti-human Fc (1:1000), PI (1:2000), BV785-CD4 (1:300), APC / Fire-CD8 (1:500) and PE / Cy7-CD25 (1:100).

[0282] A non-targeted IL-2-RAS fusion protein (comprising SEQ ID NO: 46) bound with reduced affinity to resting (FIG. 5A) and pre-activated (FIG. 5B) T cells compared to a fusion protein comprising a non-targeted VHH domain and wild-type IL-2 (comprising SEQ ID NO: 45). An isotype control without IL-2 did not bind to resting or pre-activated T cells as shown in FIG. 5A and FIG. 5B.

[0283] Example 5: IL-2-RAS has reduced affinity for Tregs Regulatory T cells ("Treg") have high endogenous expression of CD25, as well as CD122 and CD132, and are highly responsive to wild-type IL-2. Binding to Tregs of fusion proteins comprising wild-type IL-2 (comprising SEQ ID NO: 45) or the IL-2-RAS triple mutant (comprising SEQ ID NO: 46) fused to the C-terminus of the "knob" portion of the heterodimeric Fc (disulfide stabilized knob to hole) of a non-targeting VHH was measured.

[0284] EasySep Human CD4 + CD127 low CD25 + Treg and CD4 T cells were isolated using a regulatory T cell isolation kit (Stemcell) according to the manufacturer's instructions. + T responder cells (Tresp) were enriched and isolated from fresh healthy donor PBMCs. Tregs were isolated from naive CD4 T cells by culture for 7 days in ImmunoCult-XF T Cell Expansion Medium supplemented with rhTGF-B1, all-trans retinoic acid, CD3 / CD28 T Cell Activator, and IL-2. + Generated from T cells.

[0285] To distinguish the two cell populations, enriched Treg and CD4 + Responder T cells were labeled with the proliferation dyes CellTrace Violet (CTV) and CFSE, respectively, for 10 min at 37°C. After washing, Treg and CD4 +T cells were cultured at 1.5 × 10 in RPMI supplemented with 10% FBS and 1× antibiotic / antimycotic. 6 Tregs were resuspended at 10000 cells / ml. Tregs were seeded in a volume of 50 μl to obtain 75,000 Tregs / well in 96-well round-bottom plates. Tregs were incubated overnight at 37° C. in the presence of 10 nM IL-2-RAS and analyzed by flow cytometry as described in Example 1.

[0286] As shown in Figure 6, in contrast to fusion proteins containing wild-type IL-2, fusion proteins containing IL-2-RAS inhibited Tregs enriched from PBMCs (Figure 6A), induced Tregs (Figure 6B), or CD4 + It showed no observable binding to T-responders (Fig. 6C).

[0287] Example 6: IL-2-RAS reduced activity against resting T cells T cells were isolated by magnetic bead separation essentially as described in Example 4, labeled with CellTrace Violet (CTV) and treated with a fusion protein comprising wild type IL-2 (comprising SEQ ID NO: 45) or IL-2-RAS (comprising SEQ ID NO: 46) fused to the C-terminus of a non-targeting VHH linked to a heterodimeric Fc. CD4, CD8, CD71 and CTV levels were measured by flow cytometry. Proliferating T cells have reduced CTV levels.

[0288] As shown in Figures 7A and 7C, resting CD4 + T cells and CD8 + The concentration of IL-2-RAS containing fusion protein required to induce T cell proliferation was more than 100-fold higher than the concentration of wild-type IL-2 containing fusion protein or the concentration of IL-2v-analog containing fusion protein required to achieve the same induction of proliferation.

[0289] As shown in Fig. 7B and Fig. 7D, CD8 + T cells and CD4 +The concentration of IL-2-RAS containing fusion protein required to induce CD71 expression, a marker of T cell activation, on T cells was at least 100-fold higher than the concentration of wild-type IL-2 or IL-2v-analog containing fusion protein required to achieve the same induction of activation.

[0290] T cell activation can also be measured by phosphorylated STAT5 levels, which increase in activated T cells. T cells were isolated by magnetic bead separation and treated for 15 minutes with a fusion protein comprising wild type IL-2 (comprising SEQ ID NO: 45) fused to the C-terminus of a non-targeting VHH comprising a heterodimeric Fc or a fusion protein comprising IL-2-RAS (comprising SEQ ID NO: 46) fused to the C-terminus of a non-targeting VHH comprising a heterodimeric Fc. Cells were incubated with BD The cells were fixed with Cytofix / Cytoperm™ (BD Biosciences), permeabilized in 90% ice-cold methanol, and then the CD4 + T cells and CD8 + The levels of phosphorylated STAT5 ("pSTAT5") on T cells were measured using flow cytometry with anti-pSTAT5-PE antibody (1:70). Cells were incubated with the following antibodies: anti-CD3-FITC (1 :200), CD56-BV421 (1:100), CD4-BV785 (1:200), and CD8-APC-Fire (1:300).

[0291] As shown in Figures 7E and 7F, the nontargeted IL-2-RAS fusion protein did not inhibit resting CD4 + T cells and CD8 + Although only minimal phosphorylation of STAT5 was achieved in T cells, the non-targeted IL-2-wild-type fusion protein induced STAT5 phosphorylation at concentrations >1000-fold lower than the highest concentration tested.

[0292] Example 7: IL-2 mutants have reduced activity against Tregs EasySep(TM) Human CD4 + CD127low CD25 + Tregs were isolated from PBMCs using a regulatory T cell isolation kit (Stemcell). Tregs were labeled with CellTrace Violet and were cultured at 0.15 × 10 per well (96-well, U-bottom) in 100 μl of RPMI / 10% FBS. 6 Cells were plated with 100 μl of fusion protein titration starting at 100 nM. Cells were combined with 100 μl of fusion protein titration starting at 100 nM and titrated 1:4. Cells were incubated for 7 days. On day 7, proliferation and activation marker CD25 was measured by flow cytometry (Novocyte) essentially as described in Example 1, except that the following antibodies were used: BV785-CD4 (1:300), APC / Fire-CD8 (1:500), PE / Cy7-CD25 (1:100), PI (1:2000).

[0293] As shown in Figures 8A and 8B, a fusion protein comprising wild-type IL-2 (comprising SEQ ID NO: 45) fused to the C-terminus of a non-targeting VHH linked to a heterodimeric Fc induced Treg proliferation and expression of the activation marker CD25, but a fusion protein comprising IL-2-RAS instead of wild-type IL-2 (comprising SEQ ID NO: 46) did not.

[0294] Example 8: Activated T cells expressing PD-1 are stimulated by PD-1-targeted IL-2-RAS The ability to bind and stimulate PD-1-expressing T cells was tested using pembrolizumab (a conventional anti-PD-1 antibody) and a fusion protein containing a pembrolizumab analog and IL-2-RAS linked to the C-terminus of the heavy chain (see FIG. 1F).

[0295] Enriched T cells from healthy donors were activated essentially as described in Example 4. Six-well plates were coated with 1 μg / ml OKT3 antibody overnight at 4° C. The next day, plates were washed twice to remove unbound OKT3 antibody. Enriched T cells were thawed with CTL medium and cultured at 5.5×10 6The T cells were resuspended at 10 cells / mL and seeded at 3 mL per well on the coated plates. After 2 days, the activated T cells were harvested, washed once, and then seeded in medium without OKT3 antibody for 24 hours. The cells were labeled with the proliferation dye CellTrace™ Violet (CTV). The T cells were counted and then cultured at 2×10 6 Cells were resuspended at 100 μL / mL. 100 μL of resuspended cells were seeded per well in a 96-well round bottom plate. Pembrolizumab or pembrolizumab analog-IL-2-RAS fusion was added starting at a final concentration of 100 nM and titrated 1:5. On day 3, T cells were stained with the viability marker PI and the following fluorescently labeled antibodies: CD4-BV785, CD8-APC / Fire, CD25-PE / Cy7, CD71-FITC, and CD69-APC for 20 minutes at room temperature. Plates were read on a Novocyte flow cytometer essentially as described in Example 7 for measuring proliferation and as in Example 1 for binding, and data were exported to Excel for further analysis.

[0296] As shown in Figure 9, the pembrolizumab analog-IL-2-RAS fusion protein , CD8 + T cell proliferation (Figure 9A) and CD4 + 9C and 9D, pembrolizumab and pembrolizumab analog-IL-2-RAS both stimulated PD-1-targeted T cell proliferation (Figure 9B), whereas pembrolizumab alone did not. Without intending to be bound by a particular theory, the biphasic nature of the observed proliferation may suggest that the activity at low concentrations is due to PD-1-targeted activity, and the increased activity at higher concentrations is due to non-targeted activity. As shown in Figures 9C and 9D, pembrolizumab and pembrolizumab analog-IL-2-RAS both stimulated PD-1-targeted T cell proliferation (Figure 9B), whereas pembrolizumab alone did not. + T cells and CD4 + IL-2-RAS bound to T cells, although additional binding was observed for the fusion protein containing IL-2-RAS at the higher end of the dilution range above 10 nM, which may have been mediated by binding of IL-2-RAS to IL-2R.

[0297] Example 9: Preblocking PD-1 on activated T cells prevents signaling by PD-1-targeted IL-2-RAS T cells were isolated and enriched from healthy donors by magnetic bead separation and incubated on OKT3 antibody-coated plates to activate the T cells, substantially as described in Example 4. The cells were labeled with CTV. Pre-activated T cells were incubated with pembrolizumab, an anti-PD-1 antibody, to block PD-1 binding sites, or with a non-targeting antibody as a control. The cells were then incubated for 3 days with a fusion protein comprising IL-2-RAS fused to a pembrolizumab analog, or with a non-targeting antibody as a control. The extent of IL-2 signaling was measured using CD4+ / -1 as a control, substantially as described in Example 7. + T cells and CD8 + T cell proliferation was assessed by flow cytometry.

[0298] As shown in Figures 10A to 10D, wild-type IL-2 inhibited CD8 + T cells and CD4 + Treatment with pembrolizumab or a fusion protein containing IL-2-RAS and a nontargeting antibody induced robust proliferation of both CD4 T cells. + T cells and CD8 + T cells showed low levels of proliferation that were not affected by pre-blocking of PD-1. In contrast, CD4 T cells treated with a fusion protein containing IL-2-RAS and a pembrolizumab analog showed + T cells (Fig. 10B and Fig. 10D) and CD8 + Both T cells (Figures 10A and 10C) showed significant PD-1-dependent proliferation (Figures 10A and 10B), which was blocked by preincubation with anti-PD-1 antibody (Figures 10C and 10D). Thus, the fusion protein containing IL-2-RAS and anti-PD-1 antibody activated T cells only when PD-1 was expressed and accessible on the T cells.

[0299] Example 10: PD-1-targeted IL-2-RAS overcomes Treg suppression CD4 + T responder cells and Tregs were isolated as described in Example 5. CD4 + Responder cells were labeled with CTV, mixed with isolated Tregs at a 2:1 ratio, and activated with anti-CD3 beads (one bead per two T cells). The resulting mixtures were treated for 7 days with a dilution series of wild-type IL-2 fused to the C-terminus of a non-targeting VHH as shown in FIG. 1B, a fusion protein comprising IL-2-RAS fused to the C-terminus of a non-targeting VHH as shown in FIG. 1B, or a fusion protein comprising IL-2-RAS fused to an anti-PD-1 antibody (pembrolizumab analog-IL-2-RAS). Proliferation was measured by flow cytometry essentially as described in Example 7.

[0300] As shown in FIG. 11, T responder cells were suppressed by Tregs, whereas non-targeted wild-type IL-2 and a fusion protein containing IL-2-RAS and an anti-PD-1 antibody (pembrolizumab analog-IL-2-RAS) suppressed CD4+ T cells regardless of the presence of Tregs. + IL-2-RAS induced proliferation of T-responder cells. Treatment of the cells with a fusion protein containing IL-2-RAS and a non-targeting antibody did not restore proliferation to the same extent. Only non-targeting IL-2-RAS was able to induce proliferation at much higher concentrations than the PD-1-targeting IL-2-RAS fusion protein. was able to negate the suppressive effects of Tregs on T responders. Thus, PD-1-targeted IL-2-RAS overcame the suppressive effects of Tregs, and this activity was dependent on binding to PD-1 expressed on T cells.

[0301] Example 11: PD-1-targeted IL-2-RAS does not signal in trans Coat the beads at 4 x 10 x 100 µL per the manufacturer's recommended coating procedure. 8The beads are coated with 200 μg of PD-1 antigen per 10 beads. Briefly, the beads are washed once in buffer 1 (0.1 M sodium phosphate buffer, pH 7.4-8.0) and then incubated in buffer 1 containing PD-1 antigen for 18 h at room temperature on a tube rotator. The beads are then washed four times with buffer 2 (PBS, 0.1% BSA, 2 mM EDTA pH 7.4). Free tosyl groups are inactivated by incubating the beads in buffer 3 (0.2 M Tris, 0.1% BSA, pH 8.5) for 4 h at 37 °C. The beads are then washed once in buffer 2 and incubated at 37 °C ...3 and incubated at 37 °C for 4 h at 37 °C. 6 Resuspend to a concentration of 10 beads / mL.

[0302] The coated beads are incubated with wild type IL-2 or a fusion protein containing IL-2-RAS fused to an anti-PD-1 antibody and washed. The beads are then incubated with isolated resting T cells. IL-2 signaling is assessed by measuring pSTAT5 levels by flow cytometry.

[0303] A fusion protein containing wild-type IL-2 bound to beads was identified as CD8 + T cells and CD4 + Although it potently activates T cells, a fusion protein containing IL-2-RAS bound to beads did not activate CD4 + T cells or CD8 + It has no activity against T cells. Thus, T cell targeting of IL-2-RAS is required for IL-2 signaling, and signaling of targeted IL-2-RAS does not occur in trans.

[0304] Example 12: IL-2-RAS does not signal in trans A dilution series of non-targeted wild-type IL-2 and non-targeted IL-2-RAS, diluted 1:4 starting at 1000 nM, was coated onto assay plates, incubated overnight, and washed. T cells were added and incubated for 30 minutes at 37°C. CD8 +T cells and CD4 + T cell activation was measured by detecting phosphorylated STAT5 levels essentially as described in Example 6.

[0305] As shown in Figures 12A and 12B, CD8 + T cells and CD4 + T cells were trans-activated by wild-type IL-2, as measured by pSTAT5 induction, but untargeted IL-2-RAS failed to activate them in trans. Without intending to be bound by any particular theory, the reduced affinity of IL-2-RAS for CD25 and CD122 may have prevented efficient binding and clustering of IL-2R, which induces downstream signaling. Thus, only targeted IL-2-RAS fusion proteins trigger pSTAT5 signaling.

[0306] Example 13: NKp46-targeted IL-2-RAS specifically induces NK cell proliferation Fusion proteins comprising IL-2-RAS fused to the C-terminus of a heterodimeric scFv antibody targeting NKp46 as shown in FIG. 1H, fusion proteins comprising wild-type IL-2 or IL-2-RAS fused to the C-terminus of a non-targeting VHH linked to a heterodimeric Fc as shown in FIG. 1B, and NK cells, CD4 + T cells and CD8 + The effect on T cells was determined.

[0307] Fresh PBMCs from healthy donors were labeled with CellTrace™ Violet and seeded at 200,000 cells / well in round-bottom 96-well plates. Dilutions of fusion proteins and NKp46 scFv-Fc control were added to the plated cells and incubated for 7 days at 37° C. On day 7, cell proliferation was measured essentially as described in Example 7, except that the following antibodies were used: anti-CD3-BV785 (1:200), anti-CD56-APC (1:100), anti-CD4-PE (1:200), anti-CD8-APC-Fire (1:300) and PI (1:2000).

[0308] In addition, fresh PBMCs from healthy donors were treated with the same fusion proteins or NKp46 scFv-Fc control and incubated for 15 min at 37°C. + T cells, CD4 + T cells and NK cells (CD3 - , CD56 + ) was measured by detecting phosphorylated STAT5 levels essentially as described in Example 6.

[0309] Binding of the fusion proteins and NKp46 scFV-Fc control to fresh PBMC from healthy donors was measured essentially as described in Example 1, except that the following antibodies were used: anti-CD3-FITC (1:100), anti-CD56-BV421 (1:100), anti-CD4-BV785 (1:200), anti-CD8-APC-Fire (1:300), anti-human IgG-Alexa Fluor 647 (1:500), and PI (1:2000).

[0310] As shown in Figures 13A-I, NKp46-targeted IL-2-RAS potently stimulated NK cell proliferation and activation, but not CD4 + T cells or CD8 + In contrast, non-targeted wild-type IL-2 had no effect on T cells. + T cells and CD8 +NKp46 scFV-Fc (without IL-2-RAS) binding did not induce NK proliferation or pSTAT5 induction. Thus, NKp46-targeted IL-2-RAS triggered cis-signaling of IL-2 on NK cells but not CD4 + T cells or CD8 + T cells were not activated in trans.

[0311] Example 14: LAG3-targeted IL-2-RAS inhibits pre-activated LAG3 + Stimulates T cells 1G, a fusion protein comprising IL-2-RAS fused to the C-terminus of a conventional anti-LAG3 heterodimeric antibody (MAb), a fusion protein comprising IL-2-RAS fused to an anti-LAG3 VHH comprising a heterodimeric Fc, as shown in FIG. 1B, a fusion protein comprising IL-2-RAS fused to a non-targeting VHH, as shown in FIG. 1B, or a fusion protein comprising wild-type IL-2 fused to the C-terminus of a non-targeting heterodimeric Fc, as shown in FIG. 1B, or a fusion protein comprising a LAG3-targeting MAb (control), or a LAG3-targeting VHH-Fc (control) + T cells and CD8 + The effect on T cells was assayed.

[0312] Enriched T cells from healthy donors were stimulated with coated 1 μg / mL anti-CD3 (OKT3) and 10 μg / mL soluble anti-CD28 for 48 hours, followed by resting for 24 hours. Pre-activated cells were labeled with CellTrace™ Violet and seeded at 200,000 cells / well. Dilutions of fusion proteins and control proteins were added and incubated for 3 days. Proliferation and expression of activation markers CD25 and CD71 were measured essentially as in Example 7, but with additional antibodies: anti-CD25-FITC (1:100) and anti-CD71-PE / Cy7.

[0313] Stimulated CD8 + T cells express LAG3 in CD8 +Upregulation of up to 45% of T cells and CD4 + T cells express LAG3 in CD4 + In contrast, unstimulated T cells upregulated CD8 + T cells or CD4 + Nearly 0% of T cells are positive for LAG3 expression.

[0314] As shown in Figures 14A-14D, both anti-LAG3 Mab-IL-2-RAS and anti-LAG3 VHH-IL-2-RAS inhibited CD8+ / -IL-2-RAS, as indicated by the expression levels of CD25 (Figures 14C and 14D) and CD71 (Figures 14E and 14F). + and CD4 + Non-targeted wild-type IL-2 increased the proliferation (Figures 14A and 14B) and activation of CD8 + T cells and CD4 + It was a potent inducer of T cell proliferation and activation, and bound with high affinity and saturation to stimulated T cells.

[0315] Example 15: Combinations of IL-2 mutants further reduce off-target activity HEK-Blue IL-2 reporter cells (InvivoGen) were used to express non-targeted I The relative activity of the IL-2 mutants was measured: reporter cells were treated with dilutions of IL-2 mutants fused to the C-terminus of a non-targeting VHH and incubated for 20 h before Quanti-Blue analysis.

[0316] As shown in Figure 15, the IL-2 mutants exhibited a wide range of activities. The above experiments showed that IL-2-RAS (P65R, H16A, and D84S) dramatically reduced binding to IL-2R compared to wild-type IL-2 (see Figures 4-6) and reduced activity compared to wild-type IL-2 (see Figures 7A-7E). IL-2-RAS with an additional M23A mutation and IL-2-RAS with an additional E95Q mutation both showed reduced activity compared to IL-2-RAS, and the combination of IL-2-RAS with both M23A and E95Q attenuated activity even further. Experiments using PD-1-expressing reporter cells showed that all of these affinity-reduced IL-2 mutants exhibited equivalent PD-1 targeting activity (data not shown), suggesting that high affinity binding in cis to PD-1 may offset the reduced affinity of the IL-2 mutants to IL-2R. Although the HEK-Blue IL-2 reporter system was useful for measuring relative activity, the EC values ​​observed for the IL-2 mutants in the reporter system were 50 was significantly left-shifted compared to primary lymphocytes, presumably due to overexpression of IL-2R components in the reporter cells compared to lower IL-2R levels on primary cells.

[0317] The present disclosure may be embodied in other specific forms without departing from the spirit or essential characteristics of the present invention. The above-described embodiments are therefore to be considered in all respects as illustrative and not limiting of the present disclosure. The scope of the present disclosure is therefore defined by the appended claims, rather than the above detailed description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

[0318] [Table 2] TIFF2025013911000004.tif254170TIFF2025013911000005.tif254170TIFF2025013911000006.t if254170TIFF2025013911000007.tif254170TIFF2025013911000008.tif254170TIFF20250139110 00009.tif254170TIFF2025013911000010.tif253170TIFF2025013911000011.tif252170TIFF202 5013911000012.tif254170TIFF2025013911000013.tif254170TIFF2025013911000014.tif105170

[0319] In sequences with boxes or underlines, boxes around individual letters indicate amino acid substitutions relative to the corresponding wild-type or parent sequence, and boxes around groups of letters indicate linker sequences. Underlined letters indicate linker sequences.

Claims

1. A polypeptide comprising a modified IL-2, said modified IL-2 comprising at least one substitution at at least one amino acid position selected from P65, D84, E95, M23, and H16.

2. The polypeptide of claim 1, wherein the modified IL-2 is modified human IL-2.

3. The polypeptide of claim 1 or 2, wherein the amino acid positions correspond to the amino acid positions in SEQ ID NO:

1.

4. The polypeptide of any one of claims 1 to 3, wherein the modified IL-2 comprises a substitution at amino acid position P65.

5. 5. The polypeptide of claim 4, wherein the substitution is selected from P65R, P65E, P65K, P65H, P65Y, P65Q, P65D, and P65N.

6. The polypeptide of any one of claims 1 to 5, wherein the modified IL-2 comprises a substitution at amino acid position H16.

7. 7. The polypeptide of claim 6, wherein the substitution is selected from H16A, H16G, H16S, H16T, H16V, and H16P.

8. The polypeptide of any one of claims 1 to 7, wherein the modified IL-2 comprises a substitution at amino acid position D84.

9. 9. The polypeptide of claim 8, wherein the substitutions are selected from D84S, D84G, D84A, D84T, D84V, and D84P.

10. The polypeptide of any one of claims 1 to 9, wherein the modified IL-2 comprises substitutions at amino acid positions P65, H16, and D84.

11. The polypeptide of claim 10, wherein the modified IL-2 comprises the substitutions P65R, H16A, and D84S.

12. The polypeptide of any one of claims 1 to 11, wherein the modified IL-2 comprises a substitution at amino acid position M23.

13. 13. The polypeptide of claim 12, wherein the substitution is selected from M23A, M23G, M23S, M23T, M23V, and M23P.

14. The polypeptide of claim 13, wherein the modified IL-2 comprises the substitutions P65R, H16A, D84S, and M23A.

15. The polypeptide of any one of claims 1 to 14, wherein the modified IL-2 comprises a substitution at amino acid position E95.

16. 16. The polypeptide of claim 15, wherein the substitution is selected from E95Q, E95G, E95S, E95T, E95V, E95P, E95H, and E95N.

17. 17. The polypeptide of claim 16, wherein the modified IL-2 comprises the substitutions P65R, H16A, D84S, and E95Q.

18. 18. The polypeptide of claim 17, wherein the modified IL-2 comprises the substitutions P65R, H16A, D84S, M23A, and E95Q.

19. The polypeptide of any one of claims 1 to 18, wherein the modified IL-2 comprises a substitution at amino acid position F42.

20. 20. The polypeptide of claim 19, wherein the substitution at F42 is selected from F42K, F42A, F42R, F42A, F42G, F42S, and F42T.

21. The polypeptide of any one of claims 1 to 20, wherein the modified IL-2 comprises at least one substitution at at least one amino acid position selected from Y45 and L72.

22. The polypeptide of claim 21, wherein the modified IL-2 comprises at least one substitution selected from Y45A and L72G.

23. The polypeptide of any one of claims 1 to 22, wherein the modified IL-2 comprises at least one substitution at at least one amino acid position selected from T3 and C125.

24. The polypeptide of claim 23, wherein the modified IL-2 comprises at least one substitution selected from T3A and C125A.

25. The modified IL-2 includes H16A-F42K, D84S-F42K, E15S-F42K, M23A-F42K, E95Q-F42K, P65R-H16A, P65R-D84S, P65R-E15S, P65R-M23A, P65R-E95Q, T3A-C125S, T3A-P65R-C125S, T3A-H16A-C125S, T3A-D84S-C125S, T3A-H16A-P65R-C125 25. The polypeptide of any one of claims 1 to 24, comprising a set of substitutions selected from: T3A-P65R-D84S-C125S, T3A-H16A-P65R-D84S-C125S, T3A-H16A-M23A-P65R-D84S-C125S, T3A-H16A-P65R-D84S-E95Q-C125S, and T3A-H16A-M23A-P65R-D84S-E95Q-C125S.

26. 26. The polypeptide of claim 25, wherein the modified IL-2 comprises the series of substitutions and does not include any additional substitutions.

27. 27. The polypeptide of any one of claims 1 to 26, wherein the modified IL-2 comprises an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:

84.

28. 28. The polypeptide of any one of claims 1 to 27, wherein the modified IL-2 comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from SEQ ID NOs: 3 to 9, 11 to 21, and 23 to 31.

29. The polypeptide of any one of claims 1 to 28, wherein the modified IL-2 comprises an amino acid sequence selected from SEQ ID NO:3 to SEQ ID NO:9, SEQ ID NO:11 to SEQ ID NO:21, and SEQ ID NO:23 to SEQ ID NO:

31.

30. The polypeptide of any one of claims 1 to 29, wherein the polypeptide comprises an Fc region.

31. The polypeptide of claim 30, wherein the modified IL-2 is fused to the N-terminus or C-terminus of the Fc region.

32. 32. The polypeptide of claim 30 or 31, wherein the Fc region comprises a substitution at Kabat amino acid position T366.

33. 33. The polypeptide of claim 32, wherein the Fc region comprises a T366W substitution.

34. 32. The polypeptide of claim 31 , wherein the Fc region comprises at least one substitution at at least one Kabat amino acid position selected from T366, L368, and Y407.

35. 35. The polypeptide of claim 34, wherein the Fc region comprises T366S, L368A, and Y407V mutations.

36. 36. The polypeptide of any one of claims 30 to 35, wherein the Fc region comprises a substitution at a Kabat position selected from S354 and Y349.

37. 37. The polypeptide of claim 36, wherein the Fc region comprises a S354C or Y349C substitution.

38. 38. The polypeptide of any one of claims 30-37, wherein the Fc region comprises a substitution at Kabat amino acid position H435.

39. 39. The polypeptide of claim 38, wherein the Fc region comprises a substitution selected from H435R and H435K.

40. 40. The polypeptide of any one of claims 30-39, wherein the Fc region comprises at least one substitution at at least one Kabat amino acid position selected from M252 and M428.

41. 41. The polypeptide of claim 40, wherein the Fc region comprises M252Y and M428V substitutions.

42. 42. The polypeptide of any one of claims 30-41, wherein the Fc region comprises a deletion of Kabat amino acids E233, L234, and L235.

43. The polypeptide of any one of claims 30 to 41, wherein the Fc region comprises at least one substitution at at least one amino acid position selected from L234, L235, and P329.

44. 44. The polypeptide of claim 43, wherein the Fc region comprises L234A, L235A, and P329G substitutions.

45. The Fc region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of an amino acid sequence selected from SEQ ID NO:47 to SEQ ID NO:

83.

45. The polypeptide of any one of claims 30 to 44, comprising an amino acid sequence identical thereto or 100% identical thereto.

46. The polypeptide of any one of claims 30 to 44, wherein the Fc region is a portion of a heavy chain constant region.

47. 47. The polypeptide of claim 46, wherein the heavy chain constant region is an IgG constant region.

48. 48. The polypeptide of claim 47, wherein the heavy chain constant region is an IgG1, IgG2, IgG3, or IgG4 constant region.

49. The polypeptide of any one of claims 30 to 48, wherein the modified IL-2 is fused to the C-terminus of the Fc region or heavy chain constant region.

50. 50. The polypeptide of claim 49, wherein the modified IL-2 is fused to the C-terminus of the Fc region or heavy chain constant region by a linker comprising 1 to 20 amino acids.

51. 51. The polypeptide of claim 50, wherein the linker comprises a glycine amino acid.

52. 52. The polypeptide of claim 51 , wherein the linker comprises glycine and serine amino acids.

53. 53. The polypeptide of any one of claims 50 to 52, wherein most or all of the amino acids in the linker are glycine and serine.

54. 54. The polypeptide of any one of claims 30-33, 42, and 49-53, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:102, SEQ ID NO:103, or SEQ ID NO:

104.

55. The polypeptide of any one of claims 1 to 54, wherein the polypeptide comprises at least one antigen-binding domain.

56. 56. The polypeptide of claim 55, wherein the polypeptide comprises two, three, or four antigen-binding domains.

57. 57. The polypeptide of claim 55 or 56, wherein at least one antigen-binding domain specifically binds to a T cell antigen or a natural killer cell antigen.

58. At least one antigen-binding domain is a CD4 ＋ T cell antigen or CD8 ＋ 58. A polypeptide according to any one of claims 55 to 57, which specifically binds to a T cell antigen.

59. The at least one antigen-binding domain is an antigen-binding domain that binds to an activated CD4 ＋ T cells or activated CD8 ＋ 59. The polypeptide of claim 58, which specifically binds to an antigen on a T cell.

60. 60. The polypeptide of any one of claims 55 to 59, wherein at least one antigen-binding domain is agonistic.

61. The polypeptide of any one of claims 55 to 59, wherein the antigen-binding domain is an antagonist.

62. 62. The polypeptide of any one of claims 55-61, wherein at least one antigen-binding domain specifically binds to PD-1, CTLA-4, LAG3, TIM3, 4-1BB, OX40, GITR, CD8a, CD8b, CD4, NKp30, NKG2A, TIGIT, TGFβR1, TGFβR2, Fas, NKG2D, NKp46, PD-L1, CD107a, ICOS, TNFR2, or CD16a.

63. 63. The polypeptide of any one of claims 55 to 62, wherein at least one antigen-binding domain specifically binds to PD-1.

64. 64. The polypeptide of any one of claims 55 to 63, wherein at least one antigen-binding domain is a human or humanized antigen-binding domain.

65. 65. The polypeptide of claim 64, wherein each antigen-binding domain is independently a human or humanized antigen-binding domain.

66. A polypeptide according to any one of claims 55 to 65, wherein at least one antigen-binding domain comprises a VHH domain.

67. 67. The polypeptide of claim 66, wherein each antigen-binding domain comprises a VHH domain.

68. 66. A polypeptide according to any one of claims 55 to 65, wherein at least one antigen-binding domain comprises a VH domain and a VL domain.

69. 69. The polypeptide of claim 68, wherein at least one antigen-binding domain comprises the VH and VL domains of an antibody selected from pembrolizumab, nivolumab, AMP-514, TSR-042, STI-A1110, ipilimumab, tremelimumab, urelumab, utomilumab, atezolizumab, and durvalumab.

70. 70. The polypeptide of claim 68 or 69, wherein the at least one antigen-binding domain comprises a single chain Fv (scFv).

71. 70. The polypeptide of claim 68 or 69, wherein the polypeptide comprises a heavy chain constant region, the VH domain is fused to the heavy chain constant region, and the VL domain is associated with the VH domain.

72. 72. The polypeptide of claim 71, wherein the VL domain is fused to a light chain constant region.

73. 73. The polypeptide of claim 72, wherein the light chain constant region is selected from kappa and lambda.

74. A polypeptide according to any one of claims 55 to 73, wherein each of the antigen-binding domains is the same.

75. A polypeptide according to any one of claims 55 to 74, wherein each of the antigen-binding domains specifically binds to the same antigen.

76. 74. The polypeptide of any one of claims 55 to 73, wherein at least one of the antigen-binding domains specifically binds to a different antigen than at least one of the other antigen-binding domains.

77. The polypeptide of any one of claims 55 to 73, wherein at least one antigen-binding domain specifically binds to PD-1, and at least one other antigen-binding domain specifically binds to a T cell antigen or a natural killer cell antigen other than PD-1.

78. 78. The polypeptide of any one of claims 55-77, wherein at least one antigen-binding domain binds to PD-1, CTLA-4, LAG3, TIM3, 4-1BB, OX40, GITR, CD8a, CD8b, CD4, NKp30, NKG2A, TIGIT, TGFβR1, TGFβR2, Fas, NKG2D, NKp46, PD-L1, CD107a, ICOS, TNFR2, or CD16a.

79. The polypeptide of any one of claims 31 to 78, wherein the polypeptide forms a homodimer under physiological conditions.

80. 80. The polypeptide of any one of claims 1 to 79, wherein the modified IL-2 binds to human IL-2R with an affinity that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 50-fold, or at least 100-fold less than the affinity of human wild-type IL-2 for IL-2R.

81. A complex comprising a first polypeptide and a second polypeptide, wherein the first polypeptide is a polypeptide according to any one of claims 1 to 79.

82. 82. The complex of claim 81 , wherein the first polypeptide comprises a first Fc region and the second polypeptide comprises a second Fc region.

83. 83. The conjugate of claim 81 or 82, wherein each Fc region is an isotype selected from human IgG1, IgG2, IgG3, and IgG4.

84. 84. The conjugate of claim 83, wherein each Fc region is human IgG1.

85. 85. The conjugate of any one of claims 81 to 84, wherein each Fc region comprises a deletion of amino acids E233, L234, and L235.

86. A conjugate according to any one of claims 81 to 85, wherein each Fc region comprises a H435R or H435K mutation.

87. The conjugate according to any one of claims 81 to 86, wherein the Fc region comprises the mutations M252Y and M428L or the mutations M252Y and M428V.

88. 88. The conjugate of any one of claims 81 to 87, wherein the first Fc region or the second Fc region comprises a T366W mutation and the other Fc region comprises the mutations T366S, L368A, and Y407V.

89. 89. The conjugate of claim 88, wherein the first Fc region or the second Fc region comprises an S354C mutation.

90. 90. The conjugate of any one of claims 81-89, wherein each Fc region comprises an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence independently selected from SEQ ID NO:47-SEQ ID NO:

83.

91. The complex of any one of claims 81 to 90, wherein the second polypeptide does not comprise modified IL2.

92. The complex of any one of claims 81 to 91, wherein the first polypeptide comprises at least one antigen-binding domain.

93. The complex of any one of claims 81 to 92, wherein the second polypeptide comprises at least one antigen-binding domain.

94. The complex of any one of claims 81 to 93, wherein the first polypeptide comprises a first antigen-binding domain, an Fc region, and a modified IL-2.

95. The complex of claim 94, wherein the first antigen-binding domain is fused to the N-terminus of the Fc region and the modified IL-2 is fused to the C-terminus of the Fc region.

96. 96. The complex of claim 94 or 95, wherein the second polypeptide comprises a second antigen binding domain and an Fc region.

97. 97. The complex of claim 96, wherein the first antigen-binding domain and the second antigen-binding domain are the same or different.

98. a) the first antigen-binding domain and the second antigen-binding domain both bind to PD-1; b) the first antigen-binding domain binds to PD-1 and the second antigen-binding domain binds to LAG3; c) the first antigen-binding domain binds to PD-1 and the second antigen-binding domain binds to CTLA-4; d) the first antigen-binding domain binds to PD-1 and the second antigen-binding domain binds to 4-1BB; e) the first antigen-binding domain binds to PD-1 and the second antigen-binding domain binds to OX40; f) the first antigen-binding domain binds to PD-1 and the second antigen-binding domain binds to GITR; g) the first antigen-binding domain binds to PD-1 and the second antigen-binding domain binds to CD8a; h) the first antigen-binding domain binds to PD-1 and the second antigen-binding domain binds to CD8b; i) the first antigen-binding domain binds to PD-1 and the second antigen-binding domain binds to CD4; j) the first antigen-binding domain binds to PD-1 and the second antigen-binding domain binds to NKp30; k) the first antigen-binding domain binds to PD-1 and the second antigen-binding domain binds to NKG2A; l) the first antigen-binding domain binds to PD-1 and the second antigen-binding domain binds to TIGIT; m) the first antigen-binding domain binds to PD-1 and the second antigen-binding domain binds to NKG2D; n) the first antigen-binding domain binds to PD-1 and the second antigen-binding domain binds to TGFBR2; o) the first antigen-binding domain binds to PD-1 and the second antigen-binding domain binds to Fas; p) the first antigen-binding domain binds to PD-1 and the second antigen-binding domain binds to CD107a; q) the first antigen-binding domain binds to PD-1 and the second antigen-binding domain binds to NKp46; r) the first antigen-binding domain binds to CD8a and the second antigen-binding domain binds to TGFRβR2; s) the first antigen-binding domain binds to CD8a and the second antigen-binding domain binds to Fas; t) the first antigen-binding domain binds to NKG2D and the second antigen-binding domain binds to TGFRβR2; u) the first antigen-binding domain binds to NKG2D and the second antigen-binding domain binds to Fas; v) the first antigen-binding domain binds to NKG2A and the second antigen-binding domain binds to TGFRβR2; w) the first antigen-binding domain binds to NKG2A and the second antigen-binding domain binds to Fas; x) the first antigen-binding domain binds to NKp46 and the second antigen-binding domain binds to TGFRβR2; y) the first antigen-binding domain binds to NKp46 and the second antigen-binding domain binds to Fas; z) the first antigen-binding domain binds to CTLA-4 and the second antigen-binding domain binds to LAG3; aa) the first antigen-binding domain binds to CTLA-4 and the second antigen-binding domain binds to Tim3; bb) the first antigen-binding domain binds to CTLA-4 and the second antigen-binding domain binds to OX40; cc) the first antigen-binding domain binds to CTLA-4 and the second antigen-binding domain binds to GITR; dd) the first antigen-binding domain binds to CTLA-4 and the second antigen-binding domain binds to CD107a; ee) the first antigen-binding domain binds to CTLA-4 and the second antigen-binding domain binds to NKp46; or ff) the first antigen-binding domain binds to ICOS and the second antigen-binding domain binds to TNFR2.

99. 99. The conjugate of any one of claims 81 to 98, wherein the modified IL-2 binds to human IL-2R with an affinity that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 50-fold, or at least 100-fold less than the affinity of human wild-type IL-2 for IL-2R.

100. A pharmaceutical composition comprising a polypeptide according to any one of claims 1 to 80 or a complex according to any one of claims 81 to 99, and a pharma- ceutically acceptable carrier.

101. 99. An isolated nucleic acid encoding a polypeptide according to any one of claims 1 to 80 or a complex according to any one of claims 81 to 99.

102. An expression vector comprising the nucleic acid of claim 101.

103. 103. An isolated host cell comprising the nucleic acid of claim 101 or the expression vector of claim 102.

104. An isolated host cell expressing a polypeptide according to any one of claims 1 to 80 or a complex according to any one of claims 81 to 99.

105. 103. A method for producing a polypeptide according to any one of claims 1 to 80 or a complex according to any one of claims 81 to 99, comprising incubating a host cell according to claim 103 or 104 under conditions suitable for expression of said polypeptide or complex.

106. 106. The method of claim 105, further comprising isolating the polypeptide or complex.

107. CD4 ＋ T cells and / or CD8 ＋ 100. A method for increasing proliferation of T cells, comprising contacting T cells with a polypeptide according to any one of claims 1 to 80 or a complex according to any one of claims 81 to 99.

108. The CD4 ＋ T cells and / or CD8 ＋ The method of claim 107, wherein the T cell is present in vitro.

109. The CD4 ＋ T cells and / or CD8 ＋ The method of claim 107, wherein the T cell is present in vivo.

110. 110. The method of any one of claims 107 to 109, wherein the increase is at least 1.5 fold, at least 2 fold, at least 3 fold, or at least 5 fold.

111. 100. A method for increasing NK cell proliferation, comprising contacting NK cells with a polypeptide according to any one of claims 1 to 80 or a complex according to any one of claims 81 to 99.

112. 112. The method of claim 111, wherein the increase is at least 1.5-fold, at least 2-fold, at least 3-fold, or at least 5-fold.

113. A method for treating cancer, comprising administering to a subject with cancer a pharma- ceutical effective amount of a polypeptide according to any one of claims 1 to 80, or a complex according to any one of claims 81 to 99, or a pharmaceutical composition according to claim 100.

114. The cancers include basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and central nervous system cancer, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, colorectal cancer, connective tissue cancer, digestive system cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, stomach cancer, gastrointestinal cancer, glioblastoma, liver cancer, hepatocellular carcinoma, intraepithelial neoplasia, kidney cancer or renal cancer, laryngeal cancer, liver cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, melanoma, myeloma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, respiratory system cancer, salivary gland cancer, sarcoma, skin cancer, squamous cell carcinoma, stomach cancer, testicular cancer, thyroid cancer, uterine or endometrial cancer, urinary system cancer, vulvar cancer, 114. The method of claim 113, wherein the lymphoma is selected from lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, B-cell lymphoma, low-grade / follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-dividing cell NHL, bulky disease NHL, mantle cell lymphoma, AIDS-related lymphoma, Waldenstrom's macroglobulinemia, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, and chronic myeloblastic leukemia.

115. 115. The method of claim 113 or 114, further comprising administering an additional therapeutic agent.

116. 116. The method of claim 115, wherein the additional therapeutic agent is an anti-cancer agent.

117. 117. The method of claim 116, wherein the anti-cancer agent is selected from a chemotherapeutic agent, an anti-cancer biologic, a radiotherapeutic agent, a CAR-T therapeutic agent, and an oncolytic virus.

118. 118. The method of claim 116 or 117, wherein the additional therapeutic agent is an anti-cancer biologic.

119. The method of claim 118, wherein the anti-cancer biologic is an agent that inhibits PD-1 and / or PD-L1.

120. The method of claim 118, wherein the anti-cancer biologic is an agent that inhibits VISTA, gpNMB, B7H3, B7H4, HHLA2, CTLA4, or TIGIT.

121. The method of any one of claims 116 to 120, wherein the anti-cancer agent is an antibody.

122. 119. The method of claim 118, wherein the anti-cancer biologic is a cytokine.

123. The method of claim 116, wherein the anti-cancer agent is a CAR-T therapeutic agent.

124. 117. The method of claim 116, wherein the anti-cancer agent is an oncolytic virus.

125. 125. The method of any one of claims 113 to 124, further comprising tumor resection and / or radiation therapy.

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