Polypeptides Comprising Modified IL-2 Polypeptides and Uses Thereof

1 . A polypeptide comprising a modified IL-2, wherein the modified IL-2 comprises a D84Y substitution.

2 . The polypeptide of claim 1 , wherein the modified IL-2 has reduced affinity for CD122 compared to wild-type IL-2.

3 . The polypeptide of any one of claims 1 - 2 , wherein the modified IL-2 comprises at least one substitution at at least one amino acid position selected from H16, L19, M23, N88, and E95.

4 . The polypeptide of claim 3 , wherein the modified IL-2 comprises a substitution at amino acid position H16.

5 . The polypeptide of claim 4 , wherein the substitution is selected from H16A H16N, H16V, and H16T.

6 . The polypeptide of any one of claims 1 - 5 , wherein the modified IL-2 comprises a substitution at amino acid position L19.

7 . The polypeptide of claim 6 , wherein the substitution is selected from L19A, L19P, L19Q, L19Y, L19N, L19S, L19T, L19V.

8 . The polypeptide of any one of claims 1 - 7 , wherein the modified IL-2 comprises a substitution at amino acid position M23.

9 . The polypeptide of claim 8 , wherein the substitution is selected from M23A, M23G, M23S, M23T, M23V, M23D, M23E, M23I, M23K, M23L, M23N, M23Q, M23R, and M23Y.

10 . The polypeptide of any one of claims 1 - 9 , wherein the modified IL-2 comprises a substitution at amino acid position N88.

11 . The polypeptide of claim 10 , wherein the substitution is selected from N88T, N88A, and N88S.

12 . The polypeptide of any one of claims 1 - 11 , wherein the modified IL-2 comprises a substitution at amino acid position E95.

13 . The polypeptide of claim 12 , wherein the substitution is selected from E95Q, E95G, E95T, E95V, E95P, E95H, E95N, and E95Y.

14 . The polypeptide of any one of claims 1 - 13 , wherein the modified IL-2 comprises at least one substitution that reduces affinity for CD132 compared to wild-type IL-2.

15 . The polypeptide of any one of claims 1 - 14 , wherein the modified IL-2 comprises at least one substitution at at least one amino acid position selected from Q22, R120, T123, Q126, S127, I129, and S130.

16 . The polypeptide of claim 15 , wherein the modified IL-2 comprises a substitution at amino acid position Q22.

17 . The polypeptide of claim 16 , wherein the substitution is selected from Q22A, Q22D, Q22G, Q22H, Q22K, Q22N, Q22R, Q22S, Q22T, Q22V, and Q22Y.

18 . The polypeptide of any one of claims 15 - 17 , wherein the modified IL-2 comprises a substitution at amino acid position R120.

19 . The polypeptide of claim 18 , wherein the substitution is selected from R120A, R120D, R120E, R120F, R120G, R120H, R120K, R120N, R120Q, R120S, R120V, and R120Y.

20 . The polypeptide of any one of claims 15 - 19 , wherein the modified IL-2 comprises a substitution at amino acid position T123.

21 . The polypeptide of claim 20 , wherein the substitution is selected from T123D, T123E, T123H, T123K, T123N, T123Q, and T123R.

22 . The polypeptide of any one of claims 15 - 21 , wherein the modified IL-2 comprises a substitution at amino acid position Q126.

23 . The polypeptide of claim 22 , wherein the substitution is selected from Q126N, Q126A, and Q126Y.

24 . The polypeptide of any one of claims 15 - 23 , wherein the modified IL-2 comprises a substitution at amino acid position 5127.

25 . The polypeptide of claim 24 , wherein the substitution is selected from S127D, S127E, S127H, S127K, S127N, S127P, and S127R.

26 . The polypeptide of any one of claims 15 - 25 , wherein the modified IL-2 comprises a substitution at amino acid position I129.

27 . The polypeptide of claim 26 , wherein the substitution is selected from I129A, I129H, I129R, and I129S.

28 . The polypeptide of any one of claims 15 - 27 , wherein the modified IL-2 comprises a substitution at amino acid position S130.

29 . The polypeptide of claim 28 , wherein the substitution is selected from S130E, S130K, S130N, S130P, S130Q, and S130R.

30 . A polypeptide comprising a modified IL-2, wherein the modified IL-2 comprises at least one substitution at at least one amino acid position selected from Q22, R120, T123, S127, and S130.

31 . The polypeptide of claim 30 , wherein the modified IL-2 has reduced affinity for CD132 compared to wild-type IL-2.

32 . The polypeptide of any one of claims 30 - 31 , wherein the modified IL-2 comprises a substitution at amino acid position Q22.

33 . The polypeptide of claim 32 , wherein the substitution is selected from Q22A, Q22D, Q22E, Q22G, Q22H, Q22K, Q22N, Q22P, Q22R, Q22S, Q22T, Q22V, and Q22Y.

34 . The polypeptide of any one of claims 30 - 33 , wherein the modified IL-2 comprises a substitution at amino acid position R120.

35 . The polypeptide of claim 34 , wherein the substitution is selected from R120A, R120D, R120E, R120F, R120G, R120H, R120K, R120N, R120P, R120Q, R120S, R120V, and R120Y.

36 . The polypeptide of claim 30 - 35 , wherein the modified IL-2 comprises a substitution at amino acid position T123.

37 . The polypeptide of claim 36 , wherein the substitution is selected from T123D, T123E, T123H, T123K, T123N, T123Q, and T123R.

38 . The polypeptide of any one of claims 30 - 37 , wherein the modified IL-2 comprises a substitution at amino acid position 5127.

39 . The polypeptide of claim 38 , wherein the substitution is selected from S127D, S127E, S127H, S127K, S127N, S127P, S127Q, and S127R.

40 . The polypeptide of any one of claims 30 - 39 , wherein the modified IL-2 comprises a substitution at amino acid position S130.

41 . The polypeptide of claim 40 , wherein the substitution is selected from S130D, S130E, S130H, S130K, S130N, S130P, S130Q, and S130R.

42 . The polypeptide of any one of claims 30 - 41 , wherein the modified IL-2 comprises at least one substitution that reduces affinity for CD122 compared to wild-type IL-2.

43 . The polypeptide of claim 42 , wherein the modified IL-2 comprises at least one substitution at at least one amino acid position selected from H16, L19, M23, D84, N88, and E95.

44 . The polypeptide of claim 43 , wherein the modified IL-2 comprises a substitution at amino acid position H16.

45 . The polypeptide of claim 44 , wherein the substitution is selected from H16A, H16T, H16V, and H16N.

46 . The polypeptide of any one of claims 43 - 45 , wherein the modified IL-2 comprises a substitution at amino acid position L19.

47 . The polypeptide of claim 46 , wherein the substitution is selected from L19A, L19P, L19Q, L19Y, L19N, L19S, L19T, L19V.

48 . The polypeptide of any one of claims 43 - 47 , wherein the modified IL-2 comprises a substitution at amino acid position M23.

49 . The polypeptide of claim 48 , wherein the substitution is selected from M23A, M23G, M23S, M23T, M23V, M23D, M23E, M23I, M23K, M23L, M23N, M23Q, M23R, and M23Y.

50 . The polypeptide of any one of claims 43 - 49 , wherein the modified IL-2 comprises a substitution at amino acid position D84.

51 . The polypeptide of claim 50 , wherein the substitution is selected from D84S, D84G, D84A, D84T, D84V, D84Y, and D84N

52 . The polypeptide of any one of claims 43 - 51 , wherein the modified IL-2 comprises a substitution at amino acid position N88.

53 . The polypeptide of claim 52 , wherein the substitution is selected from N88T, N88A, and N88S.

54 . The polypeptide of any one of claims 43 - 53 , wherein the modified IL-2 comprises a substitution at amino acid position E95.

55 . The polypeptide of claim 54 , wherein the substitution is selected from E95Q, E95G, E95T, E95V, E95P, E95H, E95N, and E95Y.

56 . The polypeptide of any one of claims 1 - 55 , wherein the modified IL-2 comprises at least one substitution that reduces affinity for CD25 compared to wild-type IL-2.

57 . The polypeptide of any one of claims 1 - 56 , wherein the modified IL-2 comprises at least one substitution at at least one amino acid position selected from K43, Y45, E61, I114, P65, F42, R38, and L72, and/or wherein the modified IL-2 comprises a deletion of amino acid F42.

58 . The polypeptide of claim 57 , wherein the modified IL-2 comprises a substitution at amino acid F42 or comprises a deletion of amino acid F42.

59 . The polypeptide of claim 58 , wherein the modified IL-2 comprise a substitution at amino acid position F42 selected from F42K, F42A, F42R, F42G, F42S, and F42T.

60 . The polypeptide of claim 58 , wherein the modified IL-2 comprise a deletion of amino acid F42.

61 . The polypeptide of any one of claims 57 - 60 , wherein the modified IL-2 comprises a substitution at amino acid position K43.

62 . The polypeptide of claim 61 , wherein the substitution is selected from K43E and K43D.

63 . The polypeptide of any one of claims 57 - 62 , wherein the modified IL-2 comprises a substitution at amino acid position Y45.

64 . The polypeptide of claim 63 , wherein the substitution is selected from Y45R and Y45K.

65 . The polypeptide of any one of claims 57 - 64 , wherein the modified IL-2 comprises a substitution at amino acid position E61.

66 . The polypeptide of claim 65 , wherein the substitution is selected from E61R, E61G, E61H, E61N, E61P, E61S, E61T, E61Y, E61A, E61Q, and E61K.

67 . The polypeptide of any one of claims 57 - 66 , wherein the modified IL-2 comprises a substitution at amino acid position I114.

68 . The polypeptide of claim 67 , wherein the substitution is I114F, I114Y, or I114W.

69 . The polypeptide of any one of claims 57 - 68 , wherein the modified IL-2 comprises a substitution at amino acid position P65.

70 . The polypeptide of claim 69 , wherein the substitution is selected from P65R, P65E, P65K, P65H, P65Y, P65Q, P65D, and P65N.

71 . The polypeptide of any one of claims 57 - 70 , wherein the modified IL-2 comprises a substitution at amino acid position R38.

72 . The polypeptide of claim 71 , wherein the substitution at R38 is selected from R38A and R38G.

73 . The polypeptide of any one of claims 57 - 72 , wherein the modified IL-2 comprises a substitution at amino acid position L72.

74 . The polypeptide of claim 73 , wherein the substitution at L72 is and L72G.

75 . The polypeptide of any one of claims 1 - 74 , wherein the modified IL-2 comprises substitution Q22A, or substitution R120A, or substitutions Q22A and R120A.

76 . The polypeptide of any one of claims 1 - 75 , wherein the modified IL-2 comprises substitutions P65R and R38A or substitution P65R and E61R.

77 . The polypeptide of any one of claims 1 - 76 , wherein the modified IL-2 comprises at least one substitution selected from H16A, L19A, L19N, M23A, D84S or D84Y, N88S, and E95Q.

78 . The polypeptide of any one of claims 1 - 77 , wherein the modified IL-2 comprises substitutions at amino acid positions P65, H16, and D84.

79 . The polypeptide of claim 78 , wherein the modified IL-2 comprises substitutions P65R, H16A, and D84S; or substitutions P65R, H16A, and D84Y.

80 . The polypeptide of any one of claims 1 - 79 , wherein the modified IL-2 comprises at least one substitution at at least one amino acid position selected from T3 and C125, and/or comprises a deletion of the first five amino acids of IL-2.

81 . The polypeptide of claim 80 , wherein the modified IL-2 comprises at least one substitution selected from T3A, C125A, C125V, and C125S.

82 . The polypeptide of claim 81 , wherein the modified IL-2 comprises T3A and C125S substitutions, or T3A and C125V substitutions.

83 . The polypeptide of claim 81 , wherein the modified IL-2 comprises a deletion of the first five amino acids of IL-2 and a C125S substitution or C125V substitution.

84 . The polypeptide of any one of the preceding claims, wherein the modified IL-2 comprises a set of substitutions selected from [T3A, H16A, E61R, P65R, D84Y, C125S], [T3A, H16A, M23T, E61R, P65R, D84Y, E95Q, C125S], [T3A, H16A, L19N, E61R, P65R, D84Y, C125S], [T3A, H16A, L19N, M23T, E61R, P65R, D84Y, E95Q, C125S], [T3A, H16A, E61R, P65R, D84Y, C125S, S127D], [T3A, H16A, M23T, E61R, P65R, D84Y, E95Q, C125S, S127D], [T3A, H16A, L19N, E61R, P65R, D84Y, C125S, S127D], and [T3A, H16A, L19N, M23T, E61R, P65R, D84Y, E95Q, C125S, S127D].

85 . A modified polypeptide comprising a modified IL-2, wherein the modified IL-2 comprises a set of substitutions selected from [T3A, H16A, E61R, P65R, D84Y, C125S], [T3A, H16A, M23T, E61R, P65R, D84Y, E95Q, C125S], [T3A, H16A, L19N, E61R, P65R, D84Y, C125S], [T3A, H16A, L19N, M23T, E61R, P65R, D84Y, E95Q, C125S], [T3A, H16A, E61R, P65R, D84Y, C125S, S127D], [T3A, H16A, M23T, E61R, P65R, D84Y, E95Q, C125S, S127D], [T3A, H16A, L19N, E61R, P65R, D84Y, C125S, S127D], and [T3A, H16A, L19N, M23T, E61R, P65R, D84Y, E95Q, C125S, S127D].

86 . The polypeptide of any one of the preceding claims, wherein the modified IL-2 comprises the indicated substitutions, and does not comprise any additional substitutions.

87 . The polypeptide of any one of the preceding claims, wherein the modified IL-2 is a modified human IL-2.

88 . The polypeptide of any one of the preceding claims, wherein the amino acid positions correspond to the amino acid positions in SEQ ID NO: 1.

89 . The polypeptide of any one of the preceding claims, 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 and comprising corresponding substitutions of an amino acid sequence selected from SEQ ID NOs: 105-290.

90 . The polypeptide of any one of the preceding claims, wherein 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 NOs: 270-277 and comprises substitution D84Y.

91 . The polypeptide of any one of the preceding claims, wherein the modified IL-2 comprises an amino acid sequence selected from SEQ ID NOs: 105-290.

92 . The polypeptide of any one of the preceding claims, wherein the modified IL-2 comprises an amino acid sequence selected from SEQ ID NOs: 270-277.

93 . The polypeptide of any one of the preceding claims, wherein the polypeptide comprises an Fc region.

94 . The polypeptide of claim 93 , wherein the modified IL-2 is fused to the N-terminus or the C-terminus of the Fc region.

95 . The polypeptide of claim 93 or claim 94 , wherein the Fc region comprises a substitution at Kabat amino acid position T366.

96 . The polypeptide of claim 95 , wherein the Fc region comprises a T366W substitution.

97 . The polypeptide of claim 93 or claim 94 , wherein the Fc region comprises at least one substitution at at least one Kabat amino acid position selected from T366, L368, and Y407.

98 . The polypeptide of claim 108 , wherein the Fc region comprises T366S, L368A, and Y407V mutations.

99 . The polypeptide of any one of claims 93 - 98 , wherein the Fc region comprises a substitution at a Kabat position selected from S354 and Y349.

100 . The polypeptide of claim 99 , wherein the Fc region comprises a S354C or a Y349C substitution.

101 . The polypeptide of any one of claims 93 - 100 , wherein the Fc region comprises a substitution at Kabat amino acid position H435.

102 . The polypeptide of claim 101 , wherein the Fc region comprises a substitution selected from H435R and H435K.

103 . The polypeptide of any one of claims 93 - 102 , wherein the Fc region comprises at least one substitution at at least one Kabat amino acid position selected from M252 and M428.

104 . The polypeptide of claim 103 , wherein the Fc region comprises M252Y and M428V substitutions.

105 . The polypeptide of any one of claims 93 - 104 , wherein the Fc region comprises a deletion of Kabat amino acids E233, L234, and L235.

106 . The polypeptide of any one of claims 93 - 104 , wherein the Fc region comprises at least one substitution at at least one amino acid position selected from L234, L235, and P329.

107 . The polypeptide of claim 106 , wherein the Fc region comprises L234A, L235A, and P329G substitutions.

108 . The polypeptide of any one of claims 93 - 107 , wherein the 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 selected from SEQ ID NOs: 47-83, 292, and 293.

109 . The polypeptide of any one of claims 93 - 108 , wherein the Fc region is part of a heavy chain constant region.

110 . The polypeptide of claim 109 , wherein the heavy chain constant region is an IgG constant region.

111 . The polypeptide of claim 110 , wherein the heavy chain constant region is an IgG1, IgG2, IgG3, or IgG4 constant region.

112 . The polypeptide of any one of claims 93 - 111 , wherein the modified IL-2 is fused to the C-terminus of the Fc region or heavy chain constant region.

113 . The polypeptide of claim 112 , wherein the modified IL-2 is fused to the C-terminus of the Fc region or heavy chain constant region via a linker comprising 1-20 amino acids.

114 . The polypeptide of claim 113 , wherein the linker comprises glycine amino acids.

115 . The polypeptide of claim 114 , wherein the linker comprises glycine and serine amino acids.

116 . The polypeptide of any one of claims 113 - 115 , wherein a majority, or all, of the amino acids in the linker are glycine and serine.

117 . The polypeptide of any one of claims 93 - 116 , wherein the polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 105-290 and an amino acid sequence selected from SEQ ID NOs: 48, 64, 292, and 293.

118 . The polypeptide of any one of the preceding claims, wherein the polypeptide comprises at least one antigen binding domain.

119 . The polypeptide of claim 118 , wherein the polypeptide comprises two, three, or four antigen binding domains.

120 . The polypeptide of claim 118 or claim 119 , wherein at least one antigen binding domain specifically binds to a T-cell antigen or a natural killer cell antigen.

121 . The polypeptide of any one of claims 118 - 120 , wherein at least one antigen binding domain specifically binds to a CD4 + T-cell antigen or a CD8 + T-cell antigen.

122 . The polypeptide of claim 121 , wherein the at least one antigen binding domain specifically binds to an antigen on an activated CD4 + T-cell or an activated CD8 + T-cell.

123 . The polypeptide of any one of claims 118 - 122 , wherein at least one antigen binding domain is an agonist.

124 . The polypeptide of any one of claims 118 - 122 , wherein the antigen binding domain is an antagonist.

125 . The polypeptide of any one of claims 118 - 124 , 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, CD16a, or γδTCR.

126 . The polypeptide of any one of claims 118 - 124 , wherein at least one antigen binding domain specifically binds to PD-1.

127 . The polypeptide of any one of claims 118 - 126 , wherein at least one antigen binding domain is a human or humanized antigen binding domain.

128 . The polypeptide of claim 127 , wherein each antigen binding domain is, independently, a human or humanized antigen binding domain.

129 . The polypeptide of any one of claims 118 - 128 , wherein at least one antigen binding domain comprises a VHH domain.

130 . The polypeptide of claim 129 , wherein each antigen binding domain comprises a VHH domain.

131 . The polypeptide of any one of claims 118 - 128 , wherein at least one antigen binding domain comprises a VH domain and a VL domain.

132 . The polypeptide of claim 131 , wherein at least one antigen binding domain comprises the VH domain and the VL domain of an antibody selected from pembrolizumab, nivolumab, AMP-514, TSR-042, STI-A1110, ipilimumab, tremelimumab, urelumab, utomilumab, atezolizumab, and durvalumab.

133 . The polypeptide of claim 131 or 132 , wherein the at least one antigen binding domain comprises a single chain Fv (scFv).

134 . The polypeptide of claim 131 or 132 , wherein the polypeptide comprises a heavy chain constant region, wherein the VH domain is fused to the heavy chain constant region, and wherein the VL domain is associated with the VH domain.

135 . The polypeptide of claim 134 , wherein the VL domain is fused to a light chain constant region.

136 . The polypeptide of claim 135 , wherein the light chain constant region is selected from kappa and lambda.

137 . The polypeptide of any one of claims 118 - 136 , wherein each of the antigen binding domains are the same.

138 . The polypeptide of any one of claims 118 - 137 , wherein each of the antigen binding domains specifically bind to the same antigen.

139 . The polypeptide of any one of claims 118 - 136 , 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.

140 . The polypeptide of claim 139 , 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 natural killer cell antigen other than PD-1.

141 . The polypeptide of any one of claims 118 - 140 , 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, CD16a, DNAM1, or γδTCR (Vγ9, Vγ2, Vδ1).

142 . The polypeptide of any one of claims 93 - 141 , wherein the polypeptide forms a homodimer under physiological conditions.

143 . The polypeptide of any one of the preceding claims, wherein the modified IL-2 binds a human IL-2R with an affinity 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 the IL-2R.

144 . A complex comprising a first polypeptide and a second polypeptide, wherein the first polypeptide is the polypeptide of any one of the preceding claims.

145 . The complex of claim 144 , wherein the first polypeptide comprises a first Fc region and the second polypeptide comprises a second Fc region.

146 . The complex of claim 144 or claim 145 , wherein each Fc region is an isotype selected from human IgG1, IgG2, IgG3, an IgG4.

147 . The complex of claim 146 , wherein each Fc region is a human IgG1.

148 . The complex of any one of claims 144 - 147 , wherein each Fc region comprises a deletion of amino acids E233, L234, and L235.

149 . The complex of any one of claims 144 - 148 , wherein each Fc region comprises a H435R or H435K mutation.

150 . The complex of any one of claims 155 - 160 , wherein the Fc region comprises a mutations M252Y and M428L or mutations M252Y and M428V.

151 . The complex of any one of claims 144 - 150 , wherein the first Fc region or the second Fc region comprises a T366W mutation, and the other Fc region comprises mutations T366S, L368A, and Y407V.

152 . The complex of claim 151 , wherein the first Fc region or the second Fc region comprises a S354C mutation.

153 . The complex of any one of claims 144 - 152 , 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 NOs: 47-83, 292, and 293.

154 . The complex of any one of claims 144 - 153 , wherein the second polypeptide does not comprise a modified IL-2.

155 . The complex of any one of claims 144 - 154 , wherein the first polypeptide comprises at least one antigen binding domain.

156 . The complex of any one of claims 144 - 155 , wherein the second polypeptide comprises at least one antigen binding domain.

157 . The complex of any one of claims 144 - 156 , wherein the first polypeptide comprises a first antigen binding domain, an Fc region, and a modified IL-2.

158 . The complex of claim 157 , 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.

159 . The complex of claim 157 or claim 158 , wherein the second polypeptide comprises a second antigen binding domain and an Fc region.

160 . The complex of claim 159 , wherein the first antigen binding domain and the second antigen binding domain are the same or different.

161 . The complex of claim 160 , wherein:

a) the first antigen binding domain and the second antigen binding domain both bind PD-1;

b) the first antigen binding domain binds PD-1, and the second antigen binding domain binds LAG3;

c) the first antigen binding domain binds PD-1, and the second antigen binding domain binds CTLA-4;

d) the first antigen binding domain binds PD-1, and the second antigen binding domain binds 4-1BB;

e) the first antigen binding domain binds PD-1, and the second antigen binding domain binds OX40;

f) the first antigen binding domain binds PD-1, and the second antigen binding domain binds GITR;

g) the first antigen binding domain binds PD-1, and the second antigen binding domain binds CD8a;

h) the first antigen binding domain binds PD-1, and the second antigen binding domain binds CD8b;

i) the first antigen binding domain binds PD-1, and the second antigen binding domain binds CD4;

j) the first antigen binding domain binds PD-1, and the second antigen binding domain binds NKp30;

k) the first antigen binding domain binds PD-1, and the second antigen binding domain binds NKG2A;

l. the first antigen binding domain binds PD-1, and the second antigen binding domain binds TIGIT;

m) the first antigen binding domain binds PD-1, and the second antigen binding domain binds NKG2D;

n) the first antigen binding domain binds PD-1, and the second antigen binding domain binds TGFBR2;

o) the first antigen binding domain binds PD-1, and the second antigen binding domain binds Fas;

p) the first antigen binding domain binds PD-1, and the second antigen binding domain binds CD107a;

q) the first antigen binding domain binds PD-1, and the second antigen binding domain binds NKp46;

r) the first antigen binding domain binds CD8a, and the second antigen binding domain binds TGFRβR2;

s) the first antigen binding domain binds CD8a, and the second antigen binding domain binds Fas;

t) the first antigen binding domain binds NKG2D, and the second antigen binding domain binds TGFRβR2;

u) the first antigen binding domain binds NKG2D, and the second antigen binding domain binds Fas;

v) the first antigen binding domain binds NKG2A, and the second antigen binding domain binds TGFRβR2;

w) the first antigen binding domain binds NKG2A, and the second antigen binding domain binds Fas;

x) the first antigen binding domain binds NKp46, and the second antigen binding domain binds TGFRβR2;

y) the first antigen binding domain binds NKp46, and the second antigen binding domain binds Fas;

z) the first antigen binding domain binds CTLA-4, and the second antigen binding domain binds LAGS;

aa) the first antigen binding domain binds CTLA-4, and the second antigen binding domain binds Tim3;

bb) the first antigen binding domain binds CTLA-4, and the second antigen binding domain binds OX40;

cc) the first antigen binding domain binds CTLA-4, and the second antigen binding domain binds GITR;

dd) the first antigen binding domain binds CTLA-4, and the second antigen binding domain binds CD107a;

ee) the first antigen binding domain binds CTLA-4, and the second antigen binding domain binds NKp46

ff) the first antigen binding domain binds ICOS, and the second antigen binding domain binds TNFR2;

gg) the first antigen binding domain binds γδTCR, and the second antigen binding domain binds NKG2D;

hh) the first antigen binding domain binds γδTCR, and the second antigen binding domain binds DNAM1;

ii) the first antigen binding domain binds γδTCR, and the second antigen binding domain binds TIGIT;

jj) the first antigen binding domain binds γδTCR, and the second antigen binding domain binds 4-1BB;

kk) the first antigen binding domain binds γδTCR, and the second antigen binding domain binds Fas;

ll) the first antigen binding domain binds γδTCR, and the second antigen binding domain binds NKG2A; or

mm) the first antigen binding domain binds γδTCR, and the second antigen binding domain binds CD16a.

162 . The complex of any one of claims 144 - 161 , wherein the modified IL-2 binds a human IL-2R with an affinity 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 the IL-2R.

163 . A pharmaceutical composition comprising a polypeptide of any one of claims 1 - 154 or the complex of any one of claims 144 - 162 and a pharmaceutically acceptable carrier.

164 . An isolated nucleic acid the encodes a polypeptide of any one of claims 1 - 143 or the complex of any one of claims 144 - 162 .

165 . An expression vector comprising the nucleic acid of claim 164 .

166 . An isolated host cell comprising the nucleic acid of claim 164 or the expression vector of claim 165 .

167 . An isolated host cell that expresses the polypeptide of any one of claims 1 - 143 or the complex of any one of claims 144 - 162 .

168 . A method of producing the polypeptide of any one of claims 1 - 143 or the complex of any one of claims 144 - 162 comprising incubating the host cell of claim 166 or claim 167 under conditions suitable to express the polypeptide or complex.

169 . The method of claim 168 , further comprising isolating the polypeptide or complex.

170 . A method of increasing CD4+ and/or CD8+ T cell proliferation comprising contacting T cells with the polypeptide of any one of claims 1 - 154 or the complex of any one of claims 144 - 162 .

171 . The method of claim 170 , wherein the CD4+ and/or CD8+ T cells are in vitro.

172 . The method of claim 170 , wherein the CD4+ and/or CD8+ T cells are in vivo.

173 . The method of any one of claims 170 - 172 , wherein the increase is at least 1.5-fold, at least 2-fold, at least 3-fold, or by at least 5-fold.

174 . A method of increasing NK cell proliferation comprising contacting NK cells with the polypeptide of any one of claims 1 - 143 or the complex of any one of claims 144 - 162 .

175 . The method of claim 174 , wherein the increase is at least 1.5-fold, at least 2-fold, at least 3-fold, or by at least 5-fold.

176 . A method of treating cancer comprising administering to a subject with cancer a pharmaceutically effective amount of the polypeptide of any one of claims 1 - 143 or the complex of any one of claims 144 - 162 , or the pharmaceutical composition of claim 163 .

177 . The method of claim 176 , wherein the cancer is selected from basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer; gastrointestinal cancer; glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial neoplasm; kidney or renal cancer; larynx cancer; liver cancer; lung cancer; small-cell lung cancer; non-small cell lung cancer; adenocarcinoma of the lung; squamous carcinoma of the lung; melanoma; myeloma; neuroblastoma; oral cavity cancer; ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland carcinoma; sarcoma; skin cancer; squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulval 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-cleaved 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.

178 . The method of claim 176 or 177 , further comprising administering an additional therapeutic agent.

179 . The method of claim 178 , wherein the additional therapeutic agent is an anti-cancer agent.

180 . The method of claim 179 , wherein the anti-cancer agent is selected from a chemotherapeutic agent, an anti-cancer biologic, radiation therapy, CAR-T therapy, and an oncolytic virus.

181 . The method of claim 179 or claim 180 , wherein the additional therapeutic agent is an anti-cancer biologic.

182 . The method of claim 181 , wherein the anti-cancer biologic is an agent that inhibits PD-1 and/or PD-L1.

183 . The method of claim 181 , wherein the anti-cancer biologic is an agent that inhibits VISTA, gpNMB, B7H3, B7H4, HHLA2, CTLA4, or TIGIT.

184 . The method of any one of claims 179 - 183 , wherein the anti-cancer agent is an antibody.

185 . The method of claim 181 , wherein the anti-cancer biologic is a cytokine.

186 . The method of claim 179 , wherein the anti-cancer agent is CAR-T therapy.

187 . The method of claim 179 , wherein the anti-cancer agent is an oncolytic virus.

188 . The method of any one of claims 176 - 187 , further comprising tumor resection and/or radiation therapy.